| Bone defect caused by injury, tumor and infection is quite common in clinical practice. However, there is still a deficiency of ideal bone graft material. Autogenous bone grafts are often limited by their own limitations and by the injury, infection and other complications of the donor site after operation, while allograft bone has problems in immunologic rejection after transplantation and dissemination of diseases. The advent of tissue engineering provides a innovative approach for the reconstruction and repair of human tissue and organ defects. The bone replacement materials manufactured by modeling the composition and structure of natural bone can provide the cells with microenvironment similar to natural bones. With good biocompatibility and osteogenic activity, the solubility ofβ-TCP in aqueous solution and in body fluids is 10-15 times of hydroxyapatite (HAP) which makes it the main subject in the field of bone tissue repair as it can solve the problem of HAP degradation in the body. On the other hand, the shortcomings of brittle and difficult to shape severely restrict its clinical applications. Compounding the (3-TCP and the organic compound with good toughness to form inorganic/organic composite biomaterials can solve the brittleness of (3-TCP and improve the biological activity by use of organic materials. Nanoβ-TCP is the new materials under the use of nano technology in nano-structure unit or nanometer scale (1～100nm). With a small size effect and surface effect, it can improve the strength of the material three-dimensionally. Collagen is the major organic components of human bones which can significantly enhance the interaction between cells in the body, such as cell chemotaxis and cell proliferation. Gelatin is a collagen hydrolyzate. with the same amino acid as collagen and good biocompatibility, better adhesion as well as bone induction, which can be degraded and absorbed by the body with lower immunogenicity. VAP is the major pharmacologically active component of antler which can promote differentiation of bone marrow stromal cells, proliferation of chondrocytes, epidermal cells and fibroblasts and can significantly accelerate bone tissue regeneration, peripheral nerve tissue regeneration as well as wound healing. The compounding of VAP to the nano-TCP microspheres in the gelatin can not only make full use of bone conduction of nano-TCP materials but also solve the problem that peptide drugs cannot easily go through biological barriers. As to the problem that wound and wound surface can be easily damaged by hydrolase. it can conduct sustainable, stable and efficient control of drug release rate of VAP. extend half-life period, achieve targeted release of drugs, and greatly improve the bioavailability of drugs, thus can repair bone defect and drug treatment. Based on the above understandings, nano TCP/gel/VAP was developed in this study.Five parts of experimental observation are explained as follows:1. Preparation and Introduction of nano TCP/gel/VAP PropertiesBy use of reverse microemulsion. nano TCP/gel/VAP was developed. The specification of nano-composite microspheres was also observed through SEM. Examining components of composite materials by SELDI-TOF-MS mass spectrometer was also part of the experiment.2. Evaluation of biocompatibility of nano TCP/gel/VAPAccording to ISO10993 and China biological material compatibility testing requirements (GB/T 16886). the biological safety of composite materials was studied through the acute toxicity test, hemolysis test, cell proliferation test and cell toxicity test.3. The effects of nano TCP/gel/VAP on Biological characteristics of osteoblastsMTT colorimetry and Alizarin red staining were used to detect the effects of nano TCP/gel/VAP on culturing human osteoblasts proliferation, secretion of extracellular calcium matrix by cells in vitro. ALP activity was detected by automatic biochemical analyzer.4. Experimental study of nano TCP/gel/VAP on the repair of rabbit mandibular defectsBy making a rabbit mandible defect model and seting nano TCP/gel/VAP and simple nanocomposites respectively at the defect site,, the effects of nano TCP/gel/ VAP on bone repair effects by gross observation, Micro-CT, histopathology and SEM was evaluated and examined respectively 4 weeks,8 weeks and 12 weeks after surgery.5. The experimental study of nano TCP/gel/VAP in promoting early differentiation of osteogenic cells Through the effect of p38MAPK signal transduction inhibitor SB203580 on the proliferation of osteoblasts, the effect of SB203580 and nano TCP/gel/VAP on ALP, Colâ… mRNA secreting from osteogenic cells, the effects of nano TCP/gel/VAP on the process of early differentiation of osteogenic cells were observed.Main findings and conclusions of the experiments are as followings:1. This research developes nano TCP/gel/VAP. With the diameter of about 20-40μm, specification of nanocomposite microspheres looks good through SEM, with high degree of dispersion and uniformity. Meanwhile, there are nano-holes on microsphere surface. Through mass spectrometry analysis, the existence of gelatin molecules and VAP are detected. VAP coats on the inside of gelatin microspheres by electrostatic interaction. With a regular shape and controllable size, microspheres can meet the different needs of bone repair by adjusting the size.2. According to ISO10993 and China biological material compatibility testing requirements(GB/T 16886), it's proved that nano TCP/gel/VAP has no cytotoxicity, no acute toxicity, does not cause hemolysis reaction in line with corresponding standards, and ensures biological safety.3. Nano TCP/gel/VAP can promote the proliferation of osteoblasts from human embryo, secretion of extracellular matrix from osteoblasts and formation of calcium nodules which promote differentiation and maturation of osteoblasts.4.Within 12 weeks after the surgery of rabbit mandibular defects repair by the effects of nano TCP/gel/VAP. bone defects in nano TCP/gel/VAP group are repaired by bone tissues, while the ones in blank control group are covered by the fibrous tissues. Nano TCP/gel group is between the two groups in which the edges are covered by bone tissues while the central areas are covered by fibrous tissues. The examination of Micro-CT shows that the composite material group is superior to the blank control group; nano TCP/gel/VAP group is superior to nano TCP/gel group. Dense lamellar bone can be seen by histological nano TCP/gel/VAP group. Harvard tube system, medullary cavity recanalization and integration between lamellar bones and the original bone tissues are also visible. Nano TCP/gel group can repair for bones, while the defect center is still fibrous tissue. In blank control group, it is rare to see bone island generation around the defects and the center is still filled with fibrous tissues. It is dense bone-like tissue under the examination of SEM in nano TCP/gel/VAP group. New bone tissues of nano TCP/gel group are not closely integrated with the boundary, with small holes and loose structure. The blank control group is still fibrous tissue without bone tissue formation. This indicates that nano TCP/gel/VAP increase bone induction on the basis of conductivity of TCP by the introduction of VAP and improves the repair effects of rabbit mandibular defects.5. p38MAPK inhibitor SB203580 decreases ability of osteoblast proliferation. 20μM is the half density for inhibitor SB203580 to restrain the proliferation of osteoblasts. The result of ALP activity test shows that ALP activity of inhibitor group |
