Fat Injection From Filling to Regeneration

200
Part II • Regenerative Potential
Fig. 14-9
T ese lateral and mediolateral oblique bilateral mammograms o a 43-year-old patient show calcif ca-
tions o the liponecrotic areas. T e percentage o maintaining restored was evaluated using MRI.
D i scu ssi on
T is in vivo tissue-engineering approach suggests our undamental points:

. PRP sustains an optimal microenvironment that allows correct architectural adipocyte distribu-
tion, better cell-to-cell interaction, adipose tissue growth, and di erentiation rom ADSCs; the
last eature o ers early protection rom surrounding in ammatory events.

. PRP-induced early development o neoangiogenic microcapillary network acilitates the delivery
o proper nutrient and oxygen levels to gra ed cells.8,10

3. e-SVF can avor neoangiogenic vascularization and f brogenic activity o f broblasts, which avor
adipose tissue survival and three-dimensional organization.

4. e-SVF and PRP improves at gra durability in patients who undergo regenerative surgery.

201
Chapter 14 • Platelet-Rich Plasma and Fat Gra ting
New techniques in the application o PRP in tissue regeneration are explained in the literature, but to
date no articles discuss the possible use o PRP mixed with at gra s and e-SVF at gra s or the treat-
ment o so tissue de ects.
We hypothesized that the mechanism o regeneration o the tissue is this: T e damaged areas are targeted,
angiogenic and antiapoptotic actors are released, and this is ollowed by the ormation o new vessels
and enhanced oxygenation o the transplanted tissue. Gra ed adipose tissue must survive through a sim-
ple di usion mechanism until an active blood supply is reestablished. Prosurvival actors may there ore
promote long-term retention and, consequently, durability o the gra .18
T e at gra ing procedure can modi y radiologic images; however, this inter erence has been studied in
the literature,20,21 and radiologic studies suggest that imaging technologies (ultrasonography, mammog-
raphy, and MRI) can be used to identi y the microcalcif cations caused by at injection.19-21 Moreover,
recent ollow-up studies demonstrated the sa ety o the procedure, detecting no increase in new disease
or tumor recurrence.3,21-23 Maximum breast augmentation using the described technique varied among
patients and appeared to be to cc. Although these volumes may be smaller than those achieved
with large breast implants, a def nite advantage is that patients need not be concerned about postop-
erative complications induced by implants, such as rupture, in ection, capsular contracture, unnatural
contour, hardness, neurologic symptoms, and immune response.
Compared with patients who underwent conventional at gra ing to the breasts, augmentation e ects
have been higher when PRP was mixed with at. For each injection, a . to . cm increase in breast
so tissue volume is common with the conventional procedure, compared with a . to . cm increase
in our series with lipoaspirate plus PRP, although the augmentation e ect varied among patients. T e
measurement system we recently devised may help to quanti y the di erence in augmented volume in
the uture.
T e survival o a gra , particularly o a larger-volume gra , is a balance between simple di usion until
an active blood supply is reestablished and hypoxia-induced cell death.24 Long-term retention and du-
rability o the gra may be enhanced by prosurvival actors. In an animal study, this e ect was achieved
by using gene therapy to deliver vascular endothelial growth actor (a potent proangiogenic actor) to
the gra . T is resulted in increased blood vessel density within the gra and a signif cant improvement
in gra retention at weeks.
Standard cell separators and salvage devices can be used to produce PRP. T ese devices operate on a
unit o blood and typically use a continuous- ow centri uge bowl or continuous- ow disk separation
technology and both a hard ( ast) and so (slow) spin, yielding platelet concentrations rom two to our
times baseline values.25 Although all operate on a small volume o drawn blood ( to 6 ml) and on the
principle o centri ugation, these systems di er widely in their ability to collect and concentrate platelets,
with approximately % to % o the available platelets collected, and rom less than a two old to an
approximately eight old increase in the platelet concentration higher than baseline. Several devices or
PRP preparation are available, such as Fibrinet (Cascade Medical Enterprises), Regen (Regen Lab USA),
Plateltex (Plateltex SRO), Vivostat (Vivostat A/S), and Platelet Rich Lipotrans ert (PRL). Generally, most
systems, whether handling a large or small volume, do not concentrate the plasma proteins o the coag-
ulation cascade.
A variety o innovations, including advances in stem cell technology, and greater understanding o the
role o the SVF may contribute to the improvement o autologous tissue transplantation and regenera-
tion. Further improvements o the technique may lead to at gra ing becoming the f rst choice or breast
augmentation in the uture.4,24

202
Part II • Regenerative Potential
C oncl u si on
Breast reconstruction with adipose tissue mixed with the PRP can restore volume satis actorily. More-
over, this technique can improve skin quality in cases o scarring or radiotherapy outcomes. In some
cases, it is possible to give coverage to breast implants and restore residual volume de ects. In cases o
breast hypoplasia, we can restore the volume, in repeated treatments, to obtain a natural result with no
visible scars. In particular, this is an important advantage in young patients.
In our series, satis actory clinical results were generally achieved with no major complications. T us,
we can conclude that PRP mixed with at gra is su ciently sa e or continuation o the study, though
controlled investigations and accumulated long-term results are needed to determine the overall sa ety
and e cacy o the treatment. Moreover, additional studies must be undertaken to urther evaluate the
e cacy o this method.
REFERENCES
1. Rigotti G, Marchi A, Galiè M, Baroni G, Benati D, Krampera M, Pasini A, Sbarbati A. Clinical treatment
o radiotherapy tissue damage by lipoaspirate transplant: a healing process mediated by adipose-derived
adult stem cells. Plast Reconstr Surg 119:14 9; discussion 14 3, 7.
. Yoshimura K, Sato K, Aoi N, et al. Cell-assisted lipotrans er or cosmetic breast augmentation: supportive
use o adipose-derived stem/stromal cells. Aesthetic Plast Surg 3 :48; discussion 56, 8.
3. Rigotti G, Marchi A, Stringhini P, Baroni G, Galiè M, Molino AM, Mercanti A, Micciolo R, Sbarbati A.
Determining the oncological risk o autologous lipoaspirate gra ing or post-mastectomy breast recon-
struction. Aesthetic Plast Surg 34:475, 1 .
4. Yoshimura K, Asano Y, Aoi N, et al. Progenitor-enriched adipose tissue transplantation as rescue or
breast implant complications. Breast J 16:169, 1 .
5. Lendeckel S, Jodicke A, Christophis P, et al. Autologous stem cells (adipose) and f brin glue used to treat
widespread traumatic calvarial de ects: case report. J Craniomaxillo ac Surg 3 :37 , 4.
6. García-Olmo D, García-Arranz M, Herreros D, et al. A phase I clinical trial o the treatment o Crohn’s
f stula by adipose mesenchymal stem cell transplantation. Dis Colon Rectum 48:1416, 5.
7. García-Olmo D, Herreros D, De-La-Quintana P, et al. Adipose-derived stem cells in Crohn’s rectovaginal
f stula. Case Rep Med. [Epub 1 Mar 7]
8. Peterson B, Zhang J, Iglesias R, et al. Healing o critically sized emoral de ects, using genetically modif ed
mesenchymal stem cells rom human adipose tissue. issue Eng 11:1 , 5.
9. Cervelli V, Gentile P. Use o cell at mixed with platelet gel in progressive hemi acial atrophy. Aesthetic
Plast Surg 33: , 9.
1 . iryaki , Findikli N, iryaki D. Staged stem cell-enriched tissue (SE ) injections or so tissue augmen-
tation in hostile recipient areas: a preliminary report. Aesthetic Plast Surg 35:965, 11.
11. Cantarella G, Mazzola RF, Domenichini E, et al. Vocal old augmentation by autologous at injection with
lipostructure procedure. Otolaryngol Head Neck Surg 13 : 39, 5.
1 . Coleman SR. Facial recontouring with lipostructure. Clin Plast Surg 4:347, 1997.
13. Coleman SR. Long-term survival o at transplants: controlled demonstrations. Aesthetic Plast Surg
19:4 1, 1995.
14. Cervelli V, Gentile P, Scioli MG, et al. Application o platelet-rich plasma in plastic surgery: clinical and in
vitro evaluation. issue Eng Part C Methods 15:6 5, 9.
15. Cervelli V, Gentile P, Grimaldi M. Regenerative surgery: use o at gra ing combined with platelet-rich
plasma or chronic lower-extremity ulcers. Aesthetic Plast Surg 33:34 , 9.

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Chapter 14 • Platelet-Rich Plasma and Fat Gra ting
6. Gentile P, Di Pasquali C, Bocchini I, Floris M, Eleonora T, Fiaschetti V, Floris R, Cervelli V. Breast recon-
struction with autologous at gra mixed with platelet-rich plasma. Surg Innov : 7 , .
7. Gentile P, Orlandi A, Scioli MG, Di Pasquali C, Bocchini I, Curcio CB, Floris M, Fiaschetti V, Floris R,
Cervelli V. A comparative translational study: the combined use o enhanced stromal vascular raction
and platelet-rich plasma improves at gra ing maintenance in breast reconstruction. Stem Cells Transl
Med : , .
8. Gentile P, De Angelis B, Pasin M, Cervelli G, Curcio CB, Floris M, Di Pasquali C, Bocchini I, Balzani A,
Nicoli F, Insalaco C, Tati E, Lucarini L, Palla L, Pascali M, De Logu P, Di Segni C, Bottini DJ, Cervelli V.
Adipose-derived stromal vascular raction cells and platelet-rich plasma: basic and clinical evaluation or
cell-based therapies in patients with scars on the ace. J Cranio ac Surg 5: 67, .
9. Fiaschetti V, Pistolese CA, Fornari M, Liberto V, Cama V, Gentile P, Floris M, Floris R, Cervelli V, Simon-
etti G. Magnetic resonance imaging and ultrasound evaluation a er breast autologous at gra ing com-
bined with platelet-rich plasma. Plast Reconstr Surg : 98e, .
. Pulagam SR, Poulton T, Mamounas EP. Long-term clinical and radiologic results with autologous at
transplantation or breast augmentation: case reports and review o the literature. Breast J :6 , 6.
. Kwak JY, Lee SH, Park HL, et al. Sonographic f ndings in complications o cosmetic breast augmentation
with autologous at obtained by liposuction. J Clin Ultrasound : 99, .
. Fraser JK, Hedrick MH, Cohen SR. Oncologic risks o autologous at gra ing to the breast. Aesthet Surg
J :68, .
. Bielli A, Scioli MG, Gentile P, Agostinelli S, Tarquini C, Cervelli V, Orlandi A. Adult adipose-derived stem
cells and breast cancer: a controversial relationship. Springerplus 8: 5, .
. Zhu M, Zhou Z, Chen Y, et al. Supplementation o at gra s with adipose-derived regenerative cells im-
proves long-term gra retention. Ann Plast Surg 6 : , .
5. Kevy SV, Jacobson MS. Comparison o methods or point o care preparation o autologous platelet gel. J
Extra Corpor Technol 6: 8, .

(Trang không có chữ)

205
C h a p t e r 1 5
C
l , l c l b mm c l h m
c wh ch l c m ; h xc h , f l
m h c ll c . B h l c h l
w h h l m, m l ch q w l-
bl , l c h . H w , m l z w h
l q h l b c m h l - m
, b c h l l m b l h f l h h
l . W h c c
b c , h l h cl l b , b ,
h w c f m b h f m .1-3 U b l , h l-
m c c l m l c ch q w ll b f m wh
m w h h w h c m c c c .
W h h c h c c ll c ll-b
l , h h l w l b c mm -
l c c ll h c ll c b c l l c , h h c ll
w l b b m h b k ch q (c ).4,5 O
m h c w h ch q , h m
l m b l bl c l h m c l c ll , b c h
c l b c l c q .6
Cr yopr eser vat ion o Adipose
Tissue or Fat Gr a t ing
Lee L.Q. Pu, Chunmei Wang
205

206
Part II • Regenerative Potential
Fig. 15-1
A

B
Flat, spindle-shaped, processed lipoaspirate (PLA) cells a er a -week culture rom the resh control
group are shown (A) and rom the cryopreservation group (B). Phase-contrast microscopy was used
with an original magnif cation o × . Both PLA cell samples appear to have normal morphology and
may be used or cell-based therapy in the uture.
M
od ern T ech ni q ues i n C ryo p reserva ti on
T e modern technique o cryopreservation permits the long-term storage o living cells and tissues that
may have many potential clinical applications such as blood trans usion, bone marrow transplantation,
in vitro ertilization, vascular gra s, bone gra s, and skin gra s.1 T e major steps o the cryopreserva-
tion process can be summarized as ollows: ( ) add cryoprotective agents to cells/tissues be ore cooling,
( ) cool the cells/tissues in a controlled rate toward a low temperature at which the cells/tissues are
stored, (3) warm the cells/tissues, and (4) remove the cryoprotective agents rom the cells/tissues a er
thawing.7 T e optimal cooling rate or cell survival should be slow enough to avoid intracellular ice or-
mation but ast enough to minimize cell damage.
T e reezing behavior o the cells can be modif ed by the addition o cryoprotective agents, which a ect
the rates o water transport, nucleation, and crystal growth. Cells that have survived cooling still ace
the challenges o thawing, which can exert e ects on survival comparable to those o cooling. Also,
knowledge o the cell permeability to water and cryoprotective agents allows one to predict minimal and
maximal cell volume excursion during the addition and removal o cryoprotective agents in the cells to
provide a quantitatively optimal approach toward avoiding osmotic damage.

207
Chapter 15 • Cryopreservation o Adipose issue or Fat Gra ting
Dimethyl sul oxide (DMSO), a permeable agent, has been widely used as an e ective cryoprotective
agent (CPA) in the cryopreservation o living cells or tissues.7,8 T e concentration o DMSO, when used
alone as a CPA, is usually %. Because this agent is tissue toxic at normal body temperature, it should
be removed rom the previously cryopreserved cells or tissues a er thawing. In our previous study, we
attempted to lower the concentration o DMSO used in cryopreservation o adipose tissue by adding
another nontissue toxic cryoprotective agent, such as trehalose. As a CPA, trehalose can dehydrate cells
and thus reduce the amount o water present be ore reezing. It can also stabilize cellular membranes
and proteins during reezing and drying. Combining trehalose, a nonpermeable cryoprotective agent,
with DMSO, a permeable cryoprotective agent, may work synergistically to protect adipose tissue during
cryopreservation.9 T ere ore the concentration o DMSO can theoretically be reduced when it is used
in combination with trehalose. T e combined use o both DMSO and trehalose as cryoprotective agents
may be valuable to achieve optimal cryopreservation o adipose tissues or other types o tissues.
P revi ous S tud i es i n C ryo p reserva ti on o f F a t G ra f ts
A simple reezing technique has been recently used by a number o investigators to explore the possibil-
ity o long-term storage o at gra s. In one study, at gra s were rozen in liquid nitrogen and stored at
– . ° C or up to days. T e results demonstrated that the rozen at gra s showed remarkable main-
tenance o mitochondrial metabolic activity.10 However, a separate study by others indicated that a er
simple reezing, up to . % o metabolic activity o at gra s was lost, but the addition o a CPA led to
preservation o up to % o the baseline activity.11 T e authors indicated that the widely used practice
o simple reezing in a reezer leads to nonviable tissue and cell survival can be improved by the addi-
tion o a CPA. In another study, at gra s were simply rozen in liquid nitrogen and stored at – ° C or
6 months. Interestingly, these authors ound that the viability and histologic characteristics o at gra s
rozen in liquid nitrogen were similar to those o resh gra s.12 However, their f nding has been ques-
tioned by other investigators, who have ound that to achieve optimal cryopreservation o adipose tissue,
it would be necessary to add a CPA be ore reezing and at gra s mixed with a CPA should also undergo
controlled reezing and thawing during cryopreservation.1-3,13,14
In our laboratory, a preliminary study was conducted earlier to evaluate the e ect and role o tempera-
ture alone or storage o adipose tissue. In these studies, the viability o lipoaspirate that underwent
“low” temperature banking was assessed by a glycerol- -phosphate dehydrogenase (G PDH) assay at
both ° C and – ° C. T e G PDH assay was chosen in our study to assess cellular unction o adipose
tissue because it is relatively simple and the enzyme is adipose specif c. T e assay measures the level o
G PDH within the cytoplasm o adipocytes. T e higher level o the enzyme represents a better cellular
unction o adipose tissue.15

208
Part II • Regenerative Potential
REDUCTION OF G3PDH ACTIVITIES AT 4° C
0
20
40
60
80
100
1
Days of storage
2

3

4

5

6

7

8

9

10 11 12
13 14 15
P
e
r
c
e
n
t
a
g
e

o
f

a
c
t
i
v
i
t
y
Fig. 15-2
A er weeks o storage, the viability o adipose aspirates decreased by about % at 4° C.
CHANGE OF G3PDH ACTIVITIES AT –20° C
0.43
0.45
0.47
0.49
0.51
0.52
0.44
0.46
0.48
0.50
1
Days of storage
2

3

4

5

6

7

8

9

10 11 12
13 14 15
U
/
m
l
Fig. 15-3
By contrast, the viability o lipoaspirate decreased by only about 5% at – ° C in weeks. Furthermore,
a series o experiments was per ormed initially with dif erent types and concentrations o cryoprotective
agents, and their dif erent combinations to determine what would be the best CPA that can be used es-
pecially or adipose tissues.
Although a ew studies showed encouraging preliminary results or cryopreservation o autologous at
gra s or possible uture application, the technique described in the study can only be used or a short-
term preservation (a ew days or weeks) and may not be optimal due to uncontrolled cooling/warming
process and the lack o use o a cryoprotective agent. With application o modern cryopreservation
techniques, a better long-term preservation o adipose tissues can possibly be per ormed. Such a method
will likely allow at gra s to be stored or months or years (below – 5° C) or or more than 1 years (at
–196° C in liquid nitrogen).
We conducted a preliminary study with the use o modern cryopreservation technique or cryopreserva-
tion o lipoaspirates. Our initial results are quite encouraging and proper cryoprotective agents and their
optimal concentration or combination can be selected.

209
Chapter 15 • Cryopreservation o Adipose issue or Fat Gra ting
0
1.0
2.0
2.5
3.5
0.5
Fresh control
0.2 DMSO + 0.1 Trehalose
0.5 DMSO + 0.2 Trehalose
Negative control
0.25 M Trehalose
0.5 M Trehalose
1.0 DMSO
1.5 DMSO
×
1
0
6

c
e
l
l
s
/
m
l
1.5
3.0
*
RESULTS OF VIABLE ADIPOCYTE COUNT
Fig. 15-4
Results o the viable adipocyte count in dif erent cryoprotective agent–treated groups are shown:
*p > . versus all other CPA groups except the . molar (M) trehalose group; p = . (NS)
versus the . M trehalose group; p = . 4 (NS) versus the resh lipoaspirate. It becomes clear that a
combination o . M DMSO and . M trehalose can be the best selection or cryoprotective agents that
should be used during cryopreservation o adipose tissue.3 In addition, trehalose alone may also be used
or cryoprotection o adipose tissue.16 A number o reezing and thawing protocols were also evaluated
or adipose tissue during the preliminary studies.
Again in our laboratory, we conducted additional studies to determine whether trehalose alone can be
used as an ef ective cryoprotective agent. We calculated the viable adipocyte counts in dif erent concen-
trations o trehalose groups. T e highest viable adipocyte count was ound in the trehalose group with
a concentration o . mol/L, p > . versus the resh control group and p > . versus all other
trehalose groups. rehalose, as a CPA with a concentration o . mol/L, appears to provide optimal
protection o lipoaspirate during the cryopreservation in vitro and in vivo with the same reezing and
thawing protocol developed in our laboratory.17 Such protection is ound to be similar to that provided
by a combination o . M DSMO and . M trehalose as CPAs.18
0
1.0
2.0
2.5
3.5
0.5
Fresh control
0.25 M
0.30 M
0.20 M
0.35 M
0.40 M
0.50 M
0.75 M
×
1
0
6

c
e
l
l
s
/
m
l
1.5
3.0
*
VIABLE ADIPOCYTES IN DIFFERENT TREHALOSE GROUPS
Fig. 15-5
Viable adipocyte counts in dif erent concentrations o trehalose groups. T e highest viable adipocyte
count was ound in the trehalose group with a concentration o . mol/L, p > . versus the resh
control group and p > . versus all other trehalose groups.

210
Part II • Regenerative Potential
D evel o p ment o f a C ryo p reserva ti on
P rotocol f or F a t G ra f ts
CHOICES OF CRYOPROTECTIVE AGENTS
DMSO, a permeable CPA that can reduce cell injury as a result o the intracellular ice ormation and
“solution e ects,” and trehalose, a nonpermeable CPA that can protect the cell membrane, were se-
lected.7,8 A combination o .5 M ( . %) DMSO (Sigma, St. Louis, MO) and . M ( .6%) trehalose
(Sigma) was determined to be the optimal one and was used in our study.3 However, trehalose as the
only CPA with a concentration o . 5 mol/L was also ound to provide optimal protection o lipoaspi-
rate during cryopreservation and can be used clinically in the uture, since trehalose does not need to be
removed a er thawing.17 Double concentrations o the di erent CPA solutions were made f rst, and each
chosen CPA was then reshly diluted to the f nal concentration about minutes be ore it was added to
the adipose tissue.2
ESTABLISHING A FREEZING AND THAWING PROTOCOL
Fig. 15-6
T e reezing and thawing protocol described here represents the best possible method developed or
cryopreservation o adipose tissue and was used in our study. Fresh lipoaspirate, a er preparation, was
trans erred to a vial and mixed with the combined DMSO (in .5 M) and trehalose (in . M) solution or
trehalose only (in . 5 M) in a : ratio. A er adding cryoprotective agents, the vial was placed at room
temperature or minutes and then put into a methanol bath (Kinetics, Stone Ridge, NY).

211
Chapter 15 • Cryopreservation o Adipose issue or Fat Gra ting
Fig. 15-7
T e reezing system was set up at approximately ° to ° C per minute, with a slow cooling rate rom
° C to –30° C without artif cially induced ice ormation. T e vial was then trans erred to liquid nitro-
gen (– 96° C) a er it reached –30° C or long-term preservation.
Fig. 15-8
Be ore thawing, the vial containing cryopreserved lipoaspirate was taken rom the liquid nitrogen tank
and placed at room temperature or minutes to let the liquid nitrogen vapor escape rom the vial.
Fig. 15-9
T e vial was then dropped into a stirred 37° C water bath until the preserved lipoaspirate was thoroughly
thawed.

212
Part II • Regenerative Potential
FINDINGS FROM OUR STUDIES
Fig. 15-10
B
A
T e at gra s previously cryopreserved with our pre erred method a er thawing had a near-normal ap-
pearance (A), much like that o reshly aspirated/ref ned at gra s (B), and may readily be used or uture
at gra ing i indicated.3
In our studies, trehalose was used as the only CPA, and the same reezing and thawing protocol was ap-
plied. In our in vitro study, more viable adipocytes o cryopreserved at gra s were ound when trehalose
was in a concentration o 0. 5 M. T ere was no statistical di erence between the resh control group and
the cryopreserved group with trehalose in a concentration o 0. 5 M (p > 0.05). G PDH assay showed
there was no statistical signif cance in the resh control group compared with all cryopreserved groups
(all p > 0.05). Histologically, basic structure o adipose tissue was adequately maintained in most o the
cryopreserved groups. rehalose, served as a CPA with its concentration at 0. 5 M, appears to provide
optimal protection o lipoaspirate during cryopreservation based on this study.17
Fig. 15-11
A
B
Routine histology o the resh or cryopreserved lipoaspirate (hematoxylin and eosin [H&E] staining;
original magnif cation 00× ) showed the normal structure o ragmental adipose tissue rom the resh
control group (A) and adequately maintained and near normal structure o adipose tissue a er optimal
cryopreservation o lipoaspirate when the concentration o trehalose was 0. 5 M (B).
In the subsequent in vivo study, the protocol used or reezing and thawing o lipoaspirate was the same
as that used in our in vitro study. In the control group, 0.5 cc o lipoaspirate gra s was injected into the
posterior scalp o a nude mouse. In cryopreservation group , a combination o DMSO (in 0.5 M) and
trehalose (in 0. M) was used as CPAs. In the cryopreservation group , the optimal concentration o

213
Chapter 15 • Cryopreservation o Adipose issue or Fat Gra ting
trehalose (in 0. M) was used as the only CPA. In both cryopreservation groups, 0. cc o cryopreserved
at gra a er thawing was injected into the animal in the same manner as in the control group. T e f nal
volume and weight o maintained at gra s and their histology were evaluated at the end o the study.
Group had equivalently maintained volume and weight compared with group . However, the results
rom both cryopreservation groups were still in erior to those rom the control group (both p < 0.0 ).
Histologically, the basic structure o adipose tissue was maintained in all three groups.
rehalose, severed as a CPA in its optimal concentration o 0. mol/L, also appears to provide the ade-
quate protection o human at gra s during cryopreservation in vivo. Such protection is similar to that
provided by the combination o DMSO and trehalose as CPAs.18
Fig. 15-12
A
B
C
Routine histologic micrographs o the “ at gra s” are seen at weeks a er in vivo administration o resh
or cryopreserved lipoaspirate (H&E staining, original magnif cation 00× ). A maintained adipose tissue
structure within an encapsulated f brous capsule was seen primarily in all three groups. T e basic archi-
tecture o ragmental adipose tissue was maintained in all three groups, except some tissue shrinkage
was ound in both cryopreservation groups: (A) the control group, (B) cryopreservation group , and
(C) cryopreservation group .
F uture P ersp ecti ves
Because lipoaspirate may also be used or so tissue augmentation in either cosmetic or reconstructive
procedures through autologous at gra ing, it may certainly be more attractive to many patients i their
lipoaspirate could be reliably preserved a er a liposuction procedure. T is can be true or at gra ing to
the ace, breast, and buttock, where repeated gra ing procedures may be needed or better outcomes.
When uture repeated at gra ing is needed or a patient, his or her banked lipoaspirate could possibly
be used, as long as the at gra s are properly preserved and remain viable and sterile. No additional at
harvesting procedure would be required. Furthermore, i adipose-derived stem cell (ADSC)–based ther-
apy is needed at a later time, previously preserved lipoaspirate could then be processed in vitro to obtain
processed lipoaspirate cells or that particular patient.6

214
Part II • Regenerative Potential
Lipoaspirate as “raw and original” material may be preserved e ectively by an optimal cryopreservation
procedure to meet uture needs o patients, either or repeated at transplantation or or ADSC-based
therapy.17-19 Success ul long-term preservation o lipoaspirate will truly open a new era in both at gra -
ing and ADSC-related therapy in plastic and reconstructive surgery, and we look orward to this uture
o regenerative medicine.
Adipose
aspirates
Future ADSC-directed
cell therapy for
regenerative
application
Autologous
fat grafts
harvested
Future isolation
of ADSCs
Future fat grafting
for soft tissue
augmentation
Tissue banking
Fig. 15-13
T is schematic diagram shows uture autologous at gra ing or the same patient and ADSC-directed
cell therapy or regenerative application a er cryopreservation.
C oncl u si on
Our cryopreservation method developed specif cally or adipose tissues appears to provide good long-
term preservation o lipoaspirate, but so ar, the overall quality o the cryopreserved lipoaspirate is still
less ideal than reshly harvested at. T is might be true not only or adipose tissue but also or other types
o tissues a er optimal cryopreservation.7-9
Because o its sa ety and e ectiveness, trehalose only as a CPA with optimal concentration, maintained
with our cooling and thawing protocol, could possibly be used in patients or long-term preservation o
their harvested at.20,21 In addition, pretreatment o cryopreserved lipoaspirate with tissue culture me-
dium, platelet-rich plasma, or poloxamer may also improve their viability, because some o the injury to
cell membrane o adipocytes during cryopreservation can be repaired.22
Clearly, urther studies must be done to develop a reliable and clinically easible cryopreservation
method that can be used or success ul long-term preservation o adipose tissue. T e avorable results
rom our recent studies warrant urther investigations on cryopreservation o human adipose tissue or
possible uture autologous at gra ing.

215
Chapter 15 • Cryopreservation o Adipose issue or Fat Gra ting
T ech ni ca l P ea rl s
• Simple cold storage o atty tissue in a re rigerator at 4° C or even lower temperatures in a reezer—
even with liquid nitrogen—does not reliably yield viable at gra s or reimplantation and should be
avoided.
• Adding cryoprotective agents (CPAs) is necessary be ore reezing to protect atty tissues.
• DMSO, a commonly used CPA, can be toxic to humans at room temperature and there ore must be
removed rom atty tissue be ore reinjection.
• T e naturally occurring sugar trehalose is not toxic to atty tissue and dehydrates atty tissue while
stabilizing the cell membrane.
• Low-dose DMSO combined with trehalose appears to act synergistically to sa ely protect atty tissue
rom the injuries incurred during reezing, storage, and thawing.
• rehalose at a concentration o 0.3 mol/L may provide adequate protection as a single CPA and pos-
sibly be used clinically or banking o at gra s in the uture.
• Cryopreservation o at can be subject to certain tissue-banking regulations. T us appropriate ap-
proval to per orm tissue banking may be required or the long-term preservation o at harvested via
conventional liposuction.
REFERENCES
. Pu LL, Cui X, Fink BF, et al. Long-term preservation o adipose aspirates a er conventional lipoplasty.
Aesthetic Surg J 4: 36, 004.
. Pu LL, Cui XD, Li JH, et al. T e ate o cryopreserved adipose aspirates a er in vivo transplantation. Aes-
thetic Surg J 6:6 3, 006.
3. Cui XD, Gao DY, Fink BF, Vasconez HC, Pu LL. Cryopreservation o human adipose tissues. Cryobiology
: 69, 007.
4. Ashjian PH, De Ugarte DA, Katz AJ, et al. Lipoplasty: rom body contouring to tissue engineering. Aes-
thetic Surg J : , 00 .
. De Ugarte DA, Ashjian PH, Elbarbary A, et al. Future o at as a raw material or tissue regeneration. Ann
Plast Surg 0: , 003.
6. Pu LL, Cui XD, Fink BF, et al. Adipose aspirates as a source or human processed lipoaspirate cells a er
optimal cryopreservation. Plast Reconstr Surg 7: 84 , 006.
7. Gao DY, Critser JK. Mechanisms o cryoinjury in living cells. ILAR J 4 : 87, 000.
8. Pegg DE. T e history and principles o cryopreservation. Semin Reprod Med 0: , 00 .
9. Erdag G, Eroglu A, Morgan J, et al. Cryopreservation o etal skin is improved by extracellular trehalose.
Cryobiology 44: 8, 00 .
0. MacRae JW, T olpady SS, Ogle RC, et al. Ex vivo at gra preservation: ef ects and implications o cryo-
preservation. Ann Plast Surg : 8 , 004.
. Wolter P, Heimburg DV, Stof els I, Groeger A, Pallua N. Cryopreservation o mature human adipocytes:
in vitro measurement o viability. Ann Plast Surg :408, 00 .
. Atik B, Oztürk G, Erdoğan E, et al. Comparison o techniques or long-term storage o at gra s: an exper-
imental study. Plast Reconstr Surg 8: 33, 006.
3. Moscatello DK, Dougherty M, Narins RS, et al. Cryopreservation o human at or so tissue augmenta-
tion: viability requires use o cryoprotectant and controlled reezing and storage. Dermatol Surg 3 : 06,
00 .

216
Part II • Regenerative Potential
4. Pu LL. Comment on Atik B, Oztürk G, Erdoğan E, an O. Comparison o techniques or long-term stor-
age o at gra s. Plast Reconstr Surg 0:8 3, 007.
5. Pu LL, Cui XD, Fink BF, et al. T e viability o atty tissues within adipose aspirates a er conventional
liposuction: a comprehensive study. Ann Plast Surg 54: 88; discussion 9 , 005.
. Pu LL, Cui XD, Cibull ML, et al. Cryopreservation o adipose tissues: the role o trehalose. Aesthetic Surg
J 5: , 005.
7. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells rom human adipose tissue: implications or cell-
based therapies. issue Eng 7: , 00 .
8. Ashjian PH, Elbarbary AS, Edmonds B, et al. In vitro dif erentiation o human processed lipoaspirate cells
into early neural progenitors. Plast Reconstr Surg : 9 , 003.
9. De Ugarte DA, Morizono K, Elbarbary A, et al. Comparison o multi-lineage cells rom human adipose
tissue and bone marrow. Cells issues Organs 74: 0 , 003.
0. Cui XD, Pu LL. T e search or a use ul method or optimal cryopreservation o adipose aspirates: part I.
In intro study. Aesthetic Surg J 9: 48, 009.
. Cui XD, Pu LL. T e search or a use ul method or the optimal cryopreservation o adipose aspirates: part
II. In vivo study. Aesthetic Surgery J 30:45 , 0 0.
. Medina MA, Nguyen J , Kirkham JC, et al. Polymer therapy: a novel treatment to improve at gra viabil-
ity. Plast Reconstr Surg 7: 70, 0 .

CLINICAL
APPLICATIONS
P a r t I I I

(Trang không có chữ)

S e c t i o n O
n e
SKIN

(Trang không có chữ)

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Impr oving S in Qual it y:
An E per iment al and Cl inical
St udy o t he E ect s o Fat
and St r omal Cel l s on t he S in
Ali Mojallal, Jonathan Rodriguez, Ondine Rouyer,
Charlotte Lequeux, Jean-Louis Foyatier, Samia Guerid,
Odile Damour
221

222
Part III • Clinical Applications • Skin
ditions, adipose tissue was harvested, purif ed, and reinjected according to the protocol described by
Coleman.10-12 Nude immunodef cient mice were chosen or this study or their immunologic tolerance
o human cells. Histologic and immunohistologic analyses were used to investigate the structure o the
transplanted adipose tissue, the di erent subcutaneous layers, and the space between the adipose tissue
and the skin.
en male nude mice aged 4 days were used or this study. T irty cubic centimeters o adipose tissue
were harvested rom the abdominal region o a 36-year-old patient-volunteer using a 3 mm diameter
Coleman harvesting cannula attached to a ml Luer-Lok syringe. T e adipose tissue was then treated
according to the protocol described by Coleman10-12 and sent directly to the animal laboratory or the
experiment in mice. One milliliter o purif ed adipose tissue was then injected using a mm diameter
blunt-tipped cannula into the le parascapular region o each mouse. Eight weeks a er implantation, a
skin biopsy, including subcutaneous tissue, was per ormed in the injected area. A control biopsy in the
same region was per ormed on the opposite noninjected side.
Fig. 16-1
T e regions o biopsy are shown, on the le side at the site o injection, and on the right in the control
region. Note the visible growth only on the le side.
HISTOLOGIC AND IMMUNOHISTOLOGIC ANALYSES
Hematoxylin-phloxin-sa ron (HPS) staining was per ormed on 5 µm depara nated slices. T e hema-
toxylin is a basic dye that stains the nucleus o the cells blue. T e phloxin is an acid dye staining the
cytoplasm pink, and the sa ron stains the connective tissue orange.
T e samples were also embedded in issue eck; 7 µm slices were next f xed in isopropanol and then
stained with a solution o Oil Red O (Sigma), an adipocyte-specif c dye which stains the lipid-f lled vac-
uoles red.
Slices 5 µm thick were depara nated and then incubated with anti-mouse collagen I antibodies. We
used anti-mouse IgG antibodies coupled to peroxidase to detect the immunocomplexes, using diami-
nobenzidine as the substrate. Counterstaining was per ormed using Harris hematoxylin. T is labeling
enabled us to detect cells o mouse origin. Human skin was used as a negative control.
Other slices were incubated with anti-human collagen I specif c primary antibodies, ollowed by a sec-
ondary antibody coupled to a uorochrome to reveal the binding sites o the primary antibody. T ese
could thereby be observed under a uorescent microscope to visualize collagen f bers o human origin.

223
Chapter 16 • Improving Skin Quality
HISTOLOGIC AND IMMUNOHISTOLOGIC RESULTS
Eight weeks a er implantation, all 0 mice had a le parascapular growth corresponding to the site o
transplantation o the adipose tissue. Histologic analysis demonstrated the presence o mature adipo-
cytes stained by Oil Red O. T e HPS staining revealed a considerable increase in density o the extracel-
lular matrix surrounding the adipose tissue and between it and the dermis. T e dermis also showed an
increased thickness a er injection o adipose tissue. T is increase in density o the extracellular matrix
was made up o collagen I f bers, an absence o which could be noted on the control noninjected side.
200 µm
Before fat transfer
200 µm
200 µm
Before fat transfer
200 µm
Human control
Surviving fat cells
(Oil Red O stained)
A

B

C

D
E
F
Fig. 16-2
Histologic slices o control skin (A) and skin with gra ed adipose tissue (B) are shown. Note the thick-
ening o the dermis and the abundance o extracellular matrix between skin and adipose tissue, as well
as that surrounding the at lobules a er the trans er o adipose tissue. Immunohistochemistry, using an
anti-mouse collagen I antibody to label in brown the extracellular matrix, shows that the mass o colla-
gen f bers are o murine origin (C and D). T e negative control (human skin) shows an absence o label-
ing at this level (E). Adipocyte viability is conf rmed by Oil Red O staining (F).

224
Part III • Clinical Applications • Skin
Immunohistochemical analysis using a labeling technique with anti-mouse collagen I allowed us to
demonstrate the origin o the newly ormed cells. T e neosynthesized extracellular matrix was ound
to be o murine origin and not human, conf rmed by the human skin negative control. Concordantly,
immuno uorescence using an anti-human collagen I antibody conf rmed the murine origin o collagen
f bers, since no labeling was ound in the mice.
A

B
Fig. 16-3
T is immuno uorescent slice o mouse skin ollowing at tissue gra ing (A) shows the absence o hu-
man collagen f bers a er labeling with an anti-human collagen I–specif c antibody. In contrast, the hu-
man skin (B) shows a considerable density o human collagen f bers, stained green.
CONCLUSIONS
T e results o our experimental study in mice agree with clinical observations made in patients treated
by the same technique. Adipose tissue gra s survive and have a volume-increasing e ect. Moreover, the
presence o human adipose tissue increases murine dermis thickness at the recipient site by induction o
collagen neosynthesis, likely as a result o the introduction o stem cells, growth actors, and cytokines.
T is collagen, mostly type I but also types V and VI, is o murine origin. Our study also demonstrated
that adipose tissue can stimulate production o collagen by the host cells at the recipient site. T is is
supported by other studies showing that ADSCs present in adipose tissue promote proli eration and
migration o f broblasts and activation o extracellular matrix synthesis.13-16 However, our study did not
allow us to determine whether this induction is directly caused by the adipose tissue or is the result o
an indirect passage through an intermediary in ammatory reaction. T e presence o the adipose tissue
gra causes an increase in local vascularization, resulting in a new physiologic equilibrium at the recip-
ient site. ogether, these observations demonstrate active f lling properties o adipose tissue. Further
studies are needed to show the long-term e ects o at gra ing at the recipient site.

225
Chapter 16 • Improving Skin Quality
A ni ma S tud y D emon stra ti n g th e W
oun d - H ea i n g
P ro p erti es o f A d i p ose- D eri ved S tem C el
s
Cutaneous healing can be impaired by many situations such as diabetes, chronic renal ailure, irradiation
and advancing age.17 Because o our aging population, today chronic wounds constitute a real public
health problem. T e main goals o treatment are rapid closure o the wound to restore the skin’s barrier
unction and prevent in ection, suppression o pain, and recovery o unction. During the past decade,
many reconstructive therapies based on the use o stem cells have been introduced. T ese are undi er-
entiated cells with sel -renewing properties and are able to divide to generate specialized cells, including
skin cells, f broblasts, and endothelial cells.18 T e stromal vascular raction (SVF) o adipose tissue con-
tains ADSCs, and its e cacy has been shown in experimental healing models. T e suggested mechanism
o action is the modulation o in ammation and tissue regeneration by the di erentiation o stem cells.19
However, SVF is a heterogeneous cell suspension containing a small percentage ( % to 3%) o ADSCs.20
T rough cell processing, it is now possible to obtain a homogeneous cell therapy product containing
more than % ADSCs.21 T e possibility o reezing ADSCs o ers ( ) the potential to per orm all sa ety
checks be ore releasing the product and ( ) the ability to repeat the treatment i necessary.
We per ormed a study to evaluate the e ectiveness o a controlled dose o injected cultured resh or ro-
zen ADSCs compared with a stromal vascular raction (SVF) on wound healing in an animal model. o
avoid their dispersion in the subcutaneous tissue, ADSCs were injected in a noncrosslinked hyaluronic
acid gel (Cytocare 3 ).
METHOD
Human SVF was isolated rom lipoaspirate obtained rom a randomly chosen donor who was under-
going liposuction. Adipose tissue was digested with collagenase and the reaction stopped by adding
Dulbecco’s Modif ed Eagle’s Medium (DMEM). Floating adipocytes were discarded, and cells rom the
stromal vascular raction (SVF) were pelleted, rinsed, centri uged, and counted be ore being mixed with
noncrosslinked hyaluronic acid, Cytocare 3 (Cyt 3 ), or injection into nude mice. Cyt 3 is a non-
crosslinked hyaluronic acid and a rejuvenating complex containing components. It was used as a
vehicle in our study because previous published work22 showed that it was the best culture medium or
ADSCs. Crosslinked hyaluronic acid prevents stem cells rom multiplying and di erentiating.
Freshly isolated cell suspensions containing ADSCs were cultured in ADSC medium until % con u-
ence was reached. At con uence, cells were trypsinized and centri uged. A portion o the ADSCs was
obtained using the technique described earlier and these were rozen. T e cells were thawed week
be ore surgery and cultured in ADSC medium.

226
Part III • Clinical Applications • Skin
Fig. 16-4
Sixteen male nude 4 -day-old mice were anesthetized, and surgery was per ormed under standard ster-
ile conditions. wo circular ull-thickness wounds mm in diameter were created on the backs o
the mice and a ring o silicone was sutured around the wound to prevent skin retraction. Each healing
wound received . 06 ADSCs, or SVF with hal the cells intradermally injected around the wounds into
our injection sites, and the other hal applied to the wound sur ace. Each nude mouse received a di er-
ent treatment or each wound. Cyt was used as vehicle or the cells.
wo sets o experiments were designed, comprising wounds on mice (two wounds per mouse).
Five excisional wounds were carried out in each group as ollows:

. Negative control (spontaneous healing)

. Vehicle alone (Cyt 532)

. Cultured rozen ADSCs (c. r.ADSC + Cyt 532) cryopreserved at passage 1, then thawed

4. SVF (SVF + Cyt 532)

. Cultured ADSCs (passage 2) (c.ADSC + Cyt 532) without cryopreservation be ore injection
Every day the mice were observed and digital images taken. T e wound area was measured by tracing
the wound margin and calculating the pixel area using image analysis so ware. T e percentage o wound
healing was calculated using reepithelialization sur ace measurements.
At day 7, all scar tissue was harvested with a rim o healthy normal skin or histologic analyses. issue
samples were f xed, embedded in para n, and stained. In ammation was evaluated on a semiquan-
titative scale. Sections were examined in a blinded ashion by two independent observers. Per usion
evaluation was done by means o laser Doppler imaging on day 7, when all wounds had closed and
biomechanical tests were conducted to evaluate the properties o the scarring mouse tissue.
RESULTS
T e time to complete healing was signif cantly shortened by treatment with ADSCs or SVF. In the
groups treated with ADSCs, this was 4 ± 0. days without a reezing step (c.ADSC + Cyt ) and 4.4
± 0. days with a reezing step, respectively, versus .7 ± .8 days without treatment (p < 0.0 ), thus
representing a gain o more than 7 days i both ADSC groups are pooled. Furthermore, the reezing step
induced no modif cation o the ADSC e ect on healing time. A signif cant decrease in healing time was
also observed when comparing pooled ADSC groups o animals treated with Cyt alone ( 4. ± 0.
versus 7. ± .0; p < 0.0 ). T ere was also an SVF healing e ect when compared with the spontaneous

227
Chapter 16 • Improving Skin Quality
healing group ( 5. ± 0. versus . ± . days; p < 0.05). No signif cant di erence was noted between
the SVF– and Cyt 53 –alone groups.
Table 16-1 Dif erent Healing Times or Each Specimen
Spontaneous
Wound Healing ADSCs

Cytocare 532 SVF
Healing time
(days ± SEM)
. ± .8

4.4 ± 0.5 . ± .0

5. ± 0.
T us ADSCs appear to be the most e ective treatment or healing wounds, and the cryopreservation o
ADSCs does not appear to reduce their e ectiveness.
Analysis o the overall quality o scarred areas and assessment o in ammatory reaction could not be
determined because o the excessive variations in thickness ound in di erent parts o the same section.
However, dermal papillae appeared to be more developed in the samples treated with ADSCs. T e der-
mis appeared less in amed and had a more regular and organized structure in the treated groups when
compared with the spontaneous healing group, regardless o the treatment applied.
A
B
C
D
Fig. 16-5
T e histologic structure o nontreated and treated de rosted scar tissue is shown (measurement bars
show 0 µm). Representative images were obtained a er HPS staining o scarred tissues at day 8 or
spontaneous wound healing (A), ADSC (B), Cytocare (C), and SVF (D). Dermal papillae are more de-
veloped in ADSC-treated wounds (B) and seem to be less in amed in wounds treated with ADSCs (B),
Cytocare (C), and SVF (D).
In histologic sections it can be seen that the in ammatory reaction was moderate a er treatment with
ADSCs, whereas it was still high in the spontaneous healing group and those treated with SVF. T ese
di erences were signif cant between the pooled ADSC and spontaneous healing groups (p < 0.05) and
between c.ADSC and spontaneous healing groups (p < 0.05).

228
Part III • Clinical Applications • Skin
We noted that the ADSCs induced an increase in per usion o about 0% in the wounds compared with
healthy skin, whereas this increase was minimal during spontaneous healing and absent a er treatment
with Cyt 5 . T is per usion gain was statistically signif cant or c. r.ADSC compared with Cyt 5 and
the pooled ADSC group with the Cyt 5 group. Skin elasticity tended to improve, regaining the proper-
ties o normal skin control, regardless o the treatment in question, suggesting that in this model study,
the biomechanical tests chosen were possibly insu ciently sensitive to highlight signif cant di erences
between treatments.
C oncl u si on
T e e ects o using cultured ADSCs on healing were demonstrated, with a signif cantly shortened time
to complete healing o more than days when compared with spontaneous healing. T e same signif cant
e ect, but with smaller amplitude, was observed when comparing the treatment by ADSCs to that by
SVF and vehicle alone. However, it should be noted that SVF treatment is also e ective, with a shorter
healing time o about days compared with that o the spontaneous healing group. In general, these
results conf rm the data in the literature showing accelerated healing a er administration o ADSCs.23,24
Another goal being tested in healing models is to obtain a repair that is as close as possible to the healthy
skin. In this study, we analyzed the quality o healing based on histologic and unctional criteria. All
treatments tested had a positive e ect on the morphologic aspect o the scar, including less f brous der-
mis than ound in spontaneous healing, which corroborates clinical results obtained concerning healing
time.
We observed a signif cant improvement in per usion a er injection o ADSCs compared with the vehi-
cle alone, which shows that ADSCs have a distinct role. T ese results suggest that in our wound model,
ADSCs can improve per usion by directly a ecting the unction o these vessels or indirectly through a
reduction in f brosis and acceleration o healing. In addition, preliminary results suggest that the SVF,
which contains cell ractions with well-known vasotropic and vasodilator properties (endothelial cells,
macrophages), could have a greater e ect than ADSCs on blood vessel unctionality. Finally, one o the
interesting points revealed in our study was the lack o impact o reezing/thawing on the prohealing
properties o cultured ADSCs.
C l i ni ca l C a ses: S k i n I m p rovement A f ter F a t
G ra f ti n g
As noted earlier, adipose tissue acts as an active f ller. Apart rom its volumetric properties, it can stim-
ulate production o collagen and enhance vascularization. T e skin over treated zones then undergoes
visible modif cations. Some explanations o these modif cations have been presented in the previous
parts o this chapter, mostly in animal models. As we showed, adipose tissue contains a stromal vascu-
lar raction, which counts numerous mesenchymal stem cells. T ose cells have regenerative e ect by
di erentiating into various conjunctive tissue cells and by secreting enzymes with modulating e ect on
in ammation and enhancement o vascularization. In their work on ADSC antiwrinkle e ect, Kim et
al16 showed that the positive e ect o ADSCs was mainly the result o stimulation o dermal f broblasts
that in turn diminish UVB-induced apoptosis and stimulate collagen production. T ey postulated that
ADSC could also induce production o antioxidant enzymes such as superoxide dismutase in dermal
f broblasts to inhibit apoptotic cell death.

229
Chapter 16 • Improving Skin Quality
Analysis o the outcome a er one or several repeated at gra ing procedures shows that an improvement
in the trophicity o the recipient site is achieved. T ese modif cations can be described as ollows:
• An improvement in skin texture. T is may be caused by an e ect o thickening o the dermis, a
change in skin so ness, diminished local wrinkles, or a reduced pore diameter.
• An improved suppleness o the skin and notably increased ease o gliding between subcutaneous
layers. T is can be ound in dystrophic areas, under skin gra s or artif cial dermis.
• An improvement in the skin color. T is change is noted in areas o in ammation by diminishing
in ammatory scars, or ollowing stimulation o melanin synthesis in areas o dys chromia.
Fig. 16-6
T is 8-year-old patient with Romberg disease had le hemi acial atrophy that a ected his orehead. He
is seen year a er at gra ing o 7 cc in a single procedure. Note the improvement in the quality o skin
and the increased thickness o the dermis at the injection site.
Fig. 16-7
T is 5 -year-old woman with Romberg disease had right hemi acial atrophy. T ree at gra ing proce-
dures were per ormed with respectively 5, , and 4 cc injected at the level o the right malar area, 7, 5,
and 5 cc into the right cheek, 4, 3, and 3 cc at the right mandibular border, and 5, 4, and 4 cc into the
chin. She is seen years a er her third procedure. Note the improvement o the skin around the chin
and the disappearance o wrinkles.

230
Part III • Clinical Applications • Skin
Fig. 16-8
T is 56 year-old patient requested acial rejuvenation. A cervico acial li was per ormed, in addition to
one at gra ing procedure in which 6 cc was injected at the level o each malar area, 8 cc into each cheek,
and cc into each marionette groove. T e result is seen 1 year later. Note the disappearance o jugal and
perioral f ne wrinkles in the mental region.
Fig. 16-9
This 48-year-old patient presented with malar atrophy with dyschromic patches. An upper eyelid bleph-
aroplasty was performed, followed by a single fat grafting procedure in which 8 cc was grafted into the
right malar area and 6 cc into the left, 7 cc in the right cheek and 5 cc in the left, 1.5 cc in each lower eyelid
and 2 cc in the marionette grooves. Results are seen at 1 year follow-up. Note the volume-increasing effect
of the adipose tissue, as well as the regenerating effects that have led to an improved skin quality.

231
Chapter 16 • Improving Skin Quality
Fig. 16-10
T is is a split-thickness skin graf o the interior thigh. A pinch test was done to evaluate the suppleness
and ease o gliding movement between subcutaneous layers be ore surgery. T e skin is seen 4 months
af er trans er o cc o adipose tissue; note the absence o necrosis o the skin graf and the presence o
subcutaneous tissue with the pinch test.
Fig. 16-11
T is woman had sustained peribuccal and right cervical burns, and a ull-thickness skin graf o the right
upper and lower lip was per ormed. One year later, a single at graf ing procedure was, with injections
o cc into the right cheek, cc at the right mandibular border, and 4 cc at the chin level. T e results,
shown at 0 months af er at graf ing, demonstrate an overall reduction in in amed scar tissue.

232
Part III • Clinical Applications • Skin
Fig. 16-12
T is patient had localized atrophy associated with a pericicatricial hypochromia linked to a long-
acting dermocorticoid injection into a le hypertrophic lumbar scar. Fat gra ing was per ormed, with
1 cc placed under the depressed scar tissue. T e patient is seen 4 months a er at gra ing; the subcuta-
neous atrophy has been corrected, and normal pericicatricial skin color has been restored.
In reconstructive surgery, scar tissue is f brotic and o en poorly vascularized. T is results in a resistance
to at gra ing that can lead to the impairment o adipocytes. For this reason, we in orm all patients o
the possibility that a repeat at gra ing procedure may be necessary. During the f rst procedure, the in-
troduction o growth actors and stem cells, as well as the secretion o angiogenic actors such as leptin,
enhances the local trophicity and neoangiogenesis5; all actors the later improve subsequent gra sur-
vival rate.
D i scussi on
T e e ects o at gra ing on skin regeneration and wound healing share common enzymatic pathways
and cell-to-cell communication. Adipose tissue is the main source o mesenchymal stem cells, which
can now be isolated and cultured. T ese cells have been shown to di erentiate into f broblasts and stim-
ulate collagen secretion and neovascularization. Further e ects are under study so that we may clearly
understand the complex mechanisms o wound healing and skin regeneration. Animal models have
been mostly chosen because o the di culties o studying wounds in humans. Future goals o this f eld
o science will be to develop new technologies to study wound healing and tissue regeneration with in
vitro models25-28 to reveal new data on cell communication to tissue repair and to be able to prove its
e ect in humans. Comprehension o these mechanisms could open the way to modern cell or enzymatic
therapies.

233
Chapter 16 • Improving Skin Quality
REFERENCES
. von Heimburg D, Hemmrich K, Haydarlioglu S, Staiger H, Pallua N. Comparison o viable cell yield rom
excised versus aspirated adipose tissue. Cells issues Organs 78:87, 004.
. Rigotti G, Marchi A, Galiè M, Baroni G, Benati D, Krampera M, Pasini A, Sbarbati A. Clinical treatment
o radiotherapy tissue damage by lipoaspirate transplant: a healing process mediated by adipose-derived
adult stem cells. Plast Reconstr Surg 9: 409; discussion 4 , 007.
. Coleman SR. Structural at gra ing: more than a permanent f ller. Plast Reconstr Surg 8( Suppl): 08S,
00 .
4. Nakagami H, Maeda K, Morishita R, et al. Novel autologous cell therapy in ischemic limb disease through
growth actor secretion by cultured adipose tissue-derived stromal cells. Arterioscler T romb Vasc Biol
5: 54 , 005.
5. García-Olmo D, García-Arranz M, García LG, et al. Autologous stem cell transplantation or treatment o
rectovaginal f stula in perianal Crohn’s disease: a new cell-based therapy. Int J Colorectal Dis 8:45 , 00 .
. Mojallal A, Lequeux C, Shipkov C, Breton P, Foyatier JL, Braye F, Damour O. Improvement o skin quality
a er at gra ing: clinical observation and an animal study. Plast Reconstr Surg 4:7 5, 009.
7. Rodriguez J, Boucher F, Lequeux C, Josset-Lamaugarny A, Rouyer O, Ardisson O, Rutschi H, Sigaudo-
Roussel D, Damour O, Mojallal A. Intradermal injection o human adipose-derived stem cells accelerates
skin wound healing in nude mice. Stem Cell Res T er : 4 , 0 5.
8. Mojallal A, Shipkov C, Braye F, Breton P, Foyatier JL. In uence o the recipient site on the outcomes o at
gra ing in acial reconstructive surgery. Plast Reconstr Surg 4:47 , 009.
9. Foyatier JL, Mojallal A, Voulliaume D, et al. [Clinical evaluation o structural at tissue gra (Lipostruc-
ture) in volumetric acial restoration with ace-li . About 00 cases] Ann Chir Plast Esthet 49:4 7, 004.
0. Coleman SR. Long-term survival o at transplants: controlled demonstrations. Aesthetic Plast Surg
9:4 , 995.
. Coleman SR. Facial recontouring with lipostructure. Clin Plast Surg 4: 47, 997.
. Coleman SR. Structural at gra s: the ideal f ller? Clin Plast Surg 8: , 00 .
. Lequeux C, Oni G, Wong C, Damour O, Rohrich R, Mojallal A, Brown SA. Subcutaneous at tissue engi-
neering using autologous adipose-derived stem cells seeded onto a collagen sca old. Plast Reconstr Surg
0: 08, 0 .
4. Mojallal A, Lequeux C, Auxen ans C, Braye F, Damour O. Does adipose tissue cultured with collagen
matrix and preadipocytes give comparable results to the standard technique in plastic surgery? Biomed
Mater Eng 8: 87, 008.
5. Kim JH, Jung M, Kim HS, et al. Adipose-derived stem cells as a new therapeutic modality or ageing skin.
Exp Dermatol 0: 8 , 0 .
. Kim WS, Park BS, Park SH, et al. Antiwrinkle e ect o adipose-derived stem cell: activation o dermal
f broblast by secretory actors. J Dermatol Sci 5 :9 , 009.
7. Riedel K, Ryssel H, Koellensperger E, et al. [Pathogenesis o chronic wounds] Chirurg 79:5 , 008.
8. Gimble JM, Guilak F. Di erentiation potential o adipose derived adult stem (ADAS) cells. Curr op Dev
Biol 58: 7, 00 .
9. Auxen ans C, Lequeux C, Perrusel E, Mojallal A, Kinikoglu B, Damour O. Adipose-derived stem cells
(ASCs) as a source o endothelial cells in the reconstruction o endothelialized skin equivalents. J issue
Eng Regen Med :5 , 0 .
0. Oedayrajsingh-Varma MJ, van Ham SM, Knippenberg M, et al. Adipose tissue-derived mesenchymal
stem cell yield and growth characteristics are a ected by the tissue-harvesting procedure. Cytotherapy
8: , 00 .
. Mitchell JB, McIntosh K, Zvonic S, et al. Immunophenotype o human adipose-derived cells: temporal
changes in stromal-associated and stem cell-associated markers. Stem Cells 4: 7 , 00 .
. Lequeux C, Rodriguez J, Boucher F, Rouyer O, Damour O, Mojallal A, Auxen ans C. In vitro and in vivo
biocompatibility, bioavailability and tolerance o an injectable vehicle or adipose-derived stem/stromal
cells or plastic surgery indications. J Plast Reconstr Aesthet Surg 8: 49 , 0 5.

234
Part III • Clinical Applications • Skin
. Lim JS, Yoo G. Ef ects o adipose-derived stromal cells and o their extract on wound healing in a mouse
model. J Korean Med Sci 5:7 6, 010.
. Nie C, Zhang G, Yang D, et al. argeted delivery o adipose-derived stem cells via acellular dermal matrix
enhances wound repair in diabetic rats. J issue Eng Regen Med 9: , 015.
5. Lequeux C, Auxen ans C, T épot A, Géloën A, André V, Damour O, Mojallal A. A simple way to recon-
struct a human -d hypodermis: a use ul tool or pharmacological unctionality. Skin Pharmacol Physiol
5: 7, 01 .
6. Lequeux C, Auxen ans C, Mojallal A, Sergent M, Damour O. Optimization o a culture medium or the
dif erentiation o preadipocytes into adipocytes in a monolayer. Biomed Mater Eng 19: 8 , 009.
7. Lequeux C, Oni G, Mojallal A, Damour O, Brown SA. Adipose derived stem cells: e ciency, toxicity, sta-
bility o BrdU labeling and ef ects on sel -renewal and adipose dif erentiation. Mol Cell Biochem 51:65,
011.
8. Mojallal A, Lequeux C, Shipkov C, Ri in L, Rohrich R, Duclos A, Brown S, Damour O. Stem cells, ma-
ture adipocytes, and extracellular scaf old: what does each contribute to at gra survival? Aesthetic Plast
Surg 5:1061, 011.

C h a p t e r 1 7
C
m ’ m d g g g d w d. T q
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C once p ts a n d R a ti ona l es
Box 17-1 Comparison of Microfat Injection With Coleman Lipostructure
Coleman Lipostructure
A m , v d d q w 20 y ’ x
N m
Microfat Injection
S m q
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M d
T d m f d
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Micr o at In ect ions
Wit h Disposabl e Cannul as
Guy Magalon, Pierre Sébastien Nguyen
235

236
Part III • Clinical Applications • Skin
ticles measuring approximately .5 mm and containing several hundred cells, and to get closer to deeper
skin layers without the risk o causing sur ace irregularities.
Fig. 17-1
2.0 × 130 mm
0.8 × 60 mm
0.8 × 40 mm
Microgra ing techniques were made possible with the evolution o the equipment: harvesting cannulas
are now smaller ( 4-gauge, mm, mm long), and gra ing cannulas are as well ( -gauge, .8 mm,
4 or mm long). Because o the small size o the cannulas, they are impossible to clean, so single-use
cannulas are used.
Based on the work o Yoshimura and colleagues,10 the “surviving zone” is less than microns o the at
lobule. In this zone, both adipocytes and adipose-derived stromal cells (ADSCs) survive. T is explains
the very stable results with microinjection. Pallua and colleagues11 have demonstrated that the viability
and migration o isolated ADSCs, obtained with microharvested at, were signif cantly higher and that
micro at may be more suitable due to better cell viability or tissue engineering and regenerative surgery.
Harvested micro at may be mixed with platelet-rich plasma (PRP) in proportions o % to 5 % and by
counting the number o platelets to be injected using automated analyzers with impedance or uores-
cence technique. Details about the percentage o PRP volume in the mixture and the number o platelets
per volume o mixture should be systematically provided or a better understanding. I scarring appears
to be a concern, this mixed product may be injected into the previously prepared lipoaspirate according
to the concept o percutaneous aponeurotomy and lipof lling (PALF) developed by Khouri et al.12

237
Chapter 17 • Micro at Injections With Disposable Cannulas
I n d i ca ti on s
REPARATIVE AND RECONSTRUCTIVE SURGERY INDICATIONS
In reconstructive surgery, there are several indications or microgra ing: or correction o adherent or
depressed scars, atrophy a er corticosteroid therapy, skin radiodermatitis, and in e orts to improve
thinning skin, as in the hands. T e technique also allows treatment o acial atrophy, particularly or
scleroderma o the ace. T is technique is also e ective or use inside and under burn scars.
Micro at injection is indicated or small volumes—in general, when less than 50 cc is required. In pe-
diatric surgery, micro at injection is used to treat the sequelae o nasolabial and velopalatine cle s, to
improve the volume and symmetry o the lips, and to address the posterior pharyngeal wall in patients
with incompetence o the so palate.
AESTHETIC INDICATIONS
Reduced acial volume and other signs o aging can be improved by replacing the missing tissue with
at to obtain a more youth ul appearance. Microinjections are done at the cheekbones, temples, eyelids,
nasolabial olds, marionette lines, lips, and chin.
T e injection o at into the subdermal plane will reduce acial wrinkles. T is technique can be used to
correct irregularities a er rhinoplasty. Also, injection in the back o the hands and f ngers have signif -
cant benef ts or rejuvenating the appearance o the hands.
S ur g i ca l T ech ni q ue
During the f rst consultation the surgeon per orms meticulous clinical evaluation and obtains a photo-
graphic series o the areas to be treated to def ne the places to be treated and the amount o atty tissue
that will be needed. T e procedure is usually per ormed on an outpatient basis with the use o a local an-
esthetic, supplemented with conscious sedation i needed. I necessary, the procedure can be per ormed
in the hospital setting on an overnight basis.
COLLECTION AND INFILTRATION
T e donor areas are def ned during the consultation: typically, this will be the abdomen below the navel,
the anks, the thighs, the inner aspect o the knees, the back, and so on.
T e entry point in the donor site is anesthetized with pure % lidocaine with epinephrine. A cc syringe
is used with a 0-gauge needle. Next, entry is made with a 4-gauge or mm needle, and a cannula o
the same diameter is used or collection and inf ltration. T e site is inf ltrated with % lidocaine with
epinephrine, which may be diluted with normal saline solution.
Fat is harvested with a 0 cc syringe and negative-pressure suction delivering less than 00 mm o Hg.

238
Part III • Clinical Applications • Skin
PURIFICATION
Several di erent techniques can be used or preparation o the harvested lipoaspirate:

. Centri ugation: For micro at injections we recommend 1 or 2 minutes o centri ugation at 1200 g.
T e bottom raction o the lipoaspirate in the syringe, which contains inf ltration liquid and debris,
is drained away. In our experience, there is minimal oil rom disrupted at as a top layer.

. Simple decantation o the lipoaspirate is also possible, but will require additional time.

. T e inf ltration liquid raction is discarded. T ere may be no or only minimal oily layer. Lipoaspi-
rate rom 10 cc syringes is trans erred to 1 cc syringes using a Luer-Lok connector. T e connector
is f rst placed on the 10 cc syringe and f lled with atty tissue, and then connected to the 1 cc sy-
ringe to avoid air bubbles.13 T is technique is identical to the Coleman technique, but there is less
oil with this technique and the centri ugation time is shorter.

. Filtration using a closed membrane f ltration system with f lter sizes 74 micron and 800 micron
(PureGra , Cytori T erapeutics, San Diego, CA) can be used as an alternative, with the ability to
puri y at and eliminate ree lipids and red blood cells.
GRAFTING
Entry points are anesthetized with pure % lidocaine with epinephrine, but the entire recipient site does
not need to be anesthetized. T e cutaneous barrier is punctured with a -gauge or . mm needle, ol-
lowing the same direction as the mini -gauge cannula.
Fig. 17-2
Gra ing begins with entry points at a distance rom the lesion, made with the . mm needle; these are
also the entry points or the microcannulas o the same diameter, so that atty tissue can be injected in
every direction and plane. Lef , our entry points are shown with -gauge, . mm needles. It is possible
to add other entry points. Right, at is microinjected with a -gauge, . mm, mm length cannula.
Di erent levels can be injected by crossing planes.
Fatty microislands act as gra s, remaining stable over time.
Several planes may be created and intersected, bearing in mind that microgra s measure approximately
5 microns and contain several hundred cells. T ese small parcels o at can be deposited into multiple
planes—and what is very important, can be placed superf cially, as close as possible to the dermal plane.
POSTOPERATIVE RECOVERY
Postoperative recovery is routine. Pain and swelling are very minimal, and there should be very little
bruising. Results will have stabilized within the second postoperative month. Cutaneous trophicity will
continue to improve over time. With ollow-up visits at and 6 months, the surgeon can conf rm that
the desired result has been achieved or discuss additional touchup sessions with the patient, i needed.

239
Chapter 17 • Micro at Injections With Disposable Cannulas
R esul ts
Micro at graf ing may be per ormed alone or with another cosmetic surgery procedure, such as a ace
and neck lif .
Fig. 17-3
T is 0-year-old woman presented or treatment o acial scars. T e micro at graf ing technique was
used to inject 5 cc o at subdermally. Her results are shown at 6 months postoperatively.
Fig. 17-4
T is 56-year-old woman presented with a chronic wound o the neck and radiodermatitis. In one mi-
crograf ing procedure, 0 cc o at was injected around and under the wound. Her results are shown
6 months postoperatively.

240
Part III • Clinical Applications • Skin
7 cc

7 cc
3 cc

3 cc
8 cc
7 cc
3 cc
8 cc
Fig. 17-5
T is 38-year-old woman requested a more youth ul appearance to her ace. She had a loss o volume in
her cheek bones, upper lip, and chin. In one microgra ing procedure, 8 cc was injected in these areas,
as indicated. Her results are shown 1 year postoperatively demonstrating the regenerative and volumiz-
ing ef ects o microgra ing.

241
Chapter 17 • Micro at Injections With Disposable Cannulas
Fig. 17-6
T is woman, 3 years old, had irregularities o the dorsum ollowing rhinoplasty. Microgra ing o 3 cc
on the dorsum o the nose corrected the irregularity, as shown year later.
Fig. 17-7
Microgra ing is an excellent treatment or the rejuvenation o aging hands. In this patient six points
were designed as entry points. Fat was harvested with a -gauge, 0.8 mm cannula and injected with a
cc syringe, with cc placed on the dorsum o the hand in the superf cial plane above the veins. Her
results are shown 3 months postoperatively.

242
Part III • Clinical Applications • Skin
D i scussi on
T e micro at injection technique is a technical development o Coleman. An atraumatic harvesting can-
nula o 1 -gauge, mm, with terminal openings below 1 mm and an injection cannula o 1-gauge,
0.8 mm, allows the harvesting and injection o microparticles o about 600 microns. T is material, devel-
oped or the treatment o scleroderma ace, has been used in many restorative and aesthetic indications.
T e treatment is less pain ul during injections in the subcutaneous plane, due to the small diameter o
the cannulas, and because hematoma and postoperative edema are minimized. issue viability is excel-
lent and resorption is minimal.
T e current trend is toward replacing the centri ugation process with a closed f ltration system. Multiple
ongoing studies will eventually identi y the optimal protocol or preparation o the lipoaspirate to pro-
vide viable adipose tissue or gra ing. T e micro at can also be mixed with PRP or SVF to improve the
regenerative potential.
C oncl u si on
Microinjection o autologous atty tissue is a new technique that owes its success to highly e ective can-
nulas, which also o er the advantage o being disposable. T e technique has many indications, or both
reconstructive and aesthetic surgery. T e low morbidity and quick recovery make micro at gra ing a
valuable tool or surgeons and patients alike.
A video relevant to this topic can be found in the Video Contents list on p. xxvi.
REFERENCES
1. Coleman SR. Long-term survival o at transplants: controlled demonstrations. Aesthetic Plast Surg
19: 1, 1995.
. Coleman SR. Facial recontouring with lipostructure. Clin Plast Surg :3 7, 1997.
3. Coleman SR. Structural at gra s: the ideal f ller? Clin Plast Surg 8:11, 011.
. Coleman SR. Structural at gra ing: more than a permanent f ller. Plast Reconstr Surg 118(3 Suppl):108S,
006.
5. Mojallal A, Lequeux C, Shipkov C, et al. Improvement o skin quality a er at gra ing: clinical observa-
tion and an animal study. Plast Reconstr Surg 1 :765, 009.
6. Jau ret JL, Champsaur P, Robaglia-Schlupp A, Andrac-Meyer L, Magalon G. [Arguments in avor o adi-
pocyte gra s with the S.R. Coleman technique] Ann Chir Plast Esthet 6:31, 001.
7. Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source o multipotent stem cells. Mol Biol Cell
13: 79, 00 .
8. Zuk PA. Adipose-derived stem cells in tissue regeneration: a review. ISRN Stem Cells, Article ID 713959,
013.
9. Nguyen PS, Desouches C, Gay AM, Hautier A, Magalon G. Development o micro-injection as an innova-
tive autologous at gra technique: the use o adipose tissue as dermal f ller. J Plast Reconstr Aesthet Surg
65:169 , 01 .

243
Chapter 17 • Micro at Injections With Disposable Cannulas
0. 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 9: 08 , 0 .
. Alharbi Z, Opländer C, Almakadi S, Fritz A, Vogt M, Pallua N. Conventional vs. micro- at harvesting:
how at harvesting technique a ects tissue-engineering approaches using adipose tissue-derived stem/
stromal cells. J Plast Reconstr Aesthet Surg 66: , 0 .
. Khouri RK, Smit JM, Cardoso E, Pallua N, Lantieri L, Mathijssen IM, Khouri RK Jr, Rigotti G. Percuta-
neous aponeurotomy and lipof lling: a regenerative alternative to ap reconstruction? Plast Reconstr Surg
: 80, 0 .
. 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 :8 , 0 .

(Trang không có chữ)

Scar Remodel ing Wit h Fat
Gr a t ing A t er Bur n In ur y
Marco Klinger, Francesco Klinger, Fabio Caviggioli,
Silvia Giannasi, Alessandra Veronesi, Valeria Bandi,
Barbara Banzatti, Davide Forcellini, Luca Maione,
Barbara Catania, Valeriano Vinci, Andrea Lisa,
Guido Cornegliani, Micol Giaccone
P robl ems A ssoci a ted W
i th B urn S ca rs
P s w s v v s - - b s w s f b s s -
p s b v v w v s p s
s s , , ps p b s. D sp s f s
p v s p b s, - s s s b p p v .
T s q p b s s v b s p p s s, s, -
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F b s s s , p s, p , ss x-
p ss , w s k, s, , p s b p ss v
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245
C h a p t e r 18

246
Part III • Clinical Applications • Skin
R a ti ona e f or th e U
se o f F a t G ra f ti n g
i n B urned P a ti ents
reatment o deep burn injury scarring is particularly di cult because o the presence o signif cant
tissue f brosis, retractions, and scar contractures, hypertrophic areas, and keloids. In addition, there are
changes in the normal pattern and coloring o skin resulting rom in ective and in ammatory processes
caused by toxins and proteases that alter and damage the normal wound healing process. Histologic
examination o a burned tissue sample shows the presence o f brotic tissue in the reorganization phase
with no signif cant signs o regenerative activity; cell activity seems absent, as in a necrotic process.
We decided to use the at gra ing technique to treat burn scars or the ollowing reasons:

. Fat gra ing in the ace and neck with moderate shrinking has been ound to correct cicatricial
contraction.13

. An improvement in retraction and scar contraction has been demonstrated experimentally in
wounds treated with autologous f broblasts.14

. T e use o injected lipoaspirate in patients with chronic degenerative problems resulting rom
radiotherapy has provided promising outcomes.15
T he R o e o f S tem C el
s
Regenerative medicine and tissue engineering are developing in concert with recent progress in biotech-
nologies, and there is great promise or the potential to repair damage to the mesoderm by combining
biomaterials, growth actors, and stem cells. , 7
Stem cells have the capacity to sel -replicate with long-term vitality and the potential to originate and
di erentiate into more than one cell population. Well-known stem cell sources are embryonic cells and
bone marrow–derived adult cells. T e use o embryonic stem cells has ethical and political limitations,
whereas adult cells present problems or morbidity, postharvest pain, and a limit on the number o cells
obtainable. 8 Adipose mesenchymal tissue, just like bone marrow, was recently identif ed as an ideal
source o adult stem cells. In act, recent studies show how the lipoaspirate and purif ed at according to
the Coleman protocol contains a pool o mesenchymal stem cells with di erentiation potential in more
than one cell line. T ese o er the highest percentage o adult stem cells in the human body: approxi-
mately 000/g o at compared with the 00 to 000 stem cells/mm o bone marrow. It is possible to
isolate approximately 0,000 preadipocytes rom g o at. 9

247
Chapter 18 • Scar Remodeling With Fat Gra ting A ter Burn Injury
Fig. 18-1
T is microscopic photo o a stromal cell with antivimentin/Cy3 (red) and DAPI (blue) is rom an ab-
dominal lipoaspirate sample obtained via the Coleman technique. T e original image is on the le (mag-
nif cation 00× ); on the right, it has been enhanced with image-improving so ware to show the f la-
ments organized and wrapped like a ball near the core.
reatment with mesenchymal adult stem cells is a new and promising therapy proposed or the repair
o damaged tissue. In act, bone marrow–derived or at-derived adult stem cells may be o di erent cell
phenotypes, such as bone, at, cartilage, muscle, skin, and nerve tissue. Reported experiences on the use
o bone marrow–derived adult stem cells or the treatment o limb ischemia, 0 ischemic myocardium,
retina, and incontinence 3 are encouraging.
In our experience, centri ugation using the Coleman protocol is the ideal processing method to obtain
a f nal product o purif ed and enriched at with higher regenerative properties. o conf rm this act, we
conducted a clinical trial comparing the centri uged samples obtained by Coleman’s technique with non-
centri uged samples, ocusing on cell viability, the content o adipose-derived stem cell (ADSC) popula-
tions, and the requency o clonogenic precursors. T e results o our study showed that centri ugation
produces a viable cell population signif cantly enriched in mesenchymal and endothelial progenitors,
with a higher clonogenic potential than noncentri uged samples. In addition, centri uged raction sam-
ples had a lower content o contaminants, such as oil and blood cells, which may have a relevant in u-
ence on gra take. T us centri ugation concentrates a higher number o viable cells with regenerative
potential in a smaller amount o inoculum, making this method ideal or the treatment o retracted scar
tissue.

248
Part III • Clinical Applications • Skin
S tud y D esi g n
Our experience began in December 005 when we f rst treated three patients with at gra injections. 5
o date we have treated nearly 1000 patients with structural at gra ing who had either mature or im-
mature scarring caused by second- and third-degree burns. We started with a total study population o
160 patients recruited and treated through structural at gra ing. For each we per ormed a detailed
clinical evaluation, classif ed their scars according to the Vancouver Scar Scale, 6 and per ormed a series
o routine preoperative examinations. T e study subjects then underwent at liposuction with local an-
esthesia using sterile technique.
Be ore the initial surgical procedure, punch biopsies were per ormed in the burn scarred areas as well
as in the healthy surrounding tissues as a control. T e samples were f xed in para n and colored with
hematoxylin-eosin to aid assessment o the structural eatures o the tissues, the distribution o the col-
lagen, and vascularization. Nuclear MRIs, with and without contrast agent, o the burned area was per-
ormed be ore and a er the treatment to evaluate the dermohypodermic layer o the burned areas, par-
ticularly the areas in which purif ed at containing ADSCs had been injected. 7
In some patients, the same surgical procedure was repeated a er 3 to 6 months. Be ore the second in-
tervention, an MRI was obtained. During the intervention, punch biopsies and histologic examination
were per ormed, as previously described. T ree months a erward, punch biopsies and an MR scan were
obtained; 3 to 6 months later, some patients underwent a third, ourth, or f h similar operation.
In addition to this study population, we per ormed a parallel analysis on a subgroup o 0 patients
submitted to a clinical assessment, adopting the Patient and Observer Scar Assessment Scale (POSAS)
and durometer measurements to objectively demonstrate that autologous at gra ing does improve scar
quality. We excluded patients with scars retracted above bony sur aces (such as the anterior tibia, patel-
lar region, or elbow) so the durometer results would not be distorted. T e RexGauge durometer type 00
(RexGauge, Bu alo Grove, IL) is a spring instrument or measuring skin hardness, with a 5 mm diam-
eter round noninvasive gauge head and a value range o 0 to 100. T is instrument has been widely used
to evaluate sclerodermic skin, but it has been adopted in some clinical trials as an objective evaluation
tool o scarred tissue. In these patients, the scar was virtually divided into two parts: a part o the scar
was treated using at gra ing (the study portion) and the other part o the scar was inf ltrated with saline
solution (the control portion).
T ech ni ca l G ui d el i nes
ANESTHESIA
T e area chosen or liposuction was the abdomen, because it is easy to access with the patient lying
supine and because it contains a signif cant quantity o adipose tissue in most individuals. A er the pre-
liminary cut o the skin using a No. 11 blade near the umbilicus, a Coleman blunt-tipped cannula was
inserted in the chosen area to inject 100 ml o cold physiologic solution with 10 ml o levobupivacaine
7.5 mg/ml, 0 ml o mepivacaine 10 mg/ml, and 0.5 ml epinephrine 1 mg/ml. T is inf ltration ensures
good hemostasis and postoperative analgesia.