Preparation, Modification And Characterization Of Mesoporous Materials In Bone Defect Repairing

In recent years, with the development of industry and traffic, patients with multiple trauma, high-energy injury and open fracture have become an increasingly common phenomenon. These injuries are often associated with severe open comminuted fractures of limbs, and severe bone defects. So the market demand for bone repair materials is huge. The suitable scaffold for large bone defect must have specic pore architectures, mechanical strengths, bioactivity levels, degradation and antibacterial properties and must deliver drugs in a controllable way. Currently, only a few synthetic scaffolds have all of these properties. There are no scaffolds that can be used to repair large bone defects. The aim of this study is to develop the new bone repair materials, and improve the repair effect of large segmental bone defects. The high incidence of infection and delayed healing of bone defects are two critical problems in the bone repair process. We want to develop new bone graft material with anti-infective and promoting bone repair activities. The ordered mesoporous material was used as the substrate, then subjected to material modification, structural optimization, and bioactive factors loading. We evaluated the biocompatibility, antibacterial activity, osteoblast activity and repair effects in the large segmental bone defects of animal models with cell biology, histology, imaging methods. We explored the interaction between the material compositions, the structure, the growth factors and osteogenic effect. Based on the above considerations, we carried out the following studies:Mesoporous bioactive glass(MBG) has recently shown promise in the field of bone reconstruction biomaterials because of its greater specic surface area and a greater total nano-pore volume. Unfortunately, MBG is very brittle and lacks antibacterial activity. This limits its applications in the treatment of bone defects, especially large bone defects complicated by infection. In order to dispel these disadvantages, a novel hydroxypropyltrimethyl ammonium chloride chitosan(HACC) and a class of prolamine proteins found in maize, Zeins, were here used to modify the traditional MBG scaffolds. On the other hand, we aslo modify the traditional MBG scaffolds with O-acrylamidomethyl-2-hydroxypropyltrimethyl ammonium chloride chitosan(NMA-HACC) and silk. Four new types of scaffold, MBG-HACC scaffolds, MBG-HACC-Zein scaffolds, MBG-HACC-silk scaffolds(NHS) and NMA-HACC-silk-MBG scaffolds(MBG-NHS) were made. Transmission electron microscopy(TEM), small angle X-ray diffraction(SAXRD), and Barrett-Joyner-Halenda(BJH) were used to analyze the surface properties of these MBG scaffolds. Thermal gravimetric analysis(TGA), X-ray diffraction(XRD), Fourier transform infrared spectroscopy(FTIR), scanning electron microscopy(SEM), mechanical experiments, and synchrotron radiation microcomputer tomography(SRμCT) were used to compare the features of the traditional and modified scaffolds and to analyze the mineralization of the scaffold after being soaked in simulated body fluid(SBF). Confocal laser scanning microscopy(CLSM) was used to compare the antibacterial properties and biocompatibility of the scaffolds at various points in time. Our current study demonstrates that all these prepared MBG scaffolds possessed well-ordered, three-dimensional, nanometer-sized mesoporous structures and that HACC-Zein-modified MBG scaffolds(MBG-HACC-Zein) and NHS modified MBG scaffolds(MBG-NHS) are characterized by strong bioactivity and by effective, prolonged antibacterial activity. Finally, their excellent biocompatibility was demonstrated by studying the in vitro proliferation and viability of human mesenchymal stem cells(hMSCs).Nevertheless, the clinical use of rhBMP-2 is still far from optimal because of its initial burst release from the scaffolds. This reduces the bioutility of the loaded growth factors. In order to address this problem, we prepare Zein scaffold as an in vivo supportive matrix, and rhBMP-2 was incorporated into the SBA-15 nanoparticles in the Zein scaffolds, reducing its initial burst release and prolonging duration of residence at the site of bone injury and thereby overcoming the inherent problem associated with traditional scaffold materials. At the same time, we modify the former scaffolds with HACC to endow its conspicuous bacteria repellency. Prior to the incorporation of SBA-15 and rhBMP-2 into the scaffolds, a series of zein-HACC composite scaffolds with different HACC contents were evaluated first to find a balance between cellular biocompatibility and antibacterial efficacy. The porosities and mechanical properties of a set of rhBMP-2 loaded silica/HACC/zein composite scaffolds were also examined to determine the specific SBA-15 content which would be used for the following experiments. SEM were used to compare the features of the different scaffolds and the in vitro release behavior of rhBMP-2 from the different scaffolds over a period of 27 d was also examined. The study found that the optimal HACC content is 10 wt% in Zein-HACC composite scaffolds, so that it can maintain both the good antibacterial activity to the test bacterial strains, and good biocompatibility to the planted cells; the SBA-15 content should be below 30 wt% in silica/HACC/Zein composite scaffold, thus it can maintain good porosities and considerable mechanical compressive strength; the silica/HACC/Zein composite scaffold carries rhBMP-2 cytokine, the presence of SBA-15 in the scaffold can improve its release curve, achieve the effect of long-term stable release in the target area; the higher the content of SBA-15 component is, the rhBMP-2 release effect is better.On the basis of the previous research, the bioactivity of the rhBMP-2 loaded scaffolds in vitro was tested by immersing the samples in the simulated body fluid(SBF) for a few days, then before and after immersion in SBF were evaluated using SEM and energy dispersive spectroscopy(EDS) for bioactivity analysis. Human mesenchymal stem cells(hMSCs) were seeded in the different groups of scaffolds to evaluate the cell attachment, cell proliferation, cell morphology, ALP activity and ECM mineralization assay. Confocal laser scanning microscopy(CLSM) was used to quantitatively measure the bactericidal efficacy with respect to bacterial adhesion in the different scaffolds. In a mouse model of thigh muscle pouches, the ectopic bone formation of rh BMP-2 loaded silica/HACC/zein scaffolds in the thigh muscle pouches of living mice was firstly quantitatively evaluated by determining the wet, dry, and mineral ash weights of the implants. the ability of rhBMP-2-loaded silica/HACC/zein scaffolds to repair critical-sized bone defects was examined further. A radial 20 mm defect model was used in rabbits. Micro-CT, bone mineral content, bone mineral density, and histological analysis were used to evaluate the repair of bone defects with scaffolds at week 4, 8, and 12 after implantation. In this way, the zein-HACC-S20 scaffolds developed here showed bioactivity, biocompatibility, and effective antibacterial activity, especially were proven to significantly promote the bone repair. They also demonstrated considerable promise for tissue engineering. Silica/HACC/zein scaffolds with both antibacterial activity and the ability to induce osteogenesis have immense potential in orthopedics and other biomedical applications.