Phẫu Thuật Tạo Hình (Tập 1): Nguyên Lý Cơ Bản – Neligan (Ấn bản 4)

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CHAPTER 12 • Stem cells and regenerative medicine
Ideally, fully developed stem cell technology would enable
the utilization of prefabricated biomimetic scaffolds seeded
with these cells to address a wide range of soft tissue, skeletal,
muscle, cartilage, vascular, or joint defects. Recent studies
have shown that there may be differences in the osteogenic
and adipogenic potential of adipose-derived stromal cells
based on the subcutaneous region from which the cells are
harvested. Therefore, surgeons in the future may tailor
the region from which they harvest the therapeutic cells to
patient-specific needs.4
In addition, the potential for clinical utilization of
human stem and stromal cells is widely acknowledged
and has prompted some individuals to store their own
tissues for possible future use. One example of this is the
banking of umbilical cord-derived blood.5 Similarly, various
other adult stem cell types can be frozen for long-term
storage.
Access the Historical Perspective section online at

Human embryonic stem cells
Definitions
Research in hESCs is a rapidly developing field and has
attracted increasing attention over the last fifteen years. The
ability of hESCs to produce any cell type found in the human
body has yielded many encouraging new avenues of investi­
gation. Furthermore, hESC research promises to provide a
deeper understanding of cellular biology while generating
new treatment modalities for many diseases.37,38 In addition,
the ability of stem cells to repair and regenerate tissue hints
at the possible restoration of organ damage once thought to
be permanent and return of function once considered irrevers­
ibly lost. Despite recent controversy surrounding political and
ethical concerns over the research and clinical use of these
cells, hESCs continue to be a promising cell source in the field
of regenerative medicine.
The term “embryonic stem cell” was coined by Gail Martin
to distinguish them from previously described pluripotent
embryonal carcinoma (EC) cells derived from teratocarcino­
mas.29 ESCs possess three essential characteristics: (1) deriva­
tion from a pre-implantation or peri-implantation embryo;
(2) the capacity to self-renew and proliferate in a prolonged
undifferentiated state; and (3) the ability to form derivatives
of all three embryonic germ layers.39
As an extension of early work on ESCs in mice, hESCs were
first isolated in 1998.39 Donated fresh cleavage-stage human
regeneration, in short, involves the use of a biologically active
scaffold, seeded with a pluripotent, self-renewing cell type
such as stem cells (Fig. 12.2). The synergy of the biomimetic
scaffold with a competent cellular element (e.g. stem cells)
will ideally allow the regeneration of tissues in response to
injury.
Stem cells are defined functionally as a clonogenic popula­
tion of cells capable of self-renewal and differentiation into
committed progenitors. These progenitors can then differenti­
ate further into mature cell types and form functional tissues.3
Traditionally, stem cells have been divided into two categories
based on their differentiation potential: pluripotent stem cells
and multipotent stem cells. Pluripotent stem cells (also known
as embryonic stem cells) are capable of differentiating into any
cell type in the body, whereas multipotent stem cells (also
called adult stem cells) are more restricted in their potential
and can give rise to multiple, but not all, cell lineages. Recently,
in addition to this traditional stem cell classification, a new
class of stem cells has been described, called induced pluripo­
tent stem cells (iPS cells). iPS cells are derived from genetically
reprogrammed adult cells and are believed to have the same
differentiation potential as embryonic stem cells.
Why stem cells and regenerative medicine
should be of interest to plastic surgeons
Plastic surgeons are optimally positioned to utilize adult stem
cell populations in clinic as they have direct access to a plenti­
ful supply of adult stem cells. Specifically, plastic surgeons
can obtain large quantities of adipose tissue from liposuction
and body-contouring procedures. From this tissue, adipose-
derived stromal cells may be extracted for therapeutic use.
Fig. 12.1  Biomedical burden. Different tissue
reconstructive challenges faced by plastic surgeons. From
left to right, possible applications of tissue engineering
include treatment of chronic wounds, post-cancer ablation,
breast reconstruction, craniofacial reconstruction, and
traumatic injury.
Chronic
wounds
Post-cancer
ablation
Craniofacial
reconstruction
Breast
reconstruction
Traumatic
injury
Fig. 12.2  Differentiation of pluripotent stem cells down different multipotent
lineages. An embryonic stem cell is a pluripotent stem cell, whereas an adipose-
derived mesenchymal cell is a mesodermal multipotent stem cell.
• Gut
• Lung
• Thyroid
• Skin
• Hair
• Nerves
Endoderm
Ectoderm
Pluripotent
Multipotent
Unipotent
Mesoderm
• Muscle
• Bone
• Cartilage
• Fat
• Blood vessels

144.e1
Historical perspective
Historical perspective
Current understanding of clonal stem cell populations has
only been validated within the last 40 years. Prior to the 1960s
all dividing cells were thought to contribute to tissue growth
and turnover, and were generally classified as resident stem
cells.3 However, a revolutionizing publication in 1961 redefined
existing perceptions of stem cells by isolating a specific popula­
tion of self-renewing cells in the bone marrow.6 This particular
stem cell population was discovered by infusing a lethally
irradiated mouse with bone marrow cells capable of migrating
to the spleen and giving rise to hematopoietic cell nodules. The
cells from the nodules were then proven to have been derived
from a single cell. Cells capable of giving rise to clonal popula­
tions were named “colony-forming units” and eventually
shown to be short-term hematopoietic cells. Studies such as
this provided the framework for our current understanding of
the undifferentiated self-renewing cell population.
The presence of mesenchymal stem cells, a type of adult
stem cell, in the bone marrow was first suggested in the late
19th century by Cohneim, who proposed that fibroblasts
involved in peripheral wound healing were derived from the
marrow compartment.7 In the 1970s, Friedenstein et al. dem­
onstrated the existence of “fibroblastoid” cells within guinea
pig bone marrow by flushing out cells onto plastic culture
dishes and discarding the non-adherent cells.8 The remaining
spindle-like cells were heterogeneous in appearance but
appeared to be capable of forming colonies in vitro, giving rise
to the term colony-forming unit fibroblasts.8,9 When these cells
were subsequently transplanted into subcutaneous pockets,
they successfully formed heterotopic bone tissue.8 Using
similar methods, Castro-Malaspina et al. effectively isolated
colony-forming unit fibroblasts from human bone marrow.10
Since these early studies, subsequent investigations have
shown that these cells could be cultured in vitro and differenti­
ated into several cell types within the mesenchymal lineage.11,12
What Friedenstein isolated would later be aptly renamed by
Caplan to “mesenchymal stem cells.”11
The fundamental characterization of MSCs as a truly mul­
tipotent population of cells is attributed to work by Pittenger
and colleagues in 1999.13 Prior to this report, scientists were
unsure whether bone marrow MSCs represented a heteroge­
neous mixture of committed progenitor cells, each with
restricted potential, or individual cells capable of differentiat­
ing into fat, cartilage, bone, and muscle. Running human bone
marrow aspirates from the iliac crest of over 350 donors
through a density gradient isolation procedure resulted in a
phenotypically homogeneous population of adherent cells.
Flow cytometry analysis confirmed that over 98% of these
adherent cells shared the same surface marker profile. Single
cells were isolated from this population and re-expanded.
These new descendant cells were shown to retain the ability
to differentiate into adipocytes, chondrocytes, and osteo­
blasts.13 MSCs thus proven to have the ability to proliferate
extensively while maintaining their multipotent nature were
identified as true stem cells.
Historically, adipose tissues were presumed to function
solely as a metabolic reserve responsible for processing, storing,
and liberating high-energy materials in the form of cholesterol
and triglycerides. However, scientists in the early 1960s
described a stromovascular fraction (SVF) within adipose
tissue that contained fibroblastic-like cells.14 Subsequent
research into this SVF cell type led scientists to believe that they
represented an adipose progenitor cell with differentiation
potential limited to adipose tissue.15 But in 2001, Zuk et al.
demonstrated the ability of these SVF cells to undergo not only
adipogenic but also chondrogenic, myogenic, and osteogenic
differentiation.16 The cells were eventually renamed adipose-
derived stromal cells or ASCs. Similar to MSCs, ASCs have
since been shown to be capable of differentiating into cardiac
cells and even neural progenitor cells in vitro.16–18 Additional
investigation has revealed the differentiation potential of ASCs
to be much greater than what was initially thought possible.18–24
Cell surface markers on ASCs are similar to those of MSCs;
both express CD105, STRO-1, CD29, CD144, and CD166.24,25
Due to the abundance and ease of isolation of ASCs in com­
parison to other adult stem cell populations, investigators
have increasingly focused on the potential applications for
ASCs in regenerative medicine. For example, numerous
studies have demonstrated the regenerative potential of ASCs
when seeded onto a polyglycolic scaffold to create an osteoid-
like material.26–28
Establishment of embryonic stem cells was first reported in
1981 using pre-implantation mouse embryos.29 Cells from
blastocysts were explanted into tissue culture and developed
into four individual cell lines. When these cell lines were
transplanted into syngeneic murine hosts, they formed well-
differentiated teratocarcinomas, suggestive of pluripotent
capacity.30 However, early research using human embryonic
stem cells (hESCs) was complicated by contentious political
and ethical debate surrounding use of human embryos. Until
recently, the creation of hESCs necessitated destruction of a
human embryo, which inevitably raised various value-of-life
objections. Many of these embryos, however, had already
been destined for destruction as they were created for the
purpose of in vitro fertilization, but had been stored long past
their viable shelf-life.31
In 2009, President Obama expanded federal research
funding beyond what had been previously authorized to
include more than 100 existing cell lines, in addition to newly
created hESCs sponsored by private or state-level funding.32
Under this new provision, federal funds cannot be awarded
towards the creation of new stem cell lines if the creation
involves the destruction of an embryo. In response to this
restriction, scientists developed a novel method of hESC deri­
vation that does not necessitate damage to the embryo.33
Using a single cell biopsy technique similar to that used for
pre-implantation genetic diagnosis, researchers were able to
generate two new pluripotent cell lines without damaging the
original embryo.33 This approach additionally allows for
potential generation of matched tissue for children born
through in vitro fertilization.
An important concept in adult stem cell function is that of
the stem cell niche, first proposed by Schofield in 1978.34 A
stem cell niche describes the microenvironment in which
adult stem cells reside and includes signaling pathways and
intracellular communications that permit adult stem cells to
maintain their function as regenerative cells.35 This structural
unit is modulated by surrounding stimuli to sustain adult
stem cell populations while also ensuring that they are appro­
priately activated. Studies in hair follicle, intestinal lining, and
bone marrow have provided significant insight into the
precise spatiotemporal regulation of this niche.36

145
Human embryonic stem cells
(Fig. 12.3). For instance, technologies developed from stem
cell research can be used to manage spinal cord injuries,
degenerative neurologic conditions, as well as a variety of
genetic diseases. While stem cell investigators have also
made significant strides in the fields of diabetes, cardiac, and
hematopoietic/vascular research, treatment of spinal cord
trauma and Parkinson’s disease has recently received more
high-profile attention.
The ability for ESCs to undergo neuronal differentiation
was first demonstrated by Bain and colleagues in 1995.46 After
exposing mouse ESCs to retinoic acid, multiple cellular phe­
notypes were observed, a large percentage of which produced
neuron-like outgrowths. Gene expression analysis of these
cells revealed several neural-associated transcripts including
neurofilaments, glutamate receptor subunits, and nerve-
specific transcription factors. Furthermore, physiologic studies
demonstrated that these neuron-like cells could generate
action potentials. Paralleling these experiments, Schuldiner
et al. induced neuronal differentiation from hESCs using both
retinoic acid and nerve growth factor (NGF).47 These neural
progenitors were found to be capable of differentiation in vitro
into astrocytes, oligodendrocytes, and mature neurons.40,48
When transplanted into the ventricles of newborn mouse
brains, the hESC-derived neuronal cells distributed widely
throughout the brain and integrated in a region-specific
manner. These findings illustrated the ability of hESCs to
respond to local environmental cues in vivo to differentiate
into appropriate neural lineages. While only preliminary, the
results underscore the potential of hESCs to provide novel
therapies for the treatment of currently intractable neurologi­
cal diseases.
embryos produced by in vitro fertilization were obtained by
Thomson et al. and cultured to the blastocyst stage, typically
4–5 days post-fertilization. From the inner cell mass (which
ultimately forms the embryo), five separate hESC lines were
established and successfully maintained in culture for 6 months
in an undifferentiated state. All five lines retained the capacity
to form teratomas after injection into immunodeficient mice.
Histological examination of these teratomas revealed gut epi­
thelium, cartilage, bone, smooth muscle, neural epithelium,
ganglia, and stratified squamous epithelium. Similar findings
were also described by Reubinoff and colleagues, who derived
two additional hESC cell lines and demonstrated expression of
the transcription factor Oct-4 in these cells. Oct-4 had previ­
ously been shown to be essential for the maintenance of pluri­
potential capacity in mouse ESCs.40,41
The development of hESCs galvanized research into human
embryogenesis, development of birth defects, and cellular
mechanisms of a variety of pathologic states. In theory, hESC
can be used to treat a wide variety of genetic diseases, cancers,
diabetes, neurologic degenerative conditions, and spinal cord
injuries. Actual translation into clinical use, however, has been
hampered by problems with graft-versus-host disease. One
potential solution to this involves the development of several
hESC cell lines from variegated genetic backgrounds for tai­
lored use in patients to minimize risk of rejection. Other
strategies that have been proposed include the use of autolo­
gous donor adult stem cells or the more recently discovered
iPS cell.13,17,42
Concerns regarding xenogeneic contamination of stem
cells during culture have also been raised. hESCs are tradi­
tionally cultured in vitro using mouse embryonic fibroblast
(MEF) feeder layers.39 Without MEF feeder layers, hESCs
undergo rapid differentiation and lose pluripotency. A
feeder-free culture system has been presented by Xu and col­
leagues employing Matrigel in medium conditioned by MEFs.
However, even with this system, hESCs are still exposed to
murine products in order to maintain pluripotency.43 Fears of
xenogeneic contamination were further substantiated in 2005,
when Martin et al. reported the presence of a nonhuman sialic
acid Neu5Gc on the cell surface of hESCs.44 As humans are
unable to generate this particular sialic acid, this finding likely
represented uptake from media containing animal products
and incorporation through glycosylation. When these hESCs
and their derived embryoid bodies were exposed to human
serum, rapid binding of immunoglobulin and deposition of
complement occurred, ultimately resulting in cellular death.
Circumventing this problem necessitated the creation of
a new stem cell line under murine-free conditions. To
accomplish this, novel extracellular matrix-coated plates were
developed from MEFs and sterilized prior to use.45 When
hESCs were cultured using these plates, undifferentiated pro­
liferation was observed for 6 months and cells maintained the
capacity to form all three embryonic germ layers. This system
thus eliminated exposure of hESCs to potential contamination
from serum and/or live feeder cells and minimized the risk
of disease transmission through contact of cells with animal
or human pathogenic agents.
Current concepts and research
Studies utilizing hESCs continue to dramatically revamp
our contemporary understanding and treatment of disease
Fig. 12.3  Use of embryonic stem cells. Embryonic stem cells can be used to
study genetic changes, screen drugs and design novel therapies. EPC, endothelial
precursor cell; ESC, embryonic stem cell; HSC, hematopoietic stem cell; MSC,
mesenchymal stem cell.
Endoderm
Ectoderm
Mesoderm
HSC
Tissue-specific
stem cells
EPC
ESC
MSC

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CHAPTER 12 • Stem cells and regenerative medicine
The hESC-derived endothelial cells were then isolated using
fluorescence-activated cell sorting (FACS) for CD31 positive
cells.57 After expansion in culture, the cells were injected into
immunocompromised mice and found to form microvascular
networks. While their long-term stability is unknown, these
derived endothelial cells represent the first step towards
potential treatment of vascular disease and stimulation of
ischemic tissue growth using hESCs. They also provide a
foundation for future study of biomolecular mechanisms
involved in blood vessel formation.
Clinical correlates
Clinical use of hESCs remains in its early infancy. The
obstacles that must be overcome prior to wide clinical
utilization include: risk of tumorigenicity, immunocompat­
ibility, and successful isolation of a pure population of
desired cell types for therapeutic use. Nonetheless, work
continues on one of the most promising applications of
hESCs: neuroregeneration. Preliminary work performed in
the murine model has shown potential, leading to the first
Food and Drug Administration (FDA)-approved clinical trial
incorporating hESCs.
The capacity for regeneration of the central nervous system
is limited, making the possibility of repair and return of func­
tion through utilization of hESCs particularly compelling.
Following trauma, injuries to the brain and spinal cord can
result in demyelination secondary to death of local oligoden­
drocytes.58 While axons may be spared, action potential
propagation nonetheless can become disrupted, resulting in
irreversible loss of motor function. hESCs that have been dif­
ferentiated into oligodendrocytes, however, can potentially be
used to replace lost oligodendrocytes. As a proof of this
concept, Keirstead and colleagues evaluated the ability for
hESCs to incorporate, re-myelinate, and restore locomotion
after spinal cord injury in rats.59 In this study, spinal cord
contusion injuries were induced in Sprague–Dawley rats at
the T10 level using a controlled, reproducible force generator
capable of delivering a sudden desired impact. Oligodendro­
cytes derived from hESCs were purified and delivered both
above and below the place of contusion 7 days following
injury through direct injection into the spinal cord. Histologi­
cal analysis 8 weeks later revealed significantly greater density
of re-myelinated axons when compared to control animals.
More importantly, however, animals that received hESC-
derived oligodendrocytes demonstrated significantly higher
locomotor function scores, which continued to improve up to
1 month after the initial injury. These findings suggest that
transplantation of hESC-derived oligodendrocytes may be an
effective means of treating acute spinal cord injuries. Of note,
no teratomas were observed in this study, suggesting pre-
differentiation of hESCs prior to clinical use may obviate
concerns surrounding risk of tumorigenesis.60
The oligodendrocyte progenitor cell (OPC), a neural pro­
genitor derived from human embryonic stem cells, has been
identified as another promising cell population that could be
utilized for clinical CNS regeneration. OPCs have the potential
to commit to an oligodendroglial lineage and have been found
in a number of animal studies to reduce loss of function fol­
lowing spinal cord injuries.61, 62 In 2015, Asterias Biotherapeu­
tics announced the initiation of patient recruitment for a phase
I/IIa clinical trial to test the safety and efficacy of OPCs in
hESCs have likewise shown promise in the field of diabetes
research. At present, the only therapy that is potentially cura­
tive for type 1 diabetes is pancreatic islet cell replacement.49
Unfortunately, donor shortage has severely limited this
modality from becoming a widespread therapeutic solution.
Furthermore, the risk of potential disease transmission and
cellular rejection also place severe limitations on this treat­
ment modality. The generation of glucose-sensitive insulin-
secreting cells from mouse ESCs, however, has introduced a
new potential source of cells for treatment of type 1 diabetes.49
Culturing hESCs in suspension and allowing for embryoid
body formation led to the detection of insulin-producing cells
after 14 days of differentiation.50 Immunohistochemical stain­
ing suggests that 1–3% of the cells are positive for insulin.
More recent studies have cast some doubt on these numbers
however, estimating their true incidence to be less than 1 per
100 000 cells.51 While further research is necessary, these
studies nonetheless demonstrate the potential to derive cells
capable of β-islet function and insulin release that could theo­
retically be used to replace lost pancreatic cells in patients
with type 1 diabetes.
The study of cardiac tissue development has long been
handicapped by the lack of a suitable in vitro model. The
advent of hESC research, however, has also offered a potential
avenue for progress to be made in this area. Kehat and col­
leagues first noted the presence of spontaneously contracting
areas within hESCs cultured in suspension and plated to form
embryoid bodies.52 Cells from these regions, which made
up approximately 8% of the entire area, stained positively
for a variety of cardiac markers, including myosin, desmin,
troponin I, and atrial natriuretic factor. In addition, both posi­
tive and negative chronotropic effects were observed in the
contracting cells following application of isoproterenol and
carbamylcholine. Differentiation of hESCs into these cells
was found to be enhanced by the application of 5-aza-2′-
deoxycytidine and purified through density centrifugation
to obtain a population of cells comprised of 70% cardiomyo­
cyte progenitors.53 In 2003, Mummery et al. provided the
first demonstration of hESC cardiomyocyte differentiation
through co-culture techniques with visceral endoderm-like
cells.54 This approach mitigated the need for spontaneous
cardiogenesis and efficiently produced solid aggregates of
beating cells consisting of 10–200 cardiomyocytes. Interest­
ingly, these colonies could be frozen and were observed to
resume beating upon thawing. Collectively, the findings
strongly argue for the continued development of hESCs
as a model for cardiac research. hESC ability to differenti­
ate into cardiomyocytes in vitro and the development of
technology to enrich these cells represent important initial
steps towards the future clinical utilization of hESCs in heart
disease.
Investigators have also developed procedures to induce the
formation of vascular endothelial cells from hESCs. Endothe­
lial cells play a critical role in repair and regeneration, and
investigation into potential use of hESC-derived vascular
cells in the treatment of vascular disease is highly clinically
relevant.55 Zambidis et al. first described the presence
of mesodermal-hematoendothelial cluster colonies within
embryoid bodies at days 6–9.56 These colonies were found to
consist of non-adherent cells expressing CD45, and gave rise
to hematopoietic lineages, as well as adherent cells that
expressed markers characteristic for vascular endothelium.

147
Postnatal and somatic stem cells
individuals with complete cervical spinal cord injury (SCI).
The trial is a continuation of the California Institute for Regen­
erative Medicine (CIRM)-funded clinical trial begun by bio­
technology firm Geron in 2010 using the same kind of stem
cell to treat thoracic SCI. None of the five patients from the
initial Geron trial developed adverse effects such as tumors
or inflammation from the therapy, and MRI scans performed
throughout the follow-up period found decrease in the size of
injury site and suggested that OPCs may have had some
positive effect in preventing further spinal deterioration in
four out of five patients. Despite these findings, the Geron
trial was discontinued due to a reported change in business
strategy within the company.63 While it remains to be seen
whether positive findings from rat studies can be clinically
replicated, this represents an important first step towards the
implementation of translational therapies using stem cells to
treat human disease. Though full restoration of function fol­
lowing complete paralysis is not expected at this present time,
there is hope that patients with less severe injuries may one
day benefit from use of hESCs.64
Postnatal and somatic stem cells
Adipose-derived stromal cells
Definitions and harvest
Adipose tissue contains a stromal population composed of
three main types of cells: microvascular endothelial cells,
smooth muscle cells, and multipotent cells. ASCs have
the capacity to differentiate into adipocytes, osteoblasts,
and chondroblasts in vitro.16,17,19,65,66 However, the origin of
hASCs has not been clearly defined. Several laboratories
have hypothesized that these cells represent pericytes sur­
rounding blood vessels, which may explain their ability to
differentiate into endothelial cells; others suspect they are
a subpopulation of fibroblasts that reside within adipose
tissue (Fig. 12.4).
ASCs, like stem cells from the bone marrow, have extensive
proliferative potential and can self-renew, as well as undergo
osteogenic, chondrogenic, myogenic, and adipogenic differ­
entiation. This population can also be defined by its cell
surface markers. ASCs have been shown to be positive for the
following cell surface markers: CD105, STRO-1, CD29, CD144,
and CD166. They have been shown to be negative for the
following cell surface markers: CD3, CD4, CD11c, CD14,
CD15, CD16, CD19, CD31, CD33, CD38, CD56, CD62p, CD104,
and CD144.24,25
ASC harvest
Human ASC harvest can be performed on human lipoaspirate
or resected adipose tissue (Fig. 12.5). Lipoaspiration removes
the necessity for mincing adipose tissue, and ASCs have
been shown to retain differentiation potential even following
ultrasonic-assisted liposuction.67 If fat has been harvested
from multiple anatomic locations, these specimens should be
labeled accordingly and kept separate as material differences
exist in the differentiation capacity of adipose cells mined
from different subcutaneous depots (Fig. 12.6).4
Fig. 12.4  Unknown origin of human adipose-derived stromal cell (hASC).
Adipose-derived stromal cells are thought to be derived from adipocytes or
fibroblasts or from perivascular mural cells known as pericytes.
?
ASC
Adipocyte
Fibroblast
Pericyte
?
?
Fig. 12.5  Human liposuction of thigh.
Following harvest, the adipose tissue should be processed
in a sterile cell culture environment as soon as possible (Fig.
12.7). Adipose specimens should first be washed in dilute
Betadine, followed by two phosphate-buffered saline (PBS)
washes of equal volume. The tissue should then be digested
with an equal volume of 0.075% (w/v) type II collagenase in
Hank’s balanced salt solution at 37°C in a water bath with
agitation at 140 rpm for 60 minutes. Every 15 minutes during
digestion, the fat should be agitated and vented. Next, the
collagenase digest should be inactivated by adding an equal
volume of PBS with 10% fetal bovine serum (FBS) and 1%

148
CHAPTER 12 • Stem cells and regenerative medicine
• Resuspend pellet
with 10% FBS +
1% PCN in media
• Plate
• Collagenase digest
• Neutralization with 10%
FBS in PBS
• Centrifuge
Stem cell pellet
with fat solution
Lipoaspiration
harvest
Plated ASCs
Fig. 12.7  Overview of adipose-derived stromal cell harvest
process. Liposuction aspirate (left) is digested, and then
centrifuged to pellet the stromal vascular fraction (middle),
and then plated in 10 cm dishes for expansion (right). ASCs,
adipose-derived stromal cells; FBS, fetal bovine serum; PBS,
phosphate-buffered saline; PCN, penicillin.
Fig. 12.6  Different locations from which liposuction fat can be harvested. These
locations have been shown to demonstrate different osteogenic and adipogenic
potentials.
Abdomen
Buttock
Incision
Cannula
Flank
Thigh
penicillin/streptomycin. The stromal vascular fraction is then
pelleted via centrifugation at 1000 rpm for 6 minutes. The
supernatant is discarded and the cell pellet resuspended and
filtered through a 100-µm cell strainer to remove undigested
tissue fragments. The cells are pelleted and resuspended
in growth media, and primary culture is established in
tissue culture plates incubated at 37°C in an atmosphere of
5% CO2. Cell counting can be challenging given the large
number of erythrocytes in the final cell pellet. However, we
do not recommend a red cell lysis step as we believe this
decreases the viability of the ASCs. On average, 10 mL of
starting adipose tissue will allow the harvest of 1 million
hASCs.
Mouse ASC (mASC) harvests are done in a similar manner.
The first step involves harvesting the inguinal fat pad bilater­
ally from at least four mice. Here, it is imperative to mince the
adipose tissue finely prior to the digestion step. Subsequently,
the steps are the same as described above for hASCs: a
digestion step, followed by neutralization, centrifugation,
filtration, and plating.
Current concepts and research
Difference between mouse and human ASCs
The study of mASCs is attractive due to the relative ease of
cell harvest and the availability of laboratory mice. However,
important differences exist between mouse and human ASCs.
For example, hASCs have been observed to have a signifi­
cantly more robust in vitro osteogenic capacity when compared
to mASCs.68,69 In order to undergo significant in vitro osteo­
genesis, mASCs require an additional osteogenic stimulus
such as retinoic acid.70 Moreover, cytokines such as fibroblast
growth factor (FGF)-2 abolish osteogenic differentiation in
mASCs, whereas hASC osteogenic differentiation proceeds
relatively uninhibited both in the presence and absence of
FGF-2.69,71
In our laboratory, we observed that ASCs, whether derived
from mouse or human origin, contribute to osseous healing
of mouse cranial defects.72,73 For example, a critical-sized
mouse calvarial defect shows no healing without ASC engraft­
ment even up to 16 weeks post-injury. Upon hASC engraft­
ment, however, significant bony healing is observed in as little
as 4 weeks post-injury.73
Enrichment based on cell surface receptors
While substantial progress has been made in the study of
ASCs, further progress is hampered by the heterogeneity of
the cell population. We believe that a select subpopulation
of cells with robust ability to differentiate along an osteogenic
lineage can be further purified from the current ASC popula­
tion (Fig. 12.8). Selecting for this enriched subpopulation of
ASCs can help maximize outcomes of cell-based therapies in
skeletal regeneration. Borrowing from an approach used to
isolate hematopoietic stem cells (HSCs), the ASC subset can
similarly be enriched based on cell surface markers using
FACS. However, heterogeneity may persist even within a
specific subset of sorted ASCs. While stem cell populations
are known to be heterogeneous, the functional consequences
of this heterogeneity have not been fully elucidated. The
problem of undefined heterogeneity within stem cells must be
overcome before they can be used effectively for therapeutic

149
Postnatal and somatic stem cells
Cell surface
markers
Heterogeneous
population
Fluorescence-activated cell sorting
(FACS)
Enriched population
of cells
Fig. 12.8  Fluorescence-activated cell sorting (FACS) can
be used to separate cells based on cell surface receptors.
The goal would be to use these surface receptors to identify
cells that are more likely to differentiate down a specific
lineage. Here, FACS is used on a heterogeneous human
adipose-derived stromal cell (ASC) population to isolate an
osteoprogenitor population.
Fig. 12.9  Fluorescence-activated cell sorting (FACS) can be used to isolate cells
on a single cell level. Subsequently, human adipose-derived stromal cells can be
studied on a single cell level to delineate further the transcriptional properties of
each cell. PCR, polymerase chain reaction.
Human lipoaspirate harvest
(1 cell/well)
Isolation of single
cell mRNA
Single cell FACS
Reverse transcription
mRNA
PCR
Single cell cDNA
CD105+
CD105-
+
-
+
-
+
-
+
-
Methods of ASC delivery
Much remains unknown regarding the optimum method for
delivering ASC therapy. A significant and growing number of
studies have examined the intravenous (IV) administration of
ASCs. Such studies have investigated the natural distribution
of ASCs following IV injection. Interestingly, most organs
show uptake of ASCs after administration, including bone
and bone marrow tissues with some studies also showing
long-term persistence within the host without oncogenic
transformation.74–76 Studies have also examined IV injection
of ASCs for repair of the liver,75 heart,77 endothelium,78 and
even olfactory epithelium.79 An emerging area of investigation
involves IV administration of ASCs for autoimmune and
inflammatory disorders, such as experimental colitis and
abdominal sepsis,80–82 muscular dystrophy,83 experimental
arthritis,80 and encephalomyelitis.84 Two provocative case
studies have also been published showing beneficial outcomes
of IV-delivered ASCs in humans: the first in rheumatoid
arthritis, and the second in chronic autoimmune thrombocy­
topenia,85,86 indicating that IV ASCs may be a safe and benefi­
cial future therapeutic modality.
In vitro: protocols on differentiation
Osteogenic differentiation (Fig. 12.10)
For osteogenic differentiation, cells should be plated in a
six-well plate at a density of 100 000 cells per well, in 12-well
plates at a density of 50 000 cells per well, or in a 24-well
plate at 25 000 cells per well. After attachment, ASCs are
treated with osteogenic differentiation medium (ODM) con­
taining Dulbecco’s modified eagle medium (DMEM), 10%
FBS, 100 µg/mL ascorbic acid, 10 mM β-glycerophosphate,
and 100 IU/mL penicillin/streptomycin. This differentiation
medium can be enhanced with the supplementation of several
cytokines, such as insulin-like growth factor, platelet-derived
growth factor (PDGF), or BMP-2 to stimulate increased osteo­
genesis.87,88 ODM should be replenished every 3 days.
Early and late osteogenic differentiation have been defined
at time points that differ across species. Whereas mASCs
undergo early osteogenesis by day 7 and late osteogenesis
by day 14, hASCs undergo much more robust osteogenesis
with early osteogenesis by day 3 and late osteogenesis by
day 7. To assess osteogenic differentiation, RNA from ASCs
applications. The first step towards understanding this het­
erogeneity is to elucidate the gene expression profiles of these
complex cells. This can be accomplished through single cell
microfluidic technology (Fig. 12.9), which permits precise
mixing of nanoliter quantities of quantitative PCR reagents
to perform transcriptional analysis across individual cells.

150
CHAPTER 12 • Stem cells and regenerative medicine
red O staining and quantification or gene analysis. Specific
genes expressed by adipose tissue during differentiation
include GCP1, lipoprotein lipase (LPL), and peroxisome
proliferation-activated receptor γ (PPARγ ; Fig. 12.13).
Chondrogenic differentiation
Chondrogenic differentiation is difficult to perform in mono­
layer and is thus performed using micromass cell droplets.
Each 10 mL droplet contains 100 000 cells, which are seeded
in culture dishes and allowed to form cell aggregates and sub­
stratum at 37°C for 2 hours. Chondrogenic medium includes
DMEM, 1% FBS, 1% penicillin/streptomycin, 37.5 mg/mL
ascorbate-2-phosphate, ITS (insulin, human transferrin, and
selenous acid) premix, and 10 ng/mL transforming growth
factor-β1 (TGF-β1). This medium is carefully added around the
cell aggregates. By 24 hours, the aggregates should coalesce
and become spherical. Micromasses are fixed with 4%
paraformaldehyde/4% sucrose and embedded at day 3 and
day 6 for histological analysis. Alcian blue allows for stain­
ing of chondrogenic tissues and glycosaminoglycan assays
allow for quantification of chondrogenesis. Subsequent gene
analysis can be performed using chondrogenic gene analysis
such as SOX-9, aggrecan, and collagen II.
In vivo model
Nude athymic mouse 4 mm calvarial defect
To translate in vitro findings to the clinical realm, robust in
vivo data must be obtained to demonstrate the osteogenic
can be analyzed. Specific gene markers of osteogenic dif­
ferentiation include osteocalcin (OCN), osteopontin (OPN),
runt-related protein-2 (Runx2), collagen Ia (COL1A), and
alkaline phosphatase (ALP). ALP staining and quantifica­
tion can also be performed on day 3 of differentiation to
assess early osteogenic activity (Fig. 12.11). Subsequently,
late osteogenic activity can be assessed at day 7 of differen­
tiation by alizarin red staining, which detects extracellular
mineralization.
Adipogenic differentiation (Fig. 12.12)
To stimulate differentiation of ASCs towards mature adipose
tissue, cells are seeded in a similar density as described
for osteogenic differentiation. Adipogenic differentiation
medium, which contains 10 µg/mL insulin, 1 µM dexametha­
sone, 0.5 mM methylxanthine, and 200 µM indomethacin, is
added to the cells. At 3 days, the medium should be replaced
with 10 µg/mL insulin. Differentiation can be assessed by oil
Bone
Osteoinductive
factors
Lipoaspiration harvest of ASCs
Osteogenesis
Skeletal reconstruction
ASCs
Fig. 12.10  Potential clinical uses for liposuction-acquired fat. Adipose-derived stromal cells (ASCs) are harvested by liposuction (left), cultured in vitro with differentiation
medium (middle) to bone, and subsequently utilized for reconstructive procedures treating a myriad of diseases (right).
Fig. 12.11  Genes involved in differentiation of a bone progenitor cell to an
osteoblast. Lineage-specific differentiation is associated with the ordered sequence
expression of various genes. Osteoblast differentiation is associated with expression
of runt-related protein-2 (Runx2), alkaline phosphatase (ALP), osteopontin (OPN),
and osteocalcin (OCN), corresponding with early, intermediate, and late
osteogenesis.
Bone
progenitor cell
Runx2/ALP
OPN
OCN
Osteoblast
Fat
Adipogenic
induction
factors
Lipoaspiration harvest of ASCs
Adipogenesis
Breast/face reconstruction
ASCs
Fig. 12.12  Potential clinical uses for adipocyte differentiation of human adipose-derived stromal cells (ASCs). ASCs are harvested by liposuction (left), cultured in vitro
with differentiation medium (middle) to fat, and subsequently utilized for reconstructive procedures treating soft-tissue defects (right).

151
Postnatal and somatic stem cells
solubility. Na2SO4, KCl, and NaCl were added and the final
pH was adjusted to 6.5 (SBF 1). Mg2+ and HCO3
− free SBF (SBF
2) was prepared by adding CaCl2 and K2HPO4⋅3H2O in ddH2O
and pH was lowered to 6. KCl and NaCl were added and the
final pH was adjusted to 6.8. All solutions were sterile-filtered
through a 0.22-µm polyethersulfone (PES) membrane
(Nalgene, NY). The obtained PLGA scaffolds were incubated
in SBF 1 for 12 hours and switched to Mg2+ and HCO3
− free
SBF 2 for another 12 hours at 37°C under gentle stirring.
Coated scaffolds were washed with ddH2O to remove excess
ions and lyophilized prior to use.
We found 150 000 cells/scaffold to be optimal in our labora­
tory.73 The hASCs to be seeded onto each scaffold are first
suspended in 25 µL of growth media and placed onto the
scaffold for 30 minutes. The scaffold is subsequently sub­
merged in 100 µL of growth media for 12 hours of incubation.
Before implantation, cell-seeded scaffolds should be rinsed
in sterile PBS to prevent transfer of medium-derived growth
factors.
Clinical correlates
Recent case studies have focused on the use of hASCs to
replace bone loss.94 In case reports, defects of the calvaria,95
maxilla,96 and mandible97 have either healed or been enabled
to heal more rapidly with the use of hASCs. Such reconstruc­
tions eliminate the need for alloplastic materials and thus
reduce the risk of infection, breakdown, or rejection. Despite
increasing evidence from translational research, there is
a paucity of data defining the mechanisms through which
hASCs influence an osseous defect. Do hASCs directly form
bone to heal a skeletal defect? Or do engrafted hASCs func­
tion as efficient factories to produce potent pro-osteogenic
capacity of ASCs. In vivo models should include large immu­
nocompetent animals such as sheep89 and dogs,90,91 as well
as smaller immunocompromised animals such as rabbits,92
rats,93 or mice.28 Though several models exist to determine in
vivo osteogenic healing, we prefer the nude mouse model as it
is reliable and easily reproducible. Immunocompromised
animals such as nude athymic mice allow scientists to assess
the effect of ASCs on an area while decreasing the innate
immune response to xenotransplanted cells that can confound
results. Mice are readily available in large quantities and can
be studied by most small animal imaging technologies such
as micro-computed tomography (micro-CT), micro-positron
emission tomography (micro-PET), and bioluminescent
imaging (BLI). Mice, however, heal wounds rapidly and
effectively and thus scientists must demonstrate that any
result is superior to the animal’s baseline healing capacity.
One way to demonstrate an effect is to create a defect so
large that it overwhelms the animal’s innate ability to heal
the wound. This large defect is referred to as “critical-
sized” and does not heal even over time. An example of
this type of defect that we have utilized in an in vivo model
is a 4 mm defect in the parietal bone of a nude athymic
mouse.72
Non-healing, critical-sized (4 mm) calvarial defects can
be created in the right parietal bone of adult male CD-1
nude mice using a high-speed dental drill. After cleaning
the surgical site with Betadine, an incision is made just off
the sagittal midline to expose the right parietal bone. The
pericranium is then removed using a sterile cotton swab.
Using diamond-coated trephine bits and saline irrigation,
unilateral full-thickness critical-sized calvarial defects are
created in the non-suture-associated right parietal bone.
It is important that the dura mater be left undisturbed as
studies, including a 2011 study by Levi et al. suggest that
cell–cell interactions between dura mater and engrafted ASCs
may be crucial in stimulating osteogenic repair (Fig. 12.14).
In preparation for implantation, scaffolds can be seeded
with hASCs 24 hours prior to implantation. For scaffold
creation, apatite-coated PLGA scaffolds were fabricated from
85/15 poly(lactic-co-glycolic acid) by solvent casting and
a particulate leaching process. Briefly, PLGA/chloroform
solutions were mixed with 200–300 µm diameter sucrose to
obtain 92% porosity (volume fraction), and compressed into
thin sheets in a Teflon mold. After freeze-drying overnight,
scaffolds were immersed in ddH2O to dissolve the sucrose,
and gently removed from the Teflon plate for disinfection
and drying.
For apatite coating, simulated body fluid (SBF) solution
was prepared by sequentially dissolving CaCl2, MgCl2⋅6H2O,
NaHCO3, and K2HPO4⋅3H2O in ddH2O. Solution pH was
lowered to 6 by adding 1 M hydrochloric acid to increase
Fig. 12.13  Adipocyte differentiation is associated with expression of lipoprotein
lipase (LPL), peroxisome proliferator-activated receptor (PPARγ ), and enhancer-
binding proteins (EBPs).
Adipose
progenitor cell
LPL
C/EBPβ,δ
PPARγ
C/EBPα
Adipocyte
Fig. 12.14  A 4-mm non-healing critical-sized defect in the right parietal bone of a
CD-1 nude athymic mouse.

152
CHAPTER 12 • Stem cells and regenerative medicine
cytokines? In the case of our calvarial defect model, careful
examination of calvarial defects engrafted with ASCs yields
profitable insights into the potential derivation of healing.
For example, bone is often observed to mineralize from the
edges of a cranial defect inwards, which suggests that the
host calvarium may contribute to the bony regenerate. Con­
currently, small, isolated islands of bone are often observed
early postoperatively, which suggests either contribution
from engrafted ASCs or from host dura mater (another source
of significant osteogenic progenitors).98–100 Finally, uniform
and thorough healing of hASC-engrafted defects suggests
that primary osseous healing is derived from the engrafted
donor cells themselves. It is likely that all three cell types
(donor ASCs, host calvarial osteoblasts, and host dura mater)
contribute to bony healing.
Future direction of tissue engineering using ASCs
We believe that major breakthroughs in aesthetic and recon­
structive surgery using autologous tissue will not come
by incremental improvements to technical aspects such as
liposuction cannulas or injection syringes. Instead, novel
cell-based strategies must be explored to identify and isolate
specific fat precursors from hASCs and to coordinate the
physiologic induction of adipose tissue differentiation in a
biomimetic environment in vivo (Fig. 12.15). However a sig­
nificant gap exists between the current knowledge regarding
ASC biology and their future translation to clinical use. First,
the safety of hASCs must be determined. Potential oncogenic
transformation must be inhibited so that tumors of mesoder­
mal origin are not produced after cell engraftment.76
hASC-seeded biomaterial constructs may constitute an
optimal delivery method for autologous fat transfer by facili­
tating the assembly of natural three-dimensional adipose,
cartilage, or bone tissues.101 Our ultimate translational goal is
to harvest subcutaneous adipose tissue, isolate ASCs, and
implant these cells on an osteoconductive and/or osteoinduc­
tive scaffold into the skeletal defect within the course of a
single operative procedure without leaving the operating
room (Fig. 12.16).
Bone marrow mesenchymal stem cells
Definitions
Bone marrow MSCs are multipotent adult stem cells that
have been shown to differentiate into a variety of cell
types, including adipocytes, chondrocytes, osteocytes, skel­
etal myocytes, and cardiomyocytes. Numerous studies have
documented MSC plasticity, immunomodulatory properties,
and potential for use in tissue engineering strategies. MSCs
have also been found to be recruited to sites of injury, where
they may participate in the body’s own natural system of
tissue repair. These findings highlight the enormous potential
for MSCs in clinical use for an increasing range of medical
disorders. While some debate remains over the effect of
MSCs in various disease states, their prospects for use as
an “off-the-shelf” biological therapeutic remains a tangible
goal.
The role of MSCs within bone marrow has been debated,
with work by Prockop and others helping to elucidate func­
tions both intrinsic and extrinsic to the bone.7,102 By infusing
genetically marked MSCs into isogenic mice, studies revealed
a distribution of these cells to various locations, including
cortical and cancellous bone, cartilage, spleen, thymus, lung,
and liver.7 MSCs thus demonstrate the ability to circulate and
participate in normal cellular turnover even outside of hema­
topoietic tissues.
Current concepts and research
In recent years, the potential use of MSCs in therapeutic
modalities for tissue repair has become increasingly promis­
ing. As such, a number of studies have focused on phenotypi­
cally characterizing these cells both in vitro and in vivo. The
initial isolation of MSCs exploited their ability to adhere to
plastic. However, many of the clones isolated by Friedenstein’s
method proved incapable of osteogenic differentiation.103,104
This suggested that a considerably heterogeneous population
of cells exists within the marrow stroma. Percoll gradients, as
employed by Pittenger et al.,13 Caplan,105 and Haynesworth
et al.,106 allowed for a more homogeneous population of MSCs
to be harvested, as confirmed by flow cytometric analysis of
cell surface antigens. Alternative methods to obtain higher
degrees of homogeneity have also been attempted, includ­
ing culturing MSCs in low oxygen tension.107,108 Perhaps the
most rigorous means by which to define MSCs has been
through the use of cell surface antigens expressed in vitro.
The International Society for Cellular Therapy announced
A
(-) Scaffold
(-) ASCs
Fig. 12.15  Utilization of adipose-derived stromal cells (ASCs) to heal a skeletal
defect. (A–C) Micro-computed tomography images of a 4-mm critical-sized mouse
parietal bone defect in nude CD-1 mice. (A) Without either a scaffold or ASCs, this
defect will not heal by 2 weeks. (B) Scaffold only. Wounds treated with scaffold
alone also show minimal healing. (C) Scaffold with ASCs. Significant
re-mineralization is observed after only 2 weeks post-injury in wounds treated with
ASC-seeded scaffolds.
B
(+) Scaffold
(-) ASCs
C
(+) Scaffold
(+) ASCs

153
Postnatal and somatic stem cells
Fig. 12.16  Ultimate clinical translational goal: taking patient to the operating room and harvesting a multipotent, self-renewing cell population such as human adipose-
derived stromal cells (hASCs), and then placing them on a scaffold for reconstruction of a tissue defect. FACS, fluorescence-activated cell sorting.
Patients anesthetized
and lipoaspiration
performed
Seed hASCs on a
biomimetic scaffold
Single cell FACS
Construct implanted in same procedure
for in vivo tissue engineering to
regenerate calvarial bone
specific criteria to define bone marrow MSCs in 2006.109
Based on these criteria, MSCs are required to be plastic-
adherent when maintained in standard culture conditions
and to express CD73 (ecto 5′-nucleotidase), CD90 (Thy-1),
and CD105 (Endoglin) while lacking expression of CD45
(protein tyrosine phosphatase), CD34 (hematopoietic cluster
differentiation molecule 34), CD14 (macrophage/neutrophil
cluster differentiation molecule 14), CD19 (follicular dendritic
cell/B-cell cluster differentiation molecule 19), and human
leukocyte antigen (HLA)-DR. Furthermore, MSCs must be
capable of differentiation into adipocytes, chondroblasts, and
osteoblasts in vitro. While these criteria continue to evolve,
they have served as a minimum standard for more recent
investigations.
While the in vitro characterization of MSCs has progressed,
phenotypic delineation of these cells in vivo has advanced at
a much slower pace and collective data remain far from
complete. More recently, investigators enriched the STRO-1
fraction through additional positive selection for CD106
(vascular cell adhesion molecule-1: VCAM-1) and CD146
(melanoma cell adhesion molecule), obtaining a purified frac­
tion capable of self-renewal and multipotent differentiation.110
These findings lend further support to the notion that MSCs
function as an in vivo stem cell source for derivation of mes­
enchymal tissue.
The lineage of MSCs and HSCs has also been carefully
evaluated by investigators looking to determine whether
MSCs and HSCs exist as different populations within
bone marrow. Through sex-mismatched HLA-identical
bone marrow allografts, researchers have demonstrated that
marrow-derived stromal cells are exclusively host-derived
while HSC-derived macrophages originate from donors.111
This finding indicates that MSCs existing within the stromal
fraction are distinct from hematopoietic cells. Contemporary
models thus identify at least two types of stem cells within
the bone marrow, with HSCs giving rise to hematopoietic cell
types and osteoclasts, while MSCs differentiate into a multi­
tude of mesenchymal lineages. Furthermore, it is widely
believed that MSCs are derived from mesoderm and that, as
these cells stain positive for angiopoietin-1, they likely reside
with pericytes located along the walls of sinusoidal blood
vessels in bone marrow.112,113
Recent investigations have identified an extraordinary
property of MSCs with important therapeutic implications.
Although still poorly understood, MSCs have been found to
possess immunomodulatory properties.114–119 Baseline expres­
sion of MHC class I proteins has been detected in MSCs while
MHC class II proteins are entirely absent.120 This particular
profile has been associated with non-immunogenic conditions,
thus potentially allowing for MSC transplantation into alloge­
neic hosts without the requirement of immunosuppressive
therapy.121 Even more surprising, MSCs have been found to be
capable of modulating T-cell function.114,115,117,119 In co-culture
conditions, MSCs have been shown to decrease expression
levels of pro-inflammatory cytokines, including tumor
necrosis factor-alpha, interferon-gamma, interleukin-4, and
interleukin-10 in dendritic cells and helper T cells.115 MSC
secretion of interleukin-1 receptor antagonist has also been
reported to contribute to overall MSC-mediated immunosup­
pression.122 From a clinical perspective, these properties may
one day be exploited for treatment of conditions such as graft-
versus-host disease and autoimmune disorders.121,123

154
CHAPTER 12 • Stem cells and regenerative medicine
MSCs preferentially migrate to sites of inflammation, thereby
allowing for their potential participation in repair of injured
cardiac muscle.129 Myocardial ischemia may promote release
of chemo-attractive factors, increase vascular permeability,
and enhance expression of adhesion proteins to allow for
proper homing of MSCs. A 2015 randomized, double-blind,
placebo-controlled trial conducted by Mathiasen et al. found
that intra-myocardial injections of MSCs decreased left ven­
tricular end systolic volume (LVESV) in patients suffering
from New York Heart Association heart failure classes II–III
with ejection fraction <45%. In contrast, patients treated with
placebo had increased LVESV at six months follow-up. No
adverse effects were identified during the course of the study.
They concluded that intra-myocardial injections of autologous
culture-expanded MSCs were safe for clinical use and
improved myocardial function in patients with severe ische­
mic heart failure.130 How these cells truly act at post-infarct
sites, however, remains controversial.
Like myocardial infarction, injury to the central nervous
system has also contributed to significant death and disability
worldwide. As investigators have shown MSCs to be capable
of undergoing transdifferentiation into ectoderm-derived
neuronal tissue, MSC use in various animal models of stroke
has yielded provocative findings.131 Several studies have
demonstrated that injected MSCs can migrate to sites of
stroke injury and differentiate into cells expressing neuronal
markers.132–134 Preliminary reports in rats have also shown that
MSCs may potentially restore damaged neurons.133,134 From a
functional perspective, improved sensorimotor function has
been detected following injection of MSCs into sites of cortical
ischemia.135
The treatment of diabetes has been another field where use
of MSCs has provided some promising results. Presently, type
1 diabetes may be partially reversed through the replacement
of functional pancreatic β cells. Unfortunately, islet cell trans­
plantation has been limited by immune rejection and contin­
ued autoimmune-mediated destruction.136 The scarcity of
donor islet cells further obstructs treatment of this disease.
The potential ability of MSCs to modulate the immune system
and undergo in vitro β-cell transdifferentiation has led to
several preclinical investigations employing these cells in
animal models of diabetes.136–139 As with cardiac ischemia
and stroke, however, the true role of MSCs in ameliorating
hyperglycemia remains controversial. Introduction of human
MSCs into immunocompromised diabetic mice was found to
upregulate only mouse insulin production. This suggests that
MSCs promote regeneration of endogenous islets but do not
undergo transdifferentiation and integration into the host
pancreas.139 Nonetheless, these findings indicate that MSCs
may one day represent a viable therapeutic option for the
treatment of type 1 diabetes.
The potential use of MSCs most germane to the field of
plastic and reconstructive surgery remains the utilization of
these cells in bone and cartilage regeneration. Bone homeo­
stasis represents a careful balance between bone production
by mesenchymal lineage osteoblasts and bone resorption by
hematopoietic lineage osteoclasts.121 MSCs potentially alter
this balance through the modulation of receptor activator
of nuclear factor-κB ligand (RANKL) and osteoprotegrin
expression.121
It has been suggested that a loss of MSC function may
contribute to reduced bone regeneration with age.140 MSCs
In vitro: protocols on tissue harvest
and differentiation
Contemporary technologies to harvest and culture MSCs still
utilize the same fundamental property of adherence to plastic
employed by Friedenstein et al. over 40 years ago.8 More
recently, investigators have incorporated the use of density
gradients to increase homogeneity and eliminate unwanted
cell types present in marrow aspirate.13 Human MSCs are
commonly harvested from the iliac crest.124 Murine MSCs,
in contrast, can be harvested from either fibula or tibia by
first flushing the marrow contents out with a 27G syringe.125
Harvested bone marrow is then placed in an aqueous solution
of highly branched hydrophilic polysaccharides. By centrifug­
ing for 30 minutes, four layers become visible. Red blood cells
may be found in the bottom layer, polysaccharide solution
may be found in the second layer, the third layer contains
the majority of mononuclear cells, and the top layer contains
primarily acellular medium. It is the third layer (mononuclear
cells) that is isolated, re-centrifuged, and plated on to plastic
culture dishes. MSC colonies may often be observed to form
on these dishes after 6–8 days of in vitro culture.
Lineage-specific culture conditions that induce differentia­
tion of MSCs into adipocytes, chondrocytes, osteoblasts, and
myoblasts have been well described. Adipogenic differentia­
tion can be induced by treatment of MSCs with 1-methyl-3-
isobutylxanthine, dexamethasone, insulin, and indomethacin.13
Fat accumulation can be readily appreciated histologically by
the formation of lipid-rich vacuoles within cells. In addition,
differentiated MSCs have been shown to express various
markers of adipogenic differentiation, including PPARγ 2,
LPL, and fatty acid-binding protein aP2 (see Fig. 12.13). To
promote chondrogenic differentiation, MSCs are cultured
serum-free in a pelleted “micromass” with TGF-β3.13 A
proteoglycan-rich extracellular matrix can be observed along
with expression of type II collagen after 2 weeks, both of
which are typically found in articular cartilage. Osteogenic
differentiation of MSCs can be accomplished with the same
components as described in the previous section on ASCs.
Finally, culture of MSCs with dexamethasone and hydrocor­
tisone has been shown to induce expression of key regulatory
factors for myogenic differentiation, including MyoD1 and
myogenin.126
In vivo models
The ability for MSCs to differentiate along several lineages has
made these cells excellent candidates for the repair and regen­
eration of many tissues. The multipotency of MSCs, along
with their ease of isolation and expansion potential, has led
to a variety of preclinical studies evaluating their use in treat­
ing cardiovascular disease, central nervous system injury,
diabetes, and bone/cartilage regeneration.
Cardiovascular disease remains one of the leading causes
of morbidity and mortality worldwide, accounting for nearly
20 million deaths annually.127 Contemporary approaches to
treatment have centered on preservation of myocardium
through pharmacologic therapy and revascularization. The
ability for MSCs to undergo transdifferentiation along
a cardiomyogenic lineage has sparked recent interest in
their application for post-infarct myocardial regeneration.128
Importantly, in vivo studies have demonstrated that injected

155
Postnatal and somatic stem cells
begin incorporating these cells into clinical trials for an ever-
increasing range of potential medical disorders.121,123 MSCs
have been proposed for the treatment of a wide variety of
diseases and patients have already begun enrolling in
industry-sponsored studies (Osiris Therapeutics, Waltham,
MA) that investigate the use of these cells in graft-versus-host
disease, Crohn’s disease, and type 1 diabetes. The FDA has
also approved the study of autologous MSCs in middle cere­
bral artery acute stroke treatment. To date, the most prominent
application of MSCs in clinical trials has been their use in
ischemic heart disease.
Following ischemic insult to the myocardium, reperfusion
may salvage large regions of viable muscle, but this is often
incomplete, resulting in a process of adverse left ventricular
remodeling.147 As previously noted, preclinical trials have
shown that autologous MSCs improve cardiac function mod­
estly either through transdifferentiation or elaboration of
trophic factors capable of stimulating endogenous reparative
mechanisms.148,149
Similar to ischemic cardiomyopathy, MSCs have also
proven to be efficacious in early clinical studies involving
skeletal disease. Long-bone distraction osteogenesis is an
emerging field in which MSCs have been found to promote
improved healing and faster recovery rates.150 In 17 patients
undergoing distraction, injection of MSCs along with platelet-
rich plasma into the callus during both lengthening and
consolidation phases resulted in accelerated mature bone
formation and a reduction in the incidence of complica­
tions.151,152 Horwitz and colleagues have also investigated the
use of MSCs in the treatment of osteogenesis imperfecta, a
genetic disorder of type I collagen resulting in fragile bones
and skeletal deformities.153–155 While there is no current cure
for osteogenesis imperfecta, preliminary reports have shown
MSCs to be successful in enhancing bone formation in these
patients.154 Children receiving two infusions of allogeneic
MSCs were noted to demonstrate engraftment of these cells
in multiple sites, including bone and skin. Importantly, accel­
erated growth velocity was appreciated during the first 6
months following treatment with no associated complica­
tions.153 These findings reveal that MSCs may be safely
administered to children and when injected can localize to
genetically defective tissues. Furthermore, MSC therapy has
been demonstrated to be safe and effective in small numbers
of patients treated with MSCs to ameliorate clinical symptoms
of osteoarthritis. The 2015 study found that in patients with
severe knee osteoarthritis, the knee injected with MSC had
better function at 5 years compared to the knee that was not
injected.156 Another 2015 study expanded on this finding by
demonstrating long-term safety and efficacy of intra-articular
MSC injection in patients with different osteoarthritic joints
(such as hip, knee, and ankle osteoarthritis).157 In summary,
while the human data at present is promising, further studies
are needed with larger sample sizes and longer follow-up
periods to confirm the clinical benefit and safety of MSC use
in bone regeneration. There are myriad clinical trials currently
underway to address these important questions, with an
excess of 140 clinical trials at various stages of investigation
recorded in the US at present.
The breadth of clinical trials involving MSCs is rapidly
expanding, as studies have shown these cells to possess a
strong propensity to ameliorate a wide range of tissue deficits
secondary to injury or disease.
have thus been evaluated as potential cellular building blocks
for tissue engineering strategies. Using a canine femoral
critical-sized defect model, investigators have shown MSCs
to be capable of regenerating bone when loaded onto a
hydroxyapatite-tricalcium phosphate cylinder.90,91,141 Sixteen
weeks after introduction of MSCs, histological analysis
revealed new bone spanning the entire defect.91 In addition,
both autologous and allogeneic MSCs were found to yield
equivalent results with no lymphocytic infiltration or antibody
formation noted when leukocyte antigen-mismatched alloge­
neic cells were implanted.91
Vascular composite allotransplantation (VCA), includ­
ing osseous defects, is another growing area of interest
in reconstructive surgery. Interestingly, MSCs have been
shown in animal models to increase regulatory T-cell levels
and prolong survival of immunogenic skin-bearing VCA
osseous grafts when combined with a short course of
immunosuppression.142
Apart from the appendicular skeleton, investigators have
also studied the use of MSCs in promoting bone formation for
the spine. Spinal fusion remains the last resort for degenerative
disc disease and over 300 000 cases are operated on annually
in the US.121 Current research has focused on improvement
of spinal fusion through the combination of autologous bone
marrow with synthetic scaffolds and cytokine therapy. MSCs
implanted on Matrigel and placed into the lumbar fusion bed
of rats have been shown to result in a greater rate of successful
fusion compared to animals receiving only mixed-marrow
stromal cells.143 Kai et al. reported similar results in rabbits
using a combination of MSCs, calcium phosphate ceramic
blocks, and recombinant human BMP-2.144 These results
suggest that MSCs may be a suitable replacement for more
traditional methods currently employed in iliac crest bone
grafting. MSCs may also allow for greater mature osseous
tissue formation at earlier time points following spinal
fusion.
Repair and regeneration of cartilage remain another
challenging problem where MSCs may yield more compel­
ling strategies than those currently available. The ability to
treat damaged cartilage and osteoarthritis is currently quite
limited with prosthetic joint replacement representing the best
contemporary solution. Preclinical trials in goats, however,
have shown MSCs to promote cartilage regeneration in knee
joints.145 Following surgical induction of osteoarthritis by
excision of the medial meniscus and resection of the anterior
cruciate ligament, Murphy and colleagues found that a single
injection of MSCs suspended in dilute sodium hyaluronan
could result in reduction of articular cartilage degeneration,
osteophytic remodeling, and subchondral sclerosis.145 These
findings have also been observed in a mouse model for
human rheumatoid arthritis where allogeneic MSCs intro­
duced intraperitoneally were found to prevent occurrence of
severe irreversible damage to cartilage and bone.146 Rather
than directly providing a cellular contribution to cartilage
formation, in this instance MSCs were thought to modulate
immune responsiveness and expression of inflammatory
cytokines.146
Clinical correlates
The last two decades of research with MSCs in both in vitro
culture and in vivo animal models has allowed scientists to

156
CHAPTER 12 • Stem cells and regenerative medicine
division describes the generation of two daughter cells that
eventually acquire the same differentiated fate. Asymmetric
division describes the process whereby a stem cell produces
one daughter cell with a stem cell fate and another daughter
cell destined for differentiation. These models are not mutually
exclusive and are both thought to play an important role in the
maintenance of skin stem cell populations.168
Bone
Osteoblasts are derived from bone marrow MSCs and their
differentiation pathways have been well studied. These cells
form bone, regulate bone growth and remodeling, and dif­
ferentiate into mature osteocytes in response to stimuli such
as mechanical loading, hormones, and cytokines.169 A delicate
balance is maintained with their osteoclast counterparts, which
resorb bone and are derived from monocyte/macrophage
precursors. During adult osteogenesis, bone formation
proceeds through two different pathways: intramembranous
ossification (e.g., skull) or endochondral ossification (e.g.,
long bones). The former occurs directly from MSC condensa­
tion while the latter involves an intermediate MSC-mediated
cartilage formation step followed by replacement with bony
matrix (Fig. 12.18).170
Osteoblast differentiation is regulated by several major
signaling pathways such as Wnt, BMP, FGF, Hedgehog and
transcription factors such as Runx2, Osterix, ATF4, and TAZ.171
These signals determine the fate of skeletal lineage precursors
and can be manipulated to induce bone regeneration. BMP-2,
-4, and -7 have been successfully used to induce osteoblast
differentiation and matrix formation in vivo and vehicles for
BMP gene delivery such as plasmids and viruses have been
effectively employed.172 Combination gene delivery has been
Tissue-specific stem cells
Definitions
Adult stem cells are undifferentiated cells found in almost
all tissues and organs after embryonic development158 and
have the capacity for both self-renewal and differentiation
into a restricted number of specialized cell types of the tissue
or organ in which they are found. Their major function is to
maintain homeostasis and ensure that tissue damage resulting
from various forms of insult or injury is repaired. It has been
hypothesized that defects in adult stem cell function may be
responsible for a range of diseases. A study of adult stem cell
function could therefore reveal important biological mecha­
nisms that could be exploited for therapeutic purposes.159
Although rare, adult stem cells serve a critical function
within the hierarchical organization of complex tissues and
organs. They give rise to replacement cells to maintain tissue
integrity throughout development. A model of coexisting
quiescent and active progenitor subpopulations has been
proposed to describe adult stem cells with different cell cycle
states.160 It is hypothesized that they exist in tightly regu­
lated zones to ensure that differentiated cells can be rapidly
repopulated while keeping a quiescent back-up system to
maintain the longevity of adult stem cell pools. Another
concept which has been studied in adult stem cells is transdif­
ferentiation, or the ability of lineage-directed stem cells to
differentiate into tissues or organs from another embryonic
lineage.161 For example, studies in mice have demonstrated
the ability of bone marrow stem cells to differentiate into
multiple cell types.
Current concepts and research
Skin
Mammalian skin is a widely available and readily accessible
tissue for the study of adult stem cell function. Gene expres­
sion studies have identified critical regulators of epithelial
stem cell function, including Wnt/beta-catenin, BMP, notch,
and Hedgehog pathways.162 Furthermore, numerous subtypes
of epithelial progenitors have recently been identified and
contribute to different skin compartments containing specific
resident epithelial stem cells, such as the hair follicle, seba­
ceous glands, and the interfollicular epidermis (Fig. 12.17).163
Hair follicle stem cells are located in the bulge region and
cycle slowly under normal conditions. However, following
injury they rapidly activate and migrate outwards toward the
region of epithelial damage.164
These epithelial stem cells are located in discrete skin
compartments but have common features, which may reveal
unifying mechanisms in normal homeostasis. They all express
K5, K14, and delta Np63, are adjacent to an underlying base­
ment membrane, and utilize many of the same regulatory
signaling pathways.165 The complex skin environment also
houses putative melanocyte stem cells involved in the graying
of hair166 and dermal pericytes which have been implicated as
important stem cell regulators of skin regeneration.167
Many of the mechanisms underlying skin embryogenesis are
recapitulated later in adult life to maintain skin homeostasis.162
Several models have been proposed to explain the balance of
stem cell populations and function. One such model describes
asymmetric versus symmetric cell division. Symmetric cell
Fig. 12.17  Cells surrounding the hair follicle traffic to a wound site. IFE,
interfollicular epidermis; IRS, inner root sheath; MSCs, mesenchymal stem cells;
ORS, outer root sheath.
Dermis
Sweat
gland
Hair
follicle
shaft
Pilosebaceous
unit
Arrector
pili muscle
Sebaceous
gland
Hair follicle
bulge with
epi stem cells
IFE with
epi stem cells
Dermal papilla
with MSCs
ORS
IRS
Subcutaneous
tissue

157
Postnatal and somatic stem cells
(Fig. 12.19).178 However, it has been discovered that the vas­
cular programming evident during embryonic development
is recapitulated in various postnatal states during a process
known as adult vasculogenesis (see Fig. 12.19).179
Endothelial precursor cells (EPCs), which are bone marrow-
derived progenitor cells that participate in adult vasculogen­
esis, were first identified by Asahara et al.180 These cells are
recruited to the site of ischemia and divide to form syncytial
masses, which tubularize and canalize to form a patent vas­
cular network.181 Pericytes, which retain the pluripotency of
MSCs,112 are also intimately involved in vascular morphogen­
esis. They reside at the interface between endothelial cells and
the surrounding tissue and produce pro-angiogenic signals
that regulate endothelial cell differentiation and growth.182
Through both direct physical interaction and paracrine
signaling, endothelial cells and pericytes engage in complex
crosstalk that is essential for normal vasculogenesis.183
Muscle
Satellite stem cells were first described by Mauro in the 1960s
as mononucleated cells situated between muscle fibers and
their basement membrane (Fig. 12.20).184 Though different
modes of asymmetric cell division have been proposed
to regulate satellite cell self-renewal,185,186 symmetric cell
division is believed to play a major role in the response to
muscle injury or when large amounts of myogenic cells are
needed to replace missing or dysfunctional muscle mass.186
Repair mechanisms are also thought to involve recruitment
of neighboring satellite cells and circulating myogenic stem
cells. In addition, satellite cell dysfunction has been proposed
successful and is thought to recapitulate more closely the
signaling environment in vivo, providing a synergistic stimu­
lus for bone formation. This sensitization effect has been
demonstrated with combinations of BMPs, vascular endothe­
lial growth factor (VEGF), RANKL, and Runx2 delivery.172
Blood vessels
Regulated blood vessel development in response to tissue
injury or ischemia is critical for maintenance of healthy tissues.
Dysfunction in this neovascularization process often results
in disease.173 For example, adequate wound healing requires
a robust vascular response to deliver immune cells and
metabolic substrates necessary for tissue regeneration.174 In
addition, a coordinated pattern of neovascularization signal­
ing and stem cell recruitment is essential for normal organ
development during embryogenesis,175 as well as for tumor
growth and metastasis.176 During embryogenesis, mesoderm-
derived angioblasts organize via vasculogenesis to form
blood vessels.177 It was initially believed that all subsequent
blood vessel growth occurred through sprouting of pre-
existing endothelial cells in a process known as angiogenesis
Fig. 12.18  Mesenchymal stem cell (MSC) condensation into an osteoblast as in
intramembranous ossification.
Osteoblast
MSC
Bone
Fig. 12.19  Sprouting of pre-existing endothelial cells in a
process known as angiogenesis (left). Formation of new vessel
from circulating endothelial progenitor cell known as
vasculogenesis (right).
Angiogenesis
Vasculogenesis
Neovessel
Neovessel
Local sprouting
Systemic recruitment
Endothelial
cell
Endothelial
progenitor
cell
Fig. 12.20  Muscle satellite cells located between muscle fibers and their
basement membrane.
Myofiber nuclei
Satellite cell
Muscle fiber

158
CHAPTER 12 • Stem cells and regenerative medicine
Clinical correlates
Skin
Cell-based therapies have been used for decades in the treat­
ment of burn injury. Autologous epidermal sheets have been
grown ex vivo and re-implanted into patients with varying
degrees of success.191 Bone marrow and fat-derived MSC
therapies have proven useful for chronic wounds in the clini­
cal setting, although these are mostly case reports.192 Dash
et al. reported a randomized clinical trial using autologous
bone marrow MSCs to treat chronic non-healing ulcers.193 In
this study, stem cell-treated patients demonstrated improve­
ments in pain-free walking and reductions in ulcer size up to
12 weeks post-treatment.
Transplantation of HSCs from peripheral blood and
bone marrow has also demonstrated engraftment of cells
into skin.194 Histological evaluation of skin specimens from
sex-mismatched transplants showed that up to 7% of cells
were XY-positive, indicating that circulating stem cells could
differentiate into mature epithelium. However, another
study with a similar sex-mismatched transplant design
failed to show donor stem cell contribution to keratinocyte
populations.195 Badiavas and Falanga reported on 3 patients
with chronic lower extremity wounds refractory to standard
therapy who were treated with autologous bone marrow
stem cells.196 Complete wound closure was observed in all
3 patients and there was less wound fibrosis compared to
pretreatment wound specimens. It appears that numerous
adult stem cell populations can contribute to skin regenera­
tion but the optimal combination of cells and signals remains
unknown.
Bone
As previously mentioned, allogeneic bone marrow-derived
stem cells transplanted with around 2% donor cell engraft­
ment were shown to improve osteogenesis in three children
with osteogenesis imperfecta.154 Three months after treatment,
all patients had increased total bone mineral content, growth
velocity, and reduced frequency of bone fracture. In addition,
MSCs have shown great promise in early clinical trials in the
symptomatic improvement of osteoarthritis in multiple joints
as described in a previous section,156,157 and osteonecrosis of
the femoral head.197–199 For instance, a study published in 2015
by Daltro et al. found that autologous stem cell transplanta­
tion provided both pain relief and stopped the progression of
osteonecrosis of the femoral head in patients with sickle cell
disease. The focus of MSC therapy in currently active clinical
trials include: articular cartilage resurfacing with MSCs in
osteoarthritis, non-union of fractures, periodontal disease,
and spinal fusion.
Blood vessels
Studies have demonstrated a strong correlation between
clinical risk factors for cardiovascular disease and EPC
function and quantities. Disease states such as diabetes,
coronary artery disease, smoking, and hypercholesterolemia
are known to affect EPCs adversely, prompting efforts to
improve clinical outcomes through EPC augmentation.200
Direct injection of EPCs, as well as growth factors, such
as granulocyte–macrophage colony-stimulating factor have
been used with some success following cardiac ischemia. A
to underlie certain muscular diseases such as Duchenne
muscular dystrophy, but this hypothesis requires further
study.
The major components of the satellite cell niche are: the
adjacent muscle fiber (including the mechanical, electrical,
and biochemical signals which emanate from it), the basal
lamina, and the microvasculature. These interrelated compo­
nents regulate the activity of muscle stem cell populations and
include calveolin-1, sphingomyelin, calcitonin receptors, the
transmembrane protein Megf10, Notch signaling, and matrix
components such as proteoglycans.186
Peripheral nerve
Although peripheral nerves have the capacity to regenerate
axons following injury, outcomes after repair remain poor due
to direct neuronal damage, as well as chronic denervation of
the distal end.187 However, when denervated supporting
Schwann cells are replaced with healthy ones at the site of
injury, nerve regeneration is improved (Fig. 12.21). This sug­
gests that cell-based therapies may have an important role in
treating nerve injuries. Guenard et al. demonstrated that
syngeneic Schwann cells could effectively improve nerve
regeneration in rat sciatic nerve injuries.188 Murakami et al.
transplanted neural stem cells obtained from the hippocam­
pus of rat embryos into sciatic nerve defects and demonstrated
nerve regeneration after 8 weeks.189 Mean number of myelin­
ated fibers and fiber size were increased compared to
controls.
Neurotrophic growth factors have also played an impor­
tant role in experimental models of nerve repair. These
include NGF, brain-derived neurotrophic factor (BDNF),
neurotrophin-3 (NT-3), NT-4/5, FGF, and PDGF, amongst
others.190 Neurotrophic growth factors are delivered through
matrix/conduit release or mini-pump systems but bioavail­
ability throughout the necessary regenerative time scales
remains an issue.
Fig. 12.21  Schwann cell migration to peripheral nerve injury site to assist in
repair.
Schwann cell
Peripheral nerve injury

159
Prospective clinical applications of stem cell therapy
addition to safety, 12-month data analysis also revealed sus­
tained improvements in sensory function that emerged consis­
tently around three months after transplantation and persisted
until the end of the study. The patterns of sensory gains were
confirmed to involve multiple sensory pathways and were
observed more frequently in the patients with less severe
injury. HuCNS-SCs were also shown to be safe and effective in
the treatment of congenital neurodegenerative diseases.206,212
Further investigation is now underway with larger patient
numbers, to build upon promising phase I/II clinical data.
Prospective clinical applications of
stem cell therapy
Scaffolds for stem cell delivery
Stem cells exist in tightly controlled environmental niches and
alterations in this microenvironment can dramatically modify
their behavior and capabilities. Furthermore, they are often
utilized in the setting of disease or injury where toxic signals
that can impair their function are prevalent. Thus, strategies
using biomaterial scaffolds are important to stem cell therapy
as they help provide a controlled environment in which
implanted cells can be protected from harmful stimuli. Bio­
material matrices can also be used to deliver genetic material
and/or inductive biochemical cues, which allow for some
degree of developmental control over the delivered stem cells
(Fig. 12.22). In terms of tissue and organ regeneration, scaf­
folds provide the structural template on which to build new
tissues and can thus be prefabricated based on their ultimate
purpose. For example, more rigid scaffolds are much better
meta-analysis found that intracoronary bone marrow cell
infusion is a safe way to administer EPCs and is associated
with a minor improvement in left ventricular function at 3–6
months following acute myocardial infarction.201
Muscle
Satellite cells have generally been incompatible with systemic
administration and exhibit poor viability and migration when
delivered intramuscularly. Thus, the therapeutic potential of
muscle stem cells has focused more on progenitor populations
derived from the endothelial lineage. These cells, which
include mesoangioblasts, pericytes, and CD133+ cells, have
significant myogenic potential and readily cross the endothe­
lium, making intra-arterial delivery possible.185 In animal
models, these muscle progenitors have shown the ability to
restore muscle function and to reconstitute the satellite stem
cell compartment to varying degrees.202 Numerous human
trials for Duchenne muscular dystrophy demonstrated effec­
tive dystrophin production with the use of injected stem cells,
but clinical benefits have not been observed.203 Clinical trials
have also been conducted with muscle stem cells for ischemic
heart disease and stress urinary incontinence, with mixed
improvements in muscle function.204
Nervous system
Treating disorders of the central nervous system (CNS) has
been one of the most challenging areas of modern medicine.
Regenerative medicine using defined stem and progenitor
cells offers the potential for neuroprotection (to prevent
further cell loss) and/or neuronal replacement (to replace
damaged or lost neurons). Both of these strategies can be
envisioned in chronic neurodegeneration (i.e. age-related
macular degeneration and Alzheimer’s disease) and genetic
neurodegeneration, as well as in injuries to the CNS (spinal
cord injury, stroke, and traumatic brain injury).
A seminal finding in advancing regenerative medicine for
neurological disorders was the demonstration that neurogen­
esis continues to occur in the adult human brain.205 This discov­
ery, together with the identification and expansion of human
neural stem cells,206,207 has led to a plethora of studies investi­
gating neuroregeneration.206,208 One such human neural stem
cell population is the HuCNS-SC, an adult tissue-specific stem
cell, first characterized in 2000 by Stem Cells, Inc.209,210 Each
HuCNS-SC bank is created from purified neural stem cells
from a single fetal brain tissue at 16–20 weeks’ gestation by
prospective isolation using flow cytometry.206 Manufactured
under cGMP standards, the company’s HuCNS-SCs are puri­
fied, expanded in culture, cryopreserved, and then stored as
banks of cells, ready to be made into individual patient doses
when needed. Building on the promising animal studies,211
where evidence of engraftment, global CNS migration and
multi-lineage differentiation (astrocytes, oligodendrocytes,
and neurons) was demonstrated, these allogeneic cells were
then tested in phase I/II clinical trials for specific CNS disor­
ders based on neuroprotective and neuronal replacement
strategies to determine safety and preliminary efficacy of
HuCNS-SCs.206,212 As HuCNS-SCs have now been shown to
engraft and survive long term,209,213 there is possibility of a
durable clinical effect following single transplantation. In the
multi-center study of spinal cord injury, seven patients received
cell transplantation above and below the site of injury. In
Fig. 12.22  Bioscaffold construct. Bioscaffolds can be tailored based on the stem
cell type and small molecule utilized, as well as the protein type, peptide sequence,
and matrix components.
Protection
Small molecules
Bioscaffolds
Matrix components
Peptide sequences

160
CHAPTER 12 • Stem cells and regenerative medicine
in vitro. Stem cells can also be coaxed down various paths of
differentiation through gene transfer and cytokine therapies
(Fig. 12.23). However, in order for these inductive modali­
ties to be used effectively in vivo, they must be delivered in
an appropriately controlled spatiotemporal manner over the
timescales necessary to complete stem cell repair and tissue
regeneration.
Stem cell-directed gene therapy has demonstrated enor­
mous potential following the cure of children with X-linked
severe combined immune deficiency and adenosine deami­
nase deficiency.225,226 However, two of 10 children treated
subsequently developed leukemia, significantly tempering
enthusiasm for stem cell gene therapy. Another hurdle
has been the lack of relevant in vitro and animal models to
predict efficacy in humans.227 Lastly, common vector systems
currently employed in gene therapy that utilize Moloney
murine leukemia virus, human immunodeficiency virus, and
lentivirus are far from ideal as concerns regarding adverse
inflammatory responses to viral components are always
present.
Gene transfer of growth factors such as insulin-like growth
factor and FGF has improved wound healing in animal
models.228,229 Recent studies have demonstrated that lentiviral
vectors may be preferred for long-term transfection of skin
cells whereas adenoviral or adeno-associated viruses may be
better suited for short-term skin therapies.230 Margolis et al.
used replication-incompetent adenovirus to express PDGF for
chronic venous leg ulcers in a phase I clinical trial.231 Non-viral
gene therapies through liposomal gene transfer have also
been effectively used for wound regeneration.232
Iwaguro et al. found that VEGF gene transfer could be used
to augment EPC function for vascular regeneration,233 and
demonstrated improved EPC adhesion, vascular incorpora­
tion, and proliferation in a rodent limb ischemia model. EPCs
Stem cells
Viruses
Liposomes
Plasmids
Small molecules
Fig. 12.23  Induction strategies for gene therapy. Small molecules, plasmids, or
liposomes can stimulate stem cell differentiation.
suited for bone engineering, whereas soft flexible scaffolds are
ideal for skin applications.
Scaffolds can be broadly separated into two categories:
native or synthetic. Native scaffolds include those derived
from living or cadaveric donors and can be autogenous,
allogeneic, or xenogeneic. An excellent example of the utiliza­
tion of such scaffolds is the treatment of massive burns with
dermal substitutes derived from a wide range of human and
nonhuman sources. Native matrices can be obtained through
decellularization of tissues and whole organs. The matrices
can then be seeded with exogenous therapeutic cells.214,215
These techniques include the use of physical/mechanical
forces, chemicals, and enzymatic agents to remove cellular
material with minimal disruption to the matrix.216
Recently, there has been increased focus on “smart” bioma­
terials.217 These materials incorporate peptide sequences
capable of better mimicking the native cellular environment
and can significantly modulate cell differentiation, adhesion,
and proliferation.218 Smart polymers may allow scaffolds to
respond to temperature, pH, light, or ionic interactions with
altered mechanical properties, hydrophobicity, collapse, or
expansion.219
Numerous studies have demonstrated the benefits of utiliz­
ing stem cell–scaffold constructs in regenerative medicine.
For one, scaffolds provide a highly modifiable vehicle for
inductive factors. Fang et al. utilized plasmid DNA loaded
onto collagen sponges and demonstrated successful in vivo
genetic manipulation of fibroblasts to induce bone forma­
tion in rats.220 In another study, poly(lactide–coglycolide)
scaffolds loaded with DNA plasmid demonstrated enhanced
cell transfection and matrix formation compared to naked
plasmid injection alone.221 Schek et al. found that bone forma­
tion was greater with hydrogel delivery of BMP-7-expressing
adenovirus in mice compared to non-hydrogel controls.222
While there are still considerable challenges facing the
development of the ideal artificial scaffold, ongoing innova­
tion has led to significant advances combining innovative
scaffolding design with increased understanding of stem
cell biology.223 Heightened awareness of the importance of
scaffold topography and porosity, degradation rates, and
incorporation of bioactive molecules has continued abreast
of technological and stem cell advances.224 Three-dimensional
printing strategies for scaffold manufacture have also acceler­
ated personalized scaffold development, and one can envisage
adaption of this computational technology to create effective
personalized scaffolds for reconstructive surgery gener­
ated from 3D imaging in addition to scaffolds for stem cell
delivery.223,224
In summary, as researchers continue to unravel the complex
signals and interactions that act to modulate stem cell behav­
ior, advances in biomaterial techniques will be crucial to our
ability to recapitulate with accuracy the various endogenous
stem cell microenvironments.217
Genetic induction therapies
Cell-based regenerative strategies are also critically depen­
dent on the biochemical signaling environment. Exogenous
administration of specific growth factors and cytokines can
induce stem cell differentiation, which provides researchers
with a powerful means of manipulating stem cell behavior

161
Prospective clinical applications of stem cell therapy
cells with only two factors. Dr. Yamanaka, who described the
original iPS cell, used a stochastic model to illustrate the mecha­
nism behind this type of differentiation. In a stochastic model,
most or all differentiated cells have the potential to become iPS
cells with the use of four transcription factors. The idea is that
a cell represents a “ball rolling down an epigenetic landscape
from the totipotent state, going through the pluripotent state
and rolling down a lineage committed state”. With the devel­
opment of normal adult cells, pluripotent cells appear briefly
and cannot be stopped “on the slope.” In contrast, ESCs are
blocked by a “bump or roadblock formed by their epigenetic
status”. Thus, the four Yamanaka factors function to roll cells
back to a pluripotent state.241
iPS cells have a number of advantages over embryonic
stem cells for use in regenerative medicine. Firstly, the use
of iPS cells avoids the ethical conflicts associated with ESC
derivation as iPS cells are generated from adult tissues.
Secondly, since these cells can be harvested noninvasively
for autologous re-implantation, they can bypass the immune
rejection that limits the use of allogeneic cells. Thirdly, iPS
cells can be derived from many different cell types. Early
descriptions of iPS derivation used skin fibroblasts,242,243
which required only a small skin biopsy followed by 3–4
weeks of in vitro expansion. One major disadvantage of skin
fibroblasts, however, is that they have a very low reprogram­
ming efficiency (under 0.01%) when using the four Yamanaka
factors and even lower without c-Myc. Furthermore, the
length of transfection is an additional 3–4 weeks’ time before
ESC-like iPS cells begin to appear.244 Based on the epigenetic
landscape model, fibroblasts are terminally differentiated
and thus take a greater amount of time and energy to
reprogram.
Keratinocytes from human foreskin biopsies and hair
have also been used to generate iPS cells.245 These cells are
similarly easy to harvest but also require an extended time
for in vitro expansion. Compared to fibroblasts, keratinocytes
derived from neonatal/juvenile foreskin have an approximate
100-fold increase in reprogramming efficiency. This has not,
however, been seen with adult keratinocytes.
have been transfected to produce anticoagulant proteins in an
angioplasty injury model234 and VEGF-transfected EPCs have
been used to fabricate bioengineered vascular grafts.235 Gene
therapy of EPCs has included potential cancer therapy given
their avid recruitment to neovascularizing areas.236
Stem cell-mediated gene therapy has also been proposed
for a wide range of neurologic diseases such as Parkinson’s
disease, Alzheimer’s disease, amyotrophic lateral sclerosis,
and neuropathic pain.237 For the treatment of nerve injury,
Schwann cells have been transduced to express ciliary
neurotrophic factor to augment nerve growth in rats.238 An
adenoviral gene transfer system has also been developed
for Schwann cell transfection and peripheral nerve injury.239
Thus, vector-mediated strategies provide another regenera­
tive tool to control the genetic programming of delivered stem
cells with the promise of replacing missing or dysfunctional
organs.
iPS cells
Definitions
Pluripotency is the ability of a cell to differentiate into all cell
types in the body. Originally thought to be a property unique
to the blastocyst or inner cell mass of the early mammalian
embryo, this tenet was challenged by a groundbreaking 2007
study published by Yamanaka et al. In this paper scientists
definitively established a process of reprogramming or
“inducing” pluripotency in a mature cell using a few key
transcription factors in the cell (Fig. 12.24).240 The resulting
cells have been named “induced” pluripotent stem cells as
they have stem cell-like properties and can differentiate into
all cell types found in the body.
The original conditions for creating iPS cells used a set of
four transcription factors: Oct4, Sox2, Klf4, and c-Myc (Fig.
12.25) to revert mature cells into a more primitive stage and
induce pluripotency. Since these original studies, scientists
have refined the process and are now able to reprogram mature
Fig. 12.24  Stem cells can be obtained during the
different developmental stages of life. With the
development of induced pluripotent stem cell (iPS)
technology, adult cells can now be reprogrammed to
become embryonic “stem cell-like” cells.
Cord
blood
iPS technology
Resident
stem cells
Pluripotent
embryonic stem cells
Totipotent
stem cells
Fertilized egg
Morula
Blastocyst
Infant
Adult

162
CHAPTER 12 • Stem cells and regenerative medicine
More recently, laboratories have found ways to circum­
vent the use of viruses by generating a non-viral minicircle
vector to induce iPS formation.249 Minicircle vectors are
supercoiled DNA molecules composed of a eukaryotic
expression cassette. Minicircles also offer the advantages of
having a higher transfection efficiency and longer ectopic
expression due to low activation of exogenous silencing
mechanisms. Reprogramming was successfully performed
using hASCs from 3 different patients, making it an attrac­
tive option if rapid cell expansion and reprogramming are
required.249
In 2007, a human sickle-cell anemia mouse model was
successfully treated with genetically corrected hematopoietic
progenitors generated from iPS cells.250 In spite of promising
early successes such as this, however, a number of barriers
had to be overcome prior to progression of iPS cell-based
therapy to clinical use. For instance, the oncogenic transgene
integration and mutagenesis involved in iPS cell reprogram­
ming could induce cancer (Fig. 12.26). Virus-free generated
iPS cells offer a potential solution.
Also, significant safety studies are required to determine
the minimal number of undifferentiated iPS cells that can
become a teratoma as current subcutaneous injection of
iPS cells has gone on to generate teratomas. This sug­
gests that iPS cells will likely need to be differentiated in
situ prior to implantation to allow for appropriate tissue
regeneration (Fig. 12.27). Lastly, from a commercialization
standpoint, it has yet to be determined if a viable business
model for patient-specific iPS treatments is possible given
the safety considerations, as well as the challenge of their
reprogramming.244
In vitro
Culture and maintenance of iPS cells
IMR90 human fibroblasts or other cell types to be repro­
grammed can be maintained with DMEM containing 10%
FBS, L-glutamine, 4.5 g/dL glucose, 100 U/mL penicillin, and
100 µg/mL streptomycin. All cells used for reprogramming
should be passage two or less. Derived iPS cells can be main­
tained either on MEF feeder layer or on Matrigel-coated tissue
culture dishes (ES qualified; BD Biosciences) with mTESR-1
hES Growth Medium (Stemcell Technology).248
Melanocytes have also been used to generate iPS cells. This
is described by Utikal et al. in a 2009 paper, which sought to
generate iPS cells more effectively by identifying somatic cell
populations that could be reprogrammed with greater effi­
ciency.246 Melanocytes inherently express high levels of Sox2
and thus can be reprogrammed with only three factors (Oct4,
Klf4, and c-Myc). Original studies using all four Yamanaka
factors demonstrated a five-fold higher reprogramming effi­
ciency than seen with fibroblasts. In addition, the melanocytes
could be reprogrammed in only 10 days.
Two laboratories have also described iPS cell derivation
from cryopreserved umbilical cord blood cells. One labora­
tory was able to reprogram these cord blood cells successfully
using only Oct4 and Sox2.247 A benefit of this technique is that
cord blood can be stored for over 5 years and still retain the
ability to be reprogrammed.
An even more abundant source of cells for iPS applications
is human adipose tissue.248 Our laboratory has isolated adipose
cells from lipoaspirates of adult patients and successfully
reprogrammed the cells using the four Yamanaka factors.
Small-volume liposuction, in a minimally invasive outpatient
setting, allows for the harvest of millions of cells. It is esti­
mated that 10 mL of fresh lipoaspirate yields 1 million cells
after 48 hours of in vitro culture. This allows for immediate
reprogramming without prior in vitro expansion. Even in
slender patients, as little as 15–50 mL of fat can be used to
derive iPS cells. Similarly, due to their rapid expansion, hASCs
can be reprogrammed with a 20-fold greater efficiency than
fibroblasts. Klf4 and c-Myc expression are high in these cells
compared to fibroblasts and these cells do not require mouse
feeder cells. This ease of harvest and speed of expansion and
reprogramming make hASCs an exciting option for scientists
exploring translational iPS strategies.248
Despite the excitement generated around iPS cells, methods
for their derivation and use need to be further tested and
improved. The use of lentiviruses and retroviruses lead to
genomic integration of transgenes, which the host cells may
not be able to silence completely. Furthermore, both Klf4 and
c-Myc, factors commonly used to reprogram cells, are onco­
genes. Future directions may involve using small molecules
and interfering RNAs, or microRNAs to make iPS cell genera­
tion safer. With regard to cell culture, iPS cells still depend on
a layer of inactivated MEFs. A Matrigel-coated surface could
potentially allow for a feeder-free environment.
Fig. 12.25  By activation of four transcription factors, adult
somatic cells can be reprogrammed into induced pluripotent
stem cells, which then can differentiate into all the
embryonic lineages: ectoderm, mesoderm, and endoderm.
Reprogramming factors
Ectoderm:
• Skin replacement
• Nerve grafts
Mesoderm:
• Fat
• Cartilage
• Muscle
• Bone
Endoderm:
• Pancreatic islet cells
• Hepatocytes
Clinical applications
Patient
Somatic
adult cells
iPS cells

163
Prospective clinical applications of stem cell therapy
Lentivirus production and transduction
293FT cells (Invitrogen) are plated at ~80% confluence per
100-mm dish and transfected with 12 µg each of lentiviral
vectors (Oct4, Sox2, Klf4, c-Myc) plus 8 µg packaging plas­
mids and 4 µg VSVG plasmids using Lipofectamine 2000
(Invitrogen) following the manufacturer’s instructions. The
resulting supernatant should be collected 48 hours after
transfection, filtered through a 0.45-µm pore size cellulose
acetate filter (Whatman), and mixed with PEG-it Virus Con­
centration Solution (System Biosciences) overnight at 4°C.
Viruses are precipitated at 1500 g the next day and resus­
pended with Opti-MEM medium (Invitrogen).
In vitro differentiation
iPS cells cultured on Matrigel are treated with collagenase
type IV (Invitrogen) and transferred to ultra-low attachment
plates (Corning Life Sciences) in a suspension culture for 8
days with DMEM/F12 (1 : 1) containing 20% knockout serum
(Invitrogen), 4.5 g/dL glucose, L-glutamine, 1% nonessential
amino acids, 0.1 mM 2-mercaptoethanol, 50 U/mL penicillin,
and 50 µg/mL streptomycin to allow formation of embryoid
bodies (EBs).
The EBs are then seeded in 0.25% gelatin-coated tissue
culture dishes for another 3–8 days. Spontaneous differentia­
tion of iPS cells into cells of mesoderm and endoderm lineages
can be detected with appropriate markers by immunofluores­
cence. For osteogenic differentiation, similar osteogenic
medium with ascorbic acid, and β-glycerol phosphate have
been used along with supplementation of retinoic acid and
bone morphogenetic proteins. Furthermore, there remains
some dispute as to whether the EB formation step is necessary
Fig. 12.26  One potential risk of using a multipotent self-renewing cell is the
uncontrolled division of these cells leading to tumor formation. However, usually in
tissue engineering a paucity of tissue, rather than excess, is the problem.
Self-renewal
Proliferation
Differentiation
Commitment
Maturation
Tumorigenesis
i.e., teratoma
Fig. 12.27  When induced pluripotent stem cells (iPS) are
placed subcutaneously, there is differentiation into all three
embryonic lineages, resulting in the formation of a teratoma.
The hope is that these cells can be reprogrammed prior to
implantation or in situ to repair tissues that cannot be
repaired with human adipose-derived stromal cells such as
neural tissues.
Nerve plexus
injury
Nerve formation?
Teratoma formation
iPS
Direct reprogramming
in vivo?
Subcutaneous implantation
in immunocompromised mice
Neurogenic
reprogramming in vitro?
iPS

164
CHAPTER 12 • Stem cells and regenerative medicine
Thus, three main hurdles exist for iPS utilization to become
mainstream:
1. Animal proteins are still commonly used during the
reprogramming of human cells in the form of a mouse
fibroblast feeder layer. A solution would be widespread
use of a feeder-free layer or Matrigel without animal
components.
2. Currently viruses used for reprogramming can integrate
into the genome and cause mutations and possible
malignancy. What is needed is a robust and efficient
non-viral approach such as the minicircle plasmid for
reprogramming.
3. In vivo, undifferentiated iPS cells form teratomas. Direct
differentiation of iPS cells to the differentiated cells of
interest prior to placement in vivo may alleviate this
concern.
While these hurdles are being addressed, an area of great
interest in the realm of iPS cells is that of disease modeling.252
iPS cell technology has provided previously unanticipated
possibilities to model rare human diseases in vitro.252 Repro­
gramming somatic cells from affected patients into an embry­
onic stem cell-like state followed by differentiation into cell
types relevant to human disease generates an unlimited
source of human tissue carrying the genetic variations that
instigated or facilitated disease development. In addition,
recently developed gene editing technologies enable the tar­
geted modification of human cells for gene disruptions,
genetic repair, or insertion of reporter genes.252,253 The ability
to modify single base pairs, thereby seamlessly correcting or
introducing disease-causing mutations in human pluripotent
stem cells, allows the creation of genetically controlled experi­
mental model systems in which the disease-causing genetic
variation is the sole experimental variable.254–256 This strategy
could substantially simplify the analysis of the interaction
between these genomic variants and disease phenotypes,
thereby revealing new insights into the pathophysiology of
monogenic and complex diseases.252
and leads to more efficient osteogenic differentiation or
whether omission of this step is possible. Differentiation into
dopaminergic neurons can be performed using a co-culture of
hASC iPS cells with PA6 cells.
In vivo models and potential
clinical correlates
The use of iPS technology can potentially revolutionize the
field of tissue engineering by allowing the creation of any cell
type from mature cells harvested via noninvasive methods.
We believe that the derivation of iPS cells from hASCs holds
several advantages compared to other cell types such as neural
stem cells, liver cells, and skin fibroblasts. First, the lipoaspira­
tion procedure for isolating hASCs is relatively simple, fast,
and safe. Second, it is easy to obtain a large quantity of hASCs
as the starting population for reprogramming after a single
lipoaspiration operation. Millions of hASCs can be derived on
the same day of lipoaspiration, and the reprogramming can be
performed immediately. Up until recently, iPS cells required
a layer of mouse fibroblasts to survive in an undifferentiated
state, which raised concerns for potential contamination of
the cultured human cells by mouse proteins and subsequent
rejection by human recipients following transplantation.
Feeder-free derivation of iPS cells from hASCs thus represents
a more clinically applicable method for derivation of iPS cells
and should enable more efficient and rapid generation of
patient-specific and disease-specific iPS cells.248
Though the first clinical trial utilizing iPS cells is currently
underway in Japan, therapeutic utilization and delivery of iPS
cells still requires further refinement. Treatment of a focal
deficit such as those present in spinal cord injuries, Parkin­
son’s disease or type I diabetes might involve direct delivery.
More systemic diseases such as muscular dystrophies,
however, might require multiple intravenous injections for
systemic delivery.251 The use of animal models can enable
scientists to address these and other concerns about safety
and efficacy prior to use in humans.
13. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential
of adult human mesenchymal stem cells. Science. 1999;284:143–147.
25. Gronthos S, Graves SE, Ohta S, et al. The STRO-1+ fraction of
adult human bone marrow contains the osteogenic precursors.
Blood. 1994;84:4164–4173. This paper provides one of the earliest
descriptions of the stromal vascular fraction of adipose tissue and
characterizes its cell surface markers.
28. Cowan CM, Shi YY, Aalami OO, et al. Adipose-derived adult
stromal cells heal critical-size mouse calvarial defects. Nat
Biotechnol. 2004;22:560–567. This paper was the first description of
using untreated adipose-derived mesenchymal cells to heal a calvarial
defect. They use novel tissue engineering techniques and imaging
modalities.
35. Moore KA, Lemischka IR. Stem cells and their niches. Science.
2006;311:1880–1885.
48. Zhang SC, Wernig M, Duncan ID, et al. In vitro differentiation of
transplantable neural precursors from human embryonic stem
cells. Nat Biotechnol. 2001;19:1129–1133.
57. Levenberg S, Golub JS, Amit M, et al. Endothelial cells derived
from human embryonic stem cells. Proc Natl Acad Sci USA.
2002;26:26.
65. Gimble J, Guilak F. Adipose-derived adult stem cells: isolation,
characterization, and differentiation potential. Cytotherapy.
2003;5:362–369.
95. Lendeckel S, Jodicke A, Christophis P, et al. Autologous stem cells
(adipose) and fibrin glue used to treat widespread traumatic
calvarial defects: case report. J Craniomaxillofac Surg. 2004;32:370–
373. This paper describes one of the first clinical applications of
adipose-derived stromal cells to help treat a pediatric patient with an
osseous defect. Though a limited patient number, it should set the
groundwork for future studies.
241. Yamanaka S. Elite and stochastic models for induced pluripotent
stem cell generation. Nature. 2009;460:49–52. This provides an
excellent description of the concept behind iPS derivation. A nice
schematic and detailed description of the iPS induction makes a very
difficult and ground-breaking discovery appear simple.
244. Sun N, Longaker MT, Wu JC. Human iPS cell-based therapy:
considerations before clinical applications. Cell Cycle. 2010;9:880–
885. This provides an excellent review of the different cell types iPS cells
can be derived from and the benefits and pitfalls of each cell type. It also
concisely and clearly discusses current hurdles to bringing iPS cells to
the bedside.
Access the complete reference list online at

164.e1
References
References
1. Healthcare Cost and Utilization Project. Facts and Figs. 2007
[Internet]. Rockville: Agency for Healthcare Research and Quality;
2007.
2. American Society of Plastic Surgeons. 2010 Report of the 2009
Statistics: National Clearinghouse of Plastic Surgery Statistics.
Available from: <
news-resources/statistics/2009-statistics/2009-US-cosmeticreconst
ructiveplasticsurgeryminimally-invasive-statistics.pdf>; 2010.
3. Hoffman R. Hematology: Basic Principles and Practice. 5th ed.
Philadelphia: Churchill Livingstone/Elsevier; 2009.
4. Levi B, James AW, Glotzbach JP, et al. Depot-specific variation in
the osteogenic and adipogenic potential of human adipose-derived
stromal cells. Plast Reconstr Surg. 2010;126:822–834.
5. Jager M, Zilkens C, Bittersohl B, Krauspe R. Cord blood–an
alternative source for bone regeneration. Stem Cell Rev.
2009;5:266–277.
6. Till JE, McCulloch CE. A direct measurement of the radiation
sensitivity of normal mouse bone marrow cells. Radiat Res.
1961;14:213–222.
7. Prockop DJ. Marrow stromal cells as stem cells for
nonhematopoietic tissues. Science. 1997;276:71–74.
8. Friedenstein AJ, Chailakhjan RK, Lalykina KS. The development of
fibroblast colonies in monolayer cultures of guinea-pig bone
marrow and spleen cells. Cell Tissue Kinet. 1970;3:393–403.
9. Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors
in normal and irradiated mouse hematopoietic organs. Exp
Hematol. 1976;4:267–274.
10. Castro-Malaspina H, Gay RE, Resnick G, et al. Characterization of
human bone marrow fibroblast colony-forming cells (CFU-F) and
their progeny. Blood. 1980;56:289–301.
11. Caplan AI. Review: mesenchymal stem cells: cell-based
reconstructive therapy in orthopedics. Tissue Eng.
2005;11:1198–1211.
12. Caplan AI, Dennis JE. Mesenchymal stem cells as trophic
mediators. J Cell Biochem. 2006;98:1076–1084.
13. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential
of adult human mesenchymal stem cells. Science. 1999;284:143–147.
14. Rodbell M. Metabolism of isolated fat cells. I. Effects of hormones
on glucose metabolism and lipolysis. J Biol Chem. 1964;239:375–380.
15. Hauner H, Entenmann G, Wabitsch M, et al. Promoting effect of
glucocorticoids on the differentiation of human adipocyte
precursor cells cultured in a chemically defined medium. J Clin
Invest. 1989;84:1663–1670.
16. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human
adipose tissue: implications for cell-based therapies. Tissue Eng.
2001;7:211–228.
17. Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source
of multipotent stem cells. Mol Biol Cell. 2002;13:4279–4295.
18. De Ugarte DA, Morizono K, Elbarbary A, et al. Comparison of
multi-lineage cells from human adipose tissue and bone marrow.
Cells Tissues Organs. 2003;174:101–109.
19. Guilak F, Lott KE, Awad HA, et al. Clonal analysis of the
differentiation potential of human adipose-derived adult stem
cells. J Cell Physiol. 2006;206:229–237.
20. Yang M, Ma QJ, Dang GT, et al. In vitro and in vivo induction of
bone formation based on ex vivo gene therapy using rat adipose-
derived adult stem cells expressing BMP-7. Cytotherapy.
2005;7:273–281.
21. Seo MJ, Suh SY, Bae YC, Jung JS. Differentiation of human adipose
stromal cells into hepatic lineage in vitro and in vivo. Biochem
Biophys Res Commun. 2005;328:258–264.
22. Ogawa R, Mizuno H, Hyakusoku H, et al. Chondrogenic and
osteogenic differentiation of adipose-derived stem cells isolated
from GFP transgenic mice. J Nippon Med Sch. 2004;71:240–241.
23. Huang JI, Zuk PA, Jones NF, et al. Chondrogenic potential of
multipotential cells from human adipose tissue. Plast Reconstr
Surg. 2004;113:585–594.
24. Deans RJ, Moseley AB. Mesenchymal stem cells: biology and
potential clinical uses. Exp Hematol. 2000;28:875–884.
25. Gronthos S, Graves SE, Ohta S, et al. The STRO-1+ fraction of
adult human bone marrow contains the osteogenic precursors.
Blood. 1994;84:4164–4173. This paper provides one of the earliest
descriptions of the stromal vascular fraction of adipose tissue and
characterizes its cell surface markers.
26. Hicok KC, Du Laney TV, Zhou YS, et al. Human adipose-derived
adult stem cells produce osteoid in vivo. Tissue Eng.
2004;10:371–380.
27. Lee JA, Parrett BM, Conejero JA, et al. Biological alchemy:
engineering bone and fat from fat-derived stem cells. Ann Plast
Surg. 2003;50:610–617.
28. Cowan CM, Shi YY, Aalami OO, et al. Adipose-derived adult
stromal cells heal critical-size mouse calvarial defects. Nat
Biotechnol. 2004;22:560–567. This paper was the first description of
using untreated adipose-derived mesenchymal cells to heal a calvarial
defect. They use novel tissue engineering techniques and imaging
modalities.
29. Martin GR. Isolation of a pluripotent cell line from early mouse
embryos cultured in medium conditioned by teratocarcinoma stem
cells. Proc Natl Acad Sci USA. 1981;78:7634–7638.
30. Kaufman MH, Robertson EJ, Handyside AH, Evans MJ.
Establishment of pluripotential cell lines from haploid mouse
embryos. J Embryol Exp Morphol. 1983;73:249–261.
31. Murray TH. Ethical (and political) issues in research with human
stem cells. Novartis Found Symp. 2005;265:188–196, discussion
96–211.
32. Adelson JW, Weinberg JK. The California stem cell initiative:
persuasion, politics, and public science. Am J Public Health.
2010;100:446–451.
33. Klimanskaya I, Chung Y, Becker S, et al. Human embryonic stem
cell lines derived from single blastomeres. Nature. 2006;444:481–485.
34. Schofield R. The relationship between the spleen colony-forming
cell and the haemopoietic stem cell. Blood Cells. 1978;4:7–25.
35. Moore KA, Lemischka IR. Stem cells and their niches. Science.
2006;311:1880–1885.
36. Li L, Xie T. Stem cell niche: structure and function. Annu Rev Cell
Dev Biol. 2005;21:605–631.
37. Pera MF, Trounson AO. Human embryonic stem cells: prospects
for development. Development. 2004;131:5515–5525.
38. Liew CG, Moore H, Ruban L, et al. Human embryonic stem cells:
possibilities for human cell transplantation. Ann Med.
2005;37:521–532.
39. Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem
cell lines derived from human blastocysts. Science. 1998;282:1145–1147.
40. Reubinoff BE, Pera MF, Fong CY, et al. Embryonic stem cell lines
from human blastocysts: somatic differentiation in vitro. Nat
Biotechnol. 2000;18:399–404.
41. Scholer HR, Ciesiolka T, Gruss P. A nexus between Oct-4 and E1a
– implications for gene-regulation in embryonic stem-cells. Cell.
1991;66:291–304.
42. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from
mouse embryonic and adult fibroblast cultures by defined factors.
Cell. 2006;126:663–676.
43. Xu C, Inokuma MS, Denham J, et al. Feeder-free growth of
undifferentiated human embryonic stem cells. Nat Biotechnol.
2001;19:971–974.
44. Martin MJ, Muotri A, Gage F, Varki A. Human embryonic stem
cells express an immunogenic nonhuman sialic acid. Nat Med.
2005;11:228–232.
45. Klimanskaya I, Chung Y, Meisner L, et al. Human embryonic stem
cells derived without feeder cells. Lancet. 2005;365:1636–1641.
46. Bain G, Kitchens D, Yao M, et al. Embryonic stem cells express
neuronal properties in vitro. Dev Biol. 1995;168:342–357.
47. Schuldiner M, Eiges R, Eden A, et al. Induced neuronal
differentiation of human embryonic stem cells. Brain Res.
2001;913:201–205.

164.e2
CHAPTER 12 • Stem cells and regenerative medicine
48. Zhang SC, Wernig M, Duncan ID, et al. In vitro differentiation of
transplantable neural precursors from human embryonic stem
cells. Nat Biotechnol. 2001;19:1129–1133.
49. Soria B, Bedoya FJ, Martin F. Gastrointestinal stem cells. I.
Pancreatic stem cells. Am J Physiol Gastrointest Liver Physiol.
2005;289:G177–G180.
50. Assady S, Maor G, Amit M, et al. Insulin production by human
embryonic stem cells. Diabetes. 2001;50:1691–1697.
51. Rajagopal J, Anderson WJ, Kume S, et al. Insulin staining of ES
cell progeny from insulin uptake. Science. 2003;299:363.
52. Kehat I, Kenyagin-Karsenti D, Snir M, et al. Human embryonic
stem cells can differentiate into myocytes with structural and
functional properties of cardiomyocytes. J Clin Invest.
2001;108:407–414.
53. Xu C, Police S, Rao N, Carpenter MK. Characterization and
enrichment of cardiomyocytes derived from human embryonic
stem cells. Circ Res. 2002;91:501–508.
54. Mummery C, Ward-van Oostwaard D, Doevendans P, et al.
Differentiation of human embryonic stem cells to cardiomyocytes:
role of coculture with visceral endoderm-like cells. Circulation.
2003;107:2733–2740.
55. Bai H, Wang ZZ. Directing human embryonic stem cells to
generate vascular progenitor cells. Gene Ther. 2008;15:89–95.
56. Zambidis ET, Peault B, Park TS, et al. Hematopoietic
differentiation of human embryonic stem cells progresses through
sequential hematoendothelial, primitive, and definitive stages
resembling human yolk sac development. Blood. 2005;106:860–870.
57. Levenberg S, Golub JS, Amit M, et al. Endothelial cells derived
from human embryonic stem cells. Proc Natl Acad Sci USA.
2002;26:26.
58. Kim BG, Hwang DH, Lee SI, et al. Stem cell-based cell therapy for
spinal cord injury. Cell Transplant. 2007;16:355–364.
59. Keirstead HS, Nistor G, Bernal G, et al. Human embryonic stem
cell-derived oligodendrocyte progenitor cell transplants
remyelinate and restore locomotion after spinal cord injury. J
Neurosci. 2005;25:4694–4705.
60. Sharp J, Frame J, Siegenthaler M, et al. Human embryonic stem
cell-derived oligodendrocyte progenitor cell transplants improve
recovery after cervical spinal cord injury. Stem Cells.
2010;28:152–163.
61. Watson RA, Yeung TM. What is the potential of oligodendrocyte
progenitor cells to successfully treat human spinal cord injury?
BMC Neurol. 2011;11:113.
62. Nistor GI, Totoiu MO, Haque N, et al. Human embryonic stem
cells differentiate into oligodendrocytes in high purity and
myelinate after spinal cord transplantation.
Glia. 2005;49:385–396.
63. Mothe AJ, Tator CH. Advances in stem cell therapy for spinal cord
injury. J Clin Invest. 2012;122:3824–3834.
64. Alper J. Geron gets green light for human trial of ES cell-derived
product. Nat Biotechnol. 2009;27:213–214.
65. Gimble J, Guilak F. Adipose-derived adult stem cells: isolation,
characterization, and differentiation potential. Cytotherapy.
2003;5:362–369.
66. Gimble JM. Adipose tissue-derived therapeutics. Expert Opin Biol
Ther. 2003;3:705–713.
67. Panetta NJ, Gupta DM, Kwan MD, et al. Tissue harvest by means
of suction-assisted or third-generation ultrasound-assisted
lipoaspiration has no effect on osteogenic potential of human
adipose-derived stromal cells. Plast Reconstr Surg. 2009;124:65–73.
68. Quarto N, Longaker MT. Differential expression of specific FGF
ligands and receptor isoforms during osteogenic differentiation of
mouse Adipose-derived Stem Cells (mASCs) recapitulates the in
vivo osteogenic pattern. Gene. 2008;424:130–140.
69. Quarto N, Wan DC, Longaker MT. Molecular mechanisms of
FGF-2 inhibitory activity in the osteogenic context of mouse
adipose-derived stem cells (mASCs). Bone. 2008;42:1040–1052.
70. Wan DC, Shi YY, Nacamuli RP, et al. Osteogenic differentiation of
mouse adipose-derived adult stromal cells requires retinoic acid
and bone morphogenetic protein receptor type IB signaling. Proc
Natl Acad Sci USA. 2006;103:12335–12340.
71. Quarto N, Longaker MT. FGF-2 inhibits osteogenesis in mouse
adipose tissue-derived stromal cells and sustains their proliferative
and osteogenic potential state. Tissue Eng. 2006;12:1405–1418.
72. Gupta DM, Kwan MD, Slater BJ, et al. Applications of an athymic
nude mouse model of nonhealing critical-sized calvarial defects. J
Craniofac Surg. 2008;19:192–197.
73. Levi B, James AW, Nelson ER, et al. Human adipose derived
stromal cells heal critical size mouse calvarial defects. PLoS ONE.
2010;5:e11177.
74. Liao X, Li F, Wang X, et al. Distribution of murine adipose-derived
mesenchymal stem cells in vivo following transplantation in
developing mice. Stem Cells Dev. 2008;17:303–314.
75. Kim DH, Je CM, Sin JY, Jung JS. Effect of partial hepatectomy on
in vivo engraftment after intravenous administration of human
adipose tissue stromal cells in mouse. Microsurgery.
2003;23:424–431.
76. Vilalta M, Degano IR, Bago J, et al. Biodistribution, long-term
survival, and safety of human adipose tissue-derived
mesenchymal stem cells transplanted in nude mice by high
sensitivity non-invasive bioluminescence imaging. Stem Cells Dev.
2008;17:993–1003.
77. Kim U, Shin DG, Park JS, et al. Homing of adipose-derived stem
cells to radiofrequency catheter ablated canine atrium and
differentiation into cardiomyocyte-like cells. Int J Cardiol.
2011;146:371–378.
78. Miranville A, Heeschen C, Sengenes C, et al. Improvement of
postnatal neovascularization by human adipose tissue-derived
stem cells. Circulation. 2004;110:349–355.
79. Kim YM, Choi YS, Choi JW, et al. Effects of systemic
transplantation of adipose tissue-derived stem cells on olfactory
epithelium regeneration. Laryngoscope. 2009;119:993–999.
80. Gonzalez MA, Gonzalez-Rey E, Rico L, et al. Treatment of
experimental arthritis by inducing immune tolerance with human
adipose-derived mesenchymal stem cells. Arthritis Rheum.
2009;60:1006–1019.
81. Gonzalez MA, Gonzalez-Rey E, Rico L, et al. Adipose-derived
mesenchymal stem cells alleviate experimental colitis by inhibiting
inflammatory and autoimmune responses. Gastroenterology.
2009;136:978–989.
82. Gonzalez-Rey E, Anderson P, Gonzalez MA, et al. Human adult
stem cells derived from adipose tissue protect against
experimental colitis and sepsis. Gut. 2009;58:929–939.
83. Mizuno H. The potential for treatment of skeletal muscle disorders
with adipose-derived stem cells. Curr Stem Cell Res Ther.
2010;5:133–136.
84. Constantin G, Marconi S, Rossi B, et al. Adipose-derived
mesenchymal stem cells ameliorate chronic experimental
autoimmune encephalomyelitis. Stem Cells. 2009;27:2624–2635.
85. Gonzalez-Rey E, Gonzalez MA, Varela N, et al. Human adipose-
derived mesenchymal stem cells reduce inflammatory and T cell
responses and induce regulatory T cells in vitro in rheumatoid
arthritis. Ann Rheum Dis. 2010;69:241–248.
86. Fang B, Song YP, Li N, et al. Resolution of refractory chronic
autoimmune thrombocytopenic purpura following mesenchymal
stem cell transplantation: a case report. Transplant Proc.
2009;41:1827–1830.
87. Levi B, James AW, Wan DC, et al. Regulation of human adipose-
derived stromal cell osteogenic differentiation by insulin-like
growth factor-1 and platelet-derived growth factor-alpha. Plast
Reconstr Surg. 2010;126:41–52.
88. Panetta NJ, Gupta DM, Lee JK, et al. Human adipose-derived
stromal cells respond to and elaborate bone morphogenetic
protein-2 during in vitro osteogenic differentiation. Plast Reconstr
Surg. 2010;125:483–493.
89. Kon E, Muraglia A, Corsi A, et al. Autologous bone marrow
stromal cells loaded onto porous hydroxyapatite ceramic
accelerate bone repair in critical-size defects of sheep long bones. J
Biomed Mater Res. 2000;49:328–337.

164.e3
References
90. Bruder SP, Kraus KH, Goldberg VM, Kadiyala S. The effect of
implants loaded with autologous mesenchymal stem cells on the
healing of canine segmental bone defects. J Bone Joint Surg Am.
1998;80:985–996.
91. Arinzeh TL, Peter SJ, Archambault MP, et al. Allogeneic
mesenchymal stem cells regenerate bone in a critical-sized canine
segmental defect. J Bone Joint Surg Am. 2003;85-A:1927–1935.
92. Dudas JR, Marra KG, Cooper GM, et al. The osteogenic potential
of adipose-derived stem cells for the repair of rabbit calvarial
defects. Ann Plast Surg. 2006;56:543–548.
93. Yoon E, Dhar S, Chun DE, et al. In vivo osteogenic potential of
human adipose-derived stem cells/poly lactide-co-glycolic acid
constructs for bone regeneration in a rat critical-sized calvarial
defect model. Tissue Eng. 2007;13:619–627.
94. Halvorsen YC, Wilkison WO, Gimble JM. Adipose-derived stromal
cells–their utility and potential in bone formation. Int J Obes Relat
Metab Disord. 2000;24(suppl 4):S41–S44.
95. Lendeckel S, Jodicke A, Christophis P, et al. Autologous stem cells
(adipose) and fibrin glue used to treat widespread traumatic calvarial
defects: case report. J Craniomaxillofac Surg. 2004;32:370–373. This paper
describes one of the first clinical applications of adipose-derived stromal
cells to help treat a pediatric patient with an osseous defect. Though a
limited patient number, it should set the groundwork for future studies.
96. Mesimaki K, Lindroos B, Tornwall J, et al. Novel maxillary
reconstruction with ectopic bone formation by GMP adipose stem
cells. Int J Oral Maxillofac Surg. 2009;38:201–209.
97. Kulakov AA, Goldshtein DV, Grigoryan AS, et al. Clinical study of
the efficiency of combined cell transplant on the basis of
multipotent mesenchymal stromal adipose tissue cells in patients
with pronounced deficit of the maxillary and mandibulary bone
tissue. Bull Exp Biol Med. 2008;146:522–525.
98. Bradley JP, Levine JP, McCarthy JG, Longaker MT. Studies in
cranial suture biology: regional dura mater determines in vitro
cranial suture fusion. Plast Reconstr Surg. 1997;100:1091–1099,
discussion; 100–102.
99. Greenwald JA, Mehrara BJ, Spector JA, et al. Regional
differentiation of cranial suture-associated dura mater in vivo and
in vitro: implications for suture fusion and patency. J Bone Miner
Res. 2000;15:2413–2430.
100. Levine JP, Bradley JP, Roth DA, et al. Studies in cranial suture
biology: regional dura mater determines overlying suture biology.
Plast Reconstr Surg. 1998;101:1441–1447.
101. Mauney JR, Nguyen T, Gillen K, et al. Engineering adipose-like
tissue in vitro and in vivo utilizing human bone marrow and
adipose-derived mesenchymal stem cells with silk fibroin 3D
scaffolds. Biomaterials. 2007;28:5280–5290.
102. Manolagas SC, Jilka RL. Mechanisms of disease – bone-marrow,
cytokines, and bone remodeling – emerging insights into the
pathophysiology of osteoporosis. N Engl J Med. 1995;332:305–311.
103. Kuznetsov SA, Friedenstein AJ, Robey PG. Factors required for
bone marrow stromal fibroblast colony formation in vitro. Br J
Haematol. 1997;97:561–570.
104. Kuznetsov SA, Krebsbach PH, Satomura K, et al. Single-colony
derived strains of human marrow stromal fibroblasts form bone
after transplantation in vivo. J Bone Miner Res. 1997;12:1335–1347.
105. Caplan AI. Mesenchymal stem cells. J Orthop Res. 1991;9:641–650.
106. Haynesworth SE, Goshima J, Goldberg VM, Caplan AI.
Characterization of cells with osteogenic potential from human
marrow. Bone. 1992;13:81–88.
107. D’Ippolito G, Diabira S, Howard GA, et al. Marrow-isolated adult
multilineage inducible (MIAMI) cells, a unique population of
postnatal young and old human cells with extensive expansion
and differentiation potential. J Cell Sci. 2004;117:2971–2981.
108. Carrancio S, Lopez-Holgado N, Sanchez-Guijo FM, et al.
Optimization of mesenchymal stem cell expansion procedures by
cell separation and culture conditions modification. Exp Hematol.
2008;36:1014–1021.
109. Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for
defining multipotent mesenchymal stromal cells. The International
Society for Cellular Therapy position statement. Cytotherapy.
2006;8:315–317.
110. Sacchetti B, Funari A, Michienzi S, et al. Self-renewing
osteoprogenitors in bone marrow sinusoids can organize a
hematopoietic microenvironment. Cell. 2007;131:324–336.
111. Simmons PJ, Przepiorka D, Thomas ED. Host origin of marrow
stromal cells following allogeneic bone marrow transplantation.
Nature. 1987;328:429–432.
112. Crisan M, Yap S, Casteilla L, et al. A perivascular origin for
mesenchymal stem cells in multiple human organs. Cell Stem Cell.
2008;3:301–313.
113. Bianco P, Robey PG, Simmons PJ. Mesenchymal stem cells:
revisiting history, concepts, and assays. Cell Stem Cell.
2008;2:313–319.
114. Le Blanc K. Immunomodulatory effects of fetal and adult
mesenchymal stem cells. Cytotherapy. 2003;5:485–489.
115. Aggarwal S, Pittenger MF. Human mesenchymal stem cells
modulate allogeneic immune cell responses. Blood.
2005;105:1815–1822.
116. Chang CJ, Yen ML, Chen YC, et al. Placenta-derived multipotent
cells exhibit immunosuppressive properties that are enhanced in
the presence of interferon-gamma. Stem Cells. 2006;24:2466–2477.
117. Jones S, Horwood N, Cope A, Dazzzi F. The antiproliferative effect
of mesenchymal stem cells is a fundamental property shared by all
stromal cells. J Immunol. 2007;179:2824–2831.
118. Frank MH, Sayegh MH. Immunomodulatory functions of
mesenchymal stem cells. Lancet. 2004;363:1411–1412.
119. Meisel R, Zibert A, Laryea M, et al. Human bone marrow stromal
cells inhibit allogeneic T-cell responses by indoleamine
2,3-dioxygenase-mediated tryptophan degradation. Blood.
2004;103:4619–4621.
120. Ghannam S, Bouffi C, Djouad F, et al. Immunosuppression by
mesenchymal stem cells: mechanisms and clinical applications.
Stem Cell Res Ther. 2010;1:2.
121. Brooke G, Cook M, Blair C, et al. Therapeutic applications of
mesenchymal stromal cells. Semin Cell Dev Biol. 2007;18:846–858.
122. Phinney DG. Biochemical heterogeneity of mesenchymal stem cell
populations – Clues to their therapeutic efficacy. Cell Cycle.
2007;6:2884–2889.
123. Phinney DG, Prockop DJ. Concise review: mesenchymal stem/
multipotent stromal cells: the state of transdifferentiation and modes
of tissue repair–current views. Stem Cells. 2007;25:2896–2902.
124. Wagner W, Feldmann RE Jr, Seckinger A, et al. The heterogeneity
of human mesenchymal stem cell preparations–evidence from
simultaneous analysis of proteomes and transcriptomes. Exp
Hematol. 2006;34:536–548.
125. Gnecchi M, Melo LG. Bone marrow-derived mesenchymal stem
cells: isolation, expansion, characterization, viral transduction, and
production of conditioned medium. Methods Mol Biol.
2009;482:281–294.
126. Bedada FB, Braun T. Partial induction of the myogenic program in
noncommitted adult stem cells. Cells Tissues Organs.
2008;188:189–201.
127. Kochanek KD, Murphy SL, Anderson RN, Scott C. Deaths: final
data for. Natl Vital Stat Rep. 2002;53:1–115.
128. Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be
generated from marrow stromal cells in vitro. J Clin Invest.
1999;103:697–705.
129. Chapel A, Bertho JM, Bensidhoum M, et al. Mesenchymal stem
cells home to injured tissues when co-infused with hematopoietic
cells to treat a radiation-induced multi-organ failure syndrome. J
Gene Med. 2003;5:1028–1038.
130. Mathiasen AB, Qayyum AA, Jorgensen E, et al. Bone marrow-
derived mesenchymal stromal cell treatment in patients with
severe ischaemic heart failure: a randomized placebo-controlled
trial (MSC-HF trial). Eur Heart J. 2015;36:1744–1753.
131. Dezawa M, Kanno H, Hoshino M, et al. Specific induction of
neuronal cells from bone marrow stromal cells and application for
autologous transplantation. J Clin Invest. 2004;113:1701–1710.

164.e4
CHAPTER 12 • Stem cells and regenerative medicine
132. Chopp M, Zhang XH, Li Y, et al. Spinal cord injury in rat:
treatment with bone marrow stromal cell transplantation.
Neuroreport. 2000;11:3001–3005.
133. Chen J, Li Y, Wang L, et al. Therapeutic benefit of intracerebral
transplantation of bone marrow stromal cells after cerebral
ischemia in rats. J Neurol Sci. 2001;189:49–57.
134. Li Y, Chen J, Zhang CL, et al. Gliosis and brain remodeling after
treatment of stroke in rats with marrow stromal cells. Glia.
2005;49:407–417.
135. Zhao LR, Duan WM, Reyes M, et al. Human bone marrow stem
cells exhibit neural phenotypes and ameliorate neurological
deficits after grafting into the ischemic brain of rats. Exp Neurol.
2002;174:11–20.
136. Tang DQ, Cao LZ, Burkhardt BR, et al. In vivo and in vitro
characterization of insulin-producing cells obtained from murine
bone marrow. Diabetes. 2004;53:1721–1732.
137. Karnieli O, Izhar-Prato Y, Bulvik S, Efrat S. Generation of
insulin-producing cells from human bone marrow mesenchymal
stem cells by genetic manipulation. Stem Cells. 2007;25:2837–2844.
138. Li Y, Zhang R, Qiao H, et al. Generation of insulin-producing cells
from PDX-1 gene-modified human mesenchymal stem cells. J Cell
Physiol. 2007;211:36–44.
139. Lee RH, Seo MJ, Reger RL, et al. Multipotent stromal cells from
human marrow home to and promote repair of pancreatic islets
and renal glomeruli in diabetic NOD/scid mice. Proc Natl Acad Sci
USA. 2006;103:17438–17443.
140. Murphy JM, Dixon K, Beck S, et al. Reduced chondrogenic and
adipogenic activity of mesenchymal stem cells from patients with
advanced osteoarthritis. Arthritis Rheum. 2002;46:704–713.
141. Arinzeh TL. Mesenchymal stem cells for bone repair: preclinical
studies and potential orthopedic applications. Foot Ankle Clin.
2005;10:651–665, viii.
142. Plock JA, Schnider JT, Zhang W, et al. Adipose- and bone
marrow-derived mesenchymal stem cells prolong graft survival in
vascularized composite allotransplantation. Transplantation.
2015;99:1765–1773.
143. Cui QJ, Xiao ZM, Balian G, Wang GJ. Comparison of lumbar spine
fusion using mixed and cloned marrow cells. Spine.
2001;26:2305–2310.
144. Kai T, Shao-qing G, Geng-ting D. In vivo evaluation of bone
marrow stromal-derived osteoblasts-porous calcium phosphate
ceramic composites as bone graft substitute for lumbar
intervertebral spinal fusion. Spine. 2003;28:1653–1658.
145. Murphy JM, Fink DJ, Hunziker EB, Barry FP. Stem cell therapy in
a caprine model of osteoarthritis. Arthritis Rheum.
2003;48:3464–3474.
146. Augello A, Tasso R, Negrini SM, et al. Cell therapy using
allogeneic bone marrow mesenchymal stem cells prevents tissue
damage in collagen-induced arthritis. Arthritis Rheum.
2007;56:1175–1186.
147. Sutton MG, Sharpe N. Left ventricular remodeling after
myocardial infarction: pathophysiology and therapy. Circulation.
2000;101:2981–2988.
148. Lunde K, Solheim S, Aakhus S, et al. Intracoronary injection of
mononuclear bone marrow cells in acute myocardial infarction. N
Engl J Med. 2006;355:1199–1209.
149. Assmus B, Honold J, Schachinger V, et al. Transcoronary
transplantation of progenitor cells after myocardial infarction. N
Engl J Med. 2006;355:1222–1232.
150. Kitoh H, Kitakoji T, Tsuchiya H, et al. Transplantation of marrow-
derived mesenchymal stem cells and platelet-rich plasma during
distraction osteogenesis–a preliminary result of three cases. Bone.
2004;35:892–898.
151. Kitoh H, Kawasumi M, Kaneko H, Ishiguro N. Differential effects
of culture-expanded bone marrow cells on the regeneration of
bone between the femoral and the tibial lengthenings. J Pediatr
Orthop. 2009;29:643–649.
152. Kitoh H, Kitakoji T, Tsuchiya H, et al. Transplantation of culture
expanded bone marrow cells and platelet rich plasma in
distraction osteogenesis of the long bones. Bone. 2007;40:522–528.
153. Horwitz EM, Gordon PL, Koo WK, et al. Isolated allogeneic bone
marrow-derived mesenchymal cells engraft and stimulate growth
in children with osteogenesis imperfecta: Implications for cell
therapy of bone. Proc Natl Acad Sci USA. 2002;99:8932–8937.
154. Horwitz EM, Prockop DJ, Fitzpatrick LA, et al. Transplantability
and therapeutic effects of bone marrow-derived mesenchymal cells
in children with osteogenesis imperfecta. Nat Med. 1999;5:309–313.
155. Horwitz EM, Prockop DJ, Gordon PL, et al. Clinical responses to
bone marrow transplantation in children with severe osteogenesis
imperfecta. Blood. 2001;97:1227–1231.
156. Davatchi F, Sadeghi Abdollahi B, Mohyeddin M, Nikbin B.
Mesenchymal stem cell therapy for knee osteoarthritis: 5 years
follow-up of three patients. Int J Rheum Dis. 2016;19:219–225.
157. Emadedin M, Ghorbani Liastani M, Fazeli R, et al. Long-term
follow-up of intra-articular injection of autologous mesenchymal
stem cells in patients with knee, ankle, or hip osteoarthritis. Arch
Iran Med. 2015;18:336–344.
158. Weissman IL. Stem cells: units of development, units of
regeneration, and units in evolution. Cell. 2000;100:157–168.
159. Korbling M, Estrov Z. Adult stem cells for tissue repair - a new
therapeutic concept? N Engl J Med. 2003;349:570–582.
160. Li L, Clevers H. Coexistence of quiescent and active adult stem
cells in mammals. Science. 2010;327:542–545.
161. Slack JM. Origin of stem cells in organogenesis. Science.
2008;322:1498–1501.
162. Blanpain C, Fuchs E. Epidermal homeostasis: a balancing act of
stem cells in the skin. Nat Rev Mol Cell Biol. 2009;10:207–217.
163. Yan X, Owens DM. The skin: a home to multiple classes of
epithelial progenitor cells. Stem Cell Rev. 2008;4:113–118.
164. Ito M, Liu Y, Yang Z, et al. Stem cells in the hair follicle bulge
contribute to wound repair but not to homeostasis of the
epidermis. Nat Med. 2005;11:1351–1354.
165. Fuchs E, Nowak JA. Building epithelial tissues from skin stem
cells. Cold Spring Harb Symp Quant Biol. 2008;73:333–350.
166. Sarin KY, Artandi SE. Aging, graying and loss of melanocyte stem
cells. Stem Cell Rev. 2007;3:212–217.
167. Paquet-Fifield S, Schluter H, Li A, et al. A role for pericytes as
microenvironmental regulators of human skin tissue regeneration.
J Clin Invest. 2009;119:2795–2806.
168. Morrison SJ, Kimble J. Asymmetric and symmetric stem-cell
divisions in development and cancer. Nature. 2006;441:1068–1074.
169. Chau JF, Leong WF, Li B. Signaling pathways governing osteoblast
proliferation, differentiation and function. Histol Histopathol.
2009;24:1593–1606.
170. Ducy P, Schinke T, Karsenty G. The osteoblast: a sophisticated
fibroblast under central surveillance. Science. 2000;289:1501–1504.
171. Franceschi RT, Ge C, Xiao G, et al. Transcriptional regulation of
osteoblasts. Cells Tissues Organs. 2009;189:144–152.
172. Gersbach CA, Phillips JE, Garcia AJ. Genetic engineering for
skeletal regenerative medicine. Annu Rev Biomed Eng.
2007;9:87–119.
173. Yancopoulos GD, Davis S, Gale NW, et al. Vascular-specific growth
factors and blood vessel formation. Nature. 2000;407:242–248.
174. Bauer SM, Goldstein LJ, Bauer RJ, et al. The bone marrow-derived
endothelial progenitor cell response is impaired in delayed wound
healing from ischemia. J Vasc Surg. 2006;43:134–141.
175. Ferguson JE 3rd, Kelley RW, Patterson C. Mechanisms of
endothelial differentiation in embryonic vasculogenesis.
Arterioscler Thromb Vasc Biol. 2005;25:2246–2254.
176. Ferrara N. Vascular endothelial growth factor as a target for
anticancer therapy. Oncologist. 2004;9:2–10.
177. Swift ME, Kleinman HK, DiPietro LA. Impaired wound repair and
delayed angiogenesis in aged mice. Lab Invest. 1999;79:1479–1487.
178. Folkman J. Anti-angiogenesis: new concept for therapy of solid
tumors. Ann Surg. 1972;175:409–416.
179. Tepper OM, Capla JM, Galiano RD, et al. Adult vasculogenesis
occurs through in situ recruitment, proliferation, and tubulization
of circulating bone marrow-derived cells. Blood.
2005;105:1068–1077.

164.e5
References
180. Asahara T, Murohara T, Sullivan A, et al. Isolation of putative
progenitor endothelial cells for angiogenesis. Science.
1997;275:964–967.
181. Carmeliet P. VEGF gene therapy: stimulating angiogenesis or
angioma-genesis? Nat Med. 2000;6:1102–1103.
182. Gerhardt H, Betsholtz C. Endothelial-pericyte interactions in
angiogenesis. Cell Tissue Res. 2003;314:15–23.
183. Loffredo F, Lee RT. Therapeutic vasculogenesis: it takes two. Circ
Res. 2008;103:128–130.
184. Mauro A. Satellite cell of skeletal muscle fibers. J Biophys Biochem
Cytol. 1961;9:493–495.
185. Kuang S, Gillespie MA, Rudnicki MA. Niche regulation of muscle
satellite cell self-renewal and differentiation. Cell Stem Cell.
2008;2:22–31.
186. Kuang S, Rudnicki MA. The emerging biology of satellite cells and
their therapeutic potential. Trends Mol Med. 2008;14:82–91.
187. Walsh S, Midha R. Use of stem cells to augment nerve injury
repair. Neurosurgery. 2009;65:A80–A86.
188. Guenard V, Kleitman N, Morrissey TK, et al. Syngeneic Schwann
cells derived from adult nerves seeded in semipermeable guidance
channels enhance peripheral nerve regeneration. J Neurosci.
1992;12:3310–3320.
189. Murakami T, Fujimoto Y, Yasunaga Y, et al. Transplanted neuronal
progenitor cells in a peripheral nerve gap promote nerve repair.
Brain Res. 2003;974:17–24.
190. Kemp SW, Walsh SK, Midha R. Growth factor and stem cell
enhanced conduits in peripheral nerve regeneration and repair.
Neurol Res. 2008;30:1030–1038.
191. Cuono C, Langdon R, McGuire J. Use of cultured epidermal
autografts and dermal allografts as skin replacement after burn
injury. Lancet. 1986;1:1123–1124.
192. Hanson SE, Bentz ML, Hematti P. Mesenchymal stem cell therapy
for nonhealing cutaneous wounds. Plast Reconstr Surg.
2010;125:510–516.
193. Dash NR, Dash SN, Routray P, et al. Targeting nonhealing
ulcers of lower extremity in human through autologous bone
marrow-derived mesenchymal stem cells. Rejuvenation Res.
2009;12:359–366.
194. Korbling M, Katz RL, Khanna A, et al. Hepatocytes and epithelial
cells of donor origin in recipients of peripheral-blood stem cells. N
Engl J Med. 2002;346:738–746.
195. Hematti P, Sloand EM, Carvallo CA, et al. Absence of donor-
derived keratinocyte stem cells in skin tissues cultured from
patients after mobilized peripheral blood hematopoietic stem cell
transplantation. Exp Hematol. 2002;30:943–949.
196. Badiavas EV, Falanga V. Treatment of chronic wounds with bone
marrow-derived cells. Arch Dermatol. 2003;139:510–516.
197. Daltro GC, Fortuna V, de Souza ES, et al. Efficacy of autologous
stem cell-based therapy for osteonecrosis of the femoral head in
sickle cell disease: a five-year follow-up study. Stem Cell Res Ther.
2015;6:110.
198. Aoyama T, Fujita Y, Madoba K, et al. Rehabilitation program after
mesenchymal stromal cell transplantation augmented by
vascularized bone grafts for idiopathic osteonecrosis of the
femoral head: a preliminary study. Arch Phys Med Rehabil.
2015;96:532–539.
199. Liu Y, Wu J, Zhu Y, Han J. Therapeutic application of
mesenchymal stem cells in bone and joint diseases. Clin Exp Med.
2014;14:13–24.
200. Gurtner GC, Werner S, Barrandon Y, Longaker MT. Wound repair
and regeneration. Nature. 2008;453:314–321.
201. Kang S, Yang YJ, Li CJ, Gao RL. Effects of intracoronary
autologous bone marrow cells on left ventricular function in acute
myocardial infarction: a systematic review and meta-analysis for
randomized controlled trials. Coron Artery Dis. 2008;19:327–335.
202. Dellavalle A, Sampaolesi M, Tonlorenzi R, et al. Pericytes of
human skeletal muscle are myogenic precursors distinct from
satellite cells. Nat Cell Biol. 2007;9:255–267.
203. Cossu G, Sampaolesi M. New therapies for Duchenne muscular
dystrophy: challenges, prospects and clinical trials. Trends Mol
Med. 2007;13:520–526.
204. Tedesco FS, Dellavalle A, Diaz-Manera J, et al. Repairing skeletal
muscle: regenerative potential of skeletal muscle stem cells. J Clin
Invest. 2010;120:11–19.
205. Eriksson PS, Perfilieva E, Bjork-Eriksson T, et al. Neurogenesis in
the adult human hippocampus. Nat Med. 1998;4:1313–1317.
206. Tsukamoto A, Uchida N, Capela A, et al. Clinical translation of
human neural stem cells. Stem Cell Res Ther. 2013;4:102.
207. Gage FH. Mammalian neural stem cells. Science.
2000;287:1433–1438.
208. Brustle O, Choudhary K, Karram K, et al. Chimeric brains
generated by intraventricular transplantation of fetal human brain
cells into embryonic rats. Nat Biotechnol. 1998;16:1040–1044.
209. Tamaki S, Eckert K, He D, et al. Engraftment of sorted/expanded
human central nervous system stem cells from fetal brain. J
Neurosci Res. 2002;69:976–986.
210. Uchida N, Buck DW, He D, et al. Direct isolation of human central
nervous system stem cells. Proc Natl Acad Sci USA.
2000;97:14720–14725.
211. Uchida N, Chen K, Dohse M, et al. Human neural stem cells
induce functional myelination in mice with severe dysmyelination.
Sci Transl Med. 2012;4:155ra36.
212. Gupta N, Henry RG, Strober J, et al. Neural stem cell engraftment
and myelination in the human brain. Sci Transl Med.
2012;4:155ra37.
213. Selden NR, Al-Uzri A, Huhn SL, et al. Central nervous system
stem cell transplantation for children with neuronal ceroid
lipofuscinosis. J Neurosurg Pediatr. 2013;11:643–652.
214. Dahl SL, Koh J, Prabhakar V, Niklason LE. Decellularized native
and engineered arterial scaffolds for transplantation. Cell
Transplant. 2003;12:659–666.
215. Ott HC, Matthiesen TS, Goh SK, et al. Perfusion-decellularized
matrix: using nature’s platform to engineer a bioartificial heart.
Nat Med. 2008;14:213–221.
216. Gilbert TW, Sellaro TL, Badylak SF. Decellularization of tissues
and organs. Biomaterials. 2006;27:3675–3683.
217. Furth ME, Atala A, Van Dyke ME. Smart biomaterials design for
tissue engineering and regenerative medicine. Biomaterials.
2007;28:5068–5073.
218. Langer R, Tirrell DA. Designing materials for biology and
medicine. Nature. 2004;428:487–492.
219. Roy I, Gupta MN. Smart polymeric materials: emerging
biochemical applications. Chem Biol. 2003;10:1161–1171.
220. Fang J, Zhu YY, Smiley E, et al. Stimulation of new bone formation
by direct transfer of osteogenic plasmid genes. Proc Natl Acad Sci
USA. 1996;93:5753–5758.
221. Shea LD, Smiley E, Bonadio J, Mooney DJ. DNA delivery from
polymer matrices for tissue engineering. Nat Biotechnol.
1999;17:551–554.
222. Schek RM, Hollister SJ, Krebsbach PH. Delivery and protection of
adenoviruses using biocompatible hydrogels for localized gene
therapy. Mol Ther. 2004;9:130–138.
223. Tevlin R, McArdle A, Atashroo D, et al. Biomaterials for
craniofacial bone engineering. J Dent Res. 2014;93:1187–1195.
224. Harrison RH, St-Pierre JP, Stevens MM. Tissue engineering and
regenerative medicine: a year in review. Tissue Eng Part B Rev.
2014;20:1–16.
225. Aiuti A, Slavin S, Aker M, et al. Correction of ADA-SCID by stem
cell gene therapy combined with nonmyeloablative conditioning.
Science. 2002;296:2410–2413.
226. Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, et al. Gene
therapy of human severe combined immunodeficiency (SCID)-X1
disease. Science. 2000;288:669–672.
227. Horn PA, Morris JC, Neff T, Kiem HP. Stem cell gene transfer–
efficacy and safety in large animal studies. Mol Ther. 2004;10:
417–431.

164.e6
CHAPTER 12 • Stem cells and regenerative medicine
228. Sun L, Xu L, Chang H, et al. Transfection with aFGF cDNA
improves wound healing. J Invest Dermatol. 1997;108:313–318.
229. Tao Z, Herndon D, Hawkins H, et al. Insulin-like growth factor-I
cDNA gene transfer in vitro and in vivo. Biochem Genet.
2000;38:41–55.
230. Teo EH, Cross KJ, Bomsztyk ED. Gene therapy in
skin: choosing the optimal viral vector. Ann Plast Surg.
2009;62:576–580.
231. Margolis DJ, Morris LM, Papadopoulos M, et al. Phase I study of
H5.020CMV.PDGF-beta to treat venous leg ulcer disease. Mol Ther.
2009;17:1822–1829.
232. Jeschke MG, Sandmann G, Finnerty CC, et al. The structure and
composition of liposomes can affect skin regeneration,
morphology and growth factor expression in acute wounds. Gene
Ther. 2005;12:1718–1724.
233. Iwaguro H, Yamaguchi J, Kalka C, et al. Endothelial progenitor
cell vascular endothelial growth factor gene transfer for vascular
regeneration. Circulation. 2002;105:732–738.
234. Griese DP, Achatz S, Batzlsperger CA, et al. Vascular gene delivery
of anticoagulants by transplantation of retrovirally-transduced
endothelial progenitor cells. Cardiovasc Res. 2003;58:469–477.
235. Zarbiv G, Preis M, Ben-Yosef Y, Flugelman MY. Engineering blood
vessels by gene and cell therapy. Expert Opin Biol Ther.
2007;7:1183–1191.
236. Debatin KM, Wei J, Beltinger C. Endothelial progenitor cells for
cancer gene therapy. Gene Ther. 2008;15:780–786.
237. Muller FJ, Snyder EY, Loring JF. Gene therapy: can neural stem
cells deliver? Nat Rev Neurosci. 2006;7:75–84.
238. Hu Y, Leaver SG, Plant GW, et al. Lentiviral-mediated transfer of
CNTF to schwann cells within reconstructed peripheral nerve
grafts enhances adult retinal ganglion cell survival and axonal
regeneration. Mol Ther. 2005;11:906–915.
239. Joung I, Kim HS, Hong JS, et al. Effective gene transfer into
regenerating sciatic nerves by adenoviral vectors: Potentials
for gene therapy of peripheral nerve injury. Mol Cells.
2000;10:540–545.
240. Wernig M, Meissner A, Foreman R, et al. In vitro reprogramming
of fibroblasts into a pluripotent ES-cell-like state. Nature.
2007;448:318–324.
241. Yamanaka S. Elite and stochastic models for induced pluripotent
stem cell generation. Nature. 2009;460:49–52. This provides an
excellent description of the concept behind iPS derivation. A nice
schematic and detailed description of the iPS induction makes a very
difficult and ground-breaking discovery appear simple.
242. Okita K, Ichisaka T, Yamanaka S. Generation of germline-
competent induced pluripotent stem cells. Nature.
2007;448:313–317.
243. Huangfu D, Osafune K, Maehr R, et al. Induction of pluripotent
stem cells from primary human fibroblasts with only Oct4 and
Sox2. Nat Biotechnol. 2008;26:1269–1275.
244. Sun N, Longaker MT, Wu JC. Human iPS cell-based therapy:
considerations before clinical applications. Cell Cycle. 2010;9:880–
885. This provides an excellent review of the different cell types iPS cells
can be derived from and the benefits and pitfalls of each cell type. It also
concisely and clearly discusses current hurdles to bringing iPS cells to
the bedside.
245. Aasen T, Raya A, Barrero MJ, et al. Efficient and rapid generation
of induced pluripotent stem cells from human keratinocytes. Nat
Biotechnol. 2008;26:1276–1284.
246. Utikal J, Maherali N, Kulalert W, Hochedlinger K. Sox2 is
dispensable for the reprogramming of melanocytes and melanoma
cells into induced pluripotent stem cells. J Cell Sci.
2009;122:3502–3510.
247. Giorgetti A, Montserrat N, Aasen T, et al. Generation of induced
pluripotent stem cells from human cord blood using OCT4 and
SOX2. Cell Stem Cell. 2009;5:353–357.
248. Sun N, Panetta NJ, Gupta DM, et al. Feeder-free derivation of
induced pluripotent stem cells from adult human adipose stem
cells. Proc Natl Acad Sci USA. 2009;106:15720–15725.
249. Jia F, Wilson KD, Sun N, et al. A nonviral minicircle vector for
deriving human iPS cells. Nat Methods. 2010;7:197–199.
250. Hanna J, Wernig M, Markoulaki S, et al. Treatment of sickle cell
anemia mouse model with iPS cells generated from autologous
skin. Science. 2007;318:1920–1923.
251. Grabel L. Prospects for pluripotent stem cell therapies: into the
clinic and back to the bench. J Cell Biochem. 2012;113:381–387.
252. Soldner F, Jaenisch R. Medicine. iPSC disease modeling. Science.
2012;338:1155–1156.
253. Hockemeyer D, Wang H, Kiani S, et al. Genetic engineering of
human pluripotent cells using TALE nucleases. Nat Biotechnol.
2011;29:731–734.
254. Soldner F, Laganiere J, Cheng AW, et al. Generation of isogenic
pluripotent stem cells differing exclusively at two early onset
Parkinson point mutations. Cell. 2011;146:318–331.
255. Yusa K, Rashid ST, Strick-Marchand H, et al. Targeted gene
correction of alpha1-antitrypsin deficiency in induced pluripotent
stem cells. Nature. 2011;478:391–394.
256. Sebastiano V, Maeder ML, Angstman JF, et al. In situ genetic
correction of the sickle cell anemia mutation in human induced
pluripotent stem cells using engineered zinc finger nucleases. Stem
Cells. 2011;29:1717–1726.

13 
Wound healing
Chandan K. Sen, Sashwati Roy, and Gayle Gordillo
SYNOPSIS
■ There are three general techniques of wound treatment: primary
intention, secondary intention, and tertiary intention
■ The following overlapping phases drive the overall healing response:
hemostasis, inflammation, proliferation, and remodeling
■ Successful hemostasis or blood coagulation results in prevention of
blood loss by plugging the wound within seconds through
vasoconstriction and formation of a hemostatic blood clot consisting
of platelets and fibrin. The process is divided into initiation and
amplification. Initiation is caused by an extrinsic pathway, whereas
amplification is executed by an intrinsic pathway
■ The inflammatory response during normal healing is characterized by
spatially and temporally changing patterns of specific leukocyte
subsets. Dysregulated inflammation complicates wound healing.
Complete resolution of an acute inflammatory response is the ideal
outcome following an insult
■ Infection is a common problem in chronic wounds, frequently resulting
in nonhealing wounds and significant patient morbidity and mortality.
Microorganisms do not always live as pure cultures of dispersed
single cells but instead accumulate at interfaces to form polymicrobial
aggregates such as films, mats, flocs, sludge, or biofilms
■ In an open wound that has undergone contraction, restoration of an
intact epidermal barrier is enabled through wound epithelialization, also
known as re-epithelialization. During the proliferative phase of wound
healing, the granulation tissue is light red or dark pink in color because
of perfusion by new capillary loops. It is soft to the touch, moist, and
granular in appearance. The granulation tissue serves as a bed for
tissue repair.
■ Wound healing endpoint determination should include a combination of
visual inspection and measurements of skin barrier function such as
transepidermal water loss (TEWL) for accurate determination of
closure.
■ Wound vascularization may be achieved by angiogenesis or
vasculogenesis
■ A wound is generally considered chronic if it has not healed in 4
weeks. Chronic wounds can be broadly classified into three major
categories: venous and arterial ulcers, diabetic ulcers, and pressure
ulcers
■ Vascular complications commonly associated with problematic
wounds are primarily responsible for wound ischemia. Limitations in
the ability of the vasculature to deliver O2-rich blood to the wound
tissue leads to, among other consequences, hypoxia. Three major
factors may contribute to wound tissue hypoxia: (1) peripheral
vascular diseases garroting O2 supply; (2) increased O2 demand of the
healing tissue; and (3) generation of reactive oxygen species (ROS) by
way of respiratory burst and for redox signaling
■ Small RNAs are a new class of regulators of eukaryotic biology.
Alongside other small interfering RNAs (siRNAs), microRNAs
(miRNAs) execute post-transcriptional gene silencing through mRNA
destabilization as well as translational repression. miRNAs are
emerging as a key regulator of the overall wound-healing process
■ The regenerative potential of injured adult tissue suggests the
physiological existence of cells capable of participating in the
reparative process. Bone marrow-derived mesenchymal stem cells
(BM-MSCs) have been shown to promote the healing of diabetic
wounds, implying a profound therapeutic potential for skin defects
such as chronic wounds and burns
Introduction
Physical trauma represents one of the most primitive chal­
lenges that threatened survival. In other words, injury elimi­
nated the unfit. A Sumerian clay tablet (c. 2150 BC) described
early wound care that included washing the wound in beer
and hot water, using poultices from substances such as wine
dregs and lizard dung, and bandaging the wound. Ancient
scriptures depicting the science of life or Ayurveda date from
the sixth to seventh century BC, and represent the beginning
of planned physical injury with the intent to cure.1,2 Hip­
pocrates (c. 400 BC) detailed the importance of draining pus
from the wound, and Galen (c. 130–200 AD) described the
principle of first- and second-intention healing.3 Wound
healing advanced slowly over the centuries, with major
advances in the 19th century in the importance of controlling
infection, hemostasis, and necrotic tissue.4 Today, surgical
trauma, taken together with injury caused during accidents

166
CHAPTER 13 • Wound healing
vascularization, and wound closure; closure may be discussed
as wound contraction and epithelialization) and remodeling
(continues from weeks to years and encompasses collagen
deposition, acquisition of wound tensile strength, and turn­
over of extracellular matrix (ECM) components). These stages,
taken as a whole, are also referred to as the wound-healing
cascade (Fig. 13.1).
Hemostasis
For bleeding wounds, the highest priority is to stop bleeding
and this is achieved by hemostasis. Hemostasis is thus a
protective physiological response to vascular injury that
results in exposure of blood components to the sub-endothelial
layers of the vessel wall. Through successful hemostasis blood
loss is prevented by plugging the wound within seconds
through vasoconstriction and formation of a hemostatic blood
clot consisting of platelets and fibrin. Hemostasis requires
both platelets and the blood coagulation system. The process
of blood coagulation may be subdivided into initiation and
amplification. Initiation is caused by an extrinsic pathway,
whereas amplification is executed by an intrinsic pathway.
and secondary to other clinical conditions, e.g., diabetes,
represents a substantial cost to society.5 Any solution to
wound-healing problems will require a multifaceted compre­
hensive approach. First and foremost, the wound environment
will have to be made receptive to therapies. Second, the
appropriate therapeutic regimen needs to be identified and
provided while managing systemic limitations that could
secondarily limit the healing response. This chapter aims to
present an overall outline of the cutaneous wound-healing
process.
The wound-healing process
The entire wound-healing process may be viewed as a cascade
that is governed by numerous feedback and feed-forward
regulatory loops driven by signals from the wound tissue
itself, wound microenvironment, as well as interventions
under conditions where the wound is subjected to therapy. To
understand the several interdigitated biological processes
that drive the overall healing response, wound healing is
commonly discussed as the following overlapping phases:
hemostasis and inflammation, proliferation (granulation,
Neutrophils
Clot
24 hours
Scab
A
Macrophage
Fibroblast
Re-epithelialization
New
capillary
Granulation
tissue
3–7 days
B
Fig. 13.1  (A–C) Phases of cutaneous wound healing: a simplified representation.
Wound contraction
Weeks
C

167
Hemostasis
also contribute to amplification of blood coagulation through
its so-called blood-borne form, which is present as cell-derived
microparticles as well as through TF expressed within
platelets.6–8
Levels of FVIIa, a key player of the extrinsic pathway, in
the circulating blood are higher than any other activated
coagulation factor. Following injury to the blood vessel wall,
FVII comes into contact with TF expressed on TF-bearing cells
(e.g., white blood cells) and forms an activated complex
(TF–FVIIa). TF–FVIIa activates FIX and FX, resulting in FIXa
and FXa, respectively. FVII is activated by thrombin, FXIa,
FXII, and FXa. The activation of FXa by TF–FVIIa is almost
immediately inhibited by the TF pathway inhibitor. FXa and
The intrinsic pathway consists of plasma factor XI (FXI), IX,
and VIII (Fig. 13.2). Tissue factor (TF) generates a “thrombin
burst”, a process by which thrombin is released instanta­
neously. Thrombin is a key driver of the overall coagulation
cascade.
The extrinsic pathway responsible for the initiation of
blood coagulation consists of the transmembrane receptor TF
and plasma FVII or VIIa. On the other hand, the intrinsic
pathway consists of plasma FXI, FIX, and FVIII. Under physi­
ological conditions, TF is constitutively expressed by adven­
titial cells surrounding blood vessels and initiates clotting.
Examples of such adventitial cells include vascular smooth-
muscle cells, pericytes, and adventitial fibroblasts.6,7 TF may
Fig. 13.2  The blood clotting cascade. FXII, factor XII,
HK, high-molecular-weight kininogen; PF3, platelet factor
3; RBC, red blood cell; TF, tissue factor; TFPI, tissue
factor pathway inhibitor; TM, thrombomodulin; WBC,
white blood cell.
Tissue damage
Vessel injury
Healing
Collagen
Prekallikrein
Polymerization
and
stabilization
RBC
WBC
Platelet
Platelet
Common
pathway
Intrinsic
pathway
Extrinsic
pathway
Kallikrein
Phospholipids
Phospholipids
Prothrombin
Thrombin
Fibrinogen
Antithrombin
III
Protein-S
Protein-C
FXII
FXI
FXIa
TF
FIX
FIXa
FVIII
FVIIIa
FVII
FVIIa
FX
FXa
FX
FVa
FV
TM
TFPI
PF3
Ca2+
Ca2+
HK
FXIIa
FXIII
FXIIIa

168
CHAPTER 13 • Wound healing
thrombospondin. Current understanding portrays the clot as
a dynamic structural matrix containing functionally active
proteins and cells. In addition to containment of blood loss,
the clot serves as a first aid against microbial invasion. The
clot also serves as a provisional matrix for the homing of
blood-borne cells, including inflammatory as well as stem or
progenitor cells. The provisional matrix is enriched in cyto­
kines and growth factors which then regulate the function of
the homing cells.9,10
The formation of blood clot is initiated by the proteolytic
cleavage of fibrinogen by thrombin. As a result, fibrin is
produced and forms cross-links with each other. Cross-linked
fibrin entraps platelets, and together they adhere to the sub­
endothelium through adhesion molecules called integrins.
Clot fibrin plays a key role in mounting the inflammatory
process as well as in facilitating wound angiogenesis and
stromal cell proliferation. Fibrin binds to integrin CD11b/
CD18 on infiltrating monocytes and neutrophils. It also binds
to fibroblast growth factor-2 (FGF-2) and vascular endothelial
growth factor (VEGF) that help the wound tissue vascularize.
Fibrin also binds to insulin-like growth factor-I and promotes
stromal cell proliferation.11,12
A blood clot represents the seat of wound chemotaxis.
Thrombin, released by platelets at the wound site, is an early
mediator of clot development.13 Thrombin is a serine protease
that converts soluble plasma fibrinogen into an insoluble
fibrin clot. In addition, it promotes platelet aggregation.
Thrombin function may be viewed as an interface between
the hemostasis phase of wound healing and the ensuing
inflammatory phase as it plays a potent role in mounting
wound inflammation. The proinflammatory effects of throm­
bin include stimulation of vasodilation responsible for plasma
extravasation, edema, and an increased expression of endo­
thelial cell adhesion molecules that helps monocytes and
other cells extravasate and infiltrate the wound site. Thrombin
also induces the release of proinflammatory cytokines like
CCL2, interleukin-6 (IL-6), and IL-8 by endothelial cells. These
cytokines induce monocyte chemotaxis.14 Furthermore,
thrombin induces the release of inflammatory cytokines by
monocytes, including IL-6, interferon-γ, IL-1β, and tumor
necrosis factor-α (TNF-α). These early-phase cytokines are
typically proinflammatory, which may govern the differentia­
tion of blood-derived monocytes into M1 wound macro­
phages.15 Wound chemotaxis is also driven by the degradation
of fibrin and subsequent activation of the complement system.
As part of this process, several chemotactic agents and cyto­
kines are released, which in turn launch the inflammatory
phase of wound healing through chemotactic recruitment of
blood-borne immune cells (Fig. 13.3).16 Platelets are one of the
earliest sources of cytokines which execute immune cell che­
motaxis as well as macrophage activation. Once trapped in
the fibrin net, platelets release granules that function as a
reservoir for biologically active proteins, such as RANTES
(regulated on activation, normal T cell expressed, and secreted
or CCL5), thrombin, transforming growth factor-β (TGF-β),
platelet-derived growth factor (PDGF), and VEGF. CCL5 is
one of the most potent monocyte chemoattractants released
by platelets after injury. Other cytokines and chemokines that
attract monocytes to the wound bed include monocyte che­
moattractant protein-1 (MCP-1) (CCL2), MIP-1α (CCL3),
TGF-α, fibronectin, elastin, C5a, C3a, nerve growth factor, and
ECM components.17,18
its cofactor FVa form the prothrombinase complex, which
activates prothrombin to thrombin. Thrombin is a serine
protease that plays a central role in hemostasis after tissue
injury by converting soluble plasma fibrinogen into an
insoluble fibrin clot and by promoting platelet aggregation.
Thrombin activates other components of the coagulation
cascade, including FV and FVIII, which in turn activates FXI
and cascades to the activation of FIX. Thrombin also activates
and releases FVIII from being bound to von Willebrand factor.
FVIIIa is the cofactor of FIXa, and together they form the
“tenase” complex, which activates FX. In this way the cycle
continues. The intrinsic pathway begins with formation of
the primary complex on collagen by high-molecular-weight
kininogen, prekallikrein, and FXII (Hageman factor). Prekal­
likrein is converted to kallikrein, and FXII becomes FXIIa.
FXIIa converts FXI into FXIa. FXIa activates FIX, which
together with its cofactor FVIIIa forms the tenase complex.
The tenase complex activates FX to FXa (see Fig. 13.2).
The blood clot physically helps plug the wound, minimiz­
ing blood loss. It is primarily made up of cross-linked
fibrin, cells such as erythrocytes and platelets, as well as
other ECM proteins such as fibronectin, vitronectin, and
Fig. 13.3  The wound inflammatory response.
Platelets from blood release blood-clotting proteins at wound site.
Mast cells secrete factors that mediate vasodilation and vascular constriction.
Delivery of blood, plasma and cells to injured area.
Neutrophils and macrophages remove pathogens by phagocytosis.
Macrophages secrete hormones called cytokines that attract immune system cells
to the site and activate cells involved in tissue repair.
Inflammatory response continues until the foreign material is eliminated and the
wound is repaired.
Macrophage
Initiate
tissue
repair
Cytokines
signaling
path to
injury site
Mast cell
Neutrophil
Platelets
Bacteria and other pathogens enter wound.
1
2
3
4
5
6
1
2
3
4
5
6

169
Inflammation
Inflammation
Tissue injury triggers an acute-phase inflammation (Latin,
inflammare, to set on fire) response that is meant to prepare the
wound site for subsequent wound closure (see Fig. 13.3).
Inflammation encompasses a series of responses of vascular­
ized tissues of the body to injury. Local chemical mediators
that are biosynthesized during acute inflammation give rise
to the macroscopic events characterized by Celsus in the first
century, namely, rubor (redness), tumor (swelling), calor (heat),
and dolor (pain). At cellular and molecular levels, inflamma­
tion results from the coordination of manifold systems of
receptors and sensors that affect transcriptional and post-
translational programs necessary for host defense and resolu­
tion of infection. During normal healing, the inflammatory
response is characterized by spatially and temporally chang­
ing patterns of specific leukocyte subsets.
Platelets
Formation of the clot or thrombus is dependent on platelet
activation. The platelet-rich blood clot also entraps polymor­
phonuclear leukocytes (neutrophils). This helps amplify blood
coagulation and lays the foundation for the subsequent acute-
phase inflammatory response. In a matter of hours after injury,
large numbers of neutrophils extravasate by transmigrating
across the endothelial cell wall of blood capillaries to the
wound site. To enable this, local blood vessels are activated
by proinflammatory cytokines such as IL-1β, TNF-α, and
interferon-γ. These cytokines induce the expression of adhe­
sion molecules necessary for adhesion of leukocytes and
diapedesis (Fig. 13.4). Adhesion molecules such as integrins
as well as P-selectin and E-selectin play a central role in
enabling diapedesis of neutrophils (Fig. 13.5). These adhesion
molecules bind with integrins expressed on the cell surface
of neutrophils, such as CD11a/CD18 (LFA), CD 11b/CD18
(MAC-1), CD11c/CD18 (gp150, 95), and CD11d/CD18.
Alongside cytokines, chemokines play a major role in mount­
ing acute-phase inflammation after injury. Chemokines
include IL-8, MCP-1, and growth-related oncogene-α. In the
case of an infected wound, bacterial products such as lipo­
polysaccharide and formyl-methionyl peptides can enhance
neutrophil recruitment to the wound site (Fig. 13.6).
Neutrophils
Neutrophils traverse postcapillary venules at sites of inflam­
mation, degrade pathogens within phagolysosomes, and
undergo apoptosis. Neutrophils serve a wide range of func­
tions, ranging from phagocytosis of infectious agents to
cleansing of devitalized tissue. When coated with opsonins
(generally complement and/or antibody), microorganisms
bind to specific receptors on the surface of the phagocyte and
invagination of the cell membrane occurs with the incorpora­
tion of the microorganism into an intracellular phagosome.
There follows a burst of oxygen consumption, and much, if
not all, of the extra oxygen consumed is converted to highly
reactive oxygen species. This is called respiratory burst. In
addition, the cytoplasmic granules discharge their contents
into the phagosome, and death of the ingested microorganism
soon follows. Among the antimicrobial systems formed in the
Fig. 13.4  Diapedesis. In healthy permeable capillaries the endothelium lining
prevents blood cells from leaving the circulation. In response to injury, blood
vessels around the wound site undergo vasodilation, increasing the permeability of
capillaries. Such change enables inflammatory cells to extravasate through the
capillary wall and migrate to the site of injury. This process includes release of fluid
from the vessels to the extracellular space, resulting in the edematous response
commonly noted during inflammation. Blood vessels possess a built-in pathway for
such diapedesis to occur in response to tissue injury.
Normal permeability of capillary
Increased permeability of capillary
during inflammation
Capillary wall
Monocyte
Monocyte squeezing
through interstitial space
Interstitial
spaces
Small amount of fluid
More fluid and
antimicrobal chemicals
phagosome is one consisting of myeloperoxidase (MPO),
released into the phagosome during the degranulation
process, hydrogen peroxide (H2O2), formed by the respiratory
burst and a halide, particularly chloride. The initial product
of the MPO-H2O2-chloride system is hypochlorous acid,
and subsequent formation of chlorine, chloramines, hydroxyl
radicals, singlet oxygen, and ozone has been proposed. These
same toxic agents can be released to the outside of the cell,
where they may attack normal tissue and thus contribute to
the pathogenesis of disease.19 Other products delivered by
neutrophils to the wound site include antimicrobials such as
cationic peptides and eicosanoids as well as proteases such as
elastase, cathepsin G, proteinase 3, and urokinase-type plas­
minogen activator. The balance of neutrophil-released prote­
ase levels and intrinsic inhibitors in the wound environment
is important for wound healing progression.20 Chronic non-
healing wounds have elevated levels of neutrophil-derived
proteases, specifically matrix metalloproteases (MMPs), indi­
cating persistent neutrophil presence. The effects of MMPs are
specifically inhibited by the enzymatic group known as tissue
inhibitors of matrix metalloproteases (TIMPs) and these are
present in abnormally low levels in chronic wounds. The
imbalance between MMPs and TIMPs creates a hostile wound

170
CHAPTER 13 • Wound healing
Fig. 13.5  Extravasation of neutrophils in response to tissue injury. Neutrophils
move along the capillaries in a rolling motion which is facilitated by the binding
and release of L-selectin on the neutrophil surface to sialyl-Lewis, a carbohydrate
ligand expressed on the inner wall of capillaries by endothelial cells. Upon injury
and/or infection the release of lipopolysaccharides, tumor necrosis factor-alpha, and
interleukin-1 results in the shedding of L-selectin by the neutrophils which then
strongly adhere to the inner wall of the capillary by the binding of integrin on the
neutrophils to E-selectins on endothelial cells. After such adhesion, the process of
diapedesis begins, allowing the neutrophils to extravasate to the wound site.
L-selectin
Sialyl-Lewis
Integrin
E-selectin
Rolling
Shedding of
L-selectin
Lipopolysaccharides,
interleukin-1, and
tumor necrosis factor-α
C3a C5a,
chemokines,
histamine,
prostaglandins
and leukotrienes
Activating substances
released by
bacteria
and
damaged
tissues
Adhesion
Diapedesis
Neutrophil
Fig. 13.6  Diapedesis and migration of leukocytes to the wound site.
Fibrin-
platelet clot
Red blood
cell
Collagen
Fibroblast
Elastin
Cytokines &
growth factors
environment as elevated protease levels result in tissue
damage and inflammation that self-perpetuates to create a
vicious cycle that derails wound healing.
In recent years, another functional component of the neu­
trophil action response, the neutrophil extracellular traps
(NETs), have been a focus of interest. Initially identified as
being a beneficial neutrophilic response to pathogens such as
bacteria, fungi and viruses,21–25 NETs are fibrous chromatin
traps that sequester and kill a wide variety of microbes
via release of granular components such as elastase, MPO,
cathepsin G, lactoferrin and gelatinase.26 Some critical triggers
that have been implicated for this process, also called
NETosis, include ROS27 and an enzyme (peptidyl arginine
deiminase 4 (PAD4)) that post-translationally modifies argi­
nine residues on histones (H3 and H4) resulting in chromatin
condensation.28–30 An interesting NETosis response to live
Staphylococcus aureus infection was recently demonstrated
in vitro, where nuclear material was rapidly released without
cytoplasmic contents or lysis of plasma membrane.31 These
denucleated neutrophils were shown to crawl and phagocy­
tose the bacteria trapped in the created nuclear traps.32–34 The
presence of anuclear neutrophils that are biologically active
have been known since the 1980s35 and possibly reflect a popu­
lation of neutrophils that have undergone some aspect of
NETosis. However, many questions remain to be answered in
relation to specific mechanisms of activation of NETs in
response to different stimuli such as biofilm infection.
As it relates to the overall inflammatory process elicited in
response to injury, neutrophils are major players because they
can modify macrophage function and therefore regulate
innate immune response during wound healing.36 In the
absence of neutrophils, wound site macrophages seem to lack
guidance in conducting the healing process.37 In a healing
wound, neutrophil infiltration ceases after a few days of
injury. Expended neutrophils are programmed to die and
dying neutrophils are recognized by wound site macrophages
and phagocytosed. The wound site contains a small portion
of macrophages that are resident. Most macrophages at the
wound site are recruited from the peripheral circulation.
Extravasation of peripheral blood monocytes is enabled by
the interaction between endothelial vascular cell adhesion
molecule-1 and monocyte very late antigen-4 (α4β1 integrin).
Factors that guide the extravasated monocyte to the wound
site include growth factors, chemotactic proteins, proinflam­
matory cytokines, and chemokines such as macrophage
inflammatory protein 1α, MCP-1, and RANTES. The source
of these chemoattractants includes clot-associated platelets,
wound edge hyperproliferative keratinocytes, wound tissue
fibroblasts and subsets of leukocytes already at the wound
site. Once the monocyte leaves the blood vessel to transmi­
grate into the wound site through the ECM microenviron­
ment, the process of monocyte differentiation to macrophages
has started.
Mediators present in the microenvironment that the mono­
cyte traverses to reach the wound site interact with receptors
on the monocyte cell surface, bringing forth major changes in
the transcriptomic as well as proteomic make up of the cell.
Major examples of such receptors present on the monocyte
surface include Toll-like receptors (TLRs; Fig. 13.7), comple­
ment receptors, and Fc receptors. At the wound site, macro­
phages function as antigen-presenting cells and phagocytes
scavenging dead cells and debris. In addition, they deliver a
wide range of growth factors that are known for their abilities
to execute the wound-healing process. Such growth factors
include TGF-β, TGF-α, basic FGF (bFGF), VEGF, and PDGF.
These growth factors enable wound healing by causing cell
proliferation and synthesis of ECM and inducing angiogenesis.
Macrophages play a crucial role in enabling wound healing.
Macrophage depletion is known to impair wound closure
markedly.4,38

171
Inflammation
exist in macrophage activation. The proposed nomenclature
change would involve inclusion of activation standards based
on the source of the activator, be it cytokines such as IL-4,
IL-10 or bacterial lipopolysaccharide (LPS) (e.g. M(IL-4),
M(IL-10), M(LPS), etc., where M refers to macrophage) and
where relevant, a combination of macrophage-specific surface
markers or lack thereof to describe activation outcomes.51
Mast cells
Mast cells are best known for their central role in mediating
allergic responses. Beyond that function, it is now known
that mast cells are physiologically significant in recognizing
pathogens and in regulating immune response.52 Mast cells
may instantly release several proinflammatory mediators
from intracellular stores. In addition, they are localized in the
host–environment interface. These properties make mast cells
key players in fine-tuning immune responses during infec­
tion. Recent studies using mast cell activators as effective
vaccine adjuvants show the potential of harnessing these cells
to confer protective immunity against microbial pathogens.53
Mast cell activation helps initiate the inflammatory phase of
wound healing. In response to injury, mast cells at the wound
site degranulate within a matter of hours and therefore
become histologically silent at the wound tissue. After about
48 hours of injury, mast cells are again seen in the wound
tissue and their number increases as healing progresses.54 On
one hand, impaired wound healing has been reported in mast
cell-deficient mice.55 On the other hand, mast cells have been
implicated in skin wound fibrosis.56 With the aid of a wide
array of newly formed or preformed mediators released by
degranulation, the activated mast cell controls the key events
of the healing phases: triggering and modulation of the
inflammatory stage, proliferation of connective cellular ele­
ments, and final remodeling of the newly formed connective
tissue matrix. The importance of the mast cell in regulating
healing processes is also demonstrated by the fact that a
surplus or deficit of degranulated biological mediators causes
impaired repair, with the formation of exuberant granulation
tissue (e.g., keloids and hypertrophic scars), delayed closure
(dehiscence), and chronicity of the inflammatory stage.57
Resolution of inflammation
Inflammatory responses elicited by injury are only helpful to
the healing process if they are timely and transient. Dysregu­
lated inflammation complicates wound healing and prevents
progression of the wound healing cascade beyond the inflam­
matory phase.58 For resolution of inflammation to ensue,
further leukocyte recruitment must be halted and accompa­
nied by removal of leukocytes from inflammatory sites. At the
wound site successful phagocytosis of dead neutrophils by
macrophages is a key factor. Impairment in macrophage func­
tion, as is commonly seen with diabetes, derails the resolution
of inflammation.58 Lipid mediators, such as the lipoxins,
resolvins, protectins, and newly identified maresins, have
emerged as a novel genus of potent and stereoselective players
that counterregulate excessive acute inflammation and stimu­
late molecular and cellular events that define resolution.59
Successful resolution paves the path for the healing process
to progress towards successful wound closure. Prolonged
inflammation may not only compromise wound closure but
may also worsen scar outcomes.60 The inappropriate and
Macrophages
Macrophages represent the predominant cell type in a healing
wound 3–5 days following injury. The primary acute function
of wound macrophages, which arrive at an injury site hours
later than neutrophils, is to operate as voracious phagocytes
cleansing the wound of all matrix and cell debris, including
fibrin and apoptotic neutrophils. Macrophages also produce
a range of cytokines, growth and angiogenic factors that drive
fibroblast proliferation and angiogenesis.4,39–42 In a classic
study, Leibovich and Ross43 demonstrated that antimacro­
phage serum combined with hydrocortisone diminished the
accumulation of macrophages in healing skin wounds of
adult guinea pigs. Such depletion resulted in impaired dis­
posal of damaged tissue and provisional matrix, compromised
fibroblast count, and delayed healing. Today, macrophages
have emerged to be a pivotal driver of efficient skin repair.44,45
Monocyte-derived macrophages are highly plastic and can
differentiate into proinflammatory (M1) or anti-inflammatory/
pro-reparative (M2) phenotypes that can also transdifferenti­
ate into other cell types based on environmental cues and
molecular mediators.46,47 The macrophage population first
taking part in inflammation may change its phenotype to a
more anti-inflammatory macrophage and assume the role to
resolve inflammation.48,49 Macrophages from diabetic wounds
display dysfunctional inflammatory responses.50 A persistent
inflammatory state of diabetic wound macrophages is caused
by impairment in the ability of these cells to phagocytose
apoptotic cells at the wound site which in turn prevents the
switch from M1 to M2 phenotype.50 Recently, given the lack
of standard, all-encompassing descriptors for macrophages, a
collaborative effort was initiated to re-define macrophage
nomenclature to encompass the scope of diversity known to
Fig. 13.7  Toll-like receptors: responding to infectious agents and the wound
microenvironment.
Cell membrane
Cytoplasm
Lipopolysaccharide
Gram-negative
bacteria
Nucleus
Toll-like
receptor

172
CHAPTER 13 • Wound healing
towards successful healing. Platelets in the clot serve as a
source of growth factors and cytokines, which recruit several
cell types, including endothelial cells, to the wound site.
During the inflammatory phase, leukocytes at the wound site
serve as a major source of pro-angiogenic factors such as
VEGF-A and IL-8 that lay the early foundation for successful
wound tissue vascularization. As neutrophils are expended
and undergo cell death, the number of macrophages at
the wound site substantially increases. Macrophage-derived
TGF-β, TGF-α, bFGF, PDGF, and VEGF play a key role in
driving skin wound angiogenesis. Growing evidence demon­
strates that no single angiogenic factor is singularly effective
in significantly influencing wound outcomes. Wound vascu­
larization is a sophisticated process requiring dynamic, tem­
porally and spatially regulated interaction between cells,
angiogenic factors, and the ECM.
Key processes in tissue vascularization are depicted in Fig.
13.8. Angiogenic cues are elicited by microenvironmental
signals such as hypoxia and are amplified by angiogenic
factors such as VEGF expressed by and released from cells at
the wound site. VEGF was originally identified as an endo­
thelial cell-specific growth factor stimulating angiogenesis
and vascular permeability. Some family members, VEGF C
and D, are specifically involved in lymphangiogenesis. Liga­
tion of these angiogenic factors with their corresponding
receptors elicits a multitude of cell-signaling processes that
activate microvascular endothelial cells. For example, VEGFs
and their endothelial tyrosine kinase receptors are central
regulators of tissue vascularization. VEGF signaling through
VEGFR-2 is the major angiogenic pathway. VEGFR-3 has also
been shown to be important for angiogenesis, acting together
with VEGF/VEGFR-2 and Dll4/Notch signaling to control
angiogenic sprouting.66 The biological significance of other
angiogenic factors such as EGF and bFGF is mediated by their
corresponding receptors, which also belong to the family of
tyrosine kinase receptors EGFR, FGFR-1, FGFR-2, FGFR-3,
and FGFR-4.
Other tyrosine kinase receptors of outstanding significance
in this regard are Tie-1 and Tie-2. Along with the VEGF recep­
tor, these are the only known endothelial cell-specific receptor
tyrosine kinases. Tie-2 is induced on the endothelium of
neovessels in skin wounds and downregulated as newly
formed vessels regress. As an indicator of Tie-2 activation,
Tie-2 phosphorylation is detected in skin wounds at all stages
of the healing process.67 Activated microvascular endothelial
cells respond by proliferating, which is followed by directional
migration of these cells. Migration is a complex process in
which cells move in a given direction either in response to
changes in the extracellular environment or as a consequence
of an intrinsic propensity for directional movement. ECM
remodeling by proteases promotes cell migration, a critical
event in the formation of new vessels. Temporal and spatial
regulation of ECM remodeling events allows for local changes
in net matrix deposition or degradation, which in turn con­
tributes to control of cell growth, migration, and differentia­
tion during different stages of angiogenesis. Matrix-bound
growth factors released by proteases and/or by angiogenic
factors promote angiogenesis by enhancing endothelial
migration and growth. Matrix molecules promote endothelial
cell growth and morphogenesis, and/or stabilize nascent
blood vessels. Hence, ECM molecules and ECM remodeling
events play key roles in regulating angiogenesis.68
sustained levels of inflammation observed in various chronic
wound states could be induced by microbial biofilms and
certainly contribute to derailing the wound healing process.61
Bacteria come armed with various virulence factors that
stimulate a proinflammatory response in the wound microen­
vironment that interfere with the normal pattern of host
responses. Details on the role of infection, particularly bacte­
rial biofilm infection in wound healing, are covered in the
section on chronic wounds.
Proliferative phase
The proliferative phase starts around 2 days after injury and
normally lasts up to 3 weeks in a healing cutaneous wound.
This phase overlaps with the inflammatory phase and sup­
ports re-epithelialization, the formation of new blood vessels,
and the influx of fibroblasts and laying down of the ECM. By
the time this phase begins, the degradation of the fibrin clot
by the macrophages has begun and invading endothelial cells
and fibroblasts rapidly fill that space. Migrating fibroblasts
produce the cytokines that induce keratinocytes to migrate
and proliferate.62 Activated macrophages produce several
cytokines, such as PDGF and TNF-α, which also induce
fibroblasts to produce keratinocyte growth factor which in
turn induces wound re-epithelialization.63,64
Granulation tissue
The fibrin clot formed during hemostasis participates in the
early inflammatory phase and is replaced by a perfused,
fibrous connective tissue that grows from the base of a wound
and is able to fill wounds of almost any size. During the
proliferative phase of wound healing, this granulation tissue
is light red or dark pink in color because of perfusion by new
capillary loops. It is soft to the touch, moist, and granular in
appearance. The granulation tissue serves as a bed for tissue
repair. The ECM of granulation tissue is created and modified
by fibroblasts. Initially, it consists of a network of type III
collagen, a weaker form of the structural protein that can be
produced rapidly. This is later replaced by the stronger, long-
stranded type I collagen, as evidenced in scar tissue. Forma­
tion and contraction of the granulation tissue represent
integral aspects of the healing wound. In ischemic wounds,
production and contraction of the granulation tissue is
impaired because of decreased ATP production, impaired
collagen synthesis and failure to convert the fibroblast pheno­
type into a myofibroblast to promote wound contraction.65
Vascularization
Wounds complicated by underlying ischemia largely rely on
wound vascularization for their closure. Wound vasculariza­
tion may be achieved by angiogenesis or vasculogenesis.
Angiogenesis represents sprouting of capillaries from existing
blood vessels in the wound edge tissue. Vasculogenesis relies
on the formation of new blood vessels by mobilization of bone
marrow-derived endothelial stem cells.
Wound vascularization is regulated by all phases in wound
healing – hemostasis, inflammation, tissue formation, as well
as tissue remodeling. The hemostatic plug provides a bed to
attract blood-borne cells to the wound site. Once cells entangle
in this plug, the ECM environment modifies cell function

173
Wound closure
microenvironment and promoting epidermal tissue renewal
from non-stem cells.72
Wound closure
Wound contraction and re-epithelialization contribute to
closure of wounds that heal by secondary intention. Wound
contraction represents an early response to injury that is
aimed at juxtaposing the edges of an open wound.73 This early
phase of wound closure appears to be mediated by a contrac­
tile “purse-string” force produced by a circumferentially
arranged band of fusiform-shaped epidermal cells situated in
the wound margin.74 Fibroblasts at the wound edge tissue are
recognized to play a key role in enabling wound contraction.75
During the inflammatory phase of wound healing, fibroblasts
acquire smooth-muscle cell characteristics and differentiate
into contractile myofibroblasts. The first phenotypic transition
of wound edge fibroblasts into so-called protomyofibroblasts
is characterized by neoformation of contractile β-cytoplasmic
actin stress fibers and occurs in response to profibrotic cyto­
kines and to altered properties of the ECM. In the presence of
TGF-β1 in a mechanically restrained environment, these cells
express α-smooth-muscle actin de novo, which significantly
increases their contractile activity and is a hallmark of the
differentiated myofibroblast.76 Wound contraction may sig­
nificantly contribute to wound closure, although this contri­
bution is much larger in loose-skinned rodents than in
humans.
In an open wound that has undergone contraction, restora­
tion of an intact epidermal barrier is enabled through wound
epithelialization, also known as re-epithelialization.77,78 A
wound that is not epithelialized is not considered “healed”,
no matter how perfectly restored the underlying dermal
structures may be. Thus, wound epithelialization is a critical
and defining feature of wound repair. Re-epithelialization of
The formation of the capillary-like tubes is specific to
endothelial cells and integral to the process of angiogenesis.
The basement membrane represents a biologically functional
highly specialized ECM on which the basal non-luminal
surface of endothelial cells rests. This matrix forms a continu­
ous sleeve around the endothelial cells, and maintains the
tube-like structures of the blood vessels. More than 20 years
ago Kubota et al. observed that endothelial cells plated on a
reconstituted basement membrane matrix, rapidly attached,
aligned, and formed capillary-like tubules. The cells did not
proliferate. The vessels that are thus formed contain a lumen
and tight cell–cell contacts. The cells are polarized with the
nuclei basally located towards the basement membrane
matrix. Furthermore, the capillary-like structures take up
acetylated low-density lipoprotein, which is a marker of dif­
ferentiation for these cells.69 Angiogenesis not only depends
on endothelial cell invasion and proliferation, but also requires
pericyte coverage of vascular sprouts for vessel stabilization.
These processes are coordinated by VEGF and PDGF through
their cognate receptors on endothelial cells and vascular
smooth-muscle cells, respectively.70 Structural support to
blood vessels is provided to pericytes and vascular smooth-
muscle cells. Normal pericytes are embedded within the
basement membrane of capillaries, either as solitary cells or a
single-cell layer, where they coordinate intercellular signaling
with endothelial cells and other components of the blood
vessel wall to prevent leakage. In contrast, vascular smooth-
muscle cells form single or multiple layers around arteries
and veins to mediate vascular tone and contraction. Pericyte
coverage is required for the stabilization of immature endo­
thelial tubes.71 Pericytes have functions beyond angiogenesis
that are relevant to wound healing. Skin pericytes are very
plastic and may act as mesenchymal stem cells (MSCs), exhib­
iting the capacity to differentiate into bone, fat, and cartilage
lineages. Thus, pericytes represent a potent stem cell popula­
tion in the skin that is capable of modifying the ECM
Fig. 13.8  Neoangiogenesis: the formation of new
blood vessels. VEGF, vascular endothelial growth
factor; ECM, extracellular matrix.
1
3
4
5
6
10
9
8
7
2
Angiogenic
factors bind
to endothelial
cell receptors
Endothelial cell
activation
Angiogenic
factor (VEGF)
production
Release
Tie-2
αyβ3
Pericyte
Endothelial cell
proliferation
Directional
migration
Loop
formation
Vascular
stabilization
Tube
formation
ECM
remodeling

174
CHAPTER 13 • Wound healing
in subsequent sections in this chapter. The regulation of a
variety of functionally related genes by modulating a single
miRNA holds promise for miRNA-based therapeutics. As the
knowledge of specific roles of miRNAs continues to grow, the
emergence of miR-based therapies that specifically upregulate
beneficial miRs and/or downregulate potentially harmful
miRs will emerge as useful tools in the clinic. miRNA depen­
dent regulation of specific phases in wound healing are
mentioned in the section on chronic wounds.
Acute wounds
Any violation of live tissue integrity may be regarded as a
wound. Skin is the largest organ of the human body, covering
about 3000 square inches (7620 cm2) in an average adult. The
most important role of the skin for terrestrial animals is to
protect the water-rich internal organs from the dry external
environment. As a primary line of defense against external
threats, maintenance of integrity of the skin is a key prereq­
uisite for healthy survival. Thus, healthy skin can regenerate
and repair itself under most common conditions. Skin wounds
may be open when manifested as a tear, cut, or puncture.
Blunt force trauma may cause closed wounds or contusion
where the skin appears to be intact but suffers from damage
caused to underlying tissues.
Open wounds may be generally categorized as:
■ Lacerations – ragged tears and cuts; masses of torn tissue
underneath; caused by dull knife, bomb fragments and
machinery and may include crushing of tissues;
frequently contaminated (Fig. 13.9)
■ Puncture – e.g., sharp penetrations caused by nails,
needles, wire, or bullets (see Fig. 13.9). These are of great
concern in patients with diabetes, as many of these
patients have polyneuropathy and have insensate feet,
leading to occult injury. Many times these patients will
step on thumb tacks, safety pins, or other sharp
household objects and not even know it: this, coupled
with compromised vascular status, leads to chronic
wound infection
■ Abrasions – the superficial layer of the skin is removed;
skinned knee or elbows and rope burns are examples; an
abrasion lends itself to infection
the wound can be conceptually viewed as the result of three
overlapping keratinocyte functions: migration, proliferation,
and differentiation. The sequence of events by which keratin­
ocytes accomplish the task of re-epithelialization is generally
believed to begin with dissolution of cell–cell and cell–
substratum contacts. This is followed by the polarization and
initiation of directional migration in basal and a subset of
suprabasilar keratinocytes over the provisional wound matrix.
A subset of keratinocytes immediately adjacent to, but not
within, the wound bed then undergoes mitosis. Finally, there
is multi-layering of the newly formed epidermis and induc­
tion of differentiation-specific gene products to restore the
functionality of the epidermis. The most limiting factor in
wound re-epithelialization is migration, since defects in this
function, but not in proliferation or differentiation, are associ­
ated with the clinical phenotype of chronic non-healing
wounds.79 The process of epithelialization continues until the
barrier is re-established and the wound is covered. The process
of re-epithelialization is accelerated by a moist environment80,81
and is facilitated by the enzyme matrix metalloproteinase 1,
a collagenase which lessens the affinity of the collagen–integrin
contacts.82
MicroRNAs
Wound healing is largely dependent on injury-inducible
protein-coding genes as they serve as drivers of an inherent
tissue repair program that seeks to restore the injured tissue
both structurally as well as functionally. There are two key
steps that separate a protein-coding gene from its correspond­
ing protein. First, the DNA hosting the gene must transcribe
to mRNA and second, the mRNA must be translated to
protein. Work emerging during recent years demonstrates
that both of these critical steps are subject to robust and
redundant regulation by microRNAs (miRNAs: 19–22 nucleo­
tides long), which are noncoding RNAs found in all eukaryotic
cells. Work during the past decade recognizes small RNAs as
a new class of regulators of eukaryotic biology that could be
classified as epigenetic regulators of gene expression.83 Along­
side other small interfering RNAs (siRNAs), miRNAs execute
post-transcriptional gene silencing through mRNA destabili­
zation as well as translational repression. In simple terms,
whether a gene would code a protein or not is decided by
miRNAs for which the gene is a target. miRNAs form base
pairs with specific sequences in protein-coding mRNAs.
Near-perfect pairing induces cleavage of the target mRNA,
whereas partial pairing results in translational repression and
mRNA decay through deadenylation pathways.84 According
to the miRbase database, the human genome encodes 1881
miRNAs. This count is rapidly growing. These miRNAs may
regulate more than a third of all protein-coding genes and
virtually all biological processes. Mammalian cells express
cell type-specific miRNAs which silence unique subsets of
target genes within the cell. While miRNAs are mostly known
for being functional in the cytoplasm, nuclear miRNAs may
also participate in gene regulation. Initially considered an
oddity, miRNA-dependent control of gene expression is now
accepted as being integral to the normal function of cells and
organisms.85 miRNAs are emerging as key regulators of the
overall wound-healing process.86–88 miRNA dependent regu­
lation of processes as it relates to wound healing are discussed
Fig. 13.9  Laceration and puncture wounds.
Laceration
Puncture wound

175
Chronic wounds
re-epithelialization, and collagen deposition have all been
linked to the presence of adequate levels of each of these
vitamins independently.95
Micronutrients such as magnesium, copper, zinc and iron
serve as co-factors particularly for enzymes that support the
wound healing process.89,93,99
Therapeutic oxygen
Oxygen directly impacts wound healing by targeting numer­
ous molecular targets and cell types. Increased fibroblast
proliferation and migration, increased collagen synthesis and
the resulting increase in the tensile strength of collagen fibers,
stimulation of angiogenesis, promotion of immune functions
such as macrophage chemotaxis, leukocyte antibacterial and
physiological wound debridement functions are all attributed
to the essential role of oxygen in healing wounds.102,103 There­
fore, the use of direct oxygen supplementation to improve
wound healing is a natural extension of these observations.
The therapeutic oxygen modalities that are used to treat
wounds are described in more detail in later sections in this
chapter.
Chronic wounds
Factors that drive wound chronicity
Infection and biofilm
Infection is a common problem in chronic wounds, frequently
resulting in non-healing and significant patient morbidity and
mortality.104 Wound infection and the subsequent release of
proinflammatory modulators result in pain and delayed
healing. The pain, in turn, compromises the immune response
to infection.105 All wounds become contaminated by bacteria
from the surrounding skin, the local environment, and autolo­
gous patient sources. The local environment is particularly
relevant for hospitalized patients. Colonization is defined as
the presence of proliferating bacteria without a noticeable
host response. Colonization of the wound may enhance or
impede wound healing, depending upon the bacterial load.
Bacterial loads in excess of 105 organisms per gram of tissue
are a threat to wound healing, although this threshold may
be altered by the status of the host immune system and the
number and types of bacterial species present. The concept
of critical colonization, characterized by increased bacterial
burden or covert infection, is controversial and not universally
accepted. Substantial colonization may not cause obvious
signs of inflammation, but may affect wound healing with
failure to heal or slowing of progression. Signs of critical colo­
nization are atrophy or deterioration of granulation tissue,
discoloration of granulation tissue to deep red or gray,
increased wound friability, and increased drainage. The tran­
sition to infection occurs when bacterial proliferation over­
comes the host’s immune response and host injury occurs.
Several factors determine transition from colonization to
infection: the bioburden itself, the virulence of the organisms,
the synergistic action of different bacterial species, and the
ability of the host to mount an immune response.104
During the past decade, there has been rapid progress in
the understanding of innate immune recognition of microbial
■ Avulsions – sections of skin torn off either in part
(attached to body) or totally (detached from body); heavy
bleeding is common
■ Amputations – traumatic amputation results in
nonsurgical removal of limb from the body and
accompanies heavy bleeding.
There are three general techniques of wound treatment:
1. Primary intention, in which all tissues, including the
skin, are closed with suture material after completion of
the operation
2. Secondary intention, in which the wound is left open
and closes naturally
3. Tertiary intention, in which the wound is left open for a
number of days and then closed if it is found to be clean.
Factors that promote healing
Nutrition
The importance of adequate nutrition to supplement and
sustain the energy-intensive process of wound healing has
been established for many years.89–92 The need for nutrients
such as carbohydrates, protein, fat, amino acids, vitamins and
mineral supplements is underscored by the observations that
in malnutrition conditions or lack of sufficient nutritional
supplementation, processes critical to wound healing are
impeded.
These
include
delayed
neovascularization,
decreased collagen synthesis leading to impaired mechanical
strength of the skin and prolonged inflammation leading to
dysfunctional immune responses.89,93–96 Since the identifica­
tion of malnutrition as a reversible risk factor for pressure
ulcer development,97 recommendations have been made for
the inclusion of validated nutrition screening and assessment
tools to determine nutritional status. Serum pre-albumin
(transerythrin) has a shorter half-life than albumin and may
serve as a more sensitive indicator of changes in protein–
energy status. It is recommended as a good screening tool for
malnutrition if used as two measurements taken 3–5 days
apart along with CRP levels.98
Fats (particularly short chain fatty acids (SCFAs) such as
butyrates, acetate and propionate) and carbohydrates (such as
glucose) provide the primary energy source for collagen
synthesis and deposition and angiogenesis. The American
Society for Parenteral and Enteral Nutrition and the Wound
Healing Society guidelines for optimal wound healing recom­
mends 30–35 kcal.kg−1.d−1 for normal, healthy adults and
35–40 kcal.kg−1.d−1 for adults suffering from malnutrition.
Fibroblast proliferation, collagen deposition, formation of
vascular supply and immune responses are dependent on the
availability of proteins, which are the building blocks of
wound repair. The recommended range of protein intake for
wound healing is 0.8–1 g.kg−1.d−1 in normal adults and higher
(up to 2 g.kg−1.d−1) for chronic wound patients. The amino
acids arginine and glutamine have been studied for their roles
in wound healing, particularly related to collagen deposition,
angiogenesis and immune function.89,93,99–101 However, it still
remains unclear how essential these and other amino acids
are in specific aspects of wound healing.
Vitamins A, C and E have potent antioxidant and
anti-inflammatory effects that are implicated in promoting
proper tissue repair. The stimulation of immune responses,

176
CHAPTER 13 • Wound healing
broad-spectrum antibiotic therapy may predispose patients to
colonization or infection, or both, with resistant organisms,
including S. aureus (methicillin-resistant S. aureus – MRSA)
or vancomycin-resistant enterococci (VRE).108 Inflammatory
responses to microbial invasion may be diminished in persons
with diabetes or other immunosuppressive conditions.108
Microorganisms do not always live as pure cultures of
dispersed single/planktonic cells but instead accumulate at
interfaces to form polymicrobial aggregates such as films,
mats, flocs, sludge, or biofilms (Fig. 13.10). The biofilm state
of microorganisms may lead to an increase in virulence and
resistance to antibiotic therapy and evasion of host immune
response. In most biofilms, the microorganisms account for
less than 10% of the dry mass, whereas the matrix can account
for over 90%. The matrix is the extracellular material, mostly
produced by the organisms themselves, in which the biofilm
cells are embedded. It consists of a conglomeration of differ­
ent types of biopolymers – known as extracellular polymeric
substances (EPS) – that aid in adhesion to surfaces, form the
scaffold for the developing biofilm and also provide a physical
barrier that is relatively impenetrable to antimicrobial sub­
stances and immune responses. The formation of a biofilm
allows a lifestyle that is entirely different from the planktonic
state. Although the precise and molecular interactions of the
various secreted biofilm matrix polymers have not been
defined, several functions of EPS have been determined,
demonstrating a wide range of advantages for bacteria in a
biofilm state. The architecture of biofilms as determined by in
vitro studies is influenced by many factors, including hydro­
dynamic conditions, concentration of nutrients, bacterial
motility, and intercellular communication, as well as exopoly­
saccharides and proteins.109 These studies involving the use of
abiotic (e.g. Calgary biofilm device, modified Robbins device,
Bioflux systems, etc.) or biotic surfaces (e.g. reconstituted
human epithelia (RHE), Lubbock model, tissue-engineered
equivalents such as Graftskin) have helped to understand
numerous mechanistic aspects of biofilm growth such as
intercellular communication involving quorum sensing,
antimicrobial tolerance and also testing the efficacy of anti-
biofilm therapeutics. However, the clinical relevance of these
studies is limited because of the inability to address the itera­
tive host response to the presence of biofilm that defines the
establishment of chronic infection. In vivo studies on biofilms
have included a range of model organisms from the common
components and its critical role in host defense against infec­
tion. The early concept of innate immunity was that it non­
specifically recognized microbes; however, the discovery of
TLRs (Fig. 13.7) in the mid-1990s showed that pathogen rec­
ognition by the innate immune system is instead actually
specific, relying on germline-encoded pattern recognition
receptors (PRRs) that have evolved to detect components of
foreign pathogens, referred to as pathogen-associated molecu­
lar patterns (PAMPs).106 TLRs regulate innate and adaptive
immune responses and are important modulators of inflam­
mation during wound-healing responses. The finding that
there is activation of TLR signaling during tissue damage in
several disease situations in the absence of infection suggests
that endogenous molecules serve as TLR agonists, although
it is unclear whether this response is biologically important
for maintenance of homeostasis, such as tissue repair, or
whether this recognition is simply accidental. It is noteworthy
that microbial infection triggers the production of modified
endogenous molecules (such as high-mobility group protein
B1, oxidized phospholipids, β-defensin 2, and nucleic acids)
that are recognized by TLRs or other cytosolic PRRs. This may
suggest that these endogenous molecules, along with PAMPs,
act as adjuvants to activate innate immune programs via TLRs
and/or other PRRs, and have key roles in facilitating adaptive
immunity against infecting microbes.
Chronic wounds have a complex colonizing flora that
changes over time. Staphylococcus aureus and coagulase-
negative staphylococci are the most commonly isolated
organisms. Chronic wounds are colonized by multiple bacte­
rial species and many persist in the wound once they are
established. In chronic venous leg ulcers the most common
bacteria noted, in order of abundance, were S. aureus, Entero­
coccus faecalis, Pseudomonas aeruginosa, coagulase-negative
staphylococci, Proteus spp., and anaerobic bacteria. Resident
(colonizing) bacterial species are commonly present in ulcers.
The longer an ulcer remains unhealed, the more likely it will
acquire multiple aerobic organisms and a significant anaerobic
population. Chronic wounds are commonly complicated by
localized ischemia because of disruption of the vessels at the
site of tissue injury. Thus, they tend to have a low tissue
oxygen level. This facilitates the growth of anaerobes in
ischemic wounds. Adequate delivery of oxygen to the wound
tissue is vital for optimal healing and resistance to infection.107
Hospitalization, surgical procedures, and prolonged or
Fig. 13.10  Scanning electron microscopy (SEM)
images of planktonic and biofilm mode of the Gram-
negative pathogen Pseudomonas aeruginosa.

177
Ischemia and tissue oxygenation
biofilm infection is based primarily upon clinical findings.
Biofilm infection is the clinical paradigm that can be
consistently applied to all chronic wounds and likely repre­
sents a unifying pathologic feature. Therefore, there is a strong
need to understand the clinical biofilm.
Determining clinical endpoints
The FDA defines complete wound closure of chronic non-
healing wounds as “skin closure (as assessed visually) without
drainage or dressing requirements identified at two consecu­
tive study visits that are 2 weeks apart” and requires thera­
peutic trials for chronic wounds to be designed such that the
enrolled patients will be evaluated for at least 3 months fol­
lowing complete closure.125 Here, the expectation is that suc­
cessful intervention should keep the wound closed for at least
90 days. The determination of wound closure is based primar­
ily on visual assessments. However, such visually closed
wounds are sometimes prone to post-closure complications
such as recurrence. Indeed high recurrence rates have been
reported for chronic wounds (40–79% for pressure ulcers
(PUs), 24–57% for venous leg ulcers (VLUs) and upward of
60% for diabetic foot ulcers (DFUs).126–131 Recent evidence
indicates that biofilm-infected wounds appear visually closed,
but remain functionally open (high transepidermal water loss
(TEWLhi)) because of elevated levels of biofilm-induced
microRNAs (miR-146a and miR-106b) and dysregulated tight
junction proteins (ZO-1 and ZO-2) (Fig. 13.11). This challenges
the current assessment of visual wound closure and empha­
sizes the need for additional measurements such as TEWL in
the objective and comprehensive monitoring of wound
healing.113
miR regulation of skin barrier function
Evidence from Dicer ablation and microRNA overexpression
studies in the intestinal epithelium have supported roles for
miRNA regulation of epithelial barrier function.132 In the
cutaneous epithelium, recent studies have identified roles for
miRs in the regulation of skin barrier function. miR-dependent
silencing of cellular tight junction proteins resulting in com­
promised epithelial barriers was recently identified and miR-
146a was validated to directly target ZO-1 and ZO-2,113 key
tight junction proteins that are essential for the establishment
of proper junctional connections between skin cells133–135 that
contribute to proper skin barrier function. miR-146a along
with miR-106b were also identified as being biofilm inducible.
More recently, keratinocyte-specific Dicer ablation studies in
a mouse model also supported roles for the miRNA regula­
tory pathway in proper establishment of barrier function via
the p21waf1/Cip1 pathway following wounding.136
Ischemia and tissue oxygenation
Vascular complications commonly associated with problem­
atic wounds are primarily responsible for wound ischemia.
Limitations in the ability of the vasculature to deliver O2-rich
blood to the wound tissue lead to, among other consequences,
hypoxia. Hypoxia is a reduction in oxygen delivery below
tissue demand, whereas ischemia is a lack of perfusion, char­
acterized not only by hypoxia but also by insufficient nutrient
supply.103 Hypoxia, by definition, is a relative term. It is
fruit fly (Drosophila melanogaster) to vertebrates such as rats,
mice, rabbits and pigs, each with its own advantages and
disadvantages.110,111 The high homology between porcine and
human skin, the similarities in the immune responses com­
pared to rodent models and the observation that in relation
to wound therapies, porcine studies were in agreement with
humans 78% of the time compared to 53% and 57% with
rodents and in vitro, respectively, make the porcine model
translationally relevant as recommended by the Wound
Healing Society.112 Recently, a chronic wound biofilm model
was developed with the goal of understanding long term
host–biofilm interactions that drive wound chronicity. This
model was used to demonstrate that persistent biofilm infec­
tion compromised wound healing by interfering with tight
junction proteins that are critical for maintenance of skin
barrier function.113
Wounds provide an ideal environment for biofilm develop­
ment. The extracellular matrix components such as collagen,
fibronectin and laminin could serve as ligands for attachment
of bacteria to initiate colonization. Wound beds are moist and
nutritious and provide the conditions that would allow micro
colonies of bacteria to develop surrounded by the protective
barrier of the matrix provided the initial colonization is suc­
cessful. In fact, it is estimated that 60% of chronic human
wounds are infected with biofilm bacteria emphasizing an
important role for the sessile, aggregated mode of bacterial
growth as factors in chronicity.114 Compared to bacteria in the
unattached free-living planktonic form, bacteria that reside
within mature biofilms are highly resistant to traditional
antibiotic therapies. Bacteria in biofilms grow more slowly,
and slower growth may lead to decreased uptake of the
drug and other physiologic changes that could impair drug
effectiveness.109
There are several features of biofilm infection in wounds
that represent significant clinical and diagnostic challenges.
First, bacteria in a biofilm state cannot be cultured reliably
using standard culture methods and the gram stain frequently
will not show inflammatory cells, so even when bacteria are
seen on the gram stain they are diagnosed as colonizing
organisms not pathogens. In a case series of sternal wound
infections only two out of six patients with staphylococcal
biofilm infection had positive cultures, but staphylococcal
biofilm was detected on sternal wires in all six patients using
scanning electron microscopy.115 Second, debridement alone
does not effectively eradicate biofilm infection. It can drive
biofilm-producing bacteria deeper into the surrounding tissue
with recurrence of biofilm infection within 7 days.113 Addi­
tionally, aberrant host immune responses to biofilm infection
are implicated in the persistence of infection and wound
chronicity.61,116–124 Biofilms of Pseudomonas and Staphylococcus
spp. trigger the host response by recruitment of polymorpho­
nuclear leukocytes to the sites of the biofilm. However, after
recruitment, these host cells are frustrated in their expected
function of clearing the infection because of biofilm-specific
arsenal such as alginate and rhamnolipids (Pseudomonas spe­
cific) or polysaccharide intercellular adhesins (PIA, Staphylo­
coccus specific) that hold the host response at bay.123 By
definition bacteria in a biofilm state are adherent to a surface,
e.g. a catheter, an implant, soft tissue or bone in a wound124,
so they will not result in bacteremia and systemic illness. Since
scanning electron microscopy is not clinically available, and
current culture methods are unreliable, the diagnosis of

178
CHAPTER 13 • Wound healing
defined by a lower tissue partial pressure of oxygen (pO2)
compared to the pO2 to which the specific tissue element in
question is adjusted under healthy conditions in vivo. Depend­
ing on the magnitude, cells confronting hypoxic challenge
either induce an adaptive response that includes increasing
the rates of glycolysis, conservation of energy or progression
to cell death. Generally, acute, mild to moderate hypoxia sup­
ports adaptation and survival. In contrast, chronic, extreme
hypoxia leads to tissue loss.
While tumor tissue is metabolically designed to thrive
under conditions of hypoxia, hypoxia of the wound primarily
caused by vascular limitations is intensified by coincident
conditions (e.g., infection, pain, anxiety and hyperthermia)
and leads to poor healing outcomes. Oxygen and its reactive
derivatives (Fig. 13.12) are required for oxidative metabolism-
derived energy synthesis, protein synthesis, and the matura­
tion (hydroxylation) of extracellular matrices such as collagen.
Molecular oxygen is also required for nitric oxide (NO) syn­
thesis which in turn plays a key role in the regulation of
vascular tone as well as in angiogenesis. In a wound setting,
large amounts of molecular oxygen are partially reduced to
form reactive oxygen species (ROS). ROS include oxygen free
radicals such as superoxide anion as well as its non-radical
derivative, hydrogen peroxide (H2O2). Superoxide anion
radical is the one-electron reduction product of oxygen.
NADPH oxidases represent one major source of superoxide
anion radicals at the wound site. NADPH oxidases in
phagocytic cells help fight infection. Superoxide anion also
drives endothelial cell signaling such as that required during
Fig. 13.11  Induction of biofilm-inducible miRs in infected wounds followed by silencing of tight junction proteins ZO-1 and ZO-2, results in compromised skin barrier
function indicated by high transepidermal water loss (TEWL) measurements (TEWLhi). Such wounds may appear to be visually closed but are functionally open (leaky/failed
skin).
TEWLhi
Wound biofilm infection
TEWLlo
Healed wound
Visual wound closure (current clinical endpoint)
Functional closure
(proposed new endpoint
for wound closure)
Functionally open
(leaky skin)
Disrupted skin barrier
(prone to reopening / recurrence)
Tight junctions disrupted
TEWL high
Penetration high
Tight junction dysregulation
(e.g. ZO-1 ZO-2)
MicroRNA upregulation
(e.g. miR-146a miR-106b)
Fig. 13.12  Molecular oxygen and its derivatives in wound healing. ATP, adenosine
triphosphate; NO, nitric oxide.
Red blood
cells
Mature collagen
Endothelial cells
Redox
signals
H2O2
O2
Superoxide
anion radical
NO
HOCl
Infectious
pathogens
Neutrophil
Protein
synthesis
Macrophage
ATP

179
Ischemia and tissue oxygenation
to fuel the repair process. Indeed, uncontrolled expression of
VEGF and its receptors leads to insufficient skin angiogene­
sis.138 Whether cells in the pockets of extreme hypoxia are
O2-responsive is another concern. Even if such cells may have
passed the point of no return in the survival curve, correction
of tissue oxygenation is likely to help clean up the dead or
dying tissue and replace the void with proliferating neighbor­
ing cells. Pockets of moderate or mild hypoxia are likely to be
the point of origin of successful angiogenic response as long
as other barriers such as infection and epigenetic alterations
are kept to a minimum.
Limited supply and high demand:
the oxygen imbalance
Peripheral vascular disease can affect the arteries and the
veins as well as the lymph vessels. The most common and
important type of peripheral vascular disease is peripheral
arterial disease, or PAD, which affects about 8 million Ameri­
cans. The ankle brachial pressure index represents a simple
noninvasive method to detect arterial insufficiency within a
limb. Arterial diseases, especially those associated with dia­
betes, represent a major complicating factor in wound healing.
PAD is the only identifiable etiology in approximately 10% of
leg ulcers. In an ischemic limb, peripheral tissues are deprived
of blood supply as PAD progresses, causing tissue loss, ulcers,
and gangrene.
Venous insufficiency, on the other hand, is the root cause
of most leg ulcers. Chronic venous insufficiency, characterized
by the retrograde flow of blood in the lower extremity, is
associated with changes in the venous wall and valves gener­
ally caused by inflammatory disorders induced by venous
hypertension and associated fluid shear stress. Factors causing
arterial hypoxemia may also limit O2 supply to the wound
tissue. Compromised pulmonary health, loss of hepatic func­
tion, hemodialysis, anemia, altitude hypoxemia, nitroglycerin
therapy, nasal packing, critical illness, pain, and hypothermia
angiogenesis. In biological tissues, superoxide anion radical
rapidly dismutates to hydrogen peroxide, either spontane­
ously or facilitated by enzymes called superoxide dismutases.
Endogenous hydrogen peroxide drives redox signaling, a
molecular network of signal propagation that supports key
aspects of wound healing such as cell migration, proliferation,
and angiogenesis. Neutrophil-derived hydrogen peroxide
may be utilized by MPO to mediate peroxidation of chloride
ions, resulting in the formation of hypochlorous acid (HOCl),
a potent disinfectant (see Fig. 13.12).
Three major factors may contribute to wound tissue
hypoxia: (1) peripheral vascular diseases garroting O2 supply;
(2) increased O2 demand of the healing tissue; and (3) genera­
tion of ROS by way of respiratory burst and for redox signal­
ing.137 Other related factors, such as arterial hypoxia (e.g.,
pulmonary fibrosis or pneumonia, sympathetic response to
pain, hypothermia, anemia caused by major blood loss, cya­
notic heart disease, high altitude), may contribute to wound
hypoxia as well. Depending on factors such as these, it is
important to recognize that wound hypoxia may range any­
where from near anoxia to mild to modest hypoxia. In this
context it is also important to appreciate that point measure­
ments performed in the wound tissue may not provide a
complete picture of the wound tissue biology because it is
likely that the magnitude of wound hypoxia is not uniformly
distributed throughout the affected tissue, especially in large
wounds. This is most likely the case in chronic wounds pre­
sented clinically as opposed to experimental wounds, which
are more controlled and homogeneous in nature. In any single
problem wound presented in the clinic, it is likely that there
are pockets of near anoxia as well as that of different grades
of hypoxia (Fig. 13.13). As the weakest link in the chain, tissue
at the near-anoxic pockets will be vulnerable to necrosis,
which in turn may propagate secondary tissue damage and
infection. Pockets of extreme hypoxia may be flooded with
hypoxia-inducible angiogenic factors but would fail to vascu­
larize functionally because of insufficient O2 that is necessary
Fig. 13.13  Heterogeneous distribution of oxygen in the
wound tissue: pockets of graded levels of hypoxia. Shade
of blue represents graded hypoxia. Shade of red or pink
represents oxygenated tissue. Tissue around each blood
vessel is dark pink in shade, representing regions that are
well oxygenated (oxygen-rich pockets). Bacteria and
bacterial infection are presented by shades of green on
the surface of the open wound.
Pocket of oxygenation
Pocket of hypoxia
Near-anoxic necrotic core
Endothelial
progenitor cell
Endothelial
cell
Myofibroblast
Basal layer
Dermis
Keratinocyte in
epidermis
Stratum
granulosum
Stratum corneum
Neutrophil
Bacteria
Macrophage

180
CHAPTER 13 • Wound healing
of mmHg. The Km for O2 for this reaction is approximately 25
and the Vmax is approximately 250 mmHg, suggesting that
new vessels cannot even approach their greatest possible rate
of growth unless the wound tissue pO2 is high.140 Angiogen­
esis is directly proportional to pO2 in injured tissues.141,142
Hypoxic wounds deposit collagen poorly and become infected
easily, both of which are problems of considerable clinical
significance.103
Redox signaling
Additional high demand for oxygen is placed by a family of
enzymes known as NADPH oxidases, which are known to
be highly active at the wound site.143 Recent work has identi­
fied that oxygen is not only required to disinfect wounds
and fuel healing but that oxygen-dependent redox-sensitive
signaling processes represent an integral component of the
healing cascade.142 The widely held notion that biological free
radicals are necessarily agents of destruction is now facing
serious challenge.143 Over a decade ago it was proposed that
in biological systems oxidants are not necessarily always
the triggers for oxidative damage and that oxidants such as
H2O2 could actually serve as signaling messengers and drive
several aspects of cellular signaling.143 Today, that concept
is much more developed and mature. Evidence supporting
the role of oxidants such as H2O2 as signaling messengers is
compelling.144–156
Nitric oxide
At the wound site, NO is generated by an oxygen-dependent
biosynthetic process. In the late 1970s, research was unfolding
that implicated NO involvement in the process of vasodila­
tion. By 1986, research culminated in the identification of NO
as the endothelium-derived relaxing factor responsible for the
maintenance of vascular tone, thus implicating NO as a poten­
tial wound-healing agent.157 Maximal NO synthase activity is
noted early in cutaneous wound healing, with sustained
production up to 10 days after wounding. Wound macro­
phages represent a major source of NO production in the early
phase of wound healing.158 Inhibition of wound NO synthesis
lowered wound collagen accumulation and wound-breaking
strength, suggesting that NO synthesis is critical to wound
collagen accumulation and acquisition of mechanical strength.
Later it was demonstrated that wound fibroblasts are pheno­
typically altered during the healing process to synthesize NO,
which, in turn, regulates their collagen synthetic and contrac­
tile activities.159 The blockade of NO synthesis impairs cutane­
ous wound healing, acting in early and late phases of wound
repair.160 Interestingly, impaired diabetic wound healing is
associated with decreased wound NO synthesis.161
microRNA and hypoxia response
Tissue injury is often associated with disruption of vascular
supply to the injury site. Thus, the injured tissue often suffers
from insufficient oxygen supply or hypoxia. Under conditions
of additional underlying ischemia, hypoxia is severe and
seriously limits wound healing.103 Hypoxia induces specific
miRNAs, collectively referred to as hypoxamirs.162 miRNA-
210 is a classic hypoxamir. Expression of hypoxia-inducible
factor 1 (HIF-1) is also controlled by specific miRNAs. In turn,
HIF-1 controls the expression of hypoxamirs, which are
are examples of conditions associated with arterial hypox­
emia. Vasoconstricting drugs may contribute to tissue hypoxia
as well.103
Increased energy demand of the healing tissue leads to a
hypermetabolic state wherein additional energy is generated
from oxidative metabolism, increasing the O2 demand of the
healing tissue. Adenosine triphosphate (ATP) thus generated
powers tissue repair. At the injury site, extracellular ATP may
be contributed by platelets and other disintegrating cells.
Extracellular ATP liberated during hypoxia or inflammation
can either signal directly to purinergic receptors or, after
phosphohydrolytic metabolism, can activate surface adenos­
ine receptors. Purinergic signaling may influence numerous
aspects of wound biology, including immune response,
inflammation, vascular as well as epithelial biology. ATP may
be immunostimulatory or vice versa, depending on extracel­
lular concentrations as well as on expression patterns of
purinergic receptors and ectoenzymes. Extracellular ATP
induces receptor activation in epithelial cells. ATP, released
upon epithelial injury, acts as an early signal to trigger cell
responses, including an increase in heparin-binding epider­
mal growth factor (EGF)-like growth factor shedding, subse­
quent transactivation of the EGF receptor and its downstream
signaling, resulting in wound healing. ATP released from the
injured epithelial cells is now known also to turn on NADPH
oxidases, the activity of which is critically required to produce
the redox signals required for wound healing.137 Human
endothelial cells are rich in purinergic receptors and
therefore responsive to extracellular ATP as well. ATP induces
endothelium-dependent vasodilation. Both ATP as well as
adenosine regulate smooth muscle and endothelial cell prolif­
eration. Recognizing that hypoxia limits ATP synthesis in the
ischemic wound tissue, therapeutic ATP delivery systems
have been studied for their effect on wound healing. While
these approaches may compensate for the deficiency of ATP
per se in the ischemic wound tissue, they will fail to address
the other essential functions of O2 and its derivatives in wound
healing, as discussed below.
Absolute requirements for O2 arise in several points along
the angiogenic sequence. For instance, all vessels require a net
or sheath of ECM, mainly collagen and proteoglycans, to
guide tube formation and resist the pressures of blood flow.
Conditions for collagen deposition and polymerization can be
created only if molecular O2 is available to be incorporated
into the structure of nascent collagen by prolyl and lysyl
hydroxylases. Without the obligatory extracellular hydroxyl­
ated collagen, new capillary tubes assemble poorly and
remain fragile.139 This has a convincing clinical correlate in
scurvy, i.e., ascorbate deficiency. Scurvy results from insuffi­
cient intake of ascorbate which is required for correct collagen
synthesis in humans. Ascorbate is required for the post-
translational hydroxylation of collagen that enables the
matured collagen molecules to escape to the extracellular
space and provide the necessary tensile strength. In scurvy,
the collagenous sheath cannot form because, under ascorbate-
deficient conditions, collagen cannot be hydroxylated. Conse­
quently, new vessels fail to mature. Older vessels weaken and
break, and wounds fail to heal. Thus, while hypoxia is a
proved instigator of molecular signals for angiogenesis, it is
also a proven enemy of vessel growth itself in non-tumor
tissues. Collagen deposition proceeds in direct proportion to
pO2 across the entire physiologic range, from zero to hundreds

181
Stem cells
miR regulation of inflammation
Disruption of miRNA biogenesis has a major impact on the
overall immune system. Emerging studies indicate that
miRNAs, especially miR-21, miR-146a/b, and miR-155, play
a key role in regulating several hubs that orchestrate the
inflammatory process.174,175 miRNAs have been directly impli­
cated in the pathogenesis of inflammatory diseases such as
osteoarthritis and rheumatoid arthritis. Resolvin-regulated
specific miRNAs target genes involved in resolution of inflam­
mation to establish a novel resolution circuit involving RvD1
receptor-dependent regulation of specific miRNAs.176 Macro­
phage specific miR-21 induction was recently shown to
promote an anti-inflammatory phenotype by down-regulating
PTEN and PDCD4 resulting in elevated production of the
anti-inflammatory cytokine IL-10.177 miR-21 is efferocytosis-
inducible in macrophages regulating a switch to an anti-
inflammatory
phenotype
promoting
resolution
of
inflammation. Furthermore, the brain-specific microRNA-124
can tame inflammation by turning off activated microglial
cells and macrophages.178 Of relevance to tissue repair is also
the regulatory loop where cytokines, including those elicited
following injury, are regulated by miRNAs, as well as regulate
miRNA expression.179,180
Stem cells
The regenerative potential of injured adult tissue suggests the
physiological existence of cells capable of participating in the
reparative process. The epithelium of the skin, the epidermis,
is in a continuous equilibrium of growth and differentiation
and has the remarkable capacity to self-renew completely,
which relies on reservoirs of stem cells. In mammals, there are
two broad types of stem cells: embryonic stem cells (ESCs)
that are isolated from the inner cell mass of blastocysts, and
adult stem cells that are found in various tissues. In adult
organisms, stem cells and progenitor cells act as a repair
system for the body, replenished in adult tissues. Stem cells
have the property of self-renewal without differentiation and
have the potential to differentiate into any type of cell. Toti­
potent stem cells have the ability to give rise to a whole
organism due to their ability to differentiate into embryonic
and extraembryonic cells. Pluripotent stem cells are derived
from totipotent cells and can differentiate into all cell types
but cannot give rise to a new organism (Fig. 13.14). ESCs are
derived from the developing embryo, usually from the inner
cell mass of a blastocyst or earlier morula stages. Lost or
damaged cells can be replaced by differentiation, dedifferen­
tiation, or transdifferentiation (Fig. 13.15). Recent advances
have shown that the addition of a group of genes can not
only restore pluripotency in a fully differentiated cell state
(differentiation) but can also induce the cell to proliferate
(dedifferentiation) or even switch to another cell type (trans­
differentiation). Dedifferentiation is represented by a termi­
nally differentiated cell reverting back to a less-differentiated
stage from within its own lineage. This process allows the cell
to proliferate again before redifferentiating, leading to the
replacement of those cells that have been lost. Transdifferen­
tiation is another naturally occurring mechanism that takes
dedifferentiation a step further and sees cells regressing to a
point where they can switch lineages, allowing them to dif­
ferentiate into another cell type. Furthermore, reprogramming
induced in the injured tissue.163 Hypoxamirs are also induced
by HIF-independent pathways. Although hypoxamirs gener­
ally favor angiogenesis, their metabolic and cell cycle arrest
functions are in conflict with wound healing, especially in an
ischemic setting. Silencing specific hypoxamirs may therefore
represent a prudent approach to facilitate tissue repair.
miRNA-210 represses mitochondrial respiration164 and exag­
gerates production of undesired mitochondrial ROS.164 These
outcomes are not compatible with the higher energy demands
associated with tissue repair. miR-210 also silences signaling
via FGF,165 a key contributor to wound healing. The injured
tissue is highly rich in ROS.166 In addition, at the site of injury
transition metal ions are released from a protein-bound state.
Conditions such as these cause DNA damage which opposes
tissue repair. DNA repair systems are therefore of key signifi­
cance in enabling tissue repair. miR-210 antagonizes DNA
repair.166 This is another hypoxamir function that is in conflict
with wound healing. Compatible with the common observa­
tion that ischemic wounds are refractory to healing response,
elevated miRNA-210 in ischemic wounds attenuated keratin­
ocyte proliferation and impaired wound closure.86,167
miR regulation of angiogenesis
In 2005–2008, the first series of observations establishing key
significance of miRNAs in the regulation of mammalian
vascular biology came from experimental studies involved in
arresting miRNA biogenesis to deplete the miRNA pools of
vascular tissues and cells.168 Dicer-dependent biogenesis of
miRNA is required for blood vessel development during
embryogenesis. Mice with endothelial cell-specific deletion of
Dicer, a key enzyme supporting biogenesis of miRNAs,
display defective postnatal angiogenesis. NADPH oxidase-
derived ROS drive wound angiogenesis. Endothelial NADPH
oxidase is subject to control by miRNAs.169 Hypoxia is widely
recognized as a cue that drives angiogenesis as part of an
adaptive response to vascularize the oxygen-deficient host
tissue. Hypoxia-induced repression of miR-200b is involved
in such induction of angiogenesis by directly targeting GATA2,
VEGFR2 and Ets-1.170,171 Various aspects of angiogenesis, such
as proliferation, migration, and morphogenesis of endothelial
cells, can be regulated by specific miRNAs in an endothelial-
specific manner. miRNAs known to regulate angiogenesis in
vivo are referred to as angiomiRs.172 miRNA-126 is specific
to endothelial cells and regulates vascular integrity and
developmental angiogenesis. Manipulating angiomiRs in the
setting of tissue repair represents a new therapeutic approach
that could be effective in promoting wound angiogenesis.
Impaired resolution of inflammation
The inflammation stage of wound healing is meant to be
transient and self-resolving for healing to proceed normally.
Temporally and spatially controlled mechanisms tightly
control wound inflammation and resolution and these include
removal of apoptotic neutrophils by functional macrophages.
In patients with diabetes, elevated blood glucose levels results
in glycosylation of phosphatidyl serine receptors on macro­
phages, which blocks recognition of apoptotic cells. This
ineffective efferocytosis (phagocytosis of apoptotic cells) by
wound macrophages results in increased production of pro­
inflammatory cytokines such as TNF-α, IL-1α, β and IL-6173
and impaired resolution of inflammation.39

182
CHAPTER 13 • Wound healing
The normal skin has long been known to contain bone
marrow-derived cells that are involved in host defense and
inflammatory processes, including wound healing. However,
recent studies demonstrate that the bone marrow contrib­
utes not only inflammatory cells, but also keratinocytes and
fibroblast-shaped cells to the skin. Similar to leukocytes in
trafficking, stem/progenitor cells derived from the bone
marrow could home to injured tissues and participate in
the repair/regeneration. Moreover, culture-expanded bone
marrow-derived MSCs have been shown to promote the
healing of diabetic wounds, implying a profound therapeu­
tic potential for skin defects such as chronic wounds and
burns.182
During the inflammatory phase, leukocytes migrating to
the wound site are hematopoietic cells derived from the bone
marrow. In the skin the bulge of the hair follicle (Fig. 13.17)
plays a role as a reservoir of stem cells. In mice it has been
shown that the bulge region contains hematopoietic cells that
are identical to the bone marrow-derived cells and also to
those found in fetal circulation.183 Moreover this region also
serves as a reservoir for precursors of mast cells. Discovery of
these epidermal stem cells in the hair follicle bulge led to the
hypothesis that these cells are necessary for both epidermal
renewal as well as wound healing.184,185 It was shown that cells
from the bulge do not contribute to epidermal regeneration;
aims to revert differentiated cells to pluripotency. From here,
they can differentiate into almost any cell type. Although
reprogramming occurs naturally during fertilization to
produce totipotent cells that can differentiate into any cell
type, it has not yet formally been shown to be a genuine
regenerative response. Furthermore, reprogramming side­
steps the necessity of using embryos for regenerative therapies
by using differentiated cells taken from a patient. From a
clinical perspective, this comes with the additional bonus of
circumventing the immunological problems such as trans­
plant rejection and graft-versus-host disease associated with
engraftment.181
The bone marrow (Fig. 13.16), home of stem and progenitor
cells, contributes a significant proportion of cells in the skin.
Fig. 13.15  Stem cell differentiation, dedifferentiation, and transdifferentiation
White blood
cells
Muscle
Nerve
Bone
Other
tissues
Red blood
cells
Totipotent
cells
Transdifferentiation
De-differentiation
Retro-differentiation
Differentiation
Re-differentiation
Pluripotent
stem cell
Progenitor cells
Blood
stem
cells
Fig. 13.16  Bone marrow stem cells. CNS, central nervous system.
Blood cells
Skeletal
muscle
Fat cell
Bone marrow
stromal cell
Cardiac muscle
Neuron
Glial cell
Epithelial
cell
Liver
CNS
stem cells
Blood
vessel
Fig. 13.14  Stem cell renewal.
White blood cells
Platelets
Erythrocytes
Totipotent cells
Pluripotent stem cell
Other committed
stem cells
Blood
stem cells
Specialized cells

183
Stem cells
representing about 0.001–0.01% of the nucleated cells, about
10-fold less abundant than HSCs, MSCs are expandable in
culture and multipotent, capable of differentiating into
several cell types.
Because of their properties of regeneration and differentia­
tion, the use of stem cells to heal problem wounds has long
been of interest. Indeed, autologous bone marrow aspirates
and cultured cells were helpful in healing chronic wounds.192
In the case of burn wounds, bone marrow-derived stem cell
treatment shows promise as well.193 Another major source of
adult stem cells is adipose tissue.194 The capability of adipose-
derived adult stem cells to differentiate into bone, muscle, fat,
or cartilage, or into cells of mesenchymal lineage, makes
them a prime target for therapeutic use. It has been shown
that adult stem cells enhanced wound healing in a murine
model.195,196 However, a caveat to the indiscriminate use of
autologous stem cell therapy in wound healing, particularly
in the diabetic population, was revealed in a recent study that
demonstrated that diabetic adipose-derived stem cells (ASCs)
are compromised in their ability to initiate vascularization
and ineffective in wound healing.197
Hair follicles are an integral part of mammalian skin where
they help the epidermis to maintain the body’s protective
barrier against its external environment. With rare exceptions
(such as palms and eyelids), hair follicles are present all over
the skin and play an important role in physiological tissue
renewal and regeneration after injury. Hair follicles represent
an autonomous miniorgan, which provides an excellent model
system for studying the biology of adult stem cells.198 There
are obvious differences between human and mouse skin, yet
the knowledge gained from lineage-tracing experiments in
mice has greatly expanded our understanding of the cellular
behavior of the different keratinocyte populations during
physiological and injury-induced tissue regeneration. As in
most of the body’s organs, the skin experiences constant
renewal. The upper keratinized layer of the interfollicular
epidermis, which consists of terminally differentiated cells, is
shed and replaced by cells originating from the actively pro­
liferating layer beneath. In contrast, hair follicles undergo
cycles of growth (anagen) and rest (telogen). The potential for
proliferation to sustain the lifelong replenishment of normal
cell loss and to repair occasional tissue damage lies within the
population of epidermal stem cells. The hair follicle bulge
harbors stem cells that may help in renewal and repair of the
skin. The term “bulge”, originally called “der Wulst”, was
introduced in 1903 by a German morphologist, P. Stöhr, to
describe an eminent structure at the site of attachment of the
arrector pili muscle in human hair follicles.199 Similar to many
other somatic stem cells, bulge cells are slow-cycling in nature.
This feature has permitted their initial identification and isola­
tion as label-retaining cells that can retain a pulse of nucleotide
label following a long chase period and is frequently regarded
as a defining characteristic of the hair follicle stem cell. In
addition, the availability of several immunohistochemical
markers, including keratin-15 and CD34, that specifically label
murine follicular stem cells has given researchers the ability
to examine carefully the signals required for adult stem cell
activation and renewal. We now know that the bulge serves
as a repository of long-lived multipotent stem cells, imparted
with the capacity to differentiate into all cell types that consti­
tute the lower cyclic portion of the hair follicle, as well as the
interfollicular epidermis during wound repair.198–200
however, upon injury cells from the bulge are recruited into
the epidermis and migrate in a linear manner toward the
center of the wound.185 These cells were noted to be transient,
living only for a few weeks, thus representing an acute
response to injury.
There are two main branches of stem cells in the bone
marrow, including hematopoietic stem cells (HSCs) and
MSCs. Adult bone marrow-derived HSCs have long been
recognized as a precursor to all blood cell lineages, including
erythrocytes, platelets, and white blood cells. Additionally,
HSCs may also give rise to fibrocytes and endothelial pro­
genitor cells. Circulating endothelial precursor cells play a
role in neoangiogenesis which is essential for healing.186,187
Bone marrow-derived stem cells also contribute to the
deposition of collagen III in the wound188 and differentiate
into fibroblasts,189 keratinocytes,190 and fibrocytes.191 Bone
marrow-derived MSCs, which are also referred to as bone
marrow mesenchymal stromal cells or marrow stromal cells,
are self-renewing and expandable stem cells. Although
present as a rare cell population in the bone marrow,
Fig. 13.17  Stem cells in the epidermis. Hair follicles act as reservoirs of stem
cells in the skin. (A) Cross-sectional view of a hair follicle. The matrix stem cells
differentiate into various parts of the hair while the long-term stem cells are present
in the bulge region. The stem cells from the bulge maintain the sebaceous gland
and epidermal stem cells. (B) A mammalian gut crypt. Stem cells are located at the
basal region with the Paneth cells. The transit amplifying (TA) cells are stem cell
progeny which move upwards and differentiate.
Hair shaft
Outer root sheath
Inner root sheath
Dermal papilla
Basement
membrane
Matrix stem cells
“Bulge” stem cells
Sebaceous gland
A
TA cell
Paneth cell
Basement membrane
Stem cell
Mesenchymal cell
B

184
CHAPTER 13 • Wound healing
microenvironment can influence miRNA-dependent repara­
tive and regenerative processes.
Non-invasive wound measurements
Laser speckle flowmetry
Functional assessment of wound vascularization is made
possible by the application of the laser speckle imaging tech­
nology which provides dynamic response and spatial resolu­
tion resulting in both real-time graphs and video recordings
of the area of interest. The speckled patterns (dark and bright
areas) generated by the instrument reflect the degree of move­
ment in the area under consideration. Blurring in the speckle
patterns indicates motion of the particles in the blood. Blurred
(in motion) areas therefore can be contrasted from those
without blurring (no motion) and color coded to generate
perfusion maps. It is therefore a powerful approach for blood
perfusion imaging. Laser speckle imaging has been used for
the assessment of spatio-temporal hemodynamic changes
during excisional and burn wound healing.207–209 Shown in
Fig. 13.18 are longitudinal measurements of perfusion changes
in the entire wound area as healing progressed for a period
of six weeks. The increased perfusion along the wound edge
during early stages of wound healing reflect initial vasodila­
tion (day 3) followed by neovascularization, which gradually
moves towards the wound bed (days 21–42) as resolution
occurs along the edge.
High resolution harmonics ultrasound
imaging
Modern ultrasound systems have been applied to vascular
imaging, visualizing 3D structures in motion and measuring
the stiffness of tissues. For the skin, higher spatial resolution
can be obtained with low frequency sound waves which allow
the differentiation of the epidermis, dermis and subcutaneous
fat and the muscle layers. Using the color Doppler imaging
(CDI) application, longitudinal imaging of functional blood
flow parameters in a gated peripheral feeder artery supplying
the wound site were visualized for the first time indicating a
bimodal pattern of arterial hemodynamics throughout the
process of wound healing (Fig. 13.19). Additionally, B-mode
imaging and elastography of wound areas have provided 3D
visualization and measurement of wound depth, cavities/
tunnels, scar formation and mechanical features of the wound
that would otherwise go undetected by current clinical
standards.209
Transepidermal water loss (TEWL)
The TEWL measurement is a reliable indicator of skin barrier
function210 and is an important parameter to consider for
skin health and function. TEWL measurements indicate the
quantity of water that passes from inside a body through the
epidermal layer to the external environment via diffusion
and evaporation processes. These can be influenced by
humidity, temperature, hydration status of the skin, etc., and
should be interpreted cautiously. Increased TEWL measure­
ments could indicate loss or impairment of barrier function
indicating that such skin is not only vulnerable to loss of
Although research into the use of stem cells for regenera­
tive medicine is on a steep upward slope, clinical success has
not been as forthcoming. This has been primarily attributed
to a lack of information on the basic biology of stem cells,
which remains insufficient to justify clinical studies. Since
most clinical protocols use intravenous application of MSCs,
it has become important to understand their trafficking in the
blood stream. Moreover, since relatively little is known where
the transplanted MSCs might locate, a better understanding
of involved homing mechanisms will likely shed light on how
MSCs exert their therapeutic effects. For instance, it is unclear
whether mechanisms used at injured sites are location-specific
or whether this recruitment can be modulated for therapeutic
purposes. In addition, it has recently been suggested that
platelets may play an important role in stem cell recruitment
to sites of injury. A better understanding of the mechanisms
used by stem cells during tissue homing would allow us to
develop strategies to improve recruitment of these rare cells.201
Induced pluripotent stem cells
Pluripotent stem cells possess the unique property of differ­
entiating into all other cell types of the human body. The
discovery of induced pluripotent stem cells (iPSCs) in 2006
has opened up new avenues in clinical medicine.202,203 Recent
breakthrough studies using a combination of four factors to
reprogram human somatic cells into pluripotent stem cells
without using embryos or eggs have led to an important revo­
lution in stem cell research. It is now possible to convert
somatic cells, such as skin fibroblasts and B lymphocytes, into
pluripotent stem cells that closely resemble ESCs. Recently,
functional neurons, cardiomyocytes, pancreatic islet cells,
hepatocytes, and retinal cells have been derived from human
iPSCs, thus reconfirming the pluripotency and differentiation
capacity of these cells. These findings further open up the
possibility of using iPSCs in cell replacement therapy for
various disorders, including chronic wounds.
miRNA regulation of stem cells
Endogenous miRNA-binding sites have been identified in
murine embryonic stem cells (ESCs). miRNAs govern ESC
function by serving as control hubs managing regulatory
networks. A central importance of such governance is high­
lighted by the observation that ESCs lacking miRNAs lose
their “stemness”. ESCs with deficient miRNA biogenesis
systems switch to a mode of ongoing cell division. They do
not differentiate on demand because of failure to turn off the
pluripotency regulatory program.204 miRNAs conduct the
orchestra of critical gene regulatory networks controlled by
pluripotency factors within stem cells. Individual miRNA-
dependent pathways that promote the reprogramming of
somatic cells into iPSCs have now been identified. Manipula­
tion of specific cellular miRNAs helps enhance reprogram­
ming of somatic cells to an ESC-like phenotype, helping to
generate iPS cells.205 Expression of miRNAs is also subject to
control by epigenetic factors.83,206 Such control influences the
balance between proliferation and differentiation of stem
cells. In executing such control, the miRNA element of epi­
genetics cross-talks with changes in chromatin structure as
well as with changes in DNA methylation. Collectively,
this provides for a mechanism by which the tissue injury

185
Non-invasive wound measurements
allowing qualitative and quantitative measurements with
high spectral resolution. A combination of a highly sensi­
tive charge-coupled device (CCD) and infrared (IR) cameras
detect dynamic changes of tissue hemoglobin concentration
and temperature distribution in response to vascular occlu­
sion and reperfusion resulting in oxygenation maps. The
OxyVu-2 software is used to perform imaging analysis that
allows post-processing of the hyperspectral images for tissue
oxygenation (StO2). This imaging method has been used to
characterize the hypoxia component of tissue ischemia in a
mouse model167 and in combination with laser speckle and
thermal imaging modalities, it has been applied to studies
on normal human subjects following post-occlusive, reactive
hyperemia procedures.211
moisture but is now a portal of entry for microbes and other
irritants. Recent evidence from preclinical studies empha­
sizes the need for functional assessments of wound closure
using measurements such as TEWL. Biofilm-infected wounds
that appeared visually closed showed high TEWL readings
indicating functional barrier failure which was then linked
to the downregulation of critical cellular tight junction pro­
teins (ZO-1 and ZO-2) by biofilm-induced microRNAs (miR-
146a and miR-106b).113
Hyperspectral imaging
This technique is a non-invasive, non-destructive, chemical-
free assessment that provides maps of oxygenated tissue
Fig. 13.18  Laser speckle perfusion imaging
shows dynamic changes in wound-site blood flow
over time. (Reproduced from Gnyawali S, Barki KG,
Mathew-Steiner SS, et al. High-resolution
harmonics ultrasound imaging for non-invasive
characterization of wound healing in a pre-clinical
swine model. Plos One, 2015;10:e0122327.)
A
B
Wound-edge perfusion
(PU)
C
Wound-bed perfusion
(PU)