The Study Of Preadiocytes In Vitro And In Vivo And It's Clinical Application

Background The reconstruction of soft tissue defects remains as a challenge in plastic and reconstructive surgery.Examples for considerable loss of soft connective tissue especially adipose tissue are extensive deep burns,defects after tumor resection, hereditary defects and congenital defects such as Romberg's disease and Poland syndrome. Soft tissue plays a major role in maintaining contours and also serves as a mechanical cushion for muscles,tendons and bones.Among the various approaches are local and free flaps,dermal fat grafts,collagen injections,the use of synthetic materials and free adipose tissue grafts.Every method shows considerable disadvantages,such as synthetic materials always cause foreign body reactions,biologically derived materials shrink to an unpredictable extend,et.al.Thus,autologous adipose tissue is supposed to be an ideal material,as there usually is sufficient supply for grafting,in spite of the results are generally poor and unpredictable.Studies on free adipose tissue graft showed that most of it was absorbed and replaced by fibrous tissue and oil cysts.These inferior results are thought to be due to the poor rate of revascularization of the graft and the low tolerance of ischemia of fat cells.Even the reviving technique of injecting aspirated fat fragments improved the post-operation results,50%percents of the transplanted tissue shrinked or was totally absorbted.Many studies on the establishment of efficient methods for long survival of transplanted autologous fat-tissues have been performed using animal models.It has been reported that some materials and bioactive peptides can improve the graft maintenance. Researched indicated that the addition of dextran beads with bFGF improves postoperative graft weight maintenance.Preadipocytes,scattering among the mature adipocytes in adipose tissue,are a potential seed cell for soft tissue reconstruction.The proliferative activity is high in preadipocytes,whereas fully differentiated adipocytes have lost their capacity of mitosis.Since it was possible to induce adipogenesis by providing transplanted preadipocytes a suitable microenvironment,we examined the effects of bFGF on preadipocytes 'proliferation and differation in vitro,and on forming adipose tissue in nude mice,and finally on autologous fat grafting on human body.Most published studies agreed that the evaluation of vitality should be mainly based on photographic alteration at different time course,or histopathologic studies,or fatty acid analysis.Only a few studies have attempted to document the persistence of transplanted fat with imaging techniques.The changes of fatty tissue under CT scan, especially of transplanted fat,over the time course post-transplantation has never been reported.For the first time we tried to access the CT signal behavior of transplanted fat over time and compared it with native subcutaneous facial fat in the patients with temporal lipodystrophy.Objectives:1.to Isolate and culture preadipocytes from adult human adipose tissue,2.to study the effects of bFGF on preadipocytes on proliferation and differation in vitro and on forming adipose tissue in nude mice model;3.to study the effects of bFGF on autologous fat grafting;4.to observe the CT signal behavior of transplanted fat over time.Methods1.In vitro tissue culture1.1 Isolation and culture:Preadipocytes were isolated out of fragments of freshly obtained human subcutaneous adipose tissue of adults and cultured.1.2 The second passage cells with the density of 10⁴ /cm²were cultured in DMEM containing 10ng/ml bFGF(the control only in DMEM),counting the number of the cells with MTT and making the growth curve of the cells.1.3 The second passage cells with the density of 10⁴/cm²were cultured in DMEM for 2 days,After cultured with the presence of 50 nm of insulin,100 nM of dexamethasone,10 mg/ml of transferrin,200 pM of triiodet- hyronine,and bFGF(10ng/ml) for 9 days, examining the TG mass synthesized in 3^rd,6^th and 9^th.2.In vivo experiments2.1 Preparation of human preadiocytes/ADM grafts:A suspension of 100 ul, containing 310⁴cells/ml was seeded on the upper surface by gentle dropping one the pre-wetted ADM;group a,the suspension containing 10ng/ml bFGF,group b,the suspension without bFGF,group c having not any cells.2.2.Transplation and explantion:12 fabricated preadipocyte/ADM constructs were transplanted subcutaneously to the nude mice.After 8 weeks,the mice were killed and the grafts were explanted.2.3 Histology/immunohistochemistry:the explanted material was fixed in 4%buffered formaldehyde solution.Embedded,vertically sectioned,and stained.The formalin fixed fragments were dehydrated and embedded in paraffin.Slices were prepared and stained with monoclonal antibodies specific for human vimentin.2.4 Cellularity:Microscopic fields of cuts were examined at 200^* magnification.The overall cellularity was assessed.3.clinical research3.1 20 cases with temporal lipodystrophy underwent fat injection.3.2.the concentrated and purified sample containing 8000u/20ml bFGF was injected into the temporal area of both sides.3.3 CT was performed preoperation and 2days,3monthes post -operation,respectively..3.4 Invested the rate of absorption of the fat.Results1.In vitro1.1 bFGF can improve the proliferation and differentiation of preadipocytes.1.2 bFGFcan increase the synthesis of TG of preadipocytes.2.In vivo2.1 Microscopical examination of the grafts demonstrated viable adipose tissue located on the surfaces of ADM in group a,but group c showed any adipose tissue.2.2 The cellularity was 77±56/field in group a while group b showed 66±44/field.2.3 Staining for human-vimentin indicated that cells within the grafts were significantly positive and human origin in group a.3.clinical application3.1 In 20 cases,16 areas showed excellent results,20areas showed good results,4 areas showed poor results3.2 the rate of absorption of the fat was alleviated with bFGF treatment.Conclusion1.bFGF can improve the proliferation and stimulate differentiation of Pra In vitro2.bFGF can increase the adipogenesis in vivo3.ADM is abetter scaffold for preadipocyte engineer.4.bFGF can increase the survival of injection fat grafts5.CT imaging has the potential to serve as a follow-up method for fat transplantation or even for inter-study comparisons.