CN109529106B - 一种慢性创面修复基质的制备方法 - Google Patents
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Abstract
本发明公开了一种慢性创面修复基质的制备方法，包括如下步骤：（1）取3~5月龄胎牛皮，剪碎，经冻融、超声波结合化学洗脱等处理，去除其中的细胞结构及其它免疫原物质；（2）采用静电纺丝技术，将去细胞胎牛皮细胞外基质和蒙脱石粉制成微球，低温干燥制成一种慢性创面修复基质。本发明制得的慢性创面修复基质能够有效吸收创面深处，保持创面清洁，同时，胎牛皮细胞外基质含有的活性生物因子有助于组织再生修复。
Description
技术领域
本发明属于医用生物材料、组织工程修复领域，具体涉及一种慢性创面修复基质及其制备方法。
背景技术
伴随人口老龄化以及全球范围内糖尿病、肥胖的增加，慢性创面的发病率也日益增高，临床上常见的慢性创面有创伤感染、压疮、糖尿病溃疡、静脉性溃疡等等。慢性创面受组织缺氧、缺血、感染、局部生长因子数量减少、自身免疫力低下等的影响，愈合周期长，甚至常年不愈，给患者的生活带来了极大的影响。组织工程化基质的出现为慢性创面治疗提供了新的解决途径。
组织工程是以细胞生物学和材料科学为基础，多学科交叉的新型科学，其核心要素为细胞、支架和生长信息，其中一个好的支架可为细胞移植、损伤修复提供良好的载体。脱细胞真皮基质（ADM）是将真皮组织通过一系列物理化学及生物学方法去除表皮及皮内具有免疫原性的细胞成分，保留胶原等细胞外基质成分与结构，得到的一种具有三维空间结构的胶原基质材料。大量研究已经证实，ADM具有可诱导组织再生性，可为细胞提供生存及活动的微环境，促进细胞生长增殖，有助于促进新生血管和上皮形成，使组织完成愈合过程。
研究表明，胎牛皮（特指3~5月龄胎牛皮）结构与人皮相似，尤其在真皮胶原纤维束三维结构上存在较大相似性，与其它异种动物的真皮组织形态学相比较存在明显的优势。除此之外，胎牛皮的细胞外基质结构中含有大量促进组织细胞生长的蛋白，且其抗原性成分更易除去，基质成分更易被机体组织细胞作为营养物质吸收。因生物行业对胎牛血清的需求量大，导致大量胎牛皮副产物被废弃，然而胎牛皮经抗原去除处理后，却是一种优异的创面修复材料，尤其是对慢性难愈创面，更是具有特殊的应用价值。
天然蒙脱石是一种硅铝酸盐，具有层纹状结构和非均匀性电荷分布，对消化道内的病毒、病菌及其产生的毒素、气体等有极强的固定、抑制作用，使其失去致病作用；此外对消化道黏膜还具有很强的覆盖保护能力，修复、提高黏膜屏障对攻击因子的防御功能，具有平衡正常菌群和局部止痛作用。
通过组织工程技术去除组织中的抗原成分，并将蒙脱石结合在基质上制成慢性创面修复基质，最大程度地发挥胎牛皮细胞外基质的组织再生诱导功能，并利用蒙脱石的固定抑制毒素作用，为组织再生提供环境，从而辅助组织再生。
发明内容
本发明提供了一种难愈创面修复基质的制备方法，通过组织脱细胞技术将胎牛皮中的抗原成分去除，保留其组织中的活性基质成分，利用微球制备技术，将胎牛皮细胞外基质和蒙脱石有效结合在一起，形成微球颗粒，比表面积增大，能够吸收并固定不利于组织再生的液体渗出，固定毒素，招募细胞在微球上定植增生，引导组织再生，从而实现创面的修复，旨在改善慢性难愈创面患者的久治不愈的问题。
一种慢性创面修复基质的制备方法，包括以下步骤：
（1）取3~5月龄胎牛皮，剪切成不大于2mm的小块，纯化水洗涤3次，
（2）将步骤1中洗涤后的胎牛皮小块置于-196℃~-20℃环境中冷冻24h及以上。
（3）将步骤2中冷冻的胎牛皮进行恒温水浴解冻，恒温水浴解冻温度为25℃~37℃。
（4）将步骤3中解冻完成的胎牛皮水溶液沥干，纯化水洗涤3次，沥干，将胎牛皮转移至Triton X-100—PBS溶液中，浸泡1h，Triton X-100—PBS溶液的浓度为2%~4%。
（5）将步骤4中浸泡在Triton X-100—PBS溶液中的胎牛皮进行超声波处理，超声波功率选择90KW~180KW，工作时间为5min~10min，超声波处理完成后，纯化水洗涤3次。
（6）重复步骤2~5一次。
（7）将步骤6获得的去细胞胎牛皮基质浸泡在15%~18%的过氧乙酸溶液中，使用高速剪切设备进行匀浆，搅拌直至其形成凝胶态溶液。
（8）将步骤7中获得的溶液使用静电纺丝设备制备微球，凝固浴为2倍~10倍PBS溶液，凝固浴温度为35℃~39℃，其中，凝固浴中还含有5%~10%的蒙脱石超微粉，凝固浴处于搅拌状态。
（1）将步骤8中获得微球进行过滤、低温烘干处理，获得慢性创面修复基质。
本发明与现有材料相比，突出的优点在于：1、选用的胎牛皮基质材料，其中含有大量的活性蛋白和生长因子，处理过程对基质超微结构的破坏小，充分保留了其中的有效成分，是进行慢性难愈创面修复的重要条件。2、将胎牛皮基质材料与蒙脱石进行有机结合，可以吸收创面渗液，避免了对创面的浸渍，同时，及时将创面的感染性因子固定，避免对创面愈合造成不利影响。
具体实施方式
实施例1：一种慢性创面修复基质的制备方法
取5月龄胎牛皮，剪切成不大于2mm的小块，纯化水洗涤3次，将洗涤后的胎牛皮置于-196℃液氮中冷冻24h后取出，37℃恒温水浴解冻，纯化水洗涤3次，沥干转移至5%TritonX-100—PBS溶液中浸泡1h后，进行超声波处理，超声波功为90KW，工作时间为5min超声波处理完成后，纯化水洗涤3次。重复上述步骤1次。
将上述处理完成后获得的去细胞胎牛皮基质浸泡在15%~18%的过氧乙酸溶液中，使用高速剪切设备进行匀浆，搅拌直至其形成凝胶态溶液，使用静电纺丝设备制备微球，凝固浴为2倍PBS溶液，凝固浴温度为37℃，其中，凝固浴中还含有6%的蒙脱石超微粉，凝固浴搅拌状态下，将液体喷入凝固浴中，制得微球，将制得的微球37℃烘干处理，获得慢性创面修复基质。
实施例2：一种慢性创面修复基质的制备方法
取3月龄胎牛皮，剪切成不大于2mm的小块，纯化水洗涤3次，将洗涤后的胎牛皮置于-20℃冰箱冷冻室中冷冻24h后取出，37℃恒温水浴解冻，纯化水洗涤3次，沥干转移至3%Triton X-100—PBS溶液中浸泡1h后，进行超声波处理，超声波功为90KW，工作时间为5min超声波处理完成后，纯化水洗涤3次。重复上述步骤1次。
将上述处理完成后获得的去细胞胎牛皮基质浸泡在15%~18%的过氧乙酸溶液中，使用高速剪切设备进行匀浆，搅拌直至其形成凝胶态溶液，使用静电纺丝设备制备微球，凝固浴为5倍PBS溶液，凝固浴温度为37℃，其中，凝固浴中还含有8%的蒙脱石超微粉，凝固浴搅拌状态下，将液体喷入凝固浴中，制得微球，将制得的微球冷冻干燥处理，获得慢性创面修复基质。
Claims (3)
1.一种慢性创面修复基质的制备方法，其特征在于，包括以下步骤：
(1)取3~5月龄胎牛皮，剪切成不大于2mm的小块，纯化水洗涤3次;
(2)将步骤1中洗涤后的胎牛皮小块置于-196℃~-20℃环境中冷冻24h及以上;
(3)将步骤2中冷冻的胎牛皮进行恒温水浴解冻，恒温水浴解冻温度为25℃~37℃;
(4)将步骤3中解冻完成的胎牛皮水溶液沥干，纯化水洗涤3次，沥干，将胎牛皮转移至Triton X-100—PBS溶液中，浸泡搅拌1h，Triton X-100—PBS溶液的浓度为2%~4%;
(5)将步骤4中浸泡在Triton X-100—PBS溶液中的胎牛皮进行超声波处理，超声波功率选择90k W~180k W，工作时间为5min~10min，超声波处理完成后，纯化水洗涤3次;
(6)重复步骤2~5一次;
(7)将步骤6获得的去细胞胎牛皮基质浸泡在15%~18%的过氧乙酸溶液中，使用高速剪切设备进行匀浆，搅拌直至其形成凝胶态溶液;
(8)将步骤7中获得的溶液使用静电纺丝设备制备微球，凝固浴为2倍~10倍PBS溶液，凝固浴温度为35℃~39℃，其中，凝固浴中还含有5%~10%的蒙脱石超微粉，凝固浴处于搅拌状态;
(9)将步骤8中获得微球进行过滤、低温烘干处理，获得慢性创面修复基质。
2.根据权利要求1所述的制备方法，其特征在于步骤（4）中，Triton X-100—PBS溶液的制备方法为：按照体积比，将Triton X-100溶液加入到配制好的PBS缓冲溶液中，Triton X-100的体积分数为2%~4%。
3.根据权利要求1所述的制备方法，其特征在于步骤（8）中，凝固浴的配制方法为：配制2倍~10倍PBS溶液，按照蒙脱石粉与PBS缓冲液质量比5%~10%的比例，将蒙脱石粉加入到PBS溶液中。
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