CN111249529B - 一种用于软骨修复的仿生多层胶原支架及其制备方法 - Google Patents
一种用于软骨修复的仿生多层胶原支架及其制备方法 Download PDFInfo
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Abstract
本发明涉及一种医用材料，具体涉及软骨损伤再生领域，主要公开了一种用于软骨修复的仿生多层胶原支架及其制备方法。本发明所述支架产品具有多层三维空间结构，通过降低材料的免疫原性，以及包括致密层、多孔层与屏障层的多层叠加设计，促进体内细胞募集，实现无外源细胞的生物材料诱导的体内软骨再生，并优化再生策略，适用于软骨损伤的治疗和再生。
Description
技术领域
本发明涉及一种医用材料，具体涉及软骨损伤再生领域，主要公开了一种用于软骨修复的仿生多层胶原支架及其制备方法。
背景技术
由创伤或骨病所致关节部位骨软骨缺损在临床较常见，且严重影响患者生活质量，已成为目前肢体残障的主要原因之一。膝部疾病可发生在各个年龄层的人群。膝部软骨缺损可由创伤、扭伤、过度使用膝部、肌肉无力或一般磨损导致。由于关节软骨特殊的组织结构及生物学特性，虽然已知有许多治疗关节软骨缺损的方法，包括但不限于：软骨下骨钻孔，电刺激，激光，药物及细胞注射，基因治疗等等，但是上述方法都达不到满意的修复效果。
近年来，关节软骨损伤再生领域也有很多研究进展，如软骨细胞表型退化与维持、干细胞募集和分化、免疫调控、生物活性材料等，临床上也进行了诸多尝试，但总体效果仍难令人满意。
快速发展的组织工程技术，为软骨的再生修复提供了新策略。组织工程中，组织细胞依附于由生物材料所制备的支架上，植入体内的病损部位，使之最终达到修复重建组织或器官、恢复功能目的。随着细胞增殖，支架材料不断降解，被细胞外基质代替，逐渐形成具有正常功能的组织。
在目前的研究中，用于修复关节软骨的支架材料主要有透明质酸(Hyaluronate，HA)，聚乳酸(polylactic，PLA)，聚乳酸-羟基乙酸(poly(lactic-co-glycolic)，PLGA)，胶原(collagen)，壳聚糖(chitosan)等，或者其中几种材料的复合亦常见。
前述天然活性生物材料中，以胶原、透明质酸等天然软骨成分为代表，可通过纯化工艺提升生物相容性，且保持活性。人工合成材料生物相容性好，便于精确控制和原位成型，但通常来说缺乏生物活性，需加入活性分子。
一方面，理想的工程化软骨复合组织构建策略是具有功能界面的一体化三维支架机构；另一方面，理想的支架材料应具有良好的生物相容性，利于细胞粘附、增生和分化，可降解吸收性，通过人工调控可以使其降解吸收速度与体内新生组织生长速度相匹配；以及良好的机械特性，能满足植入部位的力学要求。
临床实践中，目前对软骨损伤还以微骨折和自体组织移植为主，辅助以细胞治疗、生物材料等组织工程产品，如体外添加细胞的胶原支架增强自体软骨细胞移植为胶原膜，其粗糙面用于接种体外分离、培养、扩增的软骨细胞。该产品中的自体软骨细胞来源于患者活检的膝部软骨组织，研究人员对这些细胞进行培养，并将这些细胞种植在一个纯化、可吸收、来源于猪的胶原膜。
上述胶原薄膜产品可用于修复成人的软骨缺损，但临床效果并不显著好，且有费用高、技术复杂等缺点。同时，根据研究报道，少数患者在进行治疗后出现一些副作用，主要包括关节疼痛、感冒样症状、头痛或背部疼痛。
因此，为了能够同时解决目前软骨修复实践中的不足，即生物材料诱导性低、免疫排斥风险高、有效性低和治疗费用高的问题，除了考虑支架仿生材料的选择和制备工艺，也必须考虑其仿生空间结构的设计及修复功能的实现。
发明内容
本发明研究生物医用材料学、细胞生物学、关节外科学等交叉学科，公开了一种用于软骨修复的仿生多层胶原支架及其制备方法。
本发明的目的在于创建全新的关节软骨功能性修复材料，通过材料空间结构的设计，促进体内细胞募集，实现不依赖外源细胞、生物材料诱导的体内软骨功能性再生。
从其制备方法上来说，首先优化调整猪肌腱胶原提取和纯化工艺，使得胶原蛋白含量大于99％，多糖含量小于0.1％，以提高支架材料的生物相容性、降低免疫原性。
之后通过改良现有产品技术，优化胶原支架的设计：从薄膜改成三维支架材料、从致密结构改成纵向多孔主体结构。采用液氮冷冻等工艺，制备出具有纵向孔隙结构的横向层叠胶原多孔支架，提高其细胞募集效果、增加体内软骨诱导再生活性。
最后设计具备屏障层的多层复合胶原支架，在基底层胶原层上复合羟基磷灰石屏障层，促进软骨下骨再生以及新生软骨与软骨下骨的结合。产品分别适用于局灶性软骨缺损和部分合并软骨下骨缺损。
为解决上述技术问题，本发明的技术方案是：
一种用于软骨修复的仿生多层胶原支架，其特征在于，所述仿生多层胶原支架包括上层和基底层，其中各层之间为横向平行叠加，其中上层为致密结构的胶原层，基底层为纵向交联多孔结构的胶原层。优选的，所述胶原来自猪肌腱胶原提取和纯化，胶原蛋白含量大于99％，多糖含量小于0.1％，所述百分比为质量百分比。
优选的，所述上层致密层的厚度为0.1-0.5毫米，所述基底层的厚度为2.0-5.0毫米。
更优选的，所述上层致密层的厚度为0.3毫米，所述基底层的厚度为2.5毫米。
进一步的，在所述基底层上，相对于致密层的另一端复合羟基磷灰石屏障层，形成多层结构。
所述用于软骨修复的仿生多层胶原支架的制备方法，包含以下步骤：
1.致密层-纵向交联多孔层胶原支架
步骤1将剥离的肌腱粉碎后加入10倍体积的4M盐酸胍，所述盐酸胍为pH＝7.5、0.05M Tris-HCl配制；
步骤2混悬后，粉碎匀浆，于4℃搅拌24h，12000g离心力下离心20min，分离得到上清液和沉淀；
步骤3沉淀用Tris-HCl缓冲液和0.5mM乙酸充分洗涤后再加入乙酸过夜，以除去蛋白多糖；
步骤4用含胃蛋白酶的冰乙酸溶液溶解肌腱，持续处理一段时间待混合物变成无色、透明、粘稠液体后在4℃，5000g离心力下离心20min，收集上清即为粗制肌腱胶原蛋白原液；
步骤5将氯化钠溶液加入其中，持续搅拌直至有白色絮状沉淀从溶液中析出，继续加入氯化钠溶液至沉淀不在析出为止；
步骤6在4℃温度，5000g离心力下离心20min后获得白色沉淀；
步骤7沉淀物加入一定浓度的酸溶液溶解，将溶解后的胶原蛋白溶液继续反复进行盐析处理，重复2～4次；
步骤8持续在超纯水中透析3天，每天换液3次，以除去胶原溶液中的无机盐类；
步骤9将平铺的4mg/mL胶原溶液冷冻成型后经高渗缓冲溶液浸泡脱水，挤压成膜厚度0.1-0.5毫米；
步骤10在该膜表面再平铺一层胶原溶液厚度2.0-5.0毫米，移入-80℃的冰箱低温预冷2h后，液氮中冷冻2h，真空冷冻干燥得到既有致密层又有纵向多孔结构的胶原支架。
2.多孔胶原-羟基磷灰石多层胶原支架：将纳米羟基磷灰石粉末悬于蒸馏水中，控制固液比在20/80左右，低温下超声波分散15min，迅速加到上述步骤10中在-80℃的冰箱低温预冷的胶原表面，4℃静置过夜，移入液氮中冷冻2h，真空冷冻干燥得到多孔胶原-羟基磷灰石多层支架复合材料。
进一步的，还包括上述仿生多层胶原支架材料在制备软骨修复装置方面的应用。
在本阶段，材料理化表征和安全性评价：
1)化学性能检测
a)羟基磷灰石的纯度按GB 23101.3测定；
b)胶原的总蛋白含量按凯氏定氮法测定；
c)胶原的羟脯氨酸含量按羟脯氨酸测定法测定；
d)重金属含量按《中国药典2015版第四部》中“0821-重金属检测法”测定；
e)pH值按GB9724测试
f)透明质酸钠的要求按YY/T 0606.9-2007.5测定；
g)葡萄糖醛含量按照YY/T 0606.9-2007附录A测定；
2)物理性能检测
a)尺寸用精度为0.02毫米的游标卡尺测量，颗粒参照GB/T1480测定；
b)压缩强度按GB/T1964检测；
c)气孔率、密度及容重按GB/T1966检测；
d)孔径按GB/T1967电镜扫描法检测。
3)生物安全性评价
a)遗传毒性、致癌性和生殖毒性试验按GB/T16886.3检验；
b)与血液相互作用试验按GB/T16886.4检验；
c)体外细胞毒性试验按GB/T16886.5检验；
d)植入后局部反应试验按GB/T16886.6检验；
e)降解产物的定性与定量试验按GB/T16886.9和GB/T16886.13检验；
f)刺激与致敏试验按GB/T16886.10检验；
g)全身急性、亚急性、亚慢性和慢性毒性试验按GB/T16886.11检验；
h)降解产物的毒代动力学试验按GB/T16886.16检验；
i)热原反应试验按YY/T 1500-2016检验；
j)无菌试验按GB/T 19973.2检验；
k)细菌内毒素试验按YY/T 1295-2015检验。
(2)材料的有效性评价：
1)体外有效性评价
a)细胞增殖：取材料或材料浸提液，接种骨髓间充质干细胞/软骨细胞(原代分离培养细胞)，接种后不同时间点以CCK-8试剂盒测定细胞数量，绘制细胞增殖曲线。同时设PBS处理组为空白对照，未处理组为阴性对照，用SPSS软件统计分析实验结果。对于具有明显增殖活性的材料，利用BrdU标记进一步确证其促增殖活性。
b)细胞趋化：用材料包被孔板底部，加入1×105个骨髓间充质干细胞/软骨细胞。
待细胞长满，用10μL的枪头沿皿底做一个轻微划痕划线，并用PBS清洗干净划痕处的细胞，做好标记，用无血清培养基培养细胞，每隔12h观察并采集图片，统计细胞迁移数量。
c)细胞分化：①qRT-PCR实验：将材料等份放入6孔板，每块材料上接种1×105个骨髓间充质干细胞/，接种后不同时间点移除培养基，加入Trizol试剂，将溶液转移至无RNA酶离心管中，提取RNA，反转录成cDNA。以GAPDH基因为内参，通过qRT-PCR检测干性基因和软骨标志基因在不同时间点mRNA的相对表达水平。②蛋白印迹实验：将多孔材料等份放入6孔板，每块材料上接种1×105个骨髓间充质干细胞，接种后不同时间点消化并收集细胞，加裂解液提细胞总蛋白。用BCA试剂盒对总蛋白定量，确定上样体积，然后电泳、转膜、抗原-抗体反应、显色、测定内参GAPDH及细胞特异性蛋白的条带灰度值，分析细胞特异性蛋白在不同时间点的相对表达水平。
2)体内(动物)实验有效性研究(组织学、影像学、生物力学)
以小型猪作为动物模型，小型猪双侧膝关节均进行制作模型，在小型猪膝关节股骨负重部位，使用软骨打孔装置制作膝关节直径为16毫米的全层软骨缺损，同样使用胶原支架或者凝胶填充软骨缺损处，逐层缝合手术切口，允许实验动物在笼内自由活动。于术后3个月、6个月和12个月，抽取实验动物外周血进行生化指标检测；对实验动物膝关节进行核磁共振扫描检测软骨缺损填充情况，修复组织与宿主组织的结合以及修复软骨与软骨下骨的结合情况；处死实验动物取股骨标本，使用微纳米综合力学测试***分别对软骨缺损区域以及正常软骨区域的正向垂直载荷量、剪切模量、弹性模量、摩擦系数、修复组织边界状态进行分析，以检测修复组织的生物力学特性；使用Micro CT检测软骨下骨重塑情况；并进行组织学切片进行HE染色，甲苯胺蓝染色，番红O染色，采用ICRS组织学评分***评价修复情况，将切片进行I型胶原蛋白、II型胶原蛋白、X型胶原蛋白、osteocalcin、Lubricin免疫组织化学染色，使用凋亡检测试剂盒来观察新生组织的退变；获取实验动物肝、肾、脑、心脏等重要器官检测材料降解产物残留。
本发明所述的多层胶原支架显示出符合设计目的和要求的理化性能和生物相容性，其体外细胞存活率大于对照的80％，体内研究未见周围组织坏死、明显炎症反应、感染，三月有效促进软骨再生，维持到12月，软骨没有蜕变。
通过关节镜和开放手术在小型猪关节软骨缺损植入材料，术后3个月实现诱导关节软骨形成并维持其表型，术后6个月和一年再生软骨在负重和力学刺激下无退变。
软骨缺损填充达到95％以上，修复组织均为透明软骨组织；修复软骨组织能承载6倍于体重的压力，动力摩擦系数小于0.005，伸展强度达到5-25Mpa；修复组织与宿主组织无缝连接，促进修复组织中软骨层与软骨下骨层无缝连接，现实了软骨下骨再生以及新生软骨与软骨下骨的结合。
因此，本发明的有益效果是：具备三维多层立体结构，可促进细胞募集；经纯化工艺的支架材料生物相容性好、免疫原性低；通过所述的“无细胞”生物材料修复软骨缺损可以大幅度降低成本、简化手术流程、缩短治疗时间和降低并发症。
附图说明
图1是多层胶原支架的结构示意图。
1-致密胶原层
2-纵向交联多孔胶原层
3-羟基磷灰石屏障层
图2是多孔胶原支架的形状示意图。
图3是关节软骨修复情况。
具体实施方式
实施例1
将剥离的肌腱粉碎后加入10倍体积的4M盐酸胍，所述盐酸胍为pH＝7.5、0.05MTris-HCl配制；
混悬后，粉碎匀浆，于4℃搅拌24h，12000g离心20min，分离得到上清液和沉淀；
沉淀用Tris-HCl缓冲液和0.5mM乙酸充分洗涤后再加入乙酸过夜，以除去蛋白多糖；
用含胃蛋白酶的冰乙酸溶液溶解肌腱，持续处理一段时间待混合物变成无色、透明、粘稠液体后在4℃，5000g离心力下离心20min，收集上清即为粗制肌腱胶原蛋白原液；
将氯化钠溶液加入其中，持续搅拌直至有白色絮状沉淀从溶液中析出，继续加入氯化钠溶液至沉淀不在析出为止；
4℃，5000g离心力下离心20min后获得白色沉淀；
沉淀物加入一定浓度的酸溶液溶解，将溶解后的胶原蛋白溶液继续反复进行盐析处理，重复2～4次；
持续在超纯水中透析3天，每天换液3次，以除去胶原溶液中的无机盐类；
将平铺的4mg/mL胶原溶液冷冻成型后经高渗缓冲溶液浸泡脱水，挤压成膜厚度0.5毫米；
在该膜表面再平铺一层胶原溶液厚度2.0毫米，移入-80℃的冰箱低温预冷2h后，液氮中冷冻2h，真空冷冻干燥得到既有致密层又有纵向多孔结构的胶原支架。
实施例2
将剥离的肌腱粉碎后加入10倍体积的4M盐酸胍，所述盐酸胍为pH＝7.5、0.05MTris-HCl配制；
混悬后，粉碎匀浆，于4℃搅拌24h，12000g离心20min，分离得到上清液和沉淀；
沉淀用Tris-HCl缓冲液和0.5mM乙酸充分洗涤后再加入乙酸过夜，以除去蛋白多糖；
用含胃蛋白酶的冰乙酸溶液溶解肌腱，持续处理一段时间待混合物变成无色、透明、粘稠液体后在4℃，5000g离心力下离心20min，收集上清即为粗制肌腱胶原蛋白原液；
将氯化钠溶液加入其中，持续搅拌直至有白色絮状沉淀从溶液中析出，继续加入氯化钠溶液至沉淀不在析出为止；
4℃，5000g离心力下离心20min后获得白色沉淀；
沉淀物加入一定浓度的酸溶液溶解，将溶解后的胶原蛋白溶液继续反复进行盐析处理，重复2～4次；
持续在超纯水中透析3天，每天换液3次，以除去胶原溶液中的无机盐类；
将平铺的4mg/mL胶原溶液冷冻成型后经高渗缓冲溶液浸泡脱水，挤压成膜厚度0.2毫米；
在该膜表面再平铺一层胶原溶液厚度3.0毫米，移入-80℃的冰箱低温预冷2h后，液氮中冷冻2h，真空冷冻干燥得到既有致密层又有纵向多孔结构的胶原支架。
实施例3
将剥离的肌腱粉碎后加入10倍体积的4M盐酸胍，所述盐酸胍为pH＝7.5、0.05MTris-HCl配制；
混悬后，粉碎匀浆，于4℃搅拌24h，12000g离心20min，分离得到上清液和沉淀；
沉淀用Tris-HCl缓冲液和0.5mM乙酸充分洗涤后再加入乙酸过夜，以除去蛋白多糖；
用含胃蛋白酶的冰乙酸溶液溶解肌腱，持续处理一段时间待混合物变成无色、透明、粘稠液体后在4℃，5000g离心力下离心20min，收集上清即为粗制肌腱胶原蛋白原液；
将氯化钠溶液加入其中，持续搅拌直至有白色絮状沉淀从溶液中析出，继续加入氯化钠溶液至沉淀不在析出为止；
4℃，5000g离心力下离心20min后获得白色沉淀；
沉淀物加入一定浓度的酸溶液溶解，将溶解后的胶原蛋白溶液继续反复进行盐析处理，重复2～4次；
持续在超纯水中透析3天，每天换液3次，以除去胶原溶液中的无机盐类；
将平铺的4mg/mL胶原溶液冷冻成型后经高渗缓冲溶液浸泡脱水，挤压成膜厚度0.3毫米；
在该膜表面再平铺一层胶原溶液厚度2.5毫米，移入-80℃的冰箱低温预冷2h；
将纳米羟基磷灰石粉末悬于蒸馏水中，控制固液比在20/80左右，低温下超声波分散15min，迅速加到上述-80℃的冰箱低温预冷的胶原表面，4℃静置过夜，移入液氮中冷冻2h，真空冷冻干燥得到多孔胶原-羟基磷灰石多层支架复合材料。
实施例4
将剥离的肌腱粉碎后加入10倍体积的4M盐酸胍，所述盐酸胍为pH＝7.5、0.05MTris-HCl配制；
混悬后，粉碎匀浆，于4℃搅拌24h，12000g离心20min，分离得到上清液和沉淀；
沉淀用Tris-HCl缓冲液和0.5mM乙酸充分洗涤后再加入乙酸过夜，以除去蛋白多糖；
用含胃蛋白酶的冰乙酸溶液溶解肌腱，持续处理一段时间待混合物变成无色、透明、粘稠液体后在4℃，5000g离心力下离心20min，收集上清即为粗制肌腱胶原蛋白原液；
将氯化钠溶液加入其中，持续搅拌直至有白色絮状沉淀从溶液中析出，继续加入氯化钠溶液至沉淀不在析出为止；
4℃，5000g离心力下离心20min后获得白色沉淀；
沉淀物加入一定浓度的酸溶液溶解，将溶解后的胶原蛋白溶液继续反复进行盐析处理，重复2～4次；
持续在超纯水中透析3天，每天换液3次，以除去胶原溶液中的无机盐类；
将平铺的4mg/mL胶原溶液冷冻成型后经高渗缓冲溶液浸泡脱水，挤压成膜厚度0.1毫米；
在该膜表面再平铺一层胶原溶液厚度5.0毫米，移入-80℃的冰箱低温预冷2h后；
将纳米羟基磷灰石粉末悬于蒸馏水中，控制固液比在20/80左右，低温下超声波分散15min，迅速加到上述-80℃的冰箱低温预冷的胶原表面，4℃静置过夜，移入液氮中冷冻2h，真空冷冻干燥得到多孔胶原-羟基磷灰石支架多层复合材料。
上述参照具体实施方式对该一种多层胶原支架材料及其制备方法与应用进行的详细描述，是说明性的而不是限定性的，可按照所限定范围列举出若干个实施例，因此在不脱离本发明总体构思下的变化和修改，应属本发明的保护范围之内。
Claims (3)
1.一种用于软骨修复的仿生多层胶原支架，其特征在于，所述胶原支架具备仿生多层三维空间结构，包括上层和基底层；其中各层之间为横向平行叠加，所述上层为致密结构的胶原层，所述基底层为纵向交联多孔结构的胶原层；所述胶原来自猪肌腱胶原提取和纯化，其中胶原蛋白含量大于99％，多糖含量小于0.1％，所述百分比为质量百分比；在所述基底层上，相对于致密层的另一端还复合有羟基磷灰石屏障层；
所述仿生多层胶原支架的制备方法包含以下步骤：
步骤1将剥离的肌腱粉碎后加入10倍体积的4M盐酸胍，所述盐酸胍为pH＝7.5、0.05MTris-HCl配制；
步骤2混悬后，粉碎匀浆，于4℃搅拌24h，12000g离心力下离心20min，分离得到上清液和沉淀；
步骤3将步骤2得到的沉淀用Tris-HCl缓冲液和0.5mM乙酸充分洗涤后再加入乙酸过夜，以除去蛋白多糖；
步骤4用含胃蛋白酶的冰乙酸溶液溶解，持续处理一段时间待混合物变成无色、透明、粘稠液体后在4℃，5000g离心力下离心20min，收集上清即为粗制肌腱胶原蛋白原液；
步骤5将氯化钠溶液加入其中，持续搅拌直至有白色絮状沉淀从溶液中析出，继续加入氯化钠溶液至沉淀不再 析出为止；
步骤6在4℃温度，5000g离心力下离心20min后获得白色沉淀；
步骤7将步骤6得到的沉淀物加入酸溶液溶解，将溶解后的胶原蛋白溶液继续反复进行盐析处理，重复2～4次；
步骤8持续在超纯水中透析3天，每天换液3次，以除去胶原溶液中的无机盐类；
步骤9将平铺的4mg/mL胶原溶液冷冻成型后经高渗缓冲溶液浸泡脱水，挤压成膜厚度为0.1-0.5毫米；
步骤10在步骤9得到的胶原表面再平铺一层胶原溶液，厚度为2.0-5.0毫米，移入-80℃的冰箱低温预冷2h；
步骤11将纳米羟基磷灰石粉末悬于蒸馏水中，控制固液比在20/80，低温下超声波分散15min，迅速加到步骤10中经-80℃的冰箱低温预冷的纵向多孔结构胶原表面，4℃静置过夜，再移入液氮中冷冻2h，最后真空冷冻干燥得到多孔胶原-羟基磷灰石支架复合材料。
2.一种权利要求1所述的仿生多层胶原支架，其特征在于，所述上层致密层的厚度为0.3毫米，所述基底层的厚度为2.5毫米。
3.一种权利要求1-2所述的仿生多层胶原支架在制备软骨修复装置方面的应用。
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