| BackgroundSoft tissue defects or loss can be caused by various reasons,such as trauma,surgery,congenital dysplasia and so on.Autologous lipofilling is one of the most commonly used techniques for repairing soft tissue defects due to abandunce of filling?fat?sources,the excellence effect of filling and shaping[1-3].However,physical damage to the transplanted adipocytes or insufficient tissue/vascular regeneration of the neo-adipocytes during transplantation can lead to partial adipocytes failing to survive[4,5].However,with the reported volume retention rates of 10-85%[6-9],the inconsistency around graft survival requires pertinent attention[10].In recent years,studies try to use biomaterials to provide a conductive microenvironment for fat survival and in situ fat regeneration.[24]In particular,hydrogel was successfully injected with grafted fats as effective cell carriers to enhance cell viability among various types of biomaterials.[25,26]Hydrogel is water-rich,and may promote better oxygen and nutrient exchange.[14]Hydrogel-based biomaterials,such as collagen and hyaluronan,have shown superior tissue survival compared to simple fat grafting[16,27].However,whether these biomaterials provide a suitable microenvironment remains unclear.Extracellular matrix?ECM?from different tissues can provide different microenvironment for tissue regeneration.Additionally,previous studies show that decellularized adipose tissue can provide a unique adipogenic microenvironment.[13,28,29]In our previous study,we prepared hydrogel from acellular adipose tissue?HAPA?,a natural in situ injectable thermo-responsive material,and described its adipogenic influence on ADSCs in vitro and in vivo with additional host-derived adipogenesis inducing properties.[19]An adipogenic microenvironment may improve the survival of grafted fat.Hence,we hypothesized that HAPA may provide a favorable microenvironment to improve fat survival.Herein,we report the effect of HAPA on grafted fat survival in vitro and in vivo.This study aimed to explore the effect of hydrogel from acellular porcine adipose tissue?HAPA?on angiogenesis and fat survival for lipofilling.MethodsPart I:In vitro study of HAPA promotes the survival of free transplanted adipose tissueFree transplanted adipose tissue consists of mostly adipocytes and a very small number of adipose derived stem cells?ADSCs?.Hypoxia is one of the main causes of adipocytes and ADSCs death at an early stage after autologous adipose transplantation.The effects of HAPA on the biological behavior of ADCSs and free adipose were studied in vitro by simulating the acute hypoxic environment after adipose transplantation.For ADSCs proliferation,the ADSCs suspension was cultured in a 24-well transwell plate with 3%HAPA pre-coated the upper transwell insert while the ADSCs were planted in the lower plate.Cells were cultured under normoxia and hypoxia for 4 days.Cell proliferation was measured using a Cell Counting Kit 8 assay.To assess the adipogenic capacity of ADSCs on HAPA,ADSCs were cultured in plate pre-coated with HAPA for 14 days.Oil Red O staining was performed to evaluate adipogenic differentiation of ADSCs.VEGF secration in conditioned media was detacted via Elisa assay.Part II:In vivo analysis of HAPA-assisted lipofilling in miceAnimal model of HAPA assisited lipofilling in the dorsum of nude mice was established.Control group 1?Adipose+PBS?,control group 2?Adipose+ADSCs?,experimental group 1?HAPA+Adipose?,experimental group2?HAPA+Adipose+ADSCs?and tissue engineering adipose group?HAPA+ADSCs?were grouped by the experimental protocol.The effects of HAPA on the survival of transplanted adipose tissue were preliminarily observed by volume measurement,ultrasound evaluation and histological staining.Part III:Optimazing HAPA/Adipose Volume Ratio in HAPA assisted lipofillingAdipose group,10%HAPA group,20%HAPA group,30%HAPA group,40%HAPA group and 50%HAPA group were grouped according to the diffrent HAPA/Adipose volume ratio.Adipose or HAPA/Adipose complexities was injected into the dorsum of the nude mice subcutaneously.Gross observation,volume measurement,ultrasound observation and histological evaluation of the grafts after 4 weeks of transplantation were performed to explore the effect of HAPA/Adipose volume ratio on the survival rate of the transplated adipose tissue.Results?1?HAPA promotes ADSC proliferation under normoxia environment?P<0.05??ADSCs proliferation was enhanced under hypoxia environment and this effect was more significant when HAPA exists?P<0.05?.?2?In the normoxic environment,significant improved VEGF secretion was observed in the HAPA+ADSCs group?57.36±16.80 ng/ml?when compared with the ADSCs group?26.29±10.34 ng/ml??P<0.05?.In the hypoxic environment,the VEGF secretion improved further in the HAPA+ADSCs group?95.39±22.31 ng/ml?,while that of ADSCs was 23.32±9.15 ng/ml?P<0.05?.?3?HAPA induces adipogenesis of the ADSCs and maintains the capacity even under hypoxia atomersphere.?4?Hypoxia promotes adipose tissue necrosis and apoptosis in vitro study,HAPA show a tendency of inhibiting cell apoptosis,but there is no statistical significance?P>0.05?.?5?Adipose tissue shows the tendency of increasing HIF-1?expression level under hypoxia atomersphere,while HAPA may represse HIF-1?expression in adipocytes?P>0.05?.?6?For in vivo study,volume shrinkage was observed in all groups,ADSCs assisted lipofilling improved volume retention rate compared to control group,while HAPA assisted showed significantly higher volume retention rate after 1,2 and 4 weeks'observation?P<0.05?.?7?Adipose tissue was integrated with the undegraded HAPA.Higher adipocytes integrity,fewer vacuoles,and less fibrosis were observed in the HAPA?with or without ADSCs?groups and the ADSCs group,compared to the PBS group?P<0.05?.?8?Neovascularization of HAPA assisted lipofilling group increased significantly.ADCSs has a limited pro-angiogenesis effect,while the tissue engineering adipose group?HAPA+ADSCs?has the least neo-vascularization?P<0.05?.?9?HAPA assisted lipofilling group promoted transplanted adipose tissue survival and regeneration.?10?HAPA-assisted lipofilling?with or without ADSCs?and ADSCs showed significantly more proliferative cells?P<0.05?.There were significantly more apoptotic cells in the PBS group than other groups?P<0.05?.?11?For in vivo study,grafts of all groups had a soft texture with smooth surfaces,and clear boundaries.Among them,the adipose tissue group,10%HAPA group,20%HAPA group and 30%HAPA group all had higher volume retention rate?P<0.05?.With the increase of HAPA/adipose tissue volume ratio,fat integrity improved while vacuoles and fibrosis gradually decreased?P<0.05?.?12?With the increase of HAPA/adipose tissue volume ratio,CD31 staining showed more neo-vascularization can be found,40%and 50%HAPA group showed higher vascularizations than other groups,10%,20%and 30%HAPA group showed more vessels than control group?P<0.05?.?13?With the increase of HAPA/adipose tissue volume ratio,Perilipin A staining showed more transplanted adipocytes survived and regenerated.Conclusion1.HAPA can promotes ADSCs proliferation under both normoxic and hypoxic environment,promotes VEGF secretion and maintains the adipogenic effect on the ADSCs under hypoxic conditions.All of these behaviors might be involved in the process of HAPA promoting thesurvival of transplanted Adipose tissue.2.HAPA can induces adipogenesis in situ and promotes the survival of the grafed adipose tissue.It has a pro-angiogenesis effect in an early stage and pro-proliferative and anti-apoptosis effect,which is a promising biomaterial for adipose tissue-engineering.3.As the volume ratio of HAPA/Adipose tissue gradually increased,the survival of transplanted adipose tissue also increased,but the volume retention rate decreased gradually.In this study,30%HAPA as the optimal volume ratio for HAPA assistied lipofilling when accounting the the survival of the transplanted adipose tissue and the volume retation rate.More studies are still needed to explore optimal HAPA/Adipose volume ratio. |
PDF Full Text Request