| Bone defect is a tough problem in orthopaedics.There are many troubles in the repair of the defect by using autografts,allografts,or xenografts.In1980s, repairing bone defect have entered the era of tissue engineering.Growth factor,seed cell and scaffold are three elements of tissue engineering.Today,the problem of seed cells is chief point and essential element of the construction and exploratory development of tissue engineering bone,and also is the prerequisite for further development of the bone tissue engineering.How to get the seed cells of sufficient quantity and favoruable function is a bottleneck in this field.The bone marrow stromal cells(BMSCs) have been applicated generally as seed cells of bone tissue engineering.But in clinical research,we found that the cells come from bone marrow was limited in quantity and could cause some complications in harvesting from the bone marrow.The adipose-derived stem cells(ADSCs) is a kind of multidifferentiation potential stem cells that could be isolated from adipose tissue.The ADSCs can proliferate stably and has low rate of apoptosis in vitro.It has been a new point in the study of bone tissue engineering because of its easy availability,great quantity,large-scale culture and little damage to body.Generally,we will induce seed cells to differentiate into osteoblasts in the construction of tissue engineering bone,so,to explore the best way of the mineralization of the ADSCs is a practical topic.In our study,we induce ADSCs to differentiate intoosteoblasts in different condition to investigate the optimal condition.1. Adipose-derived stem cells were differentiated into osteoblasts by rhBMP-2 in vitroTo investigate the effect of rhBMP-2 on differentiation and proliferation of cultured adipose derived stem cells in vitro, and observe whether there was a dose-dependent effect. The fat tissue that was resected from posterior cervical region of rabbits.Stem cells were isolated by type I collagenase digesting.ADSCs were cultured in vitro,and induced to differentiate into osteoblasts by rhBMP-2 of different concentrations.Osteoblasts were identified by ALP stain and type I immunohistochemistry stain. The effect of rhBMP-2 on differentiation and proliferation of the stem cells was studied by determination of ALP and MTT activity. The cells isolated from rabbit adipose tissue could rapidly expand.ALP stain and type I collagen immunohistochemistry stain was showed positive;ALP activity increased rapidly with the increase of the dose of rhBMP-2 within the range of 100-400ng/ml(P<0.05),yet there was no significant difference among the groups over 400ng/ml(P>0.05); there was no significant difference in proliferation activity below 800ng/ml rhBMP-2(P>0.05),yet over 800ng/ml of rhBMP-2 could inhibit proliferation significantly(P<0.05). In vitro, rhBMP-2 could induce adipose derived stem cells to differentiate into osteoblasts; 400-800ng/ml rhBMP-2 could promote differentiation and proliferation of adipose derived stem cells significantly.2. Studies on optimization of mineralization condition of adipose-derived adult stem cells To investigate the biological character istics of adipose derived stem cells in different mineralization condition. The fat tissue that was resected from posterior cervical region of rabbits. Stem cells were isolated by type I collagenase digesting.ADSCs were cultured in vitro,in three groups: induced to differentiate into osteoblasts by mineralization medium,BMP and the two combined. In control group,the DMEM with 10%FBS was used.Osteoblasts were identified by ALP stain and type I immunohistochemistry stain. The effect of different groups on differentiation and proliferation of the stem cells was compared between the groups by determination of ALP and MTT activity. In experimental groups,ALP stain and type I collagen immunohistoch- emistry stain was showed positive. Compared with the control group,the proliferation of ADSCs in experimental group decreased,but ALP level increased and type I collagen was positive.Of the three experimental groups,the mineralization medium-BMP combined group significantly expressed the osteoblastic marker.ADSCs in the combined group can be induced to differentiate into osteoblasts in a shorter culture time.3.Effect of age on osteogenic differentiation of adipose-derived stem cells in vitroTo determine whether or not the donor age influences osteogenic differentiation capability of adipose-derived stem cells.The ADSCs that was isolated from rabbit of different age was cultured in vitro and induced to differentiate into osteoblasts by dexamethasone.The osteogenic differentiation was investigated by ALP stain , type I immunohistochemistry stain and alizarin Bordeaux stain. The effect of age on osteogenic differentiation and proliferation of the stem cells was compared by determination of ALP and MTT activity.The result showed that ALP stain , type I immunohistochemistry stain and alizarin Bordeaux stain were positive. The determination of ALP and MTT activity showed that the capativity of osteogenic differe- ntiation of ADSCs did not significantly difference with age. 4. conclusionâ‘ In this study,the ADSCs was isolated from rabbit's adipose tissue ,and cultured successfully in vitro.ADSCs also was induced to differentiate into osteoblasts by dexamethasone or rhBMP-2.â‘¡there is a dose-effect when ADSCs was induced to differentiate into osteoblasts by rhBMP-2. ALP activity increased rapidly with the increase of the dose of rhBMP-2 within the range of 100-400 ng/ml,yet there was no significant difference among the groups over 400ng/ml; there was no significant difference in proliferation activity below 800ng/ml rhBMP-2(P>0.05),yet over 800ng/ml of rhBMP-2 could inhibit proliferation significantly.so,400-800ng/ml rhBMP-2 could promote differentiation and proliferation of adipose derived stem cells significantly.â‘¢ADSCs in the combined group can be induced to differentiate into osteoblasts in a shorter culture time.â‘£the capativity of osteogenic differentiation of ADSCs did not significantly differ with age.
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