| Bone defects due to trauma or tumor are very common in clinical practice, but the big obstacle in treating is that it's hard to repair or reconstruct the defective bone. Although there have been some methods to repair segmental bone defects by now, such as bone autograft, allograft and biomaterial implants. These methods have their own disadvantages respectively, such as limited source, immunological rejection, and can't meet the need of perfect reconstruction. Tissue engineering that aims at replicate tissue and organ provides another innovative choice for repair and regeneration of segmental limb bone defects.Great progress has been made in bone tissue engineering research about seeding cells, scaffold materials and fabrication of tissue engineered bone. Recently, the study on osteoblasts from different location and tissue shows that osteoblast, despite of it s sources, can maintain osteogenic phenotype after cultured several generations in vitro. But there are no reports about the osteogenic characteristics of osteogenic induced Mesenchymal stem cells (MSCs) during fabrication tissue-engineered bone in vitro and vivo. So we designed a co-culture model to mimic "osteogenic environment" and studied on osteogenic characteristics of induced MSCs under different culture conditions and after seeded on CPPF/PLLA scaffold. At the same time, we improved seeding efficiency (efficiency of cells attaching on the scaffold) by seeding method with complex of cell and collagen gel.1. Rabbit bone marrow mesenchymal stem cells (BMSCs) were harvested from bone marrow by puncture, then the osteogenic characteristics under different culture conditions were compared. Results showed: (1) Induced MSCs maintain steadily osteogenic differentiation phenotype during passage culture. (2) Osteoblast differentiation phenotype of induction cultured and co-cultured MSCs were verified by ALP activities, mineralized node, ALP staining and express of type I collagen and osteocalcin, but MSCs cultured in DMEM/F12 medium didn't show significant osteogenic capacity. It indicated that the MSCscultured in induction medium and cocultured with fresh bone particles for two weeks have significant osteogenic capacity. But in induce condition, MSCs express obvious osteogenic differentiation phenotype in a shorter time .2. CPPf/PLLA scaffold was fabricated with solvent-casting particulate-leaching method, and itvs capability was evaluated by osteogenic observation at different time points, mimicking process of fabrication of tissue-engineered bone. Results suggested: (l)Fiber networks were formed in the scaffold with high porosity and good cell biocompatibility; (2) the ALP activities, osteocalcin, expression of type I collagen and pretein content of osteogenic induced MSCs seeded on the CPPf/PLLA scaffold were compared with control group (passage culture). Results didn' t show significant difference in the osteogenic capcity.3. Induced MSCs were seeded on the scaffold with different planting concentration, and the seeding efficiency was compared by MTT; Seeding methods were also compared including directly seeding, pre-coating serum protein on CPPf/PLLA scaffold and seeding with complex of cell and collagen gel. Results suggested: (1) There was a good linear correlation between directly counted rabbit osteoblasts and MTT absorbency(492nm).(2) Seeding efficiency (efficiency of cells attaching on the scaffold) was different when the planting concentration of induced MSCs varied from 1 X 106cells/mlb to 10 X 106cells/ml. When planting concentration increasing, seeding efficiency reduced (3) there was not significant difference in seeding efficiency between directly seeding and pre-coating serum protein. Seeding efficiency was improved by seeding method with complex of cell and collagen gel.Conclusions: 1. MSCs have great capability of proliferation and can differentiate into osteoblasts expressing alkaline phosphatase, osteocalcin and type I collagen, which is suitable for bone tissue engineering. 2.. Induced MSCs maintain steadily osteogenic...
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