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A pilot study on ex vivo expanded autologous adipose‐derived stem cells of improving fat retention in localized scleroderma patients

Abstract In patients with localized scleroderma (LoS), facial deformity induced by subcutaneous atrophy greatly reduces life quality. Autologous fat grafting (AFG) is used for volume restoration but with low‐fat retention due to various reasons. Adipose‐derived stem cells (ADSCs) have shown potential effects in improving fat retention. We aimed to compare the feasibility and efficacy of improving fat retention in LoS patients among the ADSCs‐assisted, the stromal vascular fraction (SVF)‐assisted and conventional AFG methods. A pilot study with a 6‐month follow‐up among 18 LoS patients was conducted. Participants were randomly assigned into three AFG groups: conventional group, SVF‐assisted group, and ADSCs‐assisted group. The SVF‐assisted group received SVF‐assisted AFG at the SVF:fat ratio of 1:1. The ADSCs‐assisted group received the mixture of ADSCs‐enriched fat graft supplemented with 5 × 105 ADSCs/mL fat. Volume retention was measured by magnetic resonance imaging, and clinical photographs were taken for outcome evaluation. At sixth‐month follow‐up, the fat retention of ADSCs‐assisted group was 49.83 ± 3.61%, significantly higher than 31.75 ± 1.73% of SVF‐assisted group (P = .0004), and 21.86 ± 1.68% of the conventional group (P < .0001). A significant difference of the fat retention was also observed between the SVF‐assisted and conventional group (P = .0346). No severe adverse events occurred during the procedure and follow‐up. This pilot study suggests that ADSCs‐assisted AFG is a safe, feasible, and attractive alternative to conventional and SVF‐assisted AFG in the correction of facial atrophy of LoS patients. Future studies with large patient samples are needed for confirmation. (Chinese Clinical Trial Registry, ChiCTR1900025717).

(P = .0346). No severe adverse events occurred during the procedure and follow-up. This pilot study suggests that ADSCs-assisted AFG is a safe, feasible, and attractive alternative to conventional and SVF-assisted AFG in the correction of facial atrophy of LoS patients.

| Study design
A prospective, randomized, and controlled pilot study with three parallel comparison groups was carried out. The study followed the declaration of Helsinki guidelines, approved by the institutional ethical committee (JMLL20180902), and registered in Chinese Clinical Trial Registry (ChiCTR1900025717). The sample size of six subjects per group was determined via a power calculation. All candidates were evaluated for suitability to be enrolled in the study by physical examination and by the criteria stated in Table 1. Informed consent was obtained from each participant at study entry after an adequate explanation including the aims, methods, process, anticipated benefits, and potential risks of this study. Then participants were randomly assigned into three groups: conventional AFG group, SVF-assisted AFG group, and ADSCs-assisted AFG group. The allocation sequence was concealed by a statistician who was unrelated to the management group of this study. The radiologist who analyzed the magnetic resonance imaging for volume assessment, and five plastic surgery experts who evaluated the clinical outcome were blinded thorough the whole study.

| Intervention and outcomes
The study was performed in LoS patients with facial atrophy (forehead and cheek). All participants received AFG to correct facial atrophy according to the randomization sequence. The conventional group received conventional AFG, and the SVF-assisted group received SVF-assisted AFG according to the SVF: fat ratio of 1:1, and

Significance statement
The present study suggests that autologous fat grafting combined with adipose-derived stem cells is a safe, feasible, and attractive alternative to conventional fat grafting or fat grafting combined with stromal vascular fraction in the correction of facial atrophy of localized scleroderma patients.

Lessons learned
• Adipose-derived stem cell assisted autologous fat grafting is a safe, feasible, and attractive alternative in the correction of facial atrophy of localized scleroderma patients.
• This technique significantly improved fat retention in localized scleroderma patients during a 6-month follow-up.
the ADSCs-assisted group received fat grafting with ex vivo enriched ADSCs. The mixture of ADSCs-enriched fat grafts was supplemented with 5 × 10 5 ADSCs/mL fat. This concentration was chosen based on the protocols of previous clinical studies of ADSC transplantation. 11,12 Magnetic resonance imaging (MRI) analysis was used to measure the facial atrophy volume of each participant preoperatively. Postoperatively and in twice follow-ups of 6 months, the fat retention of each participant was also measured by MRI. Besides, clinical photos were taken preoperatively and at follow-ups. An overview flowchart of this pilot study is shown in Figure 1. The primary endpoint was the fat retention at the sixth-month follow-up. Secondary endpoints were the assessment of safety evaluation, the feasibility, and clinical outcomes of all the AFG methods.

| Liposuction, graft preparation, and grafting
At study entry, the facial atrophy volume of each participant was mea- F I G U R E 1 An overview description of the intervention in this pilot study. ADSCs, adiposederived stem cells. AFG, autologous fat grafting; MRI, magnetic resonance imaging; SVF, stromal vascular fraction Development and Clinical Trial of Stem Cell Therapy, BZ0381). Liposuction at abdomen was performed under a low, consistent negative pressure using 50-mL syringes to limit trauma to the adipocytes.
The SVF was obtained by means of collagenase digestion. Briefly, the lipoaspirate sample was diluted with an equal volume of PBS and centrifuge at 400g for 5 minutes, three times. Then dilute the aspirated lipid fraction with an equal volume of 0.2% collagenase I solution (Sigma Chemical Co) and incubate the mixture at 37 C for 30 minutes. The suspension was centrifuged at 600g for 5 min and washed twice and filtered through sterile 100 μm cell-strainers to remove any residual enzyme. The cells were centrifuged at room temperature for 10 minutes at 600g. The supernatant was discarded and the SVF was suspended in sterile saline (Dazhong, China), thereby SVF solution was gained. Approximately 6 × 10 5 SVF cells per milliliter fat were obtained and the cell viability ranged from 85% to 95% (>70% viable) measured in Trypan blue protocols using machine (Countstar, China). Immunophenotype was analyzed by flow cytometry: positive, CD13, CD29, CD44, CD73, CD90 (>40%), CD34 (>20%); negative: CD31 (<20%), CD45 (<50%).
The isolation of ADSCs was obtained by suspending the SVF into the serum-free medium (Lonza Group AG, Switzerland). The solution was again centrifuged at 600g for 5 minutes, resuspended in the culture medium, seeded into a culture flask, and cultured at 37 C under 5% CO 2 . The culture was examined the next day, and cell adherence was confirmed under a microscope. The cells were cultured to pas-

| Statistical analysis
The sample size was calculated to be significant with 18 participants allocated equally (1:1:1) to show a significant difference in retention of 20%, with a two-sided 5% significance level and a power of 80%. Statistical analyses were conducted using GraphPad Prism 8.0.2 (GraphPad Software). The difference significance was evaluated by one-way ANOVA with the Tukey's multiple comparisons test. Significance was set to the level of P < .05. All data are presented as mean ± SEM. Exploratory data analysis was further performed using Python 3.8.3 (Python) to visualize the potential relationships between exposure and outcome variables.

| RESULTS
Eighteen participants (11 female and 7 male) were enrolled into the study and retained throughout the 6 months to study completion, whose average age was 26.68 ± 1.28 years, average BMI was 21.22 ± 0.34 kg/m 2 and average LoSSI was 3.46 ± 0.11 scores. None of age, BMI, scleroderma severity (LoSSI) or facial atrophy severity (fat graft volume) was found significantly different among the three groups (Table 2, Figure 3). No participants experienced infection, hematoma, seroma, or oil cysts. No perioperative antibiotics were used. No serious adverse events or unanticipated events occurred.
At the 3-month follow-up, the fat retention of ADSCs-assisted group was 61.25 ± 2.03%, significantly higher than 45.69 ± 4.11% of SVF-assisted group (P = .0089), and 41.26 ± 3.00% of the conventional group (P = .0012). But there was no significant difference between the conventional and SVF-assisted group (P = .5929). At sixth-month follow-up, the fat retention of ADSCs-assisted group was 49.83 ± 3.61%, significantly higher than 31.75 ± 1.73% of SVFassisted group (P = .0004), and 21.86 ± 1.68% of the conventional group (P < .0001). A significant difference of the fat retention was also observed between the SVF-assisted and conventional group (P = .0346) (Figure 3). Furthermore, the AFG method was confirmed by exploratory data analysis as the only exposure variable affecting the outcome variable (fat retention) (Supporting Information Figure S1). The expert satisfaction of the ADSCs-assisted group was 3.98 ± 0.11, significantly higher than 3.07 ± 0.18 of the SVF-assisted group (P = .0092) and 2.20 ± 0.24 of the conventional fat grafting (P < .0001) (Figures 4 and 5).

| DISCUSSION
LoS is a chronic inflammatory connective tissue disease whose specific etiological factors remain unknown. LoS manifests as various plaques of different shapes and sizes with signs of skin sclerosis and subcutaneous atrophy, whose disorder classically presents benign and self-limited evolution. 17 Medical treatment can only provide partial clinical improvement (about 60% reported) with side effects such as systemic toxicity, chronic immune suppression, and bleeding, and fails to correct the facial aesthetic impairment that has occurred. 4,18 AFG has been widely used to correct the scleroderma-induced facial atrophy. 4,19,20 In addition, basic research and clinical practice also showed that AFG has a certain therapeutic effect on scleroderma, including the improvement of skin fibrosis and the reduction of pigmentation. 4,[21][22][23] However, what has been found in clinical practice is that the fat retention in scleroderma patients was significantly lower than that in normal people (approximately 25% in scleroderma patients VS 60% in normal people). 4,19 The reason for the occurrence of low-fat retention was thought to be related to the local inflammatory microenvironment, lack of local blood supply induced by vascular injuries, F I G U R E 2 Magnetic resonance images and delineation of the subcutaneous adipose tissue. A region of interest (ROI) was precisely drawn on each axial slide by comprising pre-and post-operational scans. After ensuring that the entire targeted subcutaneous adipose tissue was included in the ROIs, the volumes were calculated automatically

T A B L E 2 Description of baseline
Conventional group (n = 6) SVF-assisted group (n = 6) ADSCs-assisted group (n = 6) P value 1 P value 2 P value 3 Notes: P value 1 , the difference between ADSCs-assisted group and conventional group. P value 2 , ADSCs-assisted group to SVF-assisted group. P value 3 , conventional and SVF-assisted group. Abbreviations: BMI, body mass index; ISFV, initial subcutaneous fat volume; LoSSI, localized scleroderma severity index. and increased apoptosis of adipocytes. 5,15,23 Therefore, improving the fat retention in scleroderma patients has become an urgent problem to be solved in clinical research. 19 Our study was the first clinical study using ADSCs-assisted fat grafting to treat facial atrophy of LoS lesion and comparing the differences in fat graft retentions between ADSCs-assisted, SVF-assisted and conventional fat grafting. The results showed that the fat graft retention of ADSCs-assisted group was significantly higher than that of SVF-assisted and conventional fat grafting groups. In our study, the ADSC-assisted group showed notable graft retention over the rest groups at an early stage and exhibited the highest level of graft retention among the three groups in the relatively long run. In comparison, the SVF-assisted group did not show a statistically significant advantage of graft retention over the conventional group after 3 months.
Still, it also presented a relatively long-term efficacy that the SVFassisted group had a higher level of fat retention than the conventional group after 6 months.
ADSCs in the adipose tissue have been considered playing a crucial role in regulating the balance between apoptosis and proliferation of adipocytes in the dynamic remodeling process of the adipose tissue after lipotransfering. 24 Previous studies have revealed that ADSCs can secrete multiple cytokines to prompt vascularization, suppress the apoptosis of adipocytes, and increase fat survival and regeneration. 24,25 ADSCs also have the advantage of immunomodulatory effects to limit inflammation and reduce fibrosis in scleroderma model. 26 ADSCs (less than 3%). 28 It is reported that a concentration of approximately 1 × 10 4 ADSCs per mL is achieved with the use of an SVF: fat ratio of 1:1 in healthy people samples, which equals a doubling in the physiological number of ADSCs in adipose tissue of healthy people. 28,29 As a method of cell-assisted lipotransfer, SVF-assisted fat grafting is regarded a timesaving technique to obtain ADSCs supplementation by collagenase isolation without extra culture. 14,29 However, to date, no existing data have convincingly demonstrated that the SVF-assisted method could stably produce comparable or increased fat graft retention compared with what can be achieved in two sessions of conventional fat grafting. 14 In our study, the fat retention in the SVF-assisted group increased 45% than that in the conventional group after 6 months. However, a method could not be considered having clinical application value until it can stably improve retention by over 1.5 times. 28 Due to the composition of the SVF, the number and the activity of ADSCs vary greatly among patients, contributing to the unstable outcome of the SVF-assisted method. Though our previous study compared the biological activity of ADSCs between LoS patients and healthy people, finding no differences in morphology or adipogenic differentiation potential among the isolated ADSC lines, the number of ADSCs in scleroderma patients decreased by 50% of that in healthy people, meaning the number of ADSCs in the scleroderma SVF is also lower than that in healthy people SVF samples. 5  Due to insufficient understanding of the physiological mechanism of stem cell in human bodies, some clinicians were concerned about the potential complications of stem cell therapy. Our preclinical vivo study has presented that mesenchymal stem cell transplantation had low tumorigenicity risk. 33 We also performed MRI scans 6 months post-operation, revealing no complications or hazards associated with There are good reasons to consider the use of such technology in future clinical trials.