钛材表面纳米结构化及其对骨髓间充质干细胞的影响
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摘要
生物材料植入宿主体内后，其与生物系统的相互作用（蛋白吸附、细胞粘附/增殖等）发生在材料表面。细胞的生物学行为主要由材料表面化学成分和宏观、介观与微观多尺度的拓扑结构构成的局部微环境决定。因此，如何构建材料表面适宜的细胞外微环境，进而调控细胞的生理功能，已成为相关领域的研究热点之一。
钛及钛合金由于具有良好的物理性能已被作为植入体材料广泛应用于骨科临床领域。不足之处是，钛及钛合金材料表面有生物惰性，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，是其临床应用面临的普遍挑战。鉴于此，为了实现钛材表面原位调控细胞生物学行为，进而诱导骨组织形成，亟需研发新的钛材表面改性技术。从模拟类骨纳米结构及细胞外基质组分角度出发，本论文利用机械研磨技术、阳极氧化等方法制备了表面纳米拓扑结构的钛基材，进而沉积磷灰石/明胶，构建适宜的细胞外微环境，以期提高钛基植入体的骨整合性。主要研究内容和结论如下：
1.表面纳米结构化钛材对骨髓间充质干细胞行为的影响
为探究表面纳米结构化钛材对骨髓间充质干细胞生理行为的影响，本章利用机械研磨技术制备了表面纳米结构化钛材。通过扫描电子显微镜（SEM），透射电镜（TEM），原子力显微镜（AFM），X射线衍射仪（XRD）和接触角测试对表面特性进行表征。结果表明，经机械研磨技术处理后，在钛材表面上形成了纳米结构化薄层。通过纤连蛋白（Fn）与牛血清白蛋白（BSA）进行蛋白质吸附实验，结果表明，与未处理的纯钛相比，表面纳米结构化钛材对蛋白吸附量无显著的差异性。此外，在细胞与分子水平上，通过纽蛋白（vinculin）染色，四唑盐比色（MTT）检测，碱性磷酸酶（ALP）活性检测，骨钙素（OCN）与骨桥蛋白（OPN）染色，茜红素定量检测及骨钙素（OCN）、骨桥蛋白（OPN）、I型胶原蛋白（collagen I）与转录因子Runx2在mRNA水平表达等实验，分别研究了表面纳米结构化钛材对骨髓间充质干细胞粘附、铺展、增殖及分化的影响。结果表明，表面纳米结构化钛材促进了骨髓间充质干细胞粘附、增殖及骨发生相关的蛋白及基因在蛋白质与mRNA水平上的表达。本研究提供了一种制备表面纳米结构化钛材的新方法。
2. BMP2功能化TiO_2纳米管对骨髓间充质干细胞行为的协同效应
为探究BMP2功能化TiO_2纳米管对骨髓间充质干细胞行为的影响，本章利用聚多巴胺中间层将BMP2接枝到不同尺寸直径（30nm、60nm与100nm）的TiO_2纳米管上，并利用扫描电子显微镜（SEM），X光电子谱（XPS）及接触角对材料进行了表征。结果表明，BMP2已成功地接枝到TiO_2纳米管上。此外，进一步研究了BMP2修饰的TiO_2纳米管对骨髓间充质干细胞行为的影响。纽蛋白荧光染色结果表明，BMP2功能化的TiO_2纳米管促进了细胞粘附与生长，且经过7天与14天培养后，在BMP2功能化的TiO_2纳米管上生长的骨髓间充质干细胞表现更高的碱性磷酸酶（ALP）活性与矿化量（p<0.05或p<0.01），其中，BMP2功能化的管径为30nm的TiO_2纳米管上生长的细胞表现最高。该结果表明，BMP2功能化的TiO_2纳米管拓扑结构协同增效地促进了骨髓间充质干细胞的增殖与分化。本研究为研发高骨整合性钛植入体提供了新方法。
3.钛材表面微环境的构建及其对体外骨髓间充质干细胞成骨分化与体内成骨的影响
为模拟自然骨的细胞外基质，本章利用共沉淀方法将磷灰石/明胶沉积在纳米结构化钛材表面，这种纳米结构化钛材是通过氢氧化钾与加热处理后，在表面形成的抗腐蚀纳米结构化层。利用红外（FTIR），场发射扫描电子显微镜（FE-SEM），原子力显微镜（AFM）与薄膜X线衍射（TE-XRD）对材料进行了表征。检测结果表明，磷灰石/明胶成功地沉积在纳米结构化钛材表面。纽蛋白的荧光染色结果表明，磷灰石/明胶纳米成分促进了细胞粘附，更重要的是，在第7，14与21天时，在磷灰石/明胶纳米成分钛材上生长的骨髓间充质干细胞表现了更高的增殖与碱性磷酸酶（ALP）活性。并且，骨钙素（OCN）、骨桥蛋白（OPN）和I型胶原蛋白（p<0.05或p<0.01）的表达得到了更大地提高。通过OCN与OPN的免疫荧光染色也得到了同样的结果。通过组织切片，X光片与micro-CT（micro computedtomography，微计算机断层扫描技术）分析得知磷灰石/明胶纳米成分提高了骨密度和骨-植入体的接触率（p<0.05或p<0.01），诱导植入体与骨界面间新骨的生长。以上结果表明，磷灰石/明胶纳米成分促进了体内外成骨。本研究为制备高性能钛材植入体提供了新技术。
After implantation of a biomaterial into a host, the interaction betweenbiomaterials and biological system (protein adsorption, cell adhesion/proliferation, etc)occurs at the biomaterial surface. The biological behaviors of cells are mainlydominated by the local microenvironment of chemistry and topography on macroscle,mesoscale, and microscale sizes. Thus, how to construct desirable microenvironmentsonto the material surface, in turn mediating cells’ physiological functions, becomes oneof the hot topics in the related field.
Titanium (Ti) and its alloys have been widely applied as bone implants in clinicalapplications because of their good mechanical properties. Nevertheless，titanium basedmaterials are surface bioinert, being lack of the potential for inducing tissue formation,which results in poor osseointegration between the implant and its surrounding naturebone tissue and short lifespan of the implant. It is the common challenge in clinicalapplication. Considering those issues, to realize surface-mediating control of thebiological behaviors of cells and induction of bone tissue formation, it is urgent todevelop novel techniques. To improve the osseointegration of titanium based implants,in this study, surface mechanical attrition treatment (SMAT) technique and anodizationwere employed to fabricate surface nanostructured titanium, and then depositedapatite/gelatin composite to construct desirable microenvironments, from theperspective of mimicking the bone-like nanoscale architecture and the components ofextracellular matrix.
Main contents and conclusions of this research are listed as follows:
1. Regulation of the behaviors of mesenchymal stem cells by surface nanostructuredtitanium
The study describes the influence of surface nanostructured titanium on thebehaviors of mesenchymal stem cells (MSCs). Surface nanostructures of titanium wereproduced with surface mechanical attrition treatment (SMAT) technique. Field emissionscanning electron microscopy (FE-SEM), transmission electron microscopy (TEM),atomic force microscopy (AFM), X-ray diffraction (XRD) and contact-anglemeasurements were used to characterize the surfaces of native titanium and surfacenanostructured titanium substrates were characterized, respectively. A thinnanostructured layer was formed onto the surfaces of titanium substrates after SMAT treatment. Immunofluorescence staining of vinculin, osteocalcin (OCN), andosteopontin (OPN), MTT test, the levels of (Alkaline phosphatase) ALP and OCN andthe mRNA expressions of OCN, OPN, collagen type I (Col I) and Runx2(runt-relatedprotein2) were examined, in order to evaluate the effects of the surface nanostructuredtitanium substrates on the adhesion, spreading, proliferation and osteoblasticdifferentiation of MSCs at cellular and molecular levels in vitro. The results suggest thatthe surface nanostructured substrates were beneficial for the growth of MSCs, includingadhesion, filament orientation, proliferation and gene expression. This approach for thefabrication of surface nanostructured titanium may be exploited in the development ofhigh performance titanium-based implants.
2. Surface functionalization of TiO_2nanotubes with bone morphogenetic protein2and its synergistic effect on the differentiation of mesenchymal stem cells
To investigate the influence of surface-functionalized substrates withnanostructures on the behaviors of mesenchymal stem cells, we conjugated bonemorphogenetic protein2(BMP2) onto TiO_2nanotubes with different diameter sizes of30,60, and100nm for in vitro study. Polydopamine was employed as the intermediatelayer for the conjugation of BMP2. The successful conjugation of BMP2onto TiO_2nanotubes was revealed by field-emission scanning electron microscopy (FE-SEM),X-ray photoelectron spectroscopy (XPS), and contact angle measurements.Immunofluorescence staining of vinculin, osteocalcin (OCN), and osteopontin (OPN)revealed that BMP2-functionalized TiO_2nanotubes were favorable for cell growth.More importantly, MSCs cultured onto BMP2-functionalized TiO_2nanotubes displayedsignificantly higher (p <0.05or p <0.01) d ifferentiation levels of ALP andmineralization after7and14day cultures, respectively. The results suggested thatsurface functionalization of TiO_2nanotubes with BMP2was beneficial for cellproliferation and differentiation. The approach presented here has potential applicationfor the development of titanium-based implants for enhanced bone osseointegration.
3. Construction of microenvironment onto titanium substrates to regulate theosteoblastic differentiation of mesenchymal stem cells in vitro and osteogenesis invivo
To mimic the extracellular matrix of natural bone, apatite/gelatin composite wasdeposited onto nanostructured titanium substrates via a coprecipitation method, whichwas pretreated by potassium hydroxide and heat treatment to generate an anticorrosivenanostructured layer. The successful formation of the apatite/gelatin nanocomposite onto titanium surfaces was revealed by Fourier transform infrared spectroscopy,field-emission scanning electron microscopy, atomic force microscopy (AFM), and thinfilm X-ray diffraction (TF-XRD) measurements, respectively. The immunofluorescencestaining of vinculin revealed that the apatite/gelatin nanocomposite deposited titaniumsubstrate was favorable for cell adhesion. More importantly, mesenchymal stem cellscultured onto the apatite/gelatin nanocomposite deposited titanium displayed higher (p<0.05or p <0.01) proliferation and osteoblastic differentiation levels of alkalinephosphatase, mRNA expressions of collagen I (Col I), osteocalcin (OCN) andosteopontin (OPN), and OCN content after culture for7,14, and21days, respectively,which was also revealed by the immunofluorescence analysis of OCN and OPNexpression. The deposition of apatite/gelatin nanocomposite improved bone density (p <0.05) and bone-implant contact rate (p <0.05), which was reflected by microcomputedtomography analysis and histological evaluation in vivo using a rabbit model. This workprovides an approach to fabricate high-performance titanium-based implants withenhanced bone osseointegration.
钛及钛合金由于具有良好的物理性能已被作为植入体材料广泛应用于骨科临床领域。不足之处是，钛及钛合金材料表面有生物惰性，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，是其临床应用面临的普遍挑战。鉴于此，为了实现钛材表面原位调控细胞生物学行为，进而诱导骨组织形成，亟需研发新的钛材表面改性技术。从模拟类骨纳米结构及细胞外基质组分角度出发，本论文利用机械研磨技术、阳极氧化等方法制备了表面纳米拓扑结构的钛基材，进而沉积磷灰石/明胶，构建适宜的细胞外微环境，以期提高钛基植入体的骨整合性。主要研究内容和结论如下：
1.表面纳米结构化钛材对骨髓间充质干细胞行为的影响
为探究表面纳米结构化钛材对骨髓间充质干细胞生理行为的影响，本章利用机械研磨技术制备了表面纳米结构化钛材。通过扫描电子显微镜（SEM），透射电镜（TEM），原子力显微镜（AFM），X射线衍射仪（XRD）和接触角测试对表面特性进行表征。结果表明，经机械研磨技术处理后，在钛材表面上形成了纳米结构化薄层。通过纤连蛋白（Fn）与牛血清白蛋白（BSA）进行蛋白质吸附实验，结果表明，与未处理的纯钛相比，表面纳米结构化钛材对蛋白吸附量无显著的差异性。此外，在细胞与分子水平上，通过纽蛋白（vinculin）染色，四唑盐比色（MTT）检测，碱性磷酸酶（ALP）活性检测，骨钙素（OCN）与骨桥蛋白（OPN）染色，茜红素定量检测及骨钙素（OCN）、骨桥蛋白（OPN）、I型胶原蛋白（collagen I）与转录因子Runx2在mRNA水平表达等实验，分别研究了表面纳米结构化钛材对骨髓间充质干细胞粘附、铺展、增殖及分化的影响。结果表明，表面纳米结构化钛材促进了骨髓间充质干细胞粘附、增殖及骨发生相关的蛋白及基因在蛋白质与mRNA水平上的表达。本研究提供了一种制备表面纳米结构化钛材的新方法。
2. BMP2功能化TiO_2纳米管对骨髓间充质干细胞行为的协同效应
为探究BMP2功能化TiO_2纳米管对骨髓间充质干细胞行为的影响，本章利用聚多巴胺中间层将BMP2接枝到不同尺寸直径（30nm、60nm与100nm）的TiO_2纳米管上，并利用扫描电子显微镜（SEM），X光电子谱（XPS）及接触角对材料进行了表征。结果表明，BMP2已成功地接枝到TiO_2纳米管上。此外，进一步研究了BMP2修饰的TiO_2纳米管对骨髓间充质干细胞行为的影响。纽蛋白荧光染色结果表明，BMP2功能化的TiO_2纳米管促进了细胞粘附与生长，且经过7天与14天培养后，在BMP2功能化的TiO_2纳米管上生长的骨髓间充质干细胞表现更高的碱性磷酸酶（ALP）活性与矿化量（p<0.05或p<0.01），其中，BMP2功能化的管径为30nm的TiO_2纳米管上生长的细胞表现最高。该结果表明，BMP2功能化的TiO_2纳米管拓扑结构协同增效地促进了骨髓间充质干细胞的增殖与分化。本研究为研发高骨整合性钛植入体提供了新方法。
3.钛材表面微环境的构建及其对体外骨髓间充质干细胞成骨分化与体内成骨的影响
为模拟自然骨的细胞外基质，本章利用共沉淀方法将磷灰石/明胶沉积在纳米结构化钛材表面，这种纳米结构化钛材是通过氢氧化钾与加热处理后，在表面形成的抗腐蚀纳米结构化层。利用红外（FTIR），场发射扫描电子显微镜（FE-SEM），原子力显微镜（AFM）与薄膜X线衍射（TE-XRD）对材料进行了表征。检测结果表明，磷灰石/明胶成功地沉积在纳米结构化钛材表面。纽蛋白的荧光染色结果表明，磷灰石/明胶纳米成分促进了细胞粘附，更重要的是，在第7，14与21天时，在磷灰石/明胶纳米成分钛材上生长的骨髓间充质干细胞表现了更高的增殖与碱性磷酸酶（ALP）活性。并且，骨钙素（OCN）、骨桥蛋白（OPN）和I型胶原蛋白（p<0.05或p<0.01）的表达得到了更大地提高。通过OCN与OPN的免疫荧光染色也得到了同样的结果。通过组织切片，X光片与micro-CT（micro computedtomography，微计算机断层扫描技术）分析得知磷灰石/明胶纳米成分提高了骨密度和骨-植入体的接触率（p<0.05或p<0.01），诱导植入体与骨界面间新骨的生长。以上结果表明，磷灰石/明胶纳米成分促进了体内外成骨。本研究为制备高性能钛材植入体提供了新技术。
After implantation of a biomaterial into a host, the interaction betweenbiomaterials and biological system (protein adsorption, cell adhesion/proliferation, etc)occurs at the biomaterial surface. The biological behaviors of cells are mainlydominated by the local microenvironment of chemistry and topography on macroscle,mesoscale, and microscale sizes. Thus, how to construct desirable microenvironmentsonto the material surface, in turn mediating cells’ physiological functions, becomes oneof the hot topics in the related field.
Titanium (Ti) and its alloys have been widely applied as bone implants in clinicalapplications because of their good mechanical properties. Nevertheless，titanium basedmaterials are surface bioinert, being lack of the potential for inducing tissue formation,which results in poor osseointegration between the implant and its surrounding naturebone tissue and short lifespan of the implant. It is the common challenge in clinicalapplication. Considering those issues, to realize surface-mediating control of thebiological behaviors of cells and induction of bone tissue formation, it is urgent todevelop novel techniques. To improve the osseointegration of titanium based implants,in this study, surface mechanical attrition treatment (SMAT) technique and anodizationwere employed to fabricate surface nanostructured titanium, and then depositedapatite/gelatin composite to construct desirable microenvironments, from theperspective of mimicking the bone-like nanoscale architecture and the components ofextracellular matrix.
Main contents and conclusions of this research are listed as follows:
1. Regulation of the behaviors of mesenchymal stem cells by surface nanostructuredtitanium
The study describes the influence of surface nanostructured titanium on thebehaviors of mesenchymal stem cells (MSCs). Surface nanostructures of titanium wereproduced with surface mechanical attrition treatment (SMAT) technique. Field emissionscanning electron microscopy (FE-SEM), transmission electron microscopy (TEM),atomic force microscopy (AFM), X-ray diffraction (XRD) and contact-anglemeasurements were used to characterize the surfaces of native titanium and surfacenanostructured titanium substrates were characterized, respectively. A thinnanostructured layer was formed onto the surfaces of titanium substrates after SMAT treatment. Immunofluorescence staining of vinculin, osteocalcin (OCN), andosteopontin (OPN), MTT test, the levels of (Alkaline phosphatase) ALP and OCN andthe mRNA expressions of OCN, OPN, collagen type I (Col I) and Runx2(runt-relatedprotein2) were examined, in order to evaluate the effects of the surface nanostructuredtitanium substrates on the adhesion, spreading, proliferation and osteoblasticdifferentiation of MSCs at cellular and molecular levels in vitro. The results suggest thatthe surface nanostructured substrates were beneficial for the growth of MSCs, includingadhesion, filament orientation, proliferation and gene expression. This approach for thefabrication of surface nanostructured titanium may be exploited in the development ofhigh performance titanium-based implants.
2. Surface functionalization of TiO_2nanotubes with bone morphogenetic protein2and its synergistic effect on the differentiation of mesenchymal stem cells
To investigate the influence of surface-functionalized substrates withnanostructures on the behaviors of mesenchymal stem cells, we conjugated bonemorphogenetic protein2(BMP2) onto TiO_2nanotubes with different diameter sizes of30,60, and100nm for in vitro study. Polydopamine was employed as the intermediatelayer for the conjugation of BMP2. The successful conjugation of BMP2onto TiO_2nanotubes was revealed by field-emission scanning electron microscopy (FE-SEM),X-ray photoelectron spectroscopy (XPS), and contact angle measurements.Immunofluorescence staining of vinculin, osteocalcin (OCN), and osteopontin (OPN)revealed that BMP2-functionalized TiO_2nanotubes were favorable for cell growth.More importantly, MSCs cultured onto BMP2-functionalized TiO_2nanotubes displayedsignificantly higher (p <0.05or p <0.01) d ifferentiation levels of ALP andmineralization after7and14day cultures, respectively. The results suggested thatsurface functionalization of TiO_2nanotubes with BMP2was beneficial for cellproliferation and differentiation. The approach presented here has potential applicationfor the development of titanium-based implants for enhanced bone osseointegration.
3. Construction of microenvironment onto titanium substrates to regulate theosteoblastic differentiation of mesenchymal stem cells in vitro and osteogenesis invivo
To mimic the extracellular matrix of natural bone, apatite/gelatin composite wasdeposited onto nanostructured titanium substrates via a coprecipitation method, whichwas pretreated by potassium hydroxide and heat treatment to generate an anticorrosivenanostructured layer. The successful formation of the apatite/gelatin nanocomposite onto titanium surfaces was revealed by Fourier transform infrared spectroscopy,field-emission scanning electron microscopy, atomic force microscopy (AFM), and thinfilm X-ray diffraction (TF-XRD) measurements, respectively. The immunofluorescencestaining of vinculin revealed that the apatite/gelatin nanocomposite deposited titaniumsubstrate was favorable for cell adhesion. More importantly, mesenchymal stem cellscultured onto the apatite/gelatin nanocomposite deposited titanium displayed higher (p<0.05or p <0.01) proliferation and osteoblastic differentiation levels of alkalinephosphatase, mRNA expressions of collagen I (Col I), osteocalcin (OCN) andosteopontin (OPN), and OCN content after culture for7,14, and21days, respectively,which was also revealed by the immunofluorescence analysis of OCN and OPNexpression. The deposition of apatite/gelatin nanocomposite improved bone density (p <0.05) and bone-implant contact rate (p <0.05), which was reflected by microcomputedtomography analysis and histological evaluation in vivo using a rabbit model. This workprovides an approach to fabricate high-performance titanium-based implants withenhanced bone osseointegration.
引文
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