CN107137771B - 一种纳米磷酸钙骨水泥及其制备方法 - Google Patents
一种纳米磷酸钙骨水泥及其制备方法 Download PDFInfo
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Abstract
一种纳米磷酸钙骨水泥及其制备方法,由水溶性纳米材料添加到磷酸钙骨水泥中,调拌均匀后自固化制得。本发明选择生物相容性良好的金属纳米材料与骨水泥基质粉体混合制备纳米骨水泥,不仅使材料具有良好的机械性能,而且具有增强的成骨活性。还可以通过调节加入纳米材料的种类和数量对纳米骨水泥的性能进行调节,如通过添加磁性纳米材料来赋予骨水泥以磁性,通过在一定范围内增加纳米材料添加量来增强骨水泥的强度。本发明的纳米骨水泥材料制备方法工业简单,可操作性强,且更能满足临床使用需要,具备推广价值。
Description
技术领域
本发明属于生物医用无机材料技术领域,具体涉及一种纳米磷酸钙骨水泥及其制备方法。
背景技术
口腔颌面部先天畸形、创伤、肿瘤、炎症等所致的颌骨缺损不仅造成颜面畸形,还可引起语言、咀嚼、呼吸等口腔功能障碍,给患者生理、心理等方面带来许多不良影响。因此,颌骨缺损一直是临床治疗的重点和难点。骨移植是最常用的治疗方法,植骨材料包括自体骨、异体骨、异种骨和人工骨等。其中自体骨被认为是治疗骨缺损的“金标准”。但自体骨和异体骨来源有限,异种骨又存在抗原性强等问题,使其临床应用受到限制。骨组织工程技术以人工骨修复骨缺损,具有损伤小、无抗原性或抗原性甚微,可以准确重建缺损骨的形态等优点。
磷酸钙骨水泥是20世纪90年代初研制成功的一种具有生物活性的自固型、非陶瓷羟基磷灰石类人工骨材料。其生物相容性好,在固化结晶过程中不产热,可任意塑形和逐渐降解,并可诱导骨组织长入,符合临床骨缺损修复的需要。磷酸钙骨水泥也被用作生长因子的缓释载体和构建组织工程骨。但其存在缺点,如凝固速度慢,强度低、力学性能差,脆性大且承受载荷的能力差,成骨速度慢,大大限制了应用。
以纳米材料对磷酸钙骨水泥进行改良是一条可行之路。目前开发的纳米磷酸钙骨水泥主要集中在无机非金属类纳米材料,如纳米二氧化硅、纳米壳聚糖、纳米晶体纤维素等。这些无机非金属类纳米材料的添加使磷酸钙骨水泥中的孔隙和裂纹减少,颗粒尺寸减少,同时纳米材料可以发挥补强增韧的作用,从而提高了磷酸钙骨水泥的强度。但是对其成骨性能方面,还需要通过添加药物或者生长因子的方式进行进一步改良。基于此,开发可以同时改良物理机械性能又可增强成骨活性的纳米磷酸钙骨水泥亟待解决。
金属纳米材料在生物医学领域有着广泛的应用,其具有增强添加物和活性药物的双重功能。比如具有超顺磁性的γ三氧化二铁,作为磁共振造影剂已通过FDA的认证。近年来,其也开始被添加入骨组织工程的支架材料中以提高支架的机械强度和生物活性。添加的铁元素随支架的降解而在局部逐步释放,可提高干细胞线粒体活性和成骨相关基因的表达。γ三氧化二铁赋予支架超顺磁性,可作为磁性标记的生长因子或干细胞的靶目标而实现定向作用。磁性支架在外加磁场的辅助下可固定于植入的骨缺损区,减少移位。纳米金具有良好的纳米表面效应、量子效应以及宏观量子隧道效应,它具有很多良好的化学特性,比如抗氧性和生物相容性,因此在生物医学领域极具应用潜力。
发明内容
解决的技术问题:本发明的目的是提供一种具有良好机械性能和成骨活性的磷酸钙骨水泥。 同时还提供该纳米骨水泥的制备方法。
技术方案:一种纳米磷酸钙骨水泥,是由水溶性纳米材料添加到磷酸钙骨水泥中,调拌均匀后自固化制得。
上述磷酸钙骨水泥为磷酸四钙[Ca4(PO4)2O]和无水磷酸氢钙[CaHPO4]体系;所述磷酸四钙颗粒尺寸范围为1μm~80μm;无水磷酸氢钙尺寸范围为0.4μm~3μm;磷酸四钙与无水磷酸氢钙的摩尔比为1:1-1:3,混合后得到磷酸钙骨水泥的粉体。
上述水溶性纳米材料为表面修饰的纳米金、纳米γ三氧化二铁、纳米α三氧化二铁,及上述材料稳定的单分散性胶体溶液或干燥后得到的固体颗粒。
上述表面修饰物为二巯丁二酸(DMSA)、聚葡萄糖山梨醇羧甲醚(PSC)、聚乙二醇(PEG)或柠檬酸,尺寸为5 nm~100nm。
纳米磷酸钙骨水泥的制备方法,步骤为:准备固化液:所述固化液为水、血液、水溶性纳米材料的水溶液或5-20 wt.%的壳聚糖水溶液;制备固相:按照水溶性纳米材料占磷酸钙骨水泥质量不超过6%混合,制得纳米骨水泥固相;以水溶性纳米材料的水溶液为骨水泥固化液时,直接以磷酸四钙和无水磷酸氢钙混合物为纳米骨水泥固相;按照纳米骨水泥固相/液相质量比例2:1-4:1,将固化液加入纳米骨水泥固相中,调拌均匀;置于37℃100% 湿度环境中固化4h-7d。
上述固化液为0.5-50mg/mL水溶性纳米材料的水溶液或5-20 wt.%壳聚糖水溶液。
上述壳聚糖水溶液为壳聚糖乳酸盐或者壳聚糖马来酸盐的水溶液。
在添加固体水溶性纳米材料时,先将固体水溶性纳米材料均匀分散在磷酸钙骨水泥粉体中,再与固化液调拌均匀并固化。
有益效果:本发明选择生物相容性良好的金属纳米材料与骨水泥基质粉体混合制备纳米骨水泥,不仅使材料具有良好的机械性能,而且具有增强的成骨活性。还可以通过调节加入纳米材料的种类和数量对纳米骨水泥的性能进行调节,如通过添加磁性纳米材料来赋予骨水泥以磁性,通过在一定范围内增加纳米材料添加量来增强骨水泥的强度。本发明的纳米骨水泥材料制备方法工业简单,可操作性强,且更能满足临床使用需要,具备推广价值。
附图说明
图1为实施例4和对比例(5#)所制备纳米骨水泥的弯曲强度图;
图2 为实施例3(4#)所制备纳米骨水泥的扫描电镜图;
图3为实施例4-6(6#、7#、8#)和对比例(5#)所制备的纳米骨水泥的磁滞回线图;
图4为实施例4-6(6#、7#、8#)和对比例(5#)所制备纳米骨水泥的X线衍射图谱;
图5为实施例1-2和对比例(1#)所制备纳米骨水泥的细胞增殖结果图;
图6为实施例1-2和对比例(1#)所制备纳米骨水泥的碱性磷酸酶活性检测结果图;
图7为实施例1-2和对比例(1#)所制备纳米骨水泥的钙结节形成定量检测结果图。
具体实施方式
下面的实施例可使本专业技术人员更全面地理解本发明,但不以任何方式限制本发明。
实施例1(2#)
本实施例的纳米骨水泥固相为摩尔比为1:3的磷酸四钙和无水磷酸氢钙混合物,粉液组分组成为:2份纳米骨水泥固相粉体,1份PSC包裹的纳米γ三氧化二铁胶体溶液(Rosner MH, Auerbach M. Ferumoxytol for the treatment of iron deficiency.Expert Rev Hematol. 2011 Aug;4(4):399-406.)。
其中磷酸四钙的制备方法如下:用固态反应法合成磷酸四钙粉末,等摩尔量的无水磷酸氢钙和碳酸钙在1500℃炉中煅烧6小时,升温速度25°C/min,煅烧后冷却,在球磨机中研磨至平均粒径为5μm。无水磷酸钙的平均粒径为1μm。
其中纳米材料为PSC包裹的纳米γ-三氧化二铁胶体溶液,平均粒径9nm,并以其为骨水泥固化液。
本实施例的纳米骨水泥采用如下步骤制备:
(1)制备浓度为24mg/mL的PSC包裹的纳米γ三氧化二铁胶体溶液作为固化液;
(2)按照粉液质量比2:1取磷酸钙纳米骨水泥固相粉体2份,取纳米γ三氧化二铁胶体溶液1份,将溶液加入固相中调拌均匀,置于37℃100% 湿度环境中固化24h-7d。
实施例2(3#)
本实施例的纳米骨水泥的制备步骤与实施例1相同,区别在于纳米材料为DMSA包裹的纳米α三氧化二铁胶体溶液,浓度为2.55 mg/mL,具体的实施例内容为:本实施例的纳米骨水泥材料,是由以下粉液组分组成:2份摩尔比为1:3的磷酸四钙和无水磷酸氢钙混合物,1份DMSA包裹的纳米α三氧化二铁胶体溶液(Sun J, Su Y, Wang C, Gu N. Theinvestigation of frequency response for the magnetic nanoparticulate assemblyinduced by time-varied magnetic field. Nanoscale Res Lett. 2011 Jul 14;6:453.)。
其中磷酸四钙的制备方法如下:用固态反应法合成磷酸四钙粉末,等摩尔量的无水磷酸氢钙和碳酸钙在1500℃炉中煅烧6小时,升温速度25°C/min,煅烧后冷却,在球磨机中研磨至平均粒径为5μm。无水磷酸钙的平均粒径为1μm。
其中纳米材料为DMSA包裹的纳米α三氧化二铁胶体溶液,平均粒径50纳米,并以其为骨水泥固化液。
本实施例的纳米骨水泥采用如下步骤制备:
(1)制备浓度为2.55mg/mL的DMSA包裹的纳米α三氧化二铁胶体溶液作为固化液;
(2)按照粉液质量比比2:1取磷酸钙纳米骨水泥固相粉体2份,取DMSA包裹的纳米α三氧化二铁胶体溶液1份,将溶液加入固相中调拌均匀,置于37℃100% 湿度环境中固化24h-7d。
实施例3(4#)
本实施例的纳米骨水泥的制备步骤与实施例1相同,区别在于纳米材料为柠檬酸包裹的纳米金胶体溶液,浓度为0.5mg/mL,具体的实施例内容为:本实施例的纳米骨水泥材料,是由以下粉液组分组成:2份摩尔比为1:3的磷酸四钙和无水磷酸氢钙混合物,1份柠檬酸包裹的纳米金胶体溶液(Wang P, Sun J, Lou Z, Fan F, Hu K, Sun Y, Gu N.Assembly-Induced Thermogenesis of Gold Nanoparticles in the Presence ofAlternating Magnetic Field for Controllable Drug Release of Hydrogel. AdvMater. 2016 Dec;28(48):10801-10808.)。
其中磷酸四钙的制备方法如下:用固态反应法合成磷酸四钙粉末,等摩尔量的无水磷酸氢钙和碳酸钙在1500℃炉中煅烧6小时,升温速度25°C/min,煅烧后冷却,在球磨机中研磨至平均粒径为5μm。无水磷酸钙的平均粒径为1μm。
其中纳米材料为柠檬酸包裹的纳米金胶体溶液,平均粒径18nm,并以其为骨水泥固化液。
本实施例的纳米骨水泥采用如下步骤制备:
(1)制备浓度为0.5mg/mL的柠檬酸钠包裹的纳米金胶体溶液作为固化液;
(2)按照粉液质量比2:1取磷酸钙纳米骨水泥固相粉体2份,取柠檬酸钠包裹的纳米金胶体溶液1份,将溶液加入固相中调拌均匀,置于37℃100% 湿度环境中固化24h-7d。
实施例4
本实施例的纳米骨水泥材料,是由以下重量份的组分组成:100份摩尔比为1:3的磷酸四钙和无水磷酸氢钙混合物,PSC包裹的纳米γ三氧化二铁为1份(6#)。
其中磷酸四钙的制备方法如下:用固态反应法合成磷酸四钙粉末,等摩尔量的无水磷酸氢钙和碳酸钙在1500℃炉中煅烧6小时,升温速度25℃/min,煅烧后冷却,在球磨机中研磨至平均粒径为5μm。无水磷酸钙的平均粒径为1μm。
其中纳米材料为PSC包裹的纳米γ三氧化二铁颗粒,由其稳定胶体溶液经喷雾干燥法获得。
骨水泥固化液的制备,称取0.5克乳酸化壳聚糖,溶解于10mL水中,制备得到浓度为5wt.%乳酸壳聚糖水溶液。
本实施例的纳米骨水泥采用如下步骤制备:
(1)制备浓度为5wt.%乳酸壳聚糖溶液的骨水泥固化液;
(2)按照取磷酸钙纳米骨水泥固相粉体100份,取PSC包裹的纳米γ三氧化二铁颗粒0-6份,将纳米颗粒添加入骨水泥粉体并调拌均匀,得到纳米骨水泥固相粉体;
(3)以粉液质量比2:1,将5wt.%乳酸壳聚糖溶液加入固相中调拌均匀,并置于37℃100% 湿度环境中固化24h-7d。
实施例5
本实施例的纳米骨水泥材料,是由以下重量份的组分组成:100份摩尔比为1:3的磷酸四钙和无水磷酸氢钙混合物,PSC包裹的纳米γ三氧化二铁为3份(7#)。
其中磷酸四钙的制备方法如下:用固态反应法合成磷酸四钙粉末,等摩尔量的无水磷酸氢钙和碳酸钙在1500℃炉中煅烧6小时,升温速度25℃/min,煅烧后冷却,在球磨机中研磨至平均粒径为5μm。无水磷酸钙的平均粒径为1μm。
其中纳米材料为PSC包裹的纳米γ三氧化二铁颗粒,由其稳定胶体溶液经喷雾干燥法获得。
骨水泥固化液的制备,称取0.5克乳酸化壳聚糖,溶解于10mL水中,制备得到浓度为5wt.%乳酸壳聚糖水溶液。
本实施例的纳米骨水泥采用如下步骤制备:
(1)制备浓度为5wt.%乳酸壳聚糖溶液的骨水泥固化液;
(2)按照取磷酸钙纳米骨水泥固相粉体100份,取PSC包裹的纳米γ三氧化二铁颗粒0-6份,将纳米颗粒添加入骨水泥粉体并调拌均匀,得到纳米骨水泥固相粉体;
(3)以粉液质量比2:1,将5wt.%乳酸壳聚糖溶液加入固相中调拌均匀,并置于37℃100% 湿度环境中固化24h-7d。
实施例6
本实施例的纳米骨水泥材料,是由以下重量份的组分组成:100份摩尔比为1:3的磷酸四钙和无水磷酸氢钙混合物,PSC包裹的纳米γ三氧化二铁为6份(8#)。
其中磷酸四钙的制备方法如下:用固态反应法合成磷酸四钙粉末,等摩尔量的无水磷酸氢钙和碳酸钙在1500℃炉中煅烧6小时,升温速度25℃/min,煅烧后冷却,在球磨机中研磨至平均粒径为5μm。无水磷酸钙的平均粒径为1μm。
其中纳米材料为PSC包裹的纳米γ三氧化二铁颗粒,由其稳定胶体溶液经喷雾干燥法获得。
骨水泥固化液的制备,称取0.5克乳酸化壳聚糖,溶解于10mL水中,制备得到浓度为5wt.%乳酸壳聚糖水溶液。
本实施例的纳米骨水泥采用如下步骤制备:
(1)制备浓度为5wt.%乳酸壳聚糖溶液的骨水泥固化液;
(2)按照取磷酸钙纳米骨水泥固相粉体100份,取PSC包裹的纳米γ三氧化二铁颗粒0-6份,将纳米颗粒添加入骨水泥粉体并调拌均匀,得到纳米骨水泥固相粉体;
(3)以粉液质量比2:1,将5wt.%乳酸壳聚糖溶液加入固相中调拌均匀,并置于37℃100% 湿度环境中固化24h-7d。
对比例
本实施例的骨水泥材料与实施例1基本相同,区别在于以水(1#)或5wt.%乳酸壳聚糖溶液(5#)为骨水泥固化液。所以本对比例中骨水泥未添加纳米材料。
检测例
将实施例1-4和对比例进行如下性能试验:
1. 抗压强度和弹性模量的测定
取固相粉末与液相搅拌均匀后,将样品制成3×4×20mm的长条状试样,将其在相对湿度 100%的 37℃恒温箱内养护 72h,使用万能材料测试仪Instron 5500nR测试其抗压强度,加载速度1 mm/min,跨距为20mm。每组选取5个无损试样,测试其平均值。具体的测定结果如图1所示:纳米骨水泥的弯曲强度。可以看出,不添加纳米材料的骨水泥的弯曲强度为4MPa左右,但是本发明的纳米骨水泥材料中掺入了纳米铁,材料的力学性能得到了提高。当掺入比例为3wt.%左右,材料的抗压强度达到8MPa左右。
2. 磁性测试
将材料制成直径 6毫米,厚1毫米的圆片,使用振动样品磁强计(Lake shore7410)检测其磁性。磁滞回线可以看出,材料出现了超顺磁性,且磁性强度随纳米材料的添加量而增加(图3)。超顺磁性能提高材料的磁生热能力。
3. 材料的晶相组成。当磷酸四钙与无水磷酸钙的摩尔比为1:1时,固相粉体和液相调和后会发生反应生成羟基磷灰石[Ca5(PO4)3OH]。而当磷酸四钙与无水磷酸钙的摩尔比小于1时,会生成缺钙磷灰石。从材料的X射线衍射图谱可见,纳米材料的加入对骨水泥晶相形成没有影响(图4)。
4. 细胞增殖性能检测
将材料烘干消毒,加入培养基中,37℃预孵育后,进行细胞实验。取复苏后生长旺盛的人牙髓干细胞接种(密度为6×103个/mL)48孔培养板内。细胞培养1d、4d、7d和14d后,加入含10%CCK-8的培养基,37℃下继续培养2h,终止培养,吸取液体至96孔板内,用酶标仪在450nm波长下读取吸光度值,记录结果。做3个平行样,具体测定结果如图5。从图5可以看出,纳米骨水泥的细胞相容性良好。
5. 细胞分化性能检测(碱性磷酸酶活性检测)
用吸管吸掉96孔板内的培养液,用PBS冲洗3遍,加入含25mmol/L二乙醇胺,1mmol/L氯化镁和6.7mmol/L PNPP的反应液。每孔150μL在37℃避光放置30min,然后加0.1mol/L氢氧化钠100μL终止反应,用酶标仪于405nm波长下测定OD值。样品OD值在ALP标准曲线上读取酶活性值。将测得的OD值除以同批细胞的蛋白含量均值进行统计分析,以消除细胞总蛋白量不同对ALP值的影响。具体测定结果如图6。从图6可以看出,纳米骨水泥的碱性磷酸酶活性增高。
6. 钙化结节染色和定量检测
用茜素红法对钙化结节进行染色,吸弃原培养基,用1×PBS冲洗,用10%***在室温固定30min,再用1×PBS冲洗一次,用2%茜素红染料室温避光染色45min。用1×PBS冲洗2次。然后用10%的氯化十六烷基吡啶溶解后进行定量检测,溶解15min,用酶标仪在波长550nm读取吸光度值。具体测定结果如图7。从检测结果可以看出,纳米骨水泥的钙化结节形成量显著增加。
Claims (1)
1.纳米磷酸钙骨水泥的制备方法,其特征在于步骤为:
(1)骨水泥固化液的制备:称取0.5克乳酸化壳聚糖,溶解于10mL水中,制备得到浓度为5wt.%乳酸壳聚糖水溶液;
(2)取磷酸钙纳米骨水泥固相粉体100质量份,其中磷酸四钙和无水磷酸氢钙的摩尔比为1:3,取PSC包裹的纳米γ三氧化二铁颗粒3质量份,将PSC包裹的纳米γ三氧化二铁颗粒添加入磷酸钙纳米骨水泥固相粉体并调拌均匀,得到纳米骨水泥固相粉体;
(3)以粉液质量比2:1,将5wt.%乳酸壳聚糖水溶液加入纳米骨水泥固相粉体中调拌均匀,并置于37℃100% 湿度环境中固化24h-7d。
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