Fat Injection From Filling to Regeneration

150
Part I • Principles and Basic Concepts
Selecting Donor Sites
Decant
less-dense
oil layer
Centrifuge fat
Harvest
Wick
Release
dense aqueous
component
Transfer to
smaller syringe
Fig. 10-12
Besides the proper selection o donor sites (the lower abdomen or inner thigh or small-volume at
gra ing), at gra s should be harvested with a less traumatic method such as syringe aspiration or lower
suction pressure and then processed with proper centri ugation. Fat gra s should be placed in a small
amount (no more than . cc or equivalent amount or large volume) each pass but with multiple passes
in multiple tunnels, multiple tissue levels, and multiple directions. Anesthetic (or tumescent) solution
with low lidocaine concentration should be chosen or inf ltration o the donor site. Signif cant overcor-
rection should be avoided to minimize complications such as at necrosis. T e timing or subsequent
injection may be about 3 to 6 months a er the previous injection ( able - ). It is also critical to in orm
the patient that a subsequent procedure may be necessary a er the f rst at gra ing i the expected results
have not been achieved.

151
Chapter 10 • An Overview o Fat Gra ting echniques
Table 10-1 Important Techniques and Considerations or Fat Graf ing
Pre erred donor sites

Lower abdomen or inner thigh
Anesthesia

Low concentration o lidocaine or inf ltration
Fat gra harvesting

A less traumatic syringe technique or lower pressure suction
technique
Fat gra processing

Centri ugation with a proper setting, such as rpm ( g) or
minutes
Recipient site preparation
Use o external volume expansion device ( or large or megavolume
gra ing)
Placement o at gra s

Placed in a small amount ( . cc or equivalent amount) or each
pass in the withdrawal phase, with multiple passes in multiple
tissue planes, multiple levels, and multiple directions
Overcorrection
Not recommended
Postoperative care/expected outcome

Proper mobilization o the gra ed area
Swelling is always expected
Additional injection may be necessary
iming or subsequent injection

-6 months a er previous injection
D i scussi on
Complications o at gra ing are not common and can usually be avoided with a meticulously per-
ormed gra ing procedure.1,2,12 Complications at the donor site are the same as those expected rom
liposuction: contour depressions and an uneven body sur ace. Liposuction or gra harvest should aim
to enhance body contour by care ul assessment o the location with excess at. T e recipient site may
develop hematoma, in ection, nerve injury, or rarely, vessel injury in the immediate postoperative period
or at necrosis as a late complication. Fortunately, acute complications are very rare and sel -limiting. Fat
necrosis is most commonly ound in megavolume at gra ing and may develop continued f brosis rom
the macrophages trying to phagocytose the nonviable adipose gra in the recipient site.34 Fat necrosis
usually presents with oil cyst, or calcif cation which may require aspiration or excision depending on
severity o those conditions.
Improvement o at gra retention probably occurs through regeneration o the adipocytes within the
at gra construct by ADSCs which obtain sel -renewal capacity.37 T e sa ety issue and e cacy regard-
ing use o ADSCs in at gra ing is not yet determined and there ore needs more investigation be ore it
can be put into clinical practice. Instead, cell-assisted lipotrans er, as proposed by Matsumoto and col-
leagues,37 using stromal vascular raction (SVF), which contains a heterogenous group o cells including
ADSCs, adipose-derived progenitor cells, blood-derived cells (CD + ), endothelial progenitor cells,
and pericytes, can be practically available. Several reports have demonstrated the benef ts o its support-
ive use in experimental and clinical studies and thus the cell-assisted lipotrans er has gained increasing
popularity and the momentum as a technique or the enhancement o at gra retention recently.37 An
animal study showed cell-assisted lipotrans er to enhance at gra retention is dose dependent and con-
versely, concentration o SVF higher than the optimal level can be detrimental to at gra survival.38
However, the optimal ratio o ADSC or SVF to adipocyte or achieving maximum gra retention has
remained inconclusive, and urther studies are needed to evaluate the complex role o SVF as a stem cell
population so that such cell therapy can be used clinically to improve at gra survival.

152
Part I • Principles and Basic Concepts
C oncl usi on
Harvesting, processing, recipient site preparation and injection techniques or small- and large-volume
at gra ing procedures should be standardized based on the most recent scientif c studies. Although the
techniques or small-volume at gra ing are somewhat standardized at the present time, the techniques
or large- and megavolume at gra ing have not yet been standardized. Integration o our improved un-
derstanding o adipose tissue and stem cell biology into our practices can urther acilitate improvement
o the surgical outcome a er at gra ing. With the techniques and important considerations described
in this chapter, at gra ing can be per ormed in a more satis actory ashion or both aesthetic and recon-
structive procedures with expected good clinical outcomes and ew or no complications.
A video relevant to this topic can be found in the Video Contents list on p. xxvi.
REFERENCES
. Coleman SR. Structural at gra ing: more than a permanent f ller. Plast Reconstr Surg 8(3 Suppl): 08S,
006.
. Lin JY, Wang CM, Pu LL. Can we standardize the techniques or at gra ing? Clin Plast Surg 4 : 99, 0 .
3. Del Vecchio D, Rohrich RJ. A classif cation o clinical at gra ing: di erent problems, di erent solutions.
Plast Reconstr Surg 30: , 0 .
4. Gonzalez AM, Lobocki C, Kelly CP, Jackson I . An alternative method or harvest and processing at
gra s: an in vitro study o cell viability and survival. Plast Reconstr Surg 0: 8 , 007.
. Pu LL, Coleman SR, Cui X, et al. Autologous at gra s harvested and ref ned by the Coleman technique:
a comparative study. Plast Reconstr Surg :93 , 008.
6. Pu LL. owards more rationalized approach to autologous at gra ing. J Plast Reconstr Aesthet Surg
6 :4 3, 0 .
7. Cheriyan , Kao HK, Qiao X. Low harvest pressure enhances autologous at gra viability. Plast Reconstr
Surg 33: 36 , 0 4.
8. Ferguson REH, Cui X, Fink BF, Vasconez HC, Pu LL. T e viability o autologous at gra s harvested with
the LipiVage system: a comparative study. Ann Plast Surg 60: 94, 008.
9. Zhu M, Cohen SR, Hicok KC, et al. Comparison o three di erent at gra preparation methods: gravity
separation, centri ugation, and simultaneous washing with f ltration in a closed system. Plast Reconstr
Surg 3 :873, 0 3.
0. Ansorge H, Garza JR, McCormack MC, et al. Autologous at processing via the Revolve system: quality
and quantity o at retention evaluated in an animal model. Aesthet Surg J 34:438, 0 4.
. Kurita M, Matsumoto D, Shigeura , Sato K, Gonda K, Harii K, Yoshimura K. In uences o centri ugation
on cells and tissues in liposuction aspirates: optimized centri ugation or lipotrans er and cell isolation.
Plast Reconstr Surg : 033, 008.
. Khouri RK, Rigotti G, Cardoso E, Khouri RK Jr, Biggs M. Megavolume autologous at trans er: Part II.
Practice and techniques. Plast Reconstr Surg 33: 369, 0 4.
3. Cleveland EC, Albano NJ, Hazen A. Roll, spin, wash, or f lter? Processing o lipoaspirate or autologous at
gra ing: an updated, evidence-based review o the literature. Plast Reconstr Surg 36:706, 0 .
4. Boscher M , Beckert BW, Puckett CL, et al. Analysis o lipocyte viability a er liposuction. Plast Reconstr
Surg 09:76 , 00 .
. Pu LL, Cui X, Fink BF, et al. T e viability o atty tissues within adipose aspirates a er conventional lipo-
suction: a comprehensive study. Ann Plast Surg 4: 88, 00 .

153
Chapter 10 • An Overview o Fat Gra ting echniques
6. Pallua N, Puls ort AK, Suschek C, Wolter P. Content o the growth actors bFGF, IGF- , VEGF, and
PDGF-BB in reshly harvested lipoaspirate a er centri ugation and incubation. Plast Reconstr Surg
2 :826, 2 9.
7. Philips BJ, Grahovac L, Valentin JE, et al. Prevalence o endogenous CD 4+ adipose stem cells predicts
human at gra retention in a xenogra model. Plast Reconstr Surg 2:84 ; discussion 8 9, 2 .
8. Allen RJ Jr, Canizares O Jr, Schar C, Nguyen PD, T anik V, Saadeh PB, Coleman SR, Hazen A. Grading
lipoaspirates: is there an optimal density or at gra ing? Plast Reconstr Surg : 8, 2 .
9. Kim IH, Yang JD, Lee DG, et al. Evaluation o centri ugation technique and e ect o epinephrine on at
cell viability in autologous at injection. Aesthetic Surg J 29: , 2 9.
2 . Khouri RK, Eisenmann KM, Cardoso E, et al. Brava and autologous at trans er is a sa e and e ective
breast augmentation alternative: results o a 6-year, 8 patient, prospective multicenter study. Plast Recon-
str Surg 29: 7 , 2 2.
2 . Kato H, Suga H, Eto H, et al. Reversible adipose tissue enlargement induced by external tissue suspension:
possible contribution o basic f broblast growth actor in the preservation o enlarged tissue. issue Eng
Part A 6:2 29, 2 .
22. Heit YI, Lancerotto L, Mesteri I, et al. External volume expansion increases subcutaneous thickness, cell
proli eratio, and vascular remodeling in a murine model. Plast Reconstr Surg : 4 , 2 2.
2 . Lancerotto L, Chin MS, Freniere B, et al. Mechanism o action o external volume expansion devices. Plast
Reconst Surg 2: 69, 2 .
24. Del Vecchio DA, Bucky LP. Breast augmentation using preexpansion and autologous at transplantation:
a clinical radiographic study. Plast Reconstr Surg 27:224 , 2 .
2 . Lee JH, Kirkham JC, McCormack MC, et al. T e e ect o pressure and shear on autologous at gra ing.
Plast Reconstr Surg : 2 , 2 .
26. Xie Y, Zheng DN, Li QF, Gu B, Liu K, Shen GX, Pu LL. An integrated at gra ing technique or cosmetic
acial contouring. J Plast Reconstr Aesthetic Surg 6 :27 , 2 .
27. Lin M, Lin Y, Chou CK, et al. Application o microautologous at transplantation in the correction o
sunken upper eyelid. Plast Reconstr Surg Glob Open 2:e2 9, 2 4.
28. Chung M , Paik KJ, Atashroo DA, et al. Studies in at gra ing: Part I. E ects o injection technique on in
vitro at viability and in vivo volume retention. Plast Reconstr Surg 4:29, 2 4.
29. Geissler PJ, Davis K, Roostaeian J, Unger J, Huang J, Rohrich RJ. Improving at trans er viability: the role
o aging, body mass index, and harvest site. Plast Reconstr Surg 4:227, 2 4.
. Yoshimura K, Suga H, Eto H. Adipose-derived stem/progenitor cells: roles in adipose tissue remodeling
and potential use or so tissue augmentation. Regen Med 4:26 , 2 9.
. Padoin AV, Braga-Silva J, Martins P, et al. Sources o processed lipoaspirate cells: in uence o donor site
on cell concentration. Plast Reconstr Surg 22:6 4, 2 8.
2. Keck M, Zeyda M, Gollinger K, Burjak S, et al. Local anesthetics have a major impact on viability o
preadipocytes and their di erentiation into adipocytes. Plast Reconstr Surg 2 : , 2 .
. Carpaneda CA, Ribeiro M . Percentage o gra viability versus injected volume in adipose autotrans-
plants. Aesthetic Plast Surg 8: 7, 994.
4. Sherman JE, Fanzio PM, White H, Lei er D. Blindness and necrotizing asciitis a er liposuction and at
trans er. Plast Reconstr Surg 26: 8, 2 .
. Gutowski CA. Current applications and sa ety o autologous at gra s: a report o the ASPS Fat Gra ask
Force. Plast Reconstr Surg 24:272, 2 9.
6. Kanchwala SK, Glatt BS, Conant EF, Bucky LP. Autologous at gra ing to the reconstructed breast: the
management o acquired contour de ormities. Plast Reconstr Surg 24:4 9, 2 9.
7. Matsumoto D, Sato K, Gonda K, akai Y, Shigeura , Sato , Aiba-Kojima E, Iizuka F, Inoue K, Suga H,
Yoshimura K. Cell-assisted lipotrans er: supportive use o human adipose-derived cells or so tissue
augmentation with lipoinjection. issue Eng 2: 7 , 2 6.
8. Paik KJ, Zielins ER, Atashroo DA, et al. Studies in at gra ing: Part V. Cell-assisted lipotrans er to enhance
at gra retention is dose dependent. Plast Reconstr Surg 6: 67, 2 .

(Trang không có chữ)

REGENERATIVE
POTENTIAL
P a r t I I

(Trang không có chữ)

157
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and Regener at ive Medicine
James Christian Brown, Adam J. Katz

158
Part II • Regenerative Potential
Bone
Cartilage
Pancreas
Adipose
ADSC
Endothelium
Liver
Neuron
Muscle
Fig. 11-1
In Jarrell et al14 published a paper on the isolation and use o adipose-derived microvascular en-
dothelial cells or lining synthetic vascular gra s.15 By the turn o the century, the idea emerged that
adipose tissue contained progenitor/stem cells with the ability to di erentiate into lineages other than
adipocytes. In Halvorsen et al16 described the osteogenic di erentiation o adipose-derived stro-
mal cells, in Zuk et al17 published f ndings demonstrating that adipose tissue was a novel source o
mesenchymal stem cells (MSCs) with multilineage di erentiation potential. T ere is now a large body
o literature to suggest that a wide variety o tissues may contain an MSC raction. o this end, Crisan
et al18 and several other independent groups have proposed a “uni ying theory” o sorts that MSCs rom
various organs or tissues are located in the perivascular space or niche.19,20 Indeed, the concept that peri-
cytes (also called mural cells or Rouget cells) have multilineage potential is not new; it dates back at least
to the early s.21-25 Furthermore, the unique microvascular composition o adipose tissue has been
appreciated or at least a quarter century or longer.26-28

159
Chapter 11 • he Evolution o Adipose Biology and Regenerative Medicine
C el
I d enti ty a n d T ermi no o g y
Subcutaneous human adipose tissue is an accessible and abundant cell source or multiple applications
in regenerative medicine. Researchers and biotech companies have explored di erent methods and
point-o -care devices to isolate regenerative cells rom human adipose aspirates obtained rom lipo-
suction procedures. Upon collagenase dissociation o these lipoaspirates and subsequent exclusion o
mature adipocytes, an array o cells collectively known as the stromal vascular fraction (SVF) is obtained.
T e SVF is a primary, uncultured, heterogeneous population including f broblasts, pericytes, vascular
progenitors, and multipotent mesenchymal stem cells (MSCs).14,16,17,29
IL-4
ADSC
Regulatory
T cell
Helper
T cell
• Increased proliferation of regulatory/helper T cells.
• Subsequent suppression of inflammation and immune response.
IL-6
IL-10
GCSF
Fig. 11-2
Within the SVF, the “traditional” cells o interest express the ollowing cluster o di erentiation markers:
CD –, CD a–, CD –, CD + , and the ollowing sur ace antigens: CD , CD , CD , CD .
ADSCs, in contrast, have been plated into culture (that is, selected by plastic adherence) and possess
the multipotential capability o di erentiation into a variety o mesenchymal lineages. ADSCs express
clusters o di erentiation markers (CD , CD , CD , CD , CD , and so on), but are negative or
CD , CD , CD .29 ADSCs are there ore a distinct entity, as def ned by immunophenotypic expres-
sion and plastic adherence, and were previously assumed to have di erent developmental plasticity rom
other stromal cells. In act, several studies have validated these assumptions and have described pre er-
ential lineage models or ADSCs (which have a greater propensity o di erentiating into muscle tissue)
and MSCs (which more readily di erentiate into chondrogenic and osteogenic lineages).29,30

160
Part II • Regenerative Potential
I mmunomod ul a ti on
T e scope o adipose tissue and its uses was urther broadened when the immunomodulatory e ects o
ADSCs were discovered. Adipose stem cells increase the secretion o cytokines and growth actors (spe-
cif cally IL- , IL- , IL- , granulocyte colony–stimulating actor [G-CSF]) and modi y cell-mediated
immunity (that is, they stimulate proli eration o regulatory and helper cell phenotypes). T e abil-
ity o these cells to suppress an immune response has obvious implications or autoimmune disorders,
these antiin ammatory e ects have been exploited in studies o several pathologic processes, such as
hemorrhagic stroke, reactive airway disease, multiple sclerosis, and rheumatoid arthritis.31-36 Studies in
transplant-related immunosuppressive therapy are currently being undertaken and expanded.31,37
S ecretome
Collagenase
dissociation
Lipoaspirate
Stromal vascular
fraction
Hematopoietic
cells
Granulocytes
Phagocytes
Macrophages
Monocytes
Leukoprogenitors
C
D
4
5
(+ )
3
1
(−)
Putative mesenchymal stem cells
Mesenchymal progenitors
Pericytes
Fibroblasts
C
D
4
5
(−)
C
D
3
1
(+ )
Endothelium
Endothelial progenitors
Nonhematopoietic
cells
Mature adipocytes
Fig. 11-3
T e ability o ADSCs to modi y their environment is not restricted to direct di erentiation into target
tissue cells. A secretome is the compilation o the secretory organelles and their secreted protein prod-
ucts. T e adipose secretome generates adipokines and cytokines with both local and systemic e ects—
abilities that extend to isolated ADSCs. ADSC-conditioned media, a direct product o the ADSC secre-
tome, has been demonstrated in several studies to exert these endocrine and paracrine e ects. For exam-
ple, ADSC-conditioned media generated under proin ammatory stimuli improve wound healing rates
in rodent wound-healing models by means o the attraction o monocytes to sites o in ammation and
tissue damage. issue regeneration through extracellular matrix (ECM) remodeling, keratinocyte migra-
tion, an increase in capillary density has also been seen with the application o ADSC-secreted cytokines
(connective tissue growth actor [C GF], SERPINE , plasminogen activator inhibitor [PAI], hepatocyte
growth actor [HGF], FGF- ).38-47 T e a orementioned immunomodulation e ects o ADSCs, which
are o increasing importance in the creation o novel treatment modalities or autoimmune-mediated
disease, have been demonstrated by ADSC-conditioned media suppression o peripheral blood mono-
nuclear cells through modulation o inter eron-gamma (IFN-gamma), HGF, prostaglandin E (PGE-
), trans orming growth actor-beta ( GFB- ), indoleamine , -dioxygenase (IDO), interleukin-
(IL- ).32,35,40-43 T e e ects o ADSC-conditioned media have experimental e cacy in other pathologies,
including angiogenesis in ischemic limb tissue and acute myocardial injuries.38,39
Interestingly, the dimensional conf guration o ADSCs used to generate conditioned media modif es the
ADSC-secretome, and subsequently the intensity o media-induced endocrine and paracrine e ects.
T ree-dimensional sca old- ree spheroid preparations o ADSCs generate media that exerts more potent
e ects on wound-healing rates than media generated by ADSCs grown in two-dimensional monolayers,
perhaps because o reported synergistic e ects that can occur between ECM and growth actors.48-50

161
Chapter 11 • he Evolution o Adipose Biology and Regenerative Medicine
U ti izi n g S V F i n C el
- B a sed T hera p i es
It is o no surprise that depending on the outcome o interest, clinical applications and cell therapies
might be better served by particular derivations o lipoaspirates (that is, the stromal vascular raction
versus adipose stem cells). Although there are many actors a ecting decision-making, including regu-
latory and acility requirements, cell dose requirements, associated costs, one actor o particular impor-
tance is the processing time. T e SVF can be isolated without selection by plastic adherence, there ore,
processing times rom resh lipoaspirate to ractionated product is on the order o minutes as opposed
to the ADSC requirement o multiple incubation days. T is allows SVF cells to be implemented in a real
time, “point-o -care” manner that also provides unique advantages, including less likelihood o con-
tamination, lower associated costs, rapid therapeutic application. Several studies have demonstrated
improved results rom SVF when compared with ADSCs, including more intense inhibition o enceph-
alomyelitic progression, improved range o motion and decreased pain in canine hip dysplasia models,
augmented cartilage and subchondral bone regeneration in caprine models.51-53
Some disadvantages o using the SVF that have been previously reported are insu cient cell dose i
high cell numbers are needed, lack o e cacy. It is o note that the majority o research per ormed to
date has ocused on the characterization, biology, therapeutic potential o ADSCs, as opposed to that o
SVF cells.54-57 T ere are ew studies in the literature directly comparing (using di erent experimental
models) the e cacy and potency o SVF cells with that o ADSCs. Furthermore, a guided def nition o
adipose-derived stem cells as a means to progress toward consensus in the f eld is a relatively new en-
deavor.29 It should be emphasized that many published reports inaccurately exchange the terms ADSCs
and SVF, using them interchangeably despite their recent characterization as two distinct cellular popu-
lations. In summary, it remains to be determined whether putative unctional di erences between SVF
cells and ADSCs have relevance or specif c clinical applications. Additional preclinical data and clinical
trials comparing both cell types may help to clari y these questions.
C urrent G a p s a n d C ontroversi es: T umori g enesi s
Considerable controversy exists in the literature with regard to the behavior o ADSCs and tumors.
ADSCs have been ound to demonstrate tumortropism, leading to engra ment o cells in the tumor
mass and in the tumor’s vascular supply, an associated increase in the size o the tumor mass, the de-
velopment o novel and dense f brovascular stroma conducive to increased tumor cell viability, growth,
metastasis.58-60 T e tumor-promoting e ects o ADSCs have been demonstrated on human breast cancer
cells characterized rom malignant pleural e usions. A . - old increase in the tumor proli eration rate
was observed when active malignant cells were co-cultured with ADSCs.61
Other murine research has ocused on the tumortropic e ects o the stromal vascular raction in breast
and prostate adenocarcinoma and Kaposi sarcoma xenogra ed mice, as well as Lewis lung carcinoma
model mice. SVF cells were injected in both an intravenous and a subcutaneous ashion, cellular homing
was demonstrated despite the mode o delivery. A er homing to tumor sites, these cells subsequently
engra ed within the tumor and augmented tumor cell proli eration. Further characterization o the SVF
injected prompted investigators to distinguish two major components o SVF—ADSCs and adipose-
derived endothelial cells (AECs)—which both exhibit the a orementioned tumortropic e ects, albeit
rom separate engra ment niches within the tumor mass.62
Cellular response to the milieu o the extracellular environment is obviously important in a plethora o
cellular unctions, including the cell cycle and proli eration, metabolism, signaling, nucleic transcrip-
tion. Given the endocrine and paracrine e ects o the ADSC secretome, the possible tumorigenic prop-
erties o ADSC-conditioned media were investigated. When compared with controls, increased proli -
eration and increased secreted protein (cytokines and growth actors) production was demonstrated by
endometrial and in ammatory breast cancer cells when incubated with ADSC-conditioned media. Fur-

162
Part II • Regenerative Potential
ther elegant electromicrographic and immuno uorescent imaging o the breast cancer cells established
topographic changes o the cell sur ace (f lipodia-mediated connections with ADSCs in proximity) and
exosomal vesicular trans er (between ADSCs and tumor cells), respectively.63,64
Although these studies implicate the SVF and ADSCs and their respective secretomes in tumor pro-
gression, other research describes an opposite “antitumor” e ect o these cells. Several in vitro ADSC-
tumor co-culture studies demonstrated objective reductions in cellular proli eration in hematopoietic
and breast cancer cells.65,66 ADSCs injected into in vivo mouse breast cancer models caused not only a
reduction in tumor size but also a reduction in the incidence o lung metastases. T e purported e ects o
ADSCs were thought to be related to modif cation o the paracrine cell–signaling and caspase-induced
apoptotic biochemical pathways.67
Given the contradictory f ndings in tumor behavior in response to ADSCs, Cousin et al68 wanted to
urther explore ADSC e ects on solid tumors by studying the behavior o pancreatic ductal adenocar-
cinoma (PDAC) cells in the presence o ADSCs. Both in vitro ADSC-tumor cell co-cultures and in vivo
murine models demonstrated a signif cant decrease in proli eration rate o PDAC cells. In addition,
ADSC-conditioned media also a ected antitumorigenesis attributed to downregulation o cell cycle pro-
gression actors, specif cally cyclin-dependent kinase (CDK ).
Despite extensive experimental endeavor, there continues to be signif cant controversy regarding the
role o ADSCs in tumorigenesis. Given the mechanistic heterogeneity o tumor development and pro-
gression, compounded with the heterogeneity o ADSC paracrine-endocrine activity, con icting data
are to be expected. Furthermore, tumorigenesis is a complex amalgamation o cellular synthetic, met-
abolic, growth processes much greater than the sum o its parts; as such, SVF-ADSC tumor promoting
and inhibiting e ects are not mutually exclusive and likely co-occur. Although there continues to be
con usion regarding the role o adipose-derived stem cells and stromal vascular raction in the promo-
tion or inhibition o tumors, f ndings rom the RES ORE- clinical trial show no cancer recurrences
(in a -month period) in any o the subjects receiving SVF- and ADSC-augmented at gra s or recon-
struction a er breast-conserving therapy.69
O b sta cl es to D evel o p ment: R e g ul a tory a n d
C ommerci a l iza ti on R i sk
Cell-based regenerative medicine, as with any other medical treatment, must ulf ll minimal require-
ments to be used in humans—the most important being patient sa ety, ollowed by clinical e cacy.
T e clinical use o cells is regulated around the globe by di erent agencies and their respective rame-
works, depending on the country o interest. T e European Medicines Agency in Europe, the Food
and Drug Administration (FDA) in the United States, other regional authorities have developed di er-
ent guidelines and laws to regulate this emerging f eld to protect patient sa ety. In general, the cellular
products to be used in patients can be classif ed as those that are autologous, minimally manipulated,
reinjected during the same surgical act (such as stromal vascular raction cells), or those that are sub-
stantially manipulated (such as cultured expanded ADSCs), autologous, or allogeneic.

163
Chapter 11 • he Evolution o Adipose Biology and Regenerative Medicine
N
u
m
b
e
r

o
f

s
t
u
d
i
e
s
Time
40
38
36
34
32
30
28
26
24
22
20
18
16
14
12
10
8
6
4
2
0
38
39
25
29
24
14
5
4
1
1
0
9
















































Fig. 11-4
One rustrating theme or translational researchers is that the regulatory ramework is being continu-
ously revised and updated to be compliant with newly available clinical data and scientif c f ndings. As
an example, the European Medicines Agency (EMA), a decentralized agency servicing the European
Union, its Committee or Advanced T erapies (CA ) has recently released a re ection paper or pub-
lic consultation with some proposed changes to current regulation o cell therapies in Europe. Speci -
ically, these changes include modi ying advanced-therapy medicinal products (A MPs) classif cations
to re ect the current thinking o the committee, including the def nition o “substantial manipulation.”
Until now, in Europe the use o collagenase as a way to isolate cells during the same surgical act and or
the same individual (autologous) was classif ed as a cell separation method, hence included in the list
o nonsubstantial manipulations o Regulation EC 94/2007. In this re ection paper the EMA-CA
is studying the possibility o classi ying enzymatic digestion as a substantial manipulation. Since this
change would re ect a position more similar to current FDA policy in the United States, this may re ect
a growing momentum o e orts by global regulatory bodies to establish an “international harmoniza-
tion” o policies governing cell therapies.
C oncl usi on
T e clinical application o lipoaspirate cell ractions, such as ADSCs and SVF cells, is young but is gain-
ing momentum, with important acceleration over the past several years. Adipose-derived cells may me-
diate therapeutic e ects through a multitude o mechanisms, but endocrine- and paracrine-mediated
signaling o angiogenesis, in ammation, cell homing, cell survival, similar processes has emerged as an
important primary mechanism o action. As the study and subsequent scientif c understanding o these
cells increases, the regulatory ramework that governs their clinical use also matures. Although current
regulatory rules depend on each country and its governing body, there seems to be a trend toward the
unif cation o policies across the globe. In addition, there are now multiple companies ushering novel,
enabling cell isolation devices through the regulatory process. ogether the maturation o science, regu-
latory policy, commercial in rastructure, and scalability mark an important in ection point in the mat-
uration and translation o this promising f eld.

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Part II • Regenerative Potential
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38:382, 20 2.

167

C h a p t e r 1 2
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p .
T h e R o e o f A d i p ose- D eri ved S tem C el
s i n
A d i p ose T i ssue R emod e i n g
A p - v m c (ADSC ) m c p p c p c
p p m xp ( C p
) c v pm w , p p , p p c j c -
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p c c w p c p p c p , m
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he Physiol ogy o In ur ed,
Obese, Gr a t ed, and Dying Fat
Kotaro Yoshimura
167

168
Part II • Regenerative Potential
the adaptive repairing process, such as adipogenesis and angiogenesis, usually with collaboration with
other stem/progenitor cells recruited rom bone marrow.4,5
I n j ury to A d i p ose T i ssue
Fig. 12-1
Healthy adipose tissue

Obese adipose tissue
Injured adipose tissue
Resident cells

Adipocyte

Stem cell (Muse cell)

Progenitor (ASCs)

M2 macrophage
Recruited cells

M1 macrophage

Endothelial progenitor cell

Bone marrow-MSCs
Adipose tissue injury induces tissue damage and ollows the repair process, which involves recruitment
o in ammatory cells and release o in ammatory cytokines. A er injury, degenerative changes such
as adipocyte death occur, and primary injury actors such as basic f broblast growth actor (bFGF) and
some actors released rom aggregated platelets such as platelet-derived growth actor (PDGF), epi-
dermal growth actor (EGF), and trans orming growth actor-beta ( GF-beta) are f rst released into
the injured site and trigger a cascade o wound healing processes.1,6 Basic f broblast growth actor
(FGF) is released rom damaged connective tissue and acts through a c-Jun N-terminal kinase (JNK)–
signaling pathway to stimulate quiescent ADSCs not only to proli erate but also to secrete secondary
growth actors, such as hepatocyte growth actor (HGF) and vascular endothelial growth actor (VEGF).
T us ADSCs contribute to the regeneration o adipose tissue and suppression o f brogenesis during the
f rst week a er injury.6 In parallel, a variety o stem or progenitor cells such as endothelial progenitor
cells (EPCs) are recruited rom bone marrow and collaborate with activated ADSCs in an orchestrated
repair o the damaged adipose tissue.
Intact adipose tissue has adipocytes as well as many other types o cells such as adipose-derived stem/
stromal cells (ADSCs) and vascular endothelial cells (VECs). Obese adipose tissue occasionally has dead
adipocytes surrounded by inf ltrated M macrophages (crownlike structures) and shows low-grade
chronic in ammation. When adipose tissue is injured, ADSCs are activated and many types o progeni-
tor or stem cells are recruited rom bone marrow to repair the tissue damage.

169
Chapter 12 • he Physiology o Injured, Obese, Gra ted, and Dying Fat
M
ech a ni ca l F orces a n d A d i p ose T i ssue
Mechanical orces, whether external (shear, stretch, tension, distraction, and compression) or endogenous
(generated within the active cytoskeleton), a ect tissue growth and cellular unctions. Moreover, physical
interactions between ADSCs and the extracellular matrix in uence the cell behaviors.7 Continuous exter-
nal tissue expansion (as with the BRAVA system) can be used or the expansion o breast tissue.8
0 d
14 d
28 d
3 d
7 d
Fig. 12-2
We per ormed an experiment in mice in which 4 weeks o external expansion caused an increase in
subcutaneous tissue volume, particularly adipose tissue, although the enlargement was reversed weeks
a er the removal o the device.2 Dynamic remodeling such as adipogenesis and angiogenesis in the di-
rection o the mechanical orce was seen over time. T e regenerating potential has been attributed to the
number (density) and potential o ADSCs; thus irradiated tissue would have a very limited potential or
expansion.
W
h a t H a p p en s A f ter F a t G ra f ti n g ?
Recently, rigorous e orts in basic animal studies have been undertaken to explore the mystery o at
gra ing; these have revealed the underlying cellular mechanisms regarding how the gra ed at behaves.
Gra ed at is a nonvascularized tissue, and only superf cial cells can survive, as in a skin gra . Although
most other cells in the gra die, the degenerated tissue can be partly regenerated as a result o the contri-
bution o ADSCs and other stem/progenitor cells.

170
Part II • Regenerative Potential
In the regenerating zone, the hypoxic condition was improved by revascularization within 3 days a -
ter gra ing and ADSCs gave rise to new adipocytes, which f nally replaced the dead adipocytes by
3 months. On the other hand, in the necrotizing zone, the microenvironment was not improved within
3 days, and thus ADSCs f nally died, leading to the central necrosis o the gra tissue.
ACUTE EVENTS IMMEDIATELY AFTER FAT GRAFTING
Once injected into the recipient site, the adipose tissue is in an ischemic (hypoxic) state and is nour-
ished only by plasmatic di usion rom the surrounding host tissue or a ew days until revascularization
occurs. T is results in the death o many adipocytes within 2 hours and the release o multiple cell
death- or injury-associated actors rom both the dying gra ed tissue and the injured host tissue.4,6 In-
ammatory cells such as macrophages and lymphocytes are inf ltrated, and in ammatory cytokines such
as interleukins are secreted. Despite the death o adipocytes, ADSCs, which can remain unctional up to
2 hours, even under severe ischemia, are activated and try to repair the damaged tissue in collaboration
with inf ltrated stem/progenitor cells recruited rom bone marrow.4,5
Fig. 12-3
1 week
2 weeks
Before
Perilipin
Mac2
Hoechst
In this study o the immunohistology o gra ed at tissue in mice, harvested tissue samples were immu-
nostained or perilipin (cytoplasm o viable adipocytes, green), Mac2 (monocytes/macrophages, red),

171
Chapter 12 • he Physiology o Injured, Obese, Gra ted, and Dying Fat
and Hoechst 3334 (nuclei, blue).5 Rectangles in the low magnif cation images (le column) were ur-
ther magnif ed (right column). T e demarcation between the surviving and regenerating zone became
clear at week (dashed line); dead adipocytes (*) were perilipin negative, and surviving adipocytes were
strong-positive or perilipin. Small-sized preadipocytes with multiple intracellular lipid droplets (ar-
rows) emerged between dead adipocytes at weeks; the dead adipocytes were surrounded by a single
layer o M macrophages (stained red).
REGENERATION AFTER ISCHEMIC TISSUE DAMAGE: THREE
ZONES WITH DIFFERENTIAL CELL FATES
Based on our recent studies, the f rst 3 months a er at gra ing is a period o tissue regeneration, and
adipogenesis will not occur a er this period.9 T e gra ed at is categorized into three zones rom the
periphery to the center: ( ) surviving (superf cial), ( ) regenerating (intermediate), and (3) necrotizing
(central).5 T e demarcation o the surviving zone ( 00 to 300 µm thick) rom the regenerating zone be-
comes clear at week, whereas that between the regenerating and necrotizing zones is obvious between
and 4 weeks (see Fig. -3).
Perilipin
Mac2
Hoechst
4 weeks
Fig. 12-4
Immunohistology o a gra sample in mice at 4 weeks showed demarcated surviving, regenerating, and
necrotizing zones. A low-magnif cation image o perilipin staining showed the necrotizing zone (yellow
dashed line) with no adipogenesis. A high-magnif cation image showed demarcated (white dashed lines)
surviving (perilipin-positive adipocytes), regenerating (perilipin-positive small adipocytes), and necro-
tizing zones (no viable adipocytes).
Adipocytes superf cially located 00 to 300 µm rom the tissue edge remained alive (surviving zone),
while all the rest o the adipocytes (in the regenerating and necrotizing zones) died within 4 hours
a er gra ing. T e dead adipocytes became lipid droplets and were surrounded by M macrophages or
phagocytosis (see Fig. -3). T e lipid absorption process takes weeks and months, depending on the
droplet size; there ore the gra ed at maintained its original size or the f rst 4 weeks despite adipocyte
death. ADSCs in the regenerating and necrotizing zones were activated and started to repair the tissue.
New small preadipocytes appeared around the dead adipocytes (surrounded by a single layer o mac-
rophages) at to weeks in the regenerating zone (600 to 00 µm thick), whereas no adipogenesis
was observed in the necrotizing zone (see Fig. -4). In the regenerating zone, the hypoxic condition

172
Part II • Regenerative Potential
was improved by revascularization within 3 days a er gra ing and ADSCs gave rise to new adipocytes,
which f nally replaced the dead adipocytes by 3 months. On the other hand, in the necrotizing zone, the
microenvironment was not improved within 3 days, and thus ADSCs f nally died, leading to the central
necrosis o the gra tissue. T e ratio between necrotizing and surviving/regenerating zones, which de-
termines the f nal volume retention a er at gra ing, varies depending on actors in the recipient micro-
environment, such as vascularity, as well as the size o the gra ed at, the gra ing technique, and post-
operative care. Our experimental study using murine models revealed that oxygenation o the recipient
bed with normobaric 6 % oxygen or 3 postoperative days promoted survival, regeneration, and f nal
retention o gra ed at.10 T e thickness o surviving and regenerating zones was increased, suggesting
superior survival o adipocyte and resident ADSCs, respectively.
THE LONG-TERM STABILIZATION PROCESS: LIPID ABSORPTION
AND CICATRIZATION
In parallel with the regenerating events, stabilizing events such as lipid absorption (phagocytosis) and lipid
replacement with scar tissue (f brosis) occur in the regenerating and necrotizing zones.5 Although the ad-
ipogenesis/regeneration process in the regenerating zone peaks at 4 weeks and is completed by weeks,
the stabilizing process persists or at least several months, as suggested by clinical observations that volume
reduction a er at gra ing continues until the end o the f rst year. Small-sized oil droplets were absorbed
or temporarily f lled with multilayered M macrophages, inducing the dead space replacement with f bro-
genesis in parallel with lipid absorption. Substantially larger oil drops (larger than 8 mm) ormed oil cysts
in several months and remained permanently; this is considered the worst outcome o at gra ing (other
than complete necrosis) and may be accompanied by chronic in ammation and calcif cation.5,9,11
Fig. 12-5
8 weeks
12 weeks
Perilipin
Mac2
Hoechst
T ese images summarize the immunohistology o gra ed at tissue in mice. Harvested tissue samples
8 weeks and weeks a er gra ing were immunostained or perilipin (cytoplasm o viable adipocytes,

173
Chapter 12 • he Physiology o Injured, Obese, Gra ted, and Dying Fat
green), Mac (monocytes/macrophages, red), and Hoechst 4 (nuclei, blue). T ere are ew small new
adipocytes, which means that adipose regeneration appeared to be f nished by weeks. Large-sized
lipid drops surrounded by M macrophages are le in the tissue.
Adipocyte
ASC
Adipocyte
ASC
Adipocyte
ASC
Surviving
100-300 µm
600-1200 µm
Regenerating
Necrotizing
Adipocytes
Fat grafting
ASC
Death
Survive
Phagocytosis
by M1 Mϕ
Successful
replacement
M1 Mϕ
Death
M2 Mϕ
Fibrogenesis
by M2 Mϕ
Failed replacement
and cicatrization
Cyst wall formation
by M2 Mϕ
Oil cyst
Large necrosis and oil cyst formation
Fig. 12-6
During the f rst months o adipose tissue remodeling, transplanted adipocytes have di erential ates,
depending on their microenvironments. As shown above, complex cellular events are simplif ed and
the adipocyte ate is categorized into our patterns: survival, success ul regeneration, ailed regeneration
(cicatrization), and oil cyst ormation. Cicatrization and oil cyst ormation are o en not complete at

174
Part II • Regenerative Potential
3 months. T e most superf cial zone is the surviving zone, which is less than 300 µm thick. In the surviv-
ing zone, both adipocytes and ADSCs survive. T e second zone is the regenerating zone, the thickness
o which varies (600 to 00 µm), depending on the microenvironmental conditions. In this zone adi-
pocytes die but ADSCs survive and provide new adipocytes to replace the dead ones. T e central zone
is the necrotizing zone, in which both adipocytes and ADSCs die, no regeneration is expected, and the
dead space will be absorbed, be f lled with f brotic tissue, or develop into an oil cyst.



0
1
3
6
12
24 (months)
Postoperative sequence after fat grafting
Excellent
Good
Fair
Poor
Volume maintained
100%
T
i
s
s
u
e

v
o
l
u
m
e
Atrophy over
time
Oil drops
at 3 months
Inflammation
Calcification
Regeneration (adipogenesis/angiogenesis)
Oil absorption (scavenge)
Cyst wall formation
Tissue Repair (3M)

Stabilization (9M)
Fig. 12-7
Adipogenesis a er adipocyte death is exerted by activated ADSCs. Some o the dead adipocytes are re-
placed with new adipocytes o next generation and the adipogenesis f nishes by 3 months (tissue repair
phase). Dead adipocytes remaining at 3 months are absorbed during the next 9 months (tissue stabiliza-
tion phase). Lipid droplets (dead adipocytes) are absorbed by macrophage phagocytosis, but the absorp-
tion is very slow, and the absorption period is dependent on the diameter o the lipid droplets; when the
lipid droplet diameter is large (such as 0 mm), the cyst wall is ormed be ore completing absorption,
and the cyst wall will start to calci y over time. Final volume retention a er at gra ing is determined by
the rate o success ul replacement o adipocytes. I gra ed adipose tissue has only small lipid droplets
and absorption f nishes by 3 months, the volume will not substantially change over time (shown as “ex-
cellent”). On the other hand, i many large lipid droplets remain at 3 months, tissue will atrophy between
3 and months (shown as “poor”).
WHAT ARE THE ORIGINS OF NEXT-GENERATION CELLS AFTER
FAT GRAFTING?
Our recent study using green uorescent protein (GFP) mice revealed the origins o cell components
in gra ed at.12 Mature adipocytes are mostly derived rom ADSCs in the gra . Although vascular wall
constituents (smooth muscle cells) are chie y gra derived, capillaries (vascular endothelial cells) orig-
inate equally rom the gra and the host bone marrow. ADSCs o the regenerated at are an admixture
o gra ed, host nonbone marrow, and host bone marrow cells. T ose f ndings highlight the importance
o ADSCs contained in the gra ed at or regeneration o adipocytes. Host bone marrow and local tis-
sues contribute substantially to capillary networks and provision o new ADSCs, which are thought to
contribute to adipose tissue remodeling in the uture. T us, although ADSCs can be provided by bone
marrow or other tissues, they have to be ready in advance by staying adjacent to dying adipocytes to
become replacing adipocytes.

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Chapter 12 • he Physiology o Injured, Obese, Gra ted, and Dying Fat
H ow to I m p rove E n g ra f tment
Recent advances in understanding underlying mechanisms provide a number o clinical implications. It
is thought that the size and thickness o the surviving zone are in uenced by the surrounding recipient
tissue. Better vascularity and higher oxygen tension o the recipient tissue increase the surviving zone.
Preconditioning o recipient tissue such as application o negative pressure and/or hyperoxygenation
is believed to acilitate this. Excessively high internal pressure keeps the recipient tissue ischemic and
reduces the surviving zone. As with a skin gra , the immobilization o the recipient site should assist in
capillary growth into the gra during the f rst week, which improves the oxygen tension o the regener-
ating zone and rescues ADSCs rom ischemic death.
T e size and sur ace area o gra ed at are critical actors in minimizing the central necrotizing zone; it
is recommended that the diameter o gra ed at particles or ribbons o injected lipoaspirate should be as
small as mm. For adipogenesis a er at gra ing, it is essential to have a robust number o both viable
adipocytes and ADSCs in the gra , because recruited stem cells rom outside do not become adipocytes
e ciently. Dying adipocytes in the gra release crucial actors to activate adjacent ADSCs and lead them
to di erentiate into adipocytes. T is strongly suggests that it is worth preparing a higher number and
ratio o adipocytes and ADSCs by tissue processing be ore gra ing.
T ere are two ways o enhancing the relative number o stem cells in the gra . One is to reduce the
number o adipocytes and tissue volume, and the other is to increase the number o ASCs. Reducing
the number o adipocytes and tissue volume can be done by the mechanical removal o adipocytes, such
as mechanical crushing/mincing, aggressive centri ugation, and ultrasonic cavitation. Such destructive
processes have to be done with great care, because too much damage/heat/pressure to the tissue could
kill ASCs too. Increasing the number o ASCs can be done by supplementing reshly isolated SVF or
cultured/purif ed ASCs to the gra (cell-assisted lipotrans er [CAL]). SVF can be achieved through col-
lagenase digestion or other ways i extra liposuction aspirates are available. ASCs can be readily purif ed
and expanded by adherent culture o SVF and can also be banked in a liquid nitrogen or a long period
i needed.
C oncl u si on
ADSCs act as a main player in any type o adipose tissue regeneration, including the period a er at
gra ing, by di erentiating into adipocytes or VECs and releasing angiogenic growth actors. T e ate
o gra ed at depends on its size and the microenvironment o cellular components such as adipocytes.
Adipocytes remain alive in the surviving zone, whereas they die shortly a er gra ing in the regener-
ating and necrotizing zones. Adjacent perivascular ADSCs are activated by adipocyte death and be-
gin to proli erate and di erentiate to repair the damaged tissue, in collaboration with inf ltrated stem/
progenitor cells in the regenerating zone. Dead adipocytes are phagocytized by M macrophages and
are success ully replaced by new adipocytes without residual f brosis when the condition o the regen-
erating zone is optimal. By contrast, when the conditions in the regenerating or necrotizing zones are
suboptimal, dead adipocytes are partly replaced with f brotic tissue or oil cysts; M macrophages act in
the f brogenesis process. Dead adipocytes work as spacers, maintaining the space or new adipocytes
during the regenerating process. Final volume retention a er at gra ing is determined by the balance
between degeneration and regeneration o adipose tissue and is a ected by many actors including the
microenvironment o gra regenerating zone and surrounding recipient tissue. Adipogenesis a er at
gra ing is largely dependent on ADSCs resident in the gra tissue, suggesting the importance o tissue
processing be ore transplantation.

176
Part II • Regenerative Potential
REFERENCES
. Suga H, Eto H, Shigeura , Inoue K, Aoi N, Kato H, Nishimura S, Manabe I, Gonda K, Yoshimura K.
IFA S collection: f broblast growth actor-2 induced hepatocyte growth actor secretion by adipose-de-
rived stromal cells inhibits postinjury f brogenesis through a c-Jun N-terminal kinase-dependent mecha-
nism. Stem Cell 2 :238, 2009.
2. Kato H, Suga H, Eto H, Araki J, Aoi N, Doi K, Iida , abata Y, Yoshimura K. Reversible adipose tissue
enlargement induced by external tissue suspension: possible contribution o basic f broblast growth actor
in the preservation o enlarged tissue. issue Eng Part A :2029, 20 0.
3. Suga H, Eto H, Inoue K, Aoi N, Kato H, Araki J, Higashino , Yoshimura K. Cellular and molecular ea-
tures o lipoma tissue: comparison with normal adipose tissue. Br J Dermatol :8 9, 2009.
4. Eto H, Kato H, Suga H, Aoi N, Doi K, Kuno S, Yoshimura K. T e ate o adipocytes a er nonvascularized
at gra ing: evidence o early death and replacement o adipocytes. Plast Reconstr Surg 29: 08 , 20 2.
5. Kato H, Mineda K, Eto H, Doi K, Kuno S, Kinoshita K, Kanayama K, Yoshimura K. Degeneration, re-
generation, and cicatrization a er at gra ing: dynamic total tissue remodeling during the f rst 3 months.
Plast Reconstr Surg 33:e303, 20 4.
. Eto H, Suga H, Aoi N, Kato H, Araki J, Doi K, Higashino , Yoshimura K. Adipose injury-associated
actors activate adipose stem/stromal cells, induce neoangiogenesis, and mitigate hypoxia in ischemic
tissues. Am J Pathol 8:2322, 20 .
. Guilak F, Cohen DM, Estes B , et al. Control o stem cell ate by physical interactions with the extracellu-
lar matrix. Cell Stem Cell 5: , 2009.
8. Khouri RK, Schlenz I, Murphy BJ, et al. Nonsurgical breast enlargement using an external so -tissue ex-
pansion system. Plast Reconstr Surg 05:2500, 2000.
9. Yoshimura K, Eto H, Kato H, et al. In vivo manipulation o stem cells or adipose tissue repair/reconstruc-
tion. Regen Med ( Suppl):33, 20 .
0. Kato H, Araki J, Doi K, Kuno S, Kinoshita K, Mineda K, Kanayama K, Yoshimura K. Normobaric hyper-
oxygenation enhances initial survival, regeneration, and f nal retention in at gra ing. Plast Reconstr Surg
34:95 , 20 4.
. Mineda K, Kuno S, Kato H, Kinoshita K, Doi K, Hashimoto I, Nakanishi H, Yoshimura K. Chronic in-
ammation and progressive calcif cation as a result o at necrosis: the worst outcome in at gra ing. Plast
Reconstr Surg 33: 0 4, 20 4.
2. Doi K, Ogata F, Eto H, Kato H, Kuno S, Kinoshita K, Kanayama K, Feng J, Manabe I, Yoshimura K. Di -
erential contributions o gra - and host-derived cells in tissue regeneration/remodeling a er at gra ing.
Plast Reconstr Surg 35: 0 , 20 5.

Adipose issue
in Wound Heal ing
Janos A. Barrera, Zeshaan N. Maan,
Geof rey Cash Gurtner
W
h c m th v r hy c r t w j ry
rt t r t r h m t th t m t. T r c r q r r c t
t m r c r t m t tr c r xtr c r thw y , th c m-
x ty wh ch t b c v r . A r j ry, th b c t c c m t b
ct v t t r v t b . I r , cyt k th r tr h c m t r r r ,
th r cr t c rc t mm t ry r t r c t th w t m t
t -r t c . I mm t ry c f ht b ct r c z t q t r cr t c t -
wh cr t ct r th t r t th w -h r . Act v t r -
t r c r r wth ct r th t r m t w v rm t wh m t f br -
mm t . E th c , k r t cyt , f br b t r ct t r ct r
wh cr t ct r t r t r t t r ty.
T c m x ty w h th m r m r v wh c r th r r ty
t hy r m cy w j ry.1 I t , j r t ty c y r c
by m x f br b t r z c , c mm y r rr t c r. A th h
c rr m y rv t y r t ct v ct , c r rt t y t r t th
b m ch c r rt h thy, m t . T c y v t my c r
rct r v r c rrh , wh r th -t rm ct y ct t h r -
f c t. F r c t w , c rr t r r c th m ch c th t c
r rt h thy k . B r t t , r x m , r f r b t t
c r c tr ct r th t m t j t m b ty ct . It th r r w y ck w
th t r c t t th t v t rch t ct r c ry t t m z ct tc m
w j ry.
C h a p t e r 13
77

178
Part II • Regenerative Potential
Interestingly, some eukaryotic organisms are capable o regenerating their original tissue architecture.
Amongst mammals, early gestation etuses are capable o regeneration, but this capacity is gradually
lost during embryologic development.2-4 Understanding tissue regeneration or scarless wound healing
is a long-standing goal or both researchers and clinicians. o that end, a growing number o experi-
mental therapies are being developed to promote regeneration and to minimize f brosis, including the
delivery o stem cells, growth actors, micro RNAs, small molecules or extracellular tissue sca olds,
among others. Stem cell–based therapies have already shown signif cant promise in preclinical trials.
Adipose-derived mesenchymal stem cells (ADSCs) encompass the most promising source o stem cells
or therapeutic applications, given their relative abundance and ease o harvest rom lipoaspirate.5 As a
result, ADSCs have catapulted to the ore ront o regenerative medicine research.
In this chapter we discuss the tissue healing capacity o ADSCs in the context o the physiologic phases
o wound healing and the pathology underlying impaired wound healing, highlighting their in uence
on the cellular and molecular pathways governing these processes. ADSCs and wound healing encom-
pass an active area o research. We there ore encourage readers to re er to Medline (
nih.gov/pubmed) or the most up-to-date reviews in this f eld.
T h e P h a ses o f W
oun d H ea l i n g
Days after injury
0

5

10
15
20
25


365
INFLAMMATORY PHASE
Hemostasis
Remove dead tissue
Eliminate colonizing microbes
PROLIFERATIVE PHASE
Reepithelialization
Granulation tissue formation
Extracellular matrix deposition
Neovascularization
REMODELING PHASE
Wound contraction
Granulation tissue regression
Extracellular matrix remodeling
Fig. 13-1
Human wound healing proceeds through three distinct but overlapping phases. During the initial in-
f ammatory phase, hemostasis is achieved, while in ammatory cells are recruited to remove devitalized
tissue and combat microbial colonization. T e proli erative phase is characterized by cellular proli era-
tion, migration, and extracellular matrix (ECM) deposition. T e f nal remodeling phase is characterized
by gradual ECM remodeling to maximize the f nal structural integrity o the healed wound.

179
Chapter 13 • Adipose issue in Wound Healing
INFLAMMATORY PHASE
EPIDERMIS
DERMIS
FAT
Fibrin
Provisional matrix
Platelet
plug
SDF-1
PDGF
TGF-beta
Neutrophil
Macrophage
MCP-1
PDGF
VEGF
LPS
TGF-beta
complement
Bacteria
Macrophage
recruitment
ADSCs
Lymphocyte
proliferation
inhibited
MCP-1
T-suppressor
cell formation
TGF-beta-1
Galectin-1
Galectin-3
Fig. 13-2
T e principal processes o the in ammatory phase serve to achieve hemostasis and recruit in amma-
tory cells to remove devitalized tissue, eliminate microbes rom the wound bed, and secrete additional
soluble actors to propagate the wound-healing response. ADSCs modulate in ammation by promoting
-suppressor ormation, recruiting monocytes, and inhibiting activated lymphocyte proli eration.
T e in ammatory phase is initiated immediately ollowing tissue injury. T e principal goals o this phase
are to achieve hemostasis, as well as to recruit in ammatory cells that remove devitalized tissue, elimi-
nate colonizing microbes, and coordinate the ongoing wound-healing response.
During hemostasis, f brillar collagen and tissue actor activate the coagulation cascade, ultimately result-
ing in the conversion o f brinogen to f brin, which then polymerizes to orm a provisional matrix. T is
matrix serves as a substrate or cell recruitment and engra ment, including both in ammatory and pro-
genitor cells. In parallel, activated platelets aggregate and plug disrupted vessels. Several signaling actors
including stromal cell–derived- actor- (SDF- ), platelet-derived growth actor (PDGF) and trans orm-
ing growth actor-beta ( GF-beta) are released by platelets to activate downstream e ectors. SDF- in
particular has a well-described role in cell recruitment and governing o stem cell behavior.6 ADSCs are
capable o secreting these and other trophic actors, supplementing a normal endogenous response or
correcting an impaired response.
In ammatory cell recruitment occurs rapidly, mediated by trophic actors including GF-beta rom
degranulating platelets, complement cascade proteins, and lipopolysaccharide rom degraded bacteria.7
During the f rst or days, neutrophils are the predominant cell type present within the in ammatory
inf ltrate. Neutrophils phagocytose dead tissue and kill bacteria through ree radical- and cell-mediated
cytotoxic mechanisms. T ey also secrete proteases to degrade ECM and prepare the wound bed or cel-
lular ingress.8 While neutrophils are important or preventing acute in ection, their long-term presence
in wounds is associated with progression to chronic, non-healing wounds.9 ADSCs are known to protect
neighboring cells rom oxidative stress,10 and can support the viability and activity o neutrophils.11 T us
ADSCs may help promote early microbial clearance while protecting native tissues.
Circulating monocytes, recruited to the wound largely by monocyte chemoattractant protein- (MCP-
), di erentiate into macrophages. By days a er the injury, they represent the major cell type in the
wound. Like neutrophils, macrophages phagocytose devitalized tissue and bacteria. In addition, macro-
phages produce growth actors such as PDGF and vascular endothelial growth actor (VEGF) that are
important or ECM production and vasculogenesis, and studies have demonstrated their critical role in
wound healing.12 Interestingly, exogenously delivered ADSCs are known to express MCP- , suggesting
a potential mechanism or improved healing by supporting monocyte recruitment to the wound bed.13
Five to days a er the injury, lymphocytes enter the wound.14 Although the lymphocytes play a critical
role in eliminating oreign microbes rom the wound bed, persistent lymphocytic activation can be as-
sociated with delayed wound healing.15 ADSC delivery may help address this problem by inhibiting the

180
Part II • Regenerative Potential
proli eration o activated lymphocytes through cell-cell interactions and paracrine signaling pathways.16
ADSCs urther promote immune tolerance by secreting GF-beta- 17 and galectin- and galectin-3,18
paracrine actors that induce di erentiation o -helper cells into -suppressor cells. -suppressors pos-
itively a ect wound healing, likely by inhibiting excessive in ammation while the wound matures.19
It is clear that in ammation plays a critical role in wound healing, and ADSCs can pro oundly in uence
this process. Recent animal studies have suggested a potential therapeutic benef t o ADSCs or treating
a range o in ammatory diseases, rom gra -versus-host disease (GVHD)20 to rheumatoid arthritis21
and colitis.22 Although a ew key soluble actors produced during the in ammatory phase have been
mentioned, many other cytokines and growth actors have been shown to play important roles. T e
ollowing table highlights a ew o the most closely studied signaling actors implicated in the wound-
healing process. Notably, all o these actors are expressed by ADSCs, underscoring their therapeutic
potential or wound-healing applications.
Table 13-1 ADSC-Secreted Soluble Factors in Wound Healing
Name

Abbreviation Other Sources

Function
Vascular endothelial
growth actor
VEGF

Endothelial cells, MSCs

Promotes angiogenesis, MSC recruitment
Stromal cell–
derived- actor-
SDF-

Platelets, endothelial cells,
MSCs
Promotes angiogenesis, leukocyte and
MSC recruitment
Platelet-derived
growth actor
PDGF

Platelets, macrophages,
endothelial cells
Promotes angiogenesis and ECM
production, recruits macrophages,
pericytes, MPCs and f broblasts
Fibroblast growth
actor
FGF-

Macrophages, mast
cells, endothelial cells,
-lymphocytes
VEGF-dependent neovascularization,
MPC recruitment, vascular remodeling
Hepatocyte growth
actor
HGF

Mesenchymal cells

Promotes monocyte recruitment,
angiogenesis and reepithelialization
Keratinocyte
growth actor
KGF

Fibroblasts

Regulates keratinocyte growth,
maturation and unction
Epidermal growth
actor
EGF

Platelets, macrophages

Enhances f broblast collagenase secretion
rans orming
growth actor-beta
GF-beta

Platelets, macrophages,
lymphocytes
Promotes ECM production, angiogenesis,
MPC di erentiation
Monocyte
chemoattractant
protein-
MCP-

Monocytes, endothelial/
epithelial cells, f broblasts
Recruitment o macrophages, -cells, and
NK cells
umor necrosis
actor-alpha
NF-alpha

Macrophages,
lymphocytes, NK cells
Regulates collagen synthesis, neutrophil
recruitment and unction
Granulocyte
colony-stimulating
actor
G-CSF

Stromal cells, f broblasts,
endothelial cells,
lymphocytes
Stimulates granulocyte proli eration,
maturation and activation
Granulocyte-
macrophage
colony-stimulating
actor
GM-CSF

Keratinocytes, f broblasts,
macrophages, endothelial
cells, dendritic cells,
lymphocytes
BM-MSC recruitment, monocyte/
macrophage recruitment and activation,
immunomodulation
BM-MSC, Bone marrow–derived mesenchymal stem cell; ECM, extracellular matrix; MPC, mesenchymal progenitor cell; MSC, mesen-
chymal stem cell; NK, natural killer.

181
Chapter 13 • Adipose issue in Wound Healing
Table 13-1 ADSC-Secreted Soluble Factors in Wound Healing—cont'd
Name

Abbreviation Other Sources

Function
Inter eron alpha

IFN-alpha

Macrophages,
lymphocytes, f broblasts,
epidermal cells
Activates macrophages, inhibits f broblast
proli eration
Matrix
metalloproteinase-
MMP-

Fibroblasts, keratinocytes ECM remodeling
Matrix
metalloproteinase-9
MMP-9

Neutrophils, macrophages ECM remodeling, MPC recruitment
and mobilization, induces vasculogenic
growth actor release rom ECM
issue inhibitor
o matrix
metalloproteinase-
IMP-

Keratinocytes, f broblasts ECM remodeling, inhibits MMP
unction
PROLIFERATIVE PHASE
EPIDERMIS
DERMIS
FAT
Macrophage
Fibroblast
Keratinocyte
migration/proliferation
Type III collagen
Neovascularization
ADSCs
Reepithelialization
KGF
PDGF-BB
VEGF
SDF-1
FGF-2
TGF-beta-1
New vessel
formation
Fig. 13-3
During the proli erative phase, reepithelialization occurs, while granulation tissue produces new blood
vessels and type III collagen-rich ECM. ADSCs secrete multiple trophic actors that accelerate reepithe-
lialization and promote new vessel ormation.
T e proli erative phase o wound healing occurs rom to days a er injury, and consists o reepithe-
lialization, granulation tissue ormation, ECM deposition and neovascularization. ADSCs are known
to a ect these processes through di erentiation and secretion o paracrine actors. Reepithelialization
likely begins immediately ollowing injury. When keratinocytes lose their intercellular desmosomal and
hemidesmosomal connections, they immediately begin migrating inward rom the wound periphery via
a cytoplasmic actin polymerization-dependent mechanism. Integrin receptors acilitate migration by in-
teracting with various ECM proteins including type I collagen, f bronectin and vitronectin ound within
the provisional f brin matrix. Keratinocyte migration is dependent on the production o several ECM
remodeling enzymes, including matrix metalloproteinase- (MMP- ) and plasminogen. As cells migrate
inward, peripheral keratinocytes proli erate behind the leading edge, allowing or complete wound reep-
ithelialization. Notably, ADSCs are known to accelerate wound reepithelialization in vivo; contributory
mechanisms include their secretion o keratinocyte growth actor (KGF) and PDGF-BB.23,24 ADSCs have
also been ound to have the capacity to di erentiate along epithelial lineages.25,26

182
Part II • Regenerative Potential
Four days a er injury, the provisional f brin matrix is slowly replaced by a combination o f broblasts,
macrophages, and endothelial cells, orming granulation tissue. Granulation tissue f broblasts actively
produce, deposit and remodel the ECM that comprises the healing scar while simultaneously providing
a sca old or continuing keratinocyte migration. T e scar is composed mainly o the thinner type III
collagen, which will eventually be replaced by thick type I collagen during the remodeling phase. GF-
beta- is an important regulator o ECM deposition and remodeling, and its relative paucity in etal
wounds suggests a potential contributory mechanism to scarring during adult wound healing.27 T e
e ect o ADSCs on f brotic and scarring pathways will be discussed in the ollowing section.
As the proli erative phase continues, macrophages continue to produce growth actors such as PDGF
and FGF- , which in uence f broblast behavior and promote neovascularization by inducing endothelial
cell proli eration and migration. Hypoxia-inducible actor- -alpha (HIF- -alpha) is a master regulator
o the neovascular response, and its stabilization in the setting o hypoxia results in the expression o
several proangiogenic cytokines including VEGF and SDF- . Fibroblast growth actor (FGF- ), an-
giopoietin , and thrombospondin have also been shown to support angiogenesis during wound repair.
ADSCs signif cantly improve new blood vessel ormation during the proli erative phase. T e proangiogenic
role o ADSCs is attributable to their expression o several key growth actors including SDF- and VEGF,
among others.13,28-31 Human ADSCs have been ound to improve hind-limb ischemia in nude mice,28 and in
a similar model, were even shown to improve vascularity to an even greater extent than did bone marrow–
derived mesenchymal stem cells (BM-MSCs), another proangiogenic stem cell population.32 Preliminary
studies o autologous ADSC therapy or critical limb ischemia in humans has shown that transplanted
ADSCs are sa ely tolerated and lead to improved oxygenation and wound healing o peripheral tissues33
and improvement in pain and claudication severity.34 ADSCs are clearly able to improve blood ow and
oxygenation to wounds, supporting tissue viability and accelerating wound healing.
REMODELING PHASE
Epidermis
Dermis
Fat
Epid
Epid
Epid
Epid
Epid
Epid
Epid
Epid
Epid
pid
Epid
Epid
Epid
Epid
Epid
Epid
Epid
Epid
Epid
Epid
Epid
Epid
Epi
Epid
Epid
Epid
Epid
Epid
Epid
p
Ep
Epid
Epi
Epid
Epid
p
Epid
pid
Epid
Epid
Epid
pid
E d
Epid
Epid
pid
i
Epiderm
er
erm
erm
ermi
erm
erm
erm
rm
rmi
rmi
m
erm
erm
ermi
ermi
ermi
erm
ermi
ermi
mi
erm
ermi
er
ermi
r
erm
rm
erm
erm
ermi
ermi
m
erm
erm
ermi
erm
ermi
ermi
erm
er
ermi
m
erm
e m
e
s
Derm
De
Derm
Derm
Derm
Derm
Derm
Derm
e
Derm
Derm
Derm
Derm
Derm
Derm
erm
erm
Derm
Derm
Derm
Derm
Der
Derm
Derm
erm
Derm
erm
Der
Der
Derm
De
De
Derm
rm
rm
erm
Derm
Der
Derm
Derm
erm
Derm
Derm
Derm
er
e m
Derm
erm
Der
e m
D r
e
Der isisisisisisisisisisisisisisisisisisisisisisisisisis
Fat
Fat
Fat
Fat
Fa
Fat
Fat
EPIDERMIS
DERMIS
FAT
Blood vessel
regression
Fibroblast
Myofibroblast
Contraction
Type III
collagen
Type I
collagen
MMP
ADSCs
HGF
IGF
MMP-1
MMP-2
MMP-9
TGF-beta-1
TGF-beta-3
TIMP-1
Fibroblast
Cell-cell interactions
Decreased
Type I collagen
expression
and fibrosis
Fig. 13-4
T e remodeling phase represents the f nal and longest stage o wound healing, beginning at to weeks
a er injury and o en persisting beyond a year. During remodeling wound contraction occurs, while
ECM is gradually remodeled to improve the structural integrity o the healed wound. ADSCs attenuate
scarring through cell-cell interactions with f broblasts as well as the expression o several f bromod-
ulatory trophic actors. As with the in ammatory and proli erative phases o wound healing, there is

183
Chapter 13 • Adipose issue in Wound Healing
considerable uidity and overlap between this stage and its predecessors. During the second week o
healing, some remaining f broblasts present in granulation tissue di erentiate into myof broblasts to
lend contractility to the maturing wound. Intracellular actin microf laments provide a mechanism or
wound contraction through integrin-mediated cell-cell and cell-matrix interactions. Signaling actors
associated with this process include GF-beta- , GF-beta- , and PDGF.35,36
Another critical eature o this stage is the eventual inactivation and programmed death o cells con-
tributing to the ormation o the vascularized granulation tissue, concomitant with the ormation o
a relatively acellular scar. T is can only occur once su cient collagen matrix has been produced to f ll
the wound bed, and it must occur soon enough to prevent the ormation o excess tissue that may have
negative unctional consequences or the organism. Disruptions in this process likely play a role in the
pathogenesis o f brotic disorders such as hypertrophic scarring or keloid ormation. ADSCs have been
ound to signif cantly alter the dynamics o f brosis and scarring during the proli erative and remodeling
phases o wound healing. T e most likely mechanism or this f nding is through the secretion o para-
crine actors that a ect f broblast behavior, although cell-cell interactions may also play a role.37
ECM remodeling involves the gradual replacement o type III collagen produced during the proli erative
phase by type I collagen.38 T is process depends on continuous collagen turnover, mediated through the
expression o MMPs and tissue inhibitors o metalloproteinases ( IMPs) by a combination o keratino-
cytes, macrophages, f broblasts, and endothelial cells.39 Mechanical orce has additionally been shown to
critically regulate scarring through ocal adhesion kinase (FAK)-mediated in ammation and f brosis.40
T us scar ormation and maturation result rom a complex interplay o signaling actors, in ammation/
cells, and mechanical cues.
Preclinical studies o liver cirrhosis in rats41 and pulmonary f brosis in mice42 have demonstrated ame-
lioration o f brosis in ADSC-treated animals. T ese studies ound changes in the expression o HGF,
insulin-like growth actor (IGF) and matrix metalloproteinase- (MMP- ) as potential contributory ac-
tors. In a rabbit model o hypertrophic scarring, intralesional ADSC injection was ound to reduce col-
lagen type I deposition and improved scar histology.43 Using a porcine excisional wound model, ADSCs
were urther shown to reduce scarring and improve tissue pliability through alterations in the expression
o GF-beta- , MMP- , and IMP- .44 Additionally, at gra ing o murine burns has been ound to
decrease f brosis and expression o the f brotic markers GF-beta and MMP- .45 T ese f ndings may be
attributable to the action o ADSCs within the gra . Finally, it is noteworthy that BM-MSCs, a popula-
tion o stem cells closely related to ADSCs, have been shown to reduce hypertrophic scarring through a
postapoptotic immunoregulatory mechanism.46 Future studies involving ADSCs may uncover a similar
antif brotic mechanism. Despite the promising preclinical data outlined here, the e cacy o ADSCs or
reducing scarring in humans has yet to be tested.
F a ctors T h a t A f ect W
oun d H ea l i n g
T e wound microenvironment, including its three-dimensional matrix, as well as resident and recruited
cells that secrete cytokines and trophic actors, can have important implications or wound healing.3
Systemic and local actors such as age, diabetes, and in ection cause pathologic changes to the microen-
vironment that can signif cantly hinder the healing process. In the ollowing section, a ew o the most
common risk actors or poor wound healing are described, with brie explanations o the biology un-
derlying these processes.

184
Part II • Regenerative Potential
While ADSCs may ameliorate some def ciencies in wound healing, it is o vital importance that clini-
cians diagnose and treat all potentially modif able risk actors or poor wound healing to achieve the best
clinical result.
Table 13-2 Risk Factors for Poor Wound Healing
Systemic Factors

Local Factors
Age

In ection
Diabetes

Oxygenation/reper usion injury
Stress

Foreign body
Nutrition

Venous su ciency
Uremia

Irradiation
Jaundice

Edema
Cancer

Pressure
Obesity
Drugs: steroids, immunosuppressive agents
Chemotherapy
Smoking
Alcohol use
Genetic healing disorders
Immune status
ADIPOSE-DERIVED MESENCHYMAL STEM CELLS FOR WOUND
HEALING IN OLDER PATIENTS
Stem cell
Inflammatory cell
NON-AGED WOUND

AGED WOUND
Stem cell function
Neovascularization
Inflammation
ECM production
Stem cell function
Neovascularization
Inflammation
ECM production
Fig. 13-5
Older patients exhibit progressive def ciencies in HIF- –mediated neovascularization. Def ciencies in
the number and unction o native stem cells and the presence o poor ECM deposition and chronic
in ammation urther diminish wound-healing capacity in the wounds o aged individuals.

185
Chapter 13 • Adipose issue in Wound Healing
T e elderly exhibit decreased capillary density, impaired HIF- pathway activation, and decreased
growth actor production, resulting in poor wound vascularity.47 Progressive def ciencies in the num-
ber and unction o native stem cells, as well as the presence o chronic in ammation and poor ECM
deposition, urther hinder the normal healing process.48 Recent advances in single cell transcriptional
analysis have allowed or the study and identif cation o a subpopulation o ADSCs that is def cient in
aged mice.49 Analysis o this group’s expression prof le revealed a role or these ADSCs in expressing
proangiogenic and antioxidative genes, including HIF- -alpha and superoxide dismutase (Sod ), re-
spectively. Supplementing the wound-healing response with ADSCs in the aged patient may there ore be
relatively ine ective, though emerging strategies to isolate and replace def cient stem cell subpopulations
may help overcome their native def ciencies.
ADIPOSE-DERIVED MESENCHYMAL STEM CELLS FOR WOUND
HEALING IN PATIENTS WITH DIABETES
Deficient HIF-1-alpha
activation
VEGF
SDF-1
Impaired
neovascularization
Progenitor cells from
diabetic patient
Diminished
growth factor
secretion
Fig. 13-6
Nonhealing diabetic wounds are a signif cant public health burden, and most nontraumatic limb am-
putations are necessary as a result o such wounds. Several mechanisms have been proposed or the
impaired healing observed in diabetic patients. Hyperglycemia has been shown to hinder the activation
o HIF- -alpha, which is the master regulator o the neovascular response to tissue hypoxia. Normally,
HIF- -alpha is stabilized under low oxygen tension, allowing it to enter the nucleus and stimulate ex-
pression o several downstream genes including VEGF and SDF- .6 T ese, and other e ectors in the
HIF- -alpha pathway, play critical roles in promoting new vessel ormation and recruiting circulating
progenitor cells to areas o hypoxia. Diabetes also results in impairment o progenitor cell adhesion
and migration in response to hypoxia,50 and inhibits bone marrow–derived mesenchymal progenitor
cell (BM-MPC) dynamics by depleting provasculogenic subpopulations.51 Furthermore, diabetic ADSCs
exhibit decreased expression o HGF, VEGF, and IGF- , with a concomitant blunted therapeutic e ect
in treating wounds o diabetic mice.52 As ADSCs are tested or e cacy in treating diabetic wounds, it
will be important to account or potential di erences in subpopulation dynamics as compared to normal
ADSCs. As with the aged patient, identi ying and replacing def cient stem cell subpopulations may lead
to an improved clinical outcome.
C oncl usi on
he biomedical revolution has vastly expanded our understanding o normal and pathologic wound
healing. his new knowledge has ueled the development o an ever-growing number o experi-
mental therapies designed to enhance wound healing by modulating known molecular and cellu-
lar pathways. Given the complexity o the wound healing process, treatment strategies must take
into account the wide scope o systemic and local actors a ecting a particular wound microenvi-
ronment.

186
Part II • Regenerative Potential
Neovascularization
Inflammation
Fibrosis
VEGF
SDF-1
EGF
PDGF
KGF
HGF
TNF-alpha
ADSCs
p
VEGF
SDF-1
EGF
PDGF
TNF-al
KGF
HGF
TNF-al
PROBLEM WOUND
MMP-1
MMP-9
TIMP1
TGF-beta-1
MCP-1
FGF-2
IFN-alpha
HEALED WOUND
s
m
a
P
P
P
F-
P
F-
a
s
a
Neova
P-1
P-9
P1
-beta-1
P
-
a
HEALED WOUND
alpha
s
m
i
alpha
Neova
Inflamm
Fibrosi
P-1
-2
alpha
Fig. 13-7
Autologous at gra ing has emerged as a promising strategy or a variety o clinical applications.53 While
whole and partially processed adipose tissue is certainly use ul as a f lling agent, evidence suggests that
ADSCs alone may provide therapeutic benef ts beyond providing structural orm and volume. ADSCs
have been tested in multiple preclinical trials and have been ound to dynamically enhance cutaneous
wound healing through a variety o wound-healing pathways that would not otherwise be achievable
through delivery o a single growth actor.5,13,54 Compared with other cell populations, ADSCs have the
added benef t o being relatively abundant in adipose tissue and easy to harvest rom lipoaspirate or
excised at. Furthermore, ADSCs can be engineered to overexpress therapeutic growth actors and/or
be delivered within specialized biomimetic sca olds to urther enhance their regenerative potential,55,56
while emerging technologies such as micro uidic single cell analysis o er promising new approaches or
identi ying and isolating the most e ective cell populations or therapeutic use.57 Although numerous
clinical trials are underway to study the e ects o ADSC therapy on wound healing, no FDA-approved
therapies exist or any kind o stem cell–based wound therapy at the time o writing o this chapter.
Readers are encouraged to visit or the most current in ormation on this
area o research.
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20 .

Pl at el et -Rich Pl asma
and Fat Gr a t ing
Valerio Cervelli, Pietro Gentile
B
o u o w u o o ou b o o o y p o p o -
u y . M y v o v o bu o v op o qu ,
o v u o (SVF), o ow o , o v . T -
qu b pp o o o o b o p o o py-b
u o y,1 b u o ,2 po o y b o u o ,3
o p o o b p u y,4 v ,5 C o f u o p x p
f u ,6,7 k u ,8 P y-Ro b y o po op y,9 -
, u o u , p u x v u , of b o ,10 vo o u -
o .11
I Eu op , o y U S , w u o o ou
b oo p o u x w v o o u o
. W pub u o ou u y o u o v o b w
p - p (PRP) o p ov vo u o u b o -
u o . W o ow po u u qu b by Co 12,13; ou pu po w
o o o b po p w PRP14 o o ow
x y o u ,15 op y,9 b o u o .16,17 W o p
u o w o b o w o o o o oup w o o y -
u w j . P - o ou o w ob o
p uppo u o .
189
C h a p t e r 1 4

190
Part II • Regenerative Potential
P l a tel et- R i ch P l a sma a n d F a t G ra f ti n
P re p a ra ti on B a sed on C urrent R e g ul a ti on s
Our study was conducted with the in ormed consent o the patients and in the presence o a physician
rom the trans usion service, in accordance with the current Italian regulations. Brie y, the process o
preparing PRP consists o our phases:

. Blood collection

. Centri ugation or platelet concentration

3. Induction o gelation (i the PRP is to be used in gel orm)

. Activation
In general, most systems, whether or large- or small-volume collection, do not concentrate the plasma
proteins o the coagulation cascade. T ere ore the set o rules must be shaped using products that main-
tain the quality standards o biopharmaceutical drugs. Accordingly, the regulations are linked to the
related laws, which describe a complex pathway or authorization. Regulation No. 3 / 7 o the Eu-
ropean Parliament or Advanced T erapies has def ned bioprocess engineering products.
T is def nition excludes products that contain or are made exclusively o cells and nonvital human or
animal tissues and that do not have pharmacologic, immunologic, or metabolic action. Included among
the advanced therapy pharmaceutical products are those used or gene and somatic cell therapy (Di-
rective / 3/[European Parliament] European Community, Annex I).18 Cells and tissues are to be
considered products o bioprocess engineering i they undergo “considerable manipulation.” T e same
regulation def nes the di erence between extensive and minimal manipulation and lists those that are
considered relevant.
BLOOD COLLECTION
We prepared PRP using two methods:

. From a small volume o blood (18 ml), using the Cascade-Selphyl-Es orax system (Aesthetic Fac-
tors, LLC),9,14,15 with modif cations

. From a large volume o blood (60 to 120 ml), in accordance with the Platelet Rich Lipotrans ert
(PRL) system (Corios)
Brie y, blood was taken rom a peripheral vein using sodium citrate as an anticoagulant.
CENTRIFUGATION FOR PLATELET CONCENTRATION
T e traditional preparation o PRP consists o a slow centri ugation, which allows the platelets to remain
suspended in the plasma, whereas the leukocytes and erythrocytes are displaced to the bottom o the
tube.
In our current system or preparing platelet concentrations, we have used a wide variety o centri uges.
When PRP was prepared rom a small volume o blood ( ml) according to the method o the Cascade-
Selphyl-Es orax system, with modif cations, we centri uged the blood at g or minutes. When
PRP was prepared rom ml o blood according to the PRL system, with modif cations, using PRP
alone (CPun ; Biomed Device) without the SVF cells, we centri uged the sample at rpm or
minutes.

191
Chapter 14 • Platelet-Rich Plasma and Fat Gra ting
FAT GRAFT CENTRIFUGATION ACCORDING TO THE PLATELET
RICH LIPOTRANSFERT SYSTEM
Fig. 14-1
A

B
C
D
Be ore proceeding to the activation o PRP, with the patient under general anesthesia, we harvested atty
tissue rom the abdominal region using some specif c cannulas. o begin the PRL procedure, Fastkit
materials were assembled or processing (A). In this instance, 80 cc o at was subjected to automatic
f ltration (B). T e at harvested was centri uged at 700 rpm or 0 minutes (C), a er which 0 cc o the
suspension was extracted rom the bag. T e suspension was urther f ltered through a 0 µm f lter, and
0 cc o the enhanced SVF (e-SVF) suspension was obtained (D). Subsequently the e-SVF suspension
was added and mixed with the centri uged at gra . Using specif c microcannulas or implantation, the
e-SVF at gra was trans erred into 0 cc syringes. We mixed 0. cc o PRP with cc o e-SVF centri-
uged lipoaspirate and aseptically reinjected the mixture into the so tissue de ects.
PLATELET ACTIVATION
T e PRP protocol based on the Cascade-Selphyl-Es orax system9,14,15 uses Ca2+ to induce platelet activa-
tion and exocytosis o the alpha granules. T e PRP protocol based on the PRL system does not use Ca2+ .

192
Part II • Regenerative Potential
I m p l a nta ti on
T e recipient sites had been identif ed by thorough study o the corrections required. For acial so
tissue de ects, enhanced at gra was implanted in the zygomatic region, cheek, buccal rim, upper and
lower eyelids, temporal area, and periorbital area. T e atty tissue was implanted at di erent levels in
the small tunnels previously created by injecting the cannulas ( . mm in diameter) with accurate and
controlled movements. Small quantities o at were placed, one or two at a time, as the cannula was with-
drawn, creating a large grid to acilitate a correct vascular contact and development around each at cell.
For breast augmentation, enhanced harvested material was implanted mainly into three areas: the in-
erior breast rim, the superior and in erior regions o the areola, and the superolateral quadrant. A er
pretunneling, atty tissue was implanted (average 0 cc total; range 0 to 400 cc average 0 cc or
each breast) at di erent levels using a delivery cannula ( or mm in diameter) with precise, controlled
movements. Several layers were laid down to increase the contact sur ace between the receiving tissue
and the implant; this technique is o undamental importance to allow each layer o deposited at to
survive by di usion during the critical f rst ew days necessary or growth o blood vessels to nourish
the implant permanently.1,2,5,11 T e incisions were closed with -0 nylon sutures, and no compressive
bandage was applied.
R esul ts
PLATELET-RICH PLASMA AND INCREASES IN ADIPOSE TISSUE
In our series, PRP induced an increase o the number o adipose-derived stem cells (ADSCs) with no
morphologic changes compared with the control group. T e increase was statistically signif cant, by
approximately our old, at 4 and days, when cells were precon uent (p < 0.0 ). A er days, at con u-
ency, there was a three old increase o ADSC numbers in PRP cultures compared with the control group.
Oil Red O staining revealed no signif cant di erence in intracytoplasmic lipid accumulation between the
PRP-treated and control ADSCs.
CLINICAL EVALUATION
wo methods or the clinical evaluation o outcomes were utilized: the team evaluation and the pa-
tient sel -evaluation.16 T e team evaluation was based on clinical observation, using a scale o six values
(excellent, good, discreet enough, poor, and inadequate). T e patient sel -evaluation used the same six
values. T e actors/variables that were taken into account were pigmentation, vascularization, pliability,
thickness, itching, and pain.
T e percentage o maintenance restored was clinically evaluated using two di erent criteria: subjec-
tive evaluation and objective evaluation. Subjective evaluation was based on the personal score o each
patient, ocused on the ollowing parameters: ( ) the presence o asymmetry, de ormity, irregularity,
dyschromia, dysesthesia, paresthesia, and pain, ( ) results in the superolateral quadrant, in erolateral
quadrant, superomedial quadrant, and in eromedial quadrant in breast reconstruction, (3) resorption
o at in one or more regions, (4) the time to stabilization o the transplanted at, and ( ) the need or
retreatment. For each parameter, patients gave a yes or no (or positive or negative) response, and the per-
centage o maintenance restored was calculated as the mean o all calculated single percentages.
T e objective evaluation was based on analysis o the preoperative and postoperative photos. T e photos
were o the same size, brightness, and contrast. According to the parameters listed above, the operator
similarly calculated the percentage o restored material. Finally, the mean o patient and operator evalu-
ations was calculated.17,18

193
Chapter 14 • Platelet-Rich Plasma and Fat Gra ting
INSTRUMENTAL IMAGING EVALUATION METHOD FOR BREAST
SOFT TISSUE DEFECTS
Fig. 14-2
T ese preoperative and postoperative views show a volumetric de ect in a hypoplastic breast in a patient
with a breast implant. Fat gra ing o cc o lipoaspirate with PRP was per ormed in the in ramammary
old to restore the volume def cit and provide coverage to the implant.
Fig. 14-3
T is 2 -year-old woman, who had had no previous breast surgeries, underwent two sessions o at gra -
ing to correct hypomastia o the right breast; 20 cc o at mixed with PRP was injected in each session.
2-weighted MRI scans obtained be ore and 6 months a er the second session show the increase in
breast volume on the right.

194
Part II • Regenerative Potential
A
B
C
D
Fig. 14-4
T is patient had developed bilateral capsular contractures, and her implants were replaced (A). Fat gra -
ing was planned to correct the def cits in contour in both breasts rom the contractures. In a f rst at
gra ing procedure, 0 cc o at mixed with PRP was injected in the right breast and 85 cc in the le
breast; the improvement in her breast contour at year is evident (B). A second at gra ing session in
which an additional 70 cc was injected in the right breast and 60 cc in the le breast resulted in addi-
tional improvement, seen at 2 years (C). In a third procedure, 50 cc and 0 cc were injected, respectively.
In a ourth treatment, 0 cc injected on the right and 30 cc on the le resulted in an optimally improved
breast contour. T e patient is seen 3 years a er the f rst procedure (D).

195
Chapter 14 • Platelet-Rich Plasma and Fat Gra ting
Fig. 14-5
T is patient was treated or a bilateral volume de ect. Fat mixed with PRP was injected, 0 cc in the
right breast and 0 cc in the le breast. Her improved breast volume can be seen postoperatively.
In our series, MRI showed that gra ed at survived and ormed a signif cant thickness o a atty layer, not
only subcutaneously on and around the mammary glands, but also between the mammary glands and
the pectoralis muscles. Almost all patients were satisf ed with their enlarged breasts, which were so and
had a natural contour.
Mammography, ultrasonography, and MRI were per ormed be ore the f rst at gra ing session ( 0) and
2 months a er the last session ( 2). Ultrasonography and MRI were used 3 months a er the f rst ses-
sion ( i) and 3 and 6 months a er the last session ( 3 and 6). Volumetric evaluations were also made
through three-dimensional MRI reconstruction.19 T e average resorption percentage o injected volume
was .36% at 6 months and 28.23% at 2 months.19 ransplanted at tissue resorption was analyzed
with instrumental imaging, nuclear MRI, and ultrasound. Compared with breast reconstruction using
implants o the same size, augmentation with PRP mixed with at tissue resulted in a lower height but
a more natural contour and so ness o the breasts. All patients were satisf ed with the resulting texture,
so ness, and contour, and MRI conf rmed maintenance o the restoration.
In patients treated with at gra s mixed with PRP, ultrasonography showed oily cysts in 66.67% o the
breasts at i, 70.83% at 3, 62. % at 6, and .83% at 2, whereas MRI detected oily cysts in 8.33%
at i, 3, and 6 months and . 7% at 2. At i, 3, and 6, the cytosteatonecrotic areas identif ed
through both ultrasonography and MRI were unchanged (8.33%), whereas at 2 those cytosteatone-
crotic areas were seen to be increased on ultrasound examination ( 2. %) and even more with MRI
scans ( 6.67%).

196
Part II • Regenerative Potential
INSTRUMENTAL IMAGING EVALUATION METHOD FOR FACE
SOFT TISSUE DEFECTS
A

B
C
D
Fig. 14-6, A-D
Clinical photographs and MRI scans can be used to assess the long-term retention o graf ed at with
PRP. T is patient is seen preoperatively with sof tissue de ects in her zygomatic region (A and B). She is
shown 0 months af er two at graf ing sessions in which at mixed with PRP was injected (25 cc in each
treatment) (C and D).

197
Chapter 14 • Platelet-Rich Plasma and Fat Gra ting
E
F
G

H
Fig. 14-6, E-H
Preoperative T2-weighted imaging o the zygomatic region (E) and temporal region (F) can be con-
trasted with the scans obtained 60 months later (G and H).
Transplanted at tissue resorption was analyzed with MRI and ultrasound. Patients with acial scars who
had reconstruction with at gra ing and PRP showed lower at resorption. All patients expressed sat-
is action with the resulting texture and so ness o their skin and the f nal contour. MRI conf rmed the
absence o cyst ormation and microcalcif cations.18
In patients a ected by so tissue de ects o the ace who were treated with at gra s mixed with PRP, we
observed a 6 % maintenance o contour restoration and three-dimensional volume a er year com-
pared with that o the control group. Transplanted at tissue resorption was analyzed with instrumental
imaging (MRI and ultrasound). No complications occurred in any o the patient, and the results were
lasting in all cases (mean ollow-up 60 months).

198
Part II • Regenerative Potential
C om p l i ca ti on s
Regular patient assessment is essential or experienced radiologists to identi y breast changes a er at
gra ing and to distinguish them rom malignant alterations so that at gra ing does not make early
diagnosis o cancer di cult. Although no screening protocol exists or patients who have undergone
at gra ing to their breasts, per orming specif c evaluations during the f rst year is critical, including
ultrasound and MRI scans 3, 6, and 2 months a er the last at gra ing session, and mammography
2 months a er the last session. Patients should be ollowed annually and seen by their surgeon, even
in the absence o alterations, especially patients who have had cancer. Mammography, ultrasonography,
and MRI scans are obtained be ore the f rst at gra ing session to rule out any signs o tumor recurrence
and to have an initial point o comparison; by 3 months a er the f rst lipof lling session, these examina-
tions do not seem to o er important data.19
In patients who have had cancers or are 35 years o age or older, it is important to per orm mammogra-
phy be ore at gra ing and ollow-up annually or more requently (the requency depends on the type o
alteration ound). Mammography is the only examination that can be used to clearly identi y microcal-
cif cations and distinguish them rom those that are malignant. In addition, mammography allows us to
identi y typical “soap bubble” lesions, macrocalcif cations, and breast opacity. It is there ore a key mo-
dality or monitoring the breasts a er at gra ing; however, alone, it is not enough. In patients younger
than 35 years o age who have undergone at gra ing solely or aesthetic reasons, mammography is not
indicated.19
Ultrasonography is more reliable than MRI or detecting small cysts (it is more sensitive), but it cannot
distinguish an oily cyst rom a normal cyst (it is less specif c). Small oily cysts are best seen by ultra-
sound, because they behave like small uid ormations. In our patients, small oily cysts appeared in a
much higher percentage o breast ultrasound images than in their MRI scans, also highlighting their
resorption. In one o our patients, normal cysts that were initially mistaken or oily cysts on ultrasound
were correctly identif ed through a subsequent MRI scan.19
Ultrasonography is also extremely use ul or the evaluation o “dense breasts,” surgical scars, and com-
plex cysts, and in di erentiating between cysts and solid masses. Finally, in patients with a suspicious
f nding, an ultrasound-guided microbiopsy may be per ormed. With MRIs, oily cyst signal intensity
is suppressed in sequences with at suppression, because they are composed o at, even i necrotic.
However, small oily cysts cannot be seen properly because o their small size, and thus they are easily
con used with native at (also suppressed). MRI seems to be at least as e ective as ultrasonography and
mammography in demonstrating large cytosteatonecrotic areas, as conf rmed by the McNemar test in
our study. In addition, MRIs are extremely use ul in cases o suspected recurrence, such as clinical signs
o recurrence together with negative or equivocal standard radiologic examinations, and scar alteration.
MRI has excellent sensitivity and specif city (greater than 0%) in the diagnosis o inf ltrating recur-
rences, but because o the large variation in angiogenesis, it remains limited in pure intraductal orms
(sensitivity between 60% and 5%). Furthermore, MRI can be used or assessing breast volume and pos-
sible changes in longitudinal studies. T us it can be very use ul or better understanding the behavior o
transplanted at and the mechanisms that underlie its resorption.