| In recent decades, great advancements have achieved in the research of tissue engineering. Bone being no more complex than internal organ such as the kidney, liver and lung which consist of numerous cell types that must be arranged in the proper three-dimensional structure, the research of bone tissue engineering might be succeeded firstly, though it is still in laboratory stage at present. How to obtain the ideal seeding cells and how to improve the properties of scaffold biomaterials are of the most importance in the research of bone tissue engineering.Adult mesenchymal stem cells (MSCs) derived from bone, periosteum, bone marrow, synovium and muscle could be used in bone tissue engineering. As adult MSCs have limited potential in both proliferation and differentiation, the aims of this study were to investigate the Multilineage Potential of yolk sac Mesenchymal Stem Cells (YS-MSCs) derived from the early stage of embryonic development, the potential of YS-MSCs applied in tissue engineering research were investigated too. Yolk sac being a part of caul, YS-MSCs exist in the mesenchymal layer of yolk sac, Compared with adult MSC, YS-MSCs from the early stage of embryonic development are more primitive, have more potential in both proliferation and differentiation and do not express major histocompatibility complex (MHC) related antigen. In order to obtain ideal cell source for tissue engineering and gene therapy, murine YS-MSCs were isolated and purified, their multilineage potential was also investigated in this study.In addition, matrix scaffold biomaterial with three-dimensional structure is also needed for construction of tissue engineered bone. Hydroxyapatite (HA) and collagen type one are the dominating elements of inorganic and organic phases in bone respectively. The components and structure of HA/collagen composite being similar to normal bone tissue, this biomaterial should be suitable for construction of tissue engineered bone. Calcium phosphate cement (CPC) was surface-modified with collagen type one, its effects on the adhesion, proliferation and differentiation of YS-MSCs were also investegated in this study.The main results are as follows:1. Isolation and purification of murine yolk sac mesenchymal stemcellsThe murine yolk sacs were harvested on day 8.5 postcoitus (pc) and digested by 0.1% collagenase. YS-MSCs were existed in the adherent cells. The primary YS-MSCs were adherent cells of spindle shape, uniform in size, having colony forming ability. Purified YS-MSCs could be obtained at passages 2 or 3. Flow cytometric analysis shows the phenotype of purified YS-MSCs is uniformly positive for CD29, CD44, CD 105, and CD 166, and negative for reactivity to antigens CD34, CD45, or CD86. Both the primary and passaged YS-MSCs express collagen type one and weakly but clearly positive in AKP. The biological characteristics of YS-MSCs are coincident with the phenotype of MSCs reported by others. The proliferation ability of primary and passaged YS-MSCs is similar. The results suggest that as primitive MSCs with strong ability in proliferation and self-renewal, YS-MSCs are candidates for bone tissue engineering's ideal seeding cells.2. Multilineage Potential of murine yolk sac mesenchymal stem cellsTo investigate the differentiation potential of purified murine YS-MSCs, the cells were cultured under conditions that were favorable for osteogenic, chondrogenic, or adipogenic differentiation. Osteogenic differentiation was appeared whenYS-MSCs at passage 4th were treated with 10~8mol/L dexamethasone^ 10 mmol/L 3 -glycerophosphate and 50 u g/ml vitamin C. YS-MSCs which were of spindle shape, uniform in size could be induced to pleomorphism osteoblast-like cells which were positive in collagen type I and expressed high level of AKP. Aggregates or nodules were formed at day 7 and calcium accumulation was detected by Von Kossa's stain at week 8. BMP-2 mRNA expression of YS-MSCs was detected by RT-PCR (reverse transcriptase polymerase chain reaction) during osteogenic induction.Five...
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