镁合金及其涂层的成骨生物学性能初步研究
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摘要
研究背景
镁是一种生物可降解金属材料,但纯镁降解速率过快,产生大量氢气,临床应用受到了限制。随着合金技术的发展以及表面改性方法的应用,可降解镁合金的耐蚀性能和生物相容性有了极大的改善。
尽管镁合金具有良好的生物相容性,但是其降解速度仍较快,为了进一步减缓合金的降解速度,学者们进行了一系列合金表面改性研究,形成了不同类型的合金涂层,不仅起到延缓降解的作用,而且还改善了其生物相容性。目前研究较多的有钙磷涂层、氟化物涂层等。
但是作为骨科植入材料,其对于骨组织再生有无影响,将决定其是否能够成为一种好的骨修复材料。对于骨再生过程中的关键细胞-成骨细胞和骨髓间充质干细胞的生物学行为及成骨分化过程有无影响尚不明确,对于骨骼运动的主要动力装置一骨骼肌细胞的生物学行为影响也不明确。
本研究拟针对上述问题,以空白或钛合金为对照,对镁合金、钙磷涂层镁合金、氟涂层镁合金进行进一步的研究,研究镁合金及其涂层对成骨生物学的初步影响,明确其材料的表面性质、明确其对成骨细胞生物学行为的影响,进而明确其植入体内后的血清镁及局部骨骼的变化,明确其对骨骼肌细胞的生物影响,明确其对骨髓间充质干细胞生物学行为及成骨分化的影响。
目的
1、明确镁合金及其涂层的表面形态及性质
2、明确镁合金及其涂层对成骨细胞的生物学行为的影响
3、明确镁合金及其涂层植入体内后的血清镁变化、材料降解及成骨情况
4、明确镁合金及其涂层对骨骼肌细胞的生物学行为的影响
5、明确镁合金及其涂层对骨髓间充质干细胞的生物学行为的影响
6、明确镁合金及其涂层对骨髓间充质干细胞成骨分化的影响
研究方法
1、采用扫描电镜研究镁合金AZ31B、钙磷涂层镁合金CaP-AZ31B、氟涂层镁合金F-AZ31B的表面形态,用能谱分析研究其元素组成；通过将镁合金及其涂层浸入细胞培养基中获得浸提液,测定浸提液的PH值变化情况；通过蛋白吸附实验研究镁合金及其涂层对蛋白的吸附能力。
2、通过采用镁合金、钙磷涂层镁合金及氟涂层镁合金的浸提液培养成骨细胞,进而研究其细胞2、6及24小时的细胞黏附率,采用扫描电镜研究其黏附形态,采用倒置显微镜观察1、3、5、7天细胞形态,采用CCK法检测成骨细胞1、3、5、7天增殖活力,采用Calcein-AM和EthD-1双染观察浸提液培养24小时后的细胞存活情况,通过1、3、5、7天的磷酸酶测定及1、3、5、7天的细胞内总蛋白测定来研究镁合金及其涂层对成骨细胞功能的影响。
3、通过将镁合金组、钙磷涂层镁合金组和聚乳酸对照组3组材料植入兔股骨,术后观察兔子的大体行为及术后24小时、1周、4周、6周和8周的血清镁浓度变化；术后8周处死实验动物,通过大体形态、CT扫描及三维重建观察植入材料的形态学变化情况；通过组织学观察成骨情况；通过扫描电镜观察镁合金及骨界面处的降解情况。
4、通过采用镁合金、钙磷涂层镁合金及氟涂层镁合金的浸提液培养骨骼肌细胞,进而研究其细胞2、6及24小时的细胞黏附率,采用扫描电镜研究其黏附形态,采用倒置显微镜观察1、3、5、7天细胞形态,采用CCK法检测骨骼肌细胞1、3、5、7天增殖活力,采用细胞凋亡试剂盒检测各组细胞有无凋亡,采用Calcein-AM和EthD-1双染观察浸提液培养24小时后的细胞存活情况,采用1、3、5、7天细胞内总蛋白测定来研究镁合金及其涂层对骨骼肌细胞功能的影响。
5、从志愿者身上抽取骨髓,进行骨髓间充质干细胞的分离培养,通过免疫组织化学方法鉴定骨髓间充质干细胞；通过采用镁合金、钙磷涂层镁合金及氟涂层镁合金的浸提液培养骨髓间充质干细胞胞,进而研究其细胞2、6及24小时的细胞黏附率,采用扫描电镜研究其黏附形态,采用倒置显微镜观察1、3、5、7天细胞形态,采用CCK法检测骨髓间充质干细胞1、3、5、7天增殖活力,采用Calcein-AM和EthD-1双染观察浸提液培养24小时后的细胞存活情况。
6、通过镁合金、钙磷涂层镁合金及氟涂层镁合金置入成骨诱导液中获得成骨诱导浸提液,进而采用不同的成骨诱导液进行骨髓间充质干细胞的成骨分化诱导,通过细胞收集、引物设计、细胞RNA的提取以及逆转录等过程,以P-actin基因为内参,于成骨分化诱导后第6天和第12天研究各种浸提液中ALP、 COL I、OC、OPN和RUNX2基因的表达情况。
7、根据不同的数据类型,分别采用采用One-Way ANOVA、析因设计方差分析、重复测量方差分析及独立样本t检验进行统计学分析,多重比较应用LSD法,方差不齐时采用Dunnetts T3比较。检验水准α=0.05。
结果
1、表面形态及性质
1.1表面形态：镁合金表面形态光滑,钙磷涂层镁合金表面形态粗糙,可见大量的钙磷物质沉积及晶体状结构,氟涂层镁合金表面表面均匀、光滑,在涂层表面可见分布有少数不规则小孔。
1.2能谱分析：结果表明镁合金表面主要有镁、铝、锌组成,其中镁元素原子百分比约为96%,铝约为3%,锌约为1%；钙磷涂层镁合金CaP-AZ31B表面主要有镁、钙、磷及氧组成,其中镁元素原子百分比约为0.33%,氧为63.39%,磷为17.6%5,钙为18.63%；氟涂层镁合金F-AZ31B表面主要是镁、氟、铝及锌,其中镁元素原子百分比约为56%,氟元素约为42.34%,铝约为1.27%,锌约为0.47%。
1.3浸提液的PH值变化情况：随着时间的延长,镁合金AZ31B、钙磷涂层镁合金CaP-AZ31B、氟涂层镁合金F-AZ31B溶液的PH值都发生了改变,其逐渐向碱性环境转化,但是镁合金AZ31B浸提液的PH值升高最为显著,远氟涂层镁合金F-AZ31B溶液的PH值改变不显著,钙磷涂层镁合金CaP-AZ31B溶液的PH值改变介于二者之间。
1.4蛋白吸附能力：镁合金的蛋白吸附能力较钛合金低,氟涂层的蛋白吸附能力较镁合金稍高,钙磷涂层镁合金的蛋白吸附能力较其他三组显著增高(P<0.05),四组的蛋白吸附能力分别为26.95±3.17、25.27±2.02、70.2±5.57、27.70±2.00ug/ml。
2、对成骨细胞的生物学行为的影响
2.1早期细胞黏附率：四组材料表面MC3T3-El细胞粘附率之间有显著差异(F=259.434,P=0.000),组间比较显示钙磷涂层镁合金组细胞粘附率显著高于其他三组(P<0.05),而镁合金组细胞粘附率显著低于其他三组(P<0.05),其他两组间无显著差异(P>0.05)。针对同组材料三个不同时间点MC3T3-El细胞粘附率比较均具有显著差异(P值均<0.05)。四组之间细胞黏附率CaP-AZ31B组>F-AZ31B组>钛合金组>AZ31B组。加入四组材料浸提液后不同时间点MC3T3-El细胞粘附率有显著差异(F=630.231,P=0.000),随着时间的延长,四组材料浸提液MC3T3-El细胞粘附率显著增加,不同时间点两两比较均有显著统计学差异(P<0.05)。且同一时间点四组材料浸提液MC3T3-El细胞粘附率比较差异均具有的统计学意义(P均<0.05)其中所有组合交互效应均显著(F=21.78,P=0.000)。结合基本统计量输出结果,最大均值(62.8%)的组合为培养24h时CaP-AZ31B组MC3T3-El细胞粘附率,最小均值(24.0%)为培养2h时AZ31B细胞粘附率。
2.2细胞黏附形态观察
成骨细胞在三组材料表面黏附较好,镁合金及其涂层表面成骨细胞贴壁展开,形态不规则,大多呈梭形,有较多突起,部分细胞间突起相互连接。
2.3细胞增殖活力
加入四组材料浸提液培养后不同时间点细胞增殖活力比较有显著差异(F=1491.2,P=0.000)；镁合金及其涂层合金浸提液培养的细胞活力均随着培养时间的延长而增加,任两个不同时间其细胞活力比较差异均具有显著的统计学意义。镁合金及其涂层合金浸提液培养的细胞活力四组总体比较具有显著差异(F=143.6,P=0.000),多重比较显示四组间任何两组比较其差异均具有显著统计学意义,且总体F-AZ31B组增殖活力最大,其次是钛合金组,CaP-AZ31B组次之,AZ31B组细胞增殖活力最小。其中所有组合交互效应均显著(F=9.15,P=0.000)。
2.4相对增殖率
镁合金组的相对增殖率在四个时间点均小于80%,说明镁合金有一定毒性,按照毒性评级为2级,但钙磷涂层镁合金组培养的MC3T3-El细胞相对增值率均>80%,毒性评级为1级,氟涂层镁合金组培养的MC3T3-El细胞相对增值率均>100%,毒性评级为0级,为临床骨科植入材料所接受。
采用析因设计的方差分析结果显示：镁合金及其涂层合金浸提液培养的细胞在不同时间上细胞增殖率无显著差异(F=0.725,P=0.541)；且各个时间点之间两两比较差异均无统计学意义(P>0.05)。镁合金及其涂层合金浸提液培养的细胞增殖率四组间总体比较具有显著差异(F=534.57,P=0.000)；3个试验组的细胞增殖率均显著低于对照组,四组间细胞增值率两两比较均有显著差异,且从均值上看,其细胞增值率对照组>CaP-AZ31B组>F-AZ31B组>AZ31B组。且培养时间及浸提液之间交互效应显著(F=2.01,P=0.048)。
2.5荧光染色
见镁合金浸提液组培养的成骨细胞可见少量细胞红染,但绝大多数细胞仍具有活性(绿染),钙磷涂层镁合金及氟涂层镁合金组浸提液培养的细胞无明显红染。
2.6碱性磷酸酶定量
随着培养时间的延长,镁合金及其涂层合金浸提液培养的细胞碱性磷酸酶表达均呈上升趋势,差异具有统计学意义(F=1330.7,P=0.000),且各个时间点之间两两比较均具有显著差异(P<0.05)。镁合金及其涂层合金浸提液培养的细胞碱性磷酸酶表达水平四组间总体比较具有显著差异(F=407.3,P=0.000);且多重比较结果显示任两组浸提液培养的细胞碱性磷酸酶差异均显著,且从均值上看,四组的碱性磷酸酶表达量依次为CaP-AZ31B组>F-AZ31B组>对照组>AZ31B组。培养时间及细胞碱性磷酸酶表达水平之间交互效应显著(F=25.8,P=0.000)。
2.7细胞内总蛋白
镁合金及其涂层合金浸提液培养的细胞胞内总蛋白量随着培养时间的延长而增加,差异具有显著统计学意义(F=851.6,P=0.000)；且各个时间点之间两两比较其细胞内总蛋白量均有显著差异(P<0.05)。镁合金及其涂层合金浸提液培养的细胞胞内总蛋白量四组间总体比较具有显著差异(F=4120.7,P=0.000);且多重比较结果显示任两组浸提液培养的细胞碱性磷酸酶差异均显著,且从均值上看,四组浸提液培养的细胞胞内总蛋白量依次为CaP-AZ31B组>F-AZ31B组>对照组>AZ31B组。培养时间及细胞胞内总蛋白量之间交互效应显著(F=30.9,P=0.000)。
3、骨植入实验
3.1术后兔子一般情况良好,术后各个时间点的血清镁均在正常范围内。
3.2植入八周后聚乳酸几乎完全降解,并且植入处与新生骨贴合紧密。镁合金及钙磷涂层镁合金基本都维持较好的完整形态,其中镁合金骨质外部分可见少许降解,边界不清晰、不规则,钙磷涂层镁合金材料边界清晰,未见到明显降解痕迹。
3.3钙磷涂层镁合金组金属与骨界面出可见较多新生骨形成,骨小梁排列紧凑而规则。无涂层镁合金形成的新生骨较少。同时可见无涂层样品的扫描电镜提示其边缘不规则,发生了降解,但钙磷涂层镁合金边缘形状同植入前变化很小。
4、对骨骼肌细胞的生物学行为的影响
4.1骨骼肌细胞黏附率测定
四组材料表面兔骨骼肌细胞细胞粘附率之间有显著差异(F=157.11,P=0.000),组间比较显示钙磷涂层镁合金组细胞粘附率显著高于其他三组,而镁合金组细胞粘附率显著低于其他三组,其他两组间无显著差异。四组之间细胞黏附率CaP-AZ31B组>F-AZ31B组>钛合金组>AZ31B组。加入四组材料浸提液后不同时间点兔骨骼肌细胞粘附率有显著差异(F=874.783,P=0.000),随着时间的延长,四组材料浸提液兔骨骼肌细胞粘附率显著增加,不同时间点两两比较均有显著统计学差异(P<0.05)。其中时间与浸提液交互效应均显著(F=10.61,P=0.000)。
4.2细胞黏附形态
兔骨骼肌细胞在镁合金、钙磷涂层镁合金及氟涂层镁合金三组材料表面均表现黏附较好。
4.3细胞增殖活力
加入四组材料浸提液培养后不同时间点细胞增殖活力比较有显著差异(F=2022.53,P=0.000)；镁合金及其涂层合金浸提液培养的细胞活力均随着培养时间的延长而增加,任两个不同时间其细胞活力比较差异均具有显著的统计学意义。镁合金及其涂层合金浸提液培养的细胞活力四组总体比较具有显著差异(F=50.68,P=0.000),多重比较显示四组间任两组比较其差异显著(P<0.05),且总体CaP-AZ31B组增殖活力最大,其次是F-AZ31B及钛合金组,AZ31B组细胞增殖活力最小。其中所有组合交互效应均显著(F=5.43,P=0.000)。
4.4相对增殖率
镁合金组的相对增殖率在第5及7d两个时间点小于80%,说明镁合金有一定毒性,毒性评级为2级,但钙磷涂层镁合金及氟涂层镁合金组培养的骨骼肌细胞相对增殖率均>90%,毒性评级为1级,为临床骨科植入材料所接受。
采用析因设计的方差分析结果显示：镁合金及其涂层合金浸提液培养的细胞在不同时间上细胞增殖率有统计学差异(F=3.56,P=0.018)；且各个时间点之间比较,除第1d和第5d,第1d和第7d细胞增值率有显著差异(P<0.05)外,其余各时间点间比较均无显著差异(P>0.05)。镁合金及其涂层合金浸提液培养的细胞增殖率四组间总体比较具有显著差异(F=217.94,P=0.000)；3个试验组的细胞增殖率均显著低于对照组,四组间细胞增值率两两比较均有显著差异,且从均值上看,其细胞增值率对照组>CaP-AZ31B组>F-AZ31B组>AZ31B组且培养时间及浸提液之间无交互作用(F=0.69,P=0.71)。
4.5细胞凋亡检测
镁合金浸提液可引起骨骼肌细胞凋亡,对照组、钙磷涂层镁合金组以及氟涂层镁合金组流式细胞图未见明显细胞凋亡。
4.6荧光染色
可镁合金浸提液组培养的骨骼肌细胞可见部分细胞红染,钙磷涂层镁合金及氟涂层镁合金浸提液培养的骨骼肌细胞活性较好,如对照组一样,未见明显细胞红染。
4.7细胞内总蛋白
镁合金及其涂层合金浸提液培养的细胞胞内总蛋白量随着培养时间的延长而增加,差异具有显著统计学意义(F=1796.8,P=0.000)；且各个时间点之间两两比较其细胞内总蛋白量均有显著差异(P<0.05)。镁合金及其涂层合金浸提液培养的细胞胞内总蛋白量四组间总体比较具有显著差异(F=136.45,P=0.000)；且多重比较结果显示：除CaP-AZ31B组与F-AZ31B组细胞内总蛋白量比较无显著差异(P>0.05)外,其他各组间细胞内总蛋白量差异均显著,且从均值上看,四组浸提液培养的细胞胞内总蛋白量依次为对照组>CaP-AZ31B组>F-AZ31B组>AZ31B组。培养时间及细胞胞内总蛋白量之间交互效应显著(F=7.15,P=0.000)。
5.对骨髓间充质干细胞的生物学行为的影响
5.1骨髓间充质干细胞鉴定
流式细胞仪细胞仪检测结果提示CD34-、CD45-、CD44+、CD73+、CD90+、 CD105+。
5.2骨髓间充质干细胞黏附
四组材料表面兔骨骼肌细胞细胞粘附率之间有显著差异(F=259.43,P=0.000),组间比较显示钙磷涂层镁合金组细胞粘附率显著高于其他三组,而镁合金组细胞粘附率显著低于其他三组,氟涂层与钛合金组比较两组间无显著差异。四组之间细胞黏附率CaP-AZ31B组>F-AZ31B组>钛合金组>AZ31B组。加入四组材料浸提液后不同时间点骨髓间充质干细胞粘附率有显著差异(F=630.23,P=0.000),随着时间的延长,四组材料浸提液骨髓间充质干细胞粘附率显著增加,不同时间点两两比较均有显著统计学差异(P<0.05)。其中时间与浸提液交互效应显著(F=21.78,P=0.000)。
5.3细胞增殖
镁合金及其涂层合金浸提液培养的细胞在不同时间上细胞增殖能力比较,结果显示：加入浸提液培养后不同时间点细胞增殖活力比较有显著差异(F=399.91,P=0.000);镁合金及其涂层合金浸提液培养的细胞增殖能力均随着培养时间的延长而增加,任两个不同时间其细胞增殖能力比较差异均具有显著的统计学意义。镁合金及其涂层合金浸提液培养的细胞增殖能力四组总体比较具有显著差异(F=37.14,P=0.000),多重比较显示镁合金组细胞增殖能力均显著低于其他四组,钙磷涂层及氟涂层镁合金的细胞增殖能力较无涂层显著改善,显著优于镁合金组,与钛合金组无显著差异,且总体钙磷涂层镁合金组增殖活力最大,其次是钛合金组,氟涂层镁合金组次之,镁合金组细胞增殖活力最小。对镁合金降解液存在的PH升高问题,调节PH值组镁合金浸提液的细胞增殖能力与钛合金组、和钙磷涂层及氟涂层镁合金无显著差异(P>0.05)。其中时间降解液交互效应均显著(F=2.32,P=0.011)。
五组荧光结果提示均可见到绿色染色的细胞,未见到明显的红色染色细胞。
6、对骨髓间充质干细胞成骨分化的影响
提取的RNA较完整,纯度较高。RT-PCR结果提示：钙磷涂层镁合金对于COLI基因、ALP基因以及OC基因表达有促进作用。RUNX2基因表达量四组间无显著差异。
镁合金组的ALP表达量较低,调节PH值后ALP表达量与空白对照组无显著差异(P>0.05),镁合金的OPN基因表达量在诱导分化后第6天和第12天均显著高于其余三组(P<0.05),调整PH值后的OPN基因表达量与对照组无显著差异(P>0.05),提示PH值是引起镁合金对于ALP基因和OPN基因的表达影响的可能因素之一。
结论
1、镁合金AZ31B及氟涂层镁合金F-AZ31B表面光滑,钙磷涂层镁合金CaP-AZ31B表面粗糙,可见晶体状结构。
2、三组镁合金浸提液的PH值均出现增高,但氟涂层镁合金性质最为稳定,单纯镁合金溶液的PH值升高显著。
3、钙磷涂层镁合金对于蛋白具有更好的吸附能力,镁合金及氟涂层镁合金及钛合金对于蛋白吸附能力无显著差异。
4、镁合金对于成骨细胞存在2级毒性反应,钙磷涂层镁合金及氟涂层镁合金对于成骨细胞有着良好生物相容性。
5、镁合金及其钙磷涂层体内生物相容性良好,血清镁的浓度均为正常范围内；钙磷涂层有效地延缓了镁合金的降解,并可以促进成骨作用。
6、镁合金对于骨骼肌细胞存在2级毒性反应,钙磷涂层镁合金及涂层镁合金对于骨骼肌细胞有着良好生物相容性。
7、镁合金对于骨髓间充质干细胞存在2级毒性反应,钙磷涂层镁合金及涂层镁合金对骨髓间充质干细胞有着良好生物相容性。
8、钙磷涂层镁合金对于COLI、ALP及OC基因表达有促进作用,无涂层镁合金抑制ALP的表达,且促进OPN基因的表达,但是调节PH值后重复研究发现其与对照组无显著差异,提示其影响可能与PH值有关。
Backgrounds
Magnesium is a biodegradable metallic material, but the degradation rate of pure magnesium is too rapid and produce large amounts of hydrogen, which make the clinical application has been limited. With the the alloy technology's development, and the application of the methods of surface modification, biodegradable magnesium alloy corrosion resistance and biocompatibility has been greatly improved.
Although magnesium alloys have good biocompatibility, but its degradation rate is still faster, to further slow down the degradation rate of the alloy, the scholars have carried out a series of alloy surface modification research, and have formed different types of alloy coating, which not only to delay the degradation, but also improve its biocompatibility. Such as calcium phosphate coating and fluoride coating.
But as orthopedic implant materials, that the impact for bone tissue regeneration will decide whether to become a good bone repair materials. The key cells in the bone regeneration process are Osteoblasts and bone marrow mesenchymal stem cells.The biological behavior and osteogenic differentiation on the cells is not yet clear, and the biological behavior of skeletal muscle cells for bone movement is not clear either.
In this study intends to conduct further studies to address the problem,with blank or titanium as control, study the osteogenesis biological effects of magnesium alloy, magnesium alloy with calcium phosphate coating and fluorine-coated magnesium alloy. And then clear the nature of the surface of the material,clear impact on the biological behavior of osteoblasts,clear biological effects on skeletal muscle cells, clear serum magnesium and localized bone changes after its implanted, clear the biological behavior and osteogenic differentiation of bone marrow mesenchymal stem cells.
Objectives
1. Study surface morphology and nature of magnesium alloy and its coating.
2. Study the biological behavior of osteoblasts with magnesium alloy and its coating.
3. Study the serum magnesium level, material degradation and osteogenic of magnesium alloy and its coating in rabbits.
4. Study the biological behavior of skeletal muscle cells with magnesium alloy and its coating.
5. Study the biological behavior of bone marrow mesenchymal stem cells with magnesium alloy and its coating.
6. Study the osteogenic differentiation of bone marrow mesenchymal stem cells with magnesium alloy and its coating.
Methods
1. Using scanning electron microscopy to study the surface morphology of magnesium alloy(AZ31N), calcium and phosphorus coating of magnesium alloy(CaP-AZ31B) and fluorine coating magnesium alloy(F-AZ31B), study its elements by energy spectrum. By the magnesium alloy and the coating was immersed in the cell culture medium obtained extracts,then determined the PH value of the extracts,study the protein adsorption capacity of magnesium alloy.
2. Cultured osteoblast in extracts, then study the2,6and24hours cell adhesion rate, using scanning electron microscopy to study the adhesion morphology, using the inverted microscope to study cell morphology of1,3,5,7days, CCK assay the osteoblasts proliferation activity of1,3,5,7days, using Calcein-AM, and ETHD-1double staining to observe cell survival,study phosphatase and total protein of cells in1,3,5,7days.
3. There are3groups in implant test:, magnesium alloy group(AZ31B), calcium phosphate coating of magnesium alloy group(CaP-AZ31B), polylactic acid group(PLLA),The pins were implanted into rabbit femur,Then the general behavior of rabbits were observed, serum magnesium concentration of24hours, one week, four weeks, six weeks and eight weeks after operation were measured.The experimental animals were sacrificed after8weeks,using CT scanning and three-dimensional reconstruction of the implant material to study morphological changes, observing histological bone formation,using SEM to observe degradation.
4. Cultured skeletal muscle cells in extracts, then study the2,6and24hours cell adhesion rate, using scanning electron microscopy to study the adhesion morphology, using the inverted microscope to study cell morphology of1,3,5,7days, CCK assay the osteoblasts proliferation activity of1,3,5,7days, using apoptosis detection kit to detect apoptosis, using Calcein-AM, and ETHD-1double staining to observe cell survival,study total protein of cells in1,3,5,7days.
5. Bone marrow were drawn from volunteers, followed by the isolation and culture of mesenchymal stem cells in the bone marrow, bone marrow mesenchymal stem cells were identified by immunohistochemistry; through the use of magnesium alloy, calcium and phosphorus coating of magnesium alloy and magnesium fluoride coating alloy extractionliquid cultured bone marrow mesenchymal stem cells, then study the2,6and24hours cell adhesion rate, using scanning electron microscopy to study the adhesion morphology, using the inverted microscope to study cell morphology of1,3,5,7days, CCK assay the osteoblasts proliferation activity of1,3,5,7days, using Calcein-AM, and ETHD-1double staining to observe cell survival.
6. Using different osteogenic induction medium induced osteogenic differentiation of bone marrow mesenchymal stem cells, collected by cell primer design cell RNA extraction and reverse transcription process,(3-actin gene as an internal,study the ALP, COL I,OC, OPN and RUNX2gene expression after cells were cultured in various extracts for6days and12days.
7. The data was staticstically analysis with one-way ANOVA analysis and Univariate, and LSD test was compared in pairwise comparison. Level of significance α=0.05.
Results
1. The surface morphology and the nature
1.1The surface morphology:Magnesium alloy surface morphology was rough, smooth, calcium phosphate coating magnesium alloy surface morphology seen a lot of calcium and phosphorus material deposition and crystal-like structure, fluorine coating the surface of the magnesium alloy surface is uniform, smooth, visible distribution of a small number of irregular holes in the surface of the coating.
1.2EDS analysis:The results showed that the surface of the magnesium alloy are mainly magnesium, aluminum, zinc composition, where in the magnesium atomic percentage is approximately96%, and aluminum of about3%, zinc is about1%; calcium phosphate coating magnesium alloy surface of the CaP-AZ31B mainly consists of magnesium, calcium, phosphorus, and oxygen, where in the percentage of magnesium atom of about0.33%, oxygen63.39%,17.6%phosphorus, calcium18.63%; magnesium fluoride coating the surface of the alloy F-AZ31B magnesium,fluorine, aluminum, zinc, magnesium atom percentage of about56%, about42.34%fluorine, aluminum is about1.27%, zinc is about0.47%.
1.3PH value changes of extracts:The PH of AZ31B and its coating extracts changed gradually to alkalienvironmental transformation, but the PH value of the magnesium alloy AZ31B extraction of liquid increased most significantly, no significant changes in the solution pH value of the F-AZ31B, CaP-AZ31B solution PH value change between AZ31B and F-AZ31B.
1.4Protein adsorption ability:magnesium alloy protein adsorption capacity is lower than the titanium alloy low, the protein adsorption capacity of F-AZ31B is slightly higher than magnesium alloy, calcium phosphate coating magnesium alloy protein adsorption capacity was significantly higher compared to the other three groups (P<0.05),Protein adsorption capacity of four groups were26.95±3.17,25.27±2.02,70.2±5.57,27.70±2.00ug/ml.
2、The biological behavior of osteoblasts
2.1Early cell adhesion rate
The cell adhesion rate of MC3T3-E1cells in2h,6h and24h:titanium alloy group (27.58±1.11)%,(34.92±2.26)%,(50.21±2.11)%; magnesium alloy group (24.04±2.33)%,(29.08±1.91)%,(37.33±1.49)%; the fluorine coating magnesium alloy (28.46±1.39)%,(35.96±1.18)%,(51.75±1.94)%; calcium phosphate coating magnesium alloys(34.25±1.47)%,(43.71±1.61)%,(62.75±1.97)%; CaP-AZ31B> F-AZ31B group> titanium alloy group> AZ31B group. Significant differences exist between the groups (P<0.05).
2.2Cell adhesion morphology
Osteoblast adhesion well in three groups of materials, with osteoblasts adherent expand, irregular shape, mostly fusiform, more protruding part of the cell protrusion interconnected.
2.3Cell viability
Cell viability cultured in extracts increased with prolonged incubation time and proliferative activity of the the F-AZ31B group> control group> CaP-AZ31B group> AZ31B group, significant differences exist between the two groups (P<0.05)。
2.4Relative growth rate
The relative growth rate of AZ31B was less than80%at four time points, with a toxicity rating of2, the relative growth of CaP-AZ31B>80%, toxicity rating of1, the fluorine coating magnesium alloy group>100%, toxicity rating of0. Significant differences exist between the groups (P<0.05).
2.5Staining
a small amount of stained red cells could be seen in AZ31B extracts,but the vast majority of cells are still active (green dye), no obvious red dye could be seen in cell culture extracts of calcium and phosphorus coating magnesium alloy and magnesium fluoride coating alloy group.
2.6Alkaline phosphatase quantitative
With prolonged incubation time, the group of alkaline phosphatase expression showed an upward trend, the four groups of alkaline phosphatase expression order the CaP-AZ31B group> F-AZ31B group> control group> AZ31B group, significant differences exist between the two groups (P<0.05)。
2.7intracellular total protein
With the prolonged incubation time the total amount of protein in the cells in each group increased, the amount of protein of the four groups of1,3,5,7, d,blank control group,(60.33±2.38)ug/ml,(98.34±2.19)ug/ml,(122.41±3.92)ug/ml, (148.52±3.62)ug/ml; magnesium alloy group (46.5±1.92) ug/ml,(75.73±2.22) ug/ml,(92.29±3.09) ug/ml,(111.31±3.67)ug/ml; the F-AZ31B group (64.66±1.35) ug/ml,(105.71±1.85) ug/ml,(130.97±2.36)ug/ml, and (160.47±2.41) ug/ml; CaP-the AZ31B group(66.24±1.7) ug/ml,(108.61±1.61) ug/ml,(134.01±3.87) ug/ml,(163.03±3.84) ug/ml. Significant differences exist between the groups (P<0.05).
3. Bone implantation experiments
3.1postoperative rabbits generally in good condition, postoperative serum magnesium for each time point were within the normal range.
3.2Polylactic acid almost completely degraded after implanted eight weeks, and fitting close with the new bone. AZ31B and CaP-AZ31B basically maintain the complete form, which shows little degradation of AZ31B bone outside part, the boundary is not clear, irregular, but clear boundary was seen in CaP-AZ31B and did not see the obvious degradation traces.
3.3New bone formation could be seen in CaP-AZ31B group and bone trabeculae compact and rules. The formation of new bone is less in AZ31B group. The sem showed that the edge of AZ31B is irregular, which means degradation has occurred, the edge shape of CaP-AZ31B with preimplantation change is very small.
4、The biological behavior of skeletal muscle cells
4.1Early cell adhesion rate of skeletal muscle cells:
With the prolonged adhesion time, adhesion rate of skeletal muscle cell increased in all groups. The cell adhesion rate of skeletal muscle cells in2h,6h and24h:titanium alloy group (26.46±2.33)%,(34.92±2.26)%,(47.21±2.11)%; magnesium alloy group (23.88±2.90)%,(28.75±2.46)%,(36.79±1.66)%; the fluorine coating magnesium alloy (27.80±2.09)%,(35.33±1.49)%,(49.33±1.42)%; calcium phosphate coating magnesium alloys(33.04±2.69)%、(42.58±1.72)%、(59.29±1.62)%.Significant differences exist between the groups (P<0.05).
4.2Cell adhesion morphology
skeletal muscle cells adhesion well in three groups of materials, with skeletal muscle cells adherent expand, mostly fusiform, more protruding part of the cell protrusion interconnected.
4.3Cell viability
Cell viability cultured in extracts increased with prolonged incubation time and proliferative activity of the control group> CaP-AZ31B group>F-AZ31B group> AZ31B group, significant differences exist between the two groups (P<0.05)。
4.4Relative growth rate
The relative growth rate of AZ31B was less than80%at5and7days, with a toxicity rating of2, the relative growth of CaP-AZ31B and F-AZ31B>80%, toxicity rating of1, toxicity rating of0. Significant differences exist between the groups (P <0.05).
4.5Detection of apoptosis
Magnesium alloy extracts could cause skeletal muscle apoptosis, no significant apoptosis were found in the control group and CaP-AZ31B group and F-AZ31B group.
4.6Staining
a small amount of stained red cells could be seen in AZ31B extracts,but the vast majority of cells are still active (green dye), no obvious red dye could be seen in cell culture extracts of calcium and phosphorus coating magnesium alloy and magnesium fluoride coating alloy group.
4.7intracellular total protein
With the prolonged incubation time the total amount of protein in the cells in each group increased, the amount of protein of the four groups of1,3,5,7, d:blank control group,(75.33±2.72) ug/ml,(125.73±1.993) ug/ml,(163.27±4.197) ug/ml,(199.067±3.528) ug/ml, magnesium alloy group (60.77±2.274) ug/ml,(99.21±2.45) ug/ml,(127.35±3.214) ug/ml,(151.84±3.72) ug/ml, the F-AZ31B group (69.98±3.295)ug/ml,(116.55±3.981) ug/ml,(149.57±4.267) ug/ml,(181.89±7.79)ug/ml, CaP-the AZ31B group (70.52±3.862)ug/ml,(117.17±4.543) ug/ml,(151.68±5.429) ug/ml,(187.31±7.16) ug/ml.Significant differences exist between the groups (P<0.05).
5. The biological behavior of bone marrow mesenchymal stem cells
5.1Identification of mesenchymal stem cells in the bone marrow
Flow cytometry cell cytometry results suggest that CD34-, CD45-, CD44+, CD73+,CD90+,CD105+.
5.2Early cell adhesion rate of bone marrow mesenchymal stem cells
With the prolonged adhesion time, adhesion rate of skeletal muscle cell increased in all groups, but cell adhesion was poor in magnesium alloy surface.The cell adhesion rate of bone marrow mesenchymal stem cells in2h,6h and24h:titanium alloy group(27.08±1.95)%,(35.38±1.93)%,(49.04±2.10)%,magnesium alloy group (20.58±2.20)%,(24.63±2.03)%,(30.83±1.45)%,the fluorine coating magnesium alloy(27.58±2.29)%,(35.88±2.14)%,(50.33±2.70)%, calcium phosphate coating magnesium alloys(30.38±2.31)%、(42.58±1.72)%、(59.29±1.62)%.Significant differences exist between the groups (P<0.05).
5.3Cell viability
Cell viability cultured in extracts increased with prolonged incubation time, and proliferative capacity of magnesium alloys was relatively weak, CaP-AZ31B group and F-AZ31B group was relatively good,and the cell proliferation of adjust the PH value group magnesium alloy extracts was good too. Cell staining showed that no obvious red cells could be seen in all group.
6、Osteogenic differentiation of bone marrow mesenchymal stem cells
RNA extracted more complete, and high purity. RT-PCR results suggest that: CaP-AZ31B could promote the expression of COLI gene, ALP gene and OC gene.No significant difference in the RUNX2gene expression was found among the four groups.
ALP expression levels of magnesium alloy group was low, after adjust the PH value,the expression of ALP Promoted and no significant difference was found between blank control group (p>0.05).OPN gene expression levels of magnesium alloy was significantly higher than that of the other three groups after the induction of differentiation6days and12days(p<0.05), no significant difference was found between blank control group after adjust the PH value (P>0.05).which means that PH value could cause the ALP genes and OPN gene expression change.
Conclusions
11.The surface of AZ31B and F-AZ31B is smooth, but the surface of CaP-AZ31B is roughness with crystal-like structure.
2.PH value increased in the three groups extracts, but the fluorine coating magnesium alloy is stable, the PH value of magnesium alloy increased significantly.
3.Calcium phosphate coating magnesium alloys have better adsorption capacity for protein, there is no significant difference in protein adsorption among magnesium alloy and titanium alloy and magnesium alloy with fluorine coating.
4.Magnesium alloy have toxicity on osteoblast, but F-AZ31B and CaP-AZ31B have good biocompatibility for osteoblast.
5.Magnesium alloy and its calcium phosphate coating have good biocompatibility in vivo. Postoperative serum magnesium for each time point were within the normal range. Calcium phosphate coating effectively retard the degradation of magnesium alloy, and can promote bone formation.
6.Magnesium alloy have toxicity on skeletal muscle cells, but F-AZ31B and CaP-AZ31B have good biocompatibility for skeletal muscle cells.
7.Magnesium alloy have toxicity on bone marrow mesenchymal stem cells, but F-AZ31B and CaP-AZ31B have good biocompatibility for bone marrow mesenchymal stem cells.
8.Calcium phosphate coating of magnesium alloy can promote the expression of COLI, ALP and OC gene,the role of magnesium alloy for the ALP and OPN gene expression is related to the PH value.
镁是一种生物可降解金属材料,但纯镁降解速率过快,产生大量氢气,临床应用受到了限制。随着合金技术的发展以及表面改性方法的应用,可降解镁合金的耐蚀性能和生物相容性有了极大的改善。
尽管镁合金具有良好的生物相容性,但是其降解速度仍较快,为了进一步减缓合金的降解速度,学者们进行了一系列合金表面改性研究,形成了不同类型的合金涂层,不仅起到延缓降解的作用,而且还改善了其生物相容性。目前研究较多的有钙磷涂层、氟化物涂层等。
但是作为骨科植入材料,其对于骨组织再生有无影响,将决定其是否能够成为一种好的骨修复材料。对于骨再生过程中的关键细胞-成骨细胞和骨髓间充质干细胞的生物学行为及成骨分化过程有无影响尚不明确,对于骨骼运动的主要动力装置一骨骼肌细胞的生物学行为影响也不明确。
本研究拟针对上述问题,以空白或钛合金为对照,对镁合金、钙磷涂层镁合金、氟涂层镁合金进行进一步的研究,研究镁合金及其涂层对成骨生物学的初步影响,明确其材料的表面性质、明确其对成骨细胞生物学行为的影响,进而明确其植入体内后的血清镁及局部骨骼的变化,明确其对骨骼肌细胞的生物影响,明确其对骨髓间充质干细胞生物学行为及成骨分化的影响。
目的
1、明确镁合金及其涂层的表面形态及性质
2、明确镁合金及其涂层对成骨细胞的生物学行为的影响
3、明确镁合金及其涂层植入体内后的血清镁变化、材料降解及成骨情况
4、明确镁合金及其涂层对骨骼肌细胞的生物学行为的影响
5、明确镁合金及其涂层对骨髓间充质干细胞的生物学行为的影响
6、明确镁合金及其涂层对骨髓间充质干细胞成骨分化的影响
研究方法
1、采用扫描电镜研究镁合金AZ31B、钙磷涂层镁合金CaP-AZ31B、氟涂层镁合金F-AZ31B的表面形态,用能谱分析研究其元素组成；通过将镁合金及其涂层浸入细胞培养基中获得浸提液,测定浸提液的PH值变化情况；通过蛋白吸附实验研究镁合金及其涂层对蛋白的吸附能力。
2、通过采用镁合金、钙磷涂层镁合金及氟涂层镁合金的浸提液培养成骨细胞,进而研究其细胞2、6及24小时的细胞黏附率,采用扫描电镜研究其黏附形态,采用倒置显微镜观察1、3、5、7天细胞形态,采用CCK法检测成骨细胞1、3、5、7天增殖活力,采用Calcein-AM和EthD-1双染观察浸提液培养24小时后的细胞存活情况,通过1、3、5、7天的磷酸酶测定及1、3、5、7天的细胞内总蛋白测定来研究镁合金及其涂层对成骨细胞功能的影响。
3、通过将镁合金组、钙磷涂层镁合金组和聚乳酸对照组3组材料植入兔股骨,术后观察兔子的大体行为及术后24小时、1周、4周、6周和8周的血清镁浓度变化；术后8周处死实验动物,通过大体形态、CT扫描及三维重建观察植入材料的形态学变化情况；通过组织学观察成骨情况；通过扫描电镜观察镁合金及骨界面处的降解情况。
4、通过采用镁合金、钙磷涂层镁合金及氟涂层镁合金的浸提液培养骨骼肌细胞,进而研究其细胞2、6及24小时的细胞黏附率,采用扫描电镜研究其黏附形态,采用倒置显微镜观察1、3、5、7天细胞形态,采用CCK法检测骨骼肌细胞1、3、5、7天增殖活力,采用细胞凋亡试剂盒检测各组细胞有无凋亡,采用Calcein-AM和EthD-1双染观察浸提液培养24小时后的细胞存活情况,采用1、3、5、7天细胞内总蛋白测定来研究镁合金及其涂层对骨骼肌细胞功能的影响。
5、从志愿者身上抽取骨髓,进行骨髓间充质干细胞的分离培养,通过免疫组织化学方法鉴定骨髓间充质干细胞；通过采用镁合金、钙磷涂层镁合金及氟涂层镁合金的浸提液培养骨髓间充质干细胞胞,进而研究其细胞2、6及24小时的细胞黏附率,采用扫描电镜研究其黏附形态,采用倒置显微镜观察1、3、5、7天细胞形态,采用CCK法检测骨髓间充质干细胞1、3、5、7天增殖活力,采用Calcein-AM和EthD-1双染观察浸提液培养24小时后的细胞存活情况。
6、通过镁合金、钙磷涂层镁合金及氟涂层镁合金置入成骨诱导液中获得成骨诱导浸提液,进而采用不同的成骨诱导液进行骨髓间充质干细胞的成骨分化诱导,通过细胞收集、引物设计、细胞RNA的提取以及逆转录等过程,以P-actin基因为内参,于成骨分化诱导后第6天和第12天研究各种浸提液中ALP、 COL I、OC、OPN和RUNX2基因的表达情况。
7、根据不同的数据类型,分别采用采用One-Way ANOVA、析因设计方差分析、重复测量方差分析及独立样本t检验进行统计学分析,多重比较应用LSD法,方差不齐时采用Dunnetts T3比较。检验水准α=0.05。
结果
1、表面形态及性质
1.1表面形态：镁合金表面形态光滑,钙磷涂层镁合金表面形态粗糙,可见大量的钙磷物质沉积及晶体状结构,氟涂层镁合金表面表面均匀、光滑,在涂层表面可见分布有少数不规则小孔。
1.2能谱分析：结果表明镁合金表面主要有镁、铝、锌组成,其中镁元素原子百分比约为96%,铝约为3%,锌约为1%；钙磷涂层镁合金CaP-AZ31B表面主要有镁、钙、磷及氧组成,其中镁元素原子百分比约为0.33%,氧为63.39%,磷为17.6%5,钙为18.63%；氟涂层镁合金F-AZ31B表面主要是镁、氟、铝及锌,其中镁元素原子百分比约为56%,氟元素约为42.34%,铝约为1.27%,锌约为0.47%。
1.3浸提液的PH值变化情况：随着时间的延长,镁合金AZ31B、钙磷涂层镁合金CaP-AZ31B、氟涂层镁合金F-AZ31B溶液的PH值都发生了改变,其逐渐向碱性环境转化,但是镁合金AZ31B浸提液的PH值升高最为显著,远氟涂层镁合金F-AZ31B溶液的PH值改变不显著,钙磷涂层镁合金CaP-AZ31B溶液的PH值改变介于二者之间。
1.4蛋白吸附能力：镁合金的蛋白吸附能力较钛合金低,氟涂层的蛋白吸附能力较镁合金稍高,钙磷涂层镁合金的蛋白吸附能力较其他三组显著增高(P<0.05),四组的蛋白吸附能力分别为26.95±3.17、25.27±2.02、70.2±5.57、27.70±2.00ug/ml。
2、对成骨细胞的生物学行为的影响
2.1早期细胞黏附率：四组材料表面MC3T3-El细胞粘附率之间有显著差异(F=259.434,P=0.000),组间比较显示钙磷涂层镁合金组细胞粘附率显著高于其他三组(P<0.05),而镁合金组细胞粘附率显著低于其他三组(P<0.05),其他两组间无显著差异(P>0.05)。针对同组材料三个不同时间点MC3T3-El细胞粘附率比较均具有显著差异(P值均<0.05)。四组之间细胞黏附率CaP-AZ31B组>F-AZ31B组>钛合金组>AZ31B组。加入四组材料浸提液后不同时间点MC3T3-El细胞粘附率有显著差异(F=630.231,P=0.000),随着时间的延长,四组材料浸提液MC3T3-El细胞粘附率显著增加,不同时间点两两比较均有显著统计学差异(P<0.05)。且同一时间点四组材料浸提液MC3T3-El细胞粘附率比较差异均具有的统计学意义(P均<0.05)其中所有组合交互效应均显著(F=21.78,P=0.000)。结合基本统计量输出结果,最大均值(62.8%)的组合为培养24h时CaP-AZ31B组MC3T3-El细胞粘附率,最小均值(24.0%)为培养2h时AZ31B细胞粘附率。
2.2细胞黏附形态观察
成骨细胞在三组材料表面黏附较好,镁合金及其涂层表面成骨细胞贴壁展开,形态不规则,大多呈梭形,有较多突起,部分细胞间突起相互连接。
2.3细胞增殖活力
加入四组材料浸提液培养后不同时间点细胞增殖活力比较有显著差异(F=1491.2,P=0.000)；镁合金及其涂层合金浸提液培养的细胞活力均随着培养时间的延长而增加,任两个不同时间其细胞活力比较差异均具有显著的统计学意义。镁合金及其涂层合金浸提液培养的细胞活力四组总体比较具有显著差异(F=143.6,P=0.000),多重比较显示四组间任何两组比较其差异均具有显著统计学意义,且总体F-AZ31B组增殖活力最大,其次是钛合金组,CaP-AZ31B组次之,AZ31B组细胞增殖活力最小。其中所有组合交互效应均显著(F=9.15,P=0.000)。
2.4相对增殖率
镁合金组的相对增殖率在四个时间点均小于80%,说明镁合金有一定毒性,按照毒性评级为2级,但钙磷涂层镁合金组培养的MC3T3-El细胞相对增值率均>80%,毒性评级为1级,氟涂层镁合金组培养的MC3T3-El细胞相对增值率均>100%,毒性评级为0级,为临床骨科植入材料所接受。
采用析因设计的方差分析结果显示：镁合金及其涂层合金浸提液培养的细胞在不同时间上细胞增殖率无显著差异(F=0.725,P=0.541)；且各个时间点之间两两比较差异均无统计学意义(P>0.05)。镁合金及其涂层合金浸提液培养的细胞增殖率四组间总体比较具有显著差异(F=534.57,P=0.000)；3个试验组的细胞增殖率均显著低于对照组,四组间细胞增值率两两比较均有显著差异,且从均值上看,其细胞增值率对照组>CaP-AZ31B组>F-AZ31B组>AZ31B组。且培养时间及浸提液之间交互效应显著(F=2.01,P=0.048)。
2.5荧光染色
见镁合金浸提液组培养的成骨细胞可见少量细胞红染,但绝大多数细胞仍具有活性(绿染),钙磷涂层镁合金及氟涂层镁合金组浸提液培养的细胞无明显红染。
2.6碱性磷酸酶定量
随着培养时间的延长,镁合金及其涂层合金浸提液培养的细胞碱性磷酸酶表达均呈上升趋势,差异具有统计学意义(F=1330.7,P=0.000),且各个时间点之间两两比较均具有显著差异(P<0.05)。镁合金及其涂层合金浸提液培养的细胞碱性磷酸酶表达水平四组间总体比较具有显著差异(F=407.3,P=0.000);且多重比较结果显示任两组浸提液培养的细胞碱性磷酸酶差异均显著,且从均值上看,四组的碱性磷酸酶表达量依次为CaP-AZ31B组>F-AZ31B组>对照组>AZ31B组。培养时间及细胞碱性磷酸酶表达水平之间交互效应显著(F=25.8,P=0.000)。
2.7细胞内总蛋白
镁合金及其涂层合金浸提液培养的细胞胞内总蛋白量随着培养时间的延长而增加,差异具有显著统计学意义(F=851.6,P=0.000)；且各个时间点之间两两比较其细胞内总蛋白量均有显著差异(P<0.05)。镁合金及其涂层合金浸提液培养的细胞胞内总蛋白量四组间总体比较具有显著差异(F=4120.7,P=0.000);且多重比较结果显示任两组浸提液培养的细胞碱性磷酸酶差异均显著,且从均值上看,四组浸提液培养的细胞胞内总蛋白量依次为CaP-AZ31B组>F-AZ31B组>对照组>AZ31B组。培养时间及细胞胞内总蛋白量之间交互效应显著(F=30.9,P=0.000)。
3、骨植入实验
3.1术后兔子一般情况良好,术后各个时间点的血清镁均在正常范围内。
3.2植入八周后聚乳酸几乎完全降解,并且植入处与新生骨贴合紧密。镁合金及钙磷涂层镁合金基本都维持较好的完整形态,其中镁合金骨质外部分可见少许降解,边界不清晰、不规则,钙磷涂层镁合金材料边界清晰,未见到明显降解痕迹。
3.3钙磷涂层镁合金组金属与骨界面出可见较多新生骨形成,骨小梁排列紧凑而规则。无涂层镁合金形成的新生骨较少。同时可见无涂层样品的扫描电镜提示其边缘不规则,发生了降解,但钙磷涂层镁合金边缘形状同植入前变化很小。
4、对骨骼肌细胞的生物学行为的影响
4.1骨骼肌细胞黏附率测定
四组材料表面兔骨骼肌细胞细胞粘附率之间有显著差异(F=157.11,P=0.000),组间比较显示钙磷涂层镁合金组细胞粘附率显著高于其他三组,而镁合金组细胞粘附率显著低于其他三组,其他两组间无显著差异。四组之间细胞黏附率CaP-AZ31B组>F-AZ31B组>钛合金组>AZ31B组。加入四组材料浸提液后不同时间点兔骨骼肌细胞粘附率有显著差异(F=874.783,P=0.000),随着时间的延长,四组材料浸提液兔骨骼肌细胞粘附率显著增加,不同时间点两两比较均有显著统计学差异(P<0.05)。其中时间与浸提液交互效应均显著(F=10.61,P=0.000)。
4.2细胞黏附形态
兔骨骼肌细胞在镁合金、钙磷涂层镁合金及氟涂层镁合金三组材料表面均表现黏附较好。
4.3细胞增殖活力
加入四组材料浸提液培养后不同时间点细胞增殖活力比较有显著差异(F=2022.53,P=0.000)；镁合金及其涂层合金浸提液培养的细胞活力均随着培养时间的延长而增加,任两个不同时间其细胞活力比较差异均具有显著的统计学意义。镁合金及其涂层合金浸提液培养的细胞活力四组总体比较具有显著差异(F=50.68,P=0.000),多重比较显示四组间任两组比较其差异显著(P<0.05),且总体CaP-AZ31B组增殖活力最大,其次是F-AZ31B及钛合金组,AZ31B组细胞增殖活力最小。其中所有组合交互效应均显著(F=5.43,P=0.000)。
4.4相对增殖率
镁合金组的相对增殖率在第5及7d两个时间点小于80%,说明镁合金有一定毒性,毒性评级为2级,但钙磷涂层镁合金及氟涂层镁合金组培养的骨骼肌细胞相对增殖率均>90%,毒性评级为1级,为临床骨科植入材料所接受。
采用析因设计的方差分析结果显示：镁合金及其涂层合金浸提液培养的细胞在不同时间上细胞增殖率有统计学差异(F=3.56,P=0.018)；且各个时间点之间比较,除第1d和第5d,第1d和第7d细胞增值率有显著差异(P<0.05)外,其余各时间点间比较均无显著差异(P>0.05)。镁合金及其涂层合金浸提液培养的细胞增殖率四组间总体比较具有显著差异(F=217.94,P=0.000)；3个试验组的细胞增殖率均显著低于对照组,四组间细胞增值率两两比较均有显著差异,且从均值上看,其细胞增值率对照组>CaP-AZ31B组>F-AZ31B组>AZ31B组且培养时间及浸提液之间无交互作用(F=0.69,P=0.71)。
4.5细胞凋亡检测
镁合金浸提液可引起骨骼肌细胞凋亡,对照组、钙磷涂层镁合金组以及氟涂层镁合金组流式细胞图未见明显细胞凋亡。
4.6荧光染色
可镁合金浸提液组培养的骨骼肌细胞可见部分细胞红染,钙磷涂层镁合金及氟涂层镁合金浸提液培养的骨骼肌细胞活性较好,如对照组一样,未见明显细胞红染。
4.7细胞内总蛋白
镁合金及其涂层合金浸提液培养的细胞胞内总蛋白量随着培养时间的延长而增加,差异具有显著统计学意义(F=1796.8,P=0.000)；且各个时间点之间两两比较其细胞内总蛋白量均有显著差异(P<0.05)。镁合金及其涂层合金浸提液培养的细胞胞内总蛋白量四组间总体比较具有显著差异(F=136.45,P=0.000)；且多重比较结果显示：除CaP-AZ31B组与F-AZ31B组细胞内总蛋白量比较无显著差异(P>0.05)外,其他各组间细胞内总蛋白量差异均显著,且从均值上看,四组浸提液培养的细胞胞内总蛋白量依次为对照组>CaP-AZ31B组>F-AZ31B组>AZ31B组。培养时间及细胞胞内总蛋白量之间交互效应显著(F=7.15,P=0.000)。
5.对骨髓间充质干细胞的生物学行为的影响
5.1骨髓间充质干细胞鉴定
流式细胞仪细胞仪检测结果提示CD34-、CD45-、CD44+、CD73+、CD90+、 CD105+。
5.2骨髓间充质干细胞黏附
四组材料表面兔骨骼肌细胞细胞粘附率之间有显著差异(F=259.43,P=0.000),组间比较显示钙磷涂层镁合金组细胞粘附率显著高于其他三组,而镁合金组细胞粘附率显著低于其他三组,氟涂层与钛合金组比较两组间无显著差异。四组之间细胞黏附率CaP-AZ31B组>F-AZ31B组>钛合金组>AZ31B组。加入四组材料浸提液后不同时间点骨髓间充质干细胞粘附率有显著差异(F=630.23,P=0.000),随着时间的延长,四组材料浸提液骨髓间充质干细胞粘附率显著增加,不同时间点两两比较均有显著统计学差异(P<0.05)。其中时间与浸提液交互效应显著(F=21.78,P=0.000)。
5.3细胞增殖
镁合金及其涂层合金浸提液培养的细胞在不同时间上细胞增殖能力比较,结果显示：加入浸提液培养后不同时间点细胞增殖活力比较有显著差异(F=399.91,P=0.000);镁合金及其涂层合金浸提液培养的细胞增殖能力均随着培养时间的延长而增加,任两个不同时间其细胞增殖能力比较差异均具有显著的统计学意义。镁合金及其涂层合金浸提液培养的细胞增殖能力四组总体比较具有显著差异(F=37.14,P=0.000),多重比较显示镁合金组细胞增殖能力均显著低于其他四组,钙磷涂层及氟涂层镁合金的细胞增殖能力较无涂层显著改善,显著优于镁合金组,与钛合金组无显著差异,且总体钙磷涂层镁合金组增殖活力最大,其次是钛合金组,氟涂层镁合金组次之,镁合金组细胞增殖活力最小。对镁合金降解液存在的PH升高问题,调节PH值组镁合金浸提液的细胞增殖能力与钛合金组、和钙磷涂层及氟涂层镁合金无显著差异(P>0.05)。其中时间降解液交互效应均显著(F=2.32,P=0.011)。
五组荧光结果提示均可见到绿色染色的细胞,未见到明显的红色染色细胞。
6、对骨髓间充质干细胞成骨分化的影响
提取的RNA较完整,纯度较高。RT-PCR结果提示：钙磷涂层镁合金对于COLI基因、ALP基因以及OC基因表达有促进作用。RUNX2基因表达量四组间无显著差异。
镁合金组的ALP表达量较低,调节PH值后ALP表达量与空白对照组无显著差异(P>0.05),镁合金的OPN基因表达量在诱导分化后第6天和第12天均显著高于其余三组(P<0.05),调整PH值后的OPN基因表达量与对照组无显著差异(P>0.05),提示PH值是引起镁合金对于ALP基因和OPN基因的表达影响的可能因素之一。
结论
1、镁合金AZ31B及氟涂层镁合金F-AZ31B表面光滑,钙磷涂层镁合金CaP-AZ31B表面粗糙,可见晶体状结构。
2、三组镁合金浸提液的PH值均出现增高,但氟涂层镁合金性质最为稳定,单纯镁合金溶液的PH值升高显著。
3、钙磷涂层镁合金对于蛋白具有更好的吸附能力,镁合金及氟涂层镁合金及钛合金对于蛋白吸附能力无显著差异。
4、镁合金对于成骨细胞存在2级毒性反应,钙磷涂层镁合金及氟涂层镁合金对于成骨细胞有着良好生物相容性。
5、镁合金及其钙磷涂层体内生物相容性良好,血清镁的浓度均为正常范围内；钙磷涂层有效地延缓了镁合金的降解,并可以促进成骨作用。
6、镁合金对于骨骼肌细胞存在2级毒性反应,钙磷涂层镁合金及涂层镁合金对于骨骼肌细胞有着良好生物相容性。
7、镁合金对于骨髓间充质干细胞存在2级毒性反应,钙磷涂层镁合金及涂层镁合金对骨髓间充质干细胞有着良好生物相容性。
8、钙磷涂层镁合金对于COLI、ALP及OC基因表达有促进作用,无涂层镁合金抑制ALP的表达,且促进OPN基因的表达,但是调节PH值后重复研究发现其与对照组无显著差异,提示其影响可能与PH值有关。
Backgrounds
Magnesium is a biodegradable metallic material, but the degradation rate of pure magnesium is too rapid and produce large amounts of hydrogen, which make the clinical application has been limited. With the the alloy technology's development, and the application of the methods of surface modification, biodegradable magnesium alloy corrosion resistance and biocompatibility has been greatly improved.
Although magnesium alloys have good biocompatibility, but its degradation rate is still faster, to further slow down the degradation rate of the alloy, the scholars have carried out a series of alloy surface modification research, and have formed different types of alloy coating, which not only to delay the degradation, but also improve its biocompatibility. Such as calcium phosphate coating and fluoride coating.
But as orthopedic implant materials, that the impact for bone tissue regeneration will decide whether to become a good bone repair materials. The key cells in the bone regeneration process are Osteoblasts and bone marrow mesenchymal stem cells.The biological behavior and osteogenic differentiation on the cells is not yet clear, and the biological behavior of skeletal muscle cells for bone movement is not clear either.
In this study intends to conduct further studies to address the problem,with blank or titanium as control, study the osteogenesis biological effects of magnesium alloy, magnesium alloy with calcium phosphate coating and fluorine-coated magnesium alloy. And then clear the nature of the surface of the material,clear impact on the biological behavior of osteoblasts,clear biological effects on skeletal muscle cells, clear serum magnesium and localized bone changes after its implanted, clear the biological behavior and osteogenic differentiation of bone marrow mesenchymal stem cells.
Objectives
1. Study surface morphology and nature of magnesium alloy and its coating.
2. Study the biological behavior of osteoblasts with magnesium alloy and its coating.
3. Study the serum magnesium level, material degradation and osteogenic of magnesium alloy and its coating in rabbits.
4. Study the biological behavior of skeletal muscle cells with magnesium alloy and its coating.
5. Study the biological behavior of bone marrow mesenchymal stem cells with magnesium alloy and its coating.
6. Study the osteogenic differentiation of bone marrow mesenchymal stem cells with magnesium alloy and its coating.
Methods
1. Using scanning electron microscopy to study the surface morphology of magnesium alloy(AZ31N), calcium and phosphorus coating of magnesium alloy(CaP-AZ31B) and fluorine coating magnesium alloy(F-AZ31B), study its elements by energy spectrum. By the magnesium alloy and the coating was immersed in the cell culture medium obtained extracts,then determined the PH value of the extracts,study the protein adsorption capacity of magnesium alloy.
2. Cultured osteoblast in extracts, then study the2,6and24hours cell adhesion rate, using scanning electron microscopy to study the adhesion morphology, using the inverted microscope to study cell morphology of1,3,5,7days, CCK assay the osteoblasts proliferation activity of1,3,5,7days, using Calcein-AM, and ETHD-1double staining to observe cell survival,study phosphatase and total protein of cells in1,3,5,7days.
3. There are3groups in implant test:, magnesium alloy group(AZ31B), calcium phosphate coating of magnesium alloy group(CaP-AZ31B), polylactic acid group(PLLA),The pins were implanted into rabbit femur,Then the general behavior of rabbits were observed, serum magnesium concentration of24hours, one week, four weeks, six weeks and eight weeks after operation were measured.The experimental animals were sacrificed after8weeks,using CT scanning and three-dimensional reconstruction of the implant material to study morphological changes, observing histological bone formation,using SEM to observe degradation.
4. Cultured skeletal muscle cells in extracts, then study the2,6and24hours cell adhesion rate, using scanning electron microscopy to study the adhesion morphology, using the inverted microscope to study cell morphology of1,3,5,7days, CCK assay the osteoblasts proliferation activity of1,3,5,7days, using apoptosis detection kit to detect apoptosis, using Calcein-AM, and ETHD-1double staining to observe cell survival,study total protein of cells in1,3,5,7days.
5. Bone marrow were drawn from volunteers, followed by the isolation and culture of mesenchymal stem cells in the bone marrow, bone marrow mesenchymal stem cells were identified by immunohistochemistry; through the use of magnesium alloy, calcium and phosphorus coating of magnesium alloy and magnesium fluoride coating alloy extractionliquid cultured bone marrow mesenchymal stem cells, then study the2,6and24hours cell adhesion rate, using scanning electron microscopy to study the adhesion morphology, using the inverted microscope to study cell morphology of1,3,5,7days, CCK assay the osteoblasts proliferation activity of1,3,5,7days, using Calcein-AM, and ETHD-1double staining to observe cell survival.
6. Using different osteogenic induction medium induced osteogenic differentiation of bone marrow mesenchymal stem cells, collected by cell primer design cell RNA extraction and reverse transcription process,(3-actin gene as an internal,study the ALP, COL I,OC, OPN and RUNX2gene expression after cells were cultured in various extracts for6days and12days.
7. The data was staticstically analysis with one-way ANOVA analysis and Univariate, and LSD test was compared in pairwise comparison. Level of significance α=0.05.
Results
1. The surface morphology and the nature
1.1The surface morphology:Magnesium alloy surface morphology was rough, smooth, calcium phosphate coating magnesium alloy surface morphology seen a lot of calcium and phosphorus material deposition and crystal-like structure, fluorine coating the surface of the magnesium alloy surface is uniform, smooth, visible distribution of a small number of irregular holes in the surface of the coating.
1.2EDS analysis:The results showed that the surface of the magnesium alloy are mainly magnesium, aluminum, zinc composition, where in the magnesium atomic percentage is approximately96%, and aluminum of about3%, zinc is about1%; calcium phosphate coating magnesium alloy surface of the CaP-AZ31B mainly consists of magnesium, calcium, phosphorus, and oxygen, where in the percentage of magnesium atom of about0.33%, oxygen63.39%,17.6%phosphorus, calcium18.63%; magnesium fluoride coating the surface of the alloy F-AZ31B magnesium,fluorine, aluminum, zinc, magnesium atom percentage of about56%, about42.34%fluorine, aluminum is about1.27%, zinc is about0.47%.
1.3PH value changes of extracts:The PH of AZ31B and its coating extracts changed gradually to alkalienvironmental transformation, but the PH value of the magnesium alloy AZ31B extraction of liquid increased most significantly, no significant changes in the solution pH value of the F-AZ31B, CaP-AZ31B solution PH value change between AZ31B and F-AZ31B.
1.4Protein adsorption ability:magnesium alloy protein adsorption capacity is lower than the titanium alloy low, the protein adsorption capacity of F-AZ31B is slightly higher than magnesium alloy, calcium phosphate coating magnesium alloy protein adsorption capacity was significantly higher compared to the other three groups (P<0.05),Protein adsorption capacity of four groups were26.95±3.17,25.27±2.02,70.2±5.57,27.70±2.00ug/ml.
2、The biological behavior of osteoblasts
2.1Early cell adhesion rate
The cell adhesion rate of MC3T3-E1cells in2h,6h and24h:titanium alloy group (27.58±1.11)%,(34.92±2.26)%,(50.21±2.11)%; magnesium alloy group (24.04±2.33)%,(29.08±1.91)%,(37.33±1.49)%; the fluorine coating magnesium alloy (28.46±1.39)%,(35.96±1.18)%,(51.75±1.94)%; calcium phosphate coating magnesium alloys(34.25±1.47)%,(43.71±1.61)%,(62.75±1.97)%; CaP-AZ31B> F-AZ31B group> titanium alloy group> AZ31B group. Significant differences exist between the groups (P<0.05).
2.2Cell adhesion morphology
Osteoblast adhesion well in three groups of materials, with osteoblasts adherent expand, irregular shape, mostly fusiform, more protruding part of the cell protrusion interconnected.
2.3Cell viability
Cell viability cultured in extracts increased with prolonged incubation time and proliferative activity of the the F-AZ31B group> control group> CaP-AZ31B group> AZ31B group, significant differences exist between the two groups (P<0.05)。
2.4Relative growth rate
The relative growth rate of AZ31B was less than80%at four time points, with a toxicity rating of2, the relative growth of CaP-AZ31B>80%, toxicity rating of1, the fluorine coating magnesium alloy group>100%, toxicity rating of0. Significant differences exist between the groups (P<0.05).
2.5Staining
a small amount of stained red cells could be seen in AZ31B extracts,but the vast majority of cells are still active (green dye), no obvious red dye could be seen in cell culture extracts of calcium and phosphorus coating magnesium alloy and magnesium fluoride coating alloy group.
2.6Alkaline phosphatase quantitative
With prolonged incubation time, the group of alkaline phosphatase expression showed an upward trend, the four groups of alkaline phosphatase expression order the CaP-AZ31B group> F-AZ31B group> control group> AZ31B group, significant differences exist between the two groups (P<0.05)。
2.7intracellular total protein
With the prolonged incubation time the total amount of protein in the cells in each group increased, the amount of protein of the four groups of1,3,5,7, d,blank control group,(60.33±2.38)ug/ml,(98.34±2.19)ug/ml,(122.41±3.92)ug/ml, (148.52±3.62)ug/ml; magnesium alloy group (46.5±1.92) ug/ml,(75.73±2.22) ug/ml,(92.29±3.09) ug/ml,(111.31±3.67)ug/ml; the F-AZ31B group (64.66±1.35) ug/ml,(105.71±1.85) ug/ml,(130.97±2.36)ug/ml, and (160.47±2.41) ug/ml; CaP-the AZ31B group(66.24±1.7) ug/ml,(108.61±1.61) ug/ml,(134.01±3.87) ug/ml,(163.03±3.84) ug/ml. Significant differences exist between the groups (P<0.05).
3. Bone implantation experiments
3.1postoperative rabbits generally in good condition, postoperative serum magnesium for each time point were within the normal range.
3.2Polylactic acid almost completely degraded after implanted eight weeks, and fitting close with the new bone. AZ31B and CaP-AZ31B basically maintain the complete form, which shows little degradation of AZ31B bone outside part, the boundary is not clear, irregular, but clear boundary was seen in CaP-AZ31B and did not see the obvious degradation traces.
3.3New bone formation could be seen in CaP-AZ31B group and bone trabeculae compact and rules. The formation of new bone is less in AZ31B group. The sem showed that the edge of AZ31B is irregular, which means degradation has occurred, the edge shape of CaP-AZ31B with preimplantation change is very small.
4、The biological behavior of skeletal muscle cells
4.1Early cell adhesion rate of skeletal muscle cells:
With the prolonged adhesion time, adhesion rate of skeletal muscle cell increased in all groups. The cell adhesion rate of skeletal muscle cells in2h,6h and24h:titanium alloy group (26.46±2.33)%,(34.92±2.26)%,(47.21±2.11)%; magnesium alloy group (23.88±2.90)%,(28.75±2.46)%,(36.79±1.66)%; the fluorine coating magnesium alloy (27.80±2.09)%,(35.33±1.49)%,(49.33±1.42)%; calcium phosphate coating magnesium alloys(33.04±2.69)%、(42.58±1.72)%、(59.29±1.62)%.Significant differences exist between the groups (P<0.05).
4.2Cell adhesion morphology
skeletal muscle cells adhesion well in three groups of materials, with skeletal muscle cells adherent expand, mostly fusiform, more protruding part of the cell protrusion interconnected.
4.3Cell viability
Cell viability cultured in extracts increased with prolonged incubation time and proliferative activity of the control group> CaP-AZ31B group>F-AZ31B group> AZ31B group, significant differences exist between the two groups (P<0.05)。
4.4Relative growth rate
The relative growth rate of AZ31B was less than80%at5and7days, with a toxicity rating of2, the relative growth of CaP-AZ31B and F-AZ31B>80%, toxicity rating of1, toxicity rating of0. Significant differences exist between the groups (P <0.05).
4.5Detection of apoptosis
Magnesium alloy extracts could cause skeletal muscle apoptosis, no significant apoptosis were found in the control group and CaP-AZ31B group and F-AZ31B group.
4.6Staining
a small amount of stained red cells could be seen in AZ31B extracts,but the vast majority of cells are still active (green dye), no obvious red dye could be seen in cell culture extracts of calcium and phosphorus coating magnesium alloy and magnesium fluoride coating alloy group.
4.7intracellular total protein
With the prolonged incubation time the total amount of protein in the cells in each group increased, the amount of protein of the four groups of1,3,5,7, d:blank control group,(75.33±2.72) ug/ml,(125.73±1.993) ug/ml,(163.27±4.197) ug/ml,(199.067±3.528) ug/ml, magnesium alloy group (60.77±2.274) ug/ml,(99.21±2.45) ug/ml,(127.35±3.214) ug/ml,(151.84±3.72) ug/ml, the F-AZ31B group (69.98±3.295)ug/ml,(116.55±3.981) ug/ml,(149.57±4.267) ug/ml,(181.89±7.79)ug/ml, CaP-the AZ31B group (70.52±3.862)ug/ml,(117.17±4.543) ug/ml,(151.68±5.429) ug/ml,(187.31±7.16) ug/ml.Significant differences exist between the groups (P<0.05).
5. The biological behavior of bone marrow mesenchymal stem cells
5.1Identification of mesenchymal stem cells in the bone marrow
Flow cytometry cell cytometry results suggest that CD34-, CD45-, CD44+, CD73+,CD90+,CD105+.
5.2Early cell adhesion rate of bone marrow mesenchymal stem cells
With the prolonged adhesion time, adhesion rate of skeletal muscle cell increased in all groups, but cell adhesion was poor in magnesium alloy surface.The cell adhesion rate of bone marrow mesenchymal stem cells in2h,6h and24h:titanium alloy group(27.08±1.95)%,(35.38±1.93)%,(49.04±2.10)%,magnesium alloy group (20.58±2.20)%,(24.63±2.03)%,(30.83±1.45)%,the fluorine coating magnesium alloy(27.58±2.29)%,(35.88±2.14)%,(50.33±2.70)%, calcium phosphate coating magnesium alloys(30.38±2.31)%、(42.58±1.72)%、(59.29±1.62)%.Significant differences exist between the groups (P<0.05).
5.3Cell viability
Cell viability cultured in extracts increased with prolonged incubation time, and proliferative capacity of magnesium alloys was relatively weak, CaP-AZ31B group and F-AZ31B group was relatively good,and the cell proliferation of adjust the PH value group magnesium alloy extracts was good too. Cell staining showed that no obvious red cells could be seen in all group.
6、Osteogenic differentiation of bone marrow mesenchymal stem cells
RNA extracted more complete, and high purity. RT-PCR results suggest that: CaP-AZ31B could promote the expression of COLI gene, ALP gene and OC gene.No significant difference in the RUNX2gene expression was found among the four groups.
ALP expression levels of magnesium alloy group was low, after adjust the PH value,the expression of ALP Promoted and no significant difference was found between blank control group (p>0.05).OPN gene expression levels of magnesium alloy was significantly higher than that of the other three groups after the induction of differentiation6days and12days(p<0.05), no significant difference was found between blank control group after adjust the PH value (P>0.05).which means that PH value could cause the ALP genes and OPN gene expression change.
Conclusions
11.The surface of AZ31B and F-AZ31B is smooth, but the surface of CaP-AZ31B is roughness with crystal-like structure.
2.PH value increased in the three groups extracts, but the fluorine coating magnesium alloy is stable, the PH value of magnesium alloy increased significantly.
3.Calcium phosphate coating magnesium alloys have better adsorption capacity for protein, there is no significant difference in protein adsorption among magnesium alloy and titanium alloy and magnesium alloy with fluorine coating.
4.Magnesium alloy have toxicity on osteoblast, but F-AZ31B and CaP-AZ31B have good biocompatibility for osteoblast.
5.Magnesium alloy and its calcium phosphate coating have good biocompatibility in vivo. Postoperative serum magnesium for each time point were within the normal range. Calcium phosphate coating effectively retard the degradation of magnesium alloy, and can promote bone formation.
6.Magnesium alloy have toxicity on skeletal muscle cells, but F-AZ31B and CaP-AZ31B have good biocompatibility for skeletal muscle cells.
7.Magnesium alloy have toxicity on bone marrow mesenchymal stem cells, but F-AZ31B and CaP-AZ31B have good biocompatibility for bone marrow mesenchymal stem cells.
8.Calcium phosphate coating of magnesium alloy can promote the expression of COLI, ALP and OC gene,the role of magnesium alloy for the ALP and OPN gene expression is related to the PH value.
引文
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