CN111544653A - 一种适应多维临床需求的仿生叠层组织工程皮肤及其制备方法 - Google Patents

一种适应多维临床需求的仿生叠层组织工程皮肤及其制备方法 Download PDF

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CN111544653A
CN111544653A CN202010369031.XA CN202010369031A CN111544653A CN 111544653 A CN111544653 A CN 111544653A CN 202010369031 A CN202010369031 A CN 202010369031A CN 111544653 A CN111544653 A CN 111544653A
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tissue engineering
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hyaluronic acid
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王子健
胡伟康
陈熙
李毅祺
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Hubei Baina Huiyu Biotechnology Co ltd
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Abstract

本发明公开了一种适应多维临床需求的仿生叠层组织工程皮肤及其制备方法，属于生物医用材料与组织工程领域。所述仿生叠层组织工程皮肤由季铵化甲壳素层和FGF2‑透明质酸层组成，并交替吸附于聚乳酸‑羟基乙酸（PLGA）纳米纤维膜表面。所述季铵化甲壳素层具有广谱抗菌性和原位抗菌性；所述FGF2‑透明质酸层具有促血管生成活性；两者协同作用还具有皮肤抗炎和促胶原合成功能。本发明制备方法简单、污染小，产业附加值高；产品的临床性能优异，可适应感染性溃疡、糖尿病足、医疗美容等多种应用环境的需求。

Description

一种适应多维临床需求的仿生叠层组织工程皮肤及其制备 方法
技术领域
本发明属于生物医用材料与组织工程领域，具体涉及一种适应多维临床需求的仿生叠层组织工程皮肤及制备方法。
背景技术
皮肤组织及其附属器官是人体抵御外界损伤的重要生物屏障。当皮肤完整性受损时，患者可出现疼痛、流血、感染和延迟愈合等症状。皮肤愈合过程分为止血期、炎症期、再生期和重塑期，加速愈合的关键在于重建创面微环境并采取适宜的治疗措施。组织工程皮肤如水凝胶，3D适形打印支架等可用于维持创面湿润环境，预防病原体感染，减少继发性损伤。但现有组织工程皮肤也存在生物活性和生物降解性差、免疫原性和过敏性强、不易清洁和更换时易出血等缺陷，尚不能完全满足临床需求。纳米纤维材料可由高压静电纺丝法制备得到，具有与细胞外基质（ECM）类似的三维结构、以及高比表面积和可调控性能。因此，我们优选纳米纤维材料作为制备仿生组织工程皮肤的基底材料。
层层自组装（LBL）是一种将基底材料依次浸泡于带相反电荷的聚电解质溶液中形成聚电解质复合物涂层的改性技术。大量研究证实LBL技术是纳米纤维材料通用的改性技术之一。通过增加自组装层数可以简易的实现对材料物理、化学和生物功能的定性和定量调控。常用的改性材料包括丝素蛋白、壳聚糖、海藻酸钠等，制备得到的复合材料在抗菌活性、生物相容性、降解性等生物功能方面均有显著提高。目前，基于LBL技术的纳米纤维材料已成功应用于皮肤组织工程领域。然而，能同时满足多维临床需求的高性能组织工程皮肤仍鲜有报道。
大面积缺损，感染性溃疡、糖尿病足等难治愈创面对组织工程皮肤的生物活性，特别是广谱抗菌性和促血管生成活性提出了更高要求。现有商品抗菌皮肤仅针对革兰阳性杆菌（S.aureus）和革兰阴性菌（E.coli）感染而设计，其中可溶性抗菌试剂（抗生素或银离子）的释放对机体具有潜在的毒副作用。近年来，医院内获得性感染的发病率在逐年上升，常规抗菌敷料对医院内获得性感染（如耐甲氧西林金黄色葡萄球菌）的治疗效果不佳。因此，开发一种针对耐药菌感染且具有原位抗菌效果的新型组织工程皮肤具有重要的意义。新生血管生成不足和血液供应不良是创伤延迟愈合的另一重要因素。促血管生成活性对糖尿病足皮损的修复至关重要。新生血管的数量和成熟度受一系列物理和生物诱导因子的影响。特别的是，碱性成纤维细胞生长因子（FGF2）对血管再生具有明显的促进作用。由于FGF2的靶向性较差，注射时清除失活，实现组织工程皮肤FGF2持续缓释起效仍有一定困难。
本发明拟采用LBL技术制备一种兼具原位抗菌性和促血管生成活性的仿生叠层组织工程皮肤，旨在克服上述缺陷，并应用于治疗多种难治愈的创面。目前，针对本发明的内容和应用效果尚未见到类似的报道。
发明内容
本发明首先采用高压静电纺丝法制备得到聚乳酸-羟基乙酸共聚物（PLGA）纳米纤维膜，并将其作为组织工程皮肤的基材。在后续的LBL过程中，我们采用季铵化甲壳素作为阳性组分，FGF2-透明质酸溶液作为阴性组分，制备得到具有壳核结构的仿生叠层组织工程皮肤。我们已经证实季铵化甲壳素具有优异的广谱抗菌性，对耐甲氧西林金黄色葡萄球菌、革兰阳性菌和革兰阴性菌均有良好的抑菌效果。通过LBL技术，季铵化甲壳素固定于组织工程皮肤表面有效避免了局部释药产生的毒副作用。透明质酸是一种具有优异生物相容性的大分子物质。FGF2缓慢的从产品中释放出来实现促进新生血管生成的效果。
根据本发明的第一个方面，提供一种适应多维临床需求的仿生叠层组织工程皮肤，其特征在于，所述组织工程皮肤含有季铵化甲壳素层和FGF2-透明质酸层；所述季铵化甲壳素层和FGF2-透明质酸层通过静电作用吸附于基材表面；所述季铵化甲壳素层和FGF2-透明质酸层数相同，均不少于1层。
优选的，所述基材为聚乳酸-羟基乙酸共聚物（PLGA）纳米纤维膜。
根据本发明的另一个方面，提供一种适应多维临床需求的仿生叠层组织工程皮肤的制备方法，其特征在于，将基材依次浸泡于FGF2-透明质酸溶液和季铵化甲壳素溶液中，使带负电的FGF2-透明质酸和带正电的季铵化甲壳素通过静电作用吸附后疏水性增强，得到具有壳核结构的仿生叠层组织工程皮肤。
优选的，所述基材通过高压静电纺丝法制备得到，将PLGA溶于六氟异丙醇中得到质量浓度为5-15%预电纺溶液，纺丝机参数设置为：板间电压10-15 kV, 接收距离10-15cm，流率0.5-1.5 mL/h，电纺时间2-4 h；将产品浸泡于质量分数为0.5-5%的聚苯胺盐酸盐溶液中30-120 min，取出晾干后备用。
优选的，所述季铵化甲壳素溶液的质量浓度为2-8%；所述FGF2-透明质酸溶液的质量浓度为0.5-5%；所述浸泡时间均为10-60 min。
优选的，所述FGF2-透明质酸溶液中FGF2的浓度为1-100 mg/mL。
优选的，所述依次浸泡的轮数不少于1轮，先后分别浸泡在FGF2-透明质酸溶液和季铵化甲壳素溶液中为1轮依次浸泡。
与现有技术相比，本发明具有如下技术优点和创新点。
（1）本发明中选用带正电的季铵化甲壳素和带负电的FGF2-透明质酸作为主要活性组分，巧妙的通过层层自组装技术将两者固定于PLGA纳米纤维膜表面，制备得到具有原位抗菌性和促血管生成活性的新型组织工程皮肤。季铵化甲壳素、透明质酸和PLGA等均为生物医用高分子材料，满足FDA临床应用安全标准。相比于化学交联等传统材料加工技术，层层自组装技术具有绿色安全，操作可控等优势。在本发明中，我们可简易的通过自组装层数调控组织工程皮肤的理化和生物学性能，包括拉伸强度、水接触角、生物相容性、原位抗菌活性和体内功能等。
（2）本发明中优选季铵化甲壳素作为主要抗菌组分。前期研究中，我们已经证实季铵化甲壳素对革兰阳性菌、革兰阴性菌和耐甲氧西林金黄色葡萄球菌等均具有优异的广谱抗菌效果。进一步的，我们采用层层自组装技术将季铵化甲壳素固定于组织工程皮肤表面，原位抗菌性得到增强，有效避免了抗菌成分释放时对机体产生的毒副作用。FGF2作为另一种生物活性分子，从组织工程皮肤缓慢释放到创面内，达到促新生血管生成的功能。本发明具备的广谱抗菌性、原位抗菌性和促血管生成活性对感染创面、糖尿病足以及院内压疮等多种皮肤病损均具有治疗价值。
（3）本发明还具有未曾意料的功能和应用效果。我们体内动物实验中发现，本发明还可以显著减轻创面局部炎症（如下调CD45炎症因子表达），并促进皮肤胶原蛋白的合成（如上调I型胶原，III型胶原）。抗炎效果可归因于本发明组织工程皮肤具有优良的原位抗菌性和生物相容性。胶原蛋白是皮肤组织的主要成分之一，对维持皮肤完整性、弹性和防御功能具有重要意义。本发明可促进胶原合成，在医疗美容领域具有极大的应用潜力。
（4）本发明制备方法简单、加工设备简易、对环境污染小，产品附加值高，有望得到工业化生产。
附图说明
图1构建的仿生叠层组织工程皮肤（TESK）功能概括图。
图2是实施例1获得聚乳酸-羟基乙酸共聚物（PLGA）纳米纤维膜和仿生叠层组织工程皮肤（TESK）扫描电镜图和水接触角测试图。
图3是实施例1获得的仿生叠层组织工程皮肤（TESK）细胞流式及Transwell迁移能力测试的结果。
图4是实施例1获得的仿生叠层组织工程皮肤（TESK）应用于糖尿病皮损修复愈合图及愈合曲线。
图5是实施例1获得的仿生叠层组织工程皮肤（TESK）免疫组化定量分析的结果。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。此外，下面所描述的本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
实施例1
制备聚乳酸-羟基乙酸共聚物（PLGA）纳米纤维膜。将10gPLGA固体颗粒加入到100 mL六氟异丙醇中，充分搅拌溶解后得到预电纺溶液。将溶液添加到高压静电纺丝机的推注泵内，设置电纺丝参数：板间电压12 kV，接收距离12 cm，流率1.0 mL/h，电纺时间3 h。制备完成后取下纳米纤维膜，将其浸泡于1%聚苯胺盐酸盐溶液中，30 min后晾干备用。制备仿生叠层组织工程皮肤。将PLGA纳米纤维膜作为基材，浸泡于质量浓度为2%的FGF2-透明质酸溶液中, 30 min后用生理盐水洗净。FGF2-透明质酸溶液中FGF2的浓度为10 μg/mL。再将其浸泡于质量浓度为5%的季铵化甲壳素溶液中，30 min后用生理盐水洗净。将上述浸泡操作记为1轮，重复浸泡操作共10轮。将制备得到的仿生叠层组织工程皮肤命名为TESK，将未改性的PLGA纳米纤维膜作为对照组并命名为PLGA。
实施例2
将实施例1获得的仿生叠层组织工程皮肤（TESK）和未改性PLGA纳米纤维膜烘干后进行扫描电子显微镜观察。采用微距视频记录***检测亲疏水性。
图2是实施例1获得聚乳酸-羟基乙酸共聚物（PLGA）纳米纤维膜和仿生叠层组织工程皮肤（TESK）扫描电镜图和水接触角测试图。如图所见，PLGA纳米纤维膜呈现出相互交织的网状纳米纤维结构。经层层自组装后，获得的仿生叠层组织工程皮肤（TESK）纤维表面吸附了FGF2-透明质酸和季铵化甲壳素，TESK纤维的直径得到明显增加。与PLGA相比，TESK的水接触角明显减小，在体内应用时有利于细胞黏附和迁移。
实施例3
将实施例1获得的仿生叠层组织工程皮肤（TESK）和未改性PLGA纳米纤维膜采用紫外光灭菌后浸泡于RPMI-1640完全培养基中，24h后过滤制备得到材料浸提液。将成纤维细胞L929与材料浸提液共培养48h, 采用流式细胞术检测细胞周期分布，采用Transwell实验检测细胞的迁移能力。
图3是实施例1获得的仿生叠层组织工程皮肤（TESK）细胞流式及Transwell迁移能力测试的结果。如图所见，经TESK处理后，L929细胞中G0/G1期细胞比例大幅度降低，提示其细胞增殖活性得到了显著提高。TESK处理组细胞的迁移能力较对照组也有显著增加。
实施例4
将实施例1获得的仿生叠层组织工程皮肤（TESK）和未改性PLGA纳米纤维膜裁剪成直径20 mm的圆片，并采用糖尿病大鼠全层皮肤损伤模型验证皮肤修复效果。糖尿病大鼠购买自武汉市百开瑞生物技术有限公司，经异氟烷吸入麻醉后切除背部全层皮肤，直径20mm，深达深筋膜层。实验组将TESK敷贴在创面上，阳性对照组将医用纱布敷贴在创面上，阴性对照组将PLGA敷贴在创面上，空白对照组不做处理。统计皮损愈合曲线，收集再生皮肤组织用于病理学检测。
图4是实施例1获得的仿生叠层组织工程皮肤（TESK）应用于糖尿病皮损修复愈合图及愈合曲线。如图所见，TESK处理组伤口愈合最快，特别是在伤口愈合的早期具有显著的治疗效果。
图5是实施例1获得的仿生叠层组织工程皮肤（TESK）免疫组化定量分析的结果。如图所见，TESK可显著减轻皮肤组织炎症（下调CD45炎症因子表达），促进皮肤胶原蛋白合成（上调I型胶原，III型胶原）。
本领域的技术人员容易理解，以上所述仅为本发明的较佳实施例而已，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、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (7)

1.一种适应多维临床需求的仿生叠层组织工程皮肤，其特征在于，所述仿生叠层组织工程皮肤含有季铵化甲壳素层和FGF2-透明质酸层；所述季铵化甲壳素层和FGF2-透明质酸层通过静电作用吸附于基材表面；所述季铵化甲壳素层和FGF2-透明质酸层数相同，均不少于1层。
2.如权利要求1所述的一种适应多维临床需求的仿生叠层组织工程皮肤，其特征在于，所述基材为聚乳酸-羟基乙酸共聚物（PLGA）纳米纤维膜。
3.一种适应多维临床需求的仿生叠层组织工程皮肤的制备方法，其特征在于，将基材依次浸泡于FGF2-透明质酸溶液和季铵化甲壳素溶液中，使带负电的FGF2-透明质酸和带正电的季铵化甲壳素通过静电作用吸附后疏水性增强，得到具有壳核结构的仿生叠层组织工程皮肤。
4.如权利要求3所述的一种适应多维临床需求的仿生叠层组织工程皮肤的制备方法，其特征在于，所述基材通过高压静电纺丝法制备得到，将PLGA溶于六氟异丙醇中得到质量浓度为5-15%预电纺溶液，纺丝机参数设置为：板间电压10-15 kV, 接收距离10-15 cm，流率0.5-1.5 mL/h，电纺时间2-4 h；将产品浸泡于质量分数为0.5-5%的聚苯胺盐酸盐溶液中30-120 min，取出晾干后备用。
5.如权利要求3所述的一种适应多维临床需求的仿生叠层组织工程皮肤的制备方法，其特征在于，所述季铵化甲壳素溶液的质量浓度为2-8%；所述FGF2-透明质酸溶液的质量浓度为0.5-5%；浸泡时间均为10-60 min。
6.如权利要求5所述的一种适应多维临床需求的仿生叠层组织工程皮肤的制备方法，其特征在于，所述FGF2-透明质酸溶液中FGF2的浓度为1-100 mg/mL。
7.如权利要求3所述的一种适应多维临床需求的仿生叠层组织工程皮肤的制备方法，其特征在于，所述依次浸泡的轮数不少于1轮，先后分别浸泡在FGF2-透明质酸溶液和季铵化甲壳素溶液中为1轮依次浸泡。
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