An Experimental Studies On Construction Of Engineered Adipose Tissue Using Adipose Derived Stem Cells

ObjectivesThe purpose of this study is:(1)to explore the biological characteristics in vitro and histocompatibility in vivo of porous poly(γ-benzyl-L-glutamate)(PBLG)microspheres;(2)to construct engineered adipose tissue subcutaneously via injection of PBLG microspheres loaded with human adipose derived stem cells(h ASCs);(3)to construct epidural adipose tissue using poly(L-glutamic acid)(PLGA)/chitosan(CS)loaded with New Zealand rabbit adipose derived stem cells(r ASCs)to inhibit proliferation of scar tissue.Materials and MethodsHuman fibroblasts(FBs)were isolated from foreskin sample of patients who received circumcision at the Department of Urological Surgery of Shanghai Renji Hospital.Following seeding FBs into PBLG microspheres to construct cells/microspheres,cytotoxicity and expressions of collagen related genes,as well as cell proliferation were determined in vitro.The histocompatibility of PBLG microspheres in vivo was assessed by histological examination at 2,4,8 weeks post-injection.To further explore whether PBLG microspheres could be used as bioscaffolds for adipose tissue engineering,we constructed h ASCs/PBLG complex.Following adipogenic induction for 7 days in vitro,the adipogenic induced h ASCs/PBLG complex were injectedinto the scalp of nude mice.At 4 and 8 weeks post-injection,respectively,the neo-generated tissues were harvested from three groups.Histological examination(H&E,Masson’s trichrome,Oil red O staning and SEM)and biochemical analysis(Real-time PCR,GPDH activity and Hydroxyproline quantification)were used to evaluate the similarity of neo-generated tissues to native adipose tissue.In addition,to investigate whether PLGA/CS scaffolds could be used as bioscaffolds in adipose tissue engineering to prevent scar proliferation,we firstly isolated r ASCs from inguinal fat pads of New Zealand white rabbits.Then,we constructed cells/scaffolds complex.Following adipogenic induction for 7 days in vitro,the adipogenic cells/scaffolds complex were implanted into the surgery sites after vertebral lamina were removed.MRI images were used to observe the heal of surgical cites at 1,12,24 weeks post-operation.At 24 weeks post-operation,the New Zealand white rabbits were humanely sacrificed,histological examination(H&E,Masson’s trichrome and Oil red O staining)were used to assess tissue regeneration.ResultsWe found that porous PBLG microspheres had low cytotoxicity,which is benefit for cell proliferation.However,when inoculated within PBLG microspheres,the seeded FBs showed relatively low expression of type 1 and type 3 collagen genes,and high expression of IL-8 gene compared with non-seeded FBs cultured in normal culture dishes.Experiments in vitro and in vivo were demonstrated that injectable PBLG microspheres were benefit for adipogenic differentiation of h ASCs.In vivo neo-generated tissue was similar to native adipose tissue in fat lobule microtissues and blood supply.Implantable PLGA/CS scaffold was an excellent scaffold for cell proliferation and adipogenic differentiation of r ASCs.Epidural engineered tissue with the characteristics of adipose tissue could inhibit the proliferation of epidural scar.Conclusions(1)Porous PBLG microspheres had excellent biological characteristics,and modulated collagen related gene expression.(2)Injectable porous PBLG microspheres could be used as cell delivery vehicle in adipose tissue engineering,and were suitable for soft tissue augmentation.(3)Epidural engineered tissues using implantable PLGA/CS scaffold loaded with adipogenic differentiated r ASCs could inhibit proliferation and adherence of epidural scar.