Construction Of Mesoporous Silica Nanoparticles-Based Osteogenic Delivery System For Bone Repair

In daily life, the defect on bone tissue can be caused by trauma, tumor, infection and other diseases, which will severely affect the physical and mental health and quality of life of patients. Traditional autologous bone grafts and allogous bone grafts are still commonly used therapeutic approaches to treat bone defect in clinic. Owing to several inherent limitations dramatically constricting their wide applications, they cannot meet the huge clinical demand. Therefore, developing bone repair materials with excellent biological activity is the research direction for the researchers. Mesoporous silica nanoparticles(MSNs), a kind of nanoscale inorganic particles, possess many unique properties, such as large specific surface area and pore volume, adjustable particle size and pore size, facile surface functionalization and so on, which make them much more accessible for application in biomedical fields over the past decade. At present, most of the researches on MSNs have focused on its application in bone tissue engineering. Herein, based on the outstanding physical properties, MSNs have been used to construct the nanoparticulate osteogenic delivery systems and organic/inorganic composite scaffold for bone repair. Then, the physicochemical properties of these materials were extensively investigated, as well as the in vitro and in vivo experiments on osteogenesis. The major contents are presented as follows:(1) Polylysine-modified polyethylenimine copolymer(PEI-PLL) can be obtained by the ring-opening polymerization(ROP) using PEI as a macro-initiator and Lys(Z)-NCA as the monomer and subsequent deprotection of benzyloxycarbonyl group. We synthesized different PEI-PLL copolymers using different molecular weight of PEI(Mw = 1800, 10000, 25000), denoted as PEI-PLL-1.8k, PEI-PLL-10 k and PEI-PLL-25 k, respectively. Firstly, the successful synthesis of PEI-PLL copolymers was verified by the 1H NMR analysis and FTIR measurement. The synthesized PEI-PLL copolymers were further characterized by measuring their buffering capacity, DNA binding and DNase protection ability. The cell viability assay showed that all PEI-PLL copolymers exhibited better biocompatibility compared with pure PEI(Mw = 25000, PEI-25k). In addition, the PEI-PLL-25 k displayed the best transfection efficiency among the PEI-PLL copolymers and higher transfection efficiency than PEI-25 k as evidenced by the results of luciferase gene expression, fluorescent protein expression and flow cytometry assay. The bone morphogenetic protein-2(BMP-2) gene expression demonstrated the effective gene delivery by PEI-PLL-25 k copolymer. Furthermore, BMP-2 gene delivered into bone marrow mesenchymal stem cells(BMSCs) by PEI-PLL-25 k copolymer enhanced the osteogenic differentiation in terms of ALP activity and Von Kossa staining assays. Therefore, our results demonstrated that PEI-PLL-25 k copolymer showed high transfection efficiency and low cytotoxicity, which could be a promising gene carrier for further application in treating bone-related disease.(2) Due to its outstanding properties of synthesized PEI-PLL-25 k copolymer including high transfection efficiency and low cytotoxicity, it can be conjugated onto MSNs to render the surface with positive charge. Firstly, the prepared aminated-MSNs was functionalized with carboxyl group, and then conjugated with PEI-PLL-25 k by the EDC/NHS chemistry. Subsequently, the multifunctional mesoporous silica nanoparticles(MSNs-PPR) were achieved by further modification with RGD peptide onto the surface of nanoparticles. Then, the resulting MSNs-PPR has the ability to encapsulate the dexamethasone(DEX) into the mesopores and simultaneously carry plasmid DNA(pDNA) via electrostatic interaction between cationic MSNs-PPR and anionic DNA. The as-prepared MSNs-PPR was confirmed by TEM, FTIR zeta-potential and TGA, also the pDNA and DEX release from MSNs-PPR were investigated. The in vitro cell viability and hemolysis assays demonstrated the good biocompatibility of MSNs-PPR. In addition, the pDNA encoding BMP-2 gene can be efficiently delivered into BMSCs and expressed the BMP-2 protein. Furthermore, compared with BMSCs treated with single BMP-2 gene delivery and single DEX delivery, BMSCs treated with MSNs-PPR bearing BMP-2 gene and DEX showed higher activities of osteogenic differentiation in terms of ALP activity, osteo-related gene expression and alizarin red S staining. Therefore, simultaneous delivery of BMP-2 gene and DEX by MSNs-PPR nanocarrier could be employed as nanoparticulate osteogenic delivery systems for bone repair.(3) The peptides corresponding to residues 73-92 of BMP-2 are the bioactive domains, which become an alternative to enhance the biological activity of biomaterials. We therefore synthesized BMP-2 peptide functionalized mesoporous silica nanoparticles(MSNs-pep) by the reaction of carboxy group on peptide and amino group on aminated-MSNs using the EDC/NHS chemistry, and dexamethasone was then adsorbed into the mesopore to construct nanoparticulate osteogenic delivery systems(DEX@MSNs-pep). The in vitro cell viability of functionalized MSNs was conducted against BMSCs with different particle concentrations, revealing that the MSNs-pep had better cytocompatibility than bare MSNs. In addition, the cellular uptake of MSNs-pep against BMSCs was remarkably enhanced. The in vitro experiment also showed that MSNs-pep promoted the osteogenic differentiation of BMSCs, which was confirmed by the upregulation of ALP activity and osteo-related proteins expression, as well as the increased mineralized matrix. Also, the results demonstrated that the loading of DEX in MSNs-pep could further promote the osteogenic differentiation of BMSCs. After intramuscular implantation in rats for three weeks, the results of computed tomography(CT) images and histological examination exhibited that this nanoparticulate osteogenic delivery system could induce ectopic bone formation in vivo. Collectively, the BMP-2 peptide and DEX incorporated MSNs showed higher bioactivity for osteogenic differentiation, which have potential applications in bone tissue engineering.(4) The native bone is mainly composed of organic component(collagen) and inorganic component(hydroxyapatite). To simulate the composition of native bone, we fabricated the organic/inorganic composite scaffold by incorporating the MSNs into gelatin scaffold. The antibiotic vancomycin hydrochloride(Van) was firstly loaded into the mesopore of prepared MSNs to achieve the drug-loaded MSNs(Van@MSNs). Then Van@MSNs nanoparticles were added into the gelatin solution to fabricate the Van@MSNs incorporated composite scaffold(Van@MSNs/Gelatin). The SEM images showed that the obtained composite scaffold was porous in structure, and the pore size become larger with the increased content of MSNs(5-20% relative to the gelatin). We also studied the influence of added MSNs on the mechanical property of obtained composite scaffold. The result displayed that the compressive modulus was enhanced by the addition of MSNs, but decreased with the content of MSNs increased(5-20%) while still better than pure Gelatin scaffold. Importantly, the Van@MSNs/Gelatin composite scaffold showed a sustained drug release profile and obvious growth inhibition on Staphylococcus aureus. Additionally, in vitro experiments suggested that Van@MSNs/Gelatin composite scaffold could well support the adhesion, proliferation and differentiation of BMSCs. Moreover, in vivo results showed that the Van@MSNs/Gelatin composite scaffold could significantly promote the rabbit radius defect to heal compared with Gelatin and MSNs/Gelatin scaffolds after 12 weeks implantation. Therefore, the obtained results demonstrate that the fabricated Van@MSNs/Gelatin composite scaffold is a promising biomaterial for treating infected bone defect.In conclusion, MSNs were not only employed to constructe the nanoparticulate osteogenic delivery systems, but also acted as micocarrier to prepare the organic/inorganic composite scaffold. The potential application of these materials on bone repair was extensively explored. We hope this work will promote the application of MSNs in bone tissue engineering and provide new insights to the development of bioactive materials for bone defect repair.