| As one kind of pluripotent stem cells deriving from bone marrow, mesenchymal stem cells(MSCs) could be induced to differentiate into osteoblasts or chondroblasts, and thus being widely used in bone tissue engineering and orthopedic fields. MSCs are extremely sensitive to their survival extracellular matrix(ECM) microenvironments, and can quickly and accurately respond to any biophysical and biochemical cues supplying from ECM. A trivia change in ECM is likely to change the fates of MSCs.Therefore, how to optimize the material design and induce MSCs to differentiate into osteogenic commitment, realizing the bone regeneration and reconstruction, becomes one of the hot topics in the orthopedic related field. Titanium(Ti) and Ti alloys were widely used as bone repair materials in clinics. However, the Ti O2 layer on surfaces titanium and titanium alloys is bioinert, being lack of the potential for inducing bone tissue formation, which results in poor osseointegration between the implant and its surrounding nature bone tissue and short lifespan of the Ti-based implants. When a Ti-based impmant is implanted into a host, most osteoblasts grown onto materials surface and contribution to new bone formation originate from MSCs. Considering this fact, to improve the osseointegration property of Ti-based implant, it is urgent to develop approaches for enhancing the surface bioactivity of Ti materials, so as to induce the osteogenic differentiation of MSCs, in turn promoting bone formation and accelerating the osseointegration of implants with surrounding bone tissue.In this study, from the perspective of the hierarchical structure of natural bone, we employed simple alkali-heat treatment, ion exchange and silanization technology to fabricate Ti materials with efficient surface bioactivity, providing sesirable biophysical and/or biochemical stimuli for MSCs, inducing MSCs to differentiate into osteoblasts, in turn improving new bone formation on titanium substrates surface and osseointegration of titanium implants. Main contents and conclusions of this study are listed as follows: 1. The effects of nanosheets functional titanium on the behavior of MSCsTo explore the effect of biophysical stimulation on the osteogenic differentiation of MSCs, in this chapter, a simple alkali-heat and dilute acid treatment technology were employed to fabricate a submicron topography composed of Ti O2 nanosheets on the surface of pure titanium.Field emission scanning electron microscopy(FE-SEM), atomic force microscopy(AFM), X-ray diffraction(XRD), X-ray photoelectron spectroscopy(XPS) and contact-angle measurements were used to characterize the surfaces of native titanium and surface submicron-structured titanium substrates, respectively. Protein adsorption experiment was performed with bovine serum albumin(BSA). The result suggests that the protein adsorption capacity of topographic Ti substrates increased significantly with increasing the dimensions of Ti O2 nanosheets. Moreover, MTT test, immunofluorescence staining of vinculin, alkaline phosphatase(ALP) staining and alizarin red staining, cell mineralization, m RNA expressions of Runx2, osteogenesis related transcription factors(Osterix), ALP, Col I, OPN and OCN, and protein levels of runt-related protein 2(Runx2), collagen type I(Col I) and osteopontin(OPN), were characterized to evaluate the effects of the nanosheets functional titanium substrates on the adhesion, proliferation and osteogenic differentiation of MSCs at cellular and molecular levels in vitro. The results suggest that the nanosheets functional titanium substrates significantly promoted the adhesion, proliferation and osteogenic differentiation of MSCs. This study provides a simple approach for the fabrication of surface submicron structured titanium-based implants. 2. Synergistic effects of strontium ions incorporated nanosheets functional Ti substrates on osteogenic differentiation of MSCs in vitro and bone formation in vivoTo investigate the synergistic influences of biophysical and biochemical cues on the behavior of MSCs, we used ion-exchange technology to change the chemical elements of Ti O2 nanosheets sub-micrometer topography, introducing bioactive strontium element into the topographic structure, leading to the biofunctional nanosheets titanium containing strontium(Sr). The physical and chemical characteristics of material surface were revealed by FE-SEM, X-ray spectroscopy(EDS), XPS, AFM, XRD and contact angle measurements. The results show that Sr2+ ions were successfully introduced into the titanium surface without changing submicron topography of Ti substrates. Subsequently, MTT test immunofluorescence staining of vinculin, ALP staining and alizarin red staining, cell mineralization, the m RNA expressions of Runx2, Osterix, ALP, Col I, OPN and OCN were performed to evaluate the synergistic effects of biofunctional titanium containing strontium and sub-micrometer topography on the osteogenic differentiation of MSCs at cellular and molecular levels in vitro. The results show that the biofunctional titanium containing Sr2+ and sub-micrometer topography, on the one hand, provided MSCs strong biophysical stimulations by submicron topography to enhance the formation and maturation of focal adhesion complexes(FAs), thus inducing MSCs to differentiate into osteogenic commitment; on the other hand, the released Sr2+ ions from the biofunctional Ti substrates continuously stimulated MSCs in a certain period of time, accelerating the osteogenic differentiation of MSCs. Finally, the untreated Ti, sub-micrometer topographic Ti and the biofunctional Ti containing Sr2+ and sub-micrometer topography implants were inserted into mature New Zealand rabbit femoral bone, and at 4 and 12 weeks after implantation, X-ray photography, micro-CT and histological analysis by hematoxylin and eosin(H&E) and Masson’s trichrome staining were employed to evaluate the synergistic ability of Sr2+ and sub-micrometer topography to promote the new bone formation in vivo. The results show that the introduction of Sr2+ together with the sub-micrometer topography synergistically promoted the formation of new bone formation of implants, thus improving the osseointegration of implants with surrounding bone tissue. The approach presented in this work has great potential for the development of titanium-based implants with enhanced bone osseointegration. 3. The effects of folic acid strontium chelate functional Ti material on the osteogenic differentiation of MSCsTo endow biofunctional Ti substrates with capacityof long-term stimulation to MSCs, this work used silanization and covalent grafting methods to fix folic acid strontium chelate onto Ti substrates, leading to folic acid strontium chelate functional Ti material. The surface characteristics of Ti materials were determined by FE-SEM, AFM, XPS and contact angle measurements. The results show that folic acid strontium chelate was successfully immobilized to the Ti surface with strontium content of 3.11%. After that, CCK-8 test, immunofluorescence staining of cytoskeleton, ALP staining and alizarin red staining, cell mineralization, the m RNA expressions of Runx2, ALP, Col I and OCN were performed to evaluate the effects of folic acid strontium chelate functional titanium substrates on osteogenic differentiation of MSCs at cellular and molecular levels in vitro.The results show that folic acid strontium chelate functional titanium not only effectively promoted the osteogenic differentiation of MSCs in a short term, but also kept strong stimulation effect on MSCs for long term. The study providess a new approach for the fabrication of Ti implants with high performance. |
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