CN114732959A - 一种脱细胞瘢痕皮肤支架材料及其制备方法与应用 - Google Patents
一种脱细胞瘢痕皮肤支架材料及其制备方法与应用 Download PDFInfo
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Abstract
本发明属于医学领域,公开了一种脱细胞瘢痕皮肤支架材料及其制备方法与应用,所述脱细胞瘢痕皮肤支架材料的制备方法,包括如下步骤:步骤1:切除瘢痕标本中表皮及皮下组织,将获得的组织切成的薄片,再将薄片中的病毒进行灭活;步骤2:将步骤1处理后的薄片中细胞进行溶解;步骤3:将步骤2处理后的薄片储存于抗生素溶液中,并置于低温条件下备用,即完成脱细胞瘢痕皮肤支架材料的制备。其制备成本低,且可以实现离体瘢痕皮肤资源的合理利用,抗原性弱,且降解速度慢,使用寿命长,营养渗透性好、血管化速度快且血管化程度高,皮片成活率高。
Description
技术领域
本发明属于医学领域,尤其涉及一种脱细胞瘢痕皮肤支架材料及其制备方法与应用。
背景技术
真皮支架材料(ADM)为宿主细胞提供了三维的生长附着环境,是组织进行修复重建的关键因素。自ADM作为真皮支架材料被报道于应用于创面修复以来,ADM是皮肤重建中理想的支架材料。ADM可以作为体外支持系统调节成纤维细胞的增殖分化,同时下调其细胞外基质成分的分泌,包括胶原蛋白的过度表达,减轻创面瘢痕的产生。然而,当前市面上使用的真皮支架材料的缺点包括费用昂贵,血管化程度有限,抗原性强,来源有限及材料降解过快,营养渗透性差、血管化速度慢、皮片成活率低等,限制了临床广泛应用。我们发现,具有大面积瘢痕的患者通常会经历几次瘢痕切除术,因此能否使用上一次瘢痕切除后制备的皮肤材料来进行下一次瘢痕切除后的创面修复,从而克服当前市面上皮肤支架材料的缺点,是我们进行本专利申请的立足点。
发明内容
为了解决上述技术问题,本发明的目的之一在于提供一种成本低,且抗原性弱,且实现资源合理利用的脱细胞瘢痕皮肤支架材料的制备方法。
为了实现上述目的,本发明的技术方案如下:一种脱细胞瘢痕皮肤支架材料的制备方法,包括如下步骤:
步骤1:切除瘢痕标本中表皮及皮下组织,将获得的组织切成的薄片,再将薄片中的病毒进行灭活;
步骤2:将步骤1处理后的薄片中细胞进行溶解;
步骤3:将步骤2处理后的薄片储存于抗生素溶液中,并置于低温条件下备用,即完成脱细胞瘢痕皮肤支架材料的制备。
上述技术方案中所述步骤1中薄片病毒灭活方式是:将薄片置于消毒液中浸泡1.5-2.5h,然后将薄片取出后洗除残留的消毒液即可。
上述技术方案中所述消毒液为过氧乙酸和乙醇混合的水溶液,其中,所述消毒液中过氧乙酸的浓度为1vol%,所述消毒液中乙醇的浓度为24vol%。
上述技术方案中所述薄片洗除残留消毒液的方式是:先将薄片置于无菌的PBS缓冲液或无菌的生理盐水中浸泡20-40min,中途更换1-3次浸泡液,然后再将薄片置于纯水中清洗1-3次,每次清洗时长为5-15min。
上述技术方案中所述步骤2中薄片中细胞溶解的方式是:将薄片先置于细胞膜消解液中浸泡2h并持续振荡,再将薄片取出再置于细胞核消解液中,并在37℃下持续振荡72小时,期间每间隔24小时更换一次细胞核消解液,最后将薄片取出后洗除残留的细胞核消解液即可。
上述技术方案中所述细胞膜消解液为胰蛋白酶和EDTA-Na2混合的水溶液,所述细胞膜消解液中的胰蛋白酶浓度为2.5g/L,所述细胞膜消解液中的EDTA-Na2的浓度为0.5mmol/L。
上述技术方案中所述细胞核消解液为浓度为1vol%的Triton X-100溶液。
上述技术方案中所述薄片洗除残留细胞核消解液的方式是:先将薄片置于无菌的PBS缓冲液或无菌的生理盐水中浸泡20-40min,中途更换1-3次浸泡液,然后再将薄片置于纯水中清洗1-3次,每次清洗时长为5-15min。
本发明的目的之二在于提供一种采用如上所述制备方法所制得的脱细胞瘢痕皮肤支架材料。
本发明的目的之三在于提供一种如上所述脱细胞瘢痕皮肤支架材料在制备真皮支架中的应用。
本发明的有益效果在于:其制备成本低,且可以实现离体瘢痕皮肤资源的合理利用,抗原性弱,且降解速度慢,使用寿命长,营养渗透性好、血管化速度快且血管化程度高,皮片成活率高。
附图说明
图1为本发明实施例1所制备脱细胞瘢痕皮肤支架材料的照片图;
图2为本发明实施例2所制备脱细胞瘢痕皮肤支架材料的照片图;
图3为本发明实施例1所制备脱细胞瘢痕皮肤支架材料HE染色后的放大图;
图4为本发明实施例2所制备脱细胞瘢痕皮肤支架材料HE染色后的放大图;
图5为对照例真皮脱细胞基质HE染色后的放大图;
图6为本发明实施例1所述制备脱细胞瘢痕皮肤支架材料的电镜扫描图;
图7为本发明实施例2所述制备脱细胞瘢痕皮肤支架材料的电镜扫描图;
图8为对照例真皮脱细胞基质的电镜扫描图;
图9为本发明实施例1所述制备脱细胞瘢痕皮肤支架材料种植表皮干细胞后HE染色结果图;
图10为本发明实施例2所述制备脱细胞瘢痕皮肤支架材料种植表皮干细胞后HE染色结果图;
图11为对照例真皮脱细胞基质种植表皮干细胞后HE染色结果图;
图12为本发明实施例1所述制备脱细胞瘢痕皮肤支架材料种植皮肤成纤维细胞后HE染色结果图;
图13为本发明实施例2所述制备脱细胞瘢痕皮肤支架材料种植皮肤成纤维细胞后HE染色结果图;
图14为对照例真皮脱细胞基质种植皮肤成纤维细胞后HE染色结果图;
图15为本发明实施例1所述制备脱细胞瘢痕皮肤支架材料种植表皮干细胞后电镜放大图;
图16为本发明实施例2所述制备脱细胞瘢痕皮肤支架材料种植表皮干细胞后电镜放大图;
图17为对照例真皮脱细胞基质种植表皮干细胞后电镜放大图;
图18为本发明实施例1、实施例2及对照例的应力应变关系;
图19为本发明实施例1、实施例2及对照例的归一化松弛关系;
图20为本发明实施例1、实施例2及对照例的蠕变关系;
图21为本发明实施例2对应所述制备脱细胞瘢痕皮肤支架材料种植后不同时期胶原纤维长势图;
图22为本发明实施例1对应所述制备脱细胞瘢痕皮肤支架材料种植后不同时期胶原纤维长势图;
图23为空白对照组种植后不同时期胶原纤维长势图;
图24为本发明实施例2对应所述制备脱细胞瘢痕皮肤支架材料种植后不同时期血管长势图;
图25为本发明实施例1对应所述制备脱细胞瘢痕皮肤支架材料种植后不同时期血管长势图;
图26为空白对照组种植后不同时期血管长势图。
具体实施方式
以下结合附图对本发明的原理和特征进行描述,所举实施例只用于解释本发明,并非用于限定本发明的范围。
本实施例提供了一种脱细胞瘢痕皮肤支架材料的制备方法,其特征在于,包括如下步骤:
步骤1:切除瘢痕标本中表皮及皮下组织,将获得的组织切成0.4-0.6mm(优选的,可以时0.4mm、0.45mm、0.5mm、0.55mm或0.6mm,优选的为0.5mm)厚的薄片,再将薄片中的病毒进行灭活;
步骤2:将步骤1处理后的薄片中细胞进行溶解;
步骤3:将步骤2处理后的薄片储存于抗生素溶液(抗生素溶液的规格为100IU/L的青霉素钠溶液)中,并置于低温条件(优选的为0-4℃,可以时0℃、1℃、2℃、3℃或4℃,进一步优选的为4℃)下备用,即完成脱细胞瘢痕皮肤支架材料的制备。
上述技术方案中所述步骤1中薄片病毒灭活方式是:将薄片置于消毒液中浸泡1.5-2.5h(可以时1.5h、2h或2.5h,优选的为2h),然后将薄片取出后洗除残留的消毒液即可。
上述技术方案中所述消毒液为过氧乙酸和乙醇混合的水溶液,其中,所述消毒液中过氧乙酸的浓度为1vol%,所述消毒液中乙醇的浓度为24vol%。
上述技术方案中所述薄片洗除残留消毒液的方式是:先将薄片置于无菌的PBS缓冲液或无菌的生理盐水中浸泡20-40min(可以是20min、30min或40min,优选的为30min),中途更换1-3次(优选的,中途更换2次即可)浸泡液(浸泡液即为前述无菌的PBS缓冲液或无菌的生理盐水),然后再将薄片置于纯水中清洗1-3次,每次清洗时长为5-15min。
上述技术方案中所述步骤2中薄片中细胞溶解的方式是:将薄片先置于细胞膜消解液中浸泡2h并持续振荡,再将薄片取出再置于细胞核消解液中,并在37℃下持续振荡72小时,期间每间隔24小时更换一次细胞核消解液,最后将薄片取出后洗除残留的细胞核消解液即可。
上述技术方案中所述细胞膜消解液为胰蛋白酶和EDTA-Na2(乙二胺四乙酸二钠)混合的水溶液,所述细胞膜消解液中的胰蛋白酶浓度为2.5g/L,所述细胞膜消解液中的EDTA-Na2的浓度为0.5mmol/L。
上述技术方案中所述细胞核消解液为浓度为1vol%的Triton X-100溶液(聚乙二醇辛基苯基醚)。
其中,所述细胞膜消解液和细胞核消解液的浓度均不宜高,否则反应剧烈会导致薄片的非细胞蛋白消解,而出现脱细胞瘢痕皮肤支架材料受损。
上述技术方案中所述薄片洗除残留细胞核消解液的方式是:先将薄片置于无菌的PBS缓冲液或无菌的生理盐水中浸泡20-40min(优选的为30min),中途更换1-3次浸泡液,然后再将薄片置于纯水中清洗1-3次(优选的为2次),每次清洗时长为5-15min。其中,薄片洗除残留细胞核消解液的方式可以与薄片洗除消毒液的方式相同。
实施例1
采用增生期瘢痕细胞按照如上所述方法制备增生期瘢痕脱细胞基质。
实施例2
采用成熟期瘢痕细胞按照如上所述方法制备成熟期瘢痕脱细胞基质。
对照例
采用市购真皮脱细胞基质(ADM)作为对照例。
1、瘢痕细胞脱基质的形态图
实施例1和实施例2所得的瘢痕脱细胞基质呈瓷白色片状,大小约2cm*2cm,表面及内部均有不同大小及密度的孔径,质地柔软,有弹性,其中实施例1对应的瘢痕脱细胞基质如图1所示,实施例2对应的瘢痕脱细胞基质如图2所示。
2、组织学观察(×200)
将实施例1所得的增生期瘢痕脱细胞基质、实施例2所得的成熟期瘢痕脱细胞基质以及对照例中的真皮脱细胞基质均采用HE染色进行染色,染色结果分别如3-5所示(实施例1对应图3,实施例2对应图4,对照例对应图5),成熟期瘢痕脱细胞基质胶原纤维排列较增生期瘢痕脱细胞基质有序,纤维直径接近于正常皮肤的胶原纤维形态。
3、电镜扫描结果(×2000)
扫描电子显微镜(SEM)观察结果显示实施例1(如图6)、实施例2(如图7)和对照例(如图8)三种材料间无完整细胞核及其他细胞成分,细胞外基质结构完整、连续。ADM的胶原纤维结构均匀有序,胶原纤维间隙较增生期瘢痕脱细胞基质和成熟期瘢痕脱细胞基质稍大。
4、材料细胞种植结果观察
分别将表皮干细胞和正常皮肤成纤维细胞种植在实施例1、实施例2和对照例的三种材料上,HE染色结果(×200)显示三种材料均可供表皮干细胞及正常皮肤成纤维细胞附着生长(如图9-图14)。对照例表皮干细胞及正常皮肤成纤维细胞生长密度较两个实施例组高。表皮干细胞生长在成熟期瘢痕脱细胞基质组优于增生期瘢痕脱细胞基质组。扫描电子显微镜(×2000)观察结果显示在ADM和成熟期瘢痕脱细胞基质中表皮干细胞在材料表面和内部均见生长,ADM组细胞分布更均匀,增生期瘢痕脱细胞基质中表皮干细胞多数黏附生长在材料表面(如图15-图17)。
5、三种试件生物力学的比较
如图18-图20所示,显示了实施例1、实施例2和对照例的归一化松弛关系,应力应变关系及蠕变关系曲线,另外表1显示了三种材料生物力学之间的差异。
表1三种类型材料生物力学指标比较(平均值±标准差)
试件名称 | 实施例1 | 实施例2 | 对照例 |
伸长比 | 0.118±0.085<sup>*</sup> | 0.126±0.042<sup>*</sup> | 0.380±0.032 |
杨氏模量 | 12.689±1.732 | 16.580±3.498 | 11.589±2.913 |
松弛斜率 | -0.028±0.006<sup>*</sup> | -0.052±0.005 | -0.059±0.010 |
蠕变斜率 | 0.029±0.037 | 0.026±0.005 | 0.033±0.008 |
松弛量(MPa) | 0.372±0.062<sup>*</sup> | 0.749±0.283 | 0.856±0.119 |
蠕变量(mm/mm) | 0.018±0.009<sup>*</sup> | 0.052±0.018 | 0.051±0.016 |
最大拉伸应力(MPa) | 2.064±1.171<sup>*</sup> | 2.870±1.447 | 3.077±1.163 |
注释:实施例1和实施例2对应的材料与ADM相比较,*p<0.05
4、动物实验研究
4.1、形态学观察
移植区镜下(×100)观察:术后第3个月起,瘢痕脱细胞基质间隙逐步被新生胶原及新生成熟血管所填充,炎性细胞浸润逐步减少,至术后第6个月时两组瘢痕脱细胞基质内均未见明显的炎性细胞浸润。成熟期瘢痕脱细胞基质组内新生血管数量较其余两组多。随着创面的生长愈合,空白对照(如图23,移植时未设置任何支架材料)胶原排列更紊乱无序。成熟期瘢痕脱细胞基质组(图21)较增生期瘢痕脱细胞基质组(图22)新生胶原纤维排列规则,胶原束细。
4.2、血管形成(×200)
术后1个月时,植入的支架周围可看到血管新生。成熟期瘢痕脱细胞基质组移植区内血管的数量在术后2月时最高(图24),增生期瘢痕脱细胞基质组移植区内血管的数量在术后3月时最高(图25)。两组瘢痕脱细胞基质内新生的血管数量均较同期空白对照组(图26,移植时未设置任何支架材料)新生血管数量多,其中,图24-图26中横坐标的"mon."表示时间单位“月”。
另外,本实施例1和实施例2提供了脱细胞瘢痕皮肤支架材料可应用于制备真皮支架中(虽然性能不及市面上的ADM,但其获取更容易,同时制备成本更低)。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,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,均应包含在本发明的保护范围之内。
Claims (10)
1.一种脱细胞瘢痕皮肤支架材料的制备方法,其特征在于,包括如下步骤:
步骤1:切除瘢痕标本中表皮及皮下组织,将获得的组织切成的薄片,再将薄片中的病毒进行灭活;
步骤2:将步骤1处理后的薄片中细胞进行溶解;
步骤3:将步骤2处理后的薄片储存于抗生素溶液中,并置于低温条件下备用,即完成脱细胞瘢痕皮肤支架材料的制备。
2.根据权利要求1所述的脱细胞瘢痕皮肤支架材料的制备方法,其特征在于,所述步骤1中薄片病毒灭活方式是:将薄片置于消毒液中浸泡1.5-2.5h,然后将薄片取出后洗除残留的消毒液即可。
3.根据权利要求2所述的脱细胞瘢痕皮肤支架材料的制备方法,其特征在于,所述消毒液为过氧乙酸和乙醇混合的水溶液,其中,所述消毒液中过氧乙酸的浓度为1vol%,所述消毒液中乙醇的浓度为24vol%。
4.根据权利要求2所述的脱细胞瘢痕皮肤支架材料的制备方法,其特征在于,所述薄片洗除残留消毒液的方式是:先将薄片置于无菌的PBS缓冲液或无菌的生理盐水中浸泡20-40min,中途更换1-3次浸泡液,然后再将薄片置于纯水中清洗1-3次,每次清洗时长为5-15min。
5.根据权利要求2所述的脱细胞瘢痕皮肤支架材料的制备方法,其特征在于,所述步骤2中薄片中细胞溶解的方式是:将薄片先置于细胞膜消解液中浸泡2h并持续振荡,再将薄片取出再置于细胞核消解液中,并在37℃下持续振荡72小时,期间每间隔24小时更换一次细胞核消解液,最后将薄片取出后洗除残留的细胞核消解液即可。
6.根据权利要求5所述的脱细胞瘢痕皮肤支架材料的制备方法,其特征在于,所述细胞膜消解液为胰蛋白酶和EDTA-Na2混合的水溶液,所述细胞膜消解液中的胰蛋白酶浓度为2.5g/L,所述细胞膜消解液中的EDTA-Na2的浓度为0.5mmol/L。
7.根据权利要求5所述的脱细胞瘢痕皮肤支架材料的制备方法,其特征在于,所述细胞核消解液为浓度为1vol%的Triton X-100溶液。
8.根据权利要求5所述的脱细胞瘢痕皮肤支架材料的制备方法,其特征在于,所述薄片洗除残留细胞核消解液的方式是:先将薄片置于无菌的PBS缓冲液或无菌的生理盐水中浸泡20-40min,中途更换1-3次浸泡液,然后再将薄片置于纯水中清洗1-3次,每次清洗时长为5-15min。
9.一种采用如权利要求1-8任一项所述制备方法所制得的脱细胞瘢痕皮肤支架材料。
10.一种如权利要求9所述脱细胞瘢痕皮肤支架材料在制备真皮支架中的应用。
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