| Objective: To investigate the growth, expansion and differentiation into osteoblasts of non-human primate bone marrow mesenchymal stem cells (MSCs), the BMSCs were isolated from adult rhesus monkeys, cultured and induced in vitro. To evaluate the cell attachment and biocompatibility of valuble scaffolds, the osteoblasts differentiated by MSCs were cultured with six kinds of three-dimensional porous new tissue engineering scaffolds being consist of PDLLA, BG, β -TCP and PLA-PEG-PLA triblock copolymers. A preliminary study was also carried out to compare the different composite scaffolds seeding osteoblasts for bone tissue engineering in a critical size cranial defects in the rhesus monkey, and to evaluate the prospect of PDLLA/ PLA-PEG-PLA for bone tissue engineering.Methods: l.The rhesus monkey bone marrow-derived MSCs were isolated and purified by density gradient centrifugation. We investigated the effect of cell culture condition, such as seeding density and interval of replacing medium. Osteoblasts were induced and differentiated by cultivation of-6-confluent cells in the presence of osteogenic supplement (containing 10~7mol/l dexamethasone (Dex), 50mg/l ascorbic acid, and 10mmol/l beta-glycerophosphate) or 100 ng/ml recombinat human bone morphogenetic protein-2(rhBMP-2). Alkaline phosphatase (ALP) activity, secretion of ammo-terminal propeptide of type I procollagen, type II procollagen and osteocalcin were analyzed to characterize the osteoblasts and the effects of induction between two supplements were compared.2. Six kinds of new high-porosity foams of PDLLA, PDLLA/BG, PDLLA/P-TCP, PDLLA/PLA-PEG-PLA, PDLLA/PLA-PEG-PLA/BG and PDLLA/PLA-PEG-PLA/ P -TCP were fabricated by a solvent-casting particulate-leaching technique with Nacl as the leachable component. To find good cell scaffolds, cell culture studies using osteoblasts differentiated by MSCs were conducted to assess the biocompatibility of the foams and cell attachment to the porous substrates. The samples were examined by inverted microscope , scanning electronic microscope (SEM) and 3-(4 , 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazo-lium bromide (MTT assay) to evaluate the adhesiveness and proliferation of osteoblasts.3. Each of 24 cranial defects with 10mm diameter and complete resection of periosteum of adult rhesus monkeys was implanted with four kinds of composite scaffolds seeding osteoblasts with good cell biocompatibility in cell culture model. The specimens were harvested in 6, 12 weeks. The samples were examined by X-ray, histology and scanning electronic microscope (SEM) to evaluate the effect of defect repair.Results: 1. Inverted microscope showed the splitting and proliferation of rhesus monkey bone marrow-derived MSCs isolated and purified by density-7-gradient centrifugation. It was found that MSCs express a small quantity alkaline phosphatase (ALP) activity and secrete less calcium. The condition of cell culture with 10 104cells/cm2 to 3OX 104 cells/cm2 seeding density and semi-quantity medium replacement at 48h and total medium replacement every 3 days subsequent showed good effects on cell proliferation. Cells induced and differentiated by osteogenic differentiation or recombinat human bone morphogenetic protein-2 from MSCs have the same morphologic feature with osteoblasts in vivo. Cells were alkaline phosphatase (ALP) positive with type I procollagen secretion, and no type II procollagen secretion. There was a apparent increase in alkaline phosphatase (ALP) activity and osteocalcin content at 3d with a little increase at 14d after cell fusion. There were no statistically differences in alkaline phosphatase (ALP) activity and osteocalcin content at the same stage during MSCs differentiate into osteoblasts using osteogenic supplement (containing 10~7mol/l dexamethasone (Dex), 50mg/l ascorbic acid, and 10mmol/l beta-glycerophosphate) or 100 ng/ml recombinat human bone morphogenetic protein-2 (rhBMP-2).2. White porous biodegradable foams were manufactured using a combine... |
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