| Bone defects caused by trauma and diseases always present a dilemma for orthopaedic surgeons. Tissue engineering provides a new method to resolve the above problem. The key problem that tissue engineering will resolve is to choose seed cells which manufactured easily. Mesenchymal stem cells(MSCs) are a population of pluripotent cells within the bone marrow microenvironment of human and animals defined by their ability to differentiate into cells of osteogenic, chondrogenic, tendonogenic, adipogenic, and myogenic lineages and so on. Developmental biologist Verfaillie thinks MSCs nearly have multi-lineage potential similar to embryonic stem cells. During the long-term culture in vitro, MSCs always keeps culture explandability, self-renewal, and pluripotency, have poor host immune response. MSCs will ideally resolve the problem about seed cells of tissue engineering. Adipose-derived MSCs have multi-lineage potential similar to bone marrow-derived mesenchymal stem cells. Between Adipose-derived and bone marrow-derived MSCs, there were no significant differences for yield of adherent stem cells, growth kinetics, cell senescence, multi-lineage differentiation capacity, and gene transduction efficiency. Adipose tissue is an abundant and easily procured source of MSCs, which have a potential for use in tissue engineering applications. In the present study, human fetal bone marrow-derived MSCs and rabbit adipose-derived MSCs were isolated and cultured. In order to building a appropriate system of osteogenic differentiation of MSCs in vitro, effects of dexamethasone and conditioned medium from human fetal osteoblasts on osteogenic differentiation of human fetal bone marrow-derived and rabbit adipose-derived MSCs were investigated. This thesis consists of five parts as follow: Biological characteristics of human fetal bone marrow-derived MSCs. Effects of dexamethasone on osteogenic differentiation of human fetal bone marrow-derived MSCs. Effects of conditioned medium from human fetal osteoblasts on osteogenic differentiation of human fetal bone marrow-derived MSCs. Isolation and identification of rabbit adipose-derived MSCs. Effects of dexamethasone on osteogenic differentiation of rabbit adipose-derived MSCs. Some conclusions were drawn as follow: MSCs were isolated from fetal bone marrow by their adherence properties to plastic in tissue culture. In their undifferentiated state, human fetal bone marrow-derived MSCs were CD29+, CD44+, CD105+, and CD14–, CD34–, CD45–, and HLA-DR–. Human fetal bone marrow-derived MSCs cultured in adipogenic, osteogenic, or neurogenic media differentiated, respectively, into adipocytes, osteoblasts, and neurons. Dexamethasone inhibited the proliferation of human fetal bone marrow-derived MSCs, while induced them to differentiate into osteoblasts, and the optimal concentration of dexamethasone was 10-7mol/L. Conditioned medium(CM) from human fetal osteoblasts were collected and added to the culture medium, in which human fetal bone marrow -derived MSCs were induced to osteoblasts. There was a significant increase in alkaline phosphatase activity and bone nodules formation in the presence of CM. The same stimulus could be observed between 40%CM and 10-7mol/Ldesamethasone. Conditioned medium from human fetal osteoblasts stimulated the proliferation of human fetal bone marrow-derived MSCs, but dexamethasone inhibited the proliferation of human fetal bone marrow-derived MSCs. So 40%CM is the better method of osteogenic differentiation of human fetal bone marrow-derived MSCs. Multipotent adipose-derived MSCs were isolated from subcutaneous, ovarian, or circumrenal adipose tissue of rabbit according to our method. Briefly, minced adipose tissue were digested with 0.1% collagenase type I and 1% BSA for 90 min at 37? with intermittent shaking. In their undifferentiated state, rabbit adipose-derived MSCs were CD29+, CD44+,CD105+, and CD14–, CD34–, CD45–, and HLA-DR–. Rabbit adipose--derived MSCs cultured in adipogenic, osteogenic, or neurogenic media differentiated, respectivel... |
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