Novel Op-HA/PLGA Composites: Preparation And Investigation On Its Osteogenic Activity

Bone defect caused by Congenital defects, trauma or tumor is a common problem in clinicsal treatment. The state of the art in repairing, such as autologous or allogeneic bone transplantation, has a lot of drawbacks in varying degrees, respectively. For example, although autologous bone substitution is the best option, but the amount of autologous bone is extremely limited and the infect at donor sites, limited shape, size and amount of graft are the major drawbacks of this methods. While the allogeneic bone was used, although the size and amount of allogeneic bone could satisfy the requirement of bone transplant, its applications are limited sharply due to the suffering venture of inevitable immune response and virus disease transmission. Bone Tissue engineering aims at the repairing and restoring damaged or diseased tissue .function employing three fundamental"tools", namely biological scaffolds, cells, and bioactive molecules. Scaffolds are central components of tissue engineering because they provide a three-dimensional structure for in vitro or in vivo cells ingrowth and inducing tissue and organ regeneration[2]. They usually act as a temporary substitute for the extracellular matrix and must have both appropriate structural and functional properties. HA/PLGA has been widely applied in plastic and oral surgery ,The composition of HA and PLGA could improve the mechanical strength and osteogenic activity of PLGA.But organic macromolacule and inorganic nano-particles of the composite are blended physically, the poor interfacial binding strength of organic and inorganic components results in low strength property unsuited for bone fixation and repair. In order to overcome this disadvantages, the novel composite of modified nano-HA was prepared. Nano-HA surface was grafted by LAc oligomer to prepare modified HA (op-HA). LAc oligomer on p-HA surface could extend into PLGA and enhance the interface adhesion between the two phases (op-HA and PLGA). So the mechanical strength of the material was increased. The mechanical strengths and chemical property, biocompatibility and the ability of bone forming were performed by instrument analysis, cell culture, animal experiments detection. The comprehensive property of ungrafted HA and the composites with different graft ratio of op-HA were analyzed. The optimal graft ratio of op-HA/PLGA would be obtained. It could provide bases for the medical application and industrialization of the novel material.In the present study, the freeze-drying technique of 1,4-dioxane as a solvent was explored to fabricate porous nanocomposite scaffolds of PLGA and HA nanoparticles surface grafted with LAc oligomer (op-HA). The scaffolds with pore arrangement and highly interconnected macro/microporous structure were prepared with different freezing dry conditions. The pore microstructure, the surface topography, and the mechanical properties, as well as the cell penetration and cell viability of these scaffolds were investigated, and the optimal production condition was assessed. It provides a basis for clinical applications.Ⅰ. Different graft ratio of op-HA/PLGA bioabsorbable bone repairing material1. Preparation and Characterization of MaterialsWe will graft different ratio Lactic acid oligomer to the surface of n-HA, different graft ratio of op-HA (1%,5%,9%) were prepared. then blend with the PLGA matrix, thus op-HA/PLGA will be prepared. The scaffolds were prepared with the melt-molding/porogen (salt particles) leaching method. Op-HA grafting yield will be analyzed through thermo gravimetric instrument (TGA), pore structure and porosity will be observed through field emission scanning electron microscope (ESEM), the compressive strength of scaffold materials and bending strength will be tested by electronic Instron 1121 universal testing machine. Water contact angles indicating the wetting ability of the materials were measured by drop shape analysis. The different graft ratio of op-HA/PLGA porous scaffolds were prepared with the porogen of salt particles with certain size by melt-molding/particle leaching method. the porosity varied from 84.1% to 89.7%. Besides the macropores of 100-450μm created by the leaching of NaCl particles; HA and op-HA enhanced wettability of the composite finally the best graft ratio of op-HA of medical applications for the material which can provide a basis for industrialization was optimized.2. The preparation of simulated body fluid (SBF), the porous scaffold materials was immersed into simulated body fluid for 1 week, 2 weeks, 4 weeks and 8 weeks, respectively. The the surface morphology of samples were observation by ESEM, quality changes is measured, Ca and P content of samples were detected by ICP-OES .The results showed that the longer was the immersion time, the sample surface sediments is more, the quality of sample was increasing too. Increase the ratio of PLGA mass was the smallest in quality, 1% op-HA/PLGA was largest percentage increase in quality; Ca and P content of 1% op-HA/PLGA composite scaffold is the highest level .3. MC3T3-E1 vaccinated in op-HA/PLGA, HA/PLGA and PLGA membrane, persisting 48 hours. At different time points we will stain osteoblasts by using fluorescein isothiocyanate (FITC), observe cells form and quantity in the material by Inverted fluorescence microscope, analyze the percentage of single-cell area by NIH Image J software, detect MC3T3-E1 proliferation in the surface of material by MTT; The results show that it can exhibite better cell adhesion, spreading and proliferation of rabbit osteoblasts compared with pure PLGA. It indicate that the biocompatibility and osteogenic bioactivity of the HA/PLGA, 1%op-HA/PLGA, 5%op-HA/PLGA nanocomposite will be improved obviously than that of the pure PLGA.4. Production of rabbit middle 15mm radial bone defect model, the different rates of op-HA/PLGA stent graft material implanted bone defect at the postoperative 4 w and 12w X-ray detection of the amount of bone formation to evaluate the capacity of bone repair materials . For the animal test, there was rapid healing in the defects treated with HA/PLGA,1%op-HA/PLGA and 5%p-HA/PLGA, where bridging by a large bony callus was observed at 12 weeks post-surgery. There was nonunion of radius defects implanted with PLGA and in the untreated group.Ⅱ. Preparation of porous nanocomposite scaffolds with honeycomb monolith structure by one phase solution freeze-drying methodBiodegradable porous nanocomposite scaffolds of poly(lactide-co-glycolide) (PLGA) and LAc oligomer surface-grafted hydroxyapatite nanoparticles (op-HA) with a honeycomb monolith structure were fabricated with the single-phase solution freeze-drying method. The effects of different freezing temperatures on the properties of the scaffolds, such as microstructures, compressive strength, cell penetration and cell proliferation were studied. The highly porous and well interconnected scaffolds with a tunable pore structure were obtained. The effect of different freezing temperature (4°C, -20°C, -80°Cand-196°C) was investigated in relations to the scaffold morphology, the porosity varied from 91.2% to 83.0% and the average pore diameter varied from 167.2±62.6μm to 11.9±4.2μm while theσ10 increased significantly. The cell proliferation were decreased and associated with the above-mentioned properties. Uniform distribution of op-HA particles and homogeneous roughness of pore wall surfaces were found in the 4°C freezing scaffold. The 4°C freezing scaffold exhibited better cell penetration and increased cell proliferation because of its larger pore size, higher porosity and interconnection. conclusion: The novel type of scaffolds can be used in bone repair, op-HA/PLGA, possesses good biocompatibility, perfect cell adhesion and proliferation properties. modification of the op-HA composites improved bonding strength between two phases, reinforcing material dispersion and stability. When graft ratio Lactic acid oligomer to the surface of n-HA is high, the material mechanical properties, biocompatibility and osteogenic bioactivity and bone repair effects are bad.The new bone repair scaffolds 1% op-HA/PLGA composite bone repair material has a better cell adhesion, proliferation and osteogenic activity,Porous nanocomposite scaffolds of op-HA/PLGA with a honeycomb monolith structure were prepared by the one phase solution freeze-drying method in this study. The freezing temperature played a critical role in formation of the highly porous structure with a high degree of interconnection. The average pore diameter varied from 167.2±62.6μm to 11.9±4.2μm as the freezing temperatures were changed from 4°C to -196°C. The 4°C freezing temperature scaffold showed better cell penetration and increased cell proliferation because of its larger pore size, higher porosity and interconnection. Although its compressive mechanical property was lower than the others, it will be acceptable for replacement of nonbearing bone. The further study will focus on the preparation of scaffolds with both highly porous structures and high mechanical properties by adjusting the production conditions including solution concentration, freezing temperature and freezing rate.