CN107669371A - 三相组织工程支架 - Google Patents
三相组织工程支架 Download PDFInfo
- Publication number
- CN107669371A CN107669371A CN201710651343.8A CN201710651343A CN107669371A CN 107669371 A CN107669371 A CN 107669371A CN 201710651343 A CN201710651343 A CN 201710651343A CN 107669371 A CN107669371 A CN 107669371A
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- Prior art keywords
- cartilage
- bone
- tendon
- reference book
- book hinge
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
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Abstract
本发明公开了一种三相组织工程支架，包括骨支架、软骨支架和肌腱支架，骨支架的一端设置为骨固定包页部，骨支架的另一端切设有骨类书页片，各骨类书页片之间设有骨分化细胞片；软骨支架的一端设置为软骨固定包页部，软骨支架的另一端切设有软骨类书页片，各软骨类书页片之间设有软骨分化细胞片；肌腱支架的一端设置为肌腱固定包页部，肌腱支架的另一端切设有肌腱类书页片，各肌腱类书页片之间设有肌腱分化细胞片；骨支架最底部的骨类书页片与软骨支架最顶部的软骨类书页片连接，软骨支架最底部的软骨类书页片与肌腱支架最顶部的肌腱类书页片连接。该组织工程支架具有制备简单、使用方便、能减少去细胞时间、可提高修复质量的优点。
Description
技术领域
本发明主要涉及骨腱连接点缺损修复技术，尤其涉及一种三相组织工程支架。
背景技术
骨腱连接点损伤（如髌骨-髌腱损伤）是一种常见的运动系统疾病，其发病率有逐年上升趋势，其治疗给社会和家庭带来了沉重的经济负担。典型的骨腱连接点由骨、矿化的纤维软骨、未矿化的纤维软骨及肌腱四层结构构成。与同种组织之间（如骨-骨、腱-腱）损伤后修复不同，骨腱损伤修复发生在骨和腱两种结构完全不同的组织之间，骨腱损伤后常常伴有纤维软骨层的破坏，该部位血供差、再生能力弱，导致骨腱损伤修复十分缓慢且困难。特殊的组织梯度结构再生困难是骨腱连接点损伤难以修复的主要因素。如何有效提高特殊的组织梯度结构再生能力，特别是纤维软骨层的修复能力，从而促进骨腱连接点损伤快速而优质的愈合，已成为运动医学领域亟待解决的难题。
传统治疗骨腱连接点损伤的方法，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，骨腱连接点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断裂。近年来，随着手术方式的改进和术后康复措施的发展使骨腱连接点愈合取得了很大的进步，但损伤后的骨腱连接点仍无法完全恢复到正常的组织梯度结构，其愈合效果难以让人满意。因此，除了提高手术技巧和优化术后康复计划外，寻找一种新的有效的治疗方法促进骨腱连接点缺损修复是迫切和必要的。
发明内容
本发明要解决的技术问题是克服现有技术的不足，提供一种制备简单、使用方便、能减少去细胞时间、可提高修复质量的三相组织工程支架。
为解决上述技术问题，本发明采用以下技术方案：
一种三相组织工程支架，包括由上至下依次叠放整合的骨支架、软骨支架和肌腱支架，所述骨支架的一端设置为骨固定包页部，骨支架的另一端横向切设有多片延伸至骨固定包页部的骨类书页片，各所述骨类书页片之间设有骨分化细胞片；所述软骨支架的一端设置为软骨固定包页部，软骨支架的另一端横向切设有多片延伸至软骨固定包页部的软骨类书页片，各所述软骨类书页片之间设有软骨分化细胞片；所述肌腱支架的一端设置为肌腱固定包页部，肌腱支架的另一端横向切设有多片延伸至肌腱固定包页部的肌腱类书页片，各所述肌腱类书页片之间设有肌腱分化细胞片；所述骨支架最底部的骨类书页片与软骨支架最顶部的软骨类书页片连接，所述软骨支架最底部的软骨类书页片与肌腱支架最顶部的肌腱类书页片连接。
作为上述技术方案的进一步改进：
所述骨支架最底部的骨类书页片与软骨支架最顶部的软骨类书页片相互横向插接，该骨类书页片与软骨类书页片之间设有所述骨分化细胞片；所述软骨支架最底部的软骨类书页片与肌腱支架最顶部的肌腱类书页片相互横向插接，该软骨类书页片与肌腱类书页片之间设有所述软骨分化细胞片。
所述骨支架最底部的骨类书页片与软骨支架最顶部的软骨类书页片之间以及软骨支架最底部的软骨类书页片与肌腱支架最顶部的肌腱类书页片之间均涂附有纤维蛋白胶。
所述骨支架、软骨支架和肌腱支架沿边沿通过缝线缝制为一体。
所述缝线设置为可降解手术缝线。
所述骨支架、软骨支架和肌腱支架的四周相互平齐。
所述骨类书页片、软骨类书页片以及肌腱类书页片的上下表面均设有多肽涂层。
与现有技术相比，本发明的优点在于：
本发明的三相组织工程支架，包括由上至下依次叠放整合的骨支架、软骨支架和肌腱支架，骨支架的一端设置为骨固定包页部，骨支架的另一端横向切设有多片延伸至骨固定包页部的骨类书页片，各骨类书页片之间设有骨分化细胞片；软骨支架的一端设置为软骨固定包页部，软骨支架的另一端横向切设有多片延伸至软骨固定包页部的软骨类书页片，各软骨类书页片之间设有软骨分化细胞片；肌腱支架的一端设置为肌腱固定包页部，肌腱支架的另一端横向切设有多片延伸至肌腱固定包页部的肌腱类书页片，各肌腱类书页片之间设有肌腱分化细胞片；骨支架最底部的骨类书页片与软骨支架最顶部的软骨类书页片连接，软骨支架最底部的软骨类书页片与肌腱支架最顶部的肌腱类书页片连接。本发明的三相组织工程支架，能将体外构建的骨-软骨-肌腱组织工程复合体移植到骨腱连接点损伤部位用以修复组织缺损，在缺损部位可以再生一个正常的组织梯度结构，这样就可提高骨腱连接点损伤的修复质量；类书页结构的支架不仅可以保留组织的结构和基质成分，还利于细胞的迁移、增殖及分化，因而可以促进缺损组织的再生与愈合，而且还可以有效地加速组织的去细胞过程，减少去细胞时间；可以根据修复部位的组织形状将三相书页组织工程支架修建成适当的形状后置于缺损部位便于缝合固定，其制备简单、使用方便。
附图说明
图1是本发明的主视结构示意图。
图2是本发明的俯视结构示意图。
图3是本发明中骨支架的主视结构示意图。
图中各标号表示：
1、骨支架；11、骨固定包页部；12、骨类书页片；13、骨分化细胞片；2、软骨支架；21、软骨固定包页部；22、软骨类书页片；23、软骨分化细胞片；3、肌腱支架；31、肌腱固定包页部；32、肌腱类书页片；33、肌腱分化细胞片；4、纤维蛋白胶；5、缝线；6、多肽涂层。
具体实施方式
以下将结合说明书附图和具体实施例对本发明做进一步详细说明。
如图1至图3所示，本发明三相组织工程支架的一种实施例，包括由上至下依次叠放整合的骨支架1、软骨支架2和肌腱支架3，骨支架1的一端设置为骨固定包页部11，骨支架1的另一端横向切设有多片延伸至骨固定包页部11的骨类书页片12，各骨类书页片12之间设有骨分化细胞片13；软骨支架2的一端设置为软骨固定包页部21，软骨支架2的另一端横向切设有多片延伸至软骨固定包页部21的软骨类书页片22，各软骨类书页片22之间设有软骨分化细胞片23；肌腱支架3的一端设置为肌腱固定包页部31，肌腱支架3的另一端横向切设有多片延伸至肌腱固定包页部31的肌腱类书页片32，各肌腱类书页片32之间设有肌腱分化细胞片33；骨支架1最底部的骨类书页片12与软骨支架2最顶部的软骨类书页片22连接，软骨支架2最底部的软骨类书页片22与肌腱支架3最顶部的肌腱类书页片32连接。本发明的三相组织工程支架，能将体外构建的骨-软骨-肌腱组织工程复合体移植到骨腱连接点损伤部位用以修复组织缺损，在缺损部位可以再生一个正常的组织梯度结构，这样就可提高骨腱连接点损伤的修复质量；类书页结构的支架不仅可以保留组织的结构和基质成分，还利于细胞的迁移、增殖及分化，因而可以促进缺损组织的再生与愈合，而且还可以有效地加速组织的去细胞过程，减少去细胞时间；可以根据修复部位的组织形状将三相书页组织工程支架修建成适当的形状后置于缺损部位便于缝合固定，其制备简单、使用方便。
本实施例中，骨支架1最底部的骨类书页片12与软骨支架2最顶部的软骨类书页片22相互横向插接，该骨类书页片12与软骨类书页片22之间设有骨分化细胞片13；软骨支架2最底部的软骨类书页片22与肌腱支架3最顶部的肌腱类书页片32相互横向插接，该软骨类书页片22与肌腱类书页片32之间设有软骨分化细胞片23。该结构中，骨支架1最底部的骨类书页片12与软骨支架2最顶部的软骨类书页片22相互横向插接，形成了反向互补交叉；同样软骨支架2最底部的软骨类书页片22与肌腱支架3最顶部的肌腱类书页片32相互横向插接形成了反向互补交叉；这样设置大大提稿各相之间的整合连接性能，防止了发生各相的错位移动。
本实施例中，骨支架1最底部的骨类书页片12与软骨支架2最顶部的软骨类书页片22之间以及软骨支架2最底部的软骨类书页片22与肌腱支架3最顶部的肌腱类书页片32之间均涂附有纤维蛋白胶4。该纤维蛋白胶4的设置进一步提高了各相之间的整合力。
本实施例中，骨支架1、软骨支架2和肌腱支架3沿边沿通过缝线5缝制为一体。该缝线5的设置同样是为了使各相之间连接更为稳定可靠。
本实施例中，缝线5设置为可降解手术缝线。可降解手术缝线在修复后，能自动降解，不需要进行拆线，提高了方便性。
本实施例中，骨支架1、软骨支架2和肌腱支架3的四周相互平齐。这样设置，使得该三相组织工程支架形成了矩形体结构，便于实施。
本实施例中，骨类书页片12、软骨类书页片22以及肌腱类书页片32的上下表面均设有多肽涂层6。通过多肽涂层6的表面改性处理，能促进各类细胞片的生长。
本发明三相组织工程支架的构建原理如下：
1、骨、软骨和肌腱组织样本获取。
获取同种异体或异种骨、纤维软骨及肌腱组织，切割成长方体。
2、制备骨、软骨及肌腱组织书页支架。
2.1、骨组织脱钙：骨组织置于广口瓶中，有机酸脱钙液完全浸泡样本，37℃，每周取出样本，更换新脱钙液，直至完成脱钙。用物理检测方法判定是否脱钙完全，当骨块外观趋于透明，易弯曲，用1mL注射器针头可轻松插入样本，表明脱钙已完全。
2.2 、“书页”多层组织薄片技术制备支架：将完全脱钙的骨块、软骨及肌腱组织，用5％NaHCO3漂洗3次，每次5min，PBS液漂洗3次，每次5min。设置冰冻温度－22℃。将组织块切割成长方体，将组织块平放在冰冻切片机组织支承器上，滴加少量OCT冰冻切片包埋剂，并将骨片压平，滴加OCT包埋剂将组织块完全包埋，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，样本长轴延长线与刀片垂直。将切片厚度设置为30-150μm，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”状组织支架。支架每页厚度30-150μm，不少于10页，三种书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。
2.3、骨、软骨及肌腱支架去细胞处理：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（SDS）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0.25%胰蛋白酶, 24h， PBS漂洗 24h, 37℃；标本置于核酸酶液（含DNA酶deoxyribonuclease I， 500 U/mL，RNA酶ribonuclease A，1 mg/mL），12h，37℃；样本取出后PBS漂洗24h，样本放-80℃冰箱保存。
2.4、骨、软骨及肌腱支架表面涂层改性：将骨、软骨及肌腱书页支架置于浸渍液中进行表面改性处理，所述浸渍液为多肽溶于PBS缓冲液后配制得到，表面改性处理完成后取出得到表面改性后的支架，无菌条件下冻干备用。
2.5、将向骨、软骨及肌腱方向分化的骨髓间充质干细胞片分别种植于相应的表面改性后的支架中：将骨髓间充质干细胞以每平方厘米1×104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。向骨方向诱导分化培养基：高糖DMEM, 10% FBS, 100 nM 地塞米松，50 μM 维生素C, 10 mM 甘油磷酸钠(β-GP)，诱导2 周后形成细胞片，再将细胞片分别种植于骨书页支架的各层。向软骨方向诱导分化培养基：高糖DMEM，10% FBS，10 ng/mlTGF-β3，100 nM 地塞米松，50 μM 维生素C, 10 mM 甘油磷酸钠(β-GP) ，诱导2 周后形成细胞片，再将细胞片分别种植于软骨书页支架的各层。向肌腱方向诱导分化培养基：高糖DMEM，10% FBS，50mg/ml 抗坏血酸2-磷酸盐，100 ng/ml 生长分化因子-5 (GDF-5)，诱导2 周后形成细胞片，再将细胞片分别种植于肌腱书页支架的各层。支架在接种干细胞片前先用钴60消毒后再用相应诱导培养液浸泡6h。
2.6、将种植了细胞片的支架反向互补交叉进行叠加构建骨-软骨-肌腱三相组织工程支架复合体：将种植了干细胞片的骨、软骨及肌腱支架，采用“书页”反向互补交叉的原理，制造三相（骨、软骨和肌腱）4层复合支架，上层为一本骨多层薄片，再反向互补交叉一本软骨多层薄片，再反向互补交叉一本软骨多层薄片，最后反向互补交叉一本肌腱多层薄片，形成骨-软骨-肌腱三相组织工程支架复合体，在不同相结合部位涂纤维蛋白胶以促进不同相的整合。
2.7、用可降解手术缝线将三相组织缝合在一起：完成支架整合后，使用可降解手术缝线将三相不同支架缝合在一起，避免支架错开。
虽然本发明已以较佳实施例揭示如上，然而并非用以限定本发明。任何熟悉本领域的技术人员，在不脱离本发明技术方案范围的情况下，都可利用上述揭示的技术内容对本发明技术方案做出许多可能的变动和修饰，或修改为等同变化的等效实施例。因此，凡是未脱离本发明技术方案的内容，依据本发明技术实质对以上实施例所做的任何简单修改、等同变化及修饰，均应落在本发明技术方案保护的范围内。
Claims (9)
1.一种三相组织工程支架，其特征在于：包括由上至下依次叠放整合的骨支架（1）、软骨支架（2）和肌腱支架（3），所述骨支架（1）的一端设置为骨固定包页部（11），骨支架（1）的另一端横向切设有多片延伸至骨固定包页部（11）的骨类书页片（12），各所述骨类书页片（12）之间设有骨分化细胞片（13）；所述软骨支架（2）的一端设置为软骨固定包页部（21），软骨支架（2）的另一端横向切设有多片延伸至软骨固定包页部（21）的软骨类书页片（22），各所述软骨类书页片（22）之间设有软骨分化细胞片（23）；所述肌腱支架（3）的一端设置为肌腱固定包页部（31），肌腱支架（3）的另一端横向切设有多片延伸至肌腱固定包页部（31）的肌腱类书页片（32），各所述肌腱类书页片（32）之间设有肌腱分化细胞片（33）；所述骨支架（1）最底部的骨类书页片（12）与软骨支架（2）最顶部的软骨类书页片（22）连接，所述软骨支架（2）最底部的软骨类书页片（22）与肌腱支架（3）最顶部的肌腱类书页片（32）连接。
2.根据权利要求1所述的三相组织工程支架，其特征在于：所述骨支架（1）最底部的骨类书页片（12）与软骨支架（2）最顶部的软骨类书页片（22）相互横向插接，该骨类书页片（12）与软骨类书页片（22）之间设有所述骨分化细胞片（13）；所述软骨支架（2）最底部的软骨类书页片（22）与肌腱支架（3）最顶部的肌腱类书页片（32）相互横向插接，该软骨类书页片（22）与肌腱类书页片（32）之间设有所述软骨分化细胞片（23）。
3.根据权利要求2所述的三相组织工程支架，其特征在于：所述骨支架（1）最底部的骨类书页片（12）与软骨支架（2）最顶部的软骨类书页片（22）之间以及软骨支架（2）最底部的软骨类书页片（22）与肌腱支架（3）最顶部的肌腱类书页片（32）之间均涂附有纤维蛋白胶（4）。
4.根据权利要求1至3中任一项所述的三相组织工程支架，其特征在于：所述骨支架（1）、软骨支架（2）和肌腱支架（3）沿边沿通过缝线（5）缝制为一体。
5.根据权利要求4所述的三相组织工程支架，其特征在于：所述缝线（5）设置为可降解手术缝线。
6.根据权利要求1至3中任一项所述的三相组织工程支架，其特征在于：所述骨支架（1）、软骨支架（2）和肌腱支架（3）的四周相互平齐。
7.根据权利要求5所述的三相组织工程支架，其特征在于：所述骨支架（1）、软骨支架（2）和肌腱支架（3）的四周相互平齐。
8.根据权利要求1至3中任一项所述的三相组织工程支架，其特征在于：所述骨类书页片（12）、软骨类书页片（22）以及肌腱类书页片（32）的上下表面均设有多肽涂层（6）。
9.根据权利要求7所述的三相组织工程支架，其特征在于：所述骨类书页片（12）、软骨类书页片（22）以及肌腱类书页片（32）的上下表面均设有多肽涂层（6）。
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