The Study About Cultivation And Osteogenic Potential Of Human Adipose Derived-stromal Cells

Background Disconnection of bone fracture and bone defection are two common diseases in clinical orthopaedics . In now days the most principal methods to treat them are autogenous bone transplantation , variant bone transplantation and implantation of artificial bone, however, all these methods are not good enough. It is just the osteoblasts that play an important role in the reparative process of bone fracture, and it is also the little number of them that mostly leads to the hard reparation of disconnection of bone fracture and bulk bone defection. So given enough osteoblasts in the fracture site, there would be better accretion of bone fracture. Bone tissue engineering is also founded on the basis of this principle. Seed cells (osteoblast) is a chief link or an essential element in the construction and the applied research of bone tissue engineering and it is also the premise to ensure the bone tissue engineering to get good continuous deep study. Then it seems important to choose an ideal kind of seed cells. Ideal seed cells should have many features, such as: being easily made use of and having little damage to body, being able to differentiate directly to osteoblasts in vitro culture and have fairly productivity and having good ability to accommodate to recipient site and to keep ossification activity. Now seed cells researched are mostly made up from bone, periosteum , bone marrow and ectosteal tissues, but all these cells have their own deficients.Human adipose tissue-derived stromal cells which have competence of the adult stem cells origin from mesoblast and they can also differentiate to many kinds of cells. For the reason of all these featuresof stromal cell, it has become a field mostly researched in tissue engineering for recent years. Human adipose tissue generally exits in our bodies, so it can be looked as an adult stem cell bank easily obtained and affluently reserved. Being flexibly made use of , having little trauma and having ability of multi-directional differentiation that can be controlled, adipose tissue derived stromal cells seem to be a kind of perfect cells to tissue engineering. Furthermore, cell culture and amplification are comparatively easy so we can get a great quantity of cells applied by autografting and allografting. For these reasons it has been paid great attention by the academic circles recently. In now days , as to human adipose tissue-derived stromal cells, foreign countries have carried out comparatively more researches which , however, are mainly about animal experiments. There have never been reported about such research in the field of domestic orthopedics. Objective To isolate human adipose tissue-derived stromal cells from human adipose tissue and study the potential of osteogenic differential after induction.Method Human adipose tissues were collected during the operation and digested with collagenase type I . The obtained adipose tissue-derived stromal cells were plated in DMEM/F12 as primary culture for about seven to ten days. The cells are harvested and plated in inductive culture(supplemented with 50mg/l freshly prepared ascorbic acid, 10mmol/l Na P -glycerophosphate and 10~8mol/l dexamethasone) . Now the generally accepted criterions to identity osteoblast are the cellular morphology, alkaline phosphatase activities, type I collagen and thecontent of bone gla protein. Then we can identity the activity of the ossify obtained by differentiation applying many approaches , for instance, using observation of morphology and Gomori straining to detect alkali phosphatase, and using Von Kossa staining to detect the formation of calcium nodus . The cell-anchored slips were removed and demonstrated by Gomori and Von Kossa staining.Results It was observed that the size and shape of induced cells were much different from those of non-induced cells. Gomori and Von Kossa staining demonstracted positive expression of ALP and formation of mineralized nods in extracellular matrix. Meanwhile we still found it had different levels that cells obtained during different induced process inverted to osteoplasts. The result of the quality and the quantity of celluar inversion obtained after three weeks' induction was obviously higher than that obtained after two weeks' induction. Conclusion Recording to these results we can have an conclusion that adipose tissue-derived stromal cells have features as adult stem cells to differentiate to other cells, and they also have potential to differentiate into osteoblasts in the condition of ossify inductive culture(10mmol/l Na ï¿¡ -glycerophosphate , 10~smol/l dexamethasone and 50mg/l vitamin C). Adipose tissue-derived stromal cells will be another alternative source for cell-based engineering in bone tissue engineering and will process favorable applied perspective because they own superior conditions than other sources of seed cells.As the seed cells of bone tissue engineering, adipose tissue-derived stromal cells have absolute superiority than any other sources of seedcells. Nevertheless, bone tissue engineering needs not only seed cells but also some special brackets supplied to keep osteoplasts stick to it and grow up when explanted into the area of bone defection. Nowdays the mostly used materials are hydroxyapatite and coral and so on. However, it needs further research to find which material has superiority and which is more fit for the overgrowth of osteoblasts and after embedding which materials into bodies there will generate smaller immunization.