Experimental Research Of Nano-hydroxyapatite/Collagen/Poly(L-lactide) And Peptide P-15 Combined Bone Substitute Material

The development and application of bone graft techniques have increased the indication of oral implantology, and made impant denture a possible way for patients with inadequate bone quantity and quality. Research of bone graft material is the cutting edge of biotechnology. The ideal properties of a bone graft material should be of good osteoconductive, osteoinducive and osteogenic abilities. Autogenous bone has these three properties at the same time and without the hazard of grafting transmitted diseases, so it can be considered as the golden standard for grafting. Nevertheless, it could present some disadvantages, such as limited availability from the intraoral donor site and morbidity or other complications at extraoral donor site. Allogenic graft and xenograft posses certain immunogenicity, also cause desease transmission from the host.Currently, synthetic bone substitutes have been developing spectacularly. Nevertheless, solo kind of synthetic material, for example, bioceramics has some shortages, such as lower bending strength, fragility and poor anti-fatigue ability in the physiological environment. And this kind of material can only act as a scaffold for new bone formation without osteoinducive ability. Hence, a compound graft material with both inorganic scaffold of good physical and chemical properties and organic ingredient of osteoinducive ability seems to be a promising strategy. Inorganic scaffold part has approached to nano level, which could render novel properties. Because of the surface effect and interface effect of nano-scaffold, its strength, tenacity and plasticity improve a lot. In addition, human bone structure is a three-dimensional complex with redirecting nano-hydroxyapatite crystals in the collagen fibers. Based on the biomimics, professor Cui invents an novel nHAC/PLA biomaterial, with pores of 50-300 m in diameter and 80% porosity. The structure and chemical composition of this biomaterial are very similar to the human cancellous bone, while maintain good biocompatibility and biodegradability. Meanwhile, it is reported that rhBMP can be compouded with this biomaterial in order to obtain the osteoinducive ability.Many researches are focusing on the combination of bioactive proteins with different scaffold materials, however, the key issue is that the absorption and release of such proteins involves very complicated biological process. And they are likely to be degraded in vivo, thus losing theirs bioactivity. Heterologous proteins have the potential of immunological rejection, while the high cost of recombination protein limits their clinical application. On the contrary, peptide keeps the key sequence of bioactive protein, which may have an important role in cell adhesion, proliferation and differentiation; and its space conformation and bioactivity is more stable than protein , easier to synthesize. So, it seems as a reasonable way to combine bioactive peptide with scaffold materials. P-15(766- GTPGPQGIAGQRGVV- 780) is a short highly conserved peptide sequence of collagenⅠα1, which can bind integrin receptor on mesenchymal progenitor cells, then enhance the adhesion of human bone marrow stromal and increase their BMP-2, BMP-7 gene expression, coact with other growth factors to stimulate the differentiation of osteoblasts. Since it can provide more cell binding domain, the defect healing process of bone can be started from both the internal and external part of the graft materials in a three-dimensional way. A micro- environment that is suitable for the physiological activities and new bone formation is created, which results in the accelerated formation of new, highly vascularized bone of very high quality.In this study, we tried to combine nHAC/PLA(supplied by Tsinghua University) with P-15 peptide and test its improvement in the quality and quantity of new bone formation, and to provide experimental data for the further application of this bone substitute material.Objective: We detect the proliferation and bone forming activity of bone marrow stromal cells which are seeded on the surface of the nano- hydroxyapatite collagen and peptide P-15 combined bone substitute material. Then choose the optimum concentration of P-15, which would be uesed in vivo experiment at the skull of rabbits to test its bone healing ability.Method: nHAC/PLA was saturated with different concentrations (50 g/ml,100 g/ml,200 g/ml) of solution of the peptide in phosphate buffered saline, they were combined through the physical adsorption way, then freeze dried. The bone marrow stromal cells which were separated from rat and then the third passage cells were seeded at the surface of nHAC/PLA combined with different concentrations of P-15 peptide for 7 days. We assay the total protein and alkaline phosphatase(ALP) activity on 1d, 3d, 7d to study the effect of P-15 on stimulating the differentiation of BMSCs. Then chose one sample from each group for scanning electron microscope test. Finally, chose the optimum concentration of P-15, which will be uesed as the experiment group in vivo experiment at the skull of rabbits, nHAC/PLA alone group and negative control group were also set up. After the 4w, 8w and 12w, rabbit were executed, X ray, specimen slices technique and image analysis software were uesd to assay the quantity and quality of new bone formation and residual materials.Result: in vitro study showed cells'shapes were favourable near the material and they perform a multilayer structures. Cells which were plated on combined material with a 100 g/ml concentrations of solution of the P-15 in phosphate buffered saline performed better in the the total protein and alkaline phosphatase(ALP) activity than control groups. Scanning electron microscop shows, BMSCs adhered to the surface of combined materials, with stretching pseudopod. X ray and histological study suggested in the experiment group, newly formed bone and residual grafted materials were favorable than other groups in every check point(p<0.05). After 8w and 12w, residual grafted materials in combined groups were less than nHAC/PLA alone group(p<0.05). After 8w, at the center of skull defect in the experiment group, significantly mature bone tissue could be seen. After 12w, the radiographic density inside the material was similar to nomal bone tissue. This may imply that P-15 peptide stimulate a microenvironment that was suitable for the physiological activities and new bone formation, a three-dimension way for bone tissue regeneration could be observed.Conclusion: nano-hydroxyapatite/collagen/poly(L-lactide) and peptide P-15 combined bone substitute material maintains a good biocompatibility with a better performance in bone formation, especially in healing the skull defect of rabbit, it shows ideal vascularization and bone forming potentials, which also confirm the function of P-15 peptide on stimulating the process of bone healing.