CN112472368B - 一种具有促软骨组织形成功能涂层的关节植入物及其制备方法 - Google Patents

一种具有促软骨组织形成功能涂层的关节植入物及其制备方法 Download PDF

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CN112472368B
CN112472368B CN202011522928.8A CN202011522928A CN112472368B CN 112472368 B CN112472368 B CN 112472368B CN 202011522928 A CN202011522928 A CN 202011522928A CN 112472368 B CN112472368 B CN 112472368B
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cartilage tissue
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何佳鹏
李涵
刘宏
孙海霞
袁冰寒
李青凯
李妍
刘爽
顾静飞
唐骆
沈礼峰
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Guangdong Guangna Anyu Technology Co ltd
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Abstract

本发明涉及一种具有促软骨组织形成功能涂层的关节植入物及其制备方法，上述制备方法包括如下步骤：对关节植入物基体进行表面改性处理，随后再将溶解有功能涂料的涂层溶液均匀涂抹于改性后关节植入物基体表面，待功能溶液固化形成功能涂层后取成熟软骨组织或能分化为软骨细胞的干细胞与关节植入物基体进行共培养，以在功能涂层中生长获得软骨组织，即得一种具有促软骨组织形成功能涂层的关节植入物。实验表明，将本发明中提供的形成有功能涂层的关节植入物与兔软骨细胞共培养后植入新西兰大白兔后肢胫骨膝关节损伤模型中后一周内能形成稳定的软骨组织块，且植入后该关节植入物可让软骨细胞迅速增殖产生分泌物，从而形成可包裹软骨细胞并为软骨细胞的无氧酵解提供场所的细胞外基质。

Description

一种具有促软骨组织形成功能涂层的关节植入物及其制备方法
技术领域
本发明涉及医疗用品技术领域，尤其涉及一种具有促软骨组织形成功能涂层的关节植入物及其制备方法。
背景技术
关节结构是由硬骨、关节软骨、关节囊、关节滑膜、关节滑液组成，因而关节结构具有无神经组织、无血管分布、无淋巴组织的特点，这使得关节结构病变与损坏后，机体无法进行自我修复，只能采用人工植入物(包括自体移植与人工合成外源物)的方式进行干预修复。
目前关节损伤植入修复方式有以下两种：一、自生骨髓刺激增殖分化与自体骨组织的直接移植，这种方式会对患者产生二次伤害，且移植过程中也面临骨组织失活与感染的风险，同时由于自体骨来源有限，使得增殖分化培养费用很高；二、采用金属(钛合金)、陶瓷(羟基磷灰石)等生物材料进行替换，但是常规的金属、陶瓷材料与人体组织直接接触过程中会因弹性模量不匹配而使其与周围组织结合力差，由此可能引发植入部位组织磨损导致骨流失与骨磨损问题，且这些植入物只能替代硬骨部分功能，无法替代软骨功能，也无法诱导软骨细胞的生长与促进软骨组织的形成。
发明内容
针对上述问题，现提供具有促软骨组织形成功能涂层的关节植入物及其制备方法，旨在有效解决上述问题。
具体技术方案如下：
本发明的第一个方面是提供一种具有促软骨组织形成功能涂层的关节植入物的制备方法，具有这样的特征，包括如下步骤：对关节植入物基体进行表面改性处理，随后再将溶解有功能涂料的涂层溶液均匀涂抹于改性后关节植入物基体表面，待功能溶液固化形成功能涂层后取成熟软骨组织或能分化为软骨细胞的干细胞与关节植入物基体进行共培养，以在功能涂层中生长获得软骨组织，即得一种具有促软骨组织形成功能涂层的关节植入物。
上述的制备方法，还具有这样的特征，关节植入物基体的表面改性方法为：将关节植入物基体浸入硅烷偶联剂中3-5h后取出，烘干。
上述的制备方法，还具有这样的特征，涂层溶液中功能涂料浓度为5-10wt％，且涂层溶液中功能涂料及光引发剂的质量比为1000:(2-4)。
上述的制备方法，还具有这样的特征，功能涂料的制备方法为：以摩尔比10:(3-7)分别称取硫酸软骨素及甲基丙烯酸酐，混合并溶解于磷酸盐缓冲溶液中，调节溶液pH至pH＝8，再将溶液升温至42-58℃，反应3-4h，随后向反应后料液中加入少量强氧化剂，反应1-2h，以将硫酸软骨素分子中部分相邻羟基氧化形成醛基，从而增加涂层涂料与体内组织的相容性，停止反应，将料液于42-58℃下透析7-9d，冷冻干燥，得功能涂料。
上述的制备方法，还具有这样的特征，以硫酸软骨素摩尔量计强氧化剂的加入量为0.1-0.3％。
上述的制备方法，还具有这样的特征，强氧化剂为高碘酸钠或高锰酸钾中的一种。
上述的制备方法，还具有这样的特征，关节植入物基体为钛及其合金、钽及其合金、羟基磷灰石及以其为主要成分的烧结物、骨水泥及以其为主要成分固化物的制品中的一种。
需要说明的是，本发明中如采用干细胞共培养还需先进行诱导分化处理。
本发明的第二个方面是提供一种根据上述制备方法制备获得的具有促软骨组织形成功能涂层的关节植入物。
上述方案的有益效果是：
1)、本发明提供的关节植入物中利用硫酸软骨素促进软骨细胞铺展、增殖、粘附，体外细胞实验表明软骨细胞在关节植入物表面大量生长，3～5天内可形成软骨组织细胞团块；
2)、本发明提供的关节植入物中甲基丙烯酸酐中存在大量高能双键，在紫外光引发下可打开高能双键，以与硅烷偶联剂处理后的人工关节表面牢固结合，从而提高涂层强度；
3)、本发明提供的关节植入物可应用到髋、踝、肘、腕、肩等关节损伤场景中；
4)、实验表明，将本发明中提供的形成有功能涂层的关节植入物与兔软骨细胞共培养后植入新西兰大白兔后肢胫骨膝关节损伤模型中后一周内能形成稳定的软骨组织块，且植入后该关节植入物可让软骨细胞迅速增殖产生分泌物，从而形成可包裹软骨细胞并为软骨细胞的无氧酵解提供场所的细胞外基质；
5)、实验表明，本发明中由于功能涂层与软骨组织的存在，未发生植入物对植入部位原健康膝关节股骨端、健康髌骨的磨损损伤。
附图说明
图1为本发明的实施例1中提供的关节植入物体外细胞共培养后细胞铺展荧光染色图；
图2为本发明的实施例1中提供的关节植入物体外细胞共培养后细胞毒性荧光染色图；
图3为本发明的实施例1中提供的关节植入物体外细胞共培养后软骨细胞增殖图；
图4为本发明的实施例1中提供的关节植入物体外细胞共培养后促软骨涂层弯折、荧光染色图及植入新西兰大白兔后肢胫骨膝关节损伤模型一周后植入物基材表面剥离的促软骨涂层弯折、荧光染色图；
图5为本发明中提供的关节植入物的使用示意图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。
需要说明的是，在不冲突的情况下，本发明中的实施例及实施例中的特征可以相互组合。
下面结合附图和具体实施例对本发明作进一步说明，但不作为本发明的限定。
实施例1
一种具有促软骨组织形成功能涂层的关节植入物的制备方法，其制备包括如下步骤：将由钛制成的关节植入物基体浸入硅烷偶联剂中3h后取出，烘干，随后再将溶解有功能涂料的涂层溶液(纯水做溶剂，涂层溶液中功能涂料浓度为5wt％，且涂层溶液中功能涂料及光引发剂的质量比为1000:2，光引发剂可为Irgacure2959)均匀涂抹于改性后关节植入物基体表面，待功能溶液在紫外光照射下固化形成功能涂层后取新西兰大白兔软骨细胞与关节植入物基体进行共培养，以在功能涂层中生长获得软骨组织，即得一种具有促软骨组织形成功能涂层的关节植入物；
其中，功能涂料的制备方法为：以摩尔比10:3分别称取硫酸软骨素及甲基丙烯酸酐，混合并溶解于磷酸盐缓冲溶液中，调节溶液pH至pH＝8，再将溶液升温至42℃，反应3h，随后以硫酸软骨素摩尔量计向反应后料液中加入0.1％高碘酸钠，反应1h，以将硫酸软骨素分子中部分相邻羟基氧化形成醛基，从而增加涂层涂料与体内组织的相容性，停止反应，将料液于42-58℃下透析7d，冷冻干燥，得功能涂料。
如图1所示，软骨细胞在没有功能涂层的关节植入物基体表面(如图1(a)所示)呈现球状生长，且铺展情况很差，说明细胞生长状态较差；而软骨细胞在具有功能涂层的关节植入物基体表面(如图1(b)所示)铺展情况良好，并产生细胞铺展后特有的微管与微丝结构，这说明体外细胞共培养实验中功能涂层对软骨细胞的促进生长作用是明显的。
如图2所示，软骨细胞在没有功能涂层的关节植入物基体表面(如图2(a)所示)生长稀疏且有死细胞，而在具有功能涂层的关节植入物基体表面(如图2(b)所示)生长密集且全部为活细胞，这说明体外细胞共培养实验中功能涂层对软骨细胞的促进生长作用是明显的。
如图3所示，相比在没有功能涂层的关节植入物基体上，具有功能涂层的关节植入物基体表面细胞增殖迅速，这说明体外细胞共培养实验中功能涂层对软骨细胞的促进生长作用是明显的。
本发明中将共培养24h后所形成的软骨组织从关节植入物基体表面剥离，弯折显示其实际强度良好(如图4(a)、4(b)所示)；荧光染色实验表明，植入前软骨组织细胞生长状态良好(如图4(c)所示)。
本发明中将上述关节植入物植入新西兰大白兔膝关节人工损伤模型1周后将促软骨组织从关节植入物基体表面剥离，弯折显示其实际强度良好(如图4(d)、4(e)所示)。
本发明中将上述关节植入物植入新西兰大白兔膝关节人工损伤模型后立即对软骨细胞进行荧光染色(如图4(f)所示)，结果显示植入后细胞生长状态良好，且植入前、后功能涂层形貌完整，膝关节没有被基材所损伤，细胞生长状态良好，这表明体内植入实验中功能涂层对促进软骨组织生长作用是明显的。
实施例2
一种具有促软骨组织形成功能涂层的关节植入物的制备方法，其制备包括如下步骤：将由陶瓷制成的关节植入物基体浸入硅烷偶联剂中4h后取出，烘干，随后再将溶解有功能涂料的涂层溶液(纯水做溶剂，涂层溶液中功能涂料浓度为7wt％，且涂层溶液中功能涂料及光引发剂的质量比为1000:3，光引发剂可为Irgacure2959)均匀涂抹于改性后关节植入物基体表面，待功能溶液在紫外光照射下固化形成功能涂层后取软骨细胞(实际应用中使用患者自源性软骨细胞)与关节植入物基体进行共培养，以在功能涂层中生长获得软骨组织，即得一种具有促软骨组织形成功能涂层的关节植入物；
其中，功能涂料的制备方法为：以摩尔比10:5分别称取硫酸软骨素及甲基丙烯酸酐，混合并溶解于磷酸盐缓冲溶液中，调节溶液pH至pH＝8，再将溶液升温至50℃，反应3.5h，随后以硫酸软骨素摩尔量计向反应后料液中加入0.2％高锰酸钾，反应1.5h，以将硫酸软骨素分子中部分相邻羟基氧化形成醛基，从而增加涂层涂料与体内组织的相容性，停止反应，将料液于42-58℃下透析8d，冷冻干燥，得功能涂料。
实施例3
一种具有促软骨组织形成功能涂层的关节植入物的制备方法，其制备包括如下步骤：将由钛合金制成的关节植入物基体浸入硅烷偶联剂中5h后取出，烘干，随后再将溶解有功能涂料的涂层溶液(注射用水做溶剂，涂层溶液中功能涂料浓度为10wt％，且涂层溶液中功能涂料及光引发剂的质量比为1000:4，光引发剂可为Irgacure2959)均匀涂抹于改性后关节植入物基体表面，待功能溶液在紫外光照射下固化形成功能涂层后取大鼠骨髓间充值干细胞与关节植入物基体进行共培养(共培养过程中先诱导干细胞分化形成软骨细胞)，以在功能涂层中生长获得软骨组织，即得一种具有促软骨组织形成功能涂层的关节植入物；
其中，功能涂料的制备方法为：以摩尔比10:7分别称取硫酸软骨素及甲基丙烯酸酐，混合并溶解于磷酸盐缓冲溶液中，调节溶液pH至pH＝8，再将溶液升温至52℃，反应4h，随后以硫酸软骨素摩尔量计向反应后料液中加入0.3％高锰酸钾，反应2h，以将硫酸软骨素分子中部分相邻羟基氧化形成醛基，从而增加涂层涂料与体内组织的相容性，停止反应，将料液于42-58℃下透析9d，冷冻干燥，得功能涂料。
以上所述仅为本发明较佳的实施例，并非因此限制本发明的实施方式及保护范围，对于本领域技术人员而言，应当能够意识到凡运用本发明说明书及图示内容所作出的等同替换和显而易见的变化所得到的方案，均应当包含在本发明的保护范围内。

Claims (5)

1.一种具有促软骨组织形成功能涂层的关节植入物的制备方法，其特征在于，包括如下步骤：对关节植入物基体进行表面改性处理，随后再将溶解有功能涂料的涂层溶液均匀涂抹于改性后关节植入物基体表面，待功能溶液固化形成功能涂层后取成熟软骨组织或能分化为软骨细胞的干细胞与关节植入物基体进行共培养，以在功能涂层中生长获得软骨组织，即得一种具有促软骨组织形成功能涂层的关节植入物；
其中，功能涂料的制备方法为：以摩尔比10:(3-7)分别称取硫酸软骨素及甲基丙烯酸酐，混合并溶解于磷酸盐缓冲溶液中，调节溶液pH至pH＝8，再将溶液升温至42-58℃，反应3-4h，随后向反应后料液中以硫酸软骨素摩尔量计加入0.1-0.3％的高碘酸钠或高锰酸钾，反应1-2h，停止反应后将料液于42-58℃下透析7-9d，冷冻干燥，得功能涂料。
2.根据权利要求1所述的制备方法，其特征在于，关节植入物基体的表面改性方法为：将关节植入物基体浸入硅烷偶联剂中3-5h后取出，烘干。
3.根据权利要求2所述的制备方法，其特征在于，涂层溶液中功能涂料浓度为5-10wt％，且涂层溶液中功能涂料及光引发剂的质量比为1000:(2-4)。
4.根据权利要求1-3任一项所述的制备方法，其特征在于，关节植入物基体为钛及其合金、钽及其合金、羟基磷灰石及以其为主要成分的烧结物、骨水泥及以其为主要成分固化物的制品中的一种。
5.一种具有促软骨组织形成功能涂层的关节植入物，其特征在于，根据权利要求1-4任一项所述制备方法制备获得。
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