| Skeletal defect resulted from trauma, infection, or tumor is one of the troubling problems in clinical orthopaedics. The therapeutic efficacy treated by traditional methods, such as repair with autograft, allograft and biomaterial-filling and so on, is not optimal and hinders the widely clinical use. Tissue engineering technology has provided a promising way to solve this problem, and has been one of the hotspots recently. Seeding cells and biomaterials are the kernel of tissue engineering. In this study, adult human bone marrow stromal cells (HBMSC) as seeding cells were cultured in vitro and induced to osteoblast differentiation, composited with coral hydroxyapatite (CHA) and cultured for several days. The growth of all the cultured cells was observed. The compounds of CHA with cells were implanted into nude mouse, and the ectopia osteogenesis was detected. In addition, Green fluorescent protein (GFP) can be used as a tracer on osteoblasts transfection by an adenovirus vector, which may provide reference for further research.[Objectives]1. To explore the feasibility and application value of HBMSC, a kind of multilineage potential cells, as seeding cells for bone tissue engineering, which were isolated from adult bone marrow and cultured in a defined condition to induce to osteoblast differentiation, whose biological properties were observed.2. To explore the appropriate scaffold materials for bone tissue engineering, the biocompatibility of CHA cultured with HBMSC in vitro were observered.3. To explore the appropriate mode of fabricating tissue engineered bone, the ectopia osteogenesis in vivo of the tissue engineered bone fabricated by osteoblast cultured with CHA was studied.4. To research the feasibility of GFP as a tracer of seeding cells for tissue engineering, biological properties of osteoblasts transfected with GFP by adenovirus vector was observed.[Methods]1. Bone marrow was aspirated from the healthy adult human and culture-expanded in vitro by the method of total marrow. Subsequently, passages of HBMSC were induced to differentiate into osteoblast in the medium contain dexamethasone (10~8mol/L), beta-sodium glycerophosphate (lOmmol/L) and ascorbic acid (50 mg/L), observed continually by inverted phase contrast microscope, stained by HE, assayed by scanning and transmission electron microscope, growth curve of which was evaluated by MTT, surface marker proteins of which were analyzed by the flow cytometry. Expression of collagen type I was detected by immunohistochemistry. Alkaline phosphatase (ALP) and Calcifying nodule were strained by Gomori Calcium-Cobalt and Von Kossa methods respectively. Content of ALP was measured.2. The differentiated osteoblasts were divided into two groups, one of which were cultured together with CHA and the other not. Proliferations and differentiations of all the cultured cells were observed continually under an inverted phase contrast microscope, a light microscope with HE stained, and a scanning electron microscope. Growth curve was evaluated by MTT. Total contents of micro-protein and activities of alkaline phosphatase were quantitatively detected.3. The differentiated osteoblasts were seeded onto CHA, co-cultured for 5 to 7 days, the compounds with cells and CHA were implanted into nude mouse and CHA alone was implanted as a control. The effectiveness of bone formation was assessed separately by means of gross observation, roentgenography, histology after implantation for 4, 8 and 12 week. Human ALP and OCN of the compound were detected by RT-PCR at the end of the 4th week.4. GFP was transfected into osteoblast by adenovirus vector after being packed in 293A cells. These osteoblasts were observed by fluorescence microscope, the expression efficiency and marker proteins of their surfacewas examined by flow cytometer. Transfected osteoblasts were implanted into intramuscular nude mouse meanwhile not transfected osteoblasts were implanted as a control. The implants...
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