Fabrication Of Bioactive Materials For Bone Regeneration And Studies On Biological Responses

The damage and defect on bone tissue resulting from traumas, tumours and infections has been an important medical project affecting human health. Nowadays, it's a hot topic to develop novel biomaterials with the capabilities modulating cell behaviors and tissue regeneration. Considering the structure and extracellular matrix of the natural bone, several materials with high bioactivity for bone repair were fabricated, and the interactions between the materials and cells/tissues, the potential mechanism and applications were investigated.Magnesium phosphate/calcium phosphate cement and interactions with osteo-related cells——Trace element magnesium is reported to be essential in bone biological process. In the present study, the influences of magnesium in calcium phosphate cement (CPC) on osteoblast initial adhesion and vasculation were explored. Incorporation of magnesium into CPC was performed using Ca(H2PCO4)2 and MgO as reactants. Magnesium-calcium phosphate cement (MCPC) was developed by controlling Mg/P ratio (5,10, and 20%) and denoted as 5MCPC,10MCPC and 20MCPC, respectively. Using MG63 as in vitro model, these cements showed excellent biocompatibility. The exogenous magnesium in culture medium or ionic dissolution from cement had negligibly positive impacts on the initial cellular adhesion. However, adhesion efficiency was significantly enhanced when osteoblasts were cultured on MCPCs surface directly, especially on 5MCPC. Despite surface roughness of MCPCs decreased as compared with CPC, cell attachments were evidently enhanced on 5MCPC and 10MCPC as proved by phalloidin-cytoskeleton staining. Gene expression of integrin a5 was significantly up-regulated on 5MCPC and 10MCPC, while integrin al, a2 and?1 expressions were not positively influenced. Magnesium existing forms seem to play different roles on osteoblast adhesion:ionic Mg2+ is subsidiary in osteoblast adhesion process. But magnesium on cement surface was directly involved as a cell-inducing factor via integrin?5 signaling pathway. The present study indicated that low amount of magnesium incorporation into calcium phosphate cement was a feasible and effective way to improve osteoblast adhesion and to accelerate stable bio-interface formation for enhancing bone regeneration in orthopaedic and trauma surgery.Mesoporous silica xerogels with different calcium contents and responses on osteoblasts——Mesoporous silica xerogels with various amount of calcium oxide (0,5,10 and 15%, named m-SXCO, m-SXC5, m-SXC10 and m-SXC15, respectively) were synthesized by template sol-gel methods. Cell morphology was not affected by m-SXCs indicating good biocompatibility. Furthermore, cell proliferation ratio on the m-SXCs increased over time, among which m-SXC10 was highest. NO production obviously rose with the increase of Ca content in m-SXCs. ALP activity and PGE2 level on m-SXC5 significantly improved compared with m-SXCO while decreased with the increase of Ca content for m-SXC10 and m-SXC15. The collagen I and osteocalcin mRNA expression on m-SXC5 were up-regulated, while decreased on m-SXC15 evidently. The phosphorylation level of ERK 1/2 for the m-SXC10 was highest after 7 days. In conclusion, calcium in m-SXCs plays an important role in osteoblast activity, which indicates mesoporous silica xeroel containing appropriate calcium could stimulate osteoblast proliferation, differentiation, gene expression via the activation of ERK 1/2 signaling pathway, and shows great prospects in bone regeneration field using as a drug controlled release filler.Mesoporous silica xerogerl with different specific surface area and responses on osteoblasts——Specific surface area is a critical parameter of mesoporous silica-based biomaterials, however, little is known about its effects on osteoblast responses in vitro. In the present study, mesoporous silica xerogels (MSXs) with different surface area (401,647 and 810 m2/g, respectively) were synthesized by a sol-gel process. Surface silanol contents decreased with the increase of surface area with which protein adsorption capability positively correlated. And the apatite-like surface seemed to form faster on MSXs with higher surface area determined by XRD analysis. Using MG63 osteoblast-like cells as models, it was found that cell proliferations were promoted on MSXs with higher surface area, based on the premise that the effects of Si released from materials on osteoblast viability were excluded by real-time Transwell(?) assay. RT-PCR results indicated cell adhesion-related integrin subunits a5 were up-regulated by higher surface area at day 1, which was further confirmed by flow cytometry analysis. The data suggest that increasing SSA of MSXs could promote surface cellular affinity by adsorbing serum proteins and accelerating apatite-like layer formation, which results in promoted osteoblastic proliferation via integrin subunit a5 at initial adhesion stage. Regulating SSA, an effective approach in designing mesoporous silica-based materials, provides an alternative method to obtain desirable tissue-response in bone regeneration and drug delivery system.Modifying chitosan with sulfate group and its interaction with bone morphogenetic protein-2——Bone morphogenetic protein-2 (BMP-2) has been widely used as an effective growth factor in bone tissue engineering. However, large amounts of BMP-2 required to induce new bone formation would result in potential side-effects, which limits its clinical application. Sulfated polysaccharides, such as native heparin have been found to modulate BMP-2 bioactivity. Whereas the direct role of chitosan modified with sulfate group on BMP-2 signaling has not been reported till now. Several sulfated chitosans with different positions were synthesized by regioselective reactions firstly. Using C2C12 myoblast cells as in vitro models, the enhanced bioactivity of BMP-2 was attributed primarily to the stimulation from 6-O-sulfated chitosan (6SCS), while 2-N-sulfate was subsidiary group with less activation. Low dose of 2-N,6-O-sulfated chitosan (26SCS) showed significant enhancement on the alkaline phosphatase (ALP) activity and the mineralization formation induced by BMP-2, as well as the expression of ALP and Osteocalcin mRNA. Moreover, increased chain-length and further sulfation on 26SCS also resulted in a higher ALP activity. Dose-dependent effects on BMP-2 bioactivity were observed in both sulfated chitosan and heparin. Compared with native heparin,26SCS showed much stronger simultaneous effects on the BMP-2 bioactivity at low dose. Furthermore, simultaneous administration of BMP-2 and 26SCS in vivo dose-dependently induced larger amounts of ectopic bone formation compared with BMP-2 alone. These findings clearly indicate that 26SCS is a more potent enhancer for BMP-2 bioactivity to induce osteoblastic differentiation in vitro and in vivo by promoting BMP-2 signaling pathway, suggesting that 26SCS could be used as the synergistic factor of BMP-2 for bone regeneration.The synergistic effects of calcium silicate/calcium phosphate cement scaffolds and bone morphogenetic protein-2——Calcium silicate was incorporated into calcium phosphate cement to fabricate marcoporous scaffolds and load bone morphogenetic protein-2 (BMP-2), the possible synergistic effects were investigated. The protein absorption capability of composite scaffolds was much higher than that of CPC. Faster degradation rate was observed in composite scaffolds, which also released larger amounts of silicon, calcium and phosphate ions. Using MG63 cells as model, the indirect and direct cluture experiments demonstrated composite scaffolds in the presence of BMP-2 induced highest alkaline phosphatase activity. Same conclusion was also brought by Micro-CT and histological sections analysis on in vivo studies. These results showed that calcium silicate incorporated macroporous scaffolds have synergistic enhancing effects with BMP-2 on bone regeneration, which could be potential therapy in future.