人工关节硅酸二钙涂层离子液对成骨细胞生物活性影响的实验研究
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摘要
第一部分硅酸二钙涂层离子液对体外培养成骨细胞早期增殖、细胞周期以及凋亡的影响
     目的:探讨硅酸二钙涂层离子液对成骨细胞早期增殖及细胞周期、凋亡的影响及其机制。方法:将等离子喷涂的硅酸二钙涂层钛片于37℃浸泡在DMeM培养基中24小时,ICP测定培养基中Ca、P、Si离子浓度变化。含有硅酸二钙涂层离子液的DMEM培养基作为实验组,正常DMEM培养基作为对照组,分别接种MG63细胞培养。MTT法及细胞计数测定两组MG63细胞6、12、24、48、72小时细胞增殖情况;流式细胞仪测定MG63细胞12、24、48、72小时G0/G1、S、G2/M期细胞数量变化、增殖活性指标(PI)和细胞凋亡率;结果:在硅酸二钙涂层浸泡24小时后,培养基中Si离子浓度显著增加约17倍,有统计学意义(P＜0.001),而Ca、P离子浓度略有降低,但没有统计学意义(P＞0.05)。与正常培养基中比较,在硅酸二钙涂层条件培养基中,MG63细胞增殖在各时间点明显增强,在24和72小时有统计学差异(P＜0.05)。流式细胞仪结果显示,在12、24小时,两组中处在各期细胞数量没有明显差别,在48、72小时,实验组G0/G1期MG63细胞明显降低;在72小时,实验组S期细胞较对照组增多2倍(P＜0.05);实验组G2/M期细胞在48小时显著增多(P＜0.05)。在12、24小时,两组细胞的凋亡率都较低,未见明显差别,在48小时和72小时,实验组MG63细胞凋亡率显著高于对照组,有统计学差异(P＜0.05)。结论:硅酸二钙涂层离子液能够促进MG63细胞早期增殖,而这种作用是通过加速成骨细胞周期达到的。
     第二部分硅酸二钙涂层离子液对体外培养成骨细胞早期分化及成骨细胞特异性基因表达的影响
     目的:通过测定成骨细胞分化及成骨细胞分化相关基因的表达,探讨硅酸二钙涂层离子液对体外培养成骨细胞分化及成骨特异性基因表达的影响及其可能机制。方法:含有硅酸二钙离子溶出液的DMEM培养基作为实验组,正常DMEM培养基作为对照组,分别接种人MG63成骨细胞进行培养。分别于6、12、24、48、72小时采用生化法测定培养液中碱性磷酸酶(ALP)活性,实时定量PCR(Real time PCR)检测碱性磷酸酶(ALP)、骨钙素(OC)、Ⅰ型胶原(COL-Ⅰ)、成骨细胞特异性转录因子(Cbfal)基因表达变化,并进行比较。结果:实验组MG63细胞ALP活性在各时间点都增强,与对照组相比在6h,48h和72h有显著性差异(P＜0.05)。Real time PCR检测显示,MG63细胞Cbfal转录水平在所有时间点均较对照组上调,在6h,12h和24h有显著性差异(P＜0.05);ALP转录水平在12h和24h较对照组显著上调(P＜0.05),然后在48h和72h降低到对照组水平以下,但没有统计学差异(P＞0.05);Ⅰ型胶原转录水平在各时间点均较对照组高,在24h有显著性差异(P＜0.05);OC转录在两组早期均未检测到。结论:硅酸二钙涂层离子液能够在早期上调成骨细胞特异性基因的表达,进而促进成骨细胞的分化,加速骨的形成;硅酸二钙涂层离子液在早期增强成骨细胞的凋亡,这与成骨细胞分化增强有关。
     第三部分硅酸二钙涂层离子液对体外培养成骨细胞破骨细胞分化相关基因表达及其蛋白分泌的影响
     目的:通过测定破骨细胞分化相关基因的表达及其蛋白分泌,探讨硅酸二钙涂层离子液对破骨细胞分化的影响及可能机制。方法:含有硅酸二钙离子溶出液的DMEM培养基作为实验组,正常DMEM培养基作为对照组,分别接种人MG63成骨细胞进行培养。分别于6、12、24、48、72小时采用实时定量PCR(Real time PCR)检测OPG、RANKL、TNF-α基因表达变化,ELISA方法检测OPG、RANKL、TNF-α蛋白分泌量,并进行比较。结果:Real time PCR检测显示,实验组MG63细胞OPG转录水平在各时间点都增强并在72小时达到高峰,与对照组相比在6h,12,24和48h有显著性差异(P＜0.05)。MG63细胞RANKL转录水平在6,12h和24h非常低,在48h和72h显著增高,但实验组RANKL转录水平明显低于对照组,有显著性差异(P＜0.05);TNF-α的转录在两组中均未检测到;实验组培养基中OPG蛋白分泌量在各时间点均高于对照组,在48h和72h有统计学意义(P＜0.05);实验组RANKL蛋白分泌量在各时间点都低于对照组,但没有统计学差异(P＞0.05)。TNF-α蛋白分泌在各时间点均低于对照组,并在72小时有统计学差异。结论:硅酸二钙涂层离子液能够在早期上调OPG基因的表达,下调RANKL基因表达,同时促进OPG蛋白分泌,抑制TNF-α分泌,通过调节OPG/RANKL比率抑制破骨细胞的分化和活性,减少骨吸收,加速骨的形成。
     第四部分硅酸二钙涂层离子液对体外培养高龄来源人成骨细胞增殖、分化及对TGF-β1分泌的影响
     目的:探讨硅酸二钙涂层离子液对高龄来源的成骨细胞增殖、分化及TGF-β1分泌的影响及其机制。方法:采用原代培养技术从高龄患者(大于60岁)废弃骨组织分离培养出成骨细胞并鉴定。将等离子喷涂的硅酸二钙涂层钛片于37℃浸泡在DMEM培养基中72小时,ICP测定培养基中Ca、P、Si离子浓度变化。含有硅酸二钙涂层离子液的DMEM培养基作为实验组,正常DMEM培养基作为对照组,分别接种高龄来源成骨细胞培养。分别于1、3、7、14天采用MTT法测定两组成骨细胞细胞增殖情况;采用生化法检测ALP活性;ELISA方法检测TGF-β1蛋白分泌量;并进行比较。结果:与正常培养基中比较,条件培养基中硅离子浓度增加了19倍,有统计学差异(P＜0.05),而钙和磷离子的浓度降低,但没有统计学差异(P＞0.05)。在硅酸二钙涂层条件培养基中,高龄来源成骨细胞增殖在各时间点明显增强,在第3天有统计学差异(P＜0.05)。实验组成骨细胞ALP活性始终高于对照组,并在第7天有统计学差异(P＜0.05);ELISA结果显示在第1天和第3天,试验组中TGF-β1含量较对照组显著增高,有统计学意义(P＜0.05),而在第7天,试验组TGF-β1含量显著下降并低于对照组,但没有统计学差异(P＞0.05),在第14天,两组中TGF-β1含量没有明显差异(P＞0.05);结论:硅酸二钙涂层的离子溶出液能够促进高龄来源成骨细胞增殖和分化,而这种作用是通过促进TGF-β1分泌达到的。
     第五部分硅酸二钙涂层离子液对体外培养成骨细胞TGF-β1分泌及其受体基因表达的影响及其机制
     目的:探讨硅酸二钙涂层离子液对成骨细胞增殖、分化及TGF-β1分泌及其受体基因表达的影响及其机制。方法:含有硅酸二钙涂层离子液的DMEM培养基作为实验组,正常DMEM培养基作为对照组,分别接种MG63成骨细胞培养1、3、7、14天,成骨细胞分化通过ALP活性及骨钙素(OC)及其基因的表达检测;ELISA方法检测OC,TGF-β1蛋白分泌量;采用实时定量PCR(Real time PCR)检测TGF-β1Ⅰ型受体和Ⅱ型受体基因表达变化,并进行比较。结果:在硅酸二钙条件培养基中,硅酸二钙离子溶出液能够促进MG63细胞ALP活性及OC的分泌而促进细胞的分化,ELISA结果显示在条件培养基中TGF-β1分泌较正常培养基中有所增强,同时TGF-β受体基因表达也同样增高,与正常培养基中比较,在硅酸二钙涂层条件培养基中,MG63细胞ALP活性从第3天开始增强并高于对照组,并在第7天和14天有统计学差异(P＜0.05)。骨钙素OC的分泌在14天明显高于在正常培养基中,并有统计学差异(P＜0.05),二者的基因表达也表现为相同的趋势;ELISA结果显示在第1天和第3天,试验组中TGF-β1含量较对照组显著增高,有统计学意义(P＜0.05),而在第7天,试验组TGF-β1含量显著下降并低于对照组,有统计学差异(P＜0.05),在第14天,两组中TGF-β1含量没有明显差异(P＞0.05);Real time PCR结果显示试验组中TGF-βⅠ型受体表达在第3天开始增强,在第7天达到最高峰,并与对照组有统计学差异(P＜0.05);TGF-βⅡ型受体基因表达在各时间点均较对照组增强,但没有统计学差异(P＞0.05)。结论:硅酸二钙涂层的离子溶出液能够促进成骨细胞分化,而这种作用是通过影响TGF-β1通路达到的。
PartⅠThe effects of ionic products of dicalcium silicate coating dissolution on proliferation and cell cycle as well as cell apoptosis of osteoblasts
     Objective:To discuss the influence and possible mechanisms of ionic products of dicalcium silicate coating dissolution on proliferation,cell cycle and cell apoptosis of osteoblasts. Methods:The Ca_2SiO_4 conditioned DMEM was achieved by immersing plasma sprayed Ca_2SiO_4 coating in DMEM for 24h at 37℃.The ion concentration in DMEM was measured by ICP.MG63 cells cultured with the DMEM containing ionic products of dicalcium silicate coating dissolution was prepared to be experimental group,which cultured in normal DMEM was control groups.Cell proliferation was measure with MTT and cell counting at 6,12,24h,48h and 72h.Cell cycle distribution and cell apoptosis were investigated by flow cytometry at 12,24h,48h and 72h.Result: The results obtained from this work showed that Si ion concentration increased significantly about 17 folds in Ca_2SiO_4 conditioned DMEM(P<0.05),and Ca and P ion concentration decrease slightly and no significantly(P>0.05).MG63 cell proliferated more significantly in the group containing ionic products of Ca_2SiO_4 coatings than in the control group,and significantly at 24h and 72h.The cell cycle distribution indicated that the number of MG63 cell in each cycle phase showed no significantly difference at 12h and 24h.And the decreased osteoblast number in G_0/G_1 phase were detected in the cells cultured in the DMEM containing ionic products of Ca_2SiO_4 coatings compared with the control group at 48 and 72h.At 72h,the osteoblast number of S phase in experimental group increased about 2 folds when compared to control(P<0.05).G2/M phase osteoblast number increased significantly in experimental group at 48h(P<0.05).Cell apoptosis in experimental groups were significantly higher than control at 48 and 72h(P<0.05).Conclusion:The results suggest that the ionic products of Ca_2SiO_4 coating dissolution can promote MG63 cell proliferation which contributes to accelerating cell cycle.
     PartⅡThe influence and possible mechanisms of ionic products of dicaicium silicate coating dissolution on differentiation and osteogenic gene expression of osteoblasts
     Objective:To discuss the influence and possible mechanisms of ionic products of dicalcium silicate coating dissolution on differentiation and osteogenic gene expression of osteoblasts. Methods:MG63 cells cultured with the DMEM containing ionic products of dicalcium silicate coating dissolution was prepared to be experimental group,which cultured in normal DMEM was control groups for 6,12,24h,48h and 72h.Cell differentiation was detected by measuring ALP activity in culture media.Osteogenic gene expression(ALP,COL-Ⅰ,OC,Cbfal) was evaluated using Real time PCR technology.Result:The results obtained from this work showed that ALP activity increased in Ca_2SiO_4 conditioned DMEM when compared to control and significantly at 6h,48h and 72h(P＜0.05).Results from Real-time PCR showed that Cbfal transcriptional levels in experimental groups were up-regulated at all time points when compared with the control groups and significantly at 6h,12h and 24h(P＜0.05).When compared with control,ALP transcription in experimental groups were significantly up-regulated at 12h and 24h,and then decreased lower than control but no significantly at 48h and 72h(P＞0.05).COL-Ⅰtranscription were higher than control at all experimental periods and significantly at 24h(P＜0.05).OC transcription was not detected in both groups in early stage. Conclusion:The ionic products of Ca_2SiO_4 coating dissolution can up-regulate osteogenic-related gene expression and enhance differentiation of osteoblasts as well as new bone formation.Increased apoptosis of MG63 cells results from enhanced differentiation.
     PartⅢThe influence and possible mechanisms of ionic products of dicalcium silicate coating dissolution on osteoclastogenie gene expression and their protein synthesis of osteoblasts
     Objective:To discuss the influence and possible mechanisms of ionic products of dicalcium silicate coating dissolution on osteoclastogenic gene expression and their protein synthesis of osteoblasts.Methods:MG63 cells cultured with the DMEM containing ionic products of dicalcium silicate coating dissolution was prepared to be experimental group,which cultured in normal DMEM was control groups for 6,12,24h,48h and 72h.Osteoclastogenic gene expression(OPG,RANKL, TNF-α) was evaluated using Real time PCR technology.OPG,RANKL and TNF-αprotein synthesis in culture media were measured by ELISA assay at 12,24h,48h and 72h.Result:Results from Real-time PCR showed that OPG transcriptional levels in Ca_2SiO_4 conditioned DMEM were promoted when compared to control and significantly at 6h,12h,24h and 48h(P<0.05).RANKL transcriptional levels in both groups were low at 6h,12h and 24h.and then up-regulated at 48h and 72h,but in experimental groups,RANKL transcription were significantly lower when compared with control at 48h and 72h(P<0.05).No TNF-αtranscription was detected in both groups.OPG protein synthesis in experimental groups were higher than the control groups at all time points and significantly at 48h and 72h(P<0.05).RANKL protein synthesis in both groups showed no significantly difference but lower in experimental groups(P>0.05).TNF-αsynthesis of MG63 cells in experimental groups was lower than control and significantly at 72h(P<0.05).Conclusion:The ionic products of Ca_2SiO_4 coating dissolution can up-regulate OPG gene expression and down-regulate RANKL gene expression,more importantly increase OPG protein synthesis and inhibit TNF-αsynthesis,which influence the ratio of OPG/RANKL that inhibiting osteoclast differentiation and activity,finally lead to diminishing bone resorption and increasing bone formation.
     PartⅣThe influence and possible mechanisms of ionic products of dicalcium silicate coating dissolution on proliferation and differentiation as well as TGF-β1 synthesis of osteoblasts derived from old patients
     Objective:To discuss the influence and possible mechanisms of ionic products of dicalcium silicate coating dissolution on proliferation and differentiation as well as TGF-β1 synthesis of osteoblasts derived from old patients.Methods:The primary cultured osteoblasts were obtained from bone slice which were discarded from old patients suffered from total hip arthroplasty(older than 60 years).The Ca_2SiO_4 conditioned DMEM was achieved by immersing plasma sprayed Ca_2SiO_4 coating in DMEM for 72h at 37℃.The ion concentration in DMEM was measured by ICP.Osteoblasts cultured with the DMEM containing ionic products of dicalcium silicate coating dissolution was prepared to be experimental group,which cultured in normal DMEM was control groups for 1,3,7 and 14 days.Cell proliferation was measure with MTT assay.Osteoblast differentiation was detected by ALP activity.ELISA was used to measure TGF-β1 synthesis in culture media.Result:The results obtained from this work showed that Si ion concentration increased significantly about 19 folds in Ca_2SiO_4 conditioned DMEM(P＜0.05),and Ca and P ion concentration decrease slightly and no significantly(P＞0.05).Primary cultured osteoblast proliferated more significantly in the group containing ionic products of Ca_2SiO_4 coatings than in the control group,and significantly at 3 days. ALP activity in experimental groups were enhanced when compared to that in control groups at all time points,and significantly at 7 days(P＜0.05).ELISA results showed that TGF-β1 synthesis in experimental groups was promoted significantly when compared to control(P＜0.05) at 1 and 3 days, and decreased which lower than control but no significantly at 7 days,and no difference was found in 14 days.Conclusion:The results suggest that the ionic products of Ca_2SiO_4 coating dissolution can promote primary culture human osteoblast derived from old patient proliferation and differentiation which contributes to increasing TGF-β1 synthesis.
     PartⅤThe influence and possible mechanisms of ionic products of dicalcium silicate coating dissolution on differentiation and TGF-β1 synthesis as well as its receptors(TGF-βRⅠand TGF-βRⅡ) gene expression of MG63 cells
     Objective:To discuss the influence and possible mechanisms of ionic products of dicalcium silicate coating dissolution on differentiation and TGF-β1 synthesis as well as its receptors(TGF-βRⅠ and TGF-βRⅡ) gene expression of MG63 cells.Methods:The medium containing ionic products of dicaicium silicate(Ca_2SiO_4) for culturing MG63 cells was prepared by immersing the plasma sprayed Ca_2SiO_4 coatings in DMEM solution.The effect of the ionic products on cellular differentiation and local growth factor(TGF-β1) of osteoblast-like MG63 cell were investigated.The normal DMEM was also used to culture MG63 cells as the control group.Differentiation of cell was evaluated by detecting alkaline phosphatase(ALP) activity and osteocalcin(OC) synthesis as well as their gene expression. The levels of TGF-β1 in culture medium were measured using Enzyme-linked immunosorbent assay (ELISA).The gene expressions of TGF-β1 receptors(TGF-βRⅠand TGF-βRⅡ) were also measured by Real time PCR technology.Result:MG63 cells cultured in DMEM containing ionic products of Ca_2SiO_4 coating showed enhanced differentiation.The results obtained from ELISA showed that the levels of TGF-β1 in experimental group were higher than that in control.Compared with cultured in normal DMEM medium,ALP activity of MG63 cells in Ca_2SiO_4-DMEM was promoted from day 3 and significantly in day 7 and 14(P＜0.05);OC synthesis was significantly enhanced at day 14.Their gene expression was shown as identical pattern.ELISA results showed that TGF-β1 synthesis in experimental groups was promoted significantly when compared to control(P＜0.05) at 1 and 3 days, and decreased significantly which lower than control at 7 days,and no difference was found in 14 days. TGF-βRⅠgene expression in experimental groups was enhanced at day 3 and reached its tip at day 7 which significantly higher than control(P＜0.05).TGF-βRⅡgene expression was higher than control but no significantly(P＞0.05).Conclusion:It is concluded that ionic products of Ca_2SiO_4 coating may enhance cellular differentiation and collagen production by influencing TGF-β1 pathway.
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