钛材表面生物功能化及其对骨髓间充质干细胞的影响
发布时间：2018-08-26 12:59
【摘要】：骨髓间充质干细胞(MSCs)是一类来源于骨髓的多能干细胞,能被诱导分化为成骨细胞或者成软骨细胞,广泛应用于骨组织工程和骨外科领域。MSCs对其赖以生存的细胞外基质(ECM)微环境极其敏感,能快速而精确地响应ECM给予的任何生物物理和生物化学刺激。ECM的些许变化都有可能改变MSCs的命运。因此,如何优化材料设计并诱导MSCs向成骨系细胞分化,实现骨组织再生和重建,已成为骨科相关领域的研究热点之一。钛及钛合金是临床上广泛应用的骨修复材料。然而,钛及钛合金材料表面的二氧化钛氧化层具有生物惰性,缺乏诱导骨生成潜能,导致钛基植入体与周边自然骨组织的整合性差,使用寿命短。钛基植入体植入体内后,生长在材料表面并促进新骨生成的成骨细胞大多来自于MSCs。鉴于此,为提高钛基植入体的骨整合性能,亟需发展提高钛基材表面生物活性的方法,以诱导MSCs的成骨分化,进而促进骨组织生成和加速植入体与周围骨组织的整合。本论文从仿生自然骨微纳结构角度出发,利用简单的碱热处理技术、离子交换技术和硅烷化等方法制备了具有高效生物活性表面的钛基材,提供MSCs适宜的生物物理和/或生物化学刺激,诱导MSCs的成骨分化,进而提高钛基材表面的新骨生成能力和骨整合性。本论文主要研究内容和结论如下:1.纳米片功能化钛材对骨髓间充质干细胞行为的影响为探究生物物理刺激对MSCs成骨分化行为的影响,本章利用简单的碱热和稀酸处理,在纯钛表面制备了均匀规整的由TiO2纳米片组成的亚微米拓扑结构。通过场发射扫描电镜、原子力电镜、X光电子能谱、X射线衍射仪和接触角测试对表面特性进行表征。利用牛血清白蛋白进行蛋白质吸附实验,结果表明,随着TiO2纳米片尺寸的增大,拓扑结构化钛材对蛋白吸附的吸附能力显著增加。此外,通过四唑盐比色检测,粘着斑蛋白染色,碱性磷酸酶(ALP)染色,茜素红染色,细胞矿化钙生成量测定,成骨细胞特异转录因子Runx2、成骨相关转录因子Osterix、ALP、I型胶原蛋白(Col I)、骨钙素(OCN)和骨桥蛋白(OPN)在mRNA水平上的表达以及Runx2、Col I和OPN蛋白表达水平等实验,在细胞与分子水平上分别探究了表面纳米结构化钛材对MSCs的粘附、铺展、增殖及分化的影响。结果表明,TiO2纳米片组成的亚微米拓扑结构显著地促进了MSCs的粘附、增殖及成骨分化。本研究提供了一种制备表面亚微米复合拓扑结构化钛基植入体的新方法。2.含锶纳米片功能化钛材协同促进骨髓间充质干细胞分化及体内成骨的研究为探究生物物理和生物化学刺激对MSCs行为的协同影响,我们采用离子交换技术改变了TiO2纳米片组成的亚微米拓扑结构中的化学元素,将生物活性Sr2+巧妙地引入到拓扑结构中,制备了含锶纳米片功能化钛材。通过场发射扫描电镜、X射线能谱仪、X光电子能谱、原子力电镜、X射线衍射仪及接触角测量对材料表面物理和化学特征进行表征。在不改变亚微米拓扑结构的基础上,成功地将Sr2+引入到钛材表面。然后,通过四唑盐比色检测,粘着斑蛋白染色,ALP染色,茜素红染色,细胞矿化钙生成量测定以及Runx2、Osterix、ALP、Col I、OCN和OPN在mRNA水平上的表达等实验,从细胞和分子水平考察了引入Sr2+离子的亚微米拓扑结构对MSCs成骨分化的协同效应。结果表明,一方面,含锶纳米片功能化钛材通过亚微米拓扑结构给予MSCs强烈的生物物理刺激,增强粘着斑复合物的形成和成熟,从而诱导MSCs向成骨系分化;另一方面,从结构中释放的Sr2+,在一定时间内持续地给予MSCs生物化学刺激,加速MSCs的成骨分化。最后,将未处理的、亚微米拓扑结构的与引入Sr2+离子的亚微米拓扑结构化的钛棒分别植入到成熟新西兰兔子的股骨骨端,经过4和12周后,进行X光片,Micro-CT扫描及苏木精伊红和masson’s三色染色组织学分析,评价Sr2+离子和亚微米拓扑结构在体内协同促进新骨生成的能力。体内植入结果表明,引入了Sr2+离子的亚微米拓扑结构协同地促进了植入体的新骨生成,从而提高了植入体与周围骨组织的整合能力。本研究为研发高质量的具有优良骨整合性的钛植入体提供了新方法。3.叶酸锶螯合物功能化钛材对骨髓间充质干细胞成骨分化影响的研究为赋予生物功能化钛材长时间刺激MSCs的能力,本研究利用硅烷化和共价接枝的方法将叶酸锶螯合物固定到钛材表面,制备了叶酸锶螯合物功能化钛材。通过场发射电镜、原子力电镜、X光电子谱与接触角测量对表面特性进行表征。结果显示,叶酸锶螯合物已成功地固定到碱热处理的钛材表面,锶元素的含量达到3.11%。然后,通过CCK-8细胞活性检测,细胞骨架染色,ALP染色,茜素红染色,细胞矿化钙生成量测定以及整合素、粘着斑蛋白、Runx2、ALP、Col I和OCN在mRNA水平上的表达等实验,在细胞与分子水平上考察了叶酸锶螯合物功能化的钛材对MSCs成骨分化的影响。实验结果表明,叶酸锶螯合物功能化的钛材不仅在短时间有效地促进MSCs的成骨分化,而且能在较长时间内保持对MSCs强烈的刺激作用。本研究为制备高性能钛材植入体提供了新技术。
[Abstract]:Bone marrow mesenchymal stem cells (MSCs) are a class of pluripotent stem cells derived from bone marrow, which can be induced to differentiate into osteoblasts or chondroblasts. MSCs are widely used in bone tissue engineering and orthopedic surgery. MSCs are extremely sensitive to the extracellular matrix (ECM) microenvironment on which they live and can respond quickly and accurately to any biological substance given by ECM. Some changes in ECM may change the fate of MSCs. Therefore, how to optimize material design and induce MSCs to differentiate into osteoblasts and achieve bone tissue regeneration and reconstruction has become one of the hotspots in orthopaedic related fields. Titanium and titanium alloys are widely used bone repair materials in clinic. Titanium dioxide oxide layer on the surface of alloy materials is bio-inert and lacks the potential to induce bone formation, which leads to poor integration of titanium-based implants with surrounding natural bone tissue and short service life. The osseointegration properties of implants require the development of methods to improve the surface bioactivity of titanium substrates to induce osteogenic differentiation of MSCs, thereby promoting bone tissue formation and accelerating the integration of implants with surrounding bone tissue. Titanium substrates with high bioactive surface were prepared by chemical methods to provide suitable biophysical and/or biochemical stimuli for MSCs to induce osteogenic differentiation of MSCs, thereby improving the osteogenesis and osseointegration of titanium substrates. To investigate the effect of biophysical stimuli on osteogenic differentiation of MSCs, a uniform and regular sub-micron topology composed of titanium dioxide nanosheets was fabricated on the surface of pure titanium by alkali-heat treatment and dilute acid treatment. Antenna test was used to characterize the surface properties. Bovine serum albumin (BSA) was used to adsorb proteins. The results showed that the adsorption capacity of topographically structured titanium materials increased significantly with the increase of the size of the nanosheets. In addition, tetrazolium colorimetric assay was used to detect the adsorptive spots, alkaline phosphatase (ALP) and alizarin red. The expression of osteoblast-specific transcription factor Runx2, osteoblast-related transcription factor Osterix, ALP, collagen type I (Col I), osteocalcin (OCN) and osteopontin (OPN) at the mRNA level and the expression of Runx2, Col I and OPN protein at the cellular and molecular levels were investigated. The results show that the sub-micron topological structure of titanium dioxide nanosheets significantly promotes the adhesion, proliferation and osteogenic differentiation of MSCs. This study provides a new method for preparing surface sub-micron composite topological titanium-based implants. 2. Strontium-containing functionalized titanium nanosheets. To explore the synergistic effect of biophysical and biochemical stimuli on MSCs behavior, we used ion exchange technology to change the chemical elements in the sub-micron topology of titanium dioxide nanosheets and skillfully introduced bioactive Sr2+ into the topology. Strontium-containing nanosheets of functionalized titanium were prepared and characterized by field emission scanning electron microscopy, X-ray energy dispersive spectroscopy, X-ray photoelectron spectroscopy, atomic force electron microscopy, X-ray diffraction and contact angle measurement. The synergistic effect of Sr2+ on osteogenic differentiation of MSCs was investigated at cellular and molecular levels by azole salt colorimetric assay, adhesion spot protein staining, ALP staining, alizarin red staining, calcium mineralization assay and mRNA expression of Runx2, Osterix, ALP, Col I, OCN and OPN. On the other hand, Sr2+ released from the structure of the functionalized titanium nanosheets can continuously stimulate the osteogenic differentiation of MSCs and accelerate the osteogenic differentiation of MSCs. Finally, the untreated, submicron and Sr2+ implanted titanium rods were implanted into the femoral bones of mature New Zealand rabbits. After 4 and 12 weeks, X-ray films, micro-CT scans and hematoxylin eosin and masson's staining histological analysis were performed to evaluate Sr2+ and submicron topological junctions. In vivo implantation results show that the introduction of Sr2+ ion into the submicron topology promotes the formation of new bone and improves the ability of the implant to integrate with the surrounding bone tissue. 3. Effect of functionalized titanium folate chelate on osteogenic differentiation of bone marrow mesenchymal stem cells. In order to endow biofunctionalized titanium with the ability to stimulate MSCs for a long time, this study used silylation and covalent grafting method to fix the strontium folate chelate on the surface of titanium and prepared the functionalized titanium folate chelate. The results showed that strontium folate chelate had been successfully immobilized on the surface of alkali-treated titanium, and the content of strontium element reached 3.11%. Then, cytoskeleton staining, ALP staining, alizarin red staining were used to detect the activity of CCK-8 cells. The effects of titanium materials functionalized by strontium folate chelate on osteogenesis and differentiation of MSCs were investigated at the cellular and molecular levels by measuring integrin, adhesion spot protein, Runx2, ALP, Col I and OCN expression at the mRNA level. This study provides a new technique for the preparation of high performance titanium implants.
【学位授予单位】：重庆大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318.08
，
本文编号：2204938
[Abstract]:Bone marrow mesenchymal stem cells (MSCs) are a class of pluripotent stem cells derived from bone marrow, which can be induced to differentiate into osteoblasts or chondroblasts. MSCs are widely used in bone tissue engineering and orthopedic surgery. MSCs are extremely sensitive to the extracellular matrix (ECM) microenvironment on which they live and can respond quickly and accurately to any biological substance given by ECM. Some changes in ECM may change the fate of MSCs. Therefore, how to optimize material design and induce MSCs to differentiate into osteoblasts and achieve bone tissue regeneration and reconstruction has become one of the hotspots in orthopaedic related fields. Titanium and titanium alloys are widely used bone repair materials in clinic. Titanium dioxide oxide layer on the surface of alloy materials is bio-inert and lacks the potential to induce bone formation, which leads to poor integration of titanium-based implants with surrounding natural bone tissue and short service life. The osseointegration properties of implants require the development of methods to improve the surface bioactivity of titanium substrates to induce osteogenic differentiation of MSCs, thereby promoting bone tissue formation and accelerating the integration of implants with surrounding bone tissue. Titanium substrates with high bioactive surface were prepared by chemical methods to provide suitable biophysical and/or biochemical stimuli for MSCs to induce osteogenic differentiation of MSCs, thereby improving the osteogenesis and osseointegration of titanium substrates. To investigate the effect of biophysical stimuli on osteogenic differentiation of MSCs, a uniform and regular sub-micron topology composed of titanium dioxide nanosheets was fabricated on the surface of pure titanium by alkali-heat treatment and dilute acid treatment. Antenna test was used to characterize the surface properties. Bovine serum albumin (BSA) was used to adsorb proteins. The results showed that the adsorption capacity of topographically structured titanium materials increased significantly with the increase of the size of the nanosheets. In addition, tetrazolium colorimetric assay was used to detect the adsorptive spots, alkaline phosphatase (ALP) and alizarin red. The expression of osteoblast-specific transcription factor Runx2, osteoblast-related transcription factor Osterix, ALP, collagen type I (Col I), osteocalcin (OCN) and osteopontin (OPN) at the mRNA level and the expression of Runx2, Col I and OPN protein at the cellular and molecular levels were investigated. The results show that the sub-micron topological structure of titanium dioxide nanosheets significantly promotes the adhesion, proliferation and osteogenic differentiation of MSCs. This study provides a new method for preparing surface sub-micron composite topological titanium-based implants. 2. Strontium-containing functionalized titanium nanosheets. To explore the synergistic effect of biophysical and biochemical stimuli on MSCs behavior, we used ion exchange technology to change the chemical elements in the sub-micron topology of titanium dioxide nanosheets and skillfully introduced bioactive Sr2+ into the topology. Strontium-containing nanosheets of functionalized titanium were prepared and characterized by field emission scanning electron microscopy, X-ray energy dispersive spectroscopy, X-ray photoelectron spectroscopy, atomic force electron microscopy, X-ray diffraction and contact angle measurement. The synergistic effect of Sr2+ on osteogenic differentiation of MSCs was investigated at cellular and molecular levels by azole salt colorimetric assay, adhesion spot protein staining, ALP staining, alizarin red staining, calcium mineralization assay and mRNA expression of Runx2, Osterix, ALP, Col I, OCN and OPN. On the other hand, Sr2+ released from the structure of the functionalized titanium nanosheets can continuously stimulate the osteogenic differentiation of MSCs and accelerate the osteogenic differentiation of MSCs. Finally, the untreated, submicron and Sr2+ implanted titanium rods were implanted into the femoral bones of mature New Zealand rabbits. After 4 and 12 weeks, X-ray films, micro-CT scans and hematoxylin eosin and masson's staining histological analysis were performed to evaluate Sr2+ and submicron topological junctions. In vivo implantation results show that the introduction of Sr2+ ion into the submicron topology promotes the formation of new bone and improves the ability of the implant to integrate with the surrounding bone tissue. 3. Effect of functionalized titanium folate chelate on osteogenic differentiation of bone marrow mesenchymal stem cells. In order to endow biofunctionalized titanium with the ability to stimulate MSCs for a long time, this study used silylation and covalent grafting method to fix the strontium folate chelate on the surface of titanium and prepared the functionalized titanium folate chelate. The results showed that strontium folate chelate had been successfully immobilized on the surface of alkali-treated titanium, and the content of strontium element reached 3.11%. Then, cytoskeleton staining, ALP staining, alizarin red staining were used to detect the activity of CCK-8 cells. The effects of titanium materials functionalized by strontium folate chelate on osteogenesis and differentiation of MSCs were investigated at the cellular and molecular levels by measuring integrin, adhesion spot protein, Runx2, ALP, Col I and OCN expression at the mRNA level. This study provides a new technique for the preparation of high performance titanium implants.
【学位授予单位】：重庆大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318.08
，
本文编号：2204938
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