CN108245708A - 一种诱导肌腱组织再生的生物活性支架及其制备方法和用途 - Google Patents
一种诱导肌腱组织再生的生物活性支架及其制备方法和用途 Download PDFInfo
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Abstract
本发明公开了一种制备诱导肌腱组织再生的生物活性支架的方法，包括如下步骤：(1)取新鲜肌腱，脱细胞处理，得脱细胞肌腱压片或切片支架；(2)在支架上复合细胞外基质，即可。本发明还公开了前述方法制备的脱细胞肌腱生物活性支架及其用途。本发明方法制备的脱细胞肌腱支架生物活性高，体内修复效果良好，免疫原性低，生物相容性好，制备方法简单，成本低廉，具有较好的应用前景。
Description
技术领域
本发明涉及一种具有肌腱组织再生的生物活性支架的制备方法及其用途，属于生物材料领域。
背景技术
由于疾病、创伤、退变等原因导致的肌腱损伤已成为不可忽视的健康问题。目前肌腱损伤修复的方法多种多样，包括自体肌腱移植、同种或异种肌腱移植和人工材料替代等。自体肌腱移植会造成供区疼痛、感染、损伤，同时所能供给的肌腱来源有限；同种异体肌腱移植可能引起疾病的传播，出现不同程度的排斥反应，也存在来源有限的问题；异种肌腱移植也可能引起疾病传播，但最主要的问题是存在免疫排斥反应；人工肌腱由于力学相容性较差或降解速率难与组织再生同步等问题，使得肌腱缺损修复的长期效果有待观察，且价格昂贵。理想的肌腱修复材料应具备以下条件：(1)良好的生物相容性；(2)良好的力学相容性；(3)具有诱导肌腱组织再生的潜能；(4)形状大小可控，加工制备方法有可操作性。查阅大量文献结果表明，目前使用的肌腱损伤修复材料均存在各自的缺陷和不足，因此寻求更加理想的肌腱修复材料的脚步从未停止。
随着组织工程技术的发展，激励着研究人员试图通过组织工程肌腱来解决肌腱损伤修复困难这一难题。在国内外学者的共同努力探索下，组织工程肌腱的相关研究已取得巨大进展。然而，到目前为止，很多技术问题依然悬而未决：种子细胞的来源、数量以及质量等问题；缺乏理想的支架材料；外源性生长因子通过缓释技术用于组织工程肌腱时其浓度如何控制，多因子的协同作用及有序作用如何解决；体外力学刺激的作用机制及最佳的应力刺激条件尚需研究。而且传统的工程化肌腱的大规模应用，需要依赖于工程化肌腱的构建和长期保存及运输，其生产制备过程耗时，保存费用昂贵。总之，现有的组织工程肌腱产品的生物功能不甚理想，难于保存和运输，很难实现临床推广应用。因此，组织工程肌腱的发展遇到了技术瓶颈，利用传统的组织工程方法构建的组织工程肌腱尚难以用于临床实现肌腱缺损修复和再生的目标。
前期研究(Preparation and characterization of decellularized tendonslices for tendon tissue engineering.Journal of Biomedical Materials ResearchPart A，2012:100A:1448–1456)和发明专利(专利号：ZL201310636964.0)，已经证实脱细胞肌腱切片和压片支架是较好的肌腱组织工程的支架材料。其优越性包括：(1)这些支架主要是由平行排列的I型胶原构成，具有天然肌腱的结构和组成成分；(2)具有肌腱特有的三维空间结构，有利于细胞粘附和增殖，能为细胞提供良好的生长环境并有利于细胞与支架及细胞与细胞间的信息传递；(3)加工处理过程中除去了细胞相关物质和异种抗原，抗原性显著降低；(4)保留了肌腱的基本力学强度；(5)支架的加工和处理方法相对温和，有利于保留材料中的生物活性物质；(6)动物的肌腱组织来源丰富，无需从人体组织取材，减少了创伤，避免了自体或异体肌腱组织来源受限的问题。尽管如此，上述肌腱切片或压片支架经过一系列物理加工和酶学处理，生物活性物质部分丢失，在体外诱导干细胞腱向分化或者体内促进肌腱组织再生的能力受到限制。
因此，对上述肌腱切片或压片支架进行生物修饰，以增强其生物活性非常必要。
目前对支架进行生物修饰以提高生物活性的方法通常是直接复合生物活性因子，但是该方法制备得到的复合支架中生物活性因子种类有限，每种因子的合适浓度难以确定，因此生物活性有限，效果不尽如人意。
发明内容
为了解决上述问题，本发明提供了一种新的生物活性支架及其制备方法和用途。
本发明制备诱导肌腱组织再生的生物活性支架的方法，包括如下步骤：
(1)取新鲜肌腱，脱细胞处理，得脱细胞肌腱压片或切片支架；
(2)在上述支架上复合细胞外基质，即可。
步骤(1)所述脱细胞肌腱压片支架的制备方法如下：
1)取新鲜肌腱，清洗；
2)将肌腱沿厚度方向进行压缩，压缩率为60～90％；
3)对步骤2)压缩后的肌腱进行反复冻融；所述反复冻融是将步骤2)压缩后的肌腱置于液氮中1～3min，25～37℃放置3～10min，重复操作4～6次。
4)将步骤3)处理后的肌腱用核酸酶处理，清洗，即可；
其中，核酸酶处理的方法为：取步骤3)处理后的肌腱，置于DNase浓度为120～180IU/ml和RNase浓度为80～120μg/ml的核酸酶溶液中，室温或37℃恒温处理6～24h。
步骤(1)所述脱细胞肌腱切片支架的制备方法如下：
1)取新鲜肌腱，清洗；
2)将肌腱进行反复冻融；所述反复冻融是将步骤1)清洗后的肌腱置于液氮中1～3min，25～37℃放置3～10min，重复操作4～6次；
3)对步骤2)冻融处理后的肌腱沿长度方向进行冰冻切片，切片厚度为300～900μm；
4)将步骤3)获得的肌腱切片用核酸酶处理，清洗，即可；
其中，核酸酶处理的方法为：取步骤3)得到的肌腱切片，置于DNase浓度为120～180IU/ml和RNase浓度为80～120μg/ml的核酸酶溶液中，室温或37℃恒温处理6～24h。
步骤(2)中，复合细胞外基质的方法是：
a、制备脱细胞肌腱凝胶：取肌腱组织，脱细胞处理，冻干，粉碎，加入到1mg/ml的胃蛋白酶溶液中，室温消化24h，用碱中和成中性，然后加入终体积1/10的PBS溶液，置于37℃培养箱中孵育成肌腱凝胶；
b、取步骤a制备的脱细胞肌腱凝胶，裱籿在步骤(1)的脱细胞肌腱压片或切片支架上，使肌腱凝胶完全覆盖肌腱压片或切片支架。
步骤a中，所述脱细胞处理的步骤是：反复冻融：在液氮中1～3min，25～37℃放置3～10min，重复操作4～6次；纵向切片：沿肌腱长度方向进行冰冻切片，切片厚度为300～900μm；核酸酶溶液处理：置于120～150IU/ml DNA酶和80～100μg/ml RNA酶中，37℃恒温摇床震荡12h；
和/或，步骤a中，用0.2N NaOH中和成中性。
步骤(2)中，复合细胞外基质的方法是：
①取肌腱细胞或者干细胞，接种于步骤(1)的脱细胞肌腱压片或切片支架上培养；
②待细胞生长至融合度为100％的致密细胞片层，进行脱细胞处理。
步骤①中，所述干细胞是骨髓基质干细胞、脂肪干细胞、肌腱干细胞。
步骤②中，待细胞达到90％融合时，加入50μΜVc继续培养6-8d至融合度为100％的致密细胞片层。
步骤②中，所述脱细胞处理是先用含20mM氨水的0.5％Triton X-10037℃处理15min，再用100U/mL DNA酶37℃处理2h。
本发明还提供了前述方法制备的诱导肌腱组织再生的生物活性支架。
本发明还提供了前述的诱导肌腱组织再生的生物活性支架在制备治疗软组织缺损修复材料中的用途。
其中，所述软组织缺损修复材料是肌腱组织或韧带组织缺损修复材料。
本发明的优点在于：
(1)制备的生物活性支架基底由脱细胞肌腱切片或压片构成，是天然的肌腱来源材料；
(2)制备的生物活性支架经过脱细胞和去抗原处理，有效地降低了支架的抗原性；
(3)制备的生物活性支架充分利用了肌腱组织的胞外基质环境，能为细胞提供良好的微环境和力学支撑，利于细胞粘附、增殖和发挥功能；
(4)制备的生物活性支架具有良好的力学性能，具有与正常肌腱组织相近的力学特性，能很好的克服肌腱修复的人工材料力学相容性差的问题；
(5)制备的生物活性支架具有与肌腱再生相匹配的降解速率，且降解产物无毒无害；
(6)制备的生物活性支架通过肌腱凝胶、肌腱细胞或干细胞胞外基质修饰，含有多种生物活性因子(转化生长因子、胰岛素样生长因子、血管内皮生长因子)、结构蛋白(纤维连接蛋白、玻璃粘连蛋白)和蛋白多糖物质(Biglycan，Fibromodulin)等，有利于诱导肌腱组织再生与修复。
(7)制备的生物活性支架易于修剪，利于肌腱缺损的缝合与固定。
(8)本发明的生物活性支架的制备工艺路线简洁，可用于大规模生产，产品易于保存和运输，产品附加值高。
综上，本发明诱导肌腱组织再生的生物活性支架，通过复合肌腱细胞、干细胞分泌的基质或者天然肌腱的细胞外基质，可以有效修复肌腱缺损，修复效果良好，优于直接复合细胞因子的方式，临床应用前景良好。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1生物活性支架制备流程示意图。
图2生物活性支架裱籿肌腱凝胶中含有多种生长因子。
图3肌腱干细胞胞外基质修饰的生物活性支架组织学：H&E染色、Masson染色和DAPI染色结果证实经过脱细胞处理有效地去除了细胞组分，同时在肌腱切片支架表面有ECM组分保留。DTSs为脱细胞肌腱切片。
图4生物活性支架表面形态观察：在肌腱切片表面复合肌腱干细胞培养，形成细胞-支架复合物，对其再次脱细胞处理后，可见支架表面有大量的干细胞ECM沉积。图中DTSs为脱细胞肌腱切片。
图5肌腱干细胞胞外基质修饰肌腱切片的生物活性支架生长因子检测：ELISA定量检测结果显示肌腱干细胞ECM修饰后的生物活性支架中生长因子(TGF-β1，VEGF和IGF-1)含量显著提高。图中DTSs为脱细胞肌腱切片，ECM-DTSs为肌腱干细胞胞外基质修饰的生物活性支架。
图6干细胞胞外基质修饰的肌腱切片生物活性支架的细胞相容性观察：SEM检测证实肌腱干细胞ECM修饰的生物活性支架表面很适合骨髓基质干细胞(BMSCs)生长，具有良好的细胞相容性。图中DTSs为脱细胞肌腱切片，ECM-DTSs为肌腱干细胞胞外基质修饰的生物活性支架。
图7 West-blot定量检测肌腱干细胞ECM修饰肌腱切片支架前后胞外基质蛋白含量变化。检测结果显示肌腱干细胞ECM修饰后的生物活性支架中胞外基质蛋白组分(Biglycan，Fibromodulin，Fibronectin和Vitronectin)含量显著提高。图中DTSs为脱细胞肌腱切片，ECM-DTSs为肌腱干细胞胞外基质修饰的生物活性支架。*表示与DTSs组相比具有显著性差异。
图8定量检测肌腱干细胞ECM修饰肌腱切片得到的生物活性支架对BMSCs细胞增殖能力的影响。图中DTSs为脱细胞肌腱切片，ECM-DTSs为肌腱干细胞胞外基质修饰的生物活性支架。*表示与DTSs组相比具有显著性差异。
图9活死细胞染色结果发现BMSCs在肌腱干细胞ECM修饰的生物活性支架上可维持更高的细胞活力。图中DTSs为脱细胞肌腱切片，ECM-DTSs为肌腱干细胞胞外基质修饰的生物活性支架。
图10兔跟腱缺损模型构建及手术修复情况。SDFT为浅表性屈肌腱，DBTs为肌腱压片支架，AT为跟腱。
图11术后不同时间点不同组别的超声观察。
图12术后不同时间点不同组别的MRI观察。术后4、8、12w分别对兔子跟腱进行B超及MRI检测实验发现，三组修复和重建的跟腱随着时间的延长，其异常回声及异常信号均减少，生物活性支架从外形明显逐渐经过重塑而与自体跟腱组织信号相同，在第12w时无法分辨自体肌腱与生物活性支架修复的肌腱的差异。
图13术后不同时间点不同组别的大体观察。
图14术后不同时间点不同组别的组织学(H&E染色)观察。大体及组织学观察发现，可见自体肌腱及自体肌腱和肌腱压片支架修复处，在第4w时有瘢痕组织形成，肌腱压片支架周围炎性反应明显；术后第12w时生物活性支架组跟腱缺损已重建，纤维束连续，炎性反应明显减轻。DBTs为肌腱压片支架，ECM为肌腱凝胶。
图15术后不同时间生物活性支架修复跟腱的生物力学检测。结果显示，随时间延长，实验组最大载荷和刚度均呈逐渐增加趋势；12w时对照组和生物活性支架组最大载荷比较差异无统计学意义(P>0.05)，其余在4w时对照组较单纯肌腱压片组及肌腱凝胶复合肌腱压片组最大载荷及刚度高(P<0.05)。DBTs为肌腱压片支架，ECM为肌腱凝胶。
具体实施方式
主要材料、试剂与仪器：
新鲜肌腱、骨髓基质干细胞、肌腱干细胞、肌腱细胞；细胞培养基、核酸酶、DNA检测试剂盒；冰冻切片机、CO2培养箱、生物力学测试系统、扫描电子显微镜、荧光显微镜，冷冻干燥机(Christ，德国)、低温球磨仪(Retsch，德国)。
统计学方法：
采用SPSS16.0软件包进行数据统计分析和处理，全部数据用均数±标准差表示，数据满足正态分布，方差齐，组间两两比较采用单因素方差分析(Sceffe法)，检验水准α＝0.05，即P<0.05时差异有统计学意义。
实施例1诱导肌腱组织再生的生物活性支架
以犬后肢跟腱纵向切片为基底材料，通过肌腱干细胞胞外基质修饰肌腱切片制备生物活性支架，具体制备方法如下：
制备切片支架：1)取新鲜肌腱，清洗；2)将肌腱进行反复冻融；所述反复冻融是将步骤1)清洗后的肌腱置于液氮中1～3min，25～37℃放置3～10min，重复操作4～6次；3)对步骤2)冻融处理后的肌腱沿长度方向进行冰冻切片，切片厚度为300～900μm；4)将步骤3)获得的肌腱切片用核酸酶处理，清洗，即可；其中，核酸酶处理的方法为：取步骤3)得到的肌腱切片，置于DNase浓度为120～180IU/ml和RNase浓度为80～120μg/ml的核酸酶溶液中，室温或37℃恒温处理6～24h。冻干、消毒备用。
如图1所示，将大鼠肌腱干细胞与跟腱切片支架复合培养7d后，培养条件是5％CO2、37℃，待细胞达到90％融合时，加入50μΜVc(维生素C)继续处理8d，至肌腱干细胞在跟腱切片支架表面形成细胞融合度为100％的致密细胞片层。然后将上述培养的肌腱干细胞-跟腱切片复合物进行脱细胞处理，先用含20mM氨水的0.5％Triton X-100 37℃处理15min，再用100U/mlDNA酶37℃处理2h。
通过DNA定量和组织学观察证实干细胞已经全部脱除，在跟腱切片支架上留下肌腱干细胞的胞外基质，获得肌腱干细胞胞外基质修饰的肌腱切片支架，该支架是一种比单纯肌腱切片支架更好的生物活性支架，可用于修复肌腱缺损，重建肌腱功能。
实施例2诱导肌腱组织再生的生物活性支架
以小牛跟腱压片为基底材料，通过肌腱干细胞胞外基质修饰肌腱压片制备生物活性支架，具体制备方法如下：
制备压片支架：可以按照专利号：ZL201310636964.0的专利的权利要求1的方法制备，具体地，1)取新鲜肌腱，清洗；2)将肌腱沿厚度方向进行压缩，压缩率为60～90％，压缩成厚度约1.0～1.2mm的肌腱压片；3)对步骤2)压缩后的肌腱进行反复冻融；所述反复冻融是将步骤2)压缩后的肌腱置于液氮中1～3min，25～37℃放置3～10min，重复操作4～6次；4)将步骤3)处理后的肌腱用核酸酶处理，清洗，即可；其中，核酸酶处理的方法为：取步骤3)处理后的肌腱，置于DNase浓度为120～180IU/ml和RNase浓度为80～120μg/ml的核酸酶溶液中，室温或37℃恒温处理6～24h；冻干、消毒备用。
如图1所示，取第三代大鼠肌腱干细胞与上述跟腱压片支架复合培养7d，培养条件是5％CO2、37℃，待细胞达到90％融合时，加入50μΜVc继续培养8d，至肌腱干细胞在跟腱压片支架表面形成细胞融合度为100％的致密的细胞片层。然后将上述培养的肌腱干细胞-跟腱压片复合物进行脱细胞处理，先用含20mM氨水的0.5％Triton X-100 37℃处理15min，再用100U/ml DNA酶37℃处理2h。
通过DNA定量和组织学观察证实干细胞已经全部脱除，在跟腱压片支架上留下肌腱干细胞的胞外基质，获得肌腱干细胞胞外基质修饰的肌腱压片支架，该支架是一种比单纯肌腱压片支架更好的生物活性支架，可用于修复肌腱缺损，重建肌腱功能。
实施例3诱导肌腱组织再生的生物活性支架
1、制备方法
以小牛跟腱压片为基底材料，制备肌腱凝胶与压片支架复合物，用于修复大白兔跟腱缺损，重建跟腱功能，具体制备和使用方法如下：
制备压片支架：可以按照专利号：ZL201310636964.0的专利的权利要求1的方法制备，具体地，1)取新鲜肌腱，清洗；2)将肌腱沿厚度方向进行压缩，压缩率为60～90％，压缩成厚度约1.0～1.2mm的肌腱压片；3)对步骤2)压缩后的肌腱进行反复冻融；所述反复冻融是将步骤2)压缩后的肌腱置于液氮中1～3min，25～37℃放置3～10min，重复操作4～6次；4)将步骤3)处理后的肌腱用核酸酶处理，清洗，即可；其中，核酸酶处理的方法为：取步骤3)处理后的肌腱，置于DNase浓度为120～180IU/ml和RNase浓度为80～120μg/ml的核酸酶溶液中，室温或37℃恒温处理6～24h；冻干、消毒备用。
取新鲜恒河猴跟腱先进行脱细胞处理，即反复冻融(液氮中1～3min，25～37℃放置3～10min，重复操作4～6次)、纵向切片(沿肌腱长度方向进行冰冻切片，切片厚度为300～900μm)和核酸酶溶液(120～150IU/ml DNA酶和80～100μg/ml RNA酶)处理，37℃恒温摇床震荡12h，然后冻干，-80℃冷冻，-20℃球磨，加入到1mg/ml的胃蛋白酶溶液中，室温消化24h，用0.2N NaOH中和成中性，最后加入终体积1/10的1×PBS，置于37℃培养箱中孵育成肌腱凝胶。
将上述制备的恒河猴脱细胞肌腱凝胶，与复水的肌腱压片复合制备生物活性支架，复合的具体方式是将上述制备的肌腱凝胶裱籿在肌腱压片表面，使肌腱凝胶完全覆盖肌腱压片支架。
取成年大白兔，制备后肢双侧跟腱缺损动物模型，分别用上述单纯肌腱压片支架、生物活性支架和大白兔自体跟腱修复，重建跟腱功能。术后经大体、组织学、影像学、生物力学检测，证实生物活性支架具有诱导跟腱组织再生、重建跟腱功能的效果，详细修复实验及结果见实验例1。
2、性质检测
在肌腱凝胶中加入组织裂解液(FNN0071,Invitrogen；凝胶质量/组织裂解液体积＝1g/10ml)，混匀，冰浴条件下静置1.5h。匀浆液在4℃条件下10,000g离心20min，取上清液待测。ELISA检测方法分别按照VEGF、TGF-β1和IGF-1检测试剂盒说明书进行。
ELISA检测结果显示，肌腱凝胶中含有与肌腱修复相关的生长因子VEGF、TGF-β1和IGF-1分别为1.30±0.53pg/mg、1.56±0.24pg/mg、8.07±0.89pg/mg。
以下用实验例的方式来说明本发明的有益效果：
实验例1本发明诱导肌腱组织再生的生物活性支架的性质
1、实验方法
按照实施例1的方法制备的生物活性支架，按照下述方法检测其性质：
(1)生物活性支架组织学检测
按照文献Ning LJ,et al.Preparation and characterization ofdecellularized tendon slices for tendon tissue engineering.Journal ofBiomedical Materials Research Part A，2012:100A:1448–1456记载的方法，进行H&E染色、Masson染色和DAPI染色。
(2)表面形态观察
表面形态检测采用扫描电子显微镜(SEM)观察。具体方法是将待测样本(对照组和实验组)用2.5％戊二醛4℃固定2h以上。PBS清洗30min×3次。酒精梯度脱水：50％、70％、80％、90％、100％各15min。临界点干燥。真空喷金后扫描电镜观察。
(3)生长因子检测
同上述ELISA检测方法。按照待测生长因子检测试剂盒说明书进行。
(4)细胞相容性观察
同上述扫描电镜(SEM)观察方法。观察样本为单纯的肌腱切片支架和肌腱干细胞ECM修饰的肌腱切片支架表面接种BMSCs，样本处理方法同上。
(5)West-blot定量检测肌腱干细胞ECM修饰肌腱切片支架前后胞外基质蛋白含量变化。
Western-Blot定量检测生物活性支架中胞外基质蛋白含量的方法，包括：待测支架中总蛋白的提取，所有蛋白样本的总蛋白含量均采用BCA蛋白浓度测定试剂盒(按说明书方法)；生物活性支架相关蛋白(Biglycan，Fibromodulin，Fibronectin和Vitronectin)的电泳及抗体孵育；采用Gel-Pro Analyzer4软件对蛋白条带进行灰度分析；所有样品的目的蛋白的相对表达情况均由其内参的表达量进行归一化处理，即：目的蛋白相对表达量＝目的蛋白灰度值/GAPDH灰度值。
(6)定量检测肌腱干细胞ECM修饰肌腱切片得到的生物活性支架对BMSCs细胞增殖能力的影响
按照文献Ning LJ,et al.Preparation and characterization ofdecellularized tendon slices for tendon tissue engineering.Journal ofBiomedical Materials Research Part A，2012:100A:1448–1456记载的方法检测。
(7)活死细胞染色
将脱细胞肌腱切片和生物活性肌腱切片支架表面加无血清DMEM培养基孵育过夜。将BMSCs接种于支架表面，5％CO2、37℃条件下培养第1d和3d时行活死细胞染色：无菌PBS清洗3次；活死细胞染液(1ml无菌PBS+1μl钙黄绿素+1μl碘化丙啶)37℃避光孵育30min；无菌PBS清洗3次，荧光显微镜观察并采图。
2、体内修复实验
取实施例3制备的复合材料(其中，覆盖在肌腱压片支架上的肌腱凝胶的厚度是100～300μm)，按照如下方法进行体内修复实验：取63只雄性成年新西兰大白兔，随机选取一侧为空白组(作为对照组)、单纯肌腱压片组(DBTs)，及肌腱凝胶复合肌腱压片组(ECM+DBTs)，自跟腱跟骨止点上方1cm处向近端取2cm的跟腱组织制作跟腱缺损模型(图10)，然后利用单纯肌腱压片支架或肌腱凝胶复合肌腱压片支架重建跟腱缺损，空白组则单纯切断跟腱后进行修复。术后第4、8、12w分别进行B超和MRI检测后，再取材进一步行组织学分析(H&E染色)和生物力学检测。
研究结果：术后4、8、12w分别对兔子跟腱进行B超及MRI检测，实验发现三组修复和重建的跟腱随着时间的延长，其异常回声及异常信号均减少，生物活性支架从外形明显逐渐经过重塑而与自体跟腱组织信号相同，在第12w时无法分辨自体肌腱与生物活性支架修复的肌腱的差异(图11和图12)。取三组标本行大体观察(图13)及组织学观察和生物力学测试。大体及组织学观察显示，对照组可见自体肌腱及自体肌腱和肌腱压片支架修复处，在第4w时有瘢痕组织形成，肌腱压片支架周围炎性反应明显；术后第12w时生物活性支架组跟腱缺损已重建，纤维束连续，炎性反应明显减轻(图13和图14)。生物力学测试结果显示，随时间延长，实验组最大载荷和刚度均呈逐渐增加趋势；12w时对照组和生物活性支架组最大载荷比较差异无统计学意义(P>0.05)，其余在4w时对照组较单纯肌腱压片组及生物活性支架组最大载荷及刚度高(P<0.05)(图15)。
3、实验结果
如图3所示，H&E染色、Masson染色和DAPI染色结果证实第二次脱细胞处理有效地去除了细胞组分，同时在肌腱切片支架表面有ECM组分保留。DTSs为脱细胞肌腱切片。
如图4所示，在肌腱切片表面复合肌腱干细胞培养，形成细胞-支架复合物，对其再次脱细胞处理后，可见支架表面有大量的干细胞ECM沉积。图中DTSs为脱细胞肌腱切片。
如图5所示，ELISA定量检测结果显示肌腱干细胞ECM修饰后的生物活性支架中生长因子(TGF-β1，VEGF和IGF-1)含量显著提高。图中DTSs为脱细胞肌腱切片，ECM-DTSs为肌腱干细胞胞外基质修饰的生物活性支架。
如图6所示，SEM检测证实肌腱干细胞ECM修饰的生物活性支架表面很适合BMSCs生长，具有良好的细胞相容性。图中DTSs为脱细胞肌腱切片，ECM-DTSs为肌腱干细胞胞外基质修饰的生物活性支架。
如图7所示，检测结果显示肌腱干细胞ECM修饰后的生物活性支架中胞外基质蛋白组分(Biglycan，Fibromodulin，Fibronectin和Vitronectin)含量显著提高。图中DTSs为脱细胞肌腱切片，ECM-DTSs为肌腱干细胞胞外基质修饰的生物活性支架。*表示与DTSs组相比具有显著性差异。
如图8所示，图中DTSs为脱细胞肌腱切片，ECM-DTSs为肌腱干细胞胞外基质修饰的生物活性支架。*表示与DTSs组相比具有显著性差异。
如图9所示，活死细胞染色结果发现BMSCs在肌腱干细胞ECM修饰的生物活性支架上可维持更高的细胞活力。图中DTSs为脱细胞肌腱切片，ECM-DTSs为肌腱干细胞胞外基质修饰的生物活性支架。
如图10所示，兔跟腱缺损模型构建及手术修复情况。SDFT为浅表性屈肌腱，DBTs为肌腱压片支架，AT为跟腱。
如图11所示，术后不同时间点不同组别的超声观察；如图12所示，术后不同时间点不同组别的MRI观察。术后4、8、12w分别对兔子跟腱进行B超及MRI检测实验发现，三组修复和重建的跟腱随着时间的延长，其异常回声及异常信号均减少；生物活性支架从外形明显逐渐经过重塑而与自体跟腱组织信号相同，在第12w时无法分辨自体肌腱与生物活性支架修复的肌腱的差异
如图13所示，术后不同时间点不同组别的大体观察；如图14所示，术后不同时间点不同组别的组织学(H&E染色)观察。大体组织学观察发现，对照组可见自体肌腱及自体肌腱和肌腱压片支架修复处，在第4w时有瘢痕组织形成，肌腱压片支架周围炎性反应明显；术后第12w时生物活性支架修复跟腱缺损已重建，纤维束连续，炎性反应明显减轻。DBTs为肌腱压片支架，ECM为肌腱凝胶。
如图15所示，术后不同时间生物活性支架修复跟腱的生物力学检测。结果显示，随时间延长，实验组最大载荷和刚度均呈逐渐增加趋势；12w时对照组和生物活性支架组最大载荷比较差异无统计学意义(P>0.05)，其余在4w时对照组较单纯肌腱压片组，及生物活性支架组最大载荷及刚度高(P<0.05)。DBTs为肌腱压片支架，ECM为肌腱凝胶。
综上，本发明通过特定方法制备得到的诱导肌腱组织再生的生物活性支架，细胞因子含量高，生物相容性好，体内修复效果优良，具有较好的应用前景。
Claims (12)
1.一种制备诱导肌腱组织再生的生物活性支架的方法，其特征在于：包括如下步骤：
(1)取新鲜肌腱，脱细胞处理，得脱细胞肌腱压片或切片支架；
(2)在上述支架上复合细胞外基质，即可。
2.根据权利要求1所述的方法，其特征在于：步骤(1)所述脱细胞肌腱压片支架的制备方法如下：
1)取新鲜肌腱，清洗；
2)将肌腱沿厚度方向进行压缩，压缩率为60～90％；
3)对步骤2)压缩后的肌腱进行反复冻融；所述反复冻融是将步骤2)压缩后的肌腱置于液氮中1～3min，25～37℃放置3～10min，重复操作4～6次；
4)将步骤3)处理后的肌腱用核酸酶处理，清洗，即可；
其中，核酸酶处理的方法为：取步骤3)处理后的肌腱，置于DNase浓度为120～180IU/ml和RNase浓度为80～120μg/ml的核酸酶溶液中，室温或37℃恒温处理6～24h。
3.根据权利要求1所述的方法，其特征在于：步骤(1)所述脱细胞肌腱切片支架的制备方法如下：
1)取新鲜肌腱，清洗；
2)将肌腱进行反复冻融；所述反复冻融是将步骤1)清洗后的肌腱置于液氮中1～3min，25～37℃放置3～10min，重复操作4～6次；
3)对步骤2)冻融处理后的肌腱沿长度方向进行冰冻切片，切片厚度为300～900μm；
4)将步骤3)获得的肌腱切片用核酸酶处理，清洗，即可；
其中，核酸酶处理的方法为：取步骤3)得到的肌腱切片，置于DNase浓度为120～180IU/ml和RNase浓度为80～120μg/ml的核酸酶溶液中，室温或37℃恒温处理6～24h。
4.根据权利要求1所述的方法，其特征在于：步骤(2)中，复合细胞外基质的方法是：
a、制备脱细胞肌腱凝胶：取肌腱组织，脱细胞处理，冻干，粉碎，加入到1mg/ml的胃蛋白酶溶液中，室温消化24h，用碱中和成中性，然后加入终体积1/10的PBS溶液，置于37℃培养箱中孵育成肌腱凝胶；
b、取步骤a制备的脱细胞肌腱凝胶，裱籿在步骤(1)的脱细胞肌腱压片或切片支架上，使肌腱凝胶完全覆盖肌腱压片或切片支架。
5.根据权利要求1所述的方法，其特征在于：
步骤a中，所述脱细胞处理的步骤是：反复冻融：在液氮中1～3min，25～37℃放置3～10min，重复操作4～6次；纵向切片：沿肌腱长度方向进行冰冻切片，切片厚度为300～900μm；核酸酶溶液处理：置于120～150IU/ml DNA酶和80～100μg/ml RNA酶中，37℃恒温摇床震荡12h；
和/或，步骤a中，用0.2N NaOH中和成中性。
6.根据权利要求1所述的方法，其特征在于：步骤(2)中，复合细胞外基质的方法是：
①取肌腱细胞或者干细胞，接种于步骤(1)的脱细胞肌腱压片或切片支架上培养；
②待细胞生长至汇合度为100％的致密细胞片层，进行脱细胞处理。
7.根据权利要求6所述的方法，其特征在于：步骤①中，所述干细胞是骨髓基质干细胞、脂肪干细胞、肌腱干细胞。
8.根据权利要求6所述的方法，其特征在于：步骤②中，待细胞达到90％融合时，加入50μΜVc继续培养6-8d至融合度为100％的致密细胞片层。
9.根据权利要求6所述的方法，其特征在于：步骤②中，所述脱细胞处理是先用含20mM氨水的0.5％Triton X-100 37℃处理15min，再用100U/ml DNA酶37℃处理2h。
10.权利要求1～9任意一项所述方法制备的诱导肌腱组织再生的生物活性支架。
11.权利要求10所述的诱导肌腱组织再生的生物活性支架在制备治疗软组织缺损修复材料中的用途。
12.根据权利要求11所述的用途，其特征在于：所述软组织缺损修复材料是肌腱组织或韧带组织缺损修复材料。
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| CN114533959B (zh) * | 2022-04-02 | 2022-12-09 | 山东隽秀生物科技股份有限公司 | 一种肌腱修复材料、制备方法及在制备肌腱修复产品中的应用 |
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