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Abstract：
OBJECTIVE: The aim of this study was to evaluate the effects of collagen modification on the osteogenic performance of different surface-modified titanium, including alkaline etching, alkaline etching followed by silanization, and alkaline etching followed by dopamine modification. The proliferation, adhesion, and osteogenic differentiation abilities of MC3T3-E1 cells on the surfaces with collagen modification were analyzed and compared.
METHODS: Collagen was immobilized on the surfaces of pure titanium (Ti-C), alkaline-etched titanium (Ti-Na-C), alkaline-etched and silanized titanium (Ti-A-C), and alkaline-etched and dopamine-modified titanium (Ti-D-C), with pure titanium (Ti) as the control group. The surface morphology was observed by scanning electron microscopy (SEM), and the surface elemental composition was analyzed by X-ray photoelectron spectroscopy (XPS). Contact angle measurements were conducted to evaluate the hydrophilicity of the surfaces. MC3T3-E1 cells were cultured on the surfaces, and their proliferation, adhesion, and osteogenic differentiation abilities were assessed using CCK-8 assay, laser scanning confocal microscope, alkaline phosphatase (ALP) staining, Alizarin red staining and quantitative analysis, as well as real-time quantitative polymerase chain reaction (RT-qPCR) to evaluate the mRNA expression levels of osteogenic-related genes, including ALP, typeⅠcollagen (COL-1), osteocalcin (OCN), osteopontin (OPN).
RESULTS: SEM and XPS results confirmed the successful immobilization of collagen on the titanium surfaces, with the Ti-Na-C group exhibiting a higher amount of collagen modification. Contact angle measurements showed improved hydrophilicity of the surfaces after collagen modification. CCK-8 results indicated good compatibility of the materials with MC3T3-E1, with enhanced cell proliferation on the collagen-modified surfaces. Cell fluorescence staining revealed better cell spreading on the collagen-modified surfaces, and ALP and Alizarin red staining results suggested that the Ti-Na-C group exhibited the best osteogenic performance, with significantly higher absorbance values in the Alizarin red quantification analysis. RT-qPCR analysis showed that the Ti-Na-C group had the highest expression of the osteogenic-related gene OPN.
CONCLUSIONS: Among the different collagen modification approaches employed in this study, collagen modification on alkaline-etched titanium surfaces showed the most conducive effects on MC3T3-E1 cell adhesion, spreading, proliferation, and osteogenic differentiation. This approach can be considered as the optimal collagen modification strategy for enhancing osteogenesis on titanium surfaces.
目的: 对纯钛表面分别进行碱蚀、碱蚀后硅烷化、碱蚀后多巴胺修饰等不同方式改性后在其表面制备生物胶原蛋白涂层，评价钛表面不同改性处理后胶原修饰的方案对细胞增殖黏附与成骨分化能力的影响。方法: 胶原蛋白通过交联剂作用附着于纯钛（Ti-C）、碱蚀钛片（Ti-Na-C）、碱蚀后硅烷化修饰钛片（Ti-A-C）及碱蚀后多巴胺修饰钛片表面（Ti-D-C），以纯钛为对照组。采用扫描电子显微镜（SEM）观察材料表面微形貌，X射线光电子能谱仪（XPS）分析材料表面元素组成，表面接触角测量仪评估材料表面亲水性。体外培养MC3T3-E1细胞，通过CCK-8、激光共聚焦显微镜、碱性磷酸酶（ALP）活性检测、茜素红染色及定量检测评价材料表面成骨细胞的增殖黏附与成骨分化能力，实时定量聚合酶链反应（RT-qPCR）检测成骨相关基因ALP、Ⅰ型胶原蛋白（COL-1）、骨钙素（OCN）、骨桥蛋白（OPN）的mRNA表达水平。结果: SEM与XPS结果表明胶原已成功修饰于钛片表面，Ti-Na-C组有较厚的胶原纤维层覆盖。表面接触角测量结果显示，除了Ti-Na-C组与Ti-Na组的接触角差异无统计学意义，其余胶原修饰后的材料表面具有更好的亲水性。CCK-8结果显示各组材料均无明显细胞毒性，胶原修饰后的材料表面成骨细胞增殖高于相应的未经胶原修饰材料；共聚焦显微镜检测结果可见，胶原修饰后材料表面细胞铺展面积更大；ALP染色与茜素红染色结果均提示，Ti-Na-C组体外成骨效果最佳，茜素红定量结果显示Ti-Na-C组吸光度值最高；RT-qPCR检测结果显示Ti-Na-C组的OPN基因表达量最高。结论: 在碱蚀、碱蚀后硅烷化、碱蚀后多巴胺修饰等对钛表面进行不同改性处理后修饰胶原的方案中，钛表面经过碱蚀处理后直接修饰胶原的方法最有利于MC3T3-E1黏附铺展、增殖与成骨分化，可作为胶原修饰的方案。.
METHODS: Collagen was immobilized on the surfaces of pure titanium (Ti-C), alkaline-etched titanium (Ti-Na-C), alkaline-etched and silanized titanium (Ti-A-C), and alkaline-etched and dopamine-modified titanium (Ti-D-C), with pure titanium (Ti) as the control group. The surface morphology was observed by scanning electron microscopy (SEM), and the surface elemental composition was analyzed by X-ray photoelectron spectroscopy (XPS). Contact angle measurements were conducted to evaluate the hydrophilicity of the surfaces. MC3T3-E1 cells were cultured on the surfaces, and their proliferation, adhesion, and osteogenic differentiation abilities were assessed using CCK-8 assay, laser scanning confocal microscope, alkaline phosphatase (ALP) staining, Alizarin red staining and quantitative analysis, as well as real-time quantitative polymerase chain reaction (RT-qPCR) to evaluate the mRNA expression levels of osteogenic-related genes, including ALP, typeⅠcollagen (COL-1), osteocalcin (OCN), osteopontin (OPN).
RESULTS: SEM and XPS results confirmed the successful immobilization of collagen on the titanium surfaces, with the Ti-Na-C group exhibiting a higher amount of collagen modification. Contact angle measurements showed improved hydrophilicity of the surfaces after collagen modification. CCK-8 results indicated good compatibility of the materials with MC3T3-E1, with enhanced cell proliferation on the collagen-modified surfaces. Cell fluorescence staining revealed better cell spreading on the collagen-modified surfaces, and ALP and Alizarin red staining results suggested that the Ti-Na-C group exhibited the best osteogenic performance, with significantly higher absorbance values in the Alizarin red quantification analysis. RT-qPCR analysis showed that the Ti-Na-C group had the highest expression of the osteogenic-related gene OPN.
CONCLUSIONS: Among the different collagen modification approaches employed in this study, collagen modification on alkaline-etched titanium surfaces showed the most conducive effects on MC3T3-E1 cell adhesion, spreading, proliferation, and osteogenic differentiation. This approach can be considered as the optimal collagen modification strategy for enhancing osteogenesis on titanium surfaces.
目的: 对纯钛表面分别进行碱蚀、碱蚀后硅烷化、碱蚀后多巴胺修饰等不同方式改性后在其表面制备生物胶原蛋白涂层，评价钛表面不同改性处理后胶原修饰的方案对细胞增殖黏附与成骨分化能力的影响。方法: 胶原蛋白通过交联剂作用附着于纯钛（Ti-C）、碱蚀钛片（Ti-Na-C）、碱蚀后硅烷化修饰钛片（Ti-A-C）及碱蚀后多巴胺修饰钛片表面（Ti-D-C），以纯钛为对照组。采用扫描电子显微镜（SEM）观察材料表面微形貌，X射线光电子能谱仪（XPS）分析材料表面元素组成，表面接触角测量仪评估材料表面亲水性。体外培养MC3T3-E1细胞，通过CCK-8、激光共聚焦显微镜、碱性磷酸酶（ALP）活性检测、茜素红染色及定量检测评价材料表面成骨细胞的增殖黏附与成骨分化能力，实时定量聚合酶链反应（RT-qPCR）检测成骨相关基因ALP、Ⅰ型胶原蛋白（COL-1）、骨钙素（OCN）、骨桥蛋白（OPN）的mRNA表达水平。结果: SEM与XPS结果表明胶原已成功修饰于钛片表面，Ti-Na-C组有较厚的胶原纤维层覆盖。表面接触角测量结果显示，除了Ti-Na-C组与Ti-Na组的接触角差异无统计学意义，其余胶原修饰后的材料表面具有更好的亲水性。CCK-8结果显示各组材料均无明显细胞毒性，胶原修饰后的材料表面成骨细胞增殖高于相应的未经胶原修饰材料；共聚焦显微镜检测结果可见，胶原修饰后材料表面细胞铺展面积更大；ALP染色与茜素红染色结果均提示，Ti-Na-C组体外成骨效果最佳，茜素红定量结果显示Ti-Na-C组吸光度值最高；RT-qPCR检测结果显示Ti-Na-C组的OPN基因表达量最高。结论: 在碱蚀、碱蚀后硅烷化、碱蚀后多巴胺修饰等对钛表面进行不同改性处理后修饰胶原的方案中，钛表面经过碱蚀处理后直接修饰胶原的方法最有利于MC3T3-E1黏附铺展、增殖与成骨分化，可作为胶原修饰的方案。.
摘要：
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