Molecular Cell Biological Study On The Mineralization Of Biodegradable Calcium Phosphate Materials

Researches on biomedical materials, a significant area of materials science and technology, are involved in the essential topics in biomedicine. In order to investigate the mechanism of the bio-transformation of inorganic materials into biological organic tissues, works were carried out related to the molecular cell biological aspects of the biological mineralization of biodegradable calcium phosphate materials in this dissertation.A systemic research mode, i.e. from osteoblast isolation and cell culture, gene cloning, gene expression, purification of osteonectin, to the interaction of osteonectin between biomaterials, was established. Rat osteoblast cells were isolated from the calvarial bones of newborn Wistar rates and cultured in vitro via both enzymatic cell isolation and explant culture methods. After passage culture and purification, the harvested osteoblast cells with both enzymatic cell isolation and explant culture methods exhibited high alkaline phosphatase activity and calcified nodule-forming ability, suggesting that the osteoblast cells are of good biologic characteristics. The purified osteoblast cells in the third passage with good biological characteristics were cryopreserved. After resuscitation, 93% cryopreserved osteoblast cells survived and showed high alkaline phosphatase activity and calcified nodule-forming ability. And, the alkaline phosphatase gene was amplified from the resuscitated osteoblast cells. Compared with the cells before cryopreservation, the resuscitated cells were not subjected to obvious changes in biological characteristics.A pair of special primers, F₁ and R₁, was designed, and the full length coding cDNA sequence of rate osteonectin was amplified from the total RNA extracted from the Wistar rate's osteoblast cells obtained with the enzymatic cell isolation method. The PCR product was inserted into pBS-T vector and sequenced. Compared the sequence with the data in GeneBank, it was found that the sequence is identical with the sequence published in GenBank.The rate osteonectin gene was subcloned into pGEX-KG, an expression vector...