Adipose mesenchymal stem cell-derived exosomes promote cell proliferation, migration, and inhibit cell apoptosis via Wnt/ß-catenin signaling in cutaneous wound healing.

Cutaneous wounds, a type of soft tissue injury, are difficult to heal in aging. Differentiation, migration, proliferation, and apoptosis of skin cells are identified as key factors during wound healing processes. Mesenchymal stem cells have been documented as possible candidates for wound healing treatment because their use could augment the regenerative capacity of many tissues. However, the effects of exosomes derived from adipose-derived stem cell (ADSC-exos) on cutaneous wound healing remain to be carefully elucidated. In this present study, HaCaT cells were exposed to hydrogen peroxide (H2 O 2 ) for the establishment of the skin lesion model. Cell Counting Kit-8 assay, migration assay, and flow cytometry assay were conducted to detect the biological function of ADSC-exos in skin lesion model. Finally, the possible mechanism was further investigated using Western blot assay. The successful construction of the skin lesion model was confirmed by results of the enhanced cell apoptosis of HaCaT cells induced by H 2 O 2 , the increased Bax expression and decreased Bcl-2 expression. CD9 and CD63 expression evidenced the existence of ADSC-exos. The results of functional experiments demonstrated that ADSC-exos could prompt cell proliferation and migration of HaCaT cells, and repress cell apoptosis of HaCaT cells. In addition, the activation of Wnt/ß-catenin signaling was confirmed by the enhanced expression of ß-catenin at the protein level. Collectively, our findings suggest that ADSC-exos play a positive role in cutaneous wound healing possibly via Wnt/ß-catenin signaling. Our study may provide new insights into the therapeutic target for cutaneous wound healing.

[Application of autogenous cartilage transplantation in rhinoplasty].

OBJECTIVE: To investigate the application of autogenous cartilage transplantation in rhinoplasty. METHOD: We chose three kinds of treatment according to the shape of nasal tip and thickness of local soft tissue. Autogenous auricular cartilage transplantation combined with "L" type artificial prosthesis rhinoplasty was executed in 57 cases. Nasal alar cartilage transplantation combined with "L" type artificial prosthesis rhinoplasty was executed in 33 cases and septal cartilage transplantation combined with "willow leaf" type artificial prosthesis rhinoplasty was executed in 29 cases. RESULT: Improved nasal aesthetic effects were observed after operation in all of 119 cases, 64 cases were follow-up visited for 3 to 12 months. Both surgeons and patients were satisfied with the nasal shape. CONCLUSION: Autogenous cartilage transplantation combining with artificial prosthesis rhinoplasty could effectively rebuild the nasorostral shape. We chose different kinds of cartilage according to the nasorostral condition. We can ensure that the whole nasal shape according to aesthetic requirement.

[Transplantation of adipose tissue-derived stem cells promotes soft tissue wound repair in rats].

OBJECTIVE: To assess the effect of local and intravenous transplantation of adipose tissue-derived stem cells (ADSCs) in promoting soft tissue wound healing in rats. METHODS: ADSCs isolated from the adipose tissues of SD rats were cultured in vitro, and the third-passage cells were identified for their capacity of multipotent differentiation. Eighteen SD rats with 1.8 cm² dorsal full-thickness soft tissue defects (0.5 cm deep) were randomized into 3 groups to receive injection of 3.0×106 DiI-labeled ADSCs via the tail vein, local injection of the cells at the wound site, or injection of saline (control). The wound healing was evaluated on days 3, 7, 11, and 14 postoperatively. On day 24 after the injury, tissue samples at the wound site were collected for fluorescent microscopy and HE staining. RESULTS: The ADSCs obtained were capable of adipogenic, osteogenic, and neurogenic differentiation in vitro. ADSCs transplantation significantly promoted wound healing as compared to the control group. Obvious wound contracture was observed in the local injection group on day 3 and in the intravenous injection group on day 7. Fluorescence microscopy revealed DiI-positive cells in the healing wound, and HE staining showed a greater tissue thickness at the wound in the two ADSCs transplantation groups. Compared to the control group, the two ADSCs transplantation groups showed more gland-like structures and better neovascularization at the wound. CONCLUSION: ADSCs can significantly promote wound healing in rats, and local injection of ADSCs allows more rapid and obvious wound healing than tail veil injection of the stem cells.

[Experimental study of the effect of adipose stromal vascular fraction cells on the survival rate of fat transplantation].

OBJECTIVE: To investigate the effect of adipose stromal vascular fraction cells (SVFs) on the survival rate of fat transplantation. METHODS: 0. 5 ml autologous fat tissue was mixed with: 1) Di-labeled autologous SVFs ( Group A); 2) Di-labeled autologous adipose-derived stem cells (ASCs) (Group B); 3)Complete DMEM (Group C). And then the mixture was injected randomly under the back skin of 14 rabbits. The transplanted fat tissue in three groups was harvested at 6 months after implantation. Wet weight of fat grafts was measured for macroscopic aspects. After HE staining, blood vessel density, viable adipocytes and fibrous proliferation were counted respectively for histological evaluation. Trace of DiI-labeled ASCs in vivo was detected by fluorescent microscope. RESULTS: The wet weight of fat grafts in group A (291.0 +/- 72.1) mg and group B (269.3 +/- 67.3) mg was significantly higher than that in group C (177.8 +/- 60.0) mg, but the difference between Group A and Group B was not significant. Histological analysis revealed that the fat grafts in group A and B was consisted predominantly of adipose tissue with less fat necrosis and fibrosis, compared with the fat grafts in group C. The grafts in both group A and B had significantly higher capillary density than those in the control group. Part of vascular endothelial cells were observed to origin from ectogenic DiI-labeled SVFs and ASCs. CONCLUSIONS: The autologous isolated SVFs has a similar effect as autologous cultured ASCs to improve the survival rate of fat transplantation. And the former is more practical and safe, indicating a wide clinical application in the future.

[Rectification of nasal deviation with open operation].

OBJECTIVE: To investigate the quantitative classification and measuring method of nasal deviation and effectiveness of Septo-rhinoplasty for nasal deviation. METHOD: Fourteen selected cases of nasal deviation were rectified by Septo-rhinoplasty. The deviated parameters were measured both before and after operation. RESULT: The deviated parameter after operation was (2.75 +/- 1.67) mm in 14 cases. The preoperative parameter was (5.05 +/- 2.03) mm. The difference in changes of deviated parameters before and after operation was significant (P < 0.01). Satisfactory results were achieved after operation in 12 cases. The rate of good cosmetic outcome was 85.71%. CONCLUSION: Both quantitative grading standard of nasal deviation and measurement of deviation value contribute to clinical diagnosis and medical records. The Septo-rhinoplasty is effective for nasal deviation.

[Comparison between kinds of myofascial flap encapsulating adipose-derived stromal cells carrier complex in terms of adipogenic efficacy in vivo].

OBJECTIVE: To compare two kinds of myofascial flap encapsulating adipose-derived stromal cells (ADSCs) in adipogenic efficacy in vivo, and to provide experimental basis for the efficient transplantation of free adipose tissue. METHODS: ADSCs were isolated from the subcutaneous adipose tissue in the neck of 10 New Zealand rabbits (aged 3- 4-months-old, male and female, weighing 2.0-2.5 kg), and primary culture and subculture of ADSCs were conducted. When the cells at passage 3 covered 70%-80% of the bottom of the culture flask, BrdU (10 microg/mL) was applied to label the cells for 48 hours before performing immunofluorescence staining. Oil red O staining observation was conducted to those cells 2 weeks after being induced towards adipocyte, alizarin red staining observation was performed 3 weeks after being induced towards osteoblast, and alcian blue staining was conducted 2 weeks after being induced towards chondrocyte. Besides, after being induced towards adipocyte for 2 weeks, 1 x 10(7) ADSCs/piece at passage 3 labeled by BrdU was seeded into Col I (10 mm x 10 mm x 5 mm/piece) to prepare cell carrier complex. The experiment was divided into two groups: group A in which vascular pedicled dextral latissimus dorsi fascial flap was adopted to encapsulate the complex; group B in which dextral gluteus maximus fascial flap with no specific vessel pedicle was applied to encapsulate the complex. Rabbits in each group went through autogenous ADSCs transplant and self control. The implants were dislodged 8 weeks after operation, HE staining and immunohistochemistry staining were performed to testify cambium, the wet weight and micro vessel count of the cambium in each group were tested, immunofluorescence staining was performed to determine the origin of cambium and microvascular endothelium. RESULTS: The nucleus of ADSCs positive for BrdU labeling showed green fluorescence under fluorescence microscope, with the positive labeling ratio of ADSCs above 90%. For ADSCs at passage 3, the formation of red lipid droplets within cells was observed 2 weeks after being induced towards adipocyte, red calcium nodules were evident 3 weeks after being induced towards osteoblast, and highly congregated cell mass positive for alcian blue staining appeared 2 weeks after being induced towards chondrocyte. Eight weeks after operation, neogenetic blood vessel grew into scaffolds and no obvious fibre encapsulation was observed in group A, while few blood vessel grew into scaffolds in group B. The wet weight of cambium in group A and B was (0.149 5 +/- 0.017 3) g and (0.095 3 +/- 0.012 7) g, respectively, indicating there was a significant difference between two groups (P < 0.01). HE staining showed the formation of neogenetic adipose tissue and the growth of micrangium in the implant, and the degradation and absorption of scaffold. The micro vessel count of group A and B was 31.2 +/- 4.5 and 19.3 +/- 2.6, respectively, indicating there was a significant difference between two groups (P < 0.01). Eight weeks after operation, the immunofluorescence staining of cambium showed that the cell nucleus of regenerated adipocytes and partial capillary endothelium in groups A and B presented green fluorescence. CONCLUSION: ADSCs encapsulated by vascular pedicled latissimus dorsi fascial flap and collagen protein scaffold complex has a higher adipogenic efficacy in vivo than the gluteus maximus fascial flap with no specific vessel pedicle.

Improvement of the survival of human autologous fat transplantation by using VEGF-transfected adipose-derived stem cells.

BACKGROUND: The efficacy of autologous fat transplantation is reduced by fat absorption and fibrosis due to fat necrosis. Enhanced transplant neovascularization early after transplantation may reduce these outcomes. The authors asked whether cell and concomitant gene therapy using adipose-derived stem cells transduced with vascular endothelial growth factor (VEGF) improves fat transplant neovascularization and survival. METHODS: Human adipose-derived stem cells were expanded ex vivo for three passages, labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI), and transduced with VEGF or left untransduced. Human fat tissues were then mixed with the DiI-labeled VEGF-transduced adipose-derived stem cells, the DiI-labeled adipose-derived stem cells, the known vascularization-promoting agent insulin, or medium alone, and 18 nude mice were injected subcutaneously with all four preparations, with each of the four designated spots receiving one of these four mixtures in a random fashion. Six months later, transplanted tissue volume and histology were evaluated and neovascularization was quantified by counting the capillaries. RESULTS: Control transplant survival was 27.1 +/- 8.2 percent, but mixture with the VEGF-transduced and VEGF-untransduced stem cells significantly increased transplant survival (74.1 +/- 12.6 percent and 60.1 +/- 17.6 percent, respectively). Insulin was less effective (37.7 +/- 6.9 percent). Histological analysis revealed both types of transplants consisted predominantly of adipose tissue, unlike the control transplants, and had significantly less fat necrosis and fibrosis. The VEGF-transduced, adipose-derived stem cell-treated transplants had significantly higher capillary density than the other transplants and bore DiI-double-positive and CD31-double-positive cells (i.e., adipose-derived stem cell-derived endothelial cells). CONCLUSION: Adipose-derived stem cells together with VEGF transduction can enhance the survival and quality of transplanted fat tissues.

[Experimental study of the effect of adipose tissue derived stem cells on the survival rate of free fat transplantation].

OBJECTIVE: To investigate the feasibility of using adipose tissue derived stem cells (ASCs) to promote neovascularization and survival rate of free fat transplantation. METHODS: ASCs were isolated from aspirates from human liposuction and cultured in vitro. The cells were incubated in adipogenic, osteogenic, and chondrogenic medium for 2-4 weeks to induce adipogenesis, osteogenesis and chondrogenesis, respectively. ASCs were labelled by DiI. ASCs (A group), Insulin (B group), Medium (C group) were respectively mixed with free fat graft from aspirates. The mixtures were injected subcutaneously at the three random points on the back of eighteen 4- 6-week-old nude mice. Transplanted fat tissue was harvested after 6 months. The grafts were assessed by morphological observation, HE staining and immunohistochemistry. RESULTS: ASCs can be easily harvested from liposuction aspirates and differentiate into adipogenic, osteogenic, chondrogenic lineages. The wet weight of transplanted fat tissue in ASCs group was (165.97 +/- 5.51) mg, significantly higher than that in the insulin group (93.42 +/- 5.12) mg and control group (67.64 +/- 5.09) mg (P = 0.000). The rate of fibrosis and steatonecrosis in ASCs group was( 152.2 +/- 9.8)/10HF, significantly lower than that in the Insulin group (743.9 +/- 20.4)/10HF and control group (892.2 +/- 16.5)/10HF (P = 0.000). DiI labelled ASCs were found between adipocytes and in the connective tissue in free transplanted fat tissue, and some of these cells were immunopositive for antihuman CD31 and FITC, suggesting differentiation into vascular endothelial cells. CONCLUSIONS: ASCs can differentiate into vascular endothelial cells and contribute to angiogenesis in free transplanted fat tissue. ASCs can increase the survival rate and decrease the rate of fibrosis and steatonecrosis of free transplanted fat tissue. These findings suggest that ASCs-assisted transplantation may be an ideal cell therapy.