The Experimental Study Of The Adipose-derived Stem Cells As The Seeding Cells Of Bone Tissue Engineering

Modern tissue engineering is a new science which grew rapidly in recent years. It will make epoch-making significance to the repair and replacement of tissues and organs. The tissue engineering technology mainly consists of four parts: obtaining autologous seed cells; growth and differentiation of seed cells; move-in carrier with seed cells; integration of graft and recipient site, tissue formation. To obtain, culture and differentiate the tissue cells are the premise and basis. At the present time, many tend to use autologous or allogeneic mature cells, eg. cartilage cell and adipose stem cells, as seed cells, however, because of the poor capacity of proliferation, it is hard to get number of cells, and he harvesting can make pretty loss to the donor site. So people start to search for stem cells with strong capacity of proliferation and multi-differentiation potential, and have successfully obtained marrow mesenchymal stem cells, which are proved to be able to differentiate to various mesoblastic tissues and cells. But bone marrow aspiration can be painful, and we can only get a small amount of about 10-20ml bone marrow per person, therefore, its application is limited. In recent years, some scholars have found that in some pathological situation, there is ectopic bone formation in adipose, which indicates there may exist some cells which can differentiate into other tissue/cells. The left adipose in donor sites after aspiration can still proliferate, and there are more and more fat people, many of whom ask for being aspirated on their own initiative. So it is easier to get big amount of fat, doing little hurt to donor sites at the same time. Thus make it a hot area in seed cells of tissue engineering. This experiment studied on the biological characters, multi-differentiation potential, and the performance of being cultured in scaffold of adipose cells, to investigate whether adipose stem cells can be used as the seed cells in bone tissue engineering.Chapter 1 Experimental study of the isolation, culture and biolocal characteristics of rabbit adipose derived stem cellsObjective To master method for the isolation culture and amplification of rabbit adipose derived stem cells(ADSCs) in vitro and explore their biological characteristics.Method The inguinal fat pads were excised with sterile technique, followed by digestion with 0.075% Collagenase, then the cells were cultured in Dulbecco modified Eagle medium with 10% new-born calf serum. The morphology of ADSCs was observed with inserted microscope constantly. Passage cells were examined using immunohistochemical methods about CD antigens: 34, 44. Growth curve of passage 2,5andl0cells using MTT and seeding efficiency were detected.Result Primary cells began to be adherence after culture for 3 days and were fibroblast-shaped. Cells confluenced more than 90% after 8 days and present colony whirlpool. Passage cells began to be adherence within 2 or 4 hours after sub-culture. After temporal stage of latency phase of cells prolonged slightly and proliferation became slowly through passage 10. Immunohistochemical result showed that cells expressed CD44. In contrast, no expression of the hematopoietic lineage marker CD34. The Growth curve showed that cell proliferation ability of passage 2 and 5 was more powerful than passage 10.At 2,6,12 hours after initial seeded the seeding efficiency of cells were 33%,75%,90% respectively.Conclusion The characteristics of cells isolated from adipose were similar to MSCs. In vitro it could be cultured long-term and grow stably,proliferate rapidly. The cells could be used as seed cells for tissue engineering.Chapter 2 Culture of adipose derived stem cells from rabbit and its differentiation potential in vitro Objective To study the method of isolating and culturing stem cells from rabbit adipose tissue and to determine if adipose- derived stem cells (ADSCs) harvest from rat could differentiate into osteogenicand and chondrogenic in vitro.Methods ADSCs were isolated from rabbit inguinal fat pads after extensivewashing with phosphate- derived saline and digesting with Collagenase. After primary culture in control medium and expanded to three passages, the cells were incubated in an osteogenic medium and an chondrogenic medium for 2- 4 weeks to induce osteogenesis and chondrogenesis, respectively. Evidences of osteogenic differentiation were detected by a ALP solution and Von kossa staining, and while chondrogenic differentiation was confirmed using the toluidine blue staining and type II collagen immunohistochemistry. It exhibited a heterogeneous population of fibroblast like cells morphologically. ADSCs induced to osteogenesis were stained positively for alkaline Phosphatase activity after 2 weeks and formed mineralized nodular structures, as conformed by Von kossa staining.Conclusion Adipose- derived stem cells can be isolated from rabbit adipose tissue. Their biological characteristics are similar with mesenchymal stem cells (MSCs), and have the potential to differentiate into osteogenic and chondrogenic lineage. It may be an idea source of ADSCs for tissue engineering.Chapter 3 Chondrogenic differentiation of rabbits adipose-derived stem cells in fibrin glue/hyaluronic acid scaffoldObjective To explore chondrogenic differentiation capacity of rabbit adipose derived stem cells cultured and induced in fibrin glue/hyaluronic acid scaffold.Method Isolated and cultured cells according to chapter 1. Passages 3 cells were resuspended at concentration of 1×10~7/ml per milliliter and seeded in fibrin glue/hyaluronic acid scaffold, then was induced in chondrogenic medium which consisted of Dulbecco medium with 10%new-born calf serum supplemented with 10ng/mL TGF-β1,6.25ug/mL transferring and 6.25ug/mL insulin and 0.1nmol/mL of Dexamethasone. The scaffold was embedded in paraffin and cut in section 3 weeks after initial chondrogenic induction. Chondrogenesis was assessed using HE staining, toluidine blue staining and type II collagen immunohistochemistry. The impanted cells and scaffold were observed with scanning electron microscope.Result 3 weeks after initial induction many round cells were observed at different layer of scaffold with inverted microscope. Some cells attached to the scaffold. HE scaffold stained positively for toluidine blue staining. The cells stained positively for toluidine blue staining. Type II collagen immunohistochemistry staining showed positive staining in endochylema and some part of scaffold. Scanning electron microscope showed spherical cells distributed in pore of the scaffold.Conclusion Rabbit adipose derived stem cells seeded in fibrin glue/hyaluronic acid scaffold through chondrogenic induction could form a material with part character of cartilaginous tissue.