The Preparation Of Bone Substitute Material From The Fetal Bovine Cancellous Bone And The Researches On The Physical AndBiological Characteristics Of The Material

BACKGROUND AND OBJECTIVEBone defect is one of the major problems in oral maxillofacial surgeries. Because of the reasons of periodontal diseases, trauma or other reasons, bone graft is frequently needed. Reconstruction of oral and maxillofacial tissue is often involved in need of bone graft. Using autogenous bone is the golden standard for bone grafting, because of its good osteogenic, osteoinductive and osteoconductive properties. However, there are some limitations hindered the extensive application such as limited amounts and additional surgical wounds using autogenous bones.Therefore, it has been a hot spot in the field of biological materials to study the bone tissue engineering bone. In the field of biological material research, the cohesion between the related scientific research and achievements turning into the real usestill hassome problems. This study based in bone material basic research, and focus on the preparation of the product. So we hope we can achieve effective materials for bone tissue engineering research and development.The bone substitute materials are mainly from chemical synthesis and natural resources. Because of the differences between the natural bone and the chemical synthesis in the physical and chemical properties,there is still needed to be improved. Natural sources of bone substitute materials, due to their similar tissue and bone porous structure, are still the main materials for clinical use. Most of the current artificial bone materials only provide bases for new bone growth. Although these materials have excellent mechanical properties, they sometimes fail to integrate with the surrounding bone. When an implant fails to fully osseointegrateion, revision surgeries may be carried out, which brings agony and additional cost to the patients. Thus, lots of attempts were done including adding bioactive molecules to improve osteoconductive properties of the bone grafts. Therefore, the development of bone substitute materials, more effective and more sustained release need to be further studied.This topic will be the first comprehensive system of materials for bone tissue engineering were systematically to (1) develop a reliable biological material (2) on the production of bone of osteogenic growth factor of surface modification, improve the efficiency of bone forming; (3) preparation of bone grafts for bone surgery requires bone fixed with gel, forming perfect bone substitute material product system; (4) clearly added P-15 peptide on the mechanism of the role of bone cells.Materials and methodsThe fetal bovine cancellous bone were selected as raw materials, then prepared by chemical method to make ABM. We used MTT method. SEM scanning electron microscopy, chromosome aberration test method, to prove the Material’s biological safety.We used the ball milling and used sieving apparatus to prepare thenano-ABM, and then pressed it into tablets. The human bone marrow mesenchymal stem cells were cultured in ABM discs, and the effect of P-15 on the cells was observed. We used co-deposition to deposit calcium phosphate coating on the ABM films. By surface modification and the peptide P-15 fixed, the adhesion and osteogenic character of the ABM discs surface was improved. The AFM wasused to measure the effect of P-15 on the elastic modulus of cells, and the effect of P-15 on the adhesion of FABM particlesto HJMSC cells.The hyaluronic acid was used to form gels, and the changes of elastic modulus and the stability of the gel were detected by rheometer. The biological character of the gelwas studied by cell method. The HUVECs co-culturedwith HJMSC was induced into vascular.The calvarial critical bone defect was prepared for the implantation of bone powder, and the metabolism time of the material was studied.Results:1. The appearance of the ABM powder is white and particle size is between 0.25-2mm. X-ray diffraction showed no obvious other calcium phosphate and crystalline material peak, and the hydroxyapatite content was 96.9%. The pH value was 7.42. The measured bone calcium and phosphorus ratio:1.76+0.27.2. The biological character tests of ABM powderIn the cell toxicity test, the MTT test results showed no obvious cell toxicity of the materials. During the pyrogen reaction experiment, three animals (rabbits) temperature increases were less than 0.6℃. In the acute toxicity test, there were no differences in the test group and the control group. Hemolysis test showedthe hemolytic index of the bone material was 1.25, and the test result suggested there wasno hemolysis. The chromosome aberration analysis was performed and the results showed the bone material did not increase chromosome aberration rate.3. The effective of the polypeptide P-15 on HJMSCwe used atomic force microscope (AFM) based single cell indentation force spectroscopy to study the impact of P-15 on the stiffness and the adhesion ability of human jaw bone mesenchymal stem cells (HJMSC) to hydroxyapatite (HA). We found the stiffness of HJMSC is increasing as the concentration of P-15 grows in short culture intervals and the dissociation forces between HJMSC and FABM particles in the appearance and absence of P-15 are all around 30 pN. Moreover, by calculating the binding energy of HJMSC to FABM particles mixed with or without P-15, we proved that P-15 could increase the adhesion energy nearly four times. Scanning electron microscope (SEM) was also exploited to study the morphology of HJMSC cultured in the appearance and absence of P-15 on FABM disc surface for short term. Apparent morphological differences were observed between the cells cultured with or without P-15. These results explain the probable underlying adhesion mechanism of HJMSC promoted by P-15 and can be served as the bases for the design of bone graft substitute materials.4. The animal experimentsof bone implantWe prepared 1.8cm * 1.8cm critical bone defect in rabbit skull, and we settled 4 groups at different time points.The animals were sacrificed, and the tissue sections were collected.The histopathologic diagnosis showed good biocompatibility of the materials. There is no obvious foreign body reaction and the term of metabolism in vivo is about 22 weeks.CONCLUSION(1) The prepared ABMwas proved withproper physicochemical characters and good biological safety, which also suggesting that it has good osteoindcutivity.(2) P-15 has a significant effect on the adhesion of HJMSC to FABM.P-15 has an effect on the elastic modulus of the cells, suggesting that it may change the cell membrane structure to affect the cell adhesion. That may suggest the peptide P-15 can be used as an effective factor for surface modification of bone materials.(3) A co-deposition method can form a stable and orderly crystallization on the surface of the FABM, which can be used for the sustained release of the growth factor.(4) The animal experiment suggested that bone has good biological characters and the metabolic cycle time meet clinical needs.