CN115006592B - 一种脱细胞真皮复合材料、制备方法及用途 - Google Patents
一种脱细胞真皮复合材料、制备方法及用途 Download PDFInfo
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Abstract
本发明公开了一种脱细胞真皮复合材料的制备方法，包括：一种脱细胞真皮复合材料的制备方法，包括：步骤1，将真皮原料制备成脱细胞真皮；步骤2，制备胎盘组织浆料；步骤3，将所述脱细胞真皮浸渍于所述胎盘组织浆料中，经真空冷冻干燥和灭菌后，获得脱细胞真皮复合材料。本发明经过特殊处理的脱细胞真皮基质(ADM)复合材料含有胎盘组织浆料颗粒，含有多种溶菌酶和抗体，具有抗病毒和杀菌作用，含有碱性成纤维生长因子等多种细胞因子，有着良好的生物相容性，其覆盖在腹腔开放的肠管上方，避免与肠管的过度粘连进一步损伤肠管，促进肠浆膜层修复，从而减少空气瘘的发生。
Description
技术领域
本发明涉及医用材料领域，具体涉及一种脱细胞真皮复合材料、制备方法及用途。
背景技术
腹部严重创伤、复杂肠瘘等均可导致腹腔高压、腹腔感染灶引流困难或术后无法关腹，特殊情况下需要行腹腔开放(OA)疗法。腹腔开放可降低腹腔内压力、防止腹腔间隙综合征(ACS)、防治多器官功能障碍(MODF)，还便于感染坏死组织反复清除、通畅引流。但由于腹腔开放疗法丧失了对腹腔内脏器的保护作用，肠道、肝脏等腹腔内脏器直接暴露于各种敷料或空气中，不可避免地造成了大量体液及蛋白质的丢失、肠壁损伤出血、肠功能障碍等诸多并发症。其中，肠空气瘘无疑是最严重的。因此，在腹腔开放后早期行暂时性关腹(TAC)就尤为重要，TAC可以容纳和保护腹内脏器，避免污染，防止肠管脱出、干燥、水肿、粘连和损伤，减少体液丢失，便于再进腹，保护切口边缘避免其回缩，维持腹腔容积，为确定性腹腔关闭创造条件。因此腹腔开放疗法的关键技术在于能够采用特定的腹腔开放临时关闭保护材料及辅助装置进行安全有效的腹腔临时关闭。
TAC包括多种不同类型，其中负压辅助关腹法(VSD)是较为常用的一种类型；它主要由医用泡沫材料、黏性贴膜和负压管路系统构成。将医用泡沫材料放置于医用敷料(如凡士林纱布)覆盖的肠管上方，用黏性贴膜覆盖封闭，剪去一小片黏性贴膜并将一个接有负压管路系统的接口垫置于该缺损处。负压可使医用泡沫敷料(如聚氨酯海绵层)塌陷，从而对腹膜和筋膜施加一个向内侧的牵引力，同时可以将腹腔内的液体引流至体外。但是，由于敷料材料的限制，目前仍大量使用凡士林纱布覆盖在肠管上方，然后盖上聚氨酯海绵层接负压吸引，一方面生物相容性差，肠管易损伤面临着发生肠空气瘘的风险，另一方面，通透性不佳，引流效果受限。因此，寻求生物相容性好、通透性佳的生物材料迫在眉睫。
发明内容
本发明的目的是提供一种生物相容性好、通透性佳的生物材料。
为了达到上述目的，本发明提供了一种脱细胞真皮复合材料的制备方法，包括：
一种脱细胞真皮复合材料的制备方法，包括：
步骤1，将真皮原料制备成脱细胞真皮；
步骤2，制备胎盘组织浆料；
步骤3，将所述脱细胞真皮浸渍于所述胎盘组织浆料中，经真空冷冻干燥和灭菌后，获得脱细胞真皮复合材料。
可选地，步骤2包括：
步骤2.1，对胎盘组织预处理；
步骤2.2，向预处理后的胎盘组织添加含有抗菌剂的有机缓释介质，获得所述胎盘组织浆料；所述抗菌剂为纳米银、纳米氧化锌、纳米铜、抗菌多肽、抗菌素中的一种或多种；所述有机缓释介质为丙烯酸类共聚物、聚乙烯醇与聚乙二醇共聚物、聚山梨酯80、丙三醇、乙基纤维素、壳聚糖、海藻酸钙、海藻酸钠、明胶中的一种或多种。
可选地，步骤2.1的预处理包括：去除血污、细胞、DNA后，消毒、干燥和颗粒化处理。
可选地，还包括对所述脱细胞真皮的原料进行打孔处理的步骤，孔径为1mm-2mm，孔距为2mm-3mm。
可选地，还包括对所述脱细胞真皮进行等离子体表面改性的步骤。
可选地，还包括对所述脱细胞真皮的原料进行切片的步骤，切片后的厚度为0.12mm-0.5mm。
可选地，步骤3中浸渍时间为20h-30h。
可选地，所述脱细胞真皮的原料为同种或异种真皮。
本发明还提供了一种通过上述制备方法制备的脱细胞真皮复合材料。
本发明还提供了一种通过上述制备方法制备的脱细胞真皮复合材料的用途，所述脱细胞真皮复合材料用于腹腔开放暂时性关腹。
本发明的有益效果为：本发明经过特殊处理的脱细胞真皮基质(ADM)复合材料含有胎盘组织浆料颗粒，含有多种溶菌酶和抗体，具有抗病毒和杀菌作用，含有碱性成纤维生长因子等多种细胞因子，有着良好的生物相容性，其覆盖在腹腔开放的肠管上方，避免与肠管的过度粘连进一步损伤肠管，促进肠浆膜层修复，从而减少空气瘘的发生。优化的，可以添加含有抗菌剂的有机缓释介质，进一步增强效果。采用的脱细胞真皮基质(ADM)复合材料进行了超薄切片、打孔，同时胎盘组织浆料颗粒化处理，也有利于充分引流及降低成本。
具体实施方式
下面将对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例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。
本发明提供了一种脱细胞真皮复合材料的制备方法，包括：
1.将真皮原料制备成脱细胞真皮
步骤1.1，对真皮原料进行切片，切片后的厚度为0.12mm-0.5mm。可选地，真皮原料来源于同种或异种真皮。
步骤1.2，对真皮脱细胞处理，获得脱细胞真皮。
步骤1.3，对脱细胞真皮进行打孔处理，孔径为1mm-2mm，孔距为2mm-3mm。
步骤1.4，对脱细胞真皮进行等离子体表面改性。
2.制备胎盘组织浆料
步骤2.1，对胎盘组织预处理，包括：去除血污、细胞、DNA后，消毒、干燥和颗粒化处理。
步骤2.2，向预处理后的胎盘组织添加含有抗菌剂的有机缓释介质，获得所述胎盘组织浆料；所述抗菌剂为纳米银、纳米氧化锌、纳米铜、抗菌多肽、抗菌素中的一种或多种；所述有机缓释介质为丙烯酸类共聚物、聚乙烯醇与聚乙二醇共聚物、聚山梨酯80、丙三醇、乙基纤维素、壳聚糖、海藻酸钙、海藻酸钠、明胶中的一种或多种。
3.将所述脱细胞真皮浸渍于所述胎盘组织浆料中，浸渍时间为20h-30h，之后经真空冷冻干燥和灭菌后，获得脱细胞真皮复合材料。
实施例1
步骤1，制备脱细胞真皮。
清洗成熟大白猪(约克夏猪种)猪背部皮，去毛刀去毛后，以取皮鼓切取厚度为0.25毫米厚的皮片抛弃，除去表皮，毛发及毛囊，再用取皮鼓取厚度为0.25毫米厚真皮片备用(常规ADM的1/2厚度)。加入质量分数为0.5％的过氧乙酸消毒液里浸泡40分钟。使用磷酸盐缓冲盐水清洗3次，每次均在摇床上摇30分钟。加入脱细胞液，将猪真皮片与脱细胞液充分混合以及反应，在摇床上混合10小时，脱细胞液为4-羟乙基哌嗪乙磺酸缓冲液，其pH值为8，每升4-羟乙基哌嗪乙磺酸缓冲液含20毫摩尔4-羟乙基哌嗪乙磺酸，脱细胞液中含有脱氧胆酸钠，脱氧胆酸钠的质量体积百分比溶度为2％，脱细胞液的体积与猪真皮片体积之比为30：1。吸出脱细胞液，然后加入磷酸盐缓冲盐水充分清洗猪真皮片，使用磷酸盐缓冲盐水清洗3次，每次均在摇床上摇30分钟，得到ADM。然后，行打孔处理，打孔孔径为1.5mm，孔距为2mm，最后采用低温等离子体技术改性。
步骤2，制备胎盘组织浆料。
将获取的胎盘组织在无菌条件下送至控制处理环境，并浸泡在盛有2.0wt％氯化钠水溶液的无菌盆中进行润洗，对羊膜和绒毛膜组织进行处理，去除血污、细胞和DNA，并对去细胞的组织进行消毒：首先，用2.0wt％氯化钠水溶液反复清洗以去除血污；然后，将除去血污的组织浸泡在3.0wt％氯化钠水溶液中，在室温下摇动混合120分钟以去除细胞；之后，将去除细胞的组织放入去DNA溶液(由HEPES溶液、MgCl2溶液、CaCl2溶液、纯化水、DNA酶混合组成)中，在室温下摇动混合5小时至组织中残留DNA含量满足不大于250纳克/每毫克组织；最后，将去DNA的组织浸泡在70％异丙醇中浸泡50分钟进行消毒。然后将模具放入烘箱中，在40℃温度条件下干燥60分钟，得到干燥组织；采用低温下超声匀浆技术或者冻融破碎法，颗粒化处理。然后添加含有抗菌剂的有机缓释介质成为胎盘组织浆料，有机缓释介质选用聚乙烯醇与聚乙二醇共聚物、壳聚糖；抗菌剂为纳米银。
步骤3，将获得的多孔脱细胞真皮浸渍于胎盘组织浆料中24h，经真空冷冻干燥和灭菌后，获得脱细胞真皮复合材料。
本实施例制备的脱细胞真皮复合材料的使用方法为：将脱细胞真皮复合材料裁剪成较腹腔开放区域稍小的面积，覆盖在腹腔开放的肠管上方，接着放置医用泡沫材料(如聚氨酯海绵层)成相近大小，用黏性贴膜覆盖封闭，剪去一小片黏性贴膜并将一个接有负压管路系统的接口垫置于该缺损处。负压可使医用泡沫敷料塌陷，从而对腹腔施加一个向内侧的牵引力，同时可以将腹腔内的液体引流至体外。
实施例2
步骤1，制备脱细胞真皮。
取商品人源ADM，行超薄切片处理，厚度为原来ADM的1/2，约0.25mm。然后行打孔处理，孔径为1.5mm，孔距为2mm，最后采用低温等离子体技术改性。
步骤2，制备胎盘组织浆料。
将获取的胎盘组织在无菌条件下送至控制处理环境，并浸泡在盛有2.0wt％氯化钠水溶液的无菌盆中进行润洗，对羊膜和绒毛膜组织进行处理，去除血污、细胞和DNA，并对去细胞的组织进行消毒：首先，用2.0wt％氯化钠水溶液反复清洗以去除血污；然后，将除去血污的组织浸泡在3.0wt％氯化钠水溶液中，在室温下摇动混合120分钟以去除细胞；之后，将去除细胞的组织放入去DNA溶液(由HEPES溶液、MgCl2溶液、CaCl2溶液、纯化水、DNA酶混合组成)中，在室温下摇动混合5小时至组织中残留DNA含量满足不大于250ng/每mg组织；最后，将去DNA的组织浸泡在70％异丙醇中浸泡50分钟进行消毒。然后将模具放入烘箱中，在40℃温度条件下干燥60分钟，得到干燥组织；采用低温下超声匀浆技术或者冻融破碎法，颗粒化处理。然后添加含有抗菌剂的有机缓释介质成为胎盘组织浆料，有机缓释介质选用聚乙烯醇与聚乙二醇共聚物、壳聚糖；抗菌剂为抗菌多肽。
步骤3，将获得的多孔脱细胞真皮浸渍于胎盘组织浆料中24h，经真空冷冻干燥和灭菌后，获得脱细胞真皮复合材料。
本实施例制备的脱细胞真皮复合材料的使用方法为：经腹腔开放区域周围腹壁留置并固定1-2根乳胶引流管或双套管至腹腔，将特殊处理的ADM复合材料裁剪成较腹腔开放区域稍小的面积，覆盖在腹腔开放的肠管上方，接着放置医用泡沫材料(如聚氨酯海绵层)成相近大小，用黏性贴膜覆盖封闭，剪去一小片黏性贴膜并将一个接有负压管路系统的接口垫置于该缺损处。负压可使医用泡沫敷料塌陷，从而对腹腔施加一个向内侧的牵引力，同时可以将腹腔内的液体引流至体外。同时可以经双套管持续冲洗渗出的肠内容物。
综上所述，本发明经过特殊处理的脱细胞真皮基质(ADM)复合材料含有胎盘组织浆料颗粒，含有多种溶菌酶和抗体，具有抗病毒和杀菌作用，含有碱性成纤维生长因子等多种细胞因子，有着良好的生物相容性，其覆盖在腹腔开放的肠管上方，避免与肠管的过度粘连进一步损伤肠管，促进肠浆膜层修复，从而减少空气瘘的发生。优化的，可以添加含有抗菌剂的有机缓释介质，进一步增强效果。采用的脱细胞真皮基质(ADM)复合材料进行了超薄切片、打孔，同时胎盘组织浆料颗粒化处理，也有利于充分引流及降低成本。
尽管本发明的内容已经通过上述优选实施例作了详细介绍，但应当认识到上述的描述不应被认为是对本发明的限制。在本领域技术人员阅读了上述内容后，对于本发明的多种修改和替代都将是显而易见的。因此，本发明的保护范围应由所附的权利要求来限定。
Claims (4)
1.一种用于腹腔开放暂时性关腹的脱细胞真皮复合材料的制备方法，其特征在于，包括：
步骤1，将真皮原料制备成脱细胞真皮；
步骤2，制备胎盘组织浆料；
步骤3，将所述脱细胞真皮浸渍于所述胎盘组织浆料中，经真空冷冻干燥和灭菌后，获得脱细胞真皮复合材料；
步骤1中，制备脱细胞真皮的具体方法为，取猪背部皮，去毛刀去毛后，以取皮鼓切取厚度为0.25毫米厚的皮片抛弃，除去表皮，毛发及毛囊，再用取皮鼓取厚度为 0.25 毫米厚猪真皮片备用；加入质量分数为0.5%的过氧乙酸消毒液浸泡40分钟，使用磷酸盐缓冲盐水清洗3次，每次均在摇床上摇30分钟；加入脱细胞液，将猪真皮片与脱细胞液充分混合以及反应，在摇床上混合10小时，脱细胞液为4-羟乙基哌嗪乙磺酸缓冲液，其pH值为8，每升4-羟乙基哌嗪乙磺酸缓冲液含20毫摩尔4-羟乙基哌嗪乙磺酸，脱细胞液中含有脱氧胆酸钠，脱氧胆酸钠的质量体积百分比溶度为2%，脱细胞液的体积与猪真皮片体积之比为30：1；吸出脱细胞液，然后加入磷酸盐缓冲盐水充分清洗猪真皮片，使用磷酸盐缓冲盐水清洗3次，每次均在摇床上摇30分钟，得到脱细胞真皮；然后，行打孔处理，打孔孔径为1.5mm，孔距为2mm，最后采用低温等离子体技术改性；
步骤2中，将获取的胎盘组织在无菌条件下送至控制处理环境，并浸泡在盛有2.0wt％氯化钠水溶液的无菌盆中进行润洗，对羊膜和绒毛膜组织进行处理，去除血污、细胞和DNA，并对去细胞的组织进行消毒：首先，用2.0wt％氯化钠水溶液反复清洗以去除血污；然后，将除去血污的组织浸泡在3.0wt％氯化钠水溶液中，在室温下摇动混合120分钟以去除细胞；之后，将去除细胞的组织放入去DNA溶液，所述去DNA溶液由HEPES溶液、MgCl2溶液、CaCl2溶液、纯化水、DNA酶混合组成，在室温下摇动混合5小时至组织中残留DNA含量满足不大于250纳克/每毫克组织；最后，将去DNA的组织浸泡在70％异丙醇中浸泡50分钟进行消毒；然后将模具放入烘箱中，在40℃温度条件下干燥60分钟，得到干燥组织；采用低温下超声匀浆技术或者冻融破碎法，颗粒化处理；然后添加含有抗菌剂的有机缓释介质成为胎盘组织浆料，有机缓释介质选用聚乙烯醇与聚乙二醇共聚物、壳聚糖；抗菌剂为纳米银。
2.如权利要求1所述的制备方法，其特征在于，还包括对所述脱细胞真皮的原料进行切片的步骤，切片后的厚度为0.12mm-0.5mm。
3.如权利要求1所述的制备方法，其特征在于，步骤3中浸渍时间为20h-30h。
4.一种通过权利要求1-3任意一项所述的制备方法制备的用于腹腔开放暂时性关腹的脱细胞真皮复合材料。
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