CN111840642A - 一种软骨脱细胞基质复合支架的制备方法及其应用 - Google Patents
一种软骨脱细胞基质复合支架的制备方法及其应用 Download PDFInfo
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- CN111840642A CN111840642A CN202010679590.0A CN202010679590A CN111840642A CN 111840642 A CN111840642 A CN 111840642A CN 202010679590 A CN202010679590 A CN 202010679590A CN 111840642 A CN111840642 A CN 111840642A
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Abstract
Description
技术领域
本发明属于生物医用材料技术领域,具体为一种软骨脱细胞基质复合支架的制备方法及其应用。
背景技术
关节软骨内无血管组织、淋巴回流和神经支配,基质更新速度缓慢,软骨一旦损伤,难以自愈。随着人口的老龄化,骨关节炎患者的比例每年逐渐增加。根据世界卫生组织的报道,在60周岁以上的人群中,大约有10%的男性都有骨关节炎症状。由于激素分泌的差异,女性患者比例更为严重。根据官方统计,骨关节炎患者的人数在中国至少有1亿,其中每年需要进行手术治疗的患者人数就高达2000万。另一方面,膝关节软骨承担了大约体重的3~4倍的压力,所以由于肥胖、运动等导致的软骨损伤也逐渐增多。
目前已有很多治疗方法致力于软骨缺损修复,比如:清创术、微骨折术、自体软骨移植以及自体软骨细胞移植等,但是这些方法均有一定局限性,未从根本上实现软骨再生。组织工程为软骨缺损修复提供了新的思路,细胞、生长因子、支架材料作为软骨组织工程的三大要素在软骨再生过程中起着重要作用。目前,最复杂的合成材料和制备工艺也无法完整地模拟天然组织的细胞外基质成分和结构,而脱细胞基质作为组织的无细胞成分,储存着细胞粘附蛋白和生长因子等物质,能为细胞的增殖、迁移和分化提供理想的微环境,这是任何一种材料都无法比拟的。
发明内容
本发明的目的在于针对上述问题,提供一种用于软骨修复的软骨脱细胞基质复合支架的制备方法。该方法制备得到的软骨脱细胞基质复合支架,采用软骨细胞外基质为原材料,经物理、化学以及酶促法处理后形成脱细胞基质,该有细胞基质可储存细胞粘附蛋白和生长因子等物质,能为细胞的增殖、迁移和分化提供理想的微环境。
本发明的另外一个发明目的是提供了该软骨脱细胞基质复合支架的应用。
为了实现以上发明目的,本发明的具体技术方案为:
一种软骨脱细胞基质复合支架,其结构如式Ⅰ所示,
其中,所述天然高分子或其衍生物所占的质量百分含量为25-75%,优选25-50%;所述消化后的软骨脱细胞基质的质量百分含量为25-75%,优选50-75%。
所述天然高分子或其衍生物为透明质酸、羧甲基壳聚糖、明胶、胶原、海藻酸钠和硫酸软骨素中的任意一种或几种;优选所述天然高分子或其衍生物为胶原和胶原改性的产物。
所述的软骨细胞外基质来自兔或猪的关节软骨(死后24h内),粒径为10-50μm。
该复合支架由脱细胞后的关节软骨细胞外基质与天然高分子或其衍生物所组成;脱细胞方式为物理法、化学法以及酶促法,DNA去除率大于95%,GAG保留率大于40%。
所述脱细胞基质采用含胃蛋白酶的醋酸或盐酸溶液进行消化,胃蛋白酶与脱细胞基质的质量比为1:5-1:10。
一种如式I所述软骨脱细胞基质复合支架的制备方法,包括以下步骤:
1)将医用无菌的天然高分子或其衍生物配制成浓度为10-30mg/mL的水溶液,备用;
2)制备脱细胞基质颗粒,备用;
3)将步骤2)得到的脱细胞基质颗粒加入到含胃蛋白酶的酸性溶液中,充分搅拌48h,得到乳白色液体;
4)将步骤3)得到的乳白色液体与天然高分子材料共混,成胶后进行冷冻干燥,即可制得支架。
以上所述的软骨脱细胞基质复合支架的作为软骨修复材料、或/和生物材料的应用。
具体的生物材料可以为组织工程三维细胞支架。
利用如式I所述软骨脱细胞基质复合支架进行组织工程三维细胞支架的制备,该方法包括以下步骤:
第一步:将制得的脱细胞溶液和胶原溶液灭菌后按体积比1~3:1~3混匀,预先调节pH值至5.0~6.0,然后加入细胞悬浮液混匀,调节pH值至7.0~8.0;细胞悬液的加入量为:按照5×106~5×107cells/mL的比例向基于天然材料胶原的天然高分子复合水凝胶混合液中加入细胞悬液;
第二步:将调节pH值后的混合溶液立即注射至生物体内的待修复部位形成水凝胶,得到组织工程三维支架;或者注入模具中,静置成胶,而后将所得水凝胶从模具中取出并浸没于培养基中,置于培养箱中在34~40℃、3%~5%的CO2的条件下培养至少1天,得到组织工程三维细胞支架,培养期间定期更换培养基。
所述培养基是在DMEM基础培养基的基础上加入青霉素和链霉素混合液、抗坏血酸以及胎牛血清得到,DMEM培养基中青霉素和链霉素混合液的浓度为0.8%~1.2%,抗坏血酸的浓度为0.15%~0.25%,胎牛血清的浓度为8%~12%。
进一步地,所述天然高分子或其衍生物为I型胶原时,软骨脱细胞基质复合支架的具体制备步骤如下:
步骤1.配液:分别配置脱细胞基质溶液和I型胶原溶液;
步骤2.成胶:将步骤1配制的脱细胞基质溶液和I型胶原溶液混合均匀,通过调节pH使其形成水凝胶;
步骤3.冻干:将步骤2得到的水凝胶冻干,制得所述支架。
进一步地,所述脱细胞基质溶液的pH值为1.0~3.0,浓度为0.5wt%~6wt%;所述I型胶原溶液的pH值为2.0~3.0,浓度为1wt%~2wt%。
脱细胞基质溶液和I型胶原溶液的溶剂均为醋酸或盐酸溶液,前者溶液中含胃蛋白酶,胃蛋白酶与脱细胞基质的质量比为1:10~1:5。
进一步地,所述步骤2中,两溶液混合条件为冰浴,在37℃水浴条件下成胶。
与现有技术相比,本发明的积极效果体现在:
(一)、本发明设计科学,可控性强,有良好的生物相容性和生物降解性能,与原组织有一定的粘附能力,具有良好的软骨诱导性能。节约手术成本,手术操作更加简便,在关节软骨修复领域具有重要的应用价值。
(二)、本发明的软骨脱细胞基质复合支架,采用软骨细胞外基质为原材料,经物理、化学以及酶促法处理后形成脱细胞基质。目前,最复杂的合成材料和制备工艺也无法完整地模拟天然组织的细胞外基质成分和结构,而脱细胞基质作为组织的无细胞成分,储存着细胞粘附蛋白和生长因子等物质,能为细胞的增殖、迁移和分化提供理想的微环境。
(三)、本发明的软骨脱细胞基质复合支架,脱细胞基质通过蛋白酶消化后与天然高分子胶原材料共混后成胶,使得基质的成分,如生长因子、细胞粘附蛋白等在支架上分布均匀,避免对细胞的作用与影响出现不统一的现象。
(四)、本发明的软骨脱细胞基质复合支架,采用天然高分子胶原作为基体材料。胶原作为软骨组织的主要有机成分,具有良好的生物相容性和生物降解性能,用作复合材料的载体有利于软骨细胞、骨髓间充质干细胞快速黏附和增殖,并促进细胞的迁移和分化过程。
附图说明
图1为实施例1中软骨细胞外基质颗粒的扫描电镜图(a)及粒径分布图(b)
图2为不同化学方法脱细胞后DNA(a)与GAG(b)的对比图像
图3为实施例2中脱细胞前后染色对比图像
图4为实施例3中脱细胞基质消化液大体图像(a)及扫描电镜图(b)
图5为颗粒状与消化后脱细胞基质体外培养MSCs的GAG定量图像
图6为实施例6制得的胶原/脱细胞基质复合支架的扫描电镜图像
图7为实施例6制得的胶原/脱细胞基质复合支架的力学性能对比图
图8为实施例6制得的胶原/脱细胞基质复合支架的溶胀性能对比图
图9为实施例6制得的胶原/脱细胞基质复合支架的降解性能曲线
图10为培养不同时间得到的细胞支架的激光共聚焦扫描显微镜图
图11为实验例1中兔子一个月的Micro-CT图像。
具体实施方式
以下通过实施例对本发明提供的关节软骨脱细胞基质复合支架的制备方法及其应用作进一步说明。有必要指出,以下实施例只用于对本发明作进一步说明,不能理解为对本发明保护范围的限制,所属领域技术人员根据上述发明内容,对本发明做出一些非本质的改进和调整进行具体实施,仍属于发明保护的范围。
本申请中所使用的%,如无特殊说明,均表示其质量百分含量,即wt%。
实施例1
本实施例公开了细胞外基质颗粒的制备方法,具体步骤如下:
1)从猪关节软骨(猪屠宰后24h内)上刮下透明软骨组织,将其剪成小片,置于PBS溶液中超声5min,真空冷冻干燥后置于-20℃冰箱备用。
2)将步骤1)得到的冻干的细胞外基质放置于EP管中,加入研磨珠,在低温研磨机中研磨5~10min。其中,研磨温度为-20℃,频率为60Hz,机器转速为3000r/min。研磨后的颗粒外观和直径如图1所示,其直径大约为10~50μm。
实施例2
本实施例公开了一种温和的脱细胞方法,具体步骤如下:
1)将实施例1制备得到的颗粒依次置于低渗溶液和高渗溶液中清洗震荡3min,重复3次。然后将颗粒移至液氮中冷冻,再放入37℃热水浴中溶解,重复5次。
2)将颗粒移入到1%CHAPS溶液中清洗3次,在800r/min的转速下震荡3h;然后加入到100U/mL的DNAse1溶液中,在37℃下,转速为100r/min转速下震荡9小时,间隔3小时换一次新鲜DNAse1溶液,最后用0.5%的CHAPS溶液清洗15min。
3)用蒸馏水充分清洗基质,以去除所有洗涤剂。离心收集颗粒脱细胞基质(pdECM),冻干后存储在-20℃。
经过脱细胞处理后,无明显细胞碎片残留,细胞去除率达到95%及以上,GAG的保留率高达50%,具体前后染色对比如图2所示。
实施例3:
本实验在实施例2的基础上对比了三种不同化学试剂对脱细胞效果的影响(其余条件均实施例2),分别是聚乙二醇辛基苯基醚(Triton X-100)、十二烷基硫酸钠(SDS)、3-[(3-胆固醇氨丙基)二甲基氨基]-1-丙磺酸(CHAPS),对比方法如下:
表1
其中,三者溶液浓度均为1%,常温条件下震荡处理。
对六种不同处理方式的DNA、GAG进行定量分析,其结果如图2所示,综合来看,在不同时间条件下,CHAPS组脱细胞效果较其他两组更佳,具体表现为DNA去除率高的同时保留了更多的GAG。
实施例4
本实施例公开了一种脱细胞基质溶解和消化方法,具体步骤如下:
1)配置0.01M Hcl溶液,取1mL加入到样品玻璃瓶中,加入5mg胃蛋白酶,在磁力搅拌器上混合均匀。
2)称量40mg实施例2)所制备的脱细胞基质加入到步骤1)的混合溶液中,在37℃条件下搅拌48h,制得脱细胞基质消化液(sdECM),如附图4所示。
实施例5
本实施例比较了两种脱细胞基质的存在形式,即颗粒状和消化液体状,对MSCs增殖及向软骨分化的影响,具体步骤如下:
1)取20mg胶原溶于两管1mL 0.01M Hcl溶液中,记为A组、B组,在冰浴条件下搅拌均匀;
2)A组加入1mL含40mg pdECM的PBS溶液,B组加入1mL等质量的sdECM悬液,按5×106cells/mL的比例加入MSCs细胞悬浮液混匀,得到两组混合溶液,调节混合溶液的pH值至7.2~7.4。
3)将调节好pH值后的混合溶液分别入模具中,静置成胶,而后将所得水凝胶从模具中取出并浸没于培养基中,置于培养箱中在37℃、5%的CO2的条件下培养,培养期间定期每2~3天更换一次培养基。
所述培养基是在α-MEM基础培养基的基础上加入青霉素和链霉素混合液、抗坏血酸以及胎牛血清得到,α-MEM培养基中青霉素和链霉素混合液的质量浓度为1%,抗坏血酸的质量浓度为0.2%,胎牛血清的质量浓度为10%。本实施例中青霉素和链霉素混合液由HyClone公司提供。
分别于培养1天、7天、14天、21天后取出三维细胞支架,使用PBS缓冲液清洗2遍,将清洗后的三维细胞支架置于离心管中,冻干后进行DNA和GAG的定量,如附图5所示,随着时间的增加,两组脱细胞基质组细胞增殖情况更加明显,并且消化后的脱细胞基质对细胞增殖、促软骨分化作用更加显著。
实施例6
本实施例公开了胶原/脱细胞基质复合支架的制备方法,具体步骤如下:
1)取20mg胶原溶于1mL 0.01M Hcl溶液中,在冰浴条件下搅拌均匀;
2)取实施例4的脱细胞基质悬液,加入到步骤1)的胶原溶液中混合均匀,通过滴加1M NaOH调节pH至中性,此步骤均在冰浴条件下进行;
3)将步骤2)得到的胶原/脱细胞基质混合溶液,通过灌装设备注入模具中,在37℃水浴条件下成胶,冷冻干燥后得到所需的胶原/脱细胞基质复合支架,其具体结构如附图6所示,这种纤维结构保证了营养物质的输送,同时三维网络结构更有利于细胞的生长和增殖。
实施例7
将实施例6制成的复合支架在室温下置于动态力学分析仪上,测量支架的力学性能,结果如附图7所示,该支架的压缩模量为94.25KPa,高于纯胶原组,为细胞提供了一定的力学支撑。
实施例8
将实施例6制成的复合支架样品称重,记作Wo,然后浸入0.01M PBS溶液,置于恒温摇床(中国上海智成ZHWY-2012c)在37℃、90rpm的转速下振荡,每隔相应时间称量其重量,得到其质量最大值Wmax。
支架的溶胀行为及最大吸水率按以下公式计算:
最大吸水率=(Wmax-Wo)/Wo×100%
复合支架样品的溶胀性能附图7所示,结果显示脱细胞基质悬液的加入使得该支架的溶胀性能更佳,在实际的软骨缺损修复中能更好与周围组织贴合,降低了材料在术后不慎掉落的可能性。
实施例9
将实施例6制成的复合支架样品称重,记作Wo,然后浸入含有15μg/mL的I型胶原酶缓冲液中,置于恒温摇床(中国上海智成ZHWY-2012c)在37℃、90rpm的转速下振荡,每隔一段时间将支架取出冷冻干燥并称重,记作Wr。
支架的降解行为表示为质量损失百分数,按以下公式计算:
质量损失百分数=(Wo-Wr)/Wo×100%
根据不同时间段的质量损失百分数,绘制降解曲线。复合支架样品的降解曲线附图8所示,结果显示脱细胞基质的加入使得该支架的降解速率减慢,通过脱细胞基质的含量可以调节其降解速率,从而具备一定可控的生物降解性能。
实施例10
本实施例公开了所述组织工程细胞支架的制备方法,具体步骤为:
将实施例6所制得的支架灭菌后放入低粘附孔板中,向支架两面分别滴加细胞悬液,置于培养箱中孵育1~2h,再向孔板中加入培养基,在34~40℃、3%~5%的CO2的条件下培养,培养期间定期更换培养基。
所述培养基是在DMEM基础培养基的基础上加入青霉素和链霉素混合液、抗坏血酸以及胎牛血清得到,DMEM培养基中青霉素和链霉素混合液的浓度为0.8%~1.2%,抗坏血酸的浓度为0.15%~0.25%,胎牛血清的浓度为8%~12%。其中,细胞悬液的加入量为:按照5×106~5×107cells/mL的比例向基于天然材料胶原的天然高分子复合水凝胶混合液中加入细胞悬液。
分别于培养1天、7天、14天、21天后取出细胞支架,使用PBS缓冲液清洗2遍,将清洗后的细胞支架浸没于含FDA和PI的PBS缓冲液中染色1min,然后用PBS缓冲液清洗1次,通过共聚焦激光扫描显微镜(CLSM)观察支架中的细胞的生长状态和分布情况,如附图10所示,随着时间的增加,细胞增殖情况明显,并且出现细胞团聚现象。
实验1本发明复合支架的修复效果实验
1.实验方法
取实施例4制备的胶原/脱细胞基质复合支架,以新西兰大白兔为实验动物建立软骨缺损的微骨折模型,其中对照组不添加任何材料,实验组的缺损处置入胶原/脱细胞基质复合支架。术后常规抗炎,自由活动,单笼饲养。在整个观察期内植入部位无明显炎性反应,兔子活动度良好。进一步处死动物取材,进行影像学和组织学等检测分析,考察胶原/脱细胞基质复合支架在软骨修复领域的作用。
2.实验结果
实验结果如图8所示,与对照组相比,表观上支架组的软骨生成量更多,与周围组织贴合性更好;从组织学染色来看亦是如此,说明胶原/脱细胞基质复合支架在软骨修复初期有一定的促进作用。
以上所描述的实施例是本发明一部分实施例,而不是全部的实施例。本发明的实施例的详细描述并非旨在限制要求保护的本发明的范围,而是仅仅表示本发明的选定实施例。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前提下所获得的所有其它实施例,都属于本发明保护的范围。
Claims (10)
2.如权利要求1所述的软骨脱细胞基质复合支架,其特征在于:所述天然高分子或其衍生物为透明质酸、羧甲基壳聚糖、明胶、胶原、海藻酸钠和硫酸软骨素中的任意一种或几种;优选所述天然高分子或其衍生物为胶原和胶原改性的产物。
3.如权利要求1所述的软骨脱细胞基质复合支架,其特征在于,所述软骨细胞外基质来自兔或猪的关节软骨,粒径为10-50μm。
4.如权利要求1所述的软骨脱细胞基质复合支架,其特征在于,该复合支架由脱细胞后的软骨细胞外基质与天然高分子或其衍生物所组成;脱细胞方式为物理法、化学法以及酶促法,DNA去除率大于95%,GAG保留率大于40%。
5.如权利要求4所述的软骨脱细胞基质复合支架,其特征在于,所述脱细胞基质采用含胃蛋白酶的醋酸或盐酸溶液进行消化,胃蛋白酶与脱细胞基质的质量比为1:5-1:10。
6.一种如权利要求1-5任一所述软骨脱细胞基质复合支架的制备方法,其特征在于包括以下步骤:
1)将医用无菌的天然高分子或其衍生物配制成浓度为10-30mg/mL的水溶液,备用;
2)制备脱细胞基质颗粒,备用;
3)将步骤2)得到的脱细胞基质颗粒加入到含胃蛋白酶的酸性溶液中,充分搅拌48h,得到乳白色液体;
4)将步骤3)得到的乳白色液体与天然高分子材料共混,成胶后进行冷冻干燥,即可制得支架。
7.如权利要求1至5任一所述软骨脱细胞基质复合支架,其特征在于:该软骨脱细胞基质复合支架的作为软骨修复材料、或/和生物材料的应用。
8.如权利要求7所述软骨脱细胞基质复合支架,其特征在于:所述生物材料为组织工程三维细胞支架。
9.利用如权利要求1所述软骨脱细胞基质复合支架进行组织工程三维细胞支架的制备,其特征在于该方法包括以下步骤:
第一步:将制得的脱细胞基质消化液和胶原溶液灭菌后按体积比1~3:1~3混匀,预先调节pH值至5.0~6.0,然后加入细胞悬浮液混匀,调节pH值至7.0~8.0;细胞悬液的加入量为:按照5×106~5×107cells/mL的比例向基于天然材料胶原的天然高分子复合水凝胶混合液中加入细胞悬液;
第二步:将调节pH值后的混合溶液立即注射至生物体内的待修复部位形成水凝胶,得到组织工程三维支架;或者注入模具中,静置成胶,而后将所得水凝胶从模具中取出并浸没于培养基中,置于培养箱中在34~40℃、3%~5%的CO2的条件下培养至少1天,得到组织工程三维细胞支架,培养期间定期更换培养基。
10.如权利要求9所述方法,其特征在于:所述培养基是在DMEM基础培养基的基础上加入青霉素和链霉素混合液、抗坏血酸以及胎牛血清得到,DMEM培养基中青霉素和链霉素混合液的浓度为0.8%~1.2%,抗坏血酸的浓度为0.15%~0.25%,胎牛血清的浓度为8%~12%。
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