| Backgroud and ObjectivePRP can significantly improve the retention rate of transplanted fat.However,Its difficult to maintain an effectively optimum concentration in the objective area and continue to play a role.Therefore,how to maintain the effective concentration of PRP in the process of fat transplantation is the key to give full play to its role.The controlled release drug delivery system can effectively delay the release rate of drugs.Gelatin microspheres,as the most commonly used controlled release carriers,have good mechanical stability and degradation rate,and good biological safety.However,gelatin microspheres immobilize platelet/cytokines by physical adsorption,which results in the limitation of the platelet load and can not achieve the expected release effect.A large number of studies have proven that dopamine can promote cell adhesion,and has good biocompatibility.So we provide the hypothesis that GM microspheres coated with pDA by surface modification can significantly enhance the adhesion ability of microspheres,and attach more platelets to controlled release growth factors,and improve the retention rate of transplanted fat.MethodsGM was prepared though EDC crosslinking of gelatin solution in an emulsion state as previously reported with slight modification.pDA coating was formed through in-situ spontaneous oxidative polymerization of dopamine.Lyophilized GM.PRP were immobilized onto the GM.The morphology of the GM,GM-pDA-PRP was observed by scanning electron microscopy(SEM).The non-adhesive platelets in the remaining buffer after coating process were quantified by the automatic counter.The number of platelets immobilized on GMs was defined by the difference between the amount of initial platelets and the remaining amount in the buffer.Growth factors'release profiles in each group were quantified by ELISA using VEGF and PDGF ELISA Kits.ASCs' isolation from male Sprague-Dawley rats and cell cultures were carried out by following the reported methods by Zuk et al.and our previous work.Cell Counting Kit-8 test was carried out to investigate the cell proliferation with different samples.The fat flaps of Adult male Sprague-Dawley rats were harvested.Four groups were studied:(1)fat;(2)GM-PRP-fat;(3)PRP-fat;and(4)GM-pDA-PRP-fat.The same mixture was injected into same rats' subcutaneous dorsal pockets on the right and left sides,every side had two injection sites,and the volume was 0.5ml in each group.At a designed time(4W,8W,16W)animals were sacrificed by an overdose injection of pentobarbital,and the graft fat samples were harvested by careful removal from dorsum pockets and weight by liquid replacement.Harvested sample weight to the initial weight(Waay/W0)was used to normalize the retention ratio.Visualization of staining of GM-pDA-PRP assisted fat graft after 16 weeks post-operation and normal adipose tissue were performed.Each harvested specimen at 4,8,16 weeks after fat graft was cut into slices of the 5 ?m thick in cross-section.For histological analysis,the section was stained with H&E dyes,and the number of newly formed blood vessels in the sample was counted in 10 random high view fields in each slide by a blind fashion.Immunohistological analysis was for 4 weeks samples and performance with the following primary antibodies:mouse monoclonal CD31,rabbit anti-human Ki67,and rabbit monoclonal CD34,fluorescent antibody was used as secondary antibody.4.Statistical analysisAll the statistical data were expressed as the mean ± standard error of the mean.The data were analyzed by ANOVA to determine the statistical significance between the two mean values.P values less than 0.05 were considered to be significant.Results1.Successful preparation of GM-pDA-PRPThe condensed platelets were immobilized on the surface of microspheres with the pDA coating.SEM images showed the smooth spherical surface and good dispersion of the microspheres,and platelets were immobilized onto the pDA coated microsphere surface.pDA modification to the microsphere leads to a significant increase in the numbers of immobilized platelets compared with GM(p<0.5).2.Growth factors release detectionAfter the surface modification by poly dopamine,the adsorption of platelet as high as 68%,GM-pDA-PRP group showed higher and prolonged release in both growth factor release behaviors.In comparison with the control,the ASCs cultured with PRP group had a relatively higher level of cell viability.3.A significant increase in fat graft survival was demonstrated by analyzing the weight maintenance between GM-pDA-PRP group compared to the rest three groups.The mature vessel density in the groups PRP and GM-pDA-PRP groups were higher than that in the other groups at 4 weeks after transplantation(p<0.05).Whole-mount staining images revealed the mature adipocytes,blood vessels or capillaries were arranged in a typical manner in GM-pDA-PRP group like in normal adipose tissue.The adipocytes showed spherical or hexagonal patterns with a substantial amount of interstitial space,the mature vessels ran alongside the adipose tissue and forming a well-organized network.Compared to fat group,GM-pDA-PRP group resulted in about 6.4-fold of quantitative CD31 positive cells which demonstrated this system supported the formation of stronger and more mature vessels in the graft fat samples compares with the other three groups(p<0.05).The numbers of CD34+ cells in the graft tissues in GM-pDA-PRP group and PRP were 3.5-fold and the numbers of Ki67 fluorescence-positive cells in the granulation tissues of the GM-pDA-PRP group were 5.2 fold of the control grafts after 4 weeks(p<0.05).ConclusionWe successfully prepared GM-pDA-PRP microspheres slow-release system,which can load a large number of platelets and growth factors and has a good sustained-release effect.GM-pDA-PRP significantly improve the retention rate of grafts after fat transplantation through promoting the proliferation of ASCs and sustained releasing of growth factors,which resulted in better angiogenesis and adipogenesis. |
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