| Objectve: Repair and reconstruction of soft tissue defects is the difficult problem and challenge for plastic surgery.The traditional repair methods mainly include autologous flap transplantation,free fat graft and allograft material filling,etc,but all of these methods have drawbacks.The adipose tissue engineering constructed by seed cells and scaffolds is more suitable for soft tissue repair and reconstruction.Extracellular matrix(ECM)is some macromolecules secreted by cells into the extracellular space,which plays an important role between the cells and tissues.Intact ECM has been derived from a variety of tissues,such as porcine small intestinal submucosa and decellularized dermis matrix,which have been used in clinical.However,all of these biological products derived from animals or carcasses,it is very difficult to avoid the risk of immune rejection and pathogen transmission.Adipose tissue is loose connective tissue,which contains a lot of ECM components in addition to various cells.Adipose tissue can be obtained by liposuction from healthy human body.Liposuction is one of the most fashionable plastic surgery with the minimally invasive and fastest recovery.Scaffolds is the key part for tissue engineering,which can provide space support for tissue regeneration and involved in the adhesion,proliferation,migration,and angiogenesis of cells.Extracellular matrix derived from different tissues can be fabricate a variety of bioactive scaffolds,some have been used clinically.But These scaffolds are solid forms,which require surgical implantation and increase the surgical trauma.Soft tissue defects have more superficial location and irregular shape,therefore,fabrication of an injectable hydrogel scaffold is very important for the treatment of soft tissue defects.In this experiment,we use a decellularization method that combines physics,chemical reagents and enzymes to obtain decellularized adipose tissue(DAT)from human lipoaspirate.And then,we fabricate DAT into injectable hydrogel scaffolds and assess the completeness of decellularization,reserved ECM components,physical and chemical properties of the gel,its compatibility with cells and tissues and other related properties.To investigate whether the scaffolds can be used for adipose tissue engineering,and to provide new biological scaffolds for soft tissue repair and reconstruction.Methods: 1.Using decellularization methods combined of physical,chemical and enzymatic,we extracted the adipose tissue ECM from healthy human lipoaspirate.The ECM extracted from adipose tissue was operated HE staining,DAPI staining,oil red O staining,the specific staining of collagen fibers,reticular fibers and elastic fibers,the immunohistochemical staining of collagen I,IV,laminin,fibronectin,the quantitative detection of residual DNA and preserved collagens and GAGs.2.With collagenase digestion method,we isolated human adipose-derived stem cells(hADSCs)and subcultured.The hADSCs were determined proliferation and growth curve,induced adipogenic and osteogenic differentiation.3.DAT is dissolved in HCl and pepsin after freeze-dried and ground into powder,and the DAT solution forms gel when the pH was adjusted to neutral by NaOH.Hydrogel ultrastructure,injectability and gel process in vitro was observed.Turbidity kinetic and SDS-polyacrylamide gel electrophoresis was operated,and DAT hydrogel formed gel in rats was observed.Rat tail collagen I hydrogel scaffolds was prepared by contrast in this experiment.4.We examined the proliferation 、 toxicity and adipogenic of ADCSs after compositing with DAT hydrogel scaffold in vitro.Specimens drawn at different time periods were implemented HE staining,masson staining and immunohistochemical staining of CD45,CD68 to observe the inflammatory response in vivo when the DAT hydrogel scaffold was implanted subcutaneously in rats.Results: 1.Extraction and identification of DAT: Decellularized adipose tissue(DAT)showed soft variable shape with opaque milky white after human adipose tissue derived from liposuction operated by a series of decellularization methods.The cell components were not observed in the HE and DAPI staining and only with tiny residual DNA.Collagen fibers,reticular fibers,elastic fibers were observed in histological staining and collagen I,IV,laminin,fibronectin were observed in immunohistochemistry staining.Quantitative detection of collagens and GAGs contents showed that both components were present in the decellularized tissue with a slight decrease.2.Isolation and identification of ADSCs: ADSCs were isolated by using collagenase digestion and non-adherent cells were removed by adherent growth.ADSCs had strong self-renewal capacity so that they can keep good proliferative states when passaged to 10 generations.Isolated cells can be induced into adipocytes and osteoblasts under certain conditions.3.Fabrication of DAT hydrogel scaffolds and the detection of properties: DAT was dissolved in HCl and pepsin after freeze-dried and ground into powders,and the DAT solution formed gel when the pH was adjusted to neutral by NaOH.DAT hydrogels can pass 18 G needles at 4℃,and automatically formed gels in vivo and in vitro at 37℃.Gel was porous scaffold with different protein contents compared with type I collagen.Properties of the hydrogel scaffolds differ with the concentration,the higher concentration of the gel had the firmer appearance,earlier start time and faster rate of gelation.4.The biocompatibility testing in vitro and in vivo of DAT hydrogel: DAT hydrogel scaffolds had been verified that it was non-toxic to ADSCs,can promote the growth and proliferation of ADSCs,and induced spontaneous conversion of ADSCs to adipocytes in vitro.After being implanted subcutaneously in rats,the scaffolds can cause some early inflammation,and the inflammation gradually disappears after some weeks.DAT hydrogel can stimulate the host cells migration,promote the formation of new blood vessels and adipose tissue.Conclusion: 1.By using decellularization method combined of physical,chemical and enzymatic,we can extract DAT from healthy human lipoaspirateS.Cellular components and the grease were removed and most of the ECM components were retained in the obtained DAT.2.DAT can be fabricated to injectable hydrogel scaffolds which can automatically form gels in vivo and in vitro.Gel was porous scaffolds with complex protein contents compared with type I collagen.The properties of the hydrogel scaffolds differed with the concentration.3.DAT hydrogel scaffolds have good biocompatibility,they can promote the proliferation and adipogenesis of cells in vitro and promote the infiltration and angiogenesis of host cells by being implanted in the body.The human DAT hydrogel scaffolds were the ideal scaffolds and filling materials for soft tissue defects and adipose tissue engineering. |
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