Research On The Mechanisms Of Long-term Volume Retention After SVF-gel Grafting

BackgroundA wide range of soft tissue defect caused by such as trauma,disease and congenital malformation will be difficult to heal depending on our body itself.At present,a variety of methods like the dermis transplantation,synthetic materials,allogeneic and heterogeneous fillers are used in clinic.And due to its good biocompatibility,non-immunogenicity,convenience to obtain,and inexpensive features,autologous fat transplantation is considering the best way of repairing theoretically.Although clinical applications of autologous fat transplantation in facial profile plastic,hand rejuvenation,breast augmentation or reconstruction and the rest of the body contouring is widespread.For the reason techniques distinguish among physicians in adipose tissue acquisition,processing and transplant operation,reports of the volume absorption rate of fat transplantation range from 25%to 80%,which require excessive filling or repeated injections of 2-3 times to achieve the expected effect.Additionally,the long-term possibility of complications such as oil capsule formation and fiber calcification greatly limits its clinical application.Therefore,researchers have proposed many methods that were considered capable to improve the retention rate of fat transplantation:The Coleman techniques(including low pressure liposuction,centrifugation and three-dimensional multichannel injection),liposuction technology with larger needle diameter and low-shear,processing technology(including centrifugation,telfa gauze and washing-filtering),recipient site preconditioning by microneedle or expansion,and addition of stem cells,platelet-rich plasma(PRP)and growth factors,etc.By preserving intact fat structure as much as possible,increasing tissue perfusion,promoting angiogenesis,in order to promote cells survival in the extremely ischemic hypoxic environment before neovascular reconstruction.However,because of the lack of high quality clinical or preclinical data,it's difficult to assess these methods' relative significance.Yet there is no widely recognized optimized solution to avoid graft necrosis.As the continuous in-depth study of the volume retention mode after fat transplantation,it's found that in addition to the graft directly survive,host-derived tissue regeneration also play an important role.Researches show that normal physiological structure of adipose tissue is mainly composed of two parts:the mature adipocyte and Stromal vascular fraction.Mature adipocyte number accounts for only 7%,but occupies more than 90%of the volume in the tissue,and has high oxygen consumption.In the early after fat transplantation,blood supply has not yet been established,few mature fat cells can survive only within the scope of less than 300 mm area in the outermost layers of the graft by tissue infiltration.In the center of graft,most of mature adipocytes are unable to tolerate ischemic hypoxic environment,appear necrosis and release a large amount of lipid drops,then cause the inflammatory cells infiltrating,surrounding the droplets and cellular debris and phagocytosis,led to the volume absorption.Eventually,small-size lipid droplets will be cleared,large lipid droplets will be encased by fibrillar connective tissue and form oil cyst,some of them will end up with calcification.At the same time,in the outer layer of the graft,Adipose-derived stem cells of the SVF are more capable to tolerate ischemic hypoxic environment,which can secrete revascularization-related growth factor,promote angiogenesis outside-in,induce host-derived preadipocyte and a few remaining stromal stem cells move in,proliferate and differentiate along with the neovascular,and eventually attain local regeneration of fat lobulus.We found that necrotic fat cells is the primary cause to lower fat volume retention rate and of complications after fat transplantation.Selective elimination of mature adipocytes may result in better outcomes and a different underlying retention mode.In order to remove the "invalid volume"-mature adipocytes before transplantation,traditional method of collagenase digestion and liquid ingredients such as isopropyl alcohol are bound to introduce exogenous materials,bring the risk of biological pollution,more difficult for the routine clinical application.So we make an innovation of physical method,let the liposuction aspirate rapidly get through,a small diameter pipe system,using shear force selectively destroy relatively larger mature adipocytes,but not damage the small SVF cells.Following centrifugal remove more than 90%of the original volume of the lipid droplets.On the premise of not adding any exogenous substances,we obtained a novel adipose product,which highly concentrated the SVF cells under the protection of ECM,average concentration can reach 4.5 times higher than the original,containing the ASCs was 6.3 times higher than the original,and the osteogenic,cartilaginous,adipogenic differentiation capacity is not compromised,We named it "SVF-gel".ObjectionThis study will be around three questions:1.Can SVF-gel keeps a long-term retention rate after transplantation?Did it successfully induce adipogenesis in vivo?2.What's the concrete mechanism?3.What's the source mainly responsible for adipogenesis?For further discussion.Firstly explore the dynamic histologic changes after transplantation of a mature-adipocyte-free product named "SVF-gel".At each time point,compare effect of transplantation by volume and retention rate measure of both SVF-gel transplantation group and traditional fat transplantation group,together with HE staining,masson-trichrome staining and immunohistochemical examinations to explore the efficiency of SVF-gel as a kind of tissue filler,the mechanism and cell sources of adipogenesis after transplantation,which would provide data for clinical application of tissue filling.Methods and materials1.The traditional fat transplantation group and SVF-gel transplantation group preparation and evaluation of graft survival.Using syringe method collected liposuction from adult healthy women's abdominal and femoral subcutaneous fat.Partly use Coleman technology to prepare as traditional fat transplantation group,another part of the preparation of SVF-gel include the following steps:firstly centrifuge the lipoaspirate 1200 g/min for 3 min,then discard the liquid in the bottom,recovery the upper layer oil for backup,put the middle layer fat tissue into two 20 ml syringes,joint seal through a Luer taper,continuously push the syringe piston containing adipose tissue at the speed of 10 ml/s for 1 min.Then add the recycling oil and slowly push another 3-5 times,filter with a Nanotransfer that aperture<0.2 mm,block syringe export and pullback the syringe piston to maintain negative pressure in syringe,make mild concussion until flocculation phenomenon occur.Finally 2000 g/min centrifugal for 3 min,the middle layer product is the SVF-gel.In accordance with the principle of randomization,use 1 ml syringe connecting on a blunt end needle,to inject two groups on both sides in nude mice subcutaneous level of the ribs area.After injection(1 day,3 day,5 day,7 day,11 day,15 day,30 day,60 day and 90 day),a total of nine point in time,to observe the general situation of the graft.Graft volume was measured by applying the method of drainage.Calculate volume retention rate.Review the status of graft By HE staining and Masson staining.2.Evaluation of angiogenesis and adipogenesis in the grafts.In 1 day,3 day,5 day,7 day,11 day,15 day,30 day,60 day and 90 day after injection,detection of HE staining and immunohistochemical staining of traditional fat transplantation group and SVF-gel transplantation group was maken,(respectively using Isolectin,Perilipin antibodies and Hoechst staining to indicate blood vessels,fat cells and nucleated cells),vascular density,the change trend of necrosis area and statistical analysis between the two groups was analysed by system software.3.Evaluation of inflammation levels in the grafts.1 day,3 day,5 day,7 day,11 day,15 day,30 day,60 day and 90 day after injection,evaluate inflammation level in both groups by immunohistochemical staining(respectively use MAC2,CD206 antibodies and Hoechst staining to indicate macrophages and nucleated cells).The number of infiltrated macrophages between the two groups was analysed by system software,to compare the change trend of inflammation level over time.4.Detection of the cell sources of adipogenesis in SVF-gel transplantation.By 1 day,3 day,5 day,7 day,11 day,15 day,30 day,60 day and 90 day after transplantation,detect the cell sources of adipogenesis by immunohistochemical staining(use HLA antibodies,Perilipin and Hoechst staining respectively to indicate human-derived cells,adipocytes and nucleated cells),observe the change of the human-derived cells in SVF-gel graft overtime,evaluate the component of the cell sources of graft tissue.5.Statistical analysisUse the statistical software SPSS 22.0 for data procession.Use repeated measurement data analysis method for variance analysis of the different time points.Use paired sample t test method for comparison between the two groups difference.(P<0.05 was considered significant difference statistically),Results1.The Coleman fat and SVF-gel grafts showed a similar appearance and texture from day 1 to day 15,with both graft types covered with a thin,well-vascularized fibrous capsule.From day 15 to 90,the volume of each SVF-gel graft remained constant,and their texture was soft and similar to that of normal fat tissue at all time points.By contrast,the Coleman fat graft volumes decreased markedly from day 15 to day 90.Poor surface vascularization and visible oil cysts were found in this group.Quantification of graft volume indicated that the retention rate of the Coleman fat group remained stable from day 1 to day 15,decreased sharply from day 15 to day 30,and remained constant thereafter.Although graft retention rate in the SVF-gel group decreased over the first 3 days,it returned to initial weight on day 11 and remained constant thereafter.The retention rate was significantly higher in the SVF-gel group than in the Coleman fat group(82%± 15%vs.42%± 9%,P<0.05)2.Histological analysis showed that samples from the Coleman fat group had normal adipose structure soon after transplantation(days 1-3).Small numbers of oil cysts were observed in the central zone of these grafts after day 3,with both the number and volume of these cysts increasing over time.Although vascularized connective tissue was present in the superficial area on day 15,large amounts of necrotic tissue and large oil cysts were observed in the interior zone.From day 30-90,a few newly formed small adipocytes appeared within the vascularized connective tissue and matured,but some large oil cysts had not been completely absorbed by day 90.By contrast,both the superficial and central areas of the SVF-gel contained large numbers of small-sized preadipocytes with multiple intracellular lipid droplets,extensive well-vascularized connective tissue,and infiltrated inflammatory cells,as early as day 3 after transplantation.Beginning on day 15,the number of inflammatory cells started to gradually decrease and adipocytes began to mature.By day 90,most of the vascularized connective tissue in the grafts had been replaced by mature adipocytes,with a structure similar to that of normal tissue.Imaging of sections of complete SVF-gel and Coleman fat grafts(day 90)indicated that SVF-gel grafts had greater tissue integrity and fewer oil cysts,whereas fat grafts contained large areas of necrosis in their centers and numerous large oil cysts.By day 90,fat grafts had large areas of necrosis in the center and numerous large oil cysts,whereas SVF-gel grafts had better tissue integrity and fewer oil cysts.3.Use masson trichromatic staining to observe collagen of the traditional fat transplantation group and SVF-gel transplantation group.Results suggest 30 days after the transplantation,traditional fat transplantation group emerged a large amount of connective tissue,which contain a few small-size preadipocytes.And the volume occupied by the connective tissue in SVF-gel transplantation group had in reducing unceasingly,gradually became as the normal adipose tissue structure.Quantitative analysis for tissue fibrosis level of two groups,traditional fat transplantation group were significantly higher than SVF-gel transplantation group in 30 day,60 day and 90 day after transplantation.4.Immunostaining of Coleman fat grafts showed a large number of perilipin-positi-ve mature adipocytes on day 1.However,some of these adipocytes died after day 3 and the perilipin-negative areas gradually increased.By day 15,most of the Coleman fat grafts contained perilipin-negative areas and numerous oil cysts,similar to the results of H&E staining.By contrast,SVF-gel grafts 1 day after transplantation contained few perilipin-positive adipocytes,but contained rich lectin-positive vascular fractions.Small-sized preadipocytes with multiple intracellular lipid droplets began to appear on day 3.Each field of the SVF-gel graft sections on day 15 showed numerous perilipin-positive adipocytes and newly formed blood vessels.5.Although a number of Mac+/CD206-M1 macrophages were detected in the Coleman fat grafts during the first 2 weeks after grafting,the number of macrophages infiltrating these grafts was higher after 30 days,with most of them being M2 polarized(Mac+/CD206+).These macrophages clustered surrounded large oil droplets to form a typical "crown-like" structure.By contrast,imaging of SVF-gel showed macrophage infiltration beginning on day 7,with a decrease in infiltration on day 30.Interestingly,the "crown-like" structures in the SVF-gel grafts were small-sized,and few large oil droplets surrounded by macrophages could be detected.6.Grafts were doubly stained with antibody to HLA and the adipocyte antigen perilipin to identify the origin of newly formed adipocytes in SVF-gel grafts.By day 15,these grafts contained large numbers of immature(small-sized)and mature(large-sized)adipocytes,with nearly all being host-derived(HLA-/perilipin+).Most of the HLA-positive cells appeared in the intercellular space between mature adipocytes,with some having a vessel-like lumen structure,indicating large number of SVF cells in SVF-gel did not directly differentiate into mature adipocytes,but formed parts of the neovascular,the newborn adipocytes was mainly differentiated by host-derived cells.ConclusionSVF-gel has a high long-term retention rate and a unique adipose regeneration mode,involving prompt inflammation and infiltration of immune cells,stimulating rapid angiogenesis and inducing host-cell-mediated adipogenesis.