MiR-24-3p regulates the differentiation of adipose-derived stem cells toward pericytes and promotes fat grafting vascularization.

Adipose-derived stem cells (ADSCs) enhance fat graft survival by promoting neovascularization. The mechanism that promotes ADSCs differentiation toward pericytes was not known. We treated ADSCs with conditional medium (CM) from endothelial cells (ECs) or human recombinant transforming growth factor ß (TGF-ß) to induce differentiation into pericytes. Pericytes markers, including platelet-derived growth factor receptor ß (PDGFRß), alpha-smooth muscle actin (α-SMA), and desmin, were examined. Pericytes differentiation markers, migration, and their association with ECs were examined in ADSCs transfected with miR-24-3p mimics and inhibitors. Bioinformatics target prediction platforms and luciferase assays were used to investigate whether PDGFRß was directly targeted by miR-24-3p. In vivo, fat mixed with ADSCs transfected with miR-24-3p mimics or inhibitors was implanted subcutaneously on the lower back region of nude mice. Fat grafts were harvested and analyzed at 2, 4, 6, and 8 weeks. Results showed that endogenous TGF-ß derived from CM from EC or human recombinant TGF-ß promoted migration, association with ECs, and induced expression of pericyte markers (PDGFRß, α-SMA, Desmin) in ADSCs. MiR-24-3p directly targeted PDGFRß in ADSCs by lucifer reporter assays. Inhibition of miR-24-3p promoted pericytes differentiation, migration, and association with ECs in ADSCs. Inhibition of miR-24-3p in ADSCs promoted survival, integrity, adipocyte viability, vascularization, pericytes association with ECs, and reduced fibrosis, whereas overexpression of miR-24-3p in ADSCs yielded the opposite results. Collectively, TGF-ß released by ECs induced ADSCs differentiation toward pericytes through miR-24-3p. Downregulation of miR-24-3p in ADSCs induced survival, integrity, adipocyte viability, vascularization, pericytes association with ECs, and reduced fibrosis after fat grafting.

Improvement in autologous human fat transplant survival with SVF plus VEGF-PLA nano-sustained release microspheres.

Early neovascularization is important for autologous fat transplant survival. SVF cells are ideal seed cells. Both vascular endothelial growth factor (VEGF) and SVF cells can promote neovascularization. However, the half-life (about 50 min) of VEGF is too short to sustain an adequate local concentration. We have investigated whether VEGF-polylactic acid (PLA) nano-sustained release microspheres plus SVF cells can improve neovascularization and survival of transplanted fat tissues. SVF cells were harvested and constructed VEGF-PLA nano-sustained release microspheres in vitro. Human fat tissues was mixed with SVF cells plus VEGF-PLA, SVF cells alone or Dulbecco's modified Eagle's medium as the control. These three mixtures were injected into random sites in 18 nude mice. Two months later, the transplants were weighed and examined histologically; and capillaries were counted to quantify neovascularization. Hematoxylin-eosin (HE) and anti-VEGF stains were applied to reveal cell infiltration. The mean wet weight of fat in the SVF plus VEGF-PLA, SVF alone, and control transplants were 0.18 ± 0.013 g, 0.16 ± 0.015 g, and 0.071 ± 0.12 g, respectively; the differences between groups were statistically significant. More vessels were present in the SVF plus VEGF-PLA transplants than in the other two types. Transplants mixed with SVF cells also had an acceptable density of capillaries. Histological analysis revealed that both the SVF plus VEGF-PLA and SVF alone transplants, but not the control transplants, were composed of adipose tissue, and had less fat necrosis and less fibrosis than control specimens. SVF plus VEGF-PLA transplants had significantly greater capillary density and VEGF expression than the other two transplant groups. Thus transplanted fat tissue survival and quality can be enhanced by the addition of VEGF-PLA nano-sustained release microspheres plus SVF cells.

Creating natural double eyelids with continuous buried suture and mini-incision technique using subcutaneous absorbable suture for patients with puffy eyelids.

IMPORTANCE Incision and buried suture are 2 primary techniques for creating double eyelids. The incision method is suitable for all kinds of eyelids, but operational trauma and prolonged recovery time limit its application. The non-incision approach can shape a natural and vivid crease with a relatively short recovery time. However, it is not suitable for patients with puffy eyelids, and in those patients the duration of the supratarsal crease is not long.We propose a method of combining the techniques of continuous buried suture and mini-incision. OBJECTIVE To explore a new kind of double eyelid plasty, a corrective surgical procedure for patients with puffy eyelids. DESIGN, SETTING, AND PARTICIPANTS Observational study of 221 patients with puffy single eyelids who underwent this new blepharoplasty from May 2007 to and March 2012. INTERVENTIONS Combined continuous buried-suture and mini-incision surgery using subcutaneous absorbable suture to create natural double eyelids under local anesthesia. All procedures were performed by the same surgeon. MAIN OUTCOMES AND MEASURES All patients were observed after surgery for a period ranging from 1 to 28 months (mean follow-up, 16 months). Data collection included operative time, postoperative recovery and complications. RESULTS All double eyelids appeared natural after short operative time and rapid postoperative recovery, leaving an invisible scar and long-lasting supratarsal crease. No corneal damage or infection occurred.CONCLUSIONS AND RELEVANCE Combined continuous buried-suture and mini-incision surgery using subcutaneous absorbable suture to create natural double eyelids is a reliable,durable, and less-invasive technique for patients with puffy eyelids.

[Experimental study on fluorescent labeling and optimization method of purifying human stromal vascular fraction cells].

OBJECTIVE: To find a kind of simple and effective method for purifying and labeling stromal vascular fraction cells (SVFs) so as to provide a theoretical basis for clinical application of SVFs. METHODS: The subcutaneous adipose tissue were harvested form volunteers. The adipose tissue was digested with 0.065%, 0.125%, and 0.185% type I collagenase, respectively. SVFs were harvested after digestion and counted. After trypan blue staining, the rate of viable cells was observed. SVFs was labeled by 1, l'-dioctadecyl-3, 3, 3', 3'-2-tetramethy-lindocyanine perchlorate (DiI). The fluorescent labeling and growth was observed under an inverted fluorescence microscope. MTT assay was used to detect cell proliferation. RESULTS: The number of SVFs was (138.68 +/- 11.64) x 10(4), (183.80 +/- 10.16) x 10(4), and (293.07 +/- 8.31) x 10(4) in 0.065% group, 0.125% group, and 0.185% group, respectively, showing significant differences among 3 groups (P < 0.01). The rates of viable cells were 91% +/- 2%, 90% +/- 2%, and 81% +/- 2% in 0.065% group, 0.125% group, and 0.185% group, respectively, and it was significantly higher in 0.065% group and 0.125% group than in 0.185% group (P < 0.01), but no significant difference was found between 0.065% group and 0.125% group (P=0.881). Inverted fluorescence microscope showed that the cell membranes could be labeled by DiI with intact cell membrane, abundant cytoplasm, and good shape, but nucleus could not labeled. SVFs labeled by DiI could be cultured successfully and maintained a normal form. MTT assay showed that similar curves of the cell growth were observed before and after DiI labeled to SVFs. CONCLUSION: The optimal collagenase concentration for purifying SVFs is 0.125%. DiI is a kind of ideal fluorescent dye for SVFs.

[Experimental study of the effect of adipose stromal vascular fraction cells with VEGF on the neovascularization of free fat transplantation].

OBJECTIVE: To investigate the effect of adipose stromal vascular fraction cells (SVFs) with VEGF on the neovascularization of free fat transplantation. METHODS: SVFs were obtained from subcutaneous fat and labelled with DiI. 0.3 ml autologous fat tissue was mixed with 0.2 ml cells: 1) autologous SVFs with VEGF (Group A); 2) autologous SVFs (Group B); 3) complete DMEM (Group C) And then the mixture was injected randomly under the back skin of 12 nude mice. The transplanted fat tissue in three groups was harvested at 2 months after implantation. Wet weight and diameter of fat grafts was measured. After HE and CD31 staining,blood vessel density, viable adipocytes and fibrous proliferation were observed. RESULTS: Trace of SVFs labeled by DiI in vivo could be detected by fluorescent microscope. The wet weight of fat grafts was (191.90 +/- 9.81) mg in group A, (177.01 +/- 10.50) mg in group B, and (92.05 +/- 8.30) mg in group C (P<0.01). The diameter of fat grafts was (0.49 +/- 0.24) cm in group A, (0.40 +/- 0.26) cm in group B, and (0.32 +/- 0.28) cm in group C (P<0.01). Histological analysis showed the blood vessel density was (14.58 +/- 2.06)/HPL in group A, (11.55 +/- 2.18)/HPL in group B, (7.87 +/- 1.55)/HPL in group C. Compared with group B and group C, group A had more adipose tissue with less fat necrosis and fibrosis and had significantly higher capillary density. CONCLUSIONS: The autologous adipose stromal vascular fraction cells with VEGF could improve the neovascularization of free fat significantly. It indicates a wide clinical application in the future.