1

Title of the article:
Cell-assisted lipotransfer for facial lipoatrophy: efficacy of clinical use of
adipose-derived stem cells
Kotaro Yoshimura, M.D.1, Katsujiro Sato, M.D.2, Noriyuki Aoi, M.D.1, Masakazu
Kurita, M.D.3, Keita Inoue, M.D.1, Hirotaka Suga, M.D.1, Hitomi Eto, M.D.1,
Harunosuke Kato, M.D.1, Toshitsugu Hirohi, M.D.4, and Kiyonori Harii, M.D.3
Affiliations:
Department of Plastic Surgery, University of Tokyo School of Medicine;
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan1

2

Cellport Clinic Yokohama;
Yokohama Excellent III Building 2F, 3-35, Minami-nakadori, Naka-ku, Yokohama,
Kanagawa 231-0006, Japan3

Department of Plastic Surgery, Kyorin University School of Medicine;
6-20-2 Shinkawa, Mitaka-shi, Tokyo 181-8611, Japan
4
Ritz Cosmetic Surgery Clinic Tokyo;
Meguro Toho Building 8F, 3-1-7, Kamiosaki; Shinagawa-ku, Tokyo 141-0021,
Japan
Corresponding author:
Kotaro Yoshimura, M.D.
Department of Plastic Surgery, University of Tokyo School of Medicine;
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
Telephone: +81-3-5800-8948; Fax: +81-3-5800-9186
E-mail: yoshimura-pla@h.u-tokyo.ac.jp
Category of article: Original article
Keywords: adipose-derived stem/stromal cells, lipoinjection, cell transplantation,
lipoatrophy, lupus erythematosus profundus, Parry-Romberg syndrome
Conflict of Interest Disclosure: None declared.
No funding sources for this work.

2

Short Title:

Cell-Assisted Lipotransfer

3

Abstract
Background: Lipoinjection is a promising treatment, but its efficacy inrecontouring facial lipoatrophy remains to be established.
Objective: To evaluate the efficacy and adverse effects of lipoinjection andsupplementation of adipose-derived stem/stromal cells (ASCs) to adipose grafts.
Methods: To overcome drawbacks of autologous lipoinjection, we havedeveloped a novel strategy called cell-assisted lipotransfer (CAL). In CAL,
stromal vascular fraction containing ASCs was freshly isolated from half of anaspirated fat sample, and attached to the other half of aspirated fat sample withthe fat acting as a scaffold. This process converts relatively ASC-poor
aspirated fat into ASC-rich fat. We performed conventional lipoinjection(non-CAL; n = 3) or CAL (n = 3) on six patients with facial lipoatrophy due tolupus profundus or Parry-Romberg syndrome.
Results: All patients obtained improvement in facial contour, but the CAL group
had a better clinical improvement score than did the non-CAL patients, although
the difference did not reach statistical significance (p = 0.11). Adipose necrosis
was found in one non-CAL case who took perioperative oral corticosteroids.
Conclusion: Our results suggest that CAL is both effective and safe, andpotentially superior to conventional lipoinjection for facial recontouring.

4

Introduction
Facial lipoatrophy is a disfiguring and socially disabling problem that
accompanies several inherited and acquired diseases (see ref #1 for review).
Lupus erythematosus profundus and scleroderma ‘en coup de sabre’ (en coupde saber morphea) frequently give rise to facial lipoatrophy, which is often themost problematic manifestation of the disease for the patients even though theyrequire medication, such as oral corticosteroids, to suppress other symptoms
associated with the systemic morbidities. Facial lipoatrophy is also seen inpatients with HIV infection, and Parry-Romberg syndrome (hemifacial
progressive atrophy or idiopathic hemifacial atrophy) involves not only facial
lipoatrophy but also bony defects (2). Thus far there is no medical treatment tocorrect facial lipoatrophy.
Lipoinjection has been used for treating lipoatrophy such as thatassociated with HIV infection with moderate success (3, 4); while microsurgicaltissue transfer has been a standard surgical treatment for recontouring facial
defects seen in Parry-Romberg syndrome. However, the surgical procedure
leaves conspicuous scars on the face and the donor site (5, 6). Autologous
lipoinjection is a promising treatment for soft-tissue augmentation because there
is no associated incision scar or complications associated with foreign materials.
Although many innovative efforts to refine autologous lipoinjection have beenreported (7-14), problems such as unpredictability and a low rate of graft survival
due to partial necrosis remain. To overcome these problems, we havedeveloped a novel strategy, called cell-assisted lipotransfer (CAL; Figure. 1).
Since a fraction of stromal cells derived from the adipose tissue wasfound to have the capacity to differentiate into various cell lineages (15), thestromal progenitor cells have been designated “adipose-derived stem/stromalcells” (ASCs), and are expected to become a valuable tool in a wide range ofcell-based therapies. The therapeutic concept of CAL was described in aprevious report of our pre-clinical studies (16). In CAL, ASC-poor fat isconverted to ASC-rich fat by supplementing with cells freshly isolated from theadipose tissue during preparation of the injectable material (Figure 1). We
report the preliminary clinical results of patients who underwent CAL forrecontouring of acquired facial lipoatrophy. This is the first report of the clinicaluse of ASCs for soft tissue augmentation of the face.
Materials and Methods

5

Patients
We analyzed the outcomes of six patients with facial lipoatrophy who
underwent lipoinjection for recontouring facial defects: three patients underwent
conventional lipoinjection (non-CAL), while three patients underwent CAL. No
patient underwent more than one CAL or non-CAL procedure. Informed
consent was obtained from all patients. The clinical trial protocol conformed to
the guidelines of the 1975 Declaration of Helsinki and was approved byindividual institutional review boards.
One of the six patients had been diagnosed with Parry-Romberg
syndrome; the other five patients had been diagnosed with lupus erythematosus
profundus by their dermatologists. Two of the lupus cases (one in the non-CAL
group, and one in the CAL group) were taking oral steroids (predonisolone) at
the time of surgery. Severity grading of facial lipoatrophy was determinedaccording to the grading scale (graded 1 to 5, 5 being the most severe)developed by the Facial Lipoatrophy Panel (1). Patients were evaluatedbetween 9 and 13 months as described later. One of the three patients in eachgroup was male, and the patient’s ages ranged from 33 to 55 years (mean age ±SD, non-CAL: 46.3 ± 7.8, CAL: 38.7 ± 8.1). The mean volume of injected fat
was 133 ± 104 ml in non-CAL group, and 100 ± 10 ml in CAL group. Patient
data are summarized in Table 1.
Surgical Techniques
Before suctioning, the abdominal wall was infiltrated with saline solution
with diluted epinephrine (0.001%) with the patient under general anesthesia.
Adipose tissue was suctioned using a cannula with 2.5-mm inner diameter and aconventional liposuction machine. In the CAL group, about a half of thecollected liposuction aspirate was used for isolation of the SVF. The SVF was
isolated from both the adipose portion and the fluid portion of liposuction
aspirates as previously described (17). In brief, the adipose portion of liposuction
aspirates was digested with 0.075% collagenase in buffered saline for 30 min ona shaker at 37°C. Mature adipocytes and connective tissue were separated
from the SVF containing ASCs by centrifugation (800×g, 10 min), and thenrinsed three times with buffered saline. The fluid portion was centrifuged(800×g, 5 min), and the pellets were re-suspended in hypotonic water to lyse
erythrocytes. The cell processing procedure took about 90 minutes. During

6

the processing period, the other half of lipoaspirates was harvested as graft
material. The surgery was performed in a sterile operating room and the cellisolation was performed in a sterile cell processing room.
The adipose portion of liposuction aspirates was centrifuged at 700×g for
3 minutes without washing, and put into a metal jar (500 ml) that was placed inwater with crushed ice. In the non-CAL group, centrifuged fat was injectedwithout SVF supplementation. In CAL group, the fresh SVF isolated from boththe adipose and fluid portion was added to the graft material and, after gentle
mixing and waiting for 10-15 minutes for cell adherence to the aspirated fat, thecell-supplemented fat was then put into an injection syringe.
For the injection syringe, a 10 cc LeVeen™ inflator (Boston Scientific
Corp., MA) was used because they are screw-type syringes (with a threaded
plunger) and threaded connections that fit both the connecting tube and theneedle, to allow for precise control during injection. An 18-gauge needle (25 or
60 mm long) was used for lipoinjection and inserted in several layers (thesubcutaneous fatty layer and the muscle layer) and directions to achieve diffuse
distribution of the graft materials. Quantity of transplanted adipose tissue wasdetermined by trying to overcorrect by about 20%.
Evaluation of clinical improvement
Photographs of each patient were taken before and after treatment witha high-resolution digital camera (Canon D30, Tokyo, Japan). The percentage
of volumetric improvement of facial defects was determined via evaluation ofpreoperative and postoperative photographs by four certified plastic surgeons
blinded to clinical data. The mean data of the volumetric improvement of eachpatient were classified into four categories: excellent (80% improvement or
better), good (60% to less than 80% improvement), fair (40% to less than 60%
improvement), and poor (less than 40% improvement).
Results
The transplantation of adipose tissue was successfully performed in allcases. Subcutaneous bleeding was seen on some parts of the face, andresolved within one to two weeks.
Patients usually recovered frompost-operative swelling at about 4 weeks. Transplanted adipose tissue was
gradually absorbed during the first two postoperative months. The volumecontinued to be reduced in the non-CAL group, while it showed a minimal

7change thereafter in the CAL group. Patient data are summarized in Table 1,
and photographs of two representative cases in each group are shown in
Figures 2 to 5.
All patients showed cosmetic improvements, but the degree variedamong patients. CAL had a better clinical improvement score than non-CAL,
although the difference did not reach statistical significance (p=0.11).
The reconstructed tissue was generally soft and with natural texture,
although some fibrous tissues that had existed before surgery appeared toremain. In one case of the non-CAL group (non-CAL case #2; Figure 3),
redness and fluctuation was detected on the left cheek one month after surgery
and necrotized adipose fluids were drained through a small incision. We cultured
this fluid, but did not detect any bacteria. In other cases, no adverse effects
were seen and any nodules or cysts were not palpable.

8Discussion
In the CAL strategy, autologous ASCs are used to enhance
angiogenesis, improve the survival rate of grafts, and reduce post-operative
atrophy. In CAL, half the volume of the aspirated fat is processed for isolationof the SVF containing ASCs (Figure 1). During the isolation process, the otherhalf of the aspirated fat is prepared for grafting. Freshly isolated SVF, whichcontains ASCs as well vascular endothelial cells, pericytes, blood cells (WBCsand RBCs), and other cells as previously described (17), is attached to theaspirated fat with the fat acting as a living scaffold before transplantation.
Finally, the SVF-supplemented fat is injected into the target sites.
Using our technique, aspirated fat was harvested with a relatively
large-sized suction cannula, centrifuged at 700×g, and kept cool until
transplantation. We believe that centrifugation of aspirated fat substantiallyaffects survival rate of injected fat because centrifugation at 1,200×g
experimentally decreases the fat volume by 30%, damages 12% of theadipocytes and 0% of the ASCs, which concentrates the number of cells pervolume by 25% for adipocytes and 43% for ASCs (18). We prefer an 18- or
16-gauge sharp-tipped needle for fat injection because we have found that a
sharp needle facilitates placing fat grafts more diffusively and with higher
accuracy, even into fibrous or relatively hard tissue and into the muscle througha fascia, as compared to a blunt tipped needle.
Adipose tissue contains not only adipose progenitor cells but also
multipotent stem cells that can differentiate into fat, bone, cartilage, and othertypes of tissue (15, 19). Previously, we found that aspirated fat loses a
significant number of these precursor cells during liposuction with a suction
machine and the preparation processes, and that aspirated fat has
approximately half the number of ASCs as excised whole fat does (16). Thereseems to be two main reasons for the relative deficiency of ASCs. Firstly, amajor portion of the ASCs are located around large vessels and are left at thedonor site after liposuction (16); we have confirmed this phenomenon using
histological analysis (in preparation). Secondly, some ASCs are released into
the fluid portion of liposuction aspirates (17). The resulting relative deficiency
of precursor cells in aspirated fat may contribute to the low survival rate andlong-term atrophy of transplanted lipoaspirates.
In CAL, we compensate for this ASC deficit by supplementing theaspirated fat intended for lipoinjection with freshly-isolated SVF containing ASCs.

9

In order to maximize the biological function and avoid unexpected behavior ofASCs, we believe that it is important to ensure adherence of supplemented
ASCs to adipocytes or connective tissue (20). Centrifugation of adipose grafts
seems to be important also for reducing water content in the graft, which may
disturb the adherence of supplemented ASCs to the adipose tissue.
Preclinical studies have partly confirmed some potential mechanistic
benefits for using ASCs in CAL (16, 21, 22). ASCs can differentiate into
adipocytes and contribute to the regeneration of adipose tissue. In addition,ASCs can differentiate into endothelial cells and also probably into vascularmural cells (16, 23, 24), resulting in the promotion of angiogenesis and graft
survival. ASCs are known to release angiogenic growth factors in response tohypoxia and other conditions (25, 26), and these factors may promoteangiogenesis from surrounding host tissue. The most influential role weconsidered is that ASCs remain as original undifferentiated ASCs in surviving
adipose tissue (16). ASCs are assigned to participate in tissue turnover ofadipose, and the turnover is known to be very slow, taking two years or more(27). However, transplanted adipose tissue experiences temporary ischemia
followed by reperfusion, and so the adipose grafts probably turn over in the earlystage (up to three months) after transplantation. We hypothesize that therelative deficiency of ASCs in aspirated fat may affect the replacement process
and lead to post-operative atrophy of grafted fat, which commonly occurs during
the first 6 months after lipoinjection.
In our series, we observed no adverse side effects or complications
except for one case of lupus erythematosus profundus in the non-CAL group
who took oral corticosteroids preoperatively. The patient showed necrosis of apart of the transplanted adipose tissue and the necrotic fluids were drained. A
patient in CAL group who had taken oral corticosteroids showed a lower clinicalscore compared to non-steroid-taking patients, suggesting that steroids taken
perioperatively, or possibly the activity of lupus requiring the medication, has
negative effects on angiogenesis and graft survival.
Thus, it may berecommended that lipoinjection, either non-CAL or CAL, is performed at a periodof minimal lupus activity and after corticosteroid therapy has been discontinued.
There were notable differences between the two groups in the ages ofthe patients and in the injection volumes in this preliminary trial. Nonetheless,the data suggest that lipoinjection is an effective tool for recontouring facial
lipoatrophy, and ASC supplementation is suggested to boost effectiveness.

10

Our clinical experience using CAL for cosmetic breast augmentation with one tofour years of follow-up also suggests that CAL is safe and may be superior toconventional lipoinjection (20). We believe that CAL can improve disfigurement
and social disability of patients with facial lipoatrophy and that it has someadvantages over conventional surgical methods: lack of facial and donor site
scarring, lack of complications derived from foreign materials, and applicability to
any shape of facial defects. Larger studies and longer follow-up times areneeded to establish the superiority and the durability of the CAL-mediatedimprovements in facial lipoatrophy patients.

11

References
1. Ascher B, Coleman S, Alster T, et al. Full scope of effect of facial lipoatrophy:
a framework of disease understanding. Dermatol Surg 2006; 32:1058-69.
2. Sommer A, Gambichler T, Bacharach-Buhles M, et al. Clinical and serologicalcharacteristics of progressive facial hemiatrophy: a case series of 12 patients.J
Am Acad Dermatol 2006; 54:227-33.
3. Burnouf M, Buffet M, Schwarzinger M, et al. Evaluation of Colemanlipostructure for treatment of facial lipoatrophy in patients with humanimmunodeficiency virus and parameters associated with the efficiency of thistechnique. Arch Dermatol 2005; 141:1220-4.
4. Domergue S, Psomas C, Yachouh J, et al. Fat microinfiltration autografting for
facial restructuring in HIV patients. J Craniomaxillofac Surg 2006; 34: 484-8.
5. Longaker MT, Siebert JW. Microvascular free-flap correction of severe
hemifacial atrophy. Plast Reconstr Surg 1995; 96: 800-9.
6. Vaienti L, Soresina M, Menozzi A. Parascapular free flap and fat grafts:
combined surgical methods in morphological restoration of hemifacial
progressive atrophy. Plast Reconstr Surg 2005; 116: 699-711.
7. Carpaneda CA, Ribeiro MT. Percentage of graft viability versus injectedvolume in adipose autotransplants. Aesthetic Plast Surg 1994; 18: 17-9.
8. Ersek RA, Chang P, Salisbury MA. Lipo layering of autologous fat: an
improved technique with promising results. Plast Reconstr Surg 1998; 101:820-6.
9. Ullmann Y, Hyams M, Ramon Y, et al. Enhancing the survival of aspirated
human fat injected into nude mice. Plast Reconstr Surg 1998; 101: 1940-4.
10. Fulton JE, Suarez M, Silverton K, et al. Small volume fat transfer. Dermatol
Surg 1998; 24: 857-65.
11. Sommer B, Sattler G. Current concepts of fat graft survival: histology ofaspirated adipose tissue and review of the literature. Dermatol Surg 2000; 26:1159-66.
12. Shiffman MA, Mirrafati S. Fat transfer techniques: the effect of harvest andtransfer methods on adipocyte viability and review of the literature. Dermatol
Surg 2001; 27: 819-26.
13. Coleman SR. Structural fat grafts: the ideal filler? Clin Plast Surg 2001; 28:
111-9.
14. Butterwick KJ. Lipoaugmentation for aging hands: a comparison of the12longevity and aesthetic results of centrifuged versus noncentrifuged fat.
Dermatol Surg 2002; 28: 987-91.
15. Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source ofmultipotent stem cells. Mol Biol Cell 2002; 13: 4279-95.
16. Matsumoto D, Sato K, Gonda K, et al. Cell-assisted lipotransfer: supportive
use of human adipose-derived cells for soft tissue augmentation withlipoinjection. Tissue Eng 2006; 12: 3375-82.
17. Yoshimura K, Shigeura T, Matsumoto D, et al. Characterization of freshly
isolated and cultured cells derived from the fatty and fluid portions of liposuction
aspirates. J Cell Physiol 2006; 208: 64-76.
18. Kurita M, Matsumoto D, Shigeura T, et al. Influences of centrifugation oncells and tissues in liposuction aspirates: optimized centrifugation for lipotransfer
and cell isolation. Plast Reconstr Surg, in press.
19. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose
tissue: implications for cell-based therapies. Tissue Eng 2001; 7: 211-28.
20.Yoshimura K, Sato K, Aoi N, Kurita M, Hirohi T, Harii K. Cell-Assisted
Lipotransfer (CAL) for Cosmetic Breast Augmentation -Supportive Use of
Adipose-derived Stem/Stromal Cells-. Aesthtic Plast Surg, in press.
21. Masuda T, Furue M, Matsuda T. Novel strategy for soft tissue augmentation
based on transplantation of fragmented omentum and preadipocytes. Tissue
Eng 2004; 10: 1672-83.
22. Moseley TA, Zhu M, Hedrick MH. Adipose-derived stem and progenitor cells
as fillers in plastic and reconstructive surgery. Plast Reconstr Surg 2006; 118(3Suppl):121S-8S.
23. Miranville A, Heeschen C, Sengenes C, Curat CA, Busse R, Bouloumie A(2004) Improvement of postnatal neovascularization by human adipose
tissue-derived stem cells. Circulation 110: 349-355
24. Planat-Benard V, Silvestre JS, Cousin B, et al. Plasticity of human adipose
lineage cells toward endothelial cells: physiological and therapeutic perspectives.
Circulation 2004; 109: 656-63.
25. Rehman J, Traktuev D, Li J, Merfeld-Clauss S, Temm-Grove CJ, Bovenkerk
JE, Pell CL, Johnstone BH, Considine RV, March KL (2004) Secretion ofangiogenic and antiapoptotic factors by human adipose stromal cells. Circulation
109:1292-1298
26. Kilroy GE, Foster SJ, Wu X, et al. Cytokine profile of human adipose-derived
stem cells: Expression of angiogenic, hematopoietic, and pro-inflammatory

13

factors. J Cell Physiol 2007; May 3, Epub ahead of print.
27. Strawford A, Antelo F, Christiansen M, Hellerstein MK. Adipose tissue
triglyceride turnover, de novo lipogenesis, and cell proliferation in humans
measured with 2H2O. Am J Physiol Endocrinol Metab 2004; 286, E577-88.

14

Table 1.
Summary of patients’ data

Non-CAL

Method

CAL

Case

1

2

3

1

2

3

Sex

Male

FemaleFemaleMale

FemaleFemaleAge [year]

25

42

42

35

33

48

Diagnosis

LEP

LEP

LEP

PRS

LEP

LEP

Severity
grading

3

5

2

4

5

4

Perioperative
oral
corticosteroids

–

+

–

–

+

–

Donor site

abdomenabdomenabdomenabdomenabdomenabdomen

100

250

50

110

90

100

10

9

９

13

9

9

Clinical results

Fair

Good

Fair

Excellent

Good

Good

Complications–

Adiposenecrosis

–

–

–

–

Volume ofinjection [ml]Follow-up
periods[month]

LEP: Lupus erythematosus profundus
PRS: Parry-Romberg syndrome15

Legends

Figure 1. Scheme of cell-assisted lipotransfer (CAL). Half of the volume of
the aspirated fat is processed for isolation of the stromal vascular fraction (SVF).
During the isolation process the other half of the aspirated fat is prepared forgrafting. Freshly isolated SVF containing ASCs are attached to the aspirated
fat with the fat acting as a living scaffold. Finally, the SVF-supplemented fat is
injected into the target sites. Thus, aspirated fat, which is originally relatively
poor in ASCs, is converted to ASC-rich fat.

16

Figure 2. Clinical views of non-CAL case #1 (a 25 year-old man); Preoperative
views (Grade 3 lipoatrophy) (A), and postoperative views at one month (B) andat ten months (C). The patient was diagnosed as LEP at the age of 21 years
and started oral corticosteroid therapy. He stopped the medication one year
ago and has experienced significant change in the last 12 months. Conventional
lipoinjection (100 ml) was performed to recontour the left cheek defect, which
was successfully augmented at one month (B), but substantial absorption wasseen at ten months (C).

17

Figure 3. Clinical views of non-CAL case #2 (a 42 year-old woman);
Preoperative views (Grade 5 lipoatrophy) (A), and postoperative views at 9months (B). The patient has been taking oral corticosteroid since she wasdiagnosed with LEP at the age of 20 years. Conventional lipoinjection (250 ml)
was performed to augment facial defects on both cheeks, and she continued the
medication perioperatively. Redness and fluctuation was seen on the left cheekat one month, and necrotic adipose fluid was removed by a small skin incision.
However, the facial defect was relatively improved at nine months (B).

18

Figure 4. Clinical views of CAL case #1 (a 35 years old man); Preoperative
views (Grade 4 lipoatrophy) (A), and postoperative views at 13 months (B).
The patient noticed the right facial defect during his 20’s, was diagnosed with
PRS, and has no history of oral corticosteroid use. CAL (110 ml) was
performed to correct the facial defect, which was improved and the facial contour
maintained at 13 months follow up (B). The cheek is soft and natural appearing
with no visible scars.

19

Figure 5. Clinical views of CAL case #3 (a 48 years old woman); Preoperative
views (Grade 4 lipoatrophy) (A), and postoperative views at 13 months (B).
The patient noticed the right facial defect during her 20’s and diagnosed with
LEP. She has a history of oral corticosteroid use, but has not taken them forseveral years. CAL (100 ml) was performed to correct the facial defect, which
was improved at nine months follow up (B).