CN110448732A - 区域干细胞诱导活性去细胞骨腱界面书页支架 - Google Patents

区域干细胞诱导活性去细胞骨腱界面书页支架 Download PDF

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CN110448732A
CN110448732A CN201910796350.6A CN201910796350A CN110448732A CN 110448732 A CN110448732 A CN 110448732A CN 201910796350 A CN201910796350 A CN 201910796350A CN 110448732 A CN110448732 A CN 110448732A
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stem cell
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tendon interface
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CN110448732B (zh
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吕红斌
陈灿
陈洋
胡建中
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Xiangya Hospital of Central South University
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Abstract

本发明属于骨腱界面损伤修复技术领域，具体涉及区域干细胞诱导活性去细胞骨腱界面书页支架，具体包括：步骤一、hBMP‑2、hTGF‑β3及hGDF‑7的获取，配制成100ng/ml的hBMP‑2、hTGF‑β3及hGDF‑7的工作液；步骤二、制备hBMP‑2、hTGF‑β3及hGDF‑7缓释初乳；步骤三、双重仿生去细胞骨腱界面书页支架的制备；步骤四、区域性定向诱导缓释型去细胞骨腱界面书页支架的制备；步骤五、区域干细胞诱导活性去细胞骨腱界面书页支架的制备；为临床骨腱界面重建提供一种形态天然、力学可靠、活性优良的新型组织工程移植物，从而实现骨腱界面损伤后快速而优质的愈合。

Description

区域干细胞诱导活性去细胞骨腱界面书页支架
技术领域
本发明属于骨腱界面损伤修复技术领域，具体涉及区域干细胞诱导活性去细胞骨腱界面书页支架。
背景技术
随着全民体育健身运动的蓬勃开展，国民身体素质和健康意识得以稳步提升，但不可避免的是，各类运动损伤的发病人数也随之增加。其中，骨腱界面损伤(如交叉韧带损伤、肩袖损伤)是最为常见的一类运动损伤。据统计，仅在美国每年就有约25万例肩袖损伤和10万例前交叉韧带损伤需手术治疗。我国目前虽然缺乏相关流行病学资料，但类比美国的数据资料，骨腱界面损伤已成我国运动医学领域亟待解决的挑战，其治疗给社会和家庭带来了沉重的经济负担。典型的骨腱界面由骨、矿化的纤维软骨、未矿化的纤维软骨及肌腱四层结构构成。与同种组织骨-骨、腱-腱之间损伤后修复不同，骨腱界面愈合发生在骨和腱两种结构完全不同的组织之间，其损伤后常常伴有梯度渐变层次的破坏，且该部位血供差、再生能力弱，导致骨腱界面愈合十分缓慢且困难。因此，如何有效提高骨腱界面损伤后梯度结构再生是促进骨腱界面损伤后快速优质愈合的关键。
传统治疗骨腱界面损伤的方法，往往借助各类外科缝合技术将肌腱或韧带直接固定到骨上，骨腱界面早期被纤维瘢痕组织填充，其特征性纤维软骨层的重建不完全，缺乏梯度结构的再生，因而不能有效的分散应力和防止骨腱结合点撕脱，在恢复运动后重建的骨腱结构容易再次撕脱。近年来，随着手术方式及术后康复策略不断改进，骨腱界面愈合取得了长足的进步，但损伤后的骨腱界面仍无法完全恢复正常层次结构，导致患者早期运动功能恢复不佳，其愈合效果难以令人满意。
当今多领域的交叉融合发展为骨腱界面损伤后层次结构再生提供了新的策略。以材料科学、分子生物学及细胞生物学相结合的组织工程学成为了当今整合医学的重点研究领域。传统上，尽管单一相的生物支架或单一的种子细胞移植可以修复骨腱界面损伤，但这些方法均无法实现正常骨腱界面层次结构的优质再生。后来，多相地模拟组织的天然形态结构并结合种子细胞一起靶向修复损伤组织/器官，这为骨腱界面损伤后层次结构再生提供了新的希望。然而这些多相支架难以同时模拟生物体骨腱界面形态结构与力学性能的双重仿生，使得患者不能尽早地恢复运动，难以实现快速而优质的愈合；部分仿生支架移植物在体内需一定时间的内化吸收过程，且负载干细胞后不能提供较强的内在诱导活性，使得损伤的组织/器官局部难以达到最适宜再生微环境，不能有效地促进患者快速优质愈合。
因此，本发明吸纳“书页”状组织薄片切割方法及去细胞技术优势，并取材正常的骨腱界面组织，设计了一种形态结构与力学性能双重仿生的骨腱界面支架；然后基于骨腱界面的三层组织分布位置，对支架的肌腱区域、软骨区域及骨区域进行生物学活性的定向缓释构建；最后在支架书页间隙复合干细胞片，定向诱导干细胞在不同区域差异性精准分化，为临床骨腱界面重建提供一种形态天然、力学可靠、活性优良的新型组织工程移植物，从而实现骨腱界面损伤后快速而优质的愈合。
发明内容
为了解决上述问题，提供了区域干细胞诱导活性去细胞骨腱界面书页支架的制备方法。
具体技术方案为：区域干细胞诱导活性去细胞骨腱界面书页支架，所述支架具体制备步骤为：
步骤一、成骨生物活性因子、成软骨生物活性因子及成肌腱生物活性因子的获取；
具体为：获取hBMP-2、hTGF-β3及hGDF-7，配制成100ng/ml的hBMP-2、hTGF-β3及hGDF-7的工作液；
步骤二、制备hBMP-2、hTGF-β3及hGDF-7缓释初乳；
步骤三、双重仿生去细胞骨腱界面书页支架的制备；
步骤四、区域性定向诱导缓释型去细胞骨腱界面书页支架的制备：
冻干的去细胞双重仿生骨腱界面书页支架，无菌环境下分开书页后，在肌腱端使用夹具分开夹住每一页书页，首先将支架-纤维软骨区域浸入含有100ng/ml hTGF-β3-PLGA初乳液中2小时；取出后，将支架-骨区域浸入含有100ng/ml hBMP-2-PLGA初乳液中2小时；取出后，将支架-肌腱区域浸入含有100ng/ml hGDF-7-PLGA初乳液中2小时；获得缓释型区域干细胞定向诱导活性去细胞骨腱界面书页支架；
步骤五、区域干细胞诱导活性去细胞骨腱界面书页支架的制备：
(1)、脂肪间充质干细胞片的制备：将第3-4代脂肪间充质干细胞以每平方厘米4×105的细胞密度种植于培皿，在37℃、5％CO2饱和湿度培养箱内进行培养，每隔2-3天常规换液，待脂肪间充质干细胞增殖融合到95％左右，将培养皿置于20℃、5％CO2环境下培养20-30min，待干细胞片层脱落形成间充质干细胞片；
(2)、区域干细胞诱导活性新型书页组织工程支架复合间充质干细胞片：区域干细胞定向诱导活性去细胞骨腱界面书页支架在接种干细胞片前先用钴60消毒后，再用干细胞培养液浸泡2h，将脂肪间充质干细胞片种植于区域干细胞定向诱导活性去细胞骨腱界面书页支架的各层，完成区域干细胞诱导活性去细胞骨腱界面书页支架的制备。
优选的，步骤三中所述的核糖核酸酶液含DNA酶deoxyribonuclease I，500U/mL，RNA酶ribonuclease A，1mg/mL。
优选的，所述步骤二具体为：取20mg聚乳酸-羟基乙酸共聚物，加入二氯甲烷1mL，充分震荡使得PLGA完全溶解；分别取100ng/ml的hBMP-2、100ng/ml的hTGF-β3及hGDF-7工作液100pL，放入上述完全溶解的溶液中，冰浴条件下超声乳化1min，得到相应的hBMP-2-PLGA初乳、hTGF-β3-PLGA初乳及hGDF-7-PLGA初乳，超声过程向初乳中加入1％聚乙烯醇2mL，再次超声乳化1min，调整初乳至合适的粘稠性。
优选的，所述hBMP-2-PLGA初乳、hTGF-β3-PLGA初乳及hGDF-7-PLGA初乳均为乳白色液体。
优选的，所述步骤三具体为：获取动物骨腱界面组织，切割成长方体后，经1％双抗PBS液冲洗去除骨髓及血液，10％EDTA缓冲液脱钙15天，PBS液冲洗后冷冻在OCT包埋剂中，迅速将样本置入冰冻切片机冰冻固定；将组织支承器固定在切片机持承器上，沿着力学牵拉方向由肌腱向骨方向切割正常骨腱界面组织，使得样本长轴延长线与刀片垂直，将切片厚度设置为200-250μm，按微调按钮将标本调整到接近刀片的位置，缓慢顺时针方向旋转手动旋钮，刀片行进至组织块上端1/4处，停止并退出；逆时针方向旋转手动旋钮1/2圈，保持固定的切片厚度，再次顺时针方向旋转手动旋钮至刀片行进到骨块上端1/4处，停止并退出；逆时针方向旋转手动旋钮1/2圈，保持固定切片厚度，如此反复进行，可获得一端切开，一端带柄的“书页”状双重仿生骨腱界面支架；支架每页厚度200-250μm，不少于10页，每页支架的最终大小均一样；将切好的书页状支架置于去离子水中漂洗3次，每次5min，去除OCT包埋剂；
双重仿生支架去细胞处理：样本以4℃PBS冲洗3次，每次10min，滤纸吸除样本表面水分；将样本用纱布包裹，置入液氮冰冻，10min，取出后立即行37℃水浴，10min，反复交替3个循环；冻融循环后的书页支架置于含2％的十二烷基硫酸钠溶液中，置于摇床上，振荡4h，37℃；样本取出后PBS振荡漂洗，12h，4℃；将样本置于0.1％Triton X-100-1.5MKCL溶液中，12小时，4℃；将样本置于10mM Tris漂洗3h，PBS漂洗，3h，4℃；标本置于核糖核酸酶液，12h，37℃；样本取出后PBS漂洗24h，即可制得去细胞“书页”状双重仿生骨腱界面支架，样本无菌条件下冻干备用。
有益效果：
采用“书页”状组织薄片切割方法，沿着力学牵拉方向由肌腱向骨方向切割正常骨腱界面组织，可保留其原有抗牵拉力学性能，经去细胞处理，制备出一种兼具形态结构与力学性能双重仿生特性的骨腱界面书页支架。结合PLGA的缓释效益，采用区域缓释型设计能有效地提高支架生物学活性，又避免了生物活性因子爆发式释放，利于干细胞的黏附、增殖及差异性分化；复合细胞片，能有效地提升干细胞的密度，避免细胞流失、分布不均及支架的周边缺乏细胞等，促进肌腱-纤维软骨-骨三层渐变组织的尽早形成。可以根据修复部位的组织形状及生物力学特性，将新型书页组织工程支架修建成适当的形状及厚度后置于缺损部位便于缝合固定，其制备简单、使用方便，可有效提高骨腱界面修复质量。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为区域干细胞诱导活性新型书页组织工程支架的示意图。
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做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。
实施例1
参考图1，区域干细胞诱导活性去细胞骨腱界面书页支架，所述支架具体制备步骤为：
步骤一、成骨生物活性因子、成软骨生物活性因子及成肌腱生物活性因子的获取；
具体为：获取hBMP-2、hTGF-β3及hGDF-7，配制成100ng/ml的hBMP-2、hTGF-β3及hGDF-7的工作液；
所述hBMP-2、hTGF-β3及hGDF-7可购买商品化的人源性生物活性因子产品，如可在联科生物官网(http://www.liankebio.com/index.html)购买Human BMP-2(PeproTech96-120-02-100),Human TGF-beta3(PeproTech 96-AF-100-36E-250),Human GDF-7(PeproTech 96-120-37-100)，进一步配制100ng/ml的hBMP-2、hTGF-β3及hGDF-7工作液。
步骤二、制备hBMP-2、hTGF-β3及hGDF-7缓释初乳：
取20mg聚乳酸-羟基乙酸共聚物，加入二氯甲烷1mL，充分震荡使得PLGA完全溶解；分别取100ng/ml的hBMP-2、100ng/ml的hTGF-β3及hGDF-7工作液100pL，放入上述完全溶解的溶液中，冰浴条件下超声乳化1min，得到相应的hBMP-2-PLGA初乳、hTGF-β3-PLGA初乳及hGDF-7-PLGA初乳，所述hBMP-2-PLGA初乳、hTGF-β3-PLGA初乳及hGDF-7-PLGA初乳均为乳白色液体，超声过程向初乳中加入1％聚乙烯醇2mL，再次超声乳化1min，调整初乳至合适的粘稠性。
步骤三、双重仿生去细胞骨腱界面书页支架的制备：
获取动物骨腱界面组织，切割成长方体后，经1％双抗PBS液冲洗去除骨髓及血液，10％EDTA缓冲液脱钙15天，PBS液冲洗后冷冻在OCT包埋剂中，迅速将样本置入冰冻切片机冰冻固定；将组织支承器固定在切片机持承器上，沿着力学牵拉方向由肌腱向骨方向切割正常骨腱界面组织，使得样本长轴延长线与刀片垂直，将切片厚度设置为200-250μm，按微调按钮将标本调整到接近刀片的位置，缓慢顺时针方向旋转手动旋钮，刀片行进至组织块上端1/4处，停止并退出；逆时针方向旋转手动旋钮1/2圈，保持固定的切片厚度，再次顺时针方向旋转手动旋钮至刀片行进到骨块上端1/4处，停止并退出；逆时针方向旋转手动旋钮1/2圈，保持固定切片厚度，如此反复进行，可获得一端切开，一端带柄的“书页”状双重仿生骨腱界面支架；支架每页厚度200-250μm，不少于10页，每页支架的最终大小均一样；将切好的书页状支架置于去离子水中漂洗3次，每次5min，去除OCT包埋剂；
双重仿生支架去细胞处理：样本以4℃PBS冲洗3次，每次10min，滤纸吸除样本表面水分；将样本用纱布包裹，置入液氮冰冻，10min，取出后立即行37℃水浴，10min，反复交替3个循环；冻融循环后的书页支架置于含2％的十二烷基硫酸钠溶液中，置于摇床上，振荡4h，37℃；样本取出后PBS振荡漂洗，12h，4℃；将样本置于0.1％Triton X-100-1.5MKCL溶液中，12小时，4℃；将样本置于10mM Tris漂洗3h，PBS漂洗，3h，4℃；标本置于核糖核酸酶液，所述的核糖核酸酶液含DNA酶deoxyribonuclease I，500U/mL，RNA酶ribonuclease A，1mg/mL，核糖核酸酶液含DNA酶deoxyribonuclease I，500U/mL，RNA酶ribonuclease A，1mg/mL，12h，37℃；样本取出后PBS漂洗24h，即可制得去细胞“书页”状双重仿生骨腱界面支架，样本无菌条件下冻干备用。
步骤四、区域性定向诱导缓释型去细胞骨腱界面书页支架的制备：
冻干的去细胞双重仿生骨腱界面书页支架，无菌环境下分开书页后，在肌腱端使用夹具分开夹住每一页书页，首先将支架-纤维软骨区域浸入含有100ng/ml hTGF-β3-PLGA初乳液中2小时；取出后，将支架-骨区域浸入含有100ng/ml hBMP-2-PLGA初乳液中2小时；取出后，将支架-肌腱区域浸入含有100ng/ml hGDF-7-PLGA初乳液中2小时；获得缓释型区域干细胞定向诱导活性去细胞骨腱界面书页支架；使得去细胞骨腱界面书页支架的不同区域负载生物活性物质，从而提升支架的区域诱导活性，为后续负载的干细胞提供强有力的区域诱导活性，为损伤组织的原位修复提供适宜的微环境，实现特征性骨-纤维软骨-肌腱结构的快速再生。
步骤五、区域干细胞诱导活性去细胞骨腱界面书页支架的制备：
(1)、脂肪间充质干细胞片的制备：将第3-4代脂肪间充质干细胞以每平方厘米4×105的细胞密度种植于培皿，在37℃、5％CO2饱和湿度培养箱内进行培养，每隔2-3天常规换液，待脂肪间充质干细胞增殖融合到95％左右，将培养皿置于20℃、5％CO2环境下培养20-30min，待干细胞片层脱落形成间充质干细胞片；
(2)、区域干细胞诱导活性新型书页组织工程支架复合间充质干细胞片：区域干细胞定向诱导活性去细胞骨腱界面书页支架在接种干细胞片前先用钴60消毒后，再用干细胞培养液浸泡2h，将脂肪间充质干细胞片种植于区域干细胞定向诱导活性去细胞骨腱界面书页支架的各层，完成区域干细胞诱导活性去细胞骨腱界面书页支架的制备。
实施例2
将本申请的区域干细胞诱导活性去细胞骨腱界面书页支架以及现有技术的支架分别用于相同程度的骨腱界面损伤者，发现本申请的支架相对于现有技术的支架，骨腱界面损伤者恢复快，且愈合优质。
以上所述仅为本发明的较佳实施例而已，并非用于限定本发明的保护范围。凡在本发明的精神和原则之内所作的任何修改、等同替换、改进等，均包含在本发明的保护范围内。

Claims (5)

1.区域干细胞诱导活性去细胞骨腱界面书页支架，其特征在于，所述支架具体制备步骤为：
步骤一、成骨生物活性因子、成软骨生物活性因子及成肌腱生物活性因子的获取；
具体为：获取hBMP-2、hTGF-β3及hGDF-7，配制成100ng/ml的hBMP-2、hTGF-β3及hGDF-7的工作液；
步骤二、制备hBMP-2、hTGF-β3及hGDF-7缓释初乳；
步骤三、双重仿生去细胞骨腱界面书页支架的制备；
步骤四、区域性定向诱导缓释型去细胞骨腱界面书页支架的制备：
冻干的去细胞双重仿生骨腱界面书页支架，无菌环境下分开书页后，在肌腱端使用夹具分开夹住每一页书页，首先将支架-纤维软骨区域浸入含有100ng/ml hTGF-β3-PLGA初乳液中2小时；取出后，将支架-骨区域浸入含有100ng/ml hBMP-2-PLGA初乳液中2小时；取出后，将支架-肌腱区域浸入含有100ng/ml hGDF-7-PLGA初乳液中2小时；获得缓释型区域干细胞定向诱导活性去细胞骨腱界面书页支架；
步骤五、区域干细胞诱导活性去细胞骨腱界面书页支架的制备：
(1)、脂肪间充质干细胞片的制备：将第3-4代脂肪间充质干细胞以每平方厘米4×105的细胞密度种植于培皿，在37℃、5％CO2饱和湿度培养箱内进行培养，每隔2-3天常规换液，待脂肪间充质干细胞增殖融合到95％左右，将培养皿置于20℃、5％CO2环境下培养20-30min，待干细胞片层脱落形成间充质干细胞片；
(2)、区域干细胞诱导活性新型书页组织工程支架复合间充质干细胞片：区域干细胞定向诱导活性去细胞骨腱界面书页支架在接种干细胞片前先用钴60消毒后，再用干细胞培养液浸泡2h，将脂肪间充质干细胞片种植于区域干细胞定向诱导活性去细胞骨腱界面书页支架的各层，完成区域干细胞诱导活性去细胞骨腱界面书页支架的制备。
2.根据权利要求1所述的区域干细胞诱导活性去细胞骨腱界面书页支架，其特征在于，所述步骤二具体为：取20mg聚乳酸-羟基乙酸共聚物，加入二氯甲烷1mL，充分震荡使得PLGA完全溶解；分别取100ng/ml的hBMP-2、100ng/ml的hTGF-β3及hGDF-7工作液100pL，放入上述完全溶解的溶液中，冰浴条件下超声乳化1min，得到相应的hBMP-2-PLGA初乳、hTGF-β3-PLGA初乳及hGDF-7-PLGA初乳，超声过程向初乳中加入1％聚乙烯醇2mL，再次超声乳化1min，调整初乳至合适的粘稠性。
3.根据权利要求2所述的区域干细胞诱导活性去细胞骨腱界面书页支架，其特征在于，所述hBMP-2-PLGA初乳、hTGF-β3-PLGA初乳及hGDF-7-PLGA初乳均为乳白色液体。
4.根据权利要求1所述的区域干细胞诱导活性去细胞骨腱界面书页支架，其特征在于，所述步骤三具体为：获取动物骨腱界面组织，切割成长方体后，经1％双抗PBS液冲洗去除骨髓及血液，10％EDTA缓冲液脱钙15天，PBS液冲洗后冷冻在OCT包埋剂中，迅速将样本置入冰冻切片机冰冻固定；将组织支承器固定在切片机持承器上，沿着力学牵拉方向由肌腱向骨方向切割正常骨腱界面组织，使得样本长轴延长线与刀片垂直，将切片厚度设置为200-250μm，按微调按钮将标本调整到接近刀片的位置，缓慢顺时针方向旋转手动旋钮，刀片行进至组织块上端1/4处，停止并退出；逆时针方向旋转手动旋钮1/2圈，保持固定的切片厚度，再次顺时针方向旋转手动旋钮至刀片行进到骨块上端1/4处，停止并退出；逆时针方向旋转手动旋钮1/2圈，保持固定切片厚度，如此反复进行，可获得一端切开，一端带柄的“书页”状双重仿生骨腱界面支架；支架每页厚度200-250μm，不少于10页，每页支架的最终大小均一样；将切好的书页状支架置于去离子水中漂洗3次，每次5min，去除OCT包埋剂；
双重仿生支架去细胞处理：样本以4℃PBS冲洗3次，每次10min，滤纸吸除样本表面水分；将样本用纱布包裹，置入液氮冰冻，10min，取出后立即行37℃水浴，10min，反复交替3个循环；冻融循环后的书页支架置于含2％的十二烷基硫酸钠溶液中，置于摇床上，振荡4h，37℃；样本取出后PBS振荡漂洗，12h，4℃；将样本置于0.1％Triton X-100-1.5MKCL溶液中，12小时，4℃；将样本置于10mM Tris漂洗3h，PBS漂洗，3h，4℃；标本置于核糖核酸酶液，12h，37℃；样本取出后PBS漂洗24h，即可制得去细胞“书页”状双重仿生骨腱界面支架，样本无菌条件下冻干备用。
5.根据权利要求4所述的区域干细胞诱导活性去细胞骨腱界面书页支架，其特征在于，所述的核糖核酸酶液含DNA酶deoxyribonucleaseI，500U/mL，RNA酶ribonuclease A，1mg/mL。
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