Experimental Study On Effects Of VEGF-PLA Nano-sustained Release Mierospheres With SVFs To Fat Particles Transplantation And Collagen Scaffolds

BACKGROUNDIn recent years, with the development and application of liposuction, the autologous fat particle injection has been become more and more popular. Autologous fat transplantation has a lot of advantages, such as no rejection, low infection, good compatibility. It has been seen as an ideal way o repair soft tissue defects. As the development of adipose tissue engineering, a new way has been opened up in the field of repairing soft tissue defects without damage and in the true sense of morphological, structural and functional reconstruction completely. However, engineered adipose tissue can not be used in clinical medicine, autologous fat transplantation is still the ideal material to repair soft tissue defects.Autologous fat transplantation has always been of clinical concern, especially the negative pressure liposuction has been widely used in clinical practice. The autologous fat transplantation is accepted by more and more clinicians and patients, and the technique in obtaining, purified, injection of fat are improved, the survival rate is also high than before. However, the absorption rate of transplanted fat particles can be as high as30%to50%, there is no good way to solve the high absorption rate, the lower survival rate of transplanted fat limits its clinical application greatly. Adipose tissue is not only the organs to save energy and endocrine, it is also the ideal alternative material of soft tissue material. Adipose tissue has been believed that the effective source of adult stem cells, it has a variety of cell composition, including progenitor cell composition, the progenitor cells can be differentiated into other cell lines. Adipose tissue mainly consists of fat cells, adipose stem cells, endothelial cells, parietal cells, fibroblasts, macrophages and extracellular matrix components, among the cell compositions, adipose cells and adipose stem cells are the research focus in recent years. The adipose-derived stem cells is considered to be a manifestation of the fat cells and vascular progenitor cells, which is located between the fat cells, perivascular or extracellular matrix can promote the outcome of the adipose tissue. Adipose-derived stem cells exist in adipose tissue stromal vascular fractions(stromal vascular fractions, SVF), while the SVF cells are the most important components in autologous fat transplantation. The mixture of a variety of cells isolated from adipose tissue by collagenase digestion, the formation of cell clusters called stromal vascular fraction(SVF). Stromal vascular fraction contains a lot of mesenchymal stem cells which can differentiate into multiple lineages, it is the ideal seed cells for regenerative medicine and tissue engineering.Select the appropriate cell carrier as the extracellulat matrix is an important condition for SVFs and ADSCs proliferation and differentiation. Adipose tissue is rich in collagen, fibronectin, proteoglycans, and so on, it can provide the extracellular matrix. The technology of cell auxiliary fat transfer was proposed in recent years, while addressing the two issues of the transplanted fat of ADSCs content and lack of transplant SVF extracellular matrix. And at the same time, it simplified the SVFs extracted in vitro and repeatedly centrifugal, weight hanging steps on. It provides a solution to solve the problem of the transplanted fat absorption rate in the clinical.It can enhance the vascularization of the engineered tissue, reducing the rate of absorption and improving the survival of the transplanted tissue if there are active growth factor in the tissue engineering materials. Vascular endothelial growth factor(VEGF), also known as vascular permeability factor, can promote vascular permeability, endothelial cell proliferation and migration, angiogenesis, and prolong the life of vascular endothelial cells, enhance blood vessel through permeability and change the role of the extracellular matrix and scar tissue healing, it is closely related to inflammation and tumorigenesis. There is a positive feedback between VEGF and its receptors, it can stimulate the endothelial cells high expression of VEGF and its receptors, and thus enlarge the effects of VEGF. However, VEGF can not give full play to its biological effects, because of its short half-life, degradable easily and other factors. The sustained release technology is likely to be a solution to the problem. It could entrapped VEGF in polylactide nanospheres though the technology of entrapped microspheres, so that the cytokine could release to the surrounding environment continuously and exert its biological effects. The slow release technique has great research value in adipose tissue engineering.In recent years,with the rapid development of tissue engineering technology, people pay more attention to using tissue engineering technology to clinical practice. However, Vascularization becomes the biggest obstacle to the clinical application.Collagen scaffolds is a good choice to tissue engineering. It has been approved by FDA as artificial skin materials. It is well known that Adipose Derived Stem Cells is a good choice as a kind of ideal seed cells. However, Adipose Derived Stem Cells needs a period of time.So it is inconvenient to clinical application. Now more and more scientists have realized that SVFs as seed cells has many advantages. The research which uses collagen protein as scaffold material, SVFs as seed cells, and combines VEGF-PLA slow-release microspheres has not been found.OBJECTIVES1. Isolated SVFs from human adipose tissue using the method of enzyme digestion method, The cells were induced for three-line differentiation. Study its characteristics of stem cells and advantages as the ideal seed cells in adipose tissue engineering;2. Labeled the SVFs with DiI fluorescence in vitro, observed cells’condition with fluorescence microscope, detected the effects of Dil to PO cells coming from SVFs proliferation and adipogenic differentiation. Study the advantages of DiI as the cells’tracking markers;3. Constructed VEGF-PLA polylactide nanospheres in vitro. Detected its sample encapsulation efficiency and drug loading, and detected the sustained release capabilities. Explore the feasibility of applied assisted fat particles transplantation research;4. Design the control group, the SVFs and (or) of VEGF-PLA sustained-release microspheres combined with fat particles transplantation in vivo experiments, explore the impact of the SVFs and (or) of VEGF-PLA sustained release microspheres to survival rate of fat particles transplantation in vivo;5. Using the solide-state type I collagen as carrier material, with SVFs and (or) VEGF-PLA release microspheres to bulid engineered adipose tissue in vivo. Detected the level of the vascularized of engineering adipose tissue in vivo. So as to provided experimental basis to adipose tissue engineering.1. Isolation of SVFs from adipose tissue and multilineage differentiation SVFs were isolated from subcutaneous tissue of healthy donors who had undergone liposuction through the method of collagenase digestion. The SVFs were used to next experimental study. Draw cells proliferation curve using the method of MTT. Induced SVFs to adipogenic, osteogenic and chondrogenic differentiation with the corresponding inductive culture medium, oil O, alizarin red and alcian blue staining correspondingly.2. Labeled the SVFs with Dil fluorescence in vitro, observed cells’condition with fluorescence microscope, detected the effects of Dil to PO cells coming from SVFs proliferation and adipogenic differentiation.Labeled SVFs with Dil in vitro, observed cells growth condition with inverted microscope and fluorescence microscope. Detected Dil-labeled PO cells coming from SVFs proliferation ability and adipogenic differentiation capacity with MTT assay and Oil Red O quantitative assay, and comparied the data with unlabeled groups statistically, analysis it’s biological traits change.3. Construct VEGF-PLA polylactide nanospheres and investigated the effect of VEGF-PLA release nanospheres on proliferation and adipogenic induction of adipose-derived stem cells in vitroConstructed VEGF-PLA sustained release microspheres though the method of ultrasonic emulsification in vitro. Detected the microspheres drug loading rate and encapsulation rate and observed the condition of encapsulation with transmission electron microscope, measured the diameter of microspheres under microscope. Detected the VEGF-PLA release capability using ELISA kit.4. Effects of SVFs and VEGF-PLA release microspheres to fat particles transplantation in vivo.Group design:group1transplantation of pure fat particles; group2transplantation of fat particles+SVFs; group3transplantation of fat particles+ SVFs+VEGF-PLA sustained release microspheres. Labeled SVFs with Dil in vitro, after that mixed SVFs and (or) VEGF-PLA release microspheres and fat particles respectively in the sterile condition. Transplanted the mixture though injection into nude mice in random, each mice injected three points. At eight weeks after the injections, the grafts were explanted, weighted, and fixed with10%paraformaldehyde, recorded data and statistical comparison. Staining the tissue section with HE and immunohistochemical for CD34and VEGF. Analysis the survival rate of fat particles and observed the expression of CD34and VEGF, counted the capillary number under microscope for three sets, and sataistical comparison with the software SPSS16.0. Analysis the effects of SVFs and VEGF-PLA release microspheres to vascularization of the transplantation.5. Effects of VEGF-PLA sustained release microspheres to vascularization of adipose tissue engineering in vivoPrepared three groups of grafts:group1was SVFs+collagen scaffold; group2were SVFs+collagen scaffold; group3were SVFs+collagen scaffold+VEGF-PLA sustained release microspheres. For2and3group, adipogenic induction3days in vitro. After preparation, the grafts were implanted in nude mice subcutaneously, three groups transplanted to the same mice.4weeks after implantation, the grafts were explanted and gross observation, measured the wet weight. Observed new organizational structure and vascular formed condition through HE staining, counted the new capillary density under high magnification, and statistical analysis the data collected.STATISTICAL ANALYSISThe data obtained was represent by mean±standard deviation (x±s), using SPSS16.0software package for statistical analysis. P＜0.05represent significant differences. We used Regression, one-way ANOVA,Independent-sample T Test,General Linear Model(Repeated measures).RESULTS1. SVFs was obtained successfully from adipose tissue of liposuction patients, had strong proliferate ability, similar to fibroblasts in morphology. Regresson(Quadratic), R=0.982,R2=0.965.Rgression ANOVA, F=503.047,P=0.000.It was statistically significant.After2-3weeks induction in the adipogenic, osteogenic and chondrogenic medium, stained with oil red O, alizarin red and alcian blue respectively, the results were positive.2. Labeled SVFs with Dil in vitro successfully, the cells showed strong red fluorescence under fluorescence microscope after labeled. The rate of labeled SVFs was high, the survival rate after labeled was high too, up to (92.31±1.52)%.Comparing to the unlabeled group(92.73±0.57)%,t=-0.631, p=0.550. We used General Linear Model(Repeated measures).Mauchly’s W (Mauchly’s Test of sphericity)was0.001, P=0.018. Using Test of Between-Subjects Effects,F=0.062, p=0.810, Dil had no significant impact on cells proliferation, cells proliferation curve was similar before and after labeling, there was no statistical difference; Dil markers had no significant impact on P0cells coming from SVFs adipogenic capacity, the statistical comparison before and after labeled showed t=1.562, P=0.157.there was no significant differences.3. Constructed VEGF-PLA sustained release mierospheres in vitro successfully, the surface of mirospheres was smooth, the sphere size was uniform; the average diameter of mierospheres were (27.56±4.60) nm, the rate of encapsulation was (89.24±1.24)%, the mierospheres drug loading rate was{(17.85±0.25)×10－3}%; Detected the VEGF-PLA releasing capability using ELISA kit. The miｃrospheres could release VEGF slowly within21days. At last, the cumulative release rate was80.35%. 4.8weeks after transplantation in nude mice, the mice was survived all, the grafts were visible clearly. The wet weight are (0.077±0.119) g.(0.159±0.016) g、(0.182±0.120) and comparison of three groups of P values were:among three groups P=0.000,F=167.67;group1to group2P=0.000; group2to group3P=0.001, the wet weight of group3was much higher than the other two groups, and group2was higher than group1. Tissue sections stained with HE showed that the group3had the highest survival rate, the fibrous components was the least than the other two groups, group1had the most fibrous component in the graft. The immunohistochemical findings had the similar results as HE staining, the group3had the strongest expression, then was group2, and group1had the least expression.The vessel density was:(7.25±1.83)个／HP;(12.65±1.69)个/HP;(14.75±2.02)个/HP.The statistic analysis of vessel density was:among three groups P=0.000,F=87.048; group１to group2P=0.001, group2to group3P=0.000. Group3had the highest level of blood vessel than the other groups, and group1had the least level.5.4weeks after transplantation in nude mice, the mice was survived all, the grafts were visible clearly, and group2and group3had formed engineered adipose tissue. The wet weight are (23.28±3.86) mg.(31.16±2.19) mg、(36.67±1.60) mg. The wet weight comparison of three groups of P values were:among three groups P=0.000,F=48.768;group lto group2P=0.000; group2to group3P=0.001. The vessel density was:(1.81±0.83)个／HP;(2.62±0.89)个/HP;(3.44±1.09)个/HP.The statistic analysis of vessel density was:among three groups P=0.000,F=１1.846; group1to group2P=0.019; group2to group3P=0.019. We could see from the results above, VEGF-PLA sustained release microspheres could promote the adipogenic vascularization level of SVFs and college scaffold in vivo significantly. CONCLUSIONS1. SVFs were isolated from subcutaneous tissue of healthy donors who had undergone liposuction through the method of collagenase digestion. This method of isolation and cultivation of SVFs was convenient and easier to carry out. The SVFs isolated had the capabilities of pluripotential differentiation, we could had enough ADSCs for implantation in short time. Thus, SVFs could be used as ideal seed cells for adipose tissue engineering.2. The Dil mark was suitable as SVFs tracing, the survival rate of labeled cells were high, and it did not influence cells proliferation and adipogenic differentiation ability.3. Constructed VEGF-PLA sustained release microspheres in vitro successfully, the microspheres could release VEGF slowly, so it could affect surrounding cells continuingly.4. SVFs and VEGF-PLA sustained release microspheres could promote the survival rate of fat particles significantly in vivo, and could promote adipose granule transplant vascularization level too. So the SVFs and VEGF-PLA sustained release microspheres could as the ideal seed cells and cutokine release agent in adipose tissue engineering.5. VEGF-PLA sustained release microspheres combined with SVFs could promote engineered tissue vascularization significantly in vivo, and mixed transplantation with collagen scaffold could construct mature adipose tissue in vivo.