In this study, 9 patients with rolling acne scars were enrolled. Lesions were graded and classified by a board-certified dermatologist. Virus tests was done for all patients before treatment. Exclusion criteria were as follow: the existence of any active infectious diseases, patients who had received any procedure for acne scar treatment, patients with history of coagulopathy, skin cancer, oral steroid therapy, anticoagulant therapy, patients with systemic retinoid intake in the previous six months, patients who were pregnant or lactating were excluded from the study.
Procedures and treatment monitoring
Liposuction surgical technique
Taking into attention the individual characteristics of each patient, donor areas were selected mostly from hip, the lower abdomen, and thigh before preparing the appropriate design. On the day of surgery, lipoaspiration and injection of adipose was performed. In either general anesthesia or twilight anesthesia, anesthesia was induced by tumescent fluid (500 mL normal saline with 15 ml of 2% lidocaine [Pasteur institute of Iran] and 7 mL of bicarbonate and 1 mg of epinephrine). After that, fat tissue was gathered with a Luer Lock syringe by utilizing a 2.4 mm cannula (Figure 1). The volume of fat that was collected from each patient was about 100 ml. The aspirated fat tissue was divided into two aliquots (50 ml). 50 ml of lipoaspirated directly was grafted in the face and 50 ml was used for the SVF cells isolation.
SVF cells isolation
To remove most red blood cells and leukocytes, the tissue was washed with phosphate-buffered saline (PBS) (Miltenyi Biotech, Cologne, Germany), then digested with type I collagenase (Worthington Biochemical Corp., Lakewood, USA) at 37 ° C, 20 minutes under unbroken shaking. Solution of collagenase, final concentration of 0.2%, was made just before use by adding collagenase powder to the Hanks balanced salt solution (HBSS) (Invitrogen, Carlsbad, USA). Digestion was prevented by washing with PBS (3 times). Floating and lysed adipocytes were waste and cells of the SVF were pelleted by 10 min centrifugation at 500 g. The pellet was suspended in the HBSS and an erythrocyte lysis buffer (Sigma-Aldrich Corp, St. Louis, USA) was added and incubated 10 minutes at 37°C. This cell suspension was centrifuged (500 g, 5 min), and cells were counted and viability of cells were evaluated using Automatic cell counter (TC20TM Automatic Cell Counter, Bio-Rad). Some of the suspension was subjected to flow cytometry (Partec, Görlitz, Germany) for evaluating cell surface markers and cell viability.
Platelet‐rich plasma preparation
A sterile tube containing 1.5 mL anticoagulant (ACDA1) was arranged. Then, 10 mL of whole blood was drawn from each patient and collected in the sterile tube including 1 mL anticoagulant. In the next step the tube was centrifuged. The centrifugation process separates components of blood through their diverse specific gravities as follow; RBCs is the heaviest, after that WBCs, whereas platelets and plasma are the lightest. Centrifugation was performed in two‐step; first step was a low level centrifugation to distinct whole plasma from platelets and red cells (at 900 RPM, 10-12 minutes). Then, all the supernatant plasma was aspirated by sterile angiocatheter and transferred from the tube in a sterile pure tube. The second step was high level centrifugation (2400 RPM, 8 minutes) to separated platelet‐poor plasma from platelet‐rich pellet; about 2/3 of the supernatant platelet‐poor plasma were wasted and the platelet‐rich pellet was re‐suspended in the remaining volume of plasma. The count of platelet in the developed PRP was expected to be 3 to 4‐fold more than in the whole blood. The obtained PRP was aspirated, and in order to platelets activation, 0.1 mL of calcium chloride was added per 0.9 mL of resulting PRP. The PRP of each patient was used to re-suspend the pellet of SVF for transplantation.
Transplantation of aspirated fat tissueand SVF cells
A topical anesthetic cream (EMLA containing 2.5%lidocaine HCL and 2.5 % prilocaine) was used for 45 minutes and afterward the area was sterilized via alcohol 70% and washed through normal saline to get finally dry skin surface. The aspirated fat tissue was injected (cannula with an 18-gauge blunt needle) subcutaneously into the skin of the face. Immediately, after aspirated fat tissue injection, the prepared SVF cells was re-suspend in the PRP and injected (1 mL syringe with a 30-gauge blunt needle) intradermal at the dose of 2×107 total for whole face of each patient.
SVF cells outcome
The SVF cells counted by automatic cell counter. Also The SVF cells viability was evaluated by automatic cell counter. SVF cells surface markers and cell viability were evaluated by flow cytometry.
Before and 6 months after treatment, Visioface (D1000 ck, Cologne, Germany) photographs of each patient were taken by a high-resolution digital camera. Biometric characteristics of the patients was evaluated by Visioface 1000 D, Colorimeter, multi probe adapter Cutometer (L parameter ), Tewameter, Mexameter, (all purchased from Courage + Khazaka Electronics, Cologne, Germany) and a skin ultrasound imaging system (TPM, Luneburg, Germany) to calculate the skin layers’ thickness, the skin lightness, the skin elasticity by providing three parameters (R2, R5, R7), trans epidermal water loss (TEWL) and melanin content and the intensity of skin erythema before and after aspirated fat tissue and SVF cells transplantation, respectively. When the transplantation procedure was performed and the patients were followed up for 6 months.
The participants were requested to scale their subjective satisfaction of the treatment on a quartile grading scale (grade 1 = slightly satisfied, grade 2 = moderately satisfied, grade 3 = well satisfied).
After testing for normal distribution of data by Kolmogorov-Smirnov test, the data were analyzed using paired-samples t-test through SPSS 14.0 (SPSS Inc, Chicago, Illinois, the United States). In all tests, P value < 0.05 was considered statistically significant.