Into Bone Cells Compound Tcp Allograft Bone And Immunological Detection

OBJECTIVE To study the morphological and biologicalcharacteristics of in vitro cultured osteoblast derived from different tissues , investigate the most effective technique for the isolation and culture of osteoblast , explore the feasibility and practicable value of using osteoblast as the seed cell of osseous tissue engineering , provide stable seed cell source for osseous tissue engineering.METHOD Periosteum and bone marrow of tibia derived fromnewborne rabbits , wash periosteum three times in PBS and digest with trypsin for 20 min in order to remove rudimental blood cells. Cut periosteum cut into fragments of about 0.1×0.1mm² and put it into culture flasks (25ml) filled with 2ml high glocuse DMEM medium containing 10%FCS ,then incubate at 37℃, CO₂(5%) atmosphere.Draw marrow into a 10 ml syringe containing 7500 units of HeParin , add it into a sterile centrifuge tube containing DMEM medium with 10%FCS, centrifuge at lOOOrpm to remove the top layer of fat , count the cells , seed at the density of 1×10⁶/cm² into culture flasks(100ml) containing DMEM with 10%FCS and then incubate at 37℃, CO₂(5%)atmosphere. The medium is changed after 3 days. When the cells cover the whole bottom of the flask .digest with trypsin and subculture. The passaged cells are cultured in the selective medium containing DMEM+10%FCS supplemented with 50 mg/L ascorbic acid, 10⁸mmol/l dexamethasone and 10 mmol/L betar glycerophosphate. The 5th generation cells are trypsinized and stored by cryopreservation at the density of 106/ml. Resuscitate the cells after 1 month and seed it at the density of lOVml in 5ml selective culture medium. All the cells are obsvered under inverted phasecontrast microscope, Von Kossa stained and alkaline phosphatase stained to record matrix secretion and the process of ossification.RESULT Cells proliferate out of the Periosteum fragmentson 3rd or 4th day. The number of the cells increase as time passes. On 10th day they can cover the whole bottom of the flask. The passaged cells are triangle - like or shuttle - like cells with thick prominency and can cover the bottom in about 4 days. The primary cells and the passaged cells grow and breed rapidly , and had osteogeneric capability. On seeding the cells from marrow suspend in the medium like balls .On 4th day of culture, the nonadherent cells were removed along with the culture media. The adherent cells from marrow approached confluence in about 12 days. The passaged cells reached confluence typically 4 6 days after plating. Osteoblast of the primary culture and the subculture have strong ability to proliferate. ALP staining in subculture is stronger than in the prmiary culture cells and calcium nodule staining can be observed .CONCLUSION Osteoblast derived from both periosteum and bone marrow express osteoblastic characteristics and have strongbiological activity, so both of them can be used as a source of the seed cells for osseous tissue engineering . The morphological characteristics and biological activity of osteoblast do not change much after cryopreservation so that cryopreservation can be usedfor cell storage.