CN113456892A - 一种以人脐带沃顿胶制备软骨组织工程支架的方法及软骨组织工程支架 - Google Patents
一种以人脐带沃顿胶制备软骨组织工程支架的方法及软骨组织工程支架 Download PDFInfo
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Abstract
本发明提供一种以人脐带沃顿胶制备软骨组织工程支架的方法及软骨组织工程支架。所述以人脐带沃顿胶制备软骨组织工程支架的方法,包括以下步骤:(1)脐带标本采集与预处理;(2)沃顿胶组织块的制备;(3)沃顿胶组织块的冻融;(4)沃顿胶组织块的化学脱细胞;(5)沃顿胶组织块的冷冻干燥。本发明制备的支架可以最大限度的保留沃顿胶的细胞外基质成分,特别是促软骨分化的相关蛋白质,以及原始结构特点,同时有效清除细胞成分。沃顿胶脱细胞支架具有疏松多孔,生物相容性好,符合生物降解规律,并与软骨细胞外基质结构,成分高度相似的结构特点,为软骨组织工程支架的构建提供了新的材料体系。
Description
技术领域
本发明涉及软骨组织工程技术,特别涉及一种以人脐带沃顿胶制备软骨组织工程支架的方法及软骨组织工程支架。
背景技术
关节软骨很容易因创伤或退行性变等原因而受损。因为关节软骨不含血管和神经,细胞主要依赖组织渗透获得营养,且软骨细胞终末分化,代谢能力低下,所以软骨损伤后的自我修复能力很差。关节软骨损伤后再生困难是临床的一大挑战,也是目前研究的热点。目前用于修复关节软骨的方法有:关节腔内损伤软骨清理术、微骨折修复术、自体或异体软骨细胞植入术和自体骨软骨移植术、人工关节置换术等,但上述方法的效果却并不理想。
软骨组织工程学是一种新发展起来的有效的软骨修复方法,正在逐渐成为替代传统方法的一种很有前途的方法。培育组织工程软骨的经典方法是将种子细胞种植在特定的支架材料上,再辅以生长因子等生物活性分子进行培养,从而得到符合人体功能要求的人工软骨。支架材料其实是临时的细胞外基质,提供了软骨组织形成的微环境。软骨支架材料能够促进种子细胞的迁移、黏附,能促进干细胞顺利地向软骨细胞分化,并有利于细胞的增殖和代谢。所以,支架材料是软骨组织工程的关键一环。
目前软骨组织工程支架主要分为三种:1、天然软骨支架;2、人工合成软骨支架;3、脱细胞外基质软骨支架。天然软骨支架包括胶原、壳聚糖、透明质酸、海藻酸盐和纤维蛋白。这些天然材料具有良好的细胞亲和力和相容性,更有利于细胞的黏附和增殖,然而,由于与天然软骨相比,这些天然材料的机械性能相对较差且不稳定,目前的技术仍难以实现其临床应用。人工合成软骨支架,包括聚乳酸-羟基乙酸共聚物,聚己内酯,聚乙二醇,聚α-羟基酯:聚乳酸,通常是可生物降解的,具有良好的力学性能和较低的免疫原性,但其表面疏水,不适合细胞粘附。
脱细胞外基质软骨支架能很好的保存天然生物活性蛋白,胶原、糖胺聚糖等细胞外基质成分,其生物相容性好、细胞黏附率高,生物性降解产物易被吸收,且无炎症反应、无毒性、抗原性低,为细胞附着、增殖和再分化提供了仿生微环境,是软骨组织工程支架的理想选择。目前软骨组织工程领域的脱细胞基质材料因为来源不同而功能各异,但都存在一些问题,如关节软骨脱细胞支架具有仿生的软骨细胞外基质组成和蛋白质,它被认为是组织工程软骨的理想生物材料,但是结果发现关节软骨脱细胞支架的孔径和孔隙率往往偏小,导致细胞种植困难,且不利于营养和废物的转运;真皮脱细胞基质材料主要以I型胶原为主,其诱导干细胞向软骨细胞分化的能力不如II型胶原。另外,这类材料往往通过将捐赠的尸体标本组织经脱细胞和去免疫原性后获得,材料来源十分有限。
沃顿胶是一种包裹在人脐带血管周围的胶状物质,在很多方面与关节软骨具有高度的相似性:首先,沃顿胶虽然细胞含量很少,但是却含有非常丰富的胶原蛋白、透明质酸和硫酸糖胺聚糖,这个特点与软骨细胞外基质相似。第二,沃顿胶是疏松多孔的,其富含的蛋白多糖和透明质酸与水相互作用在基质的孔隙内形成一种高度黏性的流体。这种流体的黏性和这些孔隙结构的存在使流体在被压缩时能够运动。沃顿胶的这种多孔弹性行为使脐带能够抵抗压缩载荷,保护脐血管免受外力的伤害,保证脐静脉和动脉的血流,这在一定程度上也与软骨相似。第三,与软骨一样,沃顿胶缺少小血管、神经和淋巴。沃顿胶的营养获得及废物排泄依赖脐血管和羊水之间的组织渗透,而软骨组织的营养获得及废物排泄主要依靠与关节滑液间的组织渗透。第四,沃顿胶承载和支持脐带间充质干细胞,其广泛表达的典型透明质酸受体CD44,也表达于骨细胞、软骨细胞和造血骨髓干细胞,使这些细胞能够附着在沃顿胶上,这使得沃顿胶有支持其他未分化的间充质干细胞的能力。第五,沃顿胶还富含多肽生长因子,包括表皮生长因子(EGF)和转化生长因子-β(TGF-β)。这些多肽生长因子有利于细胞的增殖、分化和合成,以及细胞外基质的重塑,尤其是在软骨形成过程中。因此,沃顿胶在结构、生化和生物功能上与软骨相似。更重要的是,人类脐带是分娩的附件,来源广泛,没有伦理争议。
发明内容
本发明实施例的首要目的在于提供一种以人脐带沃顿胶制备软骨组织工程支架的方法。以解决目前软骨组织工程支架的缺陷,提供一种与人软骨结构成分高度相似,生物相容性好、细胞黏附率高,生物性降解产物易被吸收,且无炎症反应、无毒性、抗原性低,富含多种生长因子,原材料来源广泛的新型软骨组织工程支架。
本发明实施例的另一目的在于提供上述方法制备获得的软骨组织工程支架。
本发明的目的通过如下的技术方案实现:一种以人脐带沃顿胶制备软骨组织工程支架的方法,包括以下步骤:
(1)脐带标本采集与预处理:
在消毒后的塑料广口瓶内注入300ml含抗生素的PBS液(磷酸盐缓冲液)备用,将在产科手术室剖宫产术中离断的人脐带标本立即放入广口瓶内;在脐带离体30min内,用PBS液反复清洗,直至脐带表面及内部肉眼无可见血块及异物;将清洗后的脐带拉直,用无菌巾包裹放入普通冰箱冷冻柜-20℃预冻1h;
(2)沃顿胶组织块的制备
将预冻结束后的脐带垂直于纵向的长轴切成高9-11mm的圆柱体形组织块,精细剥离脐带外膜并切除脐带的血管,选取平整无明显缺损的部分加工成长方体的沃顿胶组织块;
(3)沃顿胶组织块的冻融
将步骤(2)获得的组织块置入含抗生素的PBS液中浸泡冲洗10min,重复3次;分别装入冻存管内,立即置入液氮(-196℃)中冷冻15min,然后将沃顿胶组织块取出并立即置入37℃水浴中复温15min,重复上述冻融过程3次;置入含抗生素的PBS液中浸泡冲洗10min,重复3次;
(4)沃顿胶组织块的化学脱细胞
将步骤(3)处理后的组织块置入1%Triton X-100+0.1%EDTA(乙二胺四乙酸)溶液中,在4℃条件下浸泡24h,含抗生素的PBS液中浸泡冲洗10min,重复3次;再置入0.1mg/mLDNA酶+1mg/mL RNA酶混合溶液,在37℃条件下消化12h,含抗生素的PBS液中浸泡冲洗10min,重复3次;
(5)沃顿胶组织块的冷冻干燥
将步骤(4)处理后的沃顿胶组织块在含抗生素的PBS液中4℃浸泡12h,将组织块放入-80℃预冻;将组织块真空冷冻干燥得到沃顿胶脱细胞软骨组织工程支架,环氧乙烷灭菌后,获得的软骨组织工程支架放入4℃冰箱密封保存备用。
进一步地,步骤(1)中所述的抗生素具体为,含青霉素1万单位/100ml,链霉素5万单位/100ml。
进一步地,步骤(2)中所述的沃顿胶组织块为,选取平整无明显缺损的部分加工成10mm×8mm×3mm大小长方体的沃顿胶组织块。
进一步地,步骤(3)中所述冻融过程是将组织块置入液氮(-196℃)中冷冻15min,然后将沃顿胶组织块取出并立即置入37℃水浴中复温15min。
进一步地,步骤(5)中所述的预冻具体为,将组织块放入-80℃的低温冰箱中预冻1h。
进一步地,步骤(5)中所述的真空冷冻干燥具体为,真空冷冻干燥机中将组织块真空冷冻干燥24h。
一种软骨组织工程支架由上述的方法制备获得。
本发明的技术方案与现有技术相比,具有以下的有益效果:
1.本发明的以人脐带沃顿胶制备软骨组织工程支架的方法,采用化学脱细胞,即组织块先是置入1%Triton X-100+0.1%EDTA溶液,然后是0.1mg/mL DNA酶+1mg/mL RNA酶混合溶液;所用的化学脱细胞方法,与之前采用的胰蛋白酶的制备方法相比,制备获得的软骨组织工程支架性能优异。
2.本发明制备获得的软骨组织工程支架,内部呈多孔状网状结构,不见细胞结构,脱细胞完全,支架壁完整度可,孔隙排列疏松,孔径大小不一,表面则呈表面均呈纵横沟壑状,沟壑表面粗糙,支架特性优良。
3.本发明制备获得的软骨组织工程支架中含有I型胶原、II型胶原、III型胶原、IV型胶原、V型胶原、VI型胶原、XII型胶原、XV型胶原、XIV型胶原和纤连蛋白,以及TGFβ、IGFBP-3和IGFBP-7、EGF等已明确证实能够促进软骨分化形成的蛋白质。
4.本发明提供了一种以人脐带沃顿胶制备软骨组织工程支架的方法,该方法制备获得的软骨组织工程支架与人软骨结构成分高度相似,孔径孔隙率高,力学性能优异,生物相容性好、细胞黏附率高,生物性降解产物易被吸收,且无炎症反应、无毒性、抗原性低,富含多种生长因子,原材料来源广泛,同时本制备方法可以最大限度的保留细胞外基质成分和结构特点。
附图说明
图1为本发明实施例1的制备流程图。
图2为本发明实施例1的沃顿胶组织块大体示意图。
图3为本发明实施例1的沃顿胶脱细胞支架大体示意图。
图4为本发明实施例1的沃顿胶脱细胞支架扫描电镜微观示意图。
图5为本发明实施例1的沃顿胶脱细胞支架三维表面形貌图。
图6为本发明实施例1的沃顿胶脱细胞支架促软骨分化相关蛋白质谱分析结果图。
图7为本发明沃顿胶未脱细胞组和沃顿胶脱细胞支架特征性傅立叶变换红外光谱图。
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的一部分实施例,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。
为解决目前软骨组织工程支架的缺陷,提供一种与人软骨结构成分高度相似,生物相容性好、细胞黏附率高,生物性降解产物易被吸收,且无炎症反应、无毒性、抗原性低,富含多种生长因子,原材料来源广泛的新型软骨组织工程支架。本发明提出了一种以人脐带沃顿胶制备软骨组织工程支架的方法及软骨组织工程支架。
实施例1
本实施例中提供一种以人脐带沃顿胶制备软骨组织工程支架的方法,具体步骤如下(如图1所示):
(1)脐带标本采集与预处理:
在消毒后的500ml塑料广口瓶内注入300ml含抗生素(青霉素:1万单位/100ml,链霉素:5万单位/100ml)的PBS液(磷酸盐缓冲液)备用,将在产科手术室剖宫产术中离断的人脐带标本立即放入广口瓶内;在脐带离体30min内,用PBS液反复清洗,直至脐带表面及内部肉眼无可见血块及异物;将清洗后的脐带拉直,用无菌巾包裹放入普通冰箱冷冻柜-20℃预冻1h;
(2)沃顿胶组织块的制备
将预冻结束后的脐带垂直其纵向的长轴切成高约10mm的圆柱体形组织块,精细剥离脐带外膜并切除脐带的血管,剩下呈长方体形状的沃顿胶组织块,选取平整无明显缺损的部分加工成10mm×8mm×3mm大小长方体的组织块如图2;
(3)沃顿胶组织块的冻融
将上述组织块置入含抗生素PBS液中浸泡冲洗10min,重复3次;分别装入冻存管内,立即置入液氮(-196℃)中冷冻15min,然后将沃顿胶组织块取出并立即置入37℃水浴中复温15min,重复上述冻融过程3次;置入含抗生素PBS液中浸泡冲洗10min,重复3次;
(4)沃顿胶组织块的化学脱细胞
将上述组织块置入1%Triton X-100+0.1%EDTA(乙二胺四乙酸)溶液中,在4℃条件下浸泡24h,含抗生素PBS液中浸泡冲洗10min,重复3次;再置入0.1mg/mL DNA酶+1mg/mLRNA酶混合溶液,在37℃条件下消化12h,含抗生素PBS液中浸泡冲洗10min,重复3次;
(5)沃顿胶组织块的冷冻干燥
将上述沃顿胶组织块在含抗生素PBS液中浸泡12h,将组织块放入-80℃的低温冰箱中预冻1h;真空冷冻干燥机中将组织块真空冷冻干燥24h得到沃顿胶脱细胞软骨组织工程支架,如图3;环氧乙烷灭菌后放入冰箱(温度为4℃)密封保存备用。
我们利用扫描电镜和原子力显微镜对沃顿胶脱细胞支架的内部以及表面微观结构进行观察测定,得到图4和图5;可以看到沃顿胶脱细胞支架内部呈多孔状网状结构,不见细胞结构,脱细胞完全,支架壁完整度可,孔隙排列疏松,孔径大小不一,表面则呈表面均呈纵横沟壑状,沟壑表面粗糙。
同时为了分析沃顿胶脱细胞支架所含与促软骨分化有关蛋白质的种类,我们进行了蛋白质质谱分析,得到图6,可以看到沃顿胶脱细胞支架中含有I型胶原、II型胶原、III型胶原、IV型胶原、V型胶原、VI型胶原、XII型胶原、XV型胶原、XIV型胶原和纤连蛋白,以及TGFβ、IGFBP-3和IGFBP-7、EGF等已明确证实能够促进软骨分化形成的蛋白质。
对比实施例1
(1)采集脐带标本:
往消毒后的500ml玻璃广口瓶内注入300ml含抗生素生理盐水(庆大霉素:1万单位/100ml,青霉素:5万单位/100ml)备用,将在剖宫产术中离断的人脐带标本立即置入上述广口瓶内;
(2)清洗脐带标本:
为保证脐带新鲜,于剖宫产离断脐带20min内将广口瓶内脐带取出进行处理,首先经过磷酸盐缓冲液(PBS液)反复冲洗,至到脐带表面无血块和异物残留,以及脐血管中无血块能挤出;
(3)预冻脐带标本:
将清洗后的脐带拉直,简单固定尽量使脐带不扭曲,放入普通冰箱冷冻室-20℃预冻30min后取出;
(4)制作分梯度的沃顿胶组织块:
将经过预冻的脐带标本垂直其长轴切成高约20mm的圆柱体,剥离脐带外膜后完整切除中心脐血管区域,剩下呈环柱体形状的沃顿胶组织块,将环柱体沃顿胶组织块沿环柱体高度线切开,将沃顿胶拉伸展开成一大块长方体,最后选取较平整无缺损的部位将沃顿胶加工成12mm×8mm×3mm大小长方体的组织块;
1)将沃顿胶组织块置入含抗生素PBS液(庆大霉素:1万单位/100ml,青霉素:5万单位/100ml)中浸泡冲洗5min,重复3次;
2)双蒸水浸泡沃顿胶组织块1min;
3)胰蛋白酶(2.5g/l)消化组织块(37℃,反复震荡)15min;
4)双蒸水震荡漂洗组织块5min;
5)将漂洗干净的组织块装入冻存管里,快速置入含液氮罐的液氮(-196℃)中冷冻15min,取出后将沃顿胶组织块立即经37℃水浴快速复温,重复上述急冻急复温过程3次;
6)再次将组织块置入含抗生素PBS液(庆大霉素:1万单位/100ml,青霉素:5万单位/100ml)中,浸泡冲洗10min次;
7)双蒸水浸泡组织块5小时,每15min震荡1次;
8)将组织块置入-80℃的低温冰箱预冻1h从而调节孔径大小;
9)真空冷冻干燥机中将组织块真空冷冻干燥12h得到沃顿胶梯度脱细胞软骨支架材料;
10)将得到的沃顿胶梯度脱细胞软骨支架材料使用环氧乙烷灭菌8h后放入冰箱(温度为4℃)密封保存备用。
实施例1和对比实施例1的脱细胞效果对比:
两者DNA清除率均在90%以上,对比实施例1获得的软骨组织工程支架的DNA清除率91.49%±1.74%,实施例1获得的软骨组织工程支架的DNA清除率为94.17%±0.73%。两种对比,经统计学分析显示有差异(P=0.026<0.05),实施例1比对比实施例1的DNA清除率更高。
实施例1和对比实施例1的细胞外基质成分保留效果:
傅立叶变换红外光谱检测结果显示沃顿胶有多糖峰(1140-985cm-1)和酰胺I峰(1720-1590cm-1),利用OMNIC9软件,分别积分(1140-985cm-1)峰面积和(1720-1590cm-1)峰面积来表征支架中糖胺聚糖和胶原的含量。在沃顿胶脱细胞支架中,和未脱细胞组比,糖胺聚糖和胶原含量,对比实施例1的软骨组织工程支架分别减少71.52%和61.72%,实施例1获得的软骨组织工程支架分别减少59.41%和4.65%;两组糖胺聚糖和胶原减少比例经统计学分析有差异(P=0.029<0.05,P<0.001),本发明实施例1糖胺聚糖和胶原含量减少比例比对比实施例1的低。
如图7所示,沃顿胶未脱细胞组和沃顿胶脱细胞支架特征性傅立叶变换红外光谱图,多糖峰和酰胺I峰分别代表糖胺聚糖和胶原。A为未脱细胞对应图,B为实施例1支架对应图,C为对比实施例1支架对应图。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (7)
1.一种以人脐带沃顿胶制备软骨组织工程支架的方法,其特征在于:包括以下步骤:
(1)脐带标本采集与预处理:
在消毒后的塑料广口瓶内注入300ml含抗生素的PBS液备用,将在产科手术室剖宫产术中离断的人脐带标本立即放入广口瓶内;在脐带离体30min内,用PBS液反复清洗,直至脐带表面及内部肉眼无可见血块及异物;将清洗后的脐带拉直,用无菌巾包裹放入普通冰箱冷冻柜-20℃预冻1h;
(2)沃顿胶组织块的制备
将预冻结束后的脐带垂直于纵向的长轴切成高9-11mm的圆柱体形组织块,精细剥离脐带外膜并切除脐带的血管,选取平整无明显缺损的部分加工成长方体的沃顿胶组织块;
(3)沃顿胶组织块的冻融
将步骤(2)获得的组织块置入含抗生素的PBS液中浸泡冲洗10min,重复3次;分别装入冻存管内,立即置入液氮中冷冻15min,然后将沃顿胶组织块取出并立即置入37℃水浴中复温15min,重复上述冻融过程3次;置入含抗生素的PBS液中浸泡冲洗10min,重复3次;
(4)沃顿胶组织块的化学脱细胞
将步骤(3)处理后的组织块置入1%TritonX-100+0.1%EDTA溶液中,在4℃条件下浸泡24h,含抗生素的PBS液中浸泡冲洗10min,重复3次;再置入0.1mg/mLDNA酶+1mg/mLRNA酶混合溶液,在37℃条件下消化12h,含抗生素的PBS液中浸泡冲洗10min,重复3次;
(5)沃顿胶组织块的冷冻干燥
将步骤(4)处理后的沃顿胶组织块在含抗生素的PBS液中4℃浸泡12h,将组织块放入-80℃预冻;将组织块真空冷冻干燥得到沃顿胶脱细胞软骨组织工程支架,环氧乙烷灭菌后,获得的软骨组织工程支架放入4℃冰箱密封保存备用。
2.根据权利要求1所述的以人脐带沃顿胶制备软骨组织工程支架的方法,其特征在于:步骤(1)中所述的抗生素具体为,含青霉素1万单位/100ml,链霉素5万单位/100ml。
3.根据权利要求1所述的以人脐带沃顿胶制备软骨组织工程支架的方法,其特征在于:步骤(2)中所述的沃顿胶组织块为,选取平整无明显缺损的部分加工成10mm×8mm×3mm大小长方体的沃顿胶组织块。
4.根据权利要求1所述的以人脐带沃顿胶制备软骨组织工程支架的方法,其特征在于:步骤(3)中所述冻融过程是将组织块置入液氮中冷冻15min,然后将沃顿胶组织块取出并立即置入37℃水浴中复温15min。
5.根据权利要求1所述的以人脐带沃顿胶制备软骨组织工程支架的方法,其特征在于:步骤(5)中所述的预冻具体为,将组织块放入-80℃的低温冰箱中预冻1h。
6.根据权利要求1所述的以人脐带沃顿胶制备软骨组织工程支架的方法,其特征在于:步骤(5)中所述的真空冷冻干燥具体为,真空冷冻干燥机中将组织块真空冷冻干燥24h。
7.一种软骨组织工程支架由权利要求1-6任一项所述的方法制备获得。
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