硅酸二钙涂层离子溶出液促进成骨的体外细胞实验研究
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摘要
第一部分硅酸二钙涂层在DMEM液中生物活性的研究
目的通过DMEM培养液浸泡实验,观测硅酸二钙涂层表面形貌、元素组成及培养液中主要离子浓度的变化情况,以了解硅酸二钙涂层在DMEM培养中的生物活性。方法将硅酸二钙涂层试样浸泡于DMEM培养液内,37℃水浴,7天时过滤除去晶体。带EDS的SEM观测涂层表面形貌及元素组成变化,ICP测定培养液中Ca、P、Si离子的浓度变化。结果浸泡后涂层表面Ca、P元素含量增多,而Si元素含量减少,并有含炭酸根的羟基磷灰石层(HCA层)形成;DMEM培养液中,Ca+、Si+离子浓度较浸泡前均有显著的升高(P<0.05),P+离子浓度较浸泡前则显著的降低(P<0.05)。结论硅酸二钙涂层于DMEM培养液中能形成HCA层,表明涂层具有好的生物活性;同时涂层溶出大量的Ca、Si离子,显著改变DMEM液的理化组成。
第二部分硅酸二钙涂层离子溶出液对体外培养成骨细胞生长及形态影响的观察
目的通过硅酸二钙涂层离子溶出液与成骨细胞复合培养,观察培养过程中成骨细胞的形态学特征的变化,以了解硅酸二钙涂层离子溶出液对成骨细胞生长的影响。方法将硅酸二钙涂层试样浸泡于DMEM培养液内,37℃水浴,7天时过滤除去晶体,获得硅酸二钙涂层离子溶出液,ICP测定培养液中Ca、P、Si离子的浓度变化情况。硅酸二钙涂层离子溶出液作为实验组,原始DMEM培养液作为对照组,分别接种人MG63成骨细胞进行培养。倒置相差显微镜观察成骨细胞的生长形态变化;台盼兰染色细胞计数法及MTT法进行活力细胞数比较。
结果实验组中Ca、Si离子浓度要显著高于对照组(P<0.05),而P离子浓度则显著低于对照组(P<0.05)。实验组细胞具有更早、更好的分化;实验组活力细胞数少于对照组,但差异无显著性意义(p>0.05)。结论硅酸二钙涂层离子溶出液具有促进细胞分化的作用,这有利于骨的形成;涂层离子溶出液对活力细胞数的增加没有促进作用,其原因及意义有待结合下部分实验结果综合分析讨论。
第三部分硅酸二钙涂层离子溶出液对体外培养成骨细胞增殖周期及凋亡的影响
目的通过观察成骨细胞生长周期及凋亡的变化,试图阐明解硅酸二钙涂层离子溶出液对成骨细胞增殖的影响。方法硅酸二钙涂层离子溶出液作为实验组,原始DMEM培养液作为对照组,分别接种人MG63成骨细胞进行培养。流式细胞仪测定3、6、12天时成骨细胞G1、S、G2期的百分比及细胞凋亡率;对细胞增殖活性指标SPF(S期细胞比率)、PI(增殖指数)及细胞凋亡率(AI)进行比较。结果3天时,实验组G1期细胞较对照组多,但差异无显著性(P>0.05),S期细胞显著少于对照组(P<0.05),反应增殖活性的PI值小于对照组,但差异无显著性(P>0.05)。6-12时,实验组G1期细胞显著少于对照组(P<0.01),S期细胞显著性多于对照组(P<0.01),PI值显著大于对照组(P<0.01)。3-12天时,实验组细胞凋亡率大于对照组,并且在3、12天时,差异有显著性意义(分别:P<0.05;P<0.01)。结论硅酸二钙涂层的离子溶出物能缩短成骨细胞的生长周期、促进细胞增殖,其作用特点为:初期对成骨细胞增殖稍有抑制作用,后期则显著刺激成骨细胞快速增殖。而实验组所表现的高细胞凋亡率可能与其有更好的分化有关。
第四部分硅酸二钙涂层离子溶出液对体外培养成骨细胞分化及成骨表型表达的影响
目的通过测定成骨细胞分化及细胞表型特征性产物及基因的表达量,观察硅酸二钙涂层离子溶出液对体外培养成骨细胞分化及成骨表型表达的影响,探讨硅酸二钙涂层离子溶出液促进成骨的可能机制。方法硅酸二钙涂层离子溶出液作为实验组,原始DMEM培养液作为对照组,分别接种人MG63成骨细胞进行培养。生化法测定培养液中ALP的活性变化,RT-PCR法检测成骨细胞Ⅰ型胶原(collagen-Ⅰ)、骨钙素(BGP)、成骨细胞特异转录因子(CBFAⅠ)、类胰岛素样生长因子Ⅱ(IGF-2)等基因表达量的变化,并进行比较。结果实验组中的ALP含量、Collagen-Ⅰ、BGP、IGF-2及CBFAⅠ基因表达量,3天时即显著大于对照组(p<0.01),此差异一直持续至第12天时。
结论硅酸二钙涂层离子溶出液具有促进成骨细胞增殖、分化、细胞周围基质的形成和矿化作用,加速骨的形成。
PartⅠBioactivity of Plasma Sprayed Dicalcium Silicate Coatings In DMEM Culture Fluid
Objective To observe bioactivity of plasma sprayed dicalcium silicate coatings in DMEM culture fluids. Methods Dicalcium silicate coatings on titanium alloys substrates were prepared by plasma spraying and immersed in DMEM culture fluid for 7 day. SEM and EDS were used to observe surface morphologies and determine the composition of dicalcium silicate coatings before and after immersion in DMEM culture fluid. The elementary content of calcium (Ca), silicon (Si) and phosphorus (P) in this solution were determined by inductively coupled plasma (ICP) analysis. Results A dense carbonate-containing hydroxyapatite (CHA) layer was formed on the surface of the plasma sprayed dicalcium silicate coating soaked in DMEM solution for 7days. There were difference in Ca, Si and P ionic concentration(p<0.05) between the dicalcium silicate coating conditioned DMEM solution and control group culture fluid. Conclutions The results obtained indicated that the plasma sprayed dicalcium silicate coating possesses excellent bioactivity.
PartⅡObservation of Ionic Products of Dicalcium Silicate Coating Dissolution to The Change of Osteoblast Morphology in Cell Culture
Objective To observe the changes of osteoblast morphology during cell culture .In order to understand the effects of Dicalcium Silicate Coating dissolution on the growth of osteoblast. Methods Dicalcium silicate coatings on titanium alloys substrates were prepared by plasma spraying and immersed in DMEM culture fluid for 7 day. The elementary content of calcium (Ca), silicon (Si) and phosphorus (P) in this solution were determined by inductively coupled plasma (ICP) analysis. The full DMEM was prepared to be the control group culture fluid, the DMEM culture adding with ionic products of dicalcium silicate coating dissolution was prepared to be the experimental group culture fluid. The human osteoblast-like MG63 cells were cultured by experimental group culture fluid and control group culture fluid respectively to observed the morphological difference in general of osteoblast in the process of growth in culture and the difference in cell population (MTT chromatometry and staining Cytometry). Results There were difference in Ca, Si and P ionic concentration(p<0.05) between experimental group culture fluid and control group culture fluid. The cells of experimental group have better cell differentiation than the osteoblast of control groups. There was no difference in cell population (p>0.05) between the experimental group culture fluid and control group culture fluid. Conclutions The ionic products of dicalcium silicate coating dissolution can increase differentiation of human osteoblasts and can not increase cell population.
PartⅢThe Effect of Ionic Products of Dicalcium Silicate Coating Dissolution to Osteoblast Growth Cycle and Apoptosis
Objective To observe the change of esteoblast growth cycle and apoptosis to understand the effect of ionic products of dicalcium silicate coating dissolution to osteoblast proliferation. Methods The full DMEM was prepared to be the control group culture fluid, the DMEM culture adding with ionic products of dicalcium silicate coating dissolution was prepared to be the experimental group culture fluid. The human osteoblast-like MG63 cells were cultured by experimental group culture fluid and control group culture fluid respectively. The osteoblast growth cycle and apoptosis was determined by flow cytometry at 3,6 and 12 day. The cell proliferative activity indicatrix[SPF(S-phase fraction) and PI(proliferous index) ] and apoptotic index(AI) were compared. Results On days 3,there were no difference in the percentage of cells in the G1 phase and PI (p>0.05) between the experimental group and control group . there was a significantly lower percentage of cells in the S phase on days 3 (P<0.05) in experimental group. In 6 to 12 day, there were significantly higher percentage of cells in the S phase and PI (P<0.01),and wae significantly lower percentage of cells in the G1 phase in experimental group. Apoptosis was markedly increased in the experimental group at days 3 and 12 (P< 0.05; P<0.01). Conclusion The ionic products of dicalcium silicate coating dissolution can increase proliferation of human osteoblasts by increased cell cycling. The feature of the ionic products of dicalcium silicate coating dissolution increased proliferation of human osteoblasts is that it has slightly depressant effect on proliferation of human osteoblasts in the initial stage, but it can significantly increase proliferation of human osteoblasts in later stage. Increase in apoptosis possibly profits to osteoblast differentiation .
PartⅣThe Effect of Ionic Products of Dicalcium Silicate Coating Dissolution to Osteoblast Differentiation and Phenotypic Expression
Objective To further investigate the possible mechanisms of ionic products of dicalcium silicate coating dissolution which enhances bone formation by observed the effect of ionic products of dicalcium silicate coating dissolution to osteoblast differentiation and phenotypic expression . Methods The full DMEM was prepared to be the control group culture fluid, the DMEM culture adding with ionic products of dicalcium silicate coating dissolution was prepared to be the experimental group culture fluid. The human osteoblast-like MG63 cells were cultured by experimental group culture fluid and control group culture fluid respectively. Detected the express of collagen-Ⅰ、bone gla-protein (BGP)、osteoblast core-binding factor alpha-1(CBFA-Ⅰ)and insulin-like growth factorⅡ(IGF-2) by polymerase chain reaction(PCR) and detected the activity of alkaline phosphatase(ALP) by biochemistry method. Results There were significantly higher express of collagen-Ⅰ,BGP,CBFA-Ⅰand IGF-2 (p<0.01) in experimental group in 3 to 12 days. Conclusion The ionic products of dicalcium silicate coating dissolution can increase proliferation , differentiation, formation of metrical and mineralization of human osteoblasts,and it profits to enhance bone formation.
目的通过DMEM培养液浸泡实验,观测硅酸二钙涂层表面形貌、元素组成及培养液中主要离子浓度的变化情况,以了解硅酸二钙涂层在DMEM培养中的生物活性。方法将硅酸二钙涂层试样浸泡于DMEM培养液内,37℃水浴,7天时过滤除去晶体。带EDS的SEM观测涂层表面形貌及元素组成变化,ICP测定培养液中Ca、P、Si离子的浓度变化。结果浸泡后涂层表面Ca、P元素含量增多,而Si元素含量减少,并有含炭酸根的羟基磷灰石层(HCA层)形成;DMEM培养液中,Ca+、Si+离子浓度较浸泡前均有显著的升高(P<0.05),P+离子浓度较浸泡前则显著的降低(P<0.05)。结论硅酸二钙涂层于DMEM培养液中能形成HCA层,表明涂层具有好的生物活性;同时涂层溶出大量的Ca、Si离子,显著改变DMEM液的理化组成。
第二部分硅酸二钙涂层离子溶出液对体外培养成骨细胞生长及形态影响的观察
目的通过硅酸二钙涂层离子溶出液与成骨细胞复合培养,观察培养过程中成骨细胞的形态学特征的变化,以了解硅酸二钙涂层离子溶出液对成骨细胞生长的影响。方法将硅酸二钙涂层试样浸泡于DMEM培养液内,37℃水浴,7天时过滤除去晶体,获得硅酸二钙涂层离子溶出液,ICP测定培养液中Ca、P、Si离子的浓度变化情况。硅酸二钙涂层离子溶出液作为实验组,原始DMEM培养液作为对照组,分别接种人MG63成骨细胞进行培养。倒置相差显微镜观察成骨细胞的生长形态变化;台盼兰染色细胞计数法及MTT法进行活力细胞数比较。
结果实验组中Ca、Si离子浓度要显著高于对照组(P<0.05),而P离子浓度则显著低于对照组(P<0.05)。实验组细胞具有更早、更好的分化;实验组活力细胞数少于对照组,但差异无显著性意义(p>0.05)。结论硅酸二钙涂层离子溶出液具有促进细胞分化的作用,这有利于骨的形成;涂层离子溶出液对活力细胞数的增加没有促进作用,其原因及意义有待结合下部分实验结果综合分析讨论。
第三部分硅酸二钙涂层离子溶出液对体外培养成骨细胞增殖周期及凋亡的影响
目的通过观察成骨细胞生长周期及凋亡的变化,试图阐明解硅酸二钙涂层离子溶出液对成骨细胞增殖的影响。方法硅酸二钙涂层离子溶出液作为实验组,原始DMEM培养液作为对照组,分别接种人MG63成骨细胞进行培养。流式细胞仪测定3、6、12天时成骨细胞G1、S、G2期的百分比及细胞凋亡率;对细胞增殖活性指标SPF(S期细胞比率)、PI(增殖指数)及细胞凋亡率(AI)进行比较。结果3天时,实验组G1期细胞较对照组多,但差异无显著性(P>0.05),S期细胞显著少于对照组(P<0.05),反应增殖活性的PI值小于对照组,但差异无显著性(P>0.05)。6-12时,实验组G1期细胞显著少于对照组(P<0.01),S期细胞显著性多于对照组(P<0.01),PI值显著大于对照组(P<0.01)。3-12天时,实验组细胞凋亡率大于对照组,并且在3、12天时,差异有显著性意义(分别:P<0.05;P<0.01)。结论硅酸二钙涂层的离子溶出物能缩短成骨细胞的生长周期、促进细胞增殖,其作用特点为:初期对成骨细胞增殖稍有抑制作用,后期则显著刺激成骨细胞快速增殖。而实验组所表现的高细胞凋亡率可能与其有更好的分化有关。
第四部分硅酸二钙涂层离子溶出液对体外培养成骨细胞分化及成骨表型表达的影响
目的通过测定成骨细胞分化及细胞表型特征性产物及基因的表达量,观察硅酸二钙涂层离子溶出液对体外培养成骨细胞分化及成骨表型表达的影响,探讨硅酸二钙涂层离子溶出液促进成骨的可能机制。方法硅酸二钙涂层离子溶出液作为实验组,原始DMEM培养液作为对照组,分别接种人MG63成骨细胞进行培养。生化法测定培养液中ALP的活性变化,RT-PCR法检测成骨细胞Ⅰ型胶原(collagen-Ⅰ)、骨钙素(BGP)、成骨细胞特异转录因子(CBFAⅠ)、类胰岛素样生长因子Ⅱ(IGF-2)等基因表达量的变化,并进行比较。结果实验组中的ALP含量、Collagen-Ⅰ、BGP、IGF-2及CBFAⅠ基因表达量,3天时即显著大于对照组(p<0.01),此差异一直持续至第12天时。
结论硅酸二钙涂层离子溶出液具有促进成骨细胞增殖、分化、细胞周围基质的形成和矿化作用,加速骨的形成。
PartⅠBioactivity of Plasma Sprayed Dicalcium Silicate Coatings In DMEM Culture Fluid
Objective To observe bioactivity of plasma sprayed dicalcium silicate coatings in DMEM culture fluids. Methods Dicalcium silicate coatings on titanium alloys substrates were prepared by plasma spraying and immersed in DMEM culture fluid for 7 day. SEM and EDS were used to observe surface morphologies and determine the composition of dicalcium silicate coatings before and after immersion in DMEM culture fluid. The elementary content of calcium (Ca), silicon (Si) and phosphorus (P) in this solution were determined by inductively coupled plasma (ICP) analysis. Results A dense carbonate-containing hydroxyapatite (CHA) layer was formed on the surface of the plasma sprayed dicalcium silicate coating soaked in DMEM solution for 7days. There were difference in Ca, Si and P ionic concentration(p<0.05) between the dicalcium silicate coating conditioned DMEM solution and control group culture fluid. Conclutions The results obtained indicated that the plasma sprayed dicalcium silicate coating possesses excellent bioactivity.
PartⅡObservation of Ionic Products of Dicalcium Silicate Coating Dissolution to The Change of Osteoblast Morphology in Cell Culture
Objective To observe the changes of osteoblast morphology during cell culture .In order to understand the effects of Dicalcium Silicate Coating dissolution on the growth of osteoblast. Methods Dicalcium silicate coatings on titanium alloys substrates were prepared by plasma spraying and immersed in DMEM culture fluid for 7 day. The elementary content of calcium (Ca), silicon (Si) and phosphorus (P) in this solution were determined by inductively coupled plasma (ICP) analysis. The full DMEM was prepared to be the control group culture fluid, the DMEM culture adding with ionic products of dicalcium silicate coating dissolution was prepared to be the experimental group culture fluid. The human osteoblast-like MG63 cells were cultured by experimental group culture fluid and control group culture fluid respectively to observed the morphological difference in general of osteoblast in the process of growth in culture and the difference in cell population (MTT chromatometry and staining Cytometry). Results There were difference in Ca, Si and P ionic concentration(p<0.05) between experimental group culture fluid and control group culture fluid. The cells of experimental group have better cell differentiation than the osteoblast of control groups. There was no difference in cell population (p>0.05) between the experimental group culture fluid and control group culture fluid. Conclutions The ionic products of dicalcium silicate coating dissolution can increase differentiation of human osteoblasts and can not increase cell population.
PartⅢThe Effect of Ionic Products of Dicalcium Silicate Coating Dissolution to Osteoblast Growth Cycle and Apoptosis
Objective To observe the change of esteoblast growth cycle and apoptosis to understand the effect of ionic products of dicalcium silicate coating dissolution to osteoblast proliferation. Methods The full DMEM was prepared to be the control group culture fluid, the DMEM culture adding with ionic products of dicalcium silicate coating dissolution was prepared to be the experimental group culture fluid. The human osteoblast-like MG63 cells were cultured by experimental group culture fluid and control group culture fluid respectively. The osteoblast growth cycle and apoptosis was determined by flow cytometry at 3,6 and 12 day. The cell proliferative activity indicatrix[SPF(S-phase fraction) and PI(proliferous index) ] and apoptotic index(AI) were compared. Results On days 3,there were no difference in the percentage of cells in the G1 phase and PI (p>0.05) between the experimental group and control group . there was a significantly lower percentage of cells in the S phase on days 3 (P<0.05) in experimental group. In 6 to 12 day, there were significantly higher percentage of cells in the S phase and PI (P<0.01),and wae significantly lower percentage of cells in the G1 phase in experimental group. Apoptosis was markedly increased in the experimental group at days 3 and 12 (P< 0.05; P<0.01). Conclusion The ionic products of dicalcium silicate coating dissolution can increase proliferation of human osteoblasts by increased cell cycling. The feature of the ionic products of dicalcium silicate coating dissolution increased proliferation of human osteoblasts is that it has slightly depressant effect on proliferation of human osteoblasts in the initial stage, but it can significantly increase proliferation of human osteoblasts in later stage. Increase in apoptosis possibly profits to osteoblast differentiation .
PartⅣThe Effect of Ionic Products of Dicalcium Silicate Coating Dissolution to Osteoblast Differentiation and Phenotypic Expression
Objective To further investigate the possible mechanisms of ionic products of dicalcium silicate coating dissolution which enhances bone formation by observed the effect of ionic products of dicalcium silicate coating dissolution to osteoblast differentiation and phenotypic expression . Methods The full DMEM was prepared to be the control group culture fluid, the DMEM culture adding with ionic products of dicalcium silicate coating dissolution was prepared to be the experimental group culture fluid. The human osteoblast-like MG63 cells were cultured by experimental group culture fluid and control group culture fluid respectively. Detected the express of collagen-Ⅰ、bone gla-protein (BGP)、osteoblast core-binding factor alpha-1(CBFA-Ⅰ)and insulin-like growth factorⅡ(IGF-2) by polymerase chain reaction(PCR) and detected the activity of alkaline phosphatase(ALP) by biochemistry method. Results There were significantly higher express of collagen-Ⅰ,BGP,CBFA-Ⅰand IGF-2 (p<0.01) in experimental group in 3 to 12 days. Conclusion The ionic products of dicalcium silicate coating dissolution can increase proliferation , differentiation, formation of metrical and mineralization of human osteoblasts,and it profits to enhance bone formation.
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