【作者】 魏立；

【导师】 孙俊英；

【作者基本信息】 苏州大学 ， 外科学， 2009， 博士

【摘要】 第一部分硅酸二钙离子溶液促进成骨细胞增殖活性的研究目的试图阐明等离子喷涂于钛基的硅酸二钙涂层的溶解能力及在不同溶液中的主要离子变化,以及这些离子溶解物对成骨细胞增殖能力的影响。方法将等离子喷涂硅酸二钙涂层的金属钛片浸泡于200ml DMEM培养基及双蒸水中,37℃下静置72h后弃钛片,以ICP检测溶液中Si、Ca、P离子浓度。将制备的条件DMEM培养基及正常DMEM培养基分别培养MG63细胞,定期观察细胞形态学特征,于第3、6、9、12天四个时间点分别以MTT法检测细胞增殖情况;以流式细胞仪分析各时间点的细胞周期情况。结果经浸泡等离子喷涂于金属钛基的硅酸二钙涂层后的DMEM培养基及双蒸水中Si离子均明显升高,与正常培养基相比差异显著（条件DMEM vs DMEM p＜0.05,双蒸水（浸泡后） vs DMEM p＜0.01）;Ca及P离子浓度在两种DMEM培养基中无明显差异。培养于条件DMEM及正常DMEM中的MG63细胞形态未见明显差异。第3天实验组MTT值明显高于对照组,其差异显著（p＜0.01）。此后三个时间点两组间MTT值差异均无统计学意义,第9、12天时实验组MTT值甚至略低于对照组。细胞周期分析结果显示在培养第3天时实验组S期细胞所占比例显著高于对照组;而实验组G0/G1期细胞百分比则明显低于对照组;两组间G2/M期细胞所占比例无统计学差异,但实验组略高于对照组。第6,9,12天时实验组S期细胞百分比仍然高于对照组,但无统计学差异。同样,第6,9,12天时两组细胞间G0/G1期及G2/M期细胞所占的百分比之间也没有显著性的差异。结论等离子喷涂硅酸二钙涂层浸泡于DMEM培养基72h后,溶液中硅离子浓度明显增高。硅酸二钙离子溶液条件培养基有促进MG63细胞早期增殖的能力,而对晚期增殖无明显促进作用。其促成骨细胞增殖的能力可能与条件培养基中较高的硅离子浓度有关,并可能是与上调S期及G2/M期细胞比例并同时降低G0/G1期细胞的比例有关的。第二部分硅酸二钙离子溶液促进成骨细胞分化能力的研究目的探讨等离子喷涂硅酸二钙涂层离子溶液对MG63成骨样细胞分化能力的影响。方法人成骨肉瘤来源的具有成骨活性的MG63细胞分别培养于正常DMEM及条件DMEM培养基中,分别于第3、6、9、12天四个时间点收集标本,检测ALP活性;以定量RT-PCR法检测转录因子Runx2及成骨细胞分化标志物Ⅰ型胶原、碱性磷酸酶及骨钙素的基因表达情况。结果对照组ALP活性随培养时间的增加呈现出逐渐增加的趋势,但增加辐度较低;实验组ALP活性从第3天起即明显高于对照组,此后随培养时间增加而逐渐增加,第6天时两组间差异更加明显,第9天时仍然明显高于对照组,第12天时较第9天略有降低,但仍然明显高于对照组。Runx2的基因表达于第3,6天两个时间点在两组之间无明显差异,但第9,12天时,实验组的表达量明显高于对照组（p＜0.05）。对照组细胞ALP mRNA的表达在整个培养过程中无明显变化,实验组从第3天起即明显高于对照组,但差异不明显（p＞0.05）。从第6天起实验组ALP基因表达量明显高于对照组,并随培养时间增加而明显增加,其表达量在其后所有时间点均显著高于对照组（p＜0.05）。Ⅰ型胶原和骨钙’素的表达变化的趋势相似,在对照组各个时间点均无明显差别,但实验组基因表达呈现逐渐增高的趋势,然后于第12天时略降低。与对照组相比,第9、12天时Ⅰ型胶原与骨钙素的基因表达量均显著高于对照组（p＜0.05）。结论硅酸二钙离子溶解物除了能促进MG63细胞早期分化外,还具有促进其晚期分化的能力,这与其它同样具有CaO-SiO₂成分并能释放硅离子的生物活性材料对成骨细胞增殖分化的影响是一致的,表明硅酸二钙也是一种良好的生物活性材料。第三部分硅酸二钙离子溶液促进成骨细胞钙矿沉积的研究目的通过形态学观察和钙矿沉积定性、定量检测,试图阐明等离子喷涂硅酸二钙涂层离子溶液促进成骨细胞矿化的能力。方法MG63细胞按前述方法培养于24孔细胞培养板中,分别于第3、6、9、12天四个时间点中止培养,以茜素红S染色方法进行染色,了解钙矿沉积情况,观察是否有钙化结节的形成,并定量检测各时间点钙矿沉积量。结果实验组第3天时已经有明显的茜素红着色,而对照组细胞只有非常微弱的茜素红着色。随着培养时间的增加,第6天时对照组细胞可以见到较为明显的茜素红S着色,但实验组细胞着色强度明显强于对照组,并明显强于实验组第3天时。第9天时对照组细胞着色强度较第6天增强,但实验组明显强于同组第6天时,亦明显强于对照组第9天时的着色强度。第12天时对照组茜素红着色程度较同组第9天有较明显的增加,但与实验组相比其着色程度仍较弱,而实验组着色非常明显,并且在部分区域有着色较浓的片状聚集。实验组在培养的第12天偶尔可发现一些着色极浓的灶状茜素红S浓集,这很可能就是钙化结节形成的中心;而在对照组则没有这样的现象可以观察到。钙矿沉积定量检测结果显示:在本实验12天的培养过程中,对照组及实验组的钙矿沉积量均随培养时间的增加而逐渐增加。对照组第3天时即有少量钙矿沉积,以后稳步上升;实验组钙矿沉积也同样随培养的时间增加而逐渐增加。但在实验组第3天时钙矿沉积量即明显大于对照组,其差异显著（p＜0.05）,此后实验组钙矿沉积量增加大于对照组,到第9、12天时两组间差异更加明显（p＜0.01）。结论硅酸二钙离子溶解物条件DMEM培养基除了有促进培养的MG63成骨细胞增殖分化的能力外,还能够促进其钙矿沉积的发生,其能力明显强于正常DMEM培养基培养的MG63细胞。这可能主要与条件培养基中较高的硅离子浓度有关。第四部分硅酸二钙离子溶液促进成骨能力机理的研究目的探讨等离子喷涂硅酸二钙涂层离子溶液促进MG63细胞增殖、分化及钙化的可能分子机理。方法MG63细胞按前法培养,于相应时间点收集标本。分别以ELISA法检测BMP2、PGE2浓度;以生化方法检测NO浓度;以定量RT-PCR检测BMP2及其信号传导通路蛋白Smad1、6、7基因水平的表达情况。结果实验组和对照组BMP2基因及蛋白水平的表达均随着培养时间的增加而增加,虽然其表达量在所有时间点均无显著差异（p＞0.05）,但实验组各时间点的表达量均大于对照组。BMP信号传导协同性Smad1通路蛋白的基因表达在对照组各个时间点没有明显的变化,但实验组第9天和第12天却有显著的升高,在两个时间点上与对照组相比均有显著差异（p＜0.05）。两种抑制性Smad蛋白即Smad6和Smad7的基因表达在实验组和对照组中均没有明显的变化。相应地,对照组NO含量随着培养时间的增加无明显变化,而实验组NO含量随培养时间增加而逐渐增加,在第12天时开始降低,在各时间点均高于对照组相应时间点,且在第6,9,12天三个时间点与对照组相比差异具有统计学意义。PGE2的含量在两组中均随培养时间呈逐渐下降的趁势,但实验组PGE2的浓度明显高于对照组,于后三个时间点其差异明显。结论硅酸二钙离子溶解物能够上调BMP2基因及蛋白水平的表达,同时也能上调BMP2信号传导通路蛋白中辅助性Smad1的表达而调节成骨细胞的代谢功能,而对两种抑制性Smad即Smad6、7的表达无明显的影响。此外,硅酸二钙对成骨细胞代谢的影响还可能与PGE2及NO的表达增加有关。更多还原

【Abstract】 PartⅠStudy of ionic dissolution products of dicalcium silicate coating on osteoblastic proliferatoinObjective To explore the dissolving properties of plasma sprayed dicalcilum silicate coating on Ti plates and accordingly the concentrations of the main ions in different dissolutions,as well as the influences of ionic dissolution products of dicalcium on proliferation of MG63 cells.Method 10 pieces of Ti plates with plasma sprayed dicalcium silicate coating were soaked in 200ml DMEM and double dilluted H₂O, respectively,at 37℃for 72h in static conditions,then ICP-OES was employed to detect the concentrations of Si,Ca and P.MG63 cells were cultured in DMEM and conditioned DMEM respectively.Cell mophorlogy was observed under microscopes,cell cycles were analyzed by flow cytometry,and MTT assay was used to detect cell proliferation on days 3, 6,9 and 12.Results Si concentrations in DMEM and double dilluted H₂O after soaking with dicalcium silicate coating were increased,markedly when compared with normal DMEM;no differences of Ca and P ionic concentrations were observed between normal DMEM and dicalcium silicate conditioned DMEM.No differences in cell morphorlogy were existed between the cells of the two groups.MTT value of experimental group was significantly higher than that of control group on day 3.With the progression of culture time,no differences statistically significant were observed between the two groups,and MTT values on days 9 and 12 in the experimental group were even a little lower than those of the control group.Flowcytometric analysis showed that the percentage of MG63 cells in S phase of the experimental group was obviously higher than that of the control group.At the same time,cells in G0/G1 phase of the experimental group was significantly lower than that of the control group.While cells in G2/M phase of the experimental group was a little higher than that of the control group with no statistically significant difference.No differences in different phases between the two groups could be observed thereafter. Conclusions Plasma sprayed dicalcium silicate coating on Ti plates lead to increase in Si ion concentration after soaked in DMEM for 72h.Dicalcium silicate conditioned DMEM was able to promote the early but not the late proliferation of MG63 cells,which may be intimately related to the high concentration of Si ion.And the ability of C2S conditioned culture medium to promote osteoblastic proliferation was intimately related to the upregulated percentage of cells in S and G2/M phases as well as the downregulated percentage in G0/G1 phase.PartⅡStudy of ionic dissolution products of dicalcium silicate coating on osteoblastic differentiationObjective To explore the influences of ionic dissolution products of dicalcium silicate coating on differentiation of MG63 cells.Method MG63 osteoblast-like cells were cultured in normal DMEM and conditioned DMEM,respcetively. After 3,6,9,12 days’ cultivation,respectively,samples were collected for detection of the ALP activity and gene expression of core binding factorα1（Runx2） and 3 markers of osteoblastic differentiation,e.g typeⅠcollagen,ALP and osteocalcin.Results From day 3 on,ALP activity in the experimental group was higher than that of the control group. With the progression of culture time,ALP activity increased in both of the two groups but was higher in the experimental group.Statistically significant differences were observed on timepoints of days 3,6 and 9.On day 12,although ALP activity was still higher in the experimental group than those in control group,the difference was already not significant. Although there were no significant differences at days 3 and 6,mRNA levels of Runx2 were higher in the experimental group on days 9 and 12.ALP mRNA expression in the control group in the culture course showed no signifant differences,but its expression markedly upregulated（p＜0.05） in the experimental group from day 3 on and increased persistently till the end of the study.When considering the mRNA levels of typeⅠcollagen and osteocalcin,the changes were similar in these two markers,which showed no significant changes in the control group but increased from 3 day on and was markedly higher on day 9 in the experimental group,then decreased slightly on day 12 but was still markedly higher than that of the control group.Conclusions Ionic dissoluiton products of dicalcium silicate coating was able to promote late differentiation as well as early proliferation of MG63 osteoblast-like cells,which indicated that dicalcium silicate may be also a favorable bioactive biomaterial.PartⅢStudy of the ionic dissolution products of dicalcium silicate coating on osteoblastic mineralisationObjective To explore the influences of ionic dissolution products of dicalcium silicate coating on osteoblastic mineralisation.Method MG63 cells were cultured in 24 wells plates accordingly and samples were collected and detected on days 3, 6,9 and 12,respectively.Alizarin Red S was used for staining of mineralisation for morphorlogic observation,and calcium mineral deposition was detected by a quantifying AR-S assay.Results AR-S staining showed that on day 3,cells in the experimental group already exhibited obvious staining,while those in the control group was very faint. On day 6,faint staining in the control group could be observed,while cells of the experimental group showed of much obvious staining compared to that in the control group,and also was stronger than that on day 3 in the same group.On day 9,cells of the control group increased a little when compared to that on day 6,while increased markedly in the experimental group during the same period.Although AR-S staining increased a lot when comparing cells in the control group on day 12 to day 9,the staining was still a little faint.While there was much stronger AR-S staining in the experimental group,and some portions with dense staining were also could be observed on day 12.Importantly,some very dense accumulation of AR-S staining which have formed focuses could be randomly observed in the experimental group that mostly like to represented the centre of nodule on day 12.What is more,calcium mineral content quantification showed that little mineral depostion was formed in the control group,while more mineral formation could be found in the experimental group on day 3.Then the mineral contents increased in both groups, but much more mineral deposition have formed in the experimental group when compared to those of the control group from day 6 to 12.Differences between the two groups on the 4 timepoints were all statistically significant.Conclusion Except for the ability of promoting proliferation and differentiation,ionic dissolution products of dicalcium silicate coating was also able to promote mineralisation of MG63 osteoblast-like cells,which may be mainly because of the high Si concentration in the conditioned DMEM.PartⅣStudy of the mechanisms of osteogenic capacity of ionic dissolution products of dicalcium silicate coatingObjective To explore the possible mechanisms of ionic dissolution products of dicalcium silicate coating in promoting osteogenic capacity in vitro.Method MG63 cells was cultured accordingly and samples were collected on the predetermined timepoints respectively.ELISA assay was used to detect BMP2 and PGE2 expression,biochemistry method was used for the detection of NO production,and quantitative RT-PCR was used to detect the gene expression of BMP2 and its’ signal transducers Smadl、6、7,respectively. Results BMP2 protein and mRNA expression were all increased in the two groups, which were higher on all of the time points in the experimental group although were not significant between the two groups.Gene expression of Smadl,which is a Co-Smad for BMPs signal tranduction increased markedly on day 9 and day 12 in the experimental group when compared with that of the control group（p＜0.05）.And no obvious changes of the two inhibitary Smads,Smad6 and 7 existed in the two groups on all of the 4 time points.Accordingly,NO production in the control group exhibited no obvious change with the progression of culture time,but was increased with the progression of culture time in the experimental group and begin to decrease on day 12,with significant differences exist on days 6,9 and 12 between the two groups.PGE2 contents in the two groups were all decreased with the progression of culture time,but which were higher in the experimental group,especially on the late 3 timepoints.Conclusion Ionic dissolution products of dicalcium silicate coating may promote the osteogenic capacity of MG63 osteoblast-like cells via upregulate expression of BMP2 and Smadl,while have no influences on the gene expression of Smad6 and Smad7.What is more,NO and PGE2 are also likely contributed to the positive effects of ionic dissolution products of dicalcium silicate coating in promoting osteoblastic metabolism.更多还原