Transplantation of Autologous Fat, Stromal Vascular Fraction (SVF) Cell and Platelet Rich Plasma (PRP) for Cell Therapy of Atrophic Acne Scars: Clinical Evaluation and Biometric Assessment

Background: Scarring is an unfortunate result of acne, because it causes psychological and cosmetic problems to patients. Unluckily no single treatment is suitable; using multiple methods may have better result. autologous fat and stromal vascular fraction (SVF) cells and their secretory factors can and enhance angiogenesis, collagen synthesis, and migration of broblasts therefore regenerate hurt tissues. Also, other treatments for acne scarring, like platelet rich plasma (PRP), induce the scare improvement. The aim of the study was to verify the effectiveness of transplantation of autologous fat, SVF cells and PRP as cell therapy technique on atrophic acne scars. Methods: This study included 9 adult patients with atrophic acne scars on the face. All patients received transplantation of autologous fat, stromal vascular fraction (SVF) cells and PRP. The treatment outcome was measured by biometric assessment (Visioface 1000 D, Colorimeter, multi probe adapter Cutometer, Tewameter, Mexameter, and skin ultrasound imaging system), also patients’ satisfaction was evaluated. The patients were followed 6 months after treatment. Results: A signicant improvement in the skin pores, spots, skin lightness and melanin content of the skin, skin elasticity and TEWL after 6 months of treatment. Also denser skin layers were observed both in the epidermis and dermis. Furthermore, 66.6 % of the patients showed well satised after treatment. Conclusion: Briey, transplantation of autologous fat, SVF cells and PRP is an effective cell therapy for atrophic acne scars. In the Pradeep V Mahajan study. Regenerative Medicine Using Platelet Rich Plasma and Stem Cells in Atrophic Acne Scars: A Case Report [37]. Our results showed that transplantation of autologous fat and SVF cells and PRP is a good technique for treatment of atrophic acne scars with signicantly increase in skin lightness and melanin content of the skin. Also the elasticity of the skin was increased. Oure results indicated that number of skin ne pores, large pores and spots were signicantly decrease after treatment. Briey transplantation of autologous fat, SVF cells and PRP is very promising strategy based on cell therapy for atrophic acne scars improvement with a minimal side effect.


Introduction
Numerous skin disorders like scars or aging signs such as wrinkles may adversely affect the appearance and beauty of the skin. Scarring occurs as a natural process after a wound healing. However, the appearance of wounds is in uenced by several factors. keloids and hypertrophic scars can treat by topical or injection application of corticosteroids [1]. However, there are some side effects like hypopigmentation and irreversible subcutaneous atrophy [2]. Scars are frequently treated by fewer invasive silicone dressing, but positive effects of them presently absence scienti c evidence [3]. Laser therapy of scars is other option that may improve the appearance, however, laser technology has some limitation like skin discoloration [4,5]. Finally, surgery can be a choice for treat overly obvious scars like dermabrasion, excision, subcision and skin grafting [6]. Nevertheless, aggressive surgical procedures are related to higher complications, leading to even larger scars with persistent contraction, which may worsen the condition [7]. By doing so, current scar treatments usually use ablative mechanisms that may have irreversible side effects. So, emphasizing the need for substitute techniques. Autologous fat injections have become very common in tissue regeneration over the past decade. Its good properties in regeneration and skin disorders have been reported in some studies [8][9][10]. Fat transplantation for repairing soft tissue defects have been applied for many years [1,2]. Also in many study fat transplantation was used for acne scare treatment. [11,12]. Particularly, the study's results on restoring fat, bone, cartilage, nerve tissue, and muscle by adipose tissue-derived stem cells (ADSCs) have been informed [3,10]. It is reported that ADSCs can repair damaged tissue by their secretory factors that can induce angiogenesis, collagen synthesis and broblasts migration [11]. Also, recently, platelet rich plasma (PRP), alone or in combination with other techniques, is considered for acne scare treatment. A combination of PRP with other treatment options was performed in the treatment of many dermatological disorders such as acne scarring [13,14]. The PRP work way depends on the presence of protein, that increases the synthesis of collagen and elastic bers lead to improves scar tissue and skin appearance [15,16]. The present study aimed to assess the e cacy and safety of transplantation of autologous fat, stromal vascular fraction (SVF) cells and PRP as cell therapy for atrophic acne scars treatment.

Patients
In this study, 9 patients with rolling acne scars were enrolled. Lesions were graded and classi ed by a board-certi ed 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.

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 uid (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, nal 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 (TC20 TM Automatic Cell Counter, Bio-Rad). Some of the suspension was subjected to ow 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 speci c gravities as follow; RBCs is the heaviest, after that WBCs, whereas platelets and plasma are the lightest. Centrifugation was performed in two-step; rst 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 nally 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 2×10 7 total for whole face of each patient.

Outcome evaluation 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 ow cytometry.

Patients outcome
Biometric assessment 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.

Patients' satisfaction
The participants were requested to scale their subjective satisfaction of the treatment on a quartile grading scale (grade 1 = slightly satis ed, grade 2 = moderately satis ed, grade 3 = well satis ed).

Statistical analysis:
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 signi cant.

Automatic cell counter
The viability and count of SVF cells harvest from fat tissue The nucleated cell harvest was counted by the automatic cell counter. Also the cell viability of the cell was assessed. The results were shown in the Table 1 for each patients. The results showed that the mean of yield cell was 22 ± 1.5 × 10 6 cells/mL of aspirated fat tissue and the mean of viability was 94.1 ± 2.5 (Table 1). Svf Cells Flow Cytometric Analysis The viability of nucleated SVF cells The viability of nucleated SVF cells isolated from all patients was evaluated by ow cytometry (Fig. 2).
According to the ow cytometry results the mean viability of cells was about 97 ± 1.2%.

The Surface Markers Of Nucleated Svf Cells
The properties of SVF cells were investigated by ow cytometry (Fig. 3). The examination of the fresh isolated SVF cells surface markers displayed the expression of endothelial markers included CD146, CD31, and CD34. Moreover, the expressions of the mesenchymal markers comprising CD44, CD73, CD90, and CD105 were determined. It should be noted that CD44 is a hyaluronic acid receptor which contributes in the dermal thickness and can be used for the molecular cues of skin rejuvenation assessment. Also, CD14, CD15, and CD45 as hematopoietic stem cell markers were found in the SVF isolated cell population. The CD56, natural killer cell marker, was expressed as well.

Biometric Characteristic
The biometric data of the patients are shown in the Table 2. Brie y, the size and number of skin pores and spots were reduced after treatment (Figs. 4 and 5). The Visioface results displayed that the transplantation can signi cantly decrease the number of skin pores and skin spots, and the percentages of change in the number of ne pores, large pores and spots were 43.01 ± 11.14, 47.37 ± 14.13 and 41.98 ± 10.19, respectively (P < 0.001; Table 2). The colorimeter results showed the skin lightness before and after treatment was 11.46 ± 2.64 and 15.94 ± 2.86 (Table 2). There was a signi cant change in skin lightness before and after treatment Fig. 6, P < 0.05 and the percentage of change in the skin lightness after treatment was 36.79 ± 10.80, ( There were not signi cant changes in erythema intensity before and after treatment (Table 2). Also, the results of skin ultrasonography exhibited denser skin layers both in the epidermis and dermis ( Fig. 6; Table 2). According to the

Patients' Satisfaction Results
The mean score of patients' satisfaction was shown in the Table 3. 22.3% of the patients showed moderately satis ed and 66.6% showed well satis ed after treatment.

Discussion
Acne is a common disorder that affects about 80% of young and 5% of adult's people [17]. One of the major mechanisms that is involved in the atrophic acne scars pathogenesis is collagen loss. So, stimulation of collagen production is the goal of several methods that have been proposed for the acne scar treatment [18]. Fat transplantation has been displayed to have bene cial effects on acne scars, which were primarily treated for volume loss. It seems, fat transplantation can repair collagen organization and reduce the brotic response to radiation and it is most likely induce improvement by many growth factors already present in lipoaspirate, which helps regenerating and remodeling of skin and scars. Also, studies indicate that tissue remodeling, the formation of fresh and new blood vessels, and in ammatory responses are possible causes of scars treatment in fat transplantation [11]. Because of biocompatible and not immunogenic properties of the adipose tissue, it is an ideal transplant for a patient with acne scar. In addition, it is easy to yield with minimal side effects in the donor site. The rst autologous fat transfer for facial volume loss was reported in 1893, when fat was applied to ll subcutaneous defects [19]. In 1912, the correction in retracted scar after mastectomy was reported by fat injections [12]. However, in recent times, the method of fat harvesting and injection has been modi ed and signi cantly improved, so it takes better results. In some studies, the plasticity and elasticity of scar tissue was increased, which was also observed microscopically in scar sample after lipo lling treatment [20,21]. In previous clinical study, regenerative effects of adipose tissue in the dermis and subcutaneous tissue was informed [22,23]. Fat transplantation has been applied in a diversity of disorders relating volume loss like panfacial global subcutaneous atrophy secondary to intrinsic aging or trauma [24].
Furthermore, stem cells have many unique properties that make them theoretically have great potential in managing a large number of diseases [25]. Some of these features include unlimited proliferation capability and plasticity [26]. It seemed recent research have shown interested to use of stem cell injection in acne scars treatment [27]. There is mutual agreement that autologous adipose tissue-derived stem cells (ADSCs) are multi-and pluripotent [28][29][30]. ADSCs are found in large amounts in the stromal vascular fraction (SVF) of adipose tissue [31]. ADSCs and their secretory factors can induce angiogenesis, collagen synthesis and broblasts migration therefore regenerate impaired tissues [11]. In the Zhang et al. study the effect of intralesionally injection of adipose tissue derived stem cells into rabbit ears are evaluated and reduction in the hypertrophic scarring formation was reported by enhancing collagen deposition [32]. In Abou Eitta study, it is shown that ADSCs is more effective than fractional carbon dioxide laser in the treatment of acne scars [11]. The e cacy of ASCs on scar appearance improvement has been studied in some studies. In the study of Lee et al. it is reported that SVF cells injections can enhance tissue regeneration via contributing stem cells and growth factors to improve the results in tissue grafts or scar revisions [33]. Lee and collogues evaluated the effect of combination of adipose tissue with SVF cells on the facial skin disorder and they declare Facial Soft Tissue signi cantly increase in the facial area grafted with adipose tissue in combination with SVF cells in compare to adipose tissue without SVF cells [34]. Also Recently, platelet-rich plasma (PRP) used alone or in combination with other therapy as an effective option for the atrophic acne scars treatment [14,35]. The PRP work way depends on the presence of protein, that increases the synthesis of collagen and elastic bers lead to improves scar tissue and skin appearance [15,16]. So in recent study we used combination of transplantation of autologous fat, SVF cells and PRP for atrophic acne scars treatment. In the study of Gentill et al., the effect of SVF cells and PRP the face scars were evaluated and they reported that the patients showed 69% maintenance of contour restoring after fat grafting and PRP treatment [36]. In the Pradeep V Mahajan study. Regenerative Medicine Using Platelet Rich Plasma and Stem Cells in Atrophic Acne Scars: A Case Report [37]. Our results showed that transplantation of autologous fat and SVF cells and PRP is a good technique for treatment of atrophic acne scars with signi cantly increase in skin lightness and melanin content of the skin. Also the elasticity of the skin was increased. Oure results indicated that number of skin ne pores, large pores and spots were signi cantly decrease after treatment. Brie y transplantation of autologous fat, SVF cells and PRP is very promising strategy based on cell therapy for atrophic acne scars improvement with a minimal side effect. Conclusion transplantation of autologous fat, SVF cells and PRP is very promising strategy based on cell therapy for atrophic acne scars improvement by repair the collagen organization, increase elastic bers and reduce the brotic response to radiation and it is most likely induce improvement by many growth factors, which helps regenerating and remodeling of skin and scars. Also, tissue remodeling, the formation of fresh and new blood vessels, and in ammatory responses are possible causes of scars treatment in this technique.