A Novel Biodegradable Polymeric Nano-composite For Bone Repair: Preparation And Study On The Repairing Of Bone Defect

Bone defect and nonunion induced by trauma or tumor are still the puzzles of reconstructive orthopaedic surgery nowadays. Although autologous and allogeneic bone substitute materials, as the common replaceable materials, play important role in treatments, they still have many intrinsic disadvantages and can not meet the demands in all situations. Autologous bone grafts are considered to be the optimal selection,but the morbidity at donor sites,limited shape,size and amount of graft are the major drawbacks of this technique. Furthermore,the additional surgery makes patient undergo more injuries to body, including pain and infection. Allogeneic bone material can be prepared into any shape,size needed in surgery without the limits of amount,however the inevitable immune response and the risk of virus disease transmission make it limit to application. In order to overcome the limitation of the two methods, it is important task to develop the better man-made biological material to repair bone defect.The biodegradable material of hadroxyapatite and polylactide(HA/PLA) owns good biocompatibility and osteoconductivity. Because of similar constitutes to bone matrix, the composite possesses great bone-bonding properties. It is a kind of promising material for bone fixation and repair. However, organic macromolacule and inorganic nano-particles of the composite are blended physically, the poor interface adhesion of two phases (p-HA and PLGA) results in ill strength property unsuited for bone fixation and repair. What's more, the degradation period of material is unequal to bone growth speed.In order to overcome the disadvantages, the novel composite of modified nano-HA and PLGA was prepared. Nano-HA surface was grafted by LAc oligomer to prepare modified HA (p-HA), then blended with PLGA to get the composite p-HA/PLGA. Modified p-HA particles owned good uniform dispersion in PLGA. LAc oligomer on p-HA surface could extend into PLGA and enhance the interface adhesion between the two phases. The mechanical strength of the material was increased. The degradation period could be adjusted by the various ratios of LA and GA in PLGA. The osteoconductivity of p-HA and degradable property of PLGA were combined, and the biocompatibility was enhanced. The novel composite was suitable for the requirement of clinical bone repair.As a novel material, biocompatibilty would be the most important index to be considered. And then the mechanical property and osteogenic activity should also be thought about. The composites with different ratio of p-HA to PLGA were prepared. Then the three-dimensional scaffolds of bone tissue engineering with certain pores and porosity were prepared by melt-molding/particle leaching method.The comprehensive evaluation of physicochemical property, mechanical strength, biocompatibility and the osteogenic ability were performed by instrument analysis, cell culture, animal experiments and molecular biological detection. The comprehensive property of ungrafted HA, grafted HA and the composites with various contents of p-HA were analyzed. The optimal blend ratio of two phases of p-HA/PLGA would be obtained. It could provide bases for the medical application and industrialization of the novel material.The experiment was divided into six parts:1. Preparation of the novel bone repair material p-HA/PLGA. The modified n-HA (p-HA) was prepared by the method of LAc oligomer grafted on the surface of n-HA particles. And the p-HA was further blended with PLGA to prepare the various blend ratios of two phases, p-HA accounted 5%, 10%, 20% and 40% in the composite respectively. The 10%HA/PLGA and pristine PLGA was prepared as controls.2. Preparation of the porous scaffolds. The porous scaffolds were prepared with the porogen of salt particles with certain size by melt-molding/particle leaching method. The mechanical strength, pore size and porosity were tested.3. Biological evaluation. According to the demands of International Standard Oganization(ISO 10993) and Technical Evaluation Standards of Biomedical Materials and Medical Instruments promulgated by Chinese Board of Health(GB/T 16886). Experiments of cell toxicity in vitro, acute toxicity, pyrogenic test and hemolysis test were investigated to evaluate the biocompatibility of the novel composite.4. Osteoblasts adherence and proliferation on the p-HA/PLGA. Rabbit osteoblasts were seeded and cultured on the material membranes for seven days, the adherent osteoblasts'number and morphological changes were observed by the fluorescence inverse microscope after dying by FITC. The cells'areas and amounts of osteoblasts were analyzed by the soft ware of NIH Image J. The different adherence abilities were compared. Osteoblasts were implanted on the membranes of the composites and cultured, the cell proliferation was tested by MTT method.5. RT-PCR of Collagen-I, Collagen-II, BMP-2 and osteocalcin. The osteoblasts were implanted on the material membranes and cultured for seven days. Total cell RNA was obtained from osteoblasts. cDNA was produced by RT-PCR according to the designed primers. Then PCR and electrophoresis were performed. At last, the gene expressions of osteoblasts on different materials were analyzed by gelatin imaging system.6. The repair of rabbit radius defects. The rabbit's 2.0 cm radius defects were made and implanted with the composites with the various p-HA ratios. At 4 and 24 weeks after operation, samples were harvested and examined by radiograph and histological section to evaluate the bone repairing property of the novel materials.Main Results and Conclusions are as follows:1. HA particles grafted with LAc oligomer (p-HA) were prepared. p-HA could disperse homogenously in the PLGA-chloroform solution or in the composite of p-HA/PLGA.2. The porous scaffolds of p-HA/PLGA with various ratios (5%,10%,20% and 40%) and HA/PLGA (10%) and PLGA as controls were prepared. The pores of scaffolds were from 100μm to 300μm, and the porosity was from 87.5 to 92.91 percent and p-HA/PLGA (10% and 20%) possessed maximal porosity. Modified HA can increase the interface adhesion of two phases in p-HA/PLGA. The compressive and bending strengths of p-HA/PLGA were higher than those of HA/PLGA. 20%p-HA/PLGA owned maximal mechanical strength. There was statistical significance compared with the control and other groups. The composites with 5% and 40% blend ratios possessed poor plasticity and low mechanical strength.3. The results of experiments on cell toxicity test in vitro, acute toxicity, pyrogenic test and hemolysis test met the demands of(ISO,10993)and GB/T, which showed the good biocompatibility.4. The results of cell culture in vitro indicated that p-HA/PLGA could promote osteoblasts adhere and proliferate better than PLGA did. The novel material of p-HA/PLGA possessed good osteoblasts compatibility. 20%p-HA/PLGA owned best adherence and 10%p-HA/PLGA owned best proliferation ability.5. RT-PCR detection illustrated that mRNA expressions of Collagen-I and Collagen-II were higher for PLGA and 5%p-HA/PLGA, BMP-2 mRNA expressions were higher for 20%p-HA/PLGA and HA/PLGA, OCN mRNA expression was higher for HA/PLGA and PLGA.6. At 24 weeks postoperatively, there were evident bone defects and nonunion in the groups of the untreated control and PLGA. Continuous bone callus filled in the defects in the groups of 5% p-HA/PLGA, 40% p-HA/PLGA and HA/PLGA. But there was no bone callus on about one third area of 5% p-HA/PLGA and one fourth area of 40% p-HA/PLGA. Continuous bone callus filled in 10% p-HA/PLGA and the defect was repaired by and large. Continuous bone callus filled completely in 20% p-HA/PLGA and the defect healed.Conclusion: The new bone repair material of p-HA/PLGA shows satisfactory biocompatibility and good ability of cell adherence and proliferation. Modified p-HA could enhance the interface adhesion between two phases, and the dispersive capacity as well as the stable ability could also be enhanced. The mechanical strength and bone repair ability are not satisfied if p-HA account more or less than the suitable ratio. The mRNA expressions of Collagen-I and Collagen-II are higher for PLGA and 5%p-HA/PLGA, BMP-2 mRNA expressions are higher for 20%p-HA/PLGA and HA/PLGA, OCN mRNA expressions are higher for HA/PLGA and PLGA. The 20% p-HA/PLGA shows good comprehensive property.