CN114479123B - 一种锶掺纳米羟基磷灰石微球/壳聚糖水凝胶及制备方法 - Google Patents
一种锶掺纳米羟基磷灰石微球/壳聚糖水凝胶及制备方法 Download PDFInfo
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- CN114479123B CN114479123B CN202210154641.7A CN202210154641A CN114479123B CN 114479123 B CN114479123 B CN 114479123B CN 202210154641 A CN202210154641 A CN 202210154641A CN 114479123 B CN114479123 B CN 114479123B
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Abstract
本发明公开了一种锶掺纳米羟基磷灰石微球/壳聚糖水凝胶及制备方法,采用微波辅助水热法合成羟基磷灰石微球备用。采用KOH/尿素水溶液与壳聚糖制成壳聚糖凝胶。将壳聚糖凝胶与羟基磷灰石微球以(0.05~0.2g):10mL的固液比混合,磁力搅拌机搅拌1h,得到均匀乳白色的复合水凝胶,即得锶掺纳米羟基磷灰石微球/壳聚糖水凝胶复合材料。采用本发明及配方制得的水凝胶有效提高了传统掺锶材料的成骨效果,制备工艺及方法简单,可注射性好。利用MTT法评价锶掺纳米羟基磷灰石微球/壳聚糖水凝胶的细胞毒性,结果细胞在不同含锶量的纳米羟基磷灰石微球/壳聚糖水凝胶浸提液培养条件下,其细胞相对增殖率在92.6%~103.1%,细胞毒性评级为0级或1级,安全性明显提升。
Description
技术领域
本发明属于生物医用材料领域,公开了一种锶掺纳米羟基磷灰石微球/壳聚糖水凝胶的制备方法以及采用该制备方法制得的锶掺纳米羟基磷灰石微球/壳聚糖水凝胶。
背景技术
羟基磷灰石(Ca10(PO4)6(OH2)作为人体骨骼中最常见的磷酸钙盐,是人体骨骼和牙齿的主要无机成分。羟基磷灰石与骨骼中矿物相的化学成分类似,具有优异的生物相容性、骨传导性和骨诱导性。羟基磷灰石的化学结构允许其掺杂一些离子以增强组织工程应用相关的物理化学及生物特性。骨骼中含有包括硅(Si)、锌(Zn)、铈(Ce)和锶(Sr)在内的多种微量元素,这些元素多骨细胞的代谢调控具有明显的生物学效应。将这些元素掺杂到羟基磷灰石晶格中可以促进植入材料的骨重建效果。
锶作为人体的一种必需微量元素,在元素周期表中与钙同族,与钙的元素性质相似,生理功能也与骨骼形成密切相关,且体内99%的锶(36~140mg/kg)均沉积在股骨、腰椎以及髂骨等部位。锶已经证明具有很强的亲和力来诱导成骨细胞活性,刺激成骨细胞增殖和分化,掺锶材料在骨表面能够吸附更多蛋白质并且增强细胞对材料的附着力,在细胞培养时添加锶,发现锶对于骨胶原的合成具有很好的作用。锶能够稳定羟基磷灰石的结构,抑制细菌酸对材料的降解作用。
壳聚糖又称脱乙酰甲壳素,是甲壳素脱乙酰化后得到的天然多糖,由随机分布的β-1,4-葡糖胺和N-乙酰-葡糖胺组成。甲壳素广泛存在于甲壳类动物(主要是虾蟹)、昆虫、真菌等生物中,是自然界储量仅次于纤维素的天然多糖;但由于结构复杂,不溶于水,萃取难度高,甲壳素的研究和应用受到严重限制。壳聚糖是甲壳素最主要的衍生物,目前主要由化学法或生物法(酶或发酵)脱乙酰化获得。除了具有良好的生物相容性和生物降解性之外,壳聚糖还具有抗菌、抗肿瘤和抗氧化等多种生物活性。其抗菌作用主要与分子量相关,低分子量壳聚糖可穿透细菌细胞膜与DNA结合,抑制DNA转录和mRNA合成;高分子量壳聚糖则可结合到细菌细胞膜负电物质上,改变膜通透性并阻断养分运输。壳聚糖还可增加白介素(interleukin,IL)1和2分泌,促进T淋巴细胞成熟和浸润,起到抗肿瘤作用,并可直接诱导肿瘤细胞凋亡。壳聚糖的抗氧化机制可能是其吡喃环上的氨基、羟基与不稳定自由基发生反应,进而形成稳定的自由基。
但通过大量的研究表明,锶单独使用在短期会达到一定良好效果,但随着锶浓度在修复部位到达极值,将失去效果,并产生一定的细胞毒性。将锶掺在骨修复材料中,减慢锶释放的速率,以防短时间内锶释放过多产生细胞毒性,同时可以提高材料的机械性能、增强细胞的增殖以及黏附性,从而达到更好的成骨效果。目前主流的掺锶骨修复材料主要有掺锶磷酸钙骨水泥、掺锶磷酸钙生物陶瓷纳米掺锶羟基磷灰石/聚乙烯醇/明胶复合水凝胶、掺锶TiO2纳米管涂层种植体等。
掺锶磷酸钙骨水泥具有良好的生物相容性和骨传导性,与骨接触更密切,骨结合强度更高。但掺锶骨水泥为纯无机材料,其浆体在注射过程中容易发生固液分离现象,导致可注射性较差,抗压强度不够,不利于临床应用。且由于其注射性较差,对技术掌握要求较高,不能随意塑性。
掺锶磷酸钙生物陶瓷将锶也可以包埋在HA微球中,通过局部缓释锶来达到骨修复的目的,具有良好的生物相容性和骨传导性,与骨接触更密切,骨结合强度更高。但目前的掺锶磷酸钙生物陶瓷不具有可注射性,制作成本较高,且植入物的力学性能不稳定。
基于上述分析,一种能够有效改善传统掺锶材料成骨效果、制备工艺复杂且存在细胞毒性的成骨材料配方与制备工艺是目前行业内急需的。
发明内容
鉴于上述不足,本发明提供了一种成骨效果高、注射性强、细胞毒性为不超过1级的锶掺纳米羟基磷灰石微球/壳聚糖水凝胶及制备方法。
本发明是通过如下手段实现的:
一种锶掺纳米羟基磷灰石微球/壳聚糖水凝胶的制备方法,包括:
(1)采用微波辅助水热法合成羟基磷灰石微球:
①将50mL物质的量浓度为的93mmol/L磷酸肌酸溶液(98wt%),缓慢滴入50mL浓度为33.33mmol/L的氯化钙溶液(99wt%)中,使用电动搅拌器(上海司乐HD2004W恒速电动搅拌器)室温搅拌30min,得到第一混合溶液;
②将96wt%的NaOH溶液滴入第一混合溶液中调节pH值,并用pH测定仪(联测SIN-PH160在线pH计)测定溶液pH=10左右;
③将不同mL的99wt%SrCl2加入到第二混合溶液中,得到第三混合溶液与锶掺羟基磷灰石微球,SrCl2的加入使(Sr+Ca)/P=1.07,与羟基磷灰石微球中的Ca/P=1.07;
计算:P的摩尔质量:4.65mmol
Ca的摩尔质量:1.6665mmol
因此:Sr的摩尔质量为:4.65×1.07-1.6665=3.309mmol
④将第三混合溶液放入微波水热反应器中120℃加热反应30min;
⑤冷却至室温,静置沉淀,用滤纸过滤,使用PBS溶液洗涤两次,冷冻干燥(海争巧FD-1A-50真空冷冻干燥机),得到羟基磷灰石微球。
(2)制作壳聚糖水凝胶:
①将16g KOH、8g尿素和71g蒸馏水加入150mL烧瓶中;
②在冰箱中预冷到-20℃,高于KOH/尿素水溶液的冰点;
③立即加入5g壳聚糖,然后猛烈搅拌约1h,得到透明的5wt%壳聚糖溶液;
④将100g 5wt%医用聚乙烯醇水溶液加入100g5wt%壳聚糖溶液中;
⑤在25℃下搅拌,得到壳聚糖/医用聚乙烯醇水溶液,再在50℃下水浴加热反应1h,即可形成部分物理交联的壳聚糖/医用聚乙烯醇水凝胶;
⑥将壳聚糖/医用聚乙烯醇水凝胶在-20℃下冷冻12h(冰箱冷冻),并在室温下解冻三次循环,以充分形成医用聚乙烯醇晶体和医用聚乙烯醇的物理交联;
⑦凝胶在蒸馏水中用直径为10mm的半透膜透析5天以除去残骸,即得壳聚糖水凝胶,壳聚糖和聚乙烯醇的浓度约为5wt%。
(3)锶掺纳米羟基磷灰石微球/壳聚糖水凝胶的制备:
将壳聚糖凝胶与羟基磷灰石微球以(0.05~0.2g):10mL的固液比混合,磁力搅拌机搅拌1h,得到均匀乳白色的复合水凝胶,即得锶掺纳米羟基磷灰石微球/壳聚糖水凝胶复合材料。
本发明还公开了一种根据上述制备工艺制得的锶掺纳米羟基磷灰石微球/壳聚糖水凝胶。
本发明的有益效果在于:
1.巧妙将四种材料混合,提高传统掺锶材料的成骨效果:与传统材料羟基磷酸钙成骨,和单纯掺锶羟基磷灰石水凝胶成骨效果提高了。
2.制备了掺锶羟基磷灰石微球,有助于锶在局部的释放。
3.制备过程简单:只需要简单混合材料就能制备,对设备要求不高。
4.可注射性:用5ml注射器吸取凝胶材料,分别在有针头和无针头的情况下注射凝胶,观察凝胶的注射情况。经实验,注射性较好。
5.具有较好的抑菌效果。
6.动物实验结果可见成骨效果良好。
7.细胞毒性低:利用CCK-8法评价锶掺纳米羟基磷灰石微球/壳聚糖水凝胶的细胞毒性。方法将制备好的含锶量分别为0%、1%、5%和10%的4种材料的浸提液培养L929细胞,1、3和5d后采用CCK-8比色法和细胞形态直接观察法测定细胞与材料浸提液作用后的生长情况。结果细胞在不同含锶量的磷酸钙骨水泥材料浸提液培养条件下,其细胞相对增殖率在92.6%~103.1%之间,锶掺纳米羟基磷灰石微球/壳聚糖水凝胶材料的细胞毒性评级为0级或1级。
附图说明
图1为锶掺纳米羟基磷灰石微球的SEM图;
图2为本发明制作的凝胶溶胀率图;
图3为锶掺纳米羟基磷灰石的EDS图;
图4为本发明的锶掺纳米羟基磷灰石微球的XRD图;
图5为本发明的锶掺纳米羟基磷灰石微球/壳聚糖在体外的注射情况;
图6为本发明的锶掺纳米羟基磷灰石微球/壳聚糖与羟基磷灰石/壳聚糖的细胞培养对比图;
图7为本发明的锶掺纳米羟基磷灰石微球/壳聚糖抑菌实验结果图。
图8为本发明的锶掺羟基磷灰石壳聚糖水凝胶与壳聚糖水凝胶溶血实验结果图,其中左侧为壳聚糖水凝胶,右侧为锶掺羟基磷灰石壳聚糖水凝胶。
具体实施方式
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解,在阅读了本发明讲授的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
实验材料:98%的磷酸肌酸溶液、99%的氯化钙溶液、96%的NaOH溶液、SrCl2(99%)、壳聚糖(脱乙酰度≥95%)、5%医用聚乙烯醇、PBS溶液、KOH、尿素。
实验仪器:PH测定仪、真空冷冻干燥机、微波水热反应器、磁力搅拌仪、冰箱、溶液配置相关仪器若干、半透膜/透析膜(直径10mm)。
实施例1
一种锶掺纳米羟基磷灰石微球/壳聚糖水凝胶的制备方法,包括:
(1)采用微波辅助水热法合成羟基磷灰石微球:
①将50mL物质的量浓度为的93mmol/L磷酸肌酸溶液(98wt%),缓慢滴入50mL浓度为33.33mmol/L的氯化钙溶液(99wt%)中,室温搅拌30min。
②将96wt%的NaOH溶液滴入混合溶液调节pH值,并用pH值测定仪测定溶液pH值=10左右。
③将不同mL的质量分数99%SrCl2加入到混合溶液中,制备Sr羟基磷灰石微球,SrCl2的加入使(Sr+Ca)/P=1.07,与羟基磷灰石微球中的Ca/P=1.07。
计算:P的摩尔质量:4.65mmol
Ca的摩尔质量:1.6665mmol
因此:Sr的摩尔质量为:4.65×1.07-1.6665=3.309mmol
④将混合溶液放入微波水热反应器中加热120℃,30min。
⑤冷却至室温,静置沉淀,用滤纸过滤,使用PBS溶液洗涤两次,冷冻干燥(真空冷冻干燥机),得到羟基磷灰石微球。
(2)制作壳聚糖水凝胶:
①将16g KOH、8g尿素和71g蒸馏水加入150mL烧瓶中。
②预冷到-20℃,(冰箱冷冻),高于KOH/尿素水溶液的冰点。
③立即加入5g壳聚糖,然后猛烈搅拌约1h,得到透明的溶液(5wt%)。
④将100g 5wt%医用聚乙烯醇水溶液加入100g壳聚糖溶液(5wt%)中。
⑤在25℃下搅拌,得壳聚糖/医用聚乙烯醇水溶液,再在50℃下反应(水浴加热)1h即可形成部分物理交联的壳聚糖/医用聚乙烯醇水凝胶。
⑥将水凝胶在-20℃下冷冻12h(冰箱冷冻),并在室温下解冻三次循环,以充分形成医用聚乙烯醇晶体和医用聚乙烯醇的物理交联。
⑦凝胶在蒸馏水中透析(半透膜直径为10mm)5天以除去残骸。壳聚糖和聚乙烯醇的浓度约为5wt%。
(3)锶掺纳米羟基磷灰石微球/壳聚糖水凝胶的制备:
将壳聚糖凝胶与羟基磷灰石微球以0.05g:10mL的固液比混合,磁力搅拌机搅拌1h,得到均匀乳白色的复合水凝胶,即得锶掺纳米羟基磷灰石微球/壳聚糖水凝胶复合材料。
对比例2
一种锶掺磷酸钙微球/壳聚糖水凝胶的制备方法,包括:
(1)锶掺磷酸钙微球合成
材料:去离子水、明胶、磷酸钠、盐酸、氢氧化钠、乙酸钙、氯化锶、无水乙醇
仪器:三口烧瓶、分析天平、药匙/镊子、烧杯若干、胶头滴管若干、玻璃棒、磁力搅拌器,pH计、恒流泵、离心机
①取200ml去离子水、20g明胶依次加入到三口烧瓶中,再置入80℃水浴中直到形成水溶胶;
②再将60ml浓度为0.25M的磷酸钠(Na2H2PO4)溶液加入上述水溶胶中,磁力搅拌10min后,用浓度为1.0M的盐酸将其pH值从5.6缓慢调节至3.0,然后再用浓度为1.0M的NaOH溶液将pH从3.0缓慢调节到4.0。
③将80ml浓度为0.25M的乙酸钙溶液用去离子水稀释至120ml,用按钙离子的摩尔浓度2.5%用天平称量SrCl2添加到乙酸钙溶液。
④利用恒流泵按1ml/min的速度滴加到上述已经调节好pH值的三口烧瓶中,在持续磁力搅拌条件下发生成核矿化反应。
⑤30min后离心,对底部沉淀用60℃的去离子水清洗3次,再对沉积物用无水乙醇洗涤一次,在60℃干燥。
(2)加入壳聚糖水凝胶:
材料:KOH(99.99%)、尿素(99%)、医用聚乙烯醇(22-88)、蒸馏水仪器:分析天平、药匙/镊子、烧杯若干、胶头滴管若干、玻璃棒、保鲜膜、
①用药匙/镊子取适量KOH放入烧杯中,托盘天平两边均放上烧杯,称取16gKOH,用药匙取出尿素然后在托盘天平上称取8g尿素、然后再分别称取5g壳聚糖、5g医用聚乙烯醇。再用量筒量取71ml、95ml的蒸馏水。
②制备100g 5wt%医用聚乙烯醇:先将蒸馏水倒入烧杯中,再将5g聚乙烯醇倒入蒸馏水中,用玻璃棒搅拌至溶解。
③将16g KOH、8g尿素和71g蒸馏水加入150mL烧瓶中,用玻璃棒缓慢搅拌。
④预冷到-20℃,(冰箱冷冻),高于KOH/尿素水溶液的冰点。
⑤立即加入5g壳聚糖,然后猛烈搅拌约1h,得到透明的壳聚糖溶液(5wt%)。
⑥将100g 5wt%医用聚乙烯醇水溶液加入100g壳聚糖溶液(5wt%)中,在25℃下搅拌,得到的壳聚糖/医用聚乙烯醇水溶液在50℃下反应(水浴加热)1h即可形成部分物理交联的壳聚糖/医用聚乙烯醇水凝胶。
⑦将水凝胶在-20℃下冷冻12h(冰箱冷冻),并在室温下解冻三次循环,以充分形成医用聚乙烯醇晶体和医用聚乙烯醇的物理交联。
⑧凝胶在蒸馏水中透析(半透膜直径为10mm)5天以除去残骸。壳聚糖和聚乙烯醇的浓度约为5wt%。将壳聚糖凝胶与羟基磷灰石微球以0.1g:10mL的固液比混合,磁力搅拌机搅拌1h,得到均匀乳白色的复合水凝胶,即得锶掺纳米羟基磷灰石微球/壳聚糖水凝胶复合材料。
对比例3
一种锶掺纳米羟基磷灰石微球/温敏水凝胶的制备方法,包括:
(1)采用微波辅助水热法合成羟基磷灰石微球:
①将50mL物质的量浓度为的93mmol/L磷酸肌酸溶液(98%),缓慢滴入50mL浓度为33.33mmol/L的氯化钙溶液(99%)中,室温搅拌30min。
②将96%的NaOH溶液滴入混合溶液调节pH值,并用pH值测定仪测定溶液pH值=10左右。
③将不同mL的99%SrCl2加入到混合溶液中,制备Sr羟基磷灰石微球,SrCl2的加入使(Sr+Ca)/P=1.07,与羟基磷灰石微球中的Ca/P一致。
计算:P的摩尔质量:4.65mmol
Ca的摩尔质量:1.6665mmol
因此:Sr的摩尔质量为:4.65×1.07-1.6665=3.309mmol
④将混合溶液放入微波水热反应器中加热120℃,30min。
⑤冷却至室温,静置沉淀,用滤纸过滤,使用PBS溶液洗涤两次,冷冻干燥(真空冷冻干燥机),得到羟基磷灰石微球。
(2)制作温敏水凝胶:
①称取5g温敏水凝胶(市售)粉末与20mL蒸馏水混合。
②放置于4℃冰箱中,每24h搅拌30min进行混合,3天后得到温敏水凝胶混合悬浮液。
(3)锶掺纳米羟基磷灰石微球/温敏水凝胶的制备:
将锶掺羟基磷灰石微球以24∶76的比例加入温敏水凝胶,磁力搅拌机搅拌1h,得到均匀乳白色的复合水凝胶,即得锶掺纳米羟基磷灰石微球/温敏水凝胶复合材料。
试验例1
取实施例1制得的锶掺纳米羟基磷灰石微球/壳聚糖水凝胶进行掺锶羟基磷灰石微球表征检测:
①X射线衍射(XRD)分析
使用X射线衍射仪(奥林巴斯小型台式XRD分析仪BTX III)对制备的样品进行物相鉴定和晶体结构分析。测定条件为:铜靶Cu-Kα辐射,光阑狭缝1mm,管电压U=40KW,管点流I=40mA。扫描范围为2θ∈[10°,70°]。具体结果详见图4,如图4所示,我们看出在2θ为31.67°时,峰最为尖锐,由此可见锶成功掺入羟基磷灰石中。
②扫描电子显微镜(SEM)分析
将样品干燥处理后,粘涂在导电胶上,喷金处理后使用扫描电子显微镜(COXEM台式扫描电镜EM-30)观察样品的形貌等相关信息。具体结果详见图1,如图1所示,在SEM图中我们可以看见锶掺羟基磷灰石呈球状,证明微球的制备成功。
③X射线能量色散仪(EDS)分析
使用JSM-7800F型场发射扫描电子显微镜配套的EDS能谱仪对样品进行元素分析。具体结果详见图3,如图3所示,我们看出该样品含有C、H、O、P、Ca、Sr,由此可见锶成功掺入羟基磷灰石中。
试验例2
取实施例1制得的锶掺纳米羟基磷灰石微球/壳聚糖水凝胶进行壳聚糖水凝胶的表征检测:
①可注射性
使用挤压试验评估水凝胶的可注射性:将凝胶转移到一个5mL的商用注射器(针头直径2mm)中。混合后90s,粘贴从注射器挤压(严格在夹具)通过应用100N的力的注入能力系数(IC)水泥被定义为IC=[(Mi-)Mr./Mi]×100。Mi是水凝胶加载到注射器的初始质量,Mr.是挤压后注射器内残留的肿块质量。每个水泥组至少测试3个样品,每个样品进行3次重复测试。结果以(均数±SD)表示。具体结果详见图5,如图5所示,我们看出凝胶注射呈一条均匀直线,由此可见凝胶具有较好的可注射性性且性能稳定。
②水凝胶溶胀试验:
将水凝胶在60℃鼓风干燥机充分干燥,称取一定质量的干燥的水凝胶,记为W1,将其在PBS溶液中浸泡一段时间,取出,用滤纸擦去样品表面液体并迅速用电子天平称重,直至重量恒定。记录数据,循环操作数次,直到其质量变化不大,取三次计算平均值,记为W2。水凝胶的溶胀度DS计算式为:DS=(W2-W1)/W1。具体结果详见图2,如图2所示,我们看出在15、30、45min时的凝胶质量变化微小,由此可见凝胶具有较好的吸水性且性能稳定。
试验例3
取实施例1制得的锶掺纳米羟基磷灰石微球/壳聚糖水凝胶进行掺锶羟基磷灰石壳聚糖水凝胶体外实验:
①抑菌试验(打孔法)
制备大肠杆菌液和琼脂平板,使用移液枪吸取一定浓度的试验菌悬液0.1ml,将其接种到已经倒好的营养琼脂平板,用涂布棒涂布均匀,盖好培养皿。在培养皿上用10~100μL的枪头打孔,打完孔后用无菌针头将琼脂孔中的培养基挑出,用微量移液枪吸取掺锶羟基磷灰石壳聚糖水凝胶20μL到琼脂孔内,盖好培养基,培养培养皿并观察抑菌效果。具体结果详见图7,如图7所示,左侧为本发明制作的掺锶羟基磷灰石壳聚糖水凝胶,右侧为生理盐水,左侧抑菌圈明显大于右侧,由此可见本发明制作的掺锶羟基磷灰石壳聚糖水凝胶具有较好的抑菌效果。
②溶血实验
将掺锶羟基磷灰石壳聚糖水凝胶用37℃的生理盐水(500mg/mL),孵育30min,然后加入抗凝液(100μL),分别37℃孵育1h。然后将反应混合物离心,用UV-vis分光光度计(UV-1200MAPADA,中国)在545nm波长下测定上清液的光密度。溶血率(HR%)计算公式如下:HR(%)=(DS-DN)/(DP-DN)×100。具体结果详见图8,左侧为壳聚糖水凝胶,右侧为锶掺羟基磷灰石壳聚糖水凝胶,两者溶血率均低于国家和国际允许的5%水平。由此可见,壳聚糖水凝胶和掺锶羟基磷灰石壳聚糖水凝胶均为非溶血性,满足骨修复的需要。
③细胞毒性分析
采用CCK-8法检测细胞毒性。称取0.9g样品浸泡在无水乙醇24h,过滤干燥后紫外消毒灭菌。再将样品浸泡在20mL的完全培养基中,379C、CO2恒温培养浸提24h,采用微孔滤膜过滤,收回浸提液。并以此浸提液浓度为基准划分浓度梯度,依次为1.0%、5.0%、10.0%、20.0%、50.0%、70.0%、80.0%、100.0%的浸提液和完全培养基9个梯度,完全培养基作为对照组,其余作为实验组。将中国仓鼠卵巢细胞(CHO)接种于96孔板,每组10孔。按照分组方法,各组加入200μL对照培养液或不同浓度的浸提液。培养72h后,每孔加入CCK-8试剂,轻微震荡后继续放入恒温细胞培养箱中培养,2~3h后用实时酶联免疫检测仪测量各孔在450nm波长处的吸光度(A)值,重复测量5次,取平均值,计算细胞相对增殖率。具体结果详见图6,如图6所示,在三天内,掺锶羟基磷灰石壳聚糖水凝胶和羟基磷灰石壳聚糖水凝胶吸光度一样,在第五到七天内掺锶羟基磷灰石壳聚糖水凝胶的吸光度略高于羟基磷灰石壳聚糖水凝胶吸光度,表明掺锶羟基磷灰石壳聚糖水凝胶活细胞数量更多。由此可见本发明制作的掺锶羟基磷灰石壳聚糖水凝胶具有细胞低毒性。
试验例4
取实施例1制得的锶掺纳米羟基磷灰石微球/壳聚糖水凝胶进行掺锶羟基磷灰石壳聚糖水凝胶体内实验:
将40只小鼠平分为A、B两组,A组为实验组,B组为对照组。用戊巴比妥钠腹腔注射麻醉这40只小鼠,用电动剃须刀去除其大腿上的毛进行备皮,用乙醇消毒。在左右两侧胫骨的位置切开皮肤,在胫骨中段进行约10mm纵向皮肤和肌肉切口,取胫骨中段2mm的骨干和骨膜,并将与切除的胫骨大小和形状类似的材料移植到该缺损处,缝合肌肉与皮肤。其中A组移植材料为掺锶羟基磷灰石壳聚糖水凝胶,B组移植材料为羟基磷灰石壳聚糖水凝胶。
术后5周,对小鼠进行腹腔麻醉,然后将小鼠固定在自制的动物板上,然后进行X-ray拍摄。在术后第6周取出右腿胫骨材料,进行micro-CT拍摄通过X射线拍摄,可见材料与骨组织连接紧密,由此可见,掺锶羟基磷灰石壳聚糖水凝胶成骨效果好。
后将所有样品在10%中性福尔马林缓冲液中室温固定约24小时,在10%EDTA中脱钙20天。室温下脱水,石蜡包埋。将包埋标本切成5um厚的组织切片。切片后用苏木精-伊红、甲苯胺蓝、亚甲基蓝-碱性品红溶液染色。观察切片比较两种材料的成骨能力。通过HE染色可见骨组织与材料间有新生骨组织生成,由此可见,掺锶羟基磷灰石壳聚糖水凝胶成骨效果好。
以上所述是本发明的优选实施方式而已,当然不能以此来限定本发明之权利范围,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和变动,这些改进和变动也视为本发明的保护范围。
Claims (6)
1.一种锶掺纳米羟基磷灰石微球/壳聚糖水凝胶的制备方法,包括:
(1)采用微波辅助水热法合成锶掺羟基磷灰石微球;
(2)制作壳聚糖水凝胶;
(3)锶掺纳米羟基磷灰石微球/壳聚糖水凝胶的制备;
其中:
步骤(2)所述壳聚糖水凝胶由如下方法制得:
①将16g KOH、8g尿素和71g蒸馏水加入150mL烧瓶中;
②预冷到-20℃冰箱冷冻;
③立即加入5g壳聚糖搅拌1h,得到5wt%的壳聚糖溶液;
④将100g 5wt%医用聚乙烯醇水溶液加入100g、5wt%的壳聚糖溶液中;
⑤25℃下搅拌,得到的壳聚糖/医用聚乙烯醇水溶液;随后在50℃下反应1h,得到壳聚糖/医用聚乙烯醇水凝胶;
⑥将壳聚糖/医用聚乙烯醇水凝胶在-20℃下冷冻12h,并在室温下解冻循环三次,结束后将凝胶在蒸馏水中用直径为10mm的半透膜透析5天,即得壳聚糖水凝胶;
步骤(3)所述锶掺纳米羟基磷灰石微球/壳聚糖水凝胶由如下方法制得:
将壳聚糖凝胶与羟基磷灰石微球按照固液比为(0.05~0.2g):10mL混合,磁力搅拌1h,即得锶掺纳米羟基磷灰石微球/壳聚糖水凝胶复合材料。
2.根据权利要求1所述的制备方法,其中:
步骤(1)所述微波辅助水热法合成羟基磷灰石微球包括:
①将50mL物质的量浓度为93mmol/L、质量分数98%的磷酸肌酸溶液滴入50mL物质的量浓度为33.33mmol/L、质量分数99%的氯化钙溶液中,室温搅拌30min,得到第一混合溶液;
②将质量分数为96%的NaOH溶液滴入第一混合溶液调节pH值,得到第二混合溶液;
③将不同mL的质量分数为99%SrCl2加入到第二混合溶液中,得到第三混合溶液与锶掺羟基磷灰石微球;
④将第三混合溶液放入微波水热反应器中加热反应;
⑤冷却至室温,静置沉淀并过滤,使用PBS溶液洗涤两次,冷冻干燥,得到羟基磷灰石微球。
3.根据权利要求2所述的制备方法,其中:
步骤②所述pH值调节至10。
4.根据权利要求2所述的制备方法,其中:
步骤③所述SrCl2的加入使(Sr+Ca)/P=1.07;
步骤③所述羟基磷灰石微球中的Ca/P=1.07。
5.根据权利要求2所述的制备方法,其中:
步骤④所述微波加热反应温度为120℃,反应时间为30min。
6.一种根据权利要求1~5所述任一制备方法制得的锶掺纳米羟基磷灰石微球/壳聚糖水凝胶。
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