CN108236739A - 一种应用于软骨损伤修复的新型复合材料 - Google Patents
一种应用于软骨损伤修复的新型复合材料 Download PDFInfo
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Abstract
本发明公开了一种应用于软骨损伤修复的新型复合材料,其由细胞外基质、软骨生成蛋白、人源性脐带间充质干细胞组合而成,所述细胞外基质取自胎盘,且与所述软骨生成蛋白组合成细胞外基质复合材料,所述人员性脐带间充质干细胞选自人源性脐带组织和胎盘组织。本发明的人源性脐带间充质干细胞对软骨损伤具有良好的修复功能,同时,胎盘来源的ECM材料富含胶原及弹力纤维,并且在制备过程中保留了各种细胞因子,能够为接种的干细胞提供良好的生长和分化的微环境,两者相结合与传统材料对比大幅度的提高了损伤软骨的修复能力。
Description
技术领域
本发明涉及属于生物材料领域,尤其是一种应用于软骨损伤修复的新型复合材料及其制备方法。
背景技术
随着全球人口老龄化、慢性疾病和运动创伤的增加,关节软骨损伤危害着数千万人的健康,逐渐成为人类致残的主要原因。近年来,修复关节软骨损伤一直是骨科研究的热点。由于关节软骨损伤后自我修复能力较弱,目前的治疗手段和方法虽能不同程度上缓解疼痛、改善功能障碍,然而疗效短暂,损伤部位新生的软骨组织最终会退化甚至坏死,难以获得满意的远期临床疗效。组织工程,再生医疗技术是近年来兴起的修复软骨损伤的方法,为治疗关节软骨损伤带来新的希望。
软骨组织工程材料的特性与天然关节软骨细胞外基质的特性相似程度越高,越有利于成功构建组织工程软骨。目前常见的细胞外基质(ECM)来源有猪软骨细胞脱细胞处理后的ECM、同种异体软骨细胞处理后的ECM以及采用II型胶原蛋白、氨基聚糖等配比产生的类似于ECM性质的ACTM生物材质等,存在治疗疗效时间短,修复能力差等问题。
发明内容
本发明的目的是克服现有技术存在的缺陷,提供一种应用于软骨损伤修复的新型复合材料及其制备方法。
实现本发明目的的技术方案是:一种应用于软骨损伤修复的新型复合材料,其由细胞外基质、软骨生成蛋白、人源性脐带间充质干细胞组合而成,所述细胞外基质取自胎盘,且与所述软骨生成蛋白组合成细胞外基质复合材料,所述人员性脐带间充质干细胞选自人源性脐带组织和胎盘组织。
作为一种应用于软骨损伤修复的新型复合材料的优化,所述细胞外基质复合材料的主要成分为:胶原(Collagen)60-75%、弹性蛋白(Elastin)10-15%、葡萄糖胺聚糖(GAGs)<1%、纤连蛋白(Fibronectin)<1%、层粘连蛋白(Laminin)<1%、骨形态发生蛋白(BMPs)<1%,同时每毫升含转化生长因子(Transforming Growth Factor Beta,TGFb)1100毫微克、碱性成纤维细胞生长因子(Basic Fibroblast Growth Factor,bFGF)1400毫微克、上表皮生长因子(Epidermal Growth Factor,EGF)200毫微克、血小板源性生长因子(platelet derived growth factor,PDGF)200毫微克、胰岛素样生长因子(InsulinlikeGrowth Factors 1,IGF1)800-900毫微克、血管内皮生长因子(vascular endothelialgrowth factor,VEGF)800毫微克。
细胞外基质复合材料的主要成分的作用:胶原(Collagen)是细胞外最重要的水不溶性纤维蛋白,是构成细胞外基质的骨架,分子量在1000道尔顿以下的acmetea修复型胶原蛋白生物性高分子物质。胶原在细胞外基质中形成半晶体的纤维,给细胞提供抗张力和弹性,并在细胞的迁移和发育中起作用;在复合材料中胶原作为最主要的成分,因其弹性和在细胞迁移和发育中的作用,作为骨架,可以辅助细胞在复合材料上着床及发育。弹性蛋白(Elastin)像胶原一样,可以为细胞外基质提供强度和韧性,但是因为其伸展度是正常长度的几倍,在复合材料中提供弹性,该成分为复合材料提供弹性和可塑性,为本材料各种塑形及迁移提供基础。葡萄糖胺聚糖(GAGs)为本复合材料中提供定向输送的材料,其原理为大量蛋白多糖聚合体形成有许多微孔的分子筛,允许水和营养物、代谢产物、激素、气体分子等通过;而大于孔隙的大分子物质、细菌等则被阻挡,使基质成为限制细菌等有害物扩散的防御屏障。纤连蛋白(Fibronectin)的主要功能是介导细胞粘着。纯化的纤连蛋白可增强细胞间粘连及细胞与基质的粘连。通过粘着,纤连蛋白可以通过细胞信号转导途径调节细胞的形状和细胞骨架的组织,促进细胞铺展,固在本品中,人源性纤连蛋白为间充质干细胞在材料支架上黏连和定向分化为软骨细胞都提供了必备的条件。层粘连蛋白(Laminin)主要存在基膜(basal lamina)中,是基膜所特有的费胶原糖蛋白,其主要作用为介导细胞与基膜结合,在本品中主要是介导细胞与骨架结合,在细胞发育中几次细胞粘着,细胞运动和促进细胞分化为软骨细胞并促进成年神经损伤后重生长和再生。骨形态发生蛋白(BMPs)在本品中,主动加入骨形态发生蛋白的目的是够诱导动物或人体间充质细胞分化为骨、软骨、韧带、肌腱和神经组织,以此来刺激负载的人间充质干细胞分化为各类细胞,从而修复软骨组织的损伤。转化生长因子(TGF)是指两类多肽类生长因子:转化生长因子-α和转化生长因子-β,本复合材料中使用人类转化生长因子-β(Transforming growth factor beta,TGF-β)是一多功能蛋白质,可以影响多种细胞的生长,分化、细胞凋亡及免疫调节等功能,在复合材料中主要促进细胞生长和分化,及调节免疫防止感染得功效。成纤维细胞生长因子(fibroblast growth factor,FGF)有几种异构体,在本复合材料中起作用的主要是bFGF(basic fibroblast growth factor),bFGF可以由内皮细胞、平滑肌细胞、巨噬细胞分泌。它的作用是促进内皮细胞的游走和平滑肌细胞的增殖,不能使平滑肌细胞游走。能够促进新血管形成,修复损害的内皮细胞,在本复合材料中主要是起促进成骨细胞大量生成,抑制破骨细胞和控制间充质干细胞转移的目的。上表皮生长因子(Epidermal Growth Factor,EGF)在本复合材料中使用Hegfr,主要作用有两项:一是EGFR复合物开始促进DNA合成,并由此趋向刺激内皮细胞、单核细胞等多种细胞分裂、增殖和分化,使之向创伤部位迁移,加速启动创伤组织再生、修复和胞外间质形成;另一方面,hEGF能增加其他内源性生长因子,促进羟脯氨酸合成,调节胶原酶和胶原的合成、分泌和沉淀,调节胶原降解,使胶原纤维以线性方式排列,增强创面抗张程度,减少疤痕形成,提高愈合质量。血小板源性生长因子(platelet derived growth factor,PDGF)能刺激停滞于G0/G1期的成纤维细胞、神经胶质细胞、平滑肌细胞等多种细胞进入分裂增殖周期,在本材料中,其主要作用是在组织受到损伤时,激活间充质干细胞并释放PDGF,其可以几次特定细胞群分裂增殖的能力,促进创伤修复处细胞生长,增殖。胰岛素样生长因子(Insulinlike Growth Factors 1,IGF1)是一类多功能细胞增殖调控因子。在细胞的分化、增殖、个体的生长发育中具有重要的促进作用,在本品中该因子主要是与其他因子一起,刺激软骨生长,其与生长激素(growth hormone)等共同作用,可以刺激软骨生长及与其他因子通过特异性的靶细胞表面受体结合而在细胞生长各阶段中刺激各类生长,分化作用。血管内皮生长因子(vascular endothelial growthfactor,VEGF)主要用于刺激血管生长,虽然软骨组织中血管组织较少,但是充足的血液供应是修复软骨的重要影响因素,在本材料中,其主要作用是刺激修复部位周围毛细血管生成,为修复区提供血液流动。
细胞外基质复合材料的基本性状为:所得支架状材料为白色、多孔状结构,扫描电镜下观察可见支架内部孔隙互相连通,孔径大小较均一,直径多分布于200um-500um。
本方案一种应用于软骨损伤修复的新型复合材料的制备方法,包括以下步骤:
(1)细胞外基质复合材料的制备;
①细胞外基质浆料的制备;
②细胞外基质与软骨生成蛋白组合成复合材料的制备;
(2)人源性脐带间充质干细胞的制备;
(3)将步骤(2)的人源性脐带间充质干细胞负载在步骤(1)细胞外基质复合材料上待使用。
作为优化,一种应用于软骨损伤修复的新型复合材料的制备方法,包括以下步骤:
(1)细胞外基质复合材料的制备:
①细胞外基质浆料的制备:
A、取得胎盘,在无菌条件下剥离胎盘上羊膜和下基膜及明显血管,余下的组织用无菌三蒸水至无血液成分流出后,用双氧水浸泡后,加入无菌三蒸水,在低温下用搅碎机粉碎成细胞外胶质匀浆;
B、取步骤A得到的匀浆,在匀浆中加入10倍体积的三蒸水混匀,冷冻后于常温融化,反复3~4次冻融,使残余细胞破碎;然后将匀浆离心,取上清加入聚乙二醇辛基苯基醚、胰蛋白酶、三羟甲基氨基甲烷盐酸盐缓冲液,于4℃条件下搅拌,洗脱后离心取上清液待用;
C、取步骤B的上清液用DNA消化酶和RNA消化酶混合液于37℃消化后离心,再用无菌三蒸水洗去细胞碎片和残留物质,然后调至pH 7.0后取上层匀浆进行离心,收集沉淀即为细胞外基质浆料;
②细胞外基质与软骨生成蛋白组合成复合材料的制备:
D、取步骤①得到的细胞外基质浆料,加入三蒸水调为混悬液,充分搅拌均匀待用;
E、取软骨生成蛋白加入步骤D待用的混悬液,充分混匀后注入模具中,预冻后放入冷冻干燥机中进行冻干,形成三维多孔海绵支架;
F、将步骤E得到的支架置于紫外线照射下进行交联,然后再放入含N-羟基琥珀酰亚胺和乙基-二甲基胺-丙基碳化二亚胺的乙醇溶液中交联,再无菌条件下用磷酸缓冲液(PBS)漂洗浸泡,最后用三蒸水漂洗去除残余交联剂,经冻干后密封保存备用;
(2)人源性脐带间充质干细胞的制备;
①选取人源性脐带组织和胎盘组织,在无菌条件下剥离胎盘上羊膜和下基膜及明显血管,余下的组织用无菌三蒸水反复冲洗直至无血液成分流出后,用双氧水浸泡后剪成片状并在F12培养基中进行贴壁培养;
②观察到人源性脐带间充质干细胞生长后,使用胰酶-EDTA消化贴壁培养皿15-30S,用培养基中止消化后离心,取沉淀细胞在6个康宁75cm矩形培养瓶中,再加入无血清培养基DEME/F-12孵育细胞继续培养到培养瓶80%面积时,用姨酶消化所有间充质干细胞,离心后用冻存液封存于外旋冻存管中待用;
(3)使用前一日,将步骤(2)冷存的人源性脐带间充质干细胞解冻,按照数量为1*106的人源性脐带间充质干细胞负载在10g支架材料上的比例,将人源性脐带间充质干细胞加入康宁培养皿中的三维多孔海绵支架上,再用二氧化碳培养箱培养,负载2小时以上就可用注治疗。
作为优化,一种应用于软骨损伤修复的新型复合材料的制备方法,包括以下步骤:
(1)细胞外基质复合材料的制备:
①细胞外基质浆料的制备:
A、取得新鲜胎盘,在无菌条件下剥离胎盘上羊膜和下基膜及明显血管,余下的组织用无菌三蒸水,反复冲洗3次,每次15min,直至无血液成分流出后,用3%双氧水浸泡30min后,加入无菌三蒸水,在低温4℃下用医用搅碎机反复粉碎成细胞外胶质匀浆;三蒸水为无热源且经过三次蒸馏后的蒸馏水。
B、取步骤A得到的匀浆,在匀浆中加入10倍体积的(匀浆体积V/三蒸水体积V)三蒸水混匀,零下20℃下冷冻后于常温融化,反复3~4次冻融,使残余细胞破碎;然后将匀浆经2000r/min的低速离心机离心20min,取上清加入1%聚乙二醇辛基苯基醚(Triton X-100)、0.25%胰蛋白酶、pH为7.5的三羟甲基氨基甲烷盐酸盐缓冲液(Tris-HCl缓冲液),于4℃条件下轻轻搅拌,洗脱24h后,经3000r/min离心20min取上清液待用;
C、取步骤B的上清液用50u/ml DNA消化酶(DNase)和1u/ml RNA消化酶(RNase)混合液于37℃消化12h,然后再4℃下经6000r/min离心15~20min,再用无菌三蒸水或磷酸盐缓冲液(PBS)充分洗去细胞碎片和残留物质,然后调至pH 7.0后取上层匀浆在4℃下经10000r/min离心40min,收集沉淀即为去细胞的纳米级细胞外基质浆料;
②细胞外基质与软骨生成蛋白组合成复合材料的制备:
D、取步骤①得到的细胞外基质浆料加入三蒸水,调为细胞外基质浆料质量比上三蒸水体积为3%的混悬液,充分搅拌均匀待用;
E、取软骨生成蛋白(BMPs)加入步骤D待用的混悬液,调为软骨生成蛋白质量比上步骤D待用的混悬液体积为0.1%的混悬液,在磁力搅拌机中充分混匀后注入聚乙烯圆筒模具中,零下20℃下预冻30min,后放入冷冻干燥机中进行冻干,48h后形成三维多孔海绵支架;
F、将步骤E得到的支架置于258nm波长紫外线照射下进行交联8h,然后再放入含20mmol/L N-羟基琥珀酰亚胺和50mmol/L乙基-二甲基胺-丙基碳化二亚胺的乙醇溶液中,在低温4℃下交联24h,再无菌条件下用磷酸缓冲液(PBS)漂洗浸泡2h,最后用三蒸水漂洗去除残余交联剂,经冻干后密封保存,保存环境为60Co照射消毒,4℃条件下保存备用;
(2)人源性脐带间充质干细胞的制备;
①选取适宜的人源性脐带组织和胎盘组织,在无菌条件下剥离胎盘上羊膜和下基膜及明显血管,余下的组织用无菌三蒸水反复冲洗,反复冲洗3次,每次15min,直至无血液成分流出后,用3%双氧水浸泡30min后剪成小型正方形片状并在F12培养基中,在37℃、二氧化碳浓度5%的条件下进行贴壁培养;
②在显微镜镜检下观察到人源性脐带间充质干细胞生长后,使用5%胰酶-EDTA消化贴壁培养皿15-30S,用培养基中止消化后,经1500rpm离心5min,取沉淀细胞在6个康宁(Corning)75cm矩形培养瓶中,再加入无血清培养基DEME/F-12孵育细胞继续培养到培养瓶80%面积时,用姨酶消化所有间充质干细胞,离心后用冻存液封存于2.5ml康宁外旋冻存管中,于零下86℃保存箱中保存待用;
(3)使用前一日,将步骤(2)冷存的人源性脐带间充质干细胞解冻,按照数量为1*106的人源性脐带间充质干细胞负载在10g支架材料上的比例,将人源性脐带间充质干细胞加入康宁培养皿中的三维多孔海绵支架上,再在37℃、二氧化碳浓度5%的条件下培养箱培养,负载2小时以上就可用注治疗。
作为优化,一种应用于软骨损伤修复的新型复合材料的制备方法,所述步骤(1)①A中的胎盘为新鲜胎盘,且经检测无各类传染病,无癌症表现,无自身免疫病。
作为优化,一种应用于软骨损伤修复的新型复合材料的制备方法,所述步骤(1)①C中也可以用磷酸盐缓冲液充分洗去细胞碎片和残留物质。
作为优化,一种应用于软骨损伤修复的新型复合材料的制备方法,所述步骤(1)②F中N-羟基琥珀酰亚胺、乙基-二甲基胺-丙基碳化二亚胺两者与乙醇的体积比为95%。
作为优化,一种应用于软骨损伤修复的新型复合材料的制备方法,所述步骤(2)②中DEME/F12无血清培养基是由10%胎牛血清替代物、1%碱性成纤维细胞生长因子、200mmol/l谷氨酰胺加入DEME/F-12培养基中混合而成。
作为优化,一种应用于软骨损伤修复的新型复合材料的制备方法,所述步骤(2)②中所述冻存液是由20%人血白蛋白、无血清培养基、二甲基亚砜(DMSO)按照5:4:1的比例混匀后而成。
本发明有以下使用效果和优势:
1、本复合材料组合包含细胞生长的必备细胞外微环境及各类促进细胞生长,分化的细胞因子,其复合组合为现阶段领先的,各类细胞因子的添加解决了目前单纯间充质干细胞在软骨修复处直接注射治疗的分化难题,同时细胞外基质3D支架的结合为间充质干细胞在修复处的黏连,着床提供了良好的生长微环境。
2、本复合材料有别于现阶段各类非生物类材料的在于其生化活性,其来源是人源性材料,从本质上各类成分与人体自有成分无差异,不存在排异反应,同时可以为创伤处的软骨炎症等提供一个抑制功能,这些都是非生物类材料所不具有的。
3、本复合材料组合因为使用了各型胶原蛋白,层粘连蛋白,纤连蛋白及弹性蛋白等,其可塑性比之同类材料有很大提升,可以多样结合患者特殊的创伤环境,有利于修复发展。
4、本组合中的细胞外支架因其生化特效可以有效的负载各种细胞因子并帮助维持各类细胞因子活性,其限制作用可以保留大量细胞因子于修复处,定向刺激组合负载的人间充质干细胞生长分化为软骨细胞,结缔组织细胞及周围毛细血管,从而使软骨损伤组织完全修复恢复其生理生化作用。
本发明人源性脐带间充质干细胞对软骨损伤具有良好的修复功能,同时,胎盘来源的ECM材料富含胶原及弹力纤维,并且在制备过程中保留了各种细胞因子,能够为接种的干细胞提供良好的生长和分化的微环境,两者相结合与传统材料对比大幅度的提高了损伤软骨的修复能力。
具体实施方式
本发明是一种应用于软骨损伤修复的新型复合材料,其由细胞外基质、软骨生成蛋白、人源性脐带间充质干细胞组合而成,所述细胞外基质取自胎盘,且与所述软骨生成蛋白组合成细胞外基质复合材料,所述人员性脐带间充质干细胞选自人源性脐带组织和胎盘组织。所述细胞外基质复合材料的主要成分为:胶原(Collagen)60-75%、弹性蛋白(Elastin)10-15%、葡萄糖胺聚糖(GAGs)<1%、纤连蛋白(Fibronectin)<1%、层粘连蛋白(Laminin)<1%、骨形态发生蛋白(BMPs)<1%,同时每毫升含转化生长因子(Transforming Growth Factor Beta,TGFb)1100毫微克、碱性成纤维细胞生长因子(BasicFibroblast Growth Factor,bFGF)1400毫微克、上表皮生长因子(Epidermal GrowthFactor,EGF)200毫微克、血小板源性生长因子(platelet derived growth factor,PDGF)200毫微克、胰岛素样生长因子(Insulinlike Growth Factors 1,IGF1)800-900毫微克、血管内皮生长因子(vascular endothelial growth factor,VEGF)800毫微克。
细胞外基质复合材料的主要成分的作用:胶原(Collagen)是细胞外最重要的水不溶性纤维蛋白,是构成细胞外基质的骨架,分子量在1000道尔顿以下的acmetea修复型胶原蛋白生物性高分子物质。胶原在细胞外基质中形成半晶体的纤维,给细胞提供抗张力和弹性,并在细胞的迁移和发育中起作用;在复合材料中胶原作为最主要的成分,因其弹性和在细胞迁移和发育中的作用,作为骨架,可以辅助细胞在复合材料上着床及发育。弹性蛋白(Elastin)像胶原一样,可以为细胞外基质提供强度和韧性,但是因为其伸展度是正常长度的几倍,在复合材料中提供弹性,该成分为复合材料提供弹性和可塑性,为本材料各种塑形及迁移提供基础。葡萄糖胺聚糖(GAGs)为本复合材料中提供定向输送的材料,其原理为大量蛋白多糖聚合体形成有许多微孔的分子筛,允许水和营养物、代谢产物、激素、气体分子等通过;而大于孔隙的大分子物质、细菌等则被阻挡,使基质成为限制细菌等有害物扩散的防御屏障。纤连蛋白(Fibronectin)的主要功能是介导细胞粘着。纯化的纤连蛋白可增强细胞间粘连及细胞与基质的粘连。通过粘着,纤连蛋白可以通过细胞信号转导途径调节细胞的形状和细胞骨架的组织,促进细胞铺展,固在本品中,人源性纤连蛋白为间充质干细胞在材料支架上黏连和定向分化为软骨细胞都提供了必备的条件。层粘连蛋白(Laminin)主要存在基膜(basal lamina)中,是基膜所特有的费胶原糖蛋白,其主要作用为介导细胞与基膜结合,在本品中主要是介导细胞与骨架结合,在细胞发育中几次细胞粘着,细胞运动和促进细胞分化为软骨细胞并促进成年神经损伤后重生长和再生。骨形态发生蛋白(BMPs)在本品中,主动加入骨形态发生蛋白的目的是够诱导动物或人体间充质细胞分化为骨、软骨、韧带、肌腱和神经组织,以此来刺激负载的人间充质干细胞分化为各类细胞,从而修复软骨组织的损伤。转化生长因子(TGF)是指两类多肽类生长因子:转化生长因子-α和转化生长因子-β,本复合材料中使用人类转化生长因子-β(Transforming growth factor beta,TGF-β)是一多功能蛋白质,可以影响多种细胞的生长,分化、细胞凋亡及免疫调节等功能,在复合材料中主要促进细胞生长和分化,及调节免疫防止感染得功效。成纤维细胞生长因子(fibroblast growth factor,FGF)有几种异构体,在本复合材料中起作用的主要是bFGF(basic fibroblast growth factor),bFGF可以由内皮细胞、平滑肌细胞、巨噬细胞分泌。它的作用是促进内皮细胞的游走和平滑肌细胞的增殖,不能使平滑肌细胞游走。能够促进新血管形成,修复损害的内皮细胞,在本复合材料中主要是起促进成骨细胞大量生成,抑制破骨细胞和控制间充质干细胞转移的目的。上表皮生长因子(Epidermal Growth Factor,EGF)在本复合材料中使用Hegfr,主要作用有两项:一是EGFR复合物开始促进DNA合成,并由此趋向刺激内皮细胞、单核细胞等多种细胞分裂、增殖和分化,使之向创伤部位迁移,加速启动创伤组织再生、修复和胞外间质形成;另一方面,hEGF能增加其他内源性生长因子,促进羟脯氨酸合成,调节胶原酶和胶原的合成、分泌和沉淀,调节胶原降解,使胶原纤维以线性方式排列,增强创面抗张程度,减少疤痕形成,提高愈合质量。血小板源性生长因子(platelet derived growth factor,PDGF)能刺激停滞于G0/G1期的成纤维细胞、神经胶质细胞、平滑肌细胞等多种细胞进入分裂增殖周期,在本材料中,其主要作用是在组织受到损伤时,激活间充质干细胞并释放PDGF,其可以几次特定细胞群分裂增殖的能力,促进创伤修复处细胞生长,增殖。胰岛素样生长因子(Insulinlike Growth Factors 1,IGF1)是一类多功能细胞增殖调控因子。在细胞的分化、增殖、个体的生长发育中具有重要的促进作用,在本品中该因子主要是与其他因子一起,刺激软骨生长,其与生长激素(growth hormone)等共同作用,可以刺激软骨生长及与其他因子通过特异性的靶细胞表面受体结合而在细胞生长各阶段中刺激各类生长,分化作用。血管内皮生长因子(vascular endothelial growthfactor,VEGF)主要用于刺激血管生长,虽然软骨组织中血管组织较少,但是充足的血液供应是修复软骨的重要影响因素,在本材料中,其主要作用是刺激修复部位周围毛细血管生成,为修复区提供血液流动。
细胞外基质复合材料的基本性状为:所得支架状材料为白色、多孔状结构,扫描电镜下观察可见支架内部孔隙互相连通,孔径大小较均一,直径多分布于200um-500um。
本发明一种应用于软骨损伤修复的新型复合材料的制备方法,包括以下步骤:
(1)细胞外基质复合材料的制备:
①细胞外基质浆料的制备:
A、取得新鲜胎盘,胎盘为新鲜胎盘,且经检测无各类传染病,无癌症表现,无自身免疫病,在无菌条件下剥离胎盘上羊膜和下基膜及明显血管,余下的组织用无菌三蒸水,反复冲洗3次,每次15min,直至无血液成分流出后,用3%双氧水浸泡30min后,加入无菌三蒸水,在低温4℃下用医用搅碎机反复粉碎成细胞外胶质匀浆;三蒸水为无热源且经过三次蒸馏后的蒸馏水。
B、取步骤A得到的匀浆,在匀浆中加入10倍体积的(匀浆体积V/三蒸水体积V)三蒸水混匀,零下20℃下冷冻后于常温融化,反复3~4次冻融,使残余细胞破碎;然后将匀浆经2000r/min的低速离心机离心20min,取上清加入1%聚乙二醇辛基苯基醚(Triton X-100)、0.25%胰蛋白酶、pH为7.5的三羟甲基氨基甲烷盐酸盐缓冲液(Tris-HCl缓冲液),于4℃条件下轻轻搅拌,洗脱24h后,经3000r/min离心20min取上清液待用;
C、取步骤B的上清液用50u/ml DNA消化酶(DNase)和1u/ml RNA消化酶(RNase)混合液于37℃消化12h,然后再4℃下经6000r/min离心15~20min,再用无菌三蒸水或磷酸盐缓冲液(PBS)充分洗去细胞碎片和残留物质,然后调至pH 7.0后取上层匀浆在4℃下经10000r/min离心40min,收集沉淀即为去细胞的纳米级细胞外基质浆料;
②细胞外基质与软骨生成蛋白组合成复合材料的制备:
D、取步骤①得到的细胞外基质浆料加入三蒸水,调为细胞外基质浆料质量比上三蒸水体积为3%的混悬液,充分搅拌均匀待用;
E、取软骨生成蛋白(BMPs)加入步骤D待用的混悬液,调为软骨生成蛋白质量比上步骤D待用的混悬液体积为0.1%的混悬液,在磁力搅拌机中充分混匀后注入聚乙烯圆筒模具中,零下20℃下预冻30min,后放入冷冻干燥机中进行冻干,48h后形成三维多孔海绵支架;
F、将步骤E得到的支架置于258nm波长紫外线照射下进行交联8h,然后再放入含20mmol/L N-羟基琥珀酰亚胺和50mmol/L乙基-二甲基胺-丙基碳化二亚胺的乙醇溶液中,在低温4℃下交联24h,再无菌条件下用磷酸缓冲液(PBS)漂洗浸泡2h,最后用三蒸水漂洗去除残余交联剂,经冻干后密封保存,保存环境为60Co照射消毒,4℃条件下保存备用;其中,N-羟基琥珀酰亚胺、乙基-二甲基胺-丙基碳化二亚胺两者与乙醇的体积比为95%。
(2)人源性脐带间充质干细胞的制备;
①选取适宜的人源性脐带组织和胎盘组织,在无菌条件下剥离胎盘上羊膜和下基膜及明显血管,余下的组织用无菌三蒸水反复冲洗,反复冲洗3次,每次15min,直至无血液成分流出后,用3%双氧水浸泡30min后剪成小型正方形片状并在F12培养基中,在37℃、二氧化碳浓度5%的条件下进行贴壁培养;
②在显微镜镜检下观察到人源性脐带间充质干细胞生长后,使用5%胰酶-EDTA消化贴壁培养皿15-30S,用培养基中止消化后,经1500rpm离心5min,取沉淀细胞在6个康宁(Corning)75cm矩形培养瓶中,再加入无血清培养基DEME/F-12孵育细胞继续培养到培养瓶80%面积时,用姨酶消化所有间充质干细胞,离心后用冻存液封存于2.5ml康宁外旋冻存管中,于零下86℃保存箱中保存待用;其中,DEME/F12无血清培养基是由10%胎牛血清替代物、1%碱性成纤维细胞生长因子、200mmol/l谷氨酰胺加入DEME/F-12培养基中混合而成,冻存液是由20%人血白蛋白、无血清培养基、二甲基亚砜(DMSO)按照5:4:1的比例混匀后而成
(3)使用前一日,将步骤(2)冷存的人源性脐带间充质干细胞解冻,按照数量为1*106的人源性脐带间充质干细胞负载在10g支架材料上的比例,将人源性脐带间充质干细胞加入康宁培养皿中的三维多孔海绵支架上,再在37℃、二氧化碳浓度5%的条件下培养箱培养,负载2小时以上就可用注治疗。
本发明治疗骨关节炎动物模型实验实施例:
本实验使用新西兰白兔为实验模型动物(国际标准骨关节损伤造模动物,购置于具有相关资质的实验动物公司,品种为新西兰白兔,雌性,年龄12-16周,体重2.0-2.5kg,接受日期为2016年9月12日)动物到达后按SOPA030的要求接收。给每只动物确定一个独立的检疫流水号。分组时为每只动物指定一个单一的研究动物号。动物及笼卡上标明动物号。原始资料中使用研究动物号来识别。
按照实验设计的要求,将12只兔子的左膝用石膏固定于伸直位,固定范围从踝关节下3cm至腹股沟下1.5cm,踝背屈30~40°,时间6周,遂后去除石膏制成骨关节炎模型,按照固定肢体使关节制动可以诱发关节软骨萎缩,致使软骨变薄、水肿,蛋白聚糖含量下降,结构改变以及合成数量的下降,同时还有胶原合成及含量的增加。因为这些软骨改变与我们所见的人类骨关节炎病理改变相似,从而模拟软骨骨关节炎,退行性性变等问题。
将动物随机分成3组,分别为:实验组(本发明治疗的移植租)、对照组(用与实验组等量生理盐水移植租)、模型组(不做任何处理)三组进行相互对照实验,每组4只动物。
根据实验设计各组给予相应的治疗,模型组立即处死,取骨关节软骨造模处组织进行观察;实验组通过关节腔注射给予1×105人源性脐带间充质干细胞(HUCMSCs)及细胞外基质复合材料0.5克,每周1次连续4次;对照组注射等量生理盐水;实验组和对照组在最后一次治疗后1周处死所有动物。
对比一:抽取关节液0.2ml,留检IL-1浓度;用ELISA双抗夹心法标记所收集关节液中的IL-1β,使呈有色反应,酶标仪450nm处读取OD值,通过绘制标准曲线,计算IL-1β浓度。
计算关节液中IL-1β浓度结果为:实验组组健侧(16.37±0.44pg/ml)、对照组健侧(14.35±0.68pg/ml)、模型组健侧(15.10±0.36pg/ml),三组比较,两两差异不显著(P>0.367);实验组患侧(24.33±2.20pg/ml)、对照组患侧(20.16±1.07pg/ml)、模型组患侧(20.70±1.02pg/ml,三组比较,两两差异不显著(P>0.727)。
对比二:各组动物总计12只,软骨组织做同样处理,即:取滑膜和股骨髁部软骨做光镜标本。滑膜4%甲醛固定,常规脱水、浸蜡包埋,切片,HE染色观察滑膜形态学变化。软骨标本4%多聚甲醛固定,15%乙二胺四乙酸脱钙10周,石蜡包埋,HE染色观察软骨形态学变化,AB-PAS染色观察多糖着色;将所得的切片进行Mankin′s评分,评分标准如下表所示。
Mankin′s评分标准:
将滑膜和股骨髁部软骨进行光镜检测结果:对照组患侧可见细胞坏死;实验组组健侧镜下与模型组无明显差异。由相应的PAS染色可以看出,实验组患侧软骨染色较浅,对照组着色最深,呈鲜紫红色,可见实验组软骨修复发生。Mankin’s评分:对光镜下滑膜及软骨进行Mankin’s评分,实验组无论健侧还是患侧,其关节软骨及滑膜的Mankin’s评分均不低于模型组及对照组,个体差异较大,可见明显的实验组患侧软骨组织修复痕迹。
对比三:各组动物总计12只,软骨组织做同样处理,即:取滑膜立刻置入0.25%戊二醛中做电镜标本,1%锇酸固定2h,梯度乙醇及丙西酮脱水,618环氧树脂包埋,超薄片机切成60nm厚薄片,醋酸铀及枸橼酸铅双重染色,透射电镜下观察。
滑膜电镜检测结果:实验组软骨细胞经过修复再生后,细胞学形态比模型组及对照组有明显改善,证实本发明复合材料组合对骨损伤的修复活动。
通过上述实验可以得出,本发明在治疗软骨损伤具有良好的修复功能,疗效时间短,修复能力好。本发明的治疗优势在于:
1、本复合材料组合包含细胞生长的必备细胞外微环境及各类促进细胞生长,分化的细胞因子,其复合组合为现阶段领先的,各类细胞因子的添加解决了目前单纯间充质干细胞在软骨修复处直接注射治疗的分化难题,同时细胞外基质3D支架的结合为间充质干细胞在修复处的黏连,着床提供了良好的生长微环境。
2、本复合材料有别于现阶段各类非生物类材料的在于其生化活性,其来源是人源性材料,从本质上各类成分与人体自有成分无差异,不存在排异反应,同时可以为创伤处的软骨炎症等提供一个抑制功能,这些都是非生物类材料所不具有的。
3、本复合材料组合因为使用了各型胶原蛋白,层粘连蛋白,纤连蛋白及弹性蛋白等,其可塑性比之同类材料有很大提升,可以多样结合患者特殊的创伤环境,有利于修复发展。
4、本组合中的细胞外支架因其生化特效可以有效的负载各种细胞因子并帮助维持各类细胞因子活性,其限制作用可以保留大量细胞因子于修复处,定向刺激组合负载的人间充质干细胞生长分化为软骨细胞,结缔组织细胞及周围毛细血管,从而使软骨损伤组织完全修复恢复其生理生化作用。
本发明的人源性脐带间充质干细胞对软骨损伤具有良好的修复功能,同时,胎盘来源的ECM材料富含胶原及弹力纤维,并且在制备过程中保留了各种细胞因子,能够为接种的干细胞提供良好的生长和分化的微环境,两者相结合与传统材料对比大幅度的提高了损伤软骨的修复能力。
以上所述的具体实施例,对本发明的目的、技术方案和有益效果进行了进一步详细说明,所应理解的是,以上所述仅为本发明的具体实施例而已,并不用于限制本发明,凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.其特征在于:其由细胞外基质、软骨生成蛋白、人源性脐带间充质干细胞组合而成,所述细胞外基质取自胎盘,且与所述软骨生成蛋白组合成细胞外基质复合材料,所述人员性脐带间充质干细胞选自人源性脐带组织和胎盘组织。
2.根据权利要求1所述的一种应用于软骨损伤修复的新型复合材料,其特征在于,所述细胞外基质复合材料的主要成分为:胶原60-75%、弹性蛋白10-15%、葡萄糖胺聚糖<1%、纤连蛋白<1%、层粘连蛋白<1%、骨形态发生蛋白<1%,同时每毫升含转化生长因子1100毫微克、碱性成纤维细胞生长因子1400毫微克、上表皮生长因子200毫微克、血小板源性生长因子200毫微克、胰岛素样生长因子800-900毫微克、血管内皮生长因子800毫微克。
3.一种应用于软骨损伤修复的新型复合材料的制备方法,其特征在于,包括以下步骤:
(1)细胞外基质复合材料的制备;
①细胞外基质浆料的制备;
②细胞外基质与软骨生成蛋白组合成复合材料的制备;
(2)人源性脐带间充质干细胞的制备;
(3)将步骤(2)的人源性脐带间充质干细胞负载在步骤(1)细胞外基质复合材料上待使用。
4.根据权利要求3所述的一种应用于软骨损伤修复的新型复合材料的制备方法,其特征在于,包括以下步骤:
(1)细胞外基质复合材料的制备:
①细胞外基质浆料的制备:
A、取得胎盘,在无菌条件下剥离胎盘上羊膜和下基膜及明显血管,余下的组织用无菌三蒸水至无血液成分流出后,用双氧水浸泡后,加入无菌三蒸水,在低温下用搅碎机粉碎成细胞外胶质匀浆;
B、取步骤A得到的匀浆,在匀浆中加入10倍体积的三蒸水混匀,冷冻后于常温融化,反复3~4次冻融,使残余细胞破碎;然后将匀浆离心,取上清加入聚乙二醇辛基苯基醚、胰蛋白酶、三羟甲基氨基甲烷盐酸盐缓冲液,于4℃条件下搅拌,洗脱后离心取上清液待用;
C、取步骤B的上清液用DNA消化酶和RNA消化酶混合液于37℃消化后离心,再用无菌三蒸水洗去细胞碎片和残留物质,然后调至pH 7.0后取上层匀浆进行离心,收集沉淀即为细胞外基质浆料;
②细胞外基质与软骨生成蛋白组合成复合材料的制备:
D、取步骤①得到的细胞外基质浆料,加入三蒸水调为混悬液,充分搅拌均匀待用;
E、取软骨生成蛋白加入步骤D待用的混悬液,充分混匀后注入模具中,预冻后放入冷冻干燥机中进行冻干,形成三维多孔海绵支架;
F、将步骤E得到的支架置于紫外线照射下进行交联,然后再放入含N-羟基琥珀酰亚胺和乙基-二甲基胺-丙基碳化二亚胺的乙醇溶液中交联,再无菌条件下用磷酸缓冲液(PBS)漂洗浸泡,最后用三蒸水漂洗去除残余交联剂,经冻干后密封保存备用;
(2)人源性脐带间充质干细胞的制备;
①选取人源性脐带组织和胎盘组织,在无菌条件下剥离胎盘上羊膜和下基膜及明显血管,余下的组织用无菌三蒸水反复冲洗直至无血液成分流出后,用双氧水浸泡后剪成片状并在F12培养基中进行贴壁培养;
②观察到人源性脐带间充质干细胞生长后,使用胰酶-EDTA消化贴壁培养皿15-30S,用培养基中止消化后离心,取沉淀细胞在6个康宁75cm矩形培养瓶中,再加入无血清培养基DEME/F-12孵育细胞继续培养到培养瓶80%面积时,用姨酶消化所有间充质干细胞,离心后用冻存液封存于外旋冻存管中待用;
(3)使用前一日,将步骤(2)冷存的人源性脐带间充质干细胞解冻,按照数量为1*106的人源性脐带间充质干细胞负载在10g支架材料上的比例,将人源性脐带间充质干细胞加入康宁培养皿中的三维多孔海绵支架上,再用二氧化碳培养箱培养,负载2小时以上就可用注治疗。
5.根据权利要求4所述的一种应用于软骨损伤修复的新型复合材料的制备方法,其特征在于,包括以下步骤:
(1)细胞外基质复合材料的制备:
①细胞外基质浆料的制备:
A、取得胎盘,在无菌条件下剥离胎盘上羊膜和下基膜及明显血管,余下的组织用无菌三蒸水反复冲洗3次,每次15min,直至无血液成分流出后,用3%双氧水浸泡30min后,加入无菌三蒸水,在低温4℃下用搅碎机反复粉碎成细胞外胶质匀浆;
B、取步骤A得到的匀浆,在匀浆中加入10倍体积的三蒸水混匀,零下20℃下冷冻后于常温融化,反复3~4次冻融,使残余细胞破碎;然后将匀浆经2000r/min的低速离心机离心20min,取上清加入1%聚乙二醇辛基苯基醚、0.25%胰蛋白酶、pH为7.5的三羟甲基氨基甲烷盐酸盐缓冲液,于4℃条件下轻轻搅拌,洗脱24h后,经3000r/min离心20min取上清液待用;
C、取步骤B的上清液用50u/mlDNA消化酶和1u/mRNA消化酶混合液于37℃消化12h,然后再4℃下经6000r/min离心15~20min,再用无菌三蒸水充分洗去细胞碎片和残留物质,然后调至pH 7.0后取上层匀浆在4℃下经10000r/min离心40min,收集沉淀即为去细胞的纳米级细胞外基质浆料;
②细胞外基质与软骨生成蛋白组合成复合材料的制备:
D、取步骤①得到的细胞外基质浆料加入三蒸水,调为细胞外基质浆料质量比上三蒸水体积为3%的混悬液,充分搅拌均匀待用;
E、取软骨生成蛋白加入步骤D待用的混悬液,调为软骨生成蛋白质量比上步骤D待用的混悬液体积为0.1%的混悬液,在磁力搅拌机中充分混匀后注入聚乙烯圆筒模具中,零下20℃下预冻30min,后放入冷冻干燥机中进行冻干,48h后形成三维多孔海绵支架;
F、将步骤E得到的支架置于258nm波长紫外线照射下进行交联8h,然后再放入含20mmol/L N-羟基琥珀酰亚胺和50mmol/L乙基-二甲基胺-丙基碳化二亚胺的乙醇溶液中,在低温4℃下交联24h,再无菌条件下用磷酸缓冲液(PBS)漂洗浸泡2h,最后用三蒸水漂洗去除残余交联剂,经冻干后密封保存备用;
(2)人源性脐带间充质干细胞的制备;
①选取适宜的人源性脐带组织和胎盘组织,在无菌条件下剥离胎盘上羊膜和下基膜及明显血管,余下的组织用无菌三蒸水反复冲洗,反复冲洗3次,每次15min,直至无血液成分流出后,用3%双氧水浸泡30min后剪成小型正方形片状并在F12培养基中,在37℃、二氧化碳浓度5%的条件下进行贴壁培养;
②在显微镜镜检下观察到人源性脐带间充质干细胞生长后,使用5%胰酶-EDTA消化贴壁培养皿15-30S,用培养基中止消化后,经1500rpm离心5min,取沉淀细胞在6个康宁75cm矩形培养瓶中,再加入无血清培养基DEME/F-12孵育细胞继续培养到培养瓶80%面积时,用姨酶消化所有间充质干细胞,离心后用冻存液封存于2.5ml康宁外旋冻存管中,于零下86℃保存箱中保存待用;
(3)使用前一日,将步骤(2)冷存的人源性脐带间充质干细胞解冻,按照数量为1*106的人源性脐带间充质干细胞负载在10g支架材料上的比例,将人源性脐带间充质干细胞加入康宁培养皿中的三维多孔海绵支架上,再在37℃、二氧化碳浓度5%的条件下培养箱培养,负载2小时以上就可用注治疗。
6.根据权利要求4或5所述的一种应用于软骨损伤修复的新型复合材料的制备方法,其特征在于:所述步骤(1)①A中的胎盘为新鲜胎盘,且经检测无各类传染病,无癌症表现,无自身免疫病。
7.根据权利要求4或5所述的一种应用于软骨损伤修复的新型复合材料的制备方法,其特征在于:所述步骤(1)①C中也可以用磷酸盐缓冲液充分洗去细胞碎片和残留物质。
8.根据权利要求5所述的一种应用于软骨损伤修复的新型复合材料的制备方法,其特征在于:所述步骤(1)②F中N-羟基琥珀酰亚胺、乙基-二甲基胺-丙基碳化二亚胺两者与乙醇的体积比为95%。
9.根据权利要求4或5所述的一种应用于软骨损伤修复的新型复合材料的制备方法,其特征在于:所述步骤(2)②中DEME/F12无血清培养基是由10%胎牛血清替代物、1%碱性成纤维细胞生长因子、200mmol/l谷氨酰胺加入DEME/F-12培养基中混合而成。
10.根据权利要求4或5所述的一种应用于软骨损伤修复的新型复合材料的制备方法,其特征在于:所述步骤(2)②中所述冻存液是由20%人血白蛋白、无血清培养基、二甲基亚砜(DMSO)按照5:4:1的比例混匀后而成。
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