Rejuvenation of Facial Skin by Transplantation of Autologous Stromal Vascular Fraction Isolated Using a New Type of Clinical-grade Collagenase


 OBJECTIVE

The purpose of this study was to detect the yield, activity and safety of the stromal vascular fraction (SVF) isolated by a new clinical-grade collagenase with a specific process and to validate the efficacy of the method.
MATERIALS AND METHODS

For the in vitro study, adipose tissue specimens were obtained from women undergoing elective liposuction (n = 5). The SVF was isolated by new clinical-grade collagenase for the experimental group and research-grade collagenase NB4 for the control group. The surface markers of the SVFs isolated by the two collagenases were detected by flow cytometry. The yield, activity and morphology of the SVFs were compared, together with their proliferation, migration and differentiation. Furthermore, we conducted a clinical trial to evaluate the improvement of skin texture after transplantation of autologous SVF isolated by clinical-grade collagenase.
RESULTS

There were no statistically significant differences in the yield, viability or morphology of the SVFs isolated by the new collagenase and collagenase NB4. Adipose tissue-derived mesenchymal stem cells (ASCs) surface marker expression was consistent between the two products. The differentiation potential of the cells was not different, which was consistent with previously published results. The SVF isolated by the new collagenase had a higher proliferation and migration. After administration of autologous SVF isolated by the new collagenase, the VISIA score improved significantly with regard to wrinkle scores and texture scores.
CONCLUSION

We concluded that this new type of clinical-grade collagenase can replace current research-grade products without any negative effect on the yield or function of the SVF, which can be applied to antiaging and rejuvenation of facial skin.


cells, and MEL-CLS-2 cells) to determine whether Rg3
has anticancer effects on human melanoma cells. Thus, we investigated whether Rg3 induces apoptosis in these cells and which signaling pathway leads to apoptosis.
In this study, we planned an in vitro test. First, we investigated cell viability, cell morphologic change, colony forming ability, and cell migration ability after treating Rg3 with 4 human melanoma cells. Then, flow cytometry, Western blot, and immunocytochemistry analysis were performed to determine whether Rg3-treated melanoma cells died through apoptosis. Finally, we investigated cell viability after treatment with various apoptotic kinase inhibitors to identify the signaling pathway leading to apoptosis.
When Rg3 was treated with 4 kinds of human melanoma cells, cell viability, cell morphologic change, colony forming ability, and cell migration ability were changed. It was also confirmed that Rg3 kills these melanoma cells through apoptosis. Finally, apoptosis is associated with the MEK signaling pathway.
The authors confirmed that Rg3 could induce apoptosis of human melanoma cell lines (A375.S2, G361, MML-1, and MEL-CLS-2 cells). BACKGROUND: Stromal vascular fraction (SVF) of fat tissue contributes to skin antiaging and rejuvenation, but standard isolation methods have not yet been established for obtaining clinical-grade cells. 1 Tissue enzymatic digestion is now the most widely used method. However, most published protocols employ research-grade collagenase containing xenogeneic components, which may cause severe anaphylaxis, immune reactions, or infections in patients. 2 And for some products, the enzymatic composition shows lot-to-lot variability that may affect reproducibility. 3 In this study, we utilized a new type of clinical-grade collagenase to determine its suitability and efficacy. Furthermore, we conducted a clinical trial to evaluate the ultrastructural improvement of facial skin after transplantation of autologous SVF isolated by the new collagenase.

MATERIALS AND METHODS:
This new type of clinicalgrade collagenase mainly consists of aseptically filled, highly purified collagenase, which has been applied to loosen hypertrophic scars. Collagenase NB4 (Sigma) was used as control. For in vitro study, adipose tissue specimens were obtained with informed consent from women undergoing elective liposuction (n = 5). SVF was isolated according to the published methods with some minor modifications, its yield and viability was detected. Then SVF was cultured and expanded to adipose stem cells (ASCs), the immunophenotype and differentiation potential was also evaluated. Our clinical trial was conducted in 13 patients aged between 30 and 56 years old. All of the cases underwent the lipoaspiration procedure from the abdomen. SVF was isolated and transplanted at dose of 2.5 × 10 7 nucleated cells in one side of the fronto-temporal area, the other side was used as self-control. The changes in the skin were analyzed by VISIA and patients' self-assessment.

RESULTS:
There are no statistically significant differences on yield and proliferation of SVF isolated by the new collagenase and Collagenase NB4. No morphologic differences were found in cells throughout the entire culturing time. ASCs surface markers expression was similar between 2 products. And the differentiation potential of the cells was not affected, which was consistent with previously published results. By administration of autologous SVF isolated by new collagenase, the elasticity and density of skin were improved significantly. The score of VISIA showed slight changes in wrinkle scores. Furthermore, most patients thought that the skin texture of treatment area was improved.

CONCLUSION:
We concluded that this new type of clinical-grade collagenase can replace current research-grade products without any negative effect in the yield or function of SVF, which can be applied to antiaging and rejuvenation of facial skin.

INTRODUCTION:
Although artificial dermis (AD) has been tested for wound bed preparation with cultured epidermal autografts (CEA), low CEA acceptance has been a challenging problem. Previously, we have reported effects of CEA overlaid on the wound bed grafted with ovine cadaver skin. The goal of the present study was to compare the efficacy of CEA overlaid on excised full-thickness burn wounds grafted with either AD or cadaver skin in sheep. We hypothesized that the effects of CEA overlaid on wound beds prepared with AD would be comparable to those prepared using cadaver skin.

METHODS:
We used our well-characterized ovine burn wound healing model. Six full thickness burn wounds (5 × 5 cm) were induced at the dorsum of sheep. After 24 hours, the eschar was excised down to the fascia and covered with ovine frozen cadaver skin (CS, n = 5) or AD (n = 5). Same day, aliquots of healthy skin were harvested for isolation of autologous keratinocytes to be cultured on temperature-responsive dishes (to avoid use of harmful enzymes for detachment) until forming the multilayer sheets. Three weeks after grafting, the keratinocyte sheets were overlaid onto the wounds covered with either CS or AD. The acceptance of cultured keratinocyte sheets and wound epithelialization were assessed for 14 days using intermittent planimetric assay and histological analysis.

CONCLUSIONS:
Our results suggest that use of AD can successfully substitute cadaver skin for preparing wound beds. Our results also demonstrate that AD and autologous keratinocyte sheets can substitute autologous skin grafts.