The study was a parallel, single-blinded, randomized, controlled clinical trial. Based on data from previous research, with 8 patients per study arm, the statistical power was >90% (two-sided, alpha = 0.05). Assuming an attrition rate of 20% over the course of the study, we enrolled 20 patients in total (10 per group.)
Patient recruitment began in March, 2014. Eligible participants met the following inclusion criteria: 18 to 65 years old without a gender preference; an indication of deterioration in expanded skin texture, such as thin/papery skin, angiotelectasia, and dermis striatum that showed no improvement after suspending expansion for 2 weeks; and a requirement for further expansion to achieve reconstruction. All patients were assessed by two independent plastic surgeons for eligibility.
The exclusion criteria were as follows: (1) a history of severe illnesses, including any cancers, hepatitis, coronary artery disease, arteriosclerosis, diabetes, and obesity (BMI > 30); (2) presence of infection in the expansion area; (3) presence of expanded skin in a haired area; and (4) current smoking or smoking cessation for less than 6 months before enrollment.
2.3 Randomization and Masking
Each eligible patient provided written informed consent and was randomly assigned to the SVF or control group in a 1:1 ratio using a computer-generated randomization schedule. The recruiting team randomized the participants, and the conduct team managed the treatments. The evaluating investigator and data collectors were masked to the group allocation. No substantial changes to the study methodology were made after commencement of the trial.
2.4 Intervention and Tissue Expansion Protocol
The SVF was isolated as in our preclinical study(14, 15). Briefly, liposuction was performed under local anesthesia with a wet-swelling solution containing lidocaine and adrenaline, and subcutaneous fat was harvested from the abdomen or posterior inner thigh region depending on the amount of fat that was preserved. The fat was aspirated using an 18-gauge liposuction needle connected to a 20-mL syringe with low pressure. The fat was centrifuged at 600 rpm for 2 minutes to remove oil droplets and the swelling solution. Each 20-mL fat sample was incubated with 600 U of collagenase (Shanghai Qiaoyuan Biological Pharmaceutical Co, LTD, Shanghai, China) at 37 °C for 1 hour. The sample was then filtered and centrifuged at 1500 rpm for 5 minutes to obtain the pelleted SVF. The SVF pellet was suspended with sterile saline twice, and the cell concentration was adjusted to 1*106 cells/ml in 2-mL syringes. The skin was pinched to facilitate the intradermal injection of the SVF suspension via a 30-G needle, with approximately 0.1 ml injected at a 1-cm interval (1 × 105 cells/cm2) (Figure 2a,b). The control group was injected with an equal volume of sterile saline in the same manner.
The expander was inflated every 3 days, and a pressure meter was used to monitor the inflation pressure; inflation was discontinued when the inflation pressure reached 100 mmHg. This procedure was repeated until sufficient tissue was obtained or the maximal regenerative assessment was achieved to perform skin flap transfer surgery.
2.5 Assessment and Endpoint
Outcomes were assessed at baseline (immediately before treatment) and at 4 weeks, 8 weeks, and 12 weeks after the first treatment. The primary endpoint was the change in skin thickness at 12 weeks posttreatment. The secondary endpoints were the changes in the skin thickness including the epidermal and dermal thicknesses, the expansion index (EI), and the subjective scores of skin texture at 4 weeks and 8 weeks during the follow-up period. Safety assessments were administered at 2 years.
2.5.1 Expanded Skin Thickness
Skin thickness was measured at eight evenly spaced measurement points along the longitudinal axis using a duplex ultrasonic scanner(26). In ultrasound images, skin appears as a well-defined, linear echogenic band between the air-epidermal band and the low echo-level hypodermal band. The epidermal thickness was defined as the distance between the most superficial, clearly visual high echo-level band, and the dermal thickness was defined as the distance between the moderate-echogenic band underneath the epidermal band.
2.5.2 Expansion Index (EI)
The inflated volumes at each visit were recorded and summated. Due to the maximum capacity of the patient's expanders, we used the EI to measure the efficiency of the inflation process; the EI was described as the ratio of the total inflated volume (ml) to the designed volume expander (ml).
2.5.3 Subjective Scores of Expanded Skin Texture
The investigator-assessed subjective scores of expanded skin texture were as follows: 3 indicated that the skin texture was significantly improved and that the optimal desired result was obtained; 2 indicated that the skin texture was significantly improved, but further treatments were required; 1 indicated that the skin texture was slightly improved; 0 indicated that the skin texture was the same as that before treatment; and -1 indicated that the skin texture was worse than that before treatment. The same blinded investigator assessed all scores.
2.5.4 Histological Examination
Expanded skin specimens were harvested during flap transfer surgery. Specimens were fixed with formalin, embedded in paraffin, and sectioned at a thickness of 4-μm. Hematoxylin and eosin (HE) staining and Masson staining were performed to assess the histologic differences. Collagen synthesis was evaluated using the collagen volume fraction (CVF). Immunohistochemistry with proliferating cell nuclear antigen (PCNA) was used to evaluate the role of SVF in promoting skin cell proliferation, and the vascular endothelial cells were identified as the surrounding vessels using an anti-CD31 antibody to evaluate angiogenesis in the skin. The number of positive cells was calculated using a laser confocal microscope (Leica, Wetzlar, Germany) in 5 high-power random fields (HPFS, 400X).
During the 2-year follow-up period, the following factors were evaluated to identify adverse reactions: redness, swelling, pain, infectious symptoms and symptoms related to the presence of a subcutaneous protuberance, mass, induration, or hyperplasia.
2.6 Study Oversight
This study was granted ethical approval from the Institutional Ethics Committee at the Shanghai Ninth People's Hospital in accordance with the principles of the Declaration of Helsinki. All treatments and follow-up visits were performed at the Department of Plastic and Reconstructive Surgery of Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine from March 2014 to December 2018. This trial was registered with Chinese Clinical Trial, ChiCTR2000039317 (registered 23 Oct 2020 - retrospectively registered, http://www.chictr.org.cn/showproj.aspx?proj=62738).
2.7 Statistical Analysis
GraphPad Prism 7 (GraphPad Software Inc., San Diego, CA, USA) was used for the statistical analysis. Continuous data were expressed as the mean ± standard deviation (SD) with a 95% confidence interval (CI), and categorical data were represented as counts. The between-group differences at each visit were performed using two-way repeated-measures ANOVA followed by Sidak’s multiple comparison tests, and the within-group differences of each follow-up visit compared to baseline were analyzed using the paired t-test. The EI increment between groups was assessed using the unpaired t-test. A value of P < 0.05 indicated that the difference was statistically significant (*p<0.05, **p<0.01).