掺锶羟基磷灰石对成骨细胞生物学行为的影响
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摘要
掺锶羟基磷灰石(Sr-HA)因锶元素的掺入对其性能产生重要影响,与目前广泛使用的羟基磷灰石(HA)相比,提高了其降解速率与力学性能,此外,低剂量的Sr还具有促进骨形成,抑制骨吸收等优点。
本实验采用微弧氧化法对纯钛表面进行改性处理,在其表面形成不同掺锶浓度的羟基磷灰石涂层,并在一定的掺锶浓度下选取三个不同的微弧氧化时间对纯钛表面进行处理,形成锶浓度相同但具有不同表面性状的涂层结构。观察不同涂层表面的物理和化学性质,并且通过体外细胞学实验对涂层的生物安全性和成骨细胞在材料表面的生物学行为进行研究,为优化锶浓度、表面处理工艺以及涂层结构提供理论依据。
研究方法:
1.采用扫描电镜观察涂层表面形貌,采用能谱分析仪分析涂层的化学成分,采用X射线衍射分析涂层的物相组成,采用粗糙度测量仪测量涂层表面粗糙程度及采用接触角测量仪测量表面液体的接触角。
2.根据国家质量技术监督局参照ISO10993标准发布的GB/T16886国家医疗器械生物学评价标准,用L929成纤维细胞与不同锶浓度涂层的浸提液复合培养,检测各个锶浓度组的细胞毒性。
3.采用原代培养的成骨细胞观察不同涂层对细胞附着、增殖、细胞形态和碱性磷酸酶(ALP)活性的影响,用Real-time PCR方法检测不同表面成骨细胞相关基因的表达水平。
结果:
1.通过改变微弧氧化电解液中[Sr/(Sr+Ca)]比例,可以在纯钛表面形成不同掺锶浓度的Sr-HA涂层,随着Sr的掺入,涂层表面HA逐渐被Sr-HA替代。不同掺锶浓度的Sr-HA涂层表面的液体接触角没有显著差异(P>0.05)。
2.不同掺锶浓度试样表面细胞毒性评级均为0级或1级,锶元素的掺入没有产生明显的细胞毒性,但随着掺锶量的增加,细胞相对增殖率有轻微下降的趋势。
3.成骨细胞在掺锶浓度为15.15%和23.78 %组涂层表面的附着和增殖明显高于掺锶浓度为2.22 %和7.24%组(P<0.05)。掺锶浓度为2.22%和7.24%组表面成骨细胞7d的ALP活性高于掺锶浓度为15.15%和23.78%组(P<0.05)。3d成骨细胞形态观察显示在不同掺锶浓度组表面成骨细胞附着和伸展状良好,表面分泌颗粒丰富,呈现良好的功能状态。
4.Realtime PCR结果显示:在细胞培养72h后掺锶浓度为7.24%组表面成骨细胞的骨形成蛋白(BMP)和Runx-2表达增加,掺锶浓度为23.78%组表面成骨细胞骨钙素(OCN)的表达增加,不同掺锶浓度组对骨桥蛋白(OPN)的表达无明显影响。
5.延长微弧氧化时间可以使涂层表面粗糙度增大,去离子水的接触角变小。此外,微弧氧化电解液中的Ca和P可以被整合到表面氧化层中且其含量随着微弧氧化时间的延长而增加。
6.成骨细胞在材料表面培养60min和90min时,微弧氧化10min和15min组的细胞附着明显高于5min组(P<0.05)。成骨细胞在5min组增殖速度最快,15min组增殖速度最慢。微弧氧化10min和15min组成骨细胞72h的ALP活性高于5min组(P<0.05)。
7. 3d成骨细胞形态观察显示不同微弧氧化时间组表面成骨细胞均表现为细胞表面分泌颗粒丰富,细胞通过板状和丝状伪足锚定在基底材料表面;在相对光滑的Ti6Al4V表面,细胞伸展及连续细胞层的形成优于粗糙表面,但是细胞的立体感不强,表面分泌颗粒少。
8. Realtime PCR结果显示在细胞培养72h后,微弧氧化10min和15min组表面成骨基因BMP和OCN的表达增加,各时间组表面对基因OPN和Runx-2的表达没有明显的影响。
结论:微弧氧化处理10min-15min形成的掺锶浓度为15.15%-23.78 %的Sr-HA涂层能更好的促进成骨细胞的附着、增殖和分化。
Strontium-containing hydroxyapatite(Sr-HA) has higher solubility and better mechanical properties because of the incorporation of strontium comparing to the pure HA and pure titanium. Additionally, low doses strontium can stimulate bone formation and reduce bone resorption .
We developed Sr-HA coatings with different doses of strontium on titanium substrates by micro-arc oxidation (MAO) method, and chose three MAO time length to developing different surface topography. The aim of this study is to observe the properties of the films and investigate the cytotoxicity and osteoblast biocompatibility of the coatings.
Methods:
The surface morphology of the coatings was analyzed by scanning electron microscope (SEM).The element composition and phase component were analyzed with energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD). Surface roughness and contact angle were measured using a surface profilometer and a contact angle measuring system.
According to the GB and YY-T, L929 fibroblasts of mouse were used in MTT assay to examine the cytotoxicity of Sr-HA containing different strontium concentrations. Osteoblast cell adhesion and proliferation were examined by the MTT method and alkaline phosphatase (ALP) activity was assessed using test kits. The gene expression of several osteogenesis-related genes was surveyed by Realtime-PCR method.
Results:
1. Sr-HA coatings with different doses of strontium on titanium substrates can be developed by changing the proportion of [Sr/(Sr+Ca)] in the MAO electrolytes. HA was substituted by Sr-HA with the incorporation of strontium. The water contact angles had no significant deviation (P>0.05).
2. The toxicity gradation of all groups was 0—1. Strontium incorporation did not induce obvious cytotoxicity, but the relative growth ratio slightly declined with the increase of strontium concentration.
3. Samples containing 15.15% and 23.78% strontium enhanced osteoblast adhesion and proliferation while 7.24% Sr-HA increased the values of ALP activity after 7 d culture (P<0.05).After 3-d culture, on the Sr-HA coatings osteoblasts exhibited a good spreading and proliferation morghology.
4. After 72h culture, the gene expression of BMP and Runx2 were increased on the Sr-HA coatings containing 7.24% strontium, while the expression of OCN was enhanced on the coating containing 23.78% strontium. No obvious variations in gene expression of OPN was observed on different strontium doses coatings.
5. After prolonging the MAO time length, the coatings’surface morghology became more irregular, surface roughness was enhanced and water contact angles became smaller. Ca and P in the MAO electrolytes were incorporated into the oxidized coatings.
6. MAO 10min and 15min coatings enhanced osteoblast adhesion after 60min and 90min-culture(P<0.05). Osteoblast exhibited fastest proliferation rate on the 5min surface, while lowest on the 15min surface. MAO 10min and 15min surface increased the values of ALP activity after 72h culture (P<0.05).
7. After 3-d culture, osteoblast on MAO surfaces exhibited the typical osteoblast phenotype, the cells displayed lamellipodia and filopodia extensions, while on the Ti6Al4V surface cells were relatively flatter and displayed less functional morghology.
8. After 72h culture, the gene expression of BMP and OCN were increased on the Sr-HA coatings after 15min MAO treatment. No obvious variations in gene expression of OPN and Runx2 were observed on different MAO time length treatments.
Conclusions:
Sr-HA coatings containing 15.15%—23.78% strontium after 10min—15min MAO treatment have better surface properties which favor the cell adhesion, proliferation, ALP activity and gene expression of osteoblast.
本实验采用微弧氧化法对纯钛表面进行改性处理,在其表面形成不同掺锶浓度的羟基磷灰石涂层,并在一定的掺锶浓度下选取三个不同的微弧氧化时间对纯钛表面进行处理,形成锶浓度相同但具有不同表面性状的涂层结构。观察不同涂层表面的物理和化学性质,并且通过体外细胞学实验对涂层的生物安全性和成骨细胞在材料表面的生物学行为进行研究,为优化锶浓度、表面处理工艺以及涂层结构提供理论依据。
研究方法:
1.采用扫描电镜观察涂层表面形貌,采用能谱分析仪分析涂层的化学成分,采用X射线衍射分析涂层的物相组成,采用粗糙度测量仪测量涂层表面粗糙程度及采用接触角测量仪测量表面液体的接触角。
2.根据国家质量技术监督局参照ISO10993标准发布的GB/T16886国家医疗器械生物学评价标准,用L929成纤维细胞与不同锶浓度涂层的浸提液复合培养,检测各个锶浓度组的细胞毒性。
3.采用原代培养的成骨细胞观察不同涂层对细胞附着、增殖、细胞形态和碱性磷酸酶(ALP)活性的影响,用Real-time PCR方法检测不同表面成骨细胞相关基因的表达水平。
结果:
1.通过改变微弧氧化电解液中[Sr/(Sr+Ca)]比例,可以在纯钛表面形成不同掺锶浓度的Sr-HA涂层,随着Sr的掺入,涂层表面HA逐渐被Sr-HA替代。不同掺锶浓度的Sr-HA涂层表面的液体接触角没有显著差异(P>0.05)。
2.不同掺锶浓度试样表面细胞毒性评级均为0级或1级,锶元素的掺入没有产生明显的细胞毒性,但随着掺锶量的增加,细胞相对增殖率有轻微下降的趋势。
3.成骨细胞在掺锶浓度为15.15%和23.78 %组涂层表面的附着和增殖明显高于掺锶浓度为2.22 %和7.24%组(P<0.05)。掺锶浓度为2.22%和7.24%组表面成骨细胞7d的ALP活性高于掺锶浓度为15.15%和23.78%组(P<0.05)。3d成骨细胞形态观察显示在不同掺锶浓度组表面成骨细胞附着和伸展状良好,表面分泌颗粒丰富,呈现良好的功能状态。
4.Realtime PCR结果显示:在细胞培养72h后掺锶浓度为7.24%组表面成骨细胞的骨形成蛋白(BMP)和Runx-2表达增加,掺锶浓度为23.78%组表面成骨细胞骨钙素(OCN)的表达增加,不同掺锶浓度组对骨桥蛋白(OPN)的表达无明显影响。
5.延长微弧氧化时间可以使涂层表面粗糙度增大,去离子水的接触角变小。此外,微弧氧化电解液中的Ca和P可以被整合到表面氧化层中且其含量随着微弧氧化时间的延长而增加。
6.成骨细胞在材料表面培养60min和90min时,微弧氧化10min和15min组的细胞附着明显高于5min组(P<0.05)。成骨细胞在5min组增殖速度最快,15min组增殖速度最慢。微弧氧化10min和15min组成骨细胞72h的ALP活性高于5min组(P<0.05)。
7. 3d成骨细胞形态观察显示不同微弧氧化时间组表面成骨细胞均表现为细胞表面分泌颗粒丰富,细胞通过板状和丝状伪足锚定在基底材料表面;在相对光滑的Ti6Al4V表面,细胞伸展及连续细胞层的形成优于粗糙表面,但是细胞的立体感不强,表面分泌颗粒少。
8. Realtime PCR结果显示在细胞培养72h后,微弧氧化10min和15min组表面成骨基因BMP和OCN的表达增加,各时间组表面对基因OPN和Runx-2的表达没有明显的影响。
结论:微弧氧化处理10min-15min形成的掺锶浓度为15.15%-23.78 %的Sr-HA涂层能更好的促进成骨细胞的附着、增殖和分化。
Strontium-containing hydroxyapatite(Sr-HA) has higher solubility and better mechanical properties because of the incorporation of strontium comparing to the pure HA and pure titanium. Additionally, low doses strontium can stimulate bone formation and reduce bone resorption .
We developed Sr-HA coatings with different doses of strontium on titanium substrates by micro-arc oxidation (MAO) method, and chose three MAO time length to developing different surface topography. The aim of this study is to observe the properties of the films and investigate the cytotoxicity and osteoblast biocompatibility of the coatings.
Methods:
The surface morphology of the coatings was analyzed by scanning electron microscope (SEM).The element composition and phase component were analyzed with energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD). Surface roughness and contact angle were measured using a surface profilometer and a contact angle measuring system.
According to the GB and YY-T, L929 fibroblasts of mouse were used in MTT assay to examine the cytotoxicity of Sr-HA containing different strontium concentrations. Osteoblast cell adhesion and proliferation were examined by the MTT method and alkaline phosphatase (ALP) activity was assessed using test kits. The gene expression of several osteogenesis-related genes was surveyed by Realtime-PCR method.
Results:
1. Sr-HA coatings with different doses of strontium on titanium substrates can be developed by changing the proportion of [Sr/(Sr+Ca)] in the MAO electrolytes. HA was substituted by Sr-HA with the incorporation of strontium. The water contact angles had no significant deviation (P>0.05).
2. The toxicity gradation of all groups was 0—1. Strontium incorporation did not induce obvious cytotoxicity, but the relative growth ratio slightly declined with the increase of strontium concentration.
3. Samples containing 15.15% and 23.78% strontium enhanced osteoblast adhesion and proliferation while 7.24% Sr-HA increased the values of ALP activity after 7 d culture (P<0.05).After 3-d culture, on the Sr-HA coatings osteoblasts exhibited a good spreading and proliferation morghology.
4. After 72h culture, the gene expression of BMP and Runx2 were increased on the Sr-HA coatings containing 7.24% strontium, while the expression of OCN was enhanced on the coating containing 23.78% strontium. No obvious variations in gene expression of OPN was observed on different strontium doses coatings.
5. After prolonging the MAO time length, the coatings’surface morghology became more irregular, surface roughness was enhanced and water contact angles became smaller. Ca and P in the MAO electrolytes were incorporated into the oxidized coatings.
6. MAO 10min and 15min coatings enhanced osteoblast adhesion after 60min and 90min-culture(P<0.05). Osteoblast exhibited fastest proliferation rate on the 5min surface, while lowest on the 15min surface. MAO 10min and 15min surface increased the values of ALP activity after 72h culture (P<0.05).
7. After 3-d culture, osteoblast on MAO surfaces exhibited the typical osteoblast phenotype, the cells displayed lamellipodia and filopodia extensions, while on the Ti6Al4V surface cells were relatively flatter and displayed less functional morghology.
8. After 72h culture, the gene expression of BMP and OCN were increased on the Sr-HA coatings after 15min MAO treatment. No obvious variations in gene expression of OPN and Runx2 were observed on different MAO time length treatments.
Conclusions:
Sr-HA coatings containing 15.15%—23.78% strontium after 10min—15min MAO treatment have better surface properties which favor the cell adhesion, proliferation, ALP activity and gene expression of osteoblast.
引文
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