镁合金（AZ60）Ca-P涂层的生物相容性初步研究
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摘要
目的
以镁为基质的生物材料近几年以来一直是生物材料研究领域极为活跃的内容。以不锈钢及钛合金为主的传统生物材料对现代骨科内固定植入材料的发展发挥了极大的作用。但传统骨科植入材料植入体内后产生应力遮挡阻碍骨折愈合；骨折愈合后需二次手术取出；材料取出后易发生再骨折等问题促使人们寻找可能的替代品。以镁为基质的生物材料由于具有良好的金属特性、力学性能及可降解性成为现在研究的热点，并极有可能成为新的骨科植入材料。
镁基质材料耐腐蚀性低，腐蚀过程中会有大量的H2产生，这些都限制了镁基质材料作为生物材料的应用。如何在提高以镁基质材料的耐腐蚀性的同时又能保证镁基质材料的生物力学特性和生物相容性，这是现阶段研究的热点。其主要研究内容有：（1）镁与其他一种或多种金属形成合金。常见的金属元素有锰、锌、铝、钙和稀土元素等。合成后的镁合金材料抗腐蚀性能较纯镁材料有了很大的提高，但作为生物可植入材料，其生物相容性表现并不理想。（2）另一种常用方法为在镁合金材料表面进行表面改性、增加表面涂层或表面保护膜。Ca和P都是骨基质的主要组成成分，研究结果显示Ca-P涂层具有良好的生物相容性，并有很好的骨传导性和诱骨活性。Ca-P涂层尤其是HA涂层在钛合金假体中已广泛应用于临床。为此，我们设计了一种简单有效地化学方法，对镁铝合金（AZ60）进行表面修饰，制得Ca-P涂层覆于镁合金材料表面，然后对制得的含有Ca-P涂层的镁合金材料进行初步的生物相容性实验研究：评价含有Ca-P涂层的镁合金在SBF溶液中的耐腐蚀性；评价含有Ca-P涂层的镁合金的细胞毒性；评价含有Ca-P涂层的镁合金植入动物体内的生物相容性。
方法
SBF溶液中的抗腐蚀实验
将有Ca-P涂层与无涂层的两种镁合金材料浸入配置好的SBF溶液中，温度维持在37±1°C，材料表面积与SBF溶液的体积比为1cm2/20mL。分别在反应后1d、3d、7d、14d与21d检测溶液中pH和Mg、Ca离子浓度的变化。体外细胞毒性实验
实验设为3组:A组，无Ca-P涂层材料组；B组，含有Ca-P涂层材料组；C组，阴性对照组
材料浸提液的制备
按照国际标准化组织的的标准（EN ISO10993-2009）进行。将A、B两组不同的材料放置于DMEM细胞培养液中，其材料面积与细胞培养液比例为1cm~2/ml。将其置于（37±1）°C的环境下24h后制得材料浸提液。
利用96孔板将MC3T3-E1成骨细胞与两组材料的DMEM浸提液培养，阴性对照组为DMEM细胞培养液。培养1d、3d、5d、7d后观察细胞生长状况，CCK-8法测定细胞增殖活性，用酶标仪在450nm处记录各孔吸光度值（OD），计算材料浸提液对细胞的毒性。
利用24孔板将MC3T3-E1成骨细胞与两组材料的DMEM浸提液培养，阴性对照组为DMEM培养液。培养5d后将事先放置于孔底的载玻片取出，酒精固定，HE染色，光镜下观察细胞在不同培养液里的生长情况，观察有无细胞形态和胞核、胞质的改变。
利用24孔板将MC3T3-E1成骨细胞与两组材料的DMEM浸提液培养，阴性对照组为DMEM培养液。培养五天后应用p-NPP法测定细胞碱性磷酸酶量，记录酶标仪在410nm处各孔吸光度值（OD），计算ALP活性，从而评价细胞分化增殖活性。
利用6孔板将MC3T3-E1成骨细胞与两组材料直接接触培养，阴性对照组为DMEM细胞培养液。培养5d后将在A、B两组材料取出，收集各孔中细胞，利用细胞凋亡检测试剂盒进行双染，应用流式细胞仪记录细胞凋亡情况。体内材料植入生物相容性实验
实验分为3组，A组，无涂层镁合金材料组；B组，Ca-P涂层镁合金材料组；C组，不锈钢材料组。
选取36只成年新西兰大白兔，体重在2.5-3.0Kg之间，雌雄不限。随机分为3组，每组12只。在其两侧股骨髁外侧植入直径3mm，长8mm的圆柱形内置物。术后观察动物的精神状态、活动、进食、创口愈合及皮下气肿等情况。分别于术后1mon、2mon和3mon空气栓塞处死12只兔子，每组4只，获得含有内置物的标本每组8个。将每组2个标本中的内置物取出，测重，剩余骨组织进行脱钙、固定后染色、观察内置物周围骨组织的细胞结构变化；3个含有内置物的标本行Micro-CT检查；3个含有内植物的标本行硬组织切片检查，立春红染色，观察内置物周围骨组织结构的变化。
结果
SBF溶液内材料耐腐蚀性实验
无Ca-P涂层的镁合金材料放入SBF溶液后降解速度很快，材料周围可见大量气泡产生，其溶液内Mg离子浓度从初始浓度1.5mmol/L，24小时后攀升到3.5mmol/L，48小时后Mg离子浓度上升到4.8mmol/L。相对于无涂层镁合金材料的较快的腐蚀速率，含有Ca-P涂层的镁合金材料置于SBF溶液中24小时后Mg离子浓度仅仅上升了0.2mol/L为1.7mol/L，最高浓度变化为72小时2.1mmol/L。Mg离子浓度的变化基本与溶液内pH值变化相对应。两组材料在SBF溶液中的抗腐蚀性能有显著差异（p<0.05）,说明Ca-P涂层的存在对增加镁合金材料的抗腐蚀性起了很大的作用。
阴性对照组与含有Ca-P涂层镁合金材料组OD值无明显差别，但与无涂层镁合金材料组间有明显差异。（p<0.05）说明含有Ca-P涂层镁合金材料对MC3T3-E1成骨细胞生长没有明显的细胞毒性。
光镜下观察到含有Ca-P涂层的镁合金材料组MC3T3-E1成骨细胞生长状态与形态结构与阴性对照组内细胞生长状态与形态结构无明显差异，细胞生长状态及分化增殖情况正常，未见明显的细胞形态结构变化。反观无涂层镁合金材料组MC3T3-E1成骨细胞生长情况，细胞生长抑制明显，分化和增值差，细胞形态与结构变化较大。
应用p-NPP法对三组培养细胞的ALP活性进行检测结果显示:含有Ca-P涂层镁合金材料组与阴性对照组内细胞ALP活性表达较高，分别达到了10.4和10.7nol/min/mg prote,而无涂层镁合金材料组细胞ALP活性仅为6.4nol/min/mg prote.Ca-P涂层镁合金材料与无涂层镁合金材料组之间差异明显。
利用AnnexinV-FITC/PI细胞双染法经流式细胞仪计数后可以明显的观察的A组即无涂层的镁合金材料组细胞数量较另外两组细胞数量减少明显。测得3组细胞凋亡率分别为A组（无涂层组），30.8%；B组（含有涂层组），17.82%；C组（阴性对照组），19.33%。其中含有Ca-P涂层镁合金材料组的细胞凋亡率与无涂层组细胞凋亡率差异明显，甚至其细胞凋亡率低于正常对照组，说明Ca-P涂层在一定程度上有利于成骨细胞的增殖与分化。体内生物相容性实验
术后所有大白兔麻醉清醒后活动良好，进食正常，术后创口愈合良，未见明显的皮下气肿。术后两周无涂层材料组一只大白兔出现了厌食，活动减少，消瘦等症状，术后1个月处死后发现其右侧手术肢体创口位置有一巨大的软组织肿物，肿物内为乳白色干酪样物质。
将标本内取出的内置物经室温干燥后称重，并与术前质量相比较。结果发现术后1个月与2个月内有涂层组与无涂层组的质量变化不明显。术后3个月时两组材料前后虽有质量变化，但两组间相比较，并没有明显的差异。
将植入后1个月和3个月的兔股骨髁部标本进行了Micro-CT检查，结果发现植入一个月后，不锈钢组与Ca-P涂层镁合金材料组中内置物材料与周围骨组织间接触紧密，未见明显的空隙，边缘较规则平滑。无涂层镁合金组内可见内置物与周围骨组织间有空隙，边缘亦见点状的腐蚀部位。术后3个月的标本Micro-CT扫描可见不锈钢材料组较1个月时无明显变化，材料周围有少量新骨形成但周围骨小梁结构排列紊乱；含有Ca-P涂层镁合金材料组内材料未见明显降解迹象，材料周围见有较多的新骨形成，并与材料结合紧密，骨小梁结构排列均匀有序；无涂层镁合金材料组可见材料降解明显，材料与周围骨组织间有较大空隙，内为无定形物质和少量的纤维组织，周围未见明显的新骨形成。
利用3D重建技术对内置物及其周围的骨组织进行重建，计算材料周围骨组织的BMD值，含有Ca-P涂层的镁合金材料周围与不锈钢材料周围的骨组织BMD值明显高于无涂层镁合金材料周围骨组织的BMD值，BMD值越大说明成骨能力越好，骨组织细胞代谢旺盛。通过系统软件计算内置物材料的腐蚀速度。A组材料术后1个月时腐蚀率为0.079mm/a,术后3个月时为0.197mm/a；B组材料术后1个月时腐蚀率为0.024mm/a术后3个月时为0.058mm/a；C组材料腐蚀率为0。A、B两组间腐蚀率比较有明显的差异性（P<0.05）。
含有内置物的标本切片后行利春红染色，观察材料表面的骨小梁结构生长和变化情况。光镜下观察B、C两组材料周围有清晰的新的骨小梁结构产生，骨小梁结构致密、粗大，B组材料周围骨小梁结构与材料表面结合紧密,骨小梁排列整齐有序。A组材料周围骨小梁结构稀疏，周围空隙较大，并含有大量的无定形物质，偶有骨组织与材料接触。
结论
含有Ca-P涂层镁合金材料可以明显的提高材料的抗腐蚀性。
含有Ca-P涂层镁合金材料无明显细胞毒性，且能促进成骨细胞的增殖分化。
体内植入后，含有Ca-P涂层镁合金材料具有很好的抗腐蚀性能，并表现出较好的生物相容性和骨诱导活性。
本研究为Mg合金Ca-P涂层作为骨科内置物应用提供了生物安全性实验依据，为进一步研究提供了理论支持。
含有Ca-P涂层的镁合金材料（AZ60）具有成为新一代骨科可降解植入材料的可能。
Object
Magnesium alloys have been recently proven as effectivebiodegradable orthopedic implants. However, the in vivo corrosion ofmagnesium alloys critically hinders the use of these materials asbiodegradable implant materials. During corrosion, the rapid degradationof magnesium alloys leads to subcutaneous bubbles from hydrogenevolution and an increase in the pH of body fluids and blood by localalkalization. Therefore, controlling the degradation rate and mechanicalintegrity of these alloys in the physiological environment is the key totheir applications.
Several strategies have been reported to overcome the low corrosionresistance and regulate the biocorrosion rate of magnesium alloys. Onestrategy is the addition of other elements such as Mn, Zn, and Al asalloying elements to develop different alloys in the past decades. Inrecent years, Ca and rare-earth elements have been used to producebinary magnesium alloys that show good corrosion resistance but limitedbone response. Another strategy for effectively reducing the corrosion ofmagnesium alloys is surface modification. Calcium phosphate (Ca-P)coating is recognized as one of the most biocompatible materials for bone replacement and regeneration and is widely used as a bioactivecoating in clinical orthopedic implants such as titanium alloys.
Recently, Ca-P coatings have been used to protect magnesium alloysfrom fast corrosion. For example, Ca-P coating on AZ30Mg andMg-Mn-Zn alloys reportedly improves corrosion resistance [10,11].
In the present study, Ca-P coating was prepared on an Mg-Al alloy(AZ60) by a two-step chemical process without pretreatment. Then, thecytotoxicity in vitro was evaluated by CCK-8test, ALP activity test, andapoptosis test. The samples were implanted into the femoral shaft ofrabbits. Analysis and evaluation of the degradation in vivo wereconducted based on corrosion measurements and micro-computedtomography (CT) technology.
3. Method
The test of corrosion resistance in SBF solution
Immerse the samples of magnesium alloys in SBF solution,the ratio ofsample surface area and SBF solution volume is1cm2/20mL,thetemperature is at37±1°C. Then observe the changes of pH value and Mgion concentration.
The cytotoxicity test in vitro
The experiment is divided into3groups:A group is uncoatedmagnesium alloy; B gruoup is Ca-P coated magnesium alloy; C group isnormal control group.
MC3T3-E1osteoblast cells are cultured in96pores plate withsample soaked solution,A group is the solution which the sample ofuncoated magnesium alloy soaked in DMEM; B group is the solutionwhich the sample of Ca-P coated magnesium alloy soaked in DMEM;Cgroup is the solution of DMEM. Observe cell growth after cultured in1d,3d,5d and7d. CCK-8test the OD value in450nm and calculate thecytotoxicity in sample soaked solution.
MC3T3-E1osteoblast cells are cultured in24pores plate with samplesoaked solution,A group is the solution which the sample of uncoatedmagnesium alloy soaked in DMEM; B group is the solution which thesample of Ca-P coated magnesium alloy soaked in DMEM;C group isthe solution of DMEM. After the cells cultured5d, the cell culture slideis removed out the plate.The cells are fixed with75%alcohol, stainedwith HE, and observed the cell’ growth with microscope.
MC3T3-E1osteoblast cells are cultured in24pores plate with samplesoaked solution,A group is the solution which the sample of uncoatedmagnesium alloy soaked in DMEM; B group is the solution which thesample of Ca-P coated magnesium alloy soaked in DMEM;C group isthe solution of DMEM. After the cells cultured5d,p-NPP test the ODvalue in410nm, observe the cells’ ALP activity and evaluate theirproliferation and differentiation.
MC3T3-E1osteoblast cells are cultured in6pores plate with samples directly.A group is the sample of uncoated magnesium alloy;B group isthe sample of Ca-P coated magnesium alloy;C group is only DMEM.After the cells cultured5d, the samples are removed from the plate.Collecting cells and double staining with Annexin V/PI, then observe thecell apoptosis with flow cytomytry.
The experiment of samples biocompatibility in vivo
The experiment is divided into3groups, A group is the group ofuncoated magnesium alloy; B group is the group of Ca-P coatedmagnesium alloy; C group is the group of stainless steel.
All animal experiments were conducted based on the animal welfarerequirements of ISO10993-2:2006. A total of36adult New Zealandrabbits with a body weight of2.5kg to3.0kg were used. The rabbits wererandomly divided into three groups. All rabbits were anesthetized with0.5pentobarbital sodium solution for surgery. After predrilling with a2.8mm hand operated drill, the samples were implanted into bothfemoral shafts of rabbits in the experimental groups. After surgery, therabbits’behavior, eating, activity, healing and body weight change wereobserved.
After1month、2month、3month，each time12rabbits were sacrificed.The sacrificed rabbit’s femoral shaft were removed. Then the specimenswere observed the sample degradation and the new bone around thesample by Micro-CT scanning. After that, the specimens were conducted with hard tissue section, stained with Ponceau S staining solution, andobserved the changes in bone tissue.
Result
The corrosion resistance in SBF solution
The corrosion degree of the coated and uncoated samples wasevaluated based on the changes in magnesium ion concentration and pHvalue in SBF. The result shows the change in magnesium ionconcentration. Uncoated magnesium alloy in SBF degraded faster, andthe magnesium ion concentration in SBF increased more quickly thancoated magnesium alloy (p<0.05).
The test result shows the variation of pH value in SBF after differentimmersion period. In the uncoated magnesium group, the pH valueincreased greatly and reached10.7after one day’s immersion.Subsequently, the pH value kept on increasing slowly. While in thecoated magnesium alloy group, the pH value reached the peak of about9.7after3days and then descended.
The tests of cytotoxicity
Cell proliferation is analyzed by viability observation of osteoblastMC3T3-E1cells using CCK-8assay for different culture periods. Asshown in Fig.3, the cell viability was no significant difference betweenthe coated magnesium alloy group and the negative control group. Butthe cell viability of uncoated magnesium alloy group was lower as compared with negative control group (p<0.05). The difference in thecell proliferation between different extract mediums was shown in Fig.4.The cells morphology in uncoated magnesium alloy group had obviousdifference with the negative control group and coated magnesium alloygroup, which showed significant reduction in both the number and thedensity of cells in uncoated magnesium alloy group and abnormal shapeand nucleus of the cells
The ALP activities of osteoblast MC3T3-E1cells in extract mediumwere measured by using aBCA Protein Assay Kit (Sigma, USA). Asexhibited in Fig5, compared with the negative control group, the ALPactivity of the cells in uncoated magnesium alloy group was observeddecreasing much evidently (p<0.05), while the ALP activity of the cellsin coated magnesium alloy group was the same as the control group.
The apoptosis and death of osteoblast MC3T3-E1cells were detectedusing flow cytometry with Annexin V-FITC/propidium iodide doublestaining of cells. Typical figures for the flow cytometric analysis areshowed in Fig.6. The cell apoptosis rate shows that after culturingdirectly with samples5days, the number of cells in uncoatedmagnesium alloy group reduced rapidly and the apoptosis rate reachedto30.8%. While in coated magnesium alloy group, the cells apoptosisrate was about17.82%, which was lower than the negative control19.33%. Biocompatibility tests in vivo
All rabbits awoke2h after surgery. All rabbits healed well, and nodifference was observed between the two groups. No obvioussubcutaneous emphysema was observed in the operated sites for allrabbits. Two weeks later, a rabbit in the uncoated Mg alloy groupappeared weakness and low activity. After the rabbit was sacrificed, ahuge mass was observed in the soft tissue of the executed site. Anoyster-white caseous substance was contained in the capsule (Fig.3).The rabbits’ weights were recorded in different periods. We observedthat the mean weight increased in the Ca-P-coated Mg alloy group,whereas the mean weight decreased in the uncoated Mg alloy group
The corrosion mass loss of the samples is shown in Fig.5. The meanmass loss after1month was0.12and0.21g for the Ca-P-coated anduncoated groups, respectively. No significant difference was observedbetween the two groups. One month after surgery, the mass lossgradually increased in the uncoated Mg alloy group; Three months aftersurgery, the mean mass loss in the uncoated Mg alloy group increased to1.42g. By contrast, the mean mass loss was0.23g in the Ca-P-coatedgroup. The difference in corrosion weight loss between the two groupswas not obvious.
The result illustrates the micro-CT reconstruction images of the rabbitfemora containing Mg implants1and3months after surgery. We observed that the surface morphologies of the samples in both groupsslightly degraded1month after surgery. Although the surface wassmooth in the uncoated magnesium alloy group, a few corrosion pitswere observed (red arrow in Fig.6). With increased implantation time,the uncoated samples exhibited more serious corrosion than theCa-P-coated samples after3months.
The3D micro-CT reconstruction images showed more obviousdegradation in the two groups of the samples than the2D reconstructionimages. Fig.8shows that the calculated corrosion rate of the uncoatedsamples was approximately three times as high as that of theCa-P-coated samples.
The spicemens were conducted hard tissue section, and stained inPonceau staining solution. Then the bone trabecula was observed. In Band C groups, the trabecula was arranged orderly、compactly and thick,while in A group, the trabecula was messy、few and scattered, Abundantamorphous mess was also observed in the gap between sample and bonetissue.
Conclution
The Ca-P coating prevent the degradation of magnesium alloy.There is not obviously cytotoxicity of Ca-P coated magnesium alloy invitro.
In vivo, the Ca-P coating has a good corrosion resistance and biocompatibility.
The experiments provide the theoretical and practical basis forbiocompatibility of Ca-P coating.
Ca-P coated magnesium alloy(AZ60) is possible for a biodegradatedmaterial in orthopedics.
以镁为基质的生物材料近几年以来一直是生物材料研究领域极为活跃的内容。以不锈钢及钛合金为主的传统生物材料对现代骨科内固定植入材料的发展发挥了极大的作用。但传统骨科植入材料植入体内后产生应力遮挡阻碍骨折愈合；骨折愈合后需二次手术取出；材料取出后易发生再骨折等问题促使人们寻找可能的替代品。以镁为基质的生物材料由于具有良好的金属特性、力学性能及可降解性成为现在研究的热点，并极有可能成为新的骨科植入材料。
镁基质材料耐腐蚀性低，腐蚀过程中会有大量的H2产生，这些都限制了镁基质材料作为生物材料的应用。如何在提高以镁基质材料的耐腐蚀性的同时又能保证镁基质材料的生物力学特性和生物相容性，这是现阶段研究的热点。其主要研究内容有：（1）镁与其他一种或多种金属形成合金。常见的金属元素有锰、锌、铝、钙和稀土元素等。合成后的镁合金材料抗腐蚀性能较纯镁材料有了很大的提高，但作为生物可植入材料，其生物相容性表现并不理想。（2）另一种常用方法为在镁合金材料表面进行表面改性、增加表面涂层或表面保护膜。Ca和P都是骨基质的主要组成成分，研究结果显示Ca-P涂层具有良好的生物相容性，并有很好的骨传导性和诱骨活性。Ca-P涂层尤其是HA涂层在钛合金假体中已广泛应用于临床。为此，我们设计了一种简单有效地化学方法，对镁铝合金（AZ60）进行表面修饰，制得Ca-P涂层覆于镁合金材料表面，然后对制得的含有Ca-P涂层的镁合金材料进行初步的生物相容性实验研究：评价含有Ca-P涂层的镁合金在SBF溶液中的耐腐蚀性；评价含有Ca-P涂层的镁合金的细胞毒性；评价含有Ca-P涂层的镁合金植入动物体内的生物相容性。
方法
SBF溶液中的抗腐蚀实验
将有Ca-P涂层与无涂层的两种镁合金材料浸入配置好的SBF溶液中，温度维持在37±1°C，材料表面积与SBF溶液的体积比为1cm2/20mL。分别在反应后1d、3d、7d、14d与21d检测溶液中pH和Mg、Ca离子浓度的变化。体外细胞毒性实验
实验设为3组:A组，无Ca-P涂层材料组；B组，含有Ca-P涂层材料组；C组，阴性对照组
材料浸提液的制备
按照国际标准化组织的的标准（EN ISO10993-2009）进行。将A、B两组不同的材料放置于DMEM细胞培养液中，其材料面积与细胞培养液比例为1cm~2/ml。将其置于（37±1）°C的环境下24h后制得材料浸提液。
利用96孔板将MC3T3-E1成骨细胞与两组材料的DMEM浸提液培养，阴性对照组为DMEM细胞培养液。培养1d、3d、5d、7d后观察细胞生长状况，CCK-8法测定细胞增殖活性，用酶标仪在450nm处记录各孔吸光度值（OD），计算材料浸提液对细胞的毒性。
利用24孔板将MC3T3-E1成骨细胞与两组材料的DMEM浸提液培养，阴性对照组为DMEM培养液。培养5d后将事先放置于孔底的载玻片取出，酒精固定，HE染色，光镜下观察细胞在不同培养液里的生长情况，观察有无细胞形态和胞核、胞质的改变。
利用24孔板将MC3T3-E1成骨细胞与两组材料的DMEM浸提液培养，阴性对照组为DMEM培养液。培养五天后应用p-NPP法测定细胞碱性磷酸酶量，记录酶标仪在410nm处各孔吸光度值（OD），计算ALP活性，从而评价细胞分化增殖活性。
利用6孔板将MC3T3-E1成骨细胞与两组材料直接接触培养，阴性对照组为DMEM细胞培养液。培养5d后将在A、B两组材料取出，收集各孔中细胞，利用细胞凋亡检测试剂盒进行双染，应用流式细胞仪记录细胞凋亡情况。体内材料植入生物相容性实验
实验分为3组，A组，无涂层镁合金材料组；B组，Ca-P涂层镁合金材料组；C组，不锈钢材料组。
选取36只成年新西兰大白兔，体重在2.5-3.0Kg之间，雌雄不限。随机分为3组，每组12只。在其两侧股骨髁外侧植入直径3mm，长8mm的圆柱形内置物。术后观察动物的精神状态、活动、进食、创口愈合及皮下气肿等情况。分别于术后1mon、2mon和3mon空气栓塞处死12只兔子，每组4只，获得含有内置物的标本每组8个。将每组2个标本中的内置物取出，测重，剩余骨组织进行脱钙、固定后染色、观察内置物周围骨组织的细胞结构变化；3个含有内置物的标本行Micro-CT检查；3个含有内植物的标本行硬组织切片检查，立春红染色，观察内置物周围骨组织结构的变化。
结果
SBF溶液内材料耐腐蚀性实验
无Ca-P涂层的镁合金材料放入SBF溶液后降解速度很快，材料周围可见大量气泡产生，其溶液内Mg离子浓度从初始浓度1.5mmol/L，24小时后攀升到3.5mmol/L，48小时后Mg离子浓度上升到4.8mmol/L。相对于无涂层镁合金材料的较快的腐蚀速率，含有Ca-P涂层的镁合金材料置于SBF溶液中24小时后Mg离子浓度仅仅上升了0.2mol/L为1.7mol/L，最高浓度变化为72小时2.1mmol/L。Mg离子浓度的变化基本与溶液内pH值变化相对应。两组材料在SBF溶液中的抗腐蚀性能有显著差异（p<0.05）,说明Ca-P涂层的存在对增加镁合金材料的抗腐蚀性起了很大的作用。
阴性对照组与含有Ca-P涂层镁合金材料组OD值无明显差别，但与无涂层镁合金材料组间有明显差异。（p<0.05）说明含有Ca-P涂层镁合金材料对MC3T3-E1成骨细胞生长没有明显的细胞毒性。
光镜下观察到含有Ca-P涂层的镁合金材料组MC3T3-E1成骨细胞生长状态与形态结构与阴性对照组内细胞生长状态与形态结构无明显差异，细胞生长状态及分化增殖情况正常，未见明显的细胞形态结构变化。反观无涂层镁合金材料组MC3T3-E1成骨细胞生长情况，细胞生长抑制明显，分化和增值差，细胞形态与结构变化较大。
应用p-NPP法对三组培养细胞的ALP活性进行检测结果显示:含有Ca-P涂层镁合金材料组与阴性对照组内细胞ALP活性表达较高，分别达到了10.4和10.7nol/min/mg prote,而无涂层镁合金材料组细胞ALP活性仅为6.4nol/min/mg prote.Ca-P涂层镁合金材料与无涂层镁合金材料组之间差异明显。
利用AnnexinV-FITC/PI细胞双染法经流式细胞仪计数后可以明显的观察的A组即无涂层的镁合金材料组细胞数量较另外两组细胞数量减少明显。测得3组细胞凋亡率分别为A组（无涂层组），30.8%；B组（含有涂层组），17.82%；C组（阴性对照组），19.33%。其中含有Ca-P涂层镁合金材料组的细胞凋亡率与无涂层组细胞凋亡率差异明显，甚至其细胞凋亡率低于正常对照组，说明Ca-P涂层在一定程度上有利于成骨细胞的增殖与分化。体内生物相容性实验
术后所有大白兔麻醉清醒后活动良好，进食正常，术后创口愈合良，未见明显的皮下气肿。术后两周无涂层材料组一只大白兔出现了厌食，活动减少，消瘦等症状，术后1个月处死后发现其右侧手术肢体创口位置有一巨大的软组织肿物，肿物内为乳白色干酪样物质。
将标本内取出的内置物经室温干燥后称重，并与术前质量相比较。结果发现术后1个月与2个月内有涂层组与无涂层组的质量变化不明显。术后3个月时两组材料前后虽有质量变化，但两组间相比较，并没有明显的差异。
将植入后1个月和3个月的兔股骨髁部标本进行了Micro-CT检查，结果发现植入一个月后，不锈钢组与Ca-P涂层镁合金材料组中内置物材料与周围骨组织间接触紧密，未见明显的空隙，边缘较规则平滑。无涂层镁合金组内可见内置物与周围骨组织间有空隙，边缘亦见点状的腐蚀部位。术后3个月的标本Micro-CT扫描可见不锈钢材料组较1个月时无明显变化，材料周围有少量新骨形成但周围骨小梁结构排列紊乱；含有Ca-P涂层镁合金材料组内材料未见明显降解迹象，材料周围见有较多的新骨形成，并与材料结合紧密，骨小梁结构排列均匀有序；无涂层镁合金材料组可见材料降解明显，材料与周围骨组织间有较大空隙，内为无定形物质和少量的纤维组织，周围未见明显的新骨形成。
利用3D重建技术对内置物及其周围的骨组织进行重建，计算材料周围骨组织的BMD值，含有Ca-P涂层的镁合金材料周围与不锈钢材料周围的骨组织BMD值明显高于无涂层镁合金材料周围骨组织的BMD值，BMD值越大说明成骨能力越好，骨组织细胞代谢旺盛。通过系统软件计算内置物材料的腐蚀速度。A组材料术后1个月时腐蚀率为0.079mm/a,术后3个月时为0.197mm/a；B组材料术后1个月时腐蚀率为0.024mm/a术后3个月时为0.058mm/a；C组材料腐蚀率为0。A、B两组间腐蚀率比较有明显的差异性（P<0.05）。
含有内置物的标本切片后行利春红染色，观察材料表面的骨小梁结构生长和变化情况。光镜下观察B、C两组材料周围有清晰的新的骨小梁结构产生，骨小梁结构致密、粗大，B组材料周围骨小梁结构与材料表面结合紧密,骨小梁排列整齐有序。A组材料周围骨小梁结构稀疏，周围空隙较大，并含有大量的无定形物质，偶有骨组织与材料接触。
结论
含有Ca-P涂层镁合金材料可以明显的提高材料的抗腐蚀性。
含有Ca-P涂层镁合金材料无明显细胞毒性，且能促进成骨细胞的增殖分化。
体内植入后，含有Ca-P涂层镁合金材料具有很好的抗腐蚀性能，并表现出较好的生物相容性和骨诱导活性。
本研究为Mg合金Ca-P涂层作为骨科内置物应用提供了生物安全性实验依据，为进一步研究提供了理论支持。
含有Ca-P涂层的镁合金材料（AZ60）具有成为新一代骨科可降解植入材料的可能。
Object
Magnesium alloys have been recently proven as effectivebiodegradable orthopedic implants. However, the in vivo corrosion ofmagnesium alloys critically hinders the use of these materials asbiodegradable implant materials. During corrosion, the rapid degradationof magnesium alloys leads to subcutaneous bubbles from hydrogenevolution and an increase in the pH of body fluids and blood by localalkalization. Therefore, controlling the degradation rate and mechanicalintegrity of these alloys in the physiological environment is the key totheir applications.
Several strategies have been reported to overcome the low corrosionresistance and regulate the biocorrosion rate of magnesium alloys. Onestrategy is the addition of other elements such as Mn, Zn, and Al asalloying elements to develop different alloys in the past decades. Inrecent years, Ca and rare-earth elements have been used to producebinary magnesium alloys that show good corrosion resistance but limitedbone response. Another strategy for effectively reducing the corrosion ofmagnesium alloys is surface modification. Calcium phosphate (Ca-P)coating is recognized as one of the most biocompatible materials for bone replacement and regeneration and is widely used as a bioactivecoating in clinical orthopedic implants such as titanium alloys.
Recently, Ca-P coatings have been used to protect magnesium alloysfrom fast corrosion. For example, Ca-P coating on AZ30Mg andMg-Mn-Zn alloys reportedly improves corrosion resistance [10,11].
In the present study, Ca-P coating was prepared on an Mg-Al alloy(AZ60) by a two-step chemical process without pretreatment. Then, thecytotoxicity in vitro was evaluated by CCK-8test, ALP activity test, andapoptosis test. The samples were implanted into the femoral shaft ofrabbits. Analysis and evaluation of the degradation in vivo wereconducted based on corrosion measurements and micro-computedtomography (CT) technology.
3. Method
The test of corrosion resistance in SBF solution
Immerse the samples of magnesium alloys in SBF solution,the ratio ofsample surface area and SBF solution volume is1cm2/20mL,thetemperature is at37±1°C. Then observe the changes of pH value and Mgion concentration.
The cytotoxicity test in vitro
The experiment is divided into3groups:A group is uncoatedmagnesium alloy; B gruoup is Ca-P coated magnesium alloy; C group isnormal control group.
MC3T3-E1osteoblast cells are cultured in96pores plate withsample soaked solution,A group is the solution which the sample ofuncoated magnesium alloy soaked in DMEM; B group is the solutionwhich the sample of Ca-P coated magnesium alloy soaked in DMEM;Cgroup is the solution of DMEM. Observe cell growth after cultured in1d,3d,5d and7d. CCK-8test the OD value in450nm and calculate thecytotoxicity in sample soaked solution.
MC3T3-E1osteoblast cells are cultured in24pores plate with samplesoaked solution,A group is the solution which the sample of uncoatedmagnesium alloy soaked in DMEM; B group is the solution which thesample of Ca-P coated magnesium alloy soaked in DMEM;C group isthe solution of DMEM. After the cells cultured5d, the cell culture slideis removed out the plate.The cells are fixed with75%alcohol, stainedwith HE, and observed the cell’ growth with microscope.
MC3T3-E1osteoblast cells are cultured in24pores plate with samplesoaked solution,A group is the solution which the sample of uncoatedmagnesium alloy soaked in DMEM; B group is the solution which thesample of Ca-P coated magnesium alloy soaked in DMEM;C group isthe solution of DMEM. After the cells cultured5d,p-NPP test the ODvalue in410nm, observe the cells’ ALP activity and evaluate theirproliferation and differentiation.
MC3T3-E1osteoblast cells are cultured in6pores plate with samples directly.A group is the sample of uncoated magnesium alloy;B group isthe sample of Ca-P coated magnesium alloy;C group is only DMEM.After the cells cultured5d, the samples are removed from the plate.Collecting cells and double staining with Annexin V/PI, then observe thecell apoptosis with flow cytomytry.
The experiment of samples biocompatibility in vivo
The experiment is divided into3groups, A group is the group ofuncoated magnesium alloy; B group is the group of Ca-P coatedmagnesium alloy; C group is the group of stainless steel.
All animal experiments were conducted based on the animal welfarerequirements of ISO10993-2:2006. A total of36adult New Zealandrabbits with a body weight of2.5kg to3.0kg were used. The rabbits wererandomly divided into three groups. All rabbits were anesthetized with0.5pentobarbital sodium solution for surgery. After predrilling with a2.8mm hand operated drill, the samples were implanted into bothfemoral shafts of rabbits in the experimental groups. After surgery, therabbits’behavior, eating, activity, healing and body weight change wereobserved.
After1month、2month、3month，each time12rabbits were sacrificed.The sacrificed rabbit’s femoral shaft were removed. Then the specimenswere observed the sample degradation and the new bone around thesample by Micro-CT scanning. After that, the specimens were conducted with hard tissue section, stained with Ponceau S staining solution, andobserved the changes in bone tissue.
Result
The corrosion resistance in SBF solution
The corrosion degree of the coated and uncoated samples wasevaluated based on the changes in magnesium ion concentration and pHvalue in SBF. The result shows the change in magnesium ionconcentration. Uncoated magnesium alloy in SBF degraded faster, andthe magnesium ion concentration in SBF increased more quickly thancoated magnesium alloy (p<0.05).
The test result shows the variation of pH value in SBF after differentimmersion period. In the uncoated magnesium group, the pH valueincreased greatly and reached10.7after one day’s immersion.Subsequently, the pH value kept on increasing slowly. While in thecoated magnesium alloy group, the pH value reached the peak of about9.7after3days and then descended.
The tests of cytotoxicity
Cell proliferation is analyzed by viability observation of osteoblastMC3T3-E1cells using CCK-8assay for different culture periods. Asshown in Fig.3, the cell viability was no significant difference betweenthe coated magnesium alloy group and the negative control group. Butthe cell viability of uncoated magnesium alloy group was lower as compared with negative control group (p<0.05). The difference in thecell proliferation between different extract mediums was shown in Fig.4.The cells morphology in uncoated magnesium alloy group had obviousdifference with the negative control group and coated magnesium alloygroup, which showed significant reduction in both the number and thedensity of cells in uncoated magnesium alloy group and abnormal shapeand nucleus of the cells
The ALP activities of osteoblast MC3T3-E1cells in extract mediumwere measured by using aBCA Protein Assay Kit (Sigma, USA). Asexhibited in Fig5, compared with the negative control group, the ALPactivity of the cells in uncoated magnesium alloy group was observeddecreasing much evidently (p<0.05), while the ALP activity of the cellsin coated magnesium alloy group was the same as the control group.
The apoptosis and death of osteoblast MC3T3-E1cells were detectedusing flow cytometry with Annexin V-FITC/propidium iodide doublestaining of cells. Typical figures for the flow cytometric analysis areshowed in Fig.6. The cell apoptosis rate shows that after culturingdirectly with samples5days, the number of cells in uncoatedmagnesium alloy group reduced rapidly and the apoptosis rate reachedto30.8%. While in coated magnesium alloy group, the cells apoptosisrate was about17.82%, which was lower than the negative control19.33%. Biocompatibility tests in vivo
All rabbits awoke2h after surgery. All rabbits healed well, and nodifference was observed between the two groups. No obvioussubcutaneous emphysema was observed in the operated sites for allrabbits. Two weeks later, a rabbit in the uncoated Mg alloy groupappeared weakness and low activity. After the rabbit was sacrificed, ahuge mass was observed in the soft tissue of the executed site. Anoyster-white caseous substance was contained in the capsule (Fig.3).The rabbits’ weights were recorded in different periods. We observedthat the mean weight increased in the Ca-P-coated Mg alloy group,whereas the mean weight decreased in the uncoated Mg alloy group
The corrosion mass loss of the samples is shown in Fig.5. The meanmass loss after1month was0.12and0.21g for the Ca-P-coated anduncoated groups, respectively. No significant difference was observedbetween the two groups. One month after surgery, the mass lossgradually increased in the uncoated Mg alloy group; Three months aftersurgery, the mean mass loss in the uncoated Mg alloy group increased to1.42g. By contrast, the mean mass loss was0.23g in the Ca-P-coatedgroup. The difference in corrosion weight loss between the two groupswas not obvious.
The result illustrates the micro-CT reconstruction images of the rabbitfemora containing Mg implants1and3months after surgery. We observed that the surface morphologies of the samples in both groupsslightly degraded1month after surgery. Although the surface wassmooth in the uncoated magnesium alloy group, a few corrosion pitswere observed (red arrow in Fig.6). With increased implantation time,the uncoated samples exhibited more serious corrosion than theCa-P-coated samples after3months.
The3D micro-CT reconstruction images showed more obviousdegradation in the two groups of the samples than the2D reconstructionimages. Fig.8shows that the calculated corrosion rate of the uncoatedsamples was approximately three times as high as that of theCa-P-coated samples.
The spicemens were conducted hard tissue section, and stained inPonceau staining solution. Then the bone trabecula was observed. In Band C groups, the trabecula was arranged orderly、compactly and thick,while in A group, the trabecula was messy、few and scattered, Abundantamorphous mess was also observed in the gap between sample and bonetissue.
Conclution
The Ca-P coating prevent the degradation of magnesium alloy.There is not obviously cytotoxicity of Ca-P coated magnesium alloy invitro.
In vivo, the Ca-P coating has a good corrosion resistance and biocompatibility.
The experiments provide the theoretical and practical basis forbiocompatibility of Ca-P coating.
Ca-P coated magnesium alloy(AZ60) is possible for a biodegradatedmaterial in orthopedics.
引文
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