| Bone defection caused by trauma, tumor and infection is one of troubling problems in clinical orthopaedics. Biomaterial such as autologus bone, allogenic bone and bone xenograft and non-biomaterial such as bone cement, metal and ceramics were implanted into defection, but which wasn't a optimal treatment. Tissue engineering research was a new way to solve the problem, and became one of hotspots recently. Seed cells and biomaterial are fundamental problems in tissue engineering. In this study adult bone marrow mesenchymal stem cells as seed cells were cultured in vitro, induced to differentiate into osteoblast, cultured with allogenic partially decalcified bone in rotary cell culture system, and transfected by green fluorescent protein as a tracer on adenovirus vector.[Objectives]1. To explore the feasibility of MSC, a kind of multilineage potential cells, as seed cells of tissue engineering, which were isolated from adult bone marrow, cultured and observed on biological properties2. To explore the feasibility and application value of MSC being seed cells of bone tissue engineering, which were induced to differentiate into osteoblast.3. To construct tissue-engineered bone, MSCs were seeded on allogenic partially decalcified bone and cultured in rotary cell culture system.4. To explore the feasibility of GFP as a tracer of cell which was transfered into MSC by adenovirus vector.[Methods]1. MSCs were isolated from adult bone marrow and cultured in vitro,whose morphology were observed by inverted phase contrast microscope, whose ultrastructure were observed by transmission electron microscopy, whose growth curve was evaluated by MTT, and whose surface antigen were examined by flow cytometer.2. MSCs were cultured in the medium contain dexamethasone (10-8mol/L),β-sodium glycerylphosphate(10mniol/L) and ascorbic acid(50mg/L), observed by inverted phase contrast microscope and transmission electron microscopy, growth curve was evaluated by MTT, collagen type I was examined by immunohistochemistry, alkaline phosphatase (ALP) was strained by Calcium-Cobalt methods, calcifying nodule was strained by Von Kossa methods, and the content of ALP were measured.3. The bone from fresh cadaver donor was seeded with MSCs and cultured in rotary cell culture system for a week after being defated, decalcified, and sterilized, whose structure and cell on which were observed by scanning electron microscope.4. GFP was transfered into MSCs by adenovirus vector after being packed in 293-A cells. MSCs were observed by fluorescence microscope after transfection, and whose surface antigen were examined by flow cytometer. Efficiency of transfection was calculated.[Results]1. MSC isolated from adult bone marrow was undifferentiated state with active proliferative abilities and high purity, whose surface antigen showed the positive expression of CD29, CD44 and CD105, while CD14, CD34 and HLA-DR negative.2. MSCs proliferated rapidly after being induced to differentiate into osteoblast and have behaviors of the cell which can secrete protein. Collagen type I examined by immunohistochemistry was positive expression The result of stain reaction of ALP and calcifying nodule were positive respectively. The abilities of secreting ALP were enhanced.3. The structure of allogenic partially decalcified bone was polyporou. MSCs grow well and proliferate rapidly on it.4. Green fluorescent can be seen in MSC after being transfected for 24h and became brighter after 48h. Efficiency of transfection was 72%. Surface antigen of MSC transfected showed the positive expression of CD44 and CD29.[Conclusions]1. MSCs from bone marrow with active proliferative abilities and high purity were optimal seed cells of tissue engineering.2. MSCs can be induced to differentiate into osteoblast with typical characteristic on morphology and function. They were optimal cell source for bone tissue engineering.3. Allogenic partially decalcified bone is optimal biomaterial for tissue engineering. Dynamic three-di... |
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