| Nano-composites can be simulated is similar to human tissue micro-environment of the cell matrix, which is the tissue engineering of the materials widely used. A majority of biological tissues in living creature can be regarded as organizations of a variety of matrix materials from the composite materials , especially the inorganic - organic nano-bio-composite materials, Such as bone, teeth, etc. which formed by the nano-hydroxyapatite crystals and organic composition of polymer matrix. Nano-traditional Chinese-medicine is an active component and effective fraction of Chinese herbs, an original drug or a compound preparation ,particle size less than 100 nm of which produced by nano-technology manufacturing. With increasing of nanotechnology, nano-composites have attracted much attention for becoming an important bone repairing materials in recent years. Antler is a unique regeneration organization of bone used as a traditional medicine in China for thousands of years. A polypeptide extracted from fresh Sika Deer can significantly speed up the experimental rat fracture callus formation, shorten the time of fracture healing , improved biomechanical properties of fracture sites, promoted the accumulation of collagen and calcium deposition in bony callus. We combines both nanotechnology and biological medicine to prepare a new type of stuff which formed of biological activity velvet antler polypeptides and nano TCP / gelatin, hoping to improve the therapy and the therapeutic efficacy of bone defect.There is a tendency which loading biological active component to bone tissue engineering scaffold materials as a novel bone repairing materials to facilitate bone regeneration at present. Velvet antler polypeptides could enhance MSCs differentiation in a dose-dependent manner, stimulate the proliferation of cultured chondrocytes and epidermic cells and collagenoblasts in vitro, which also showed pronounced efficiency of bone tissue regeneration ,peripheral nerve trauma and ulcer healing.hus, compounding biological activity factors such as velvet antler polypeptides with nano-materials, can not only give full play to bone conductivity of nano-TCP, but also resolve those problems: peptide drugs through biological barriers is not easy, but apt to be damaged by hydrolase in wounds and raw surfaces; releases of velvet antler polypeptides continuously, steadily and efficiently, prolong its half-decay period; the target release of active factors; raise the biological availability. Accordingly to achieve the double effects of repairations of bone defects and the drug treatment.The material of nano tricalcium phosphate/ gelatin/velvet antler polypeptide developed by Jilin University and Physical and Chemical Research Institute, Chinese Academy of Sciences, which composition and structural properties are similar to natural bone ,mainly behaves with favourable biocompatibility and cranial conduction, also shows the bonding of bone and other biological characteristics. Compared with domestic and foreign traditional bone repair materials, this in situ synthesis of multihole bone-repairing material has a drug delayed releasing and transporting function, its structural design highlighted the advantages of solving the chemical bonding between materials, increasing the mechanical properties; the use of nano-tricalcium phosphate as a carrier, outstanding its nano-properties, having a greater specific surface area and higher specific surface energy , absorbing the necessary osteogenesis factor (VAP); The research and development of conformation of rules of the compound microspheres, bone repairing will come true at the same time, with a further set of drugs and drug delivery function to bone repairation and that is expected to achieve the dual objective of drug therapy; In addition, this complex has a natural microspheres porous structure is conducive to the control of drug release and new bone in the material cling internal growth, so as to promote new bone formation to enhance the effect.Firstly ,our study evaluate the biocompatibility of the nano tricalcium phosphate/ gelatin/velvet antler polypeptide material, With reference to standard of Biological evaluation of dental materials- YY/T 0127.2-19983,We prepared leaching liquor of the nano-material, after mice by intraperitoneal injection 4 h, 24 h, 48 h and 72 h , weigh all of the animals, observe and record the toxicity performance and the number of dead animals for acute toxicity tests, in the 72 h observation period , injection with 200 mg / L extract composite material (50 mL / kg) in mice does not have signs of listlessness, closed eyes, uneasiness, tremor, convulsions, breath with difficulty and death. With reference to standard of Biological evaluation of dental materials- YY/T 0127.1-93,add the diluted solution of anticoagulative rabbit blood to composite solution, centrifuge and draw the supernatant to determine the absorbance value, calculated composite's rate of hemolysis; With reference to standard of Biological evaluation of dental materials- YY/T 0127.94-2001,use molecular filtration method to proceed the cytotoxicity test, the toxicity of composite material for the grade 0, no cytotoxicity. Experimental results showed that nano-tricalcium phosphate / gelatin / VAP composite materials with good biocompatibility, the initial clinical application of the material to verify the safety, development and utilization of its next step to provide a theoretical and experimental basis.The mesenchymal stem cells of experimental rabbits of in vitro primary generation isolating culture showed fusiform, spindle and polygonal, formed fibrocyte, didn't found the phenomenon of contact inhibition, was vortex-like distribution after convergence. To the 3rd generation, the morphology of cells was homogeneous. Identification the surface markers of the 3rd generation mesenchymal stem cells of in vitro primary generation isolating culture showed that the surface antigen of CD44 was positive and the department of hematopoietic stem cell-specific antigen was negative, so that the cells of isolating culture were mesenchymal stem ones. 20% nano tricalcium phosphate/ 0.5μg/mLvelvet antler polypeptides crosslinking composites (shot for composites) could improve the proliferation of the mesenchymal stem cells. After the Staining of AO/EB, could be observed under fluorescence microscope, the composites could suppression of bone marrow-derived mesenchymal stem cell apoptosis. Detected by flow cytometry, When the composites were given to the MSCs, the apoptosis rate of cells were 2.08%, which were significantly higher than that of control group(13.68 %),increased the proportion of S phase cells significantly, from 7.96 %( control group) to 45.84 %. These results suggest that composite materials can inhibit apoptosis, promote cell proliferation. ALP is a sign of osteoblasts and osteoblast enzyme metabolism of sensitive and specific indicators. Culture of MSCs with sustained composite , with the extension of time, ALP activity gradually increased, after 21d, the ALP activity compared with the control group significantly elevated(P<0.001).Suggest that composite materials can be induced by mesenchymal stem cells to osteoblast differentiation. The formation of calcified nodules on behalf of the bone marrow-derived mesenchymal stem cells to osteoblast differentiation induced successfully. Composite materials culture the mesenchymal stem cells for 21 days, can significantly increase the formation of calcified nodules. Suggest that composite materials can promote mesenchymal stem cells to osteoblast differentiation and maturation. |
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