CN111001042A - 可完全降解组织工程皮肤支架材料及其制备方法 - Google Patents
可完全降解组织工程皮肤支架材料及其制备方法 Download PDFInfo
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- CN111001042A CN111001042A CN201911259503.XA CN201911259503A CN111001042A CN 111001042 A CN111001042 A CN 111001042A CN 201911259503 A CN201911259503 A CN 201911259503A CN 111001042 A CN111001042 A CN 111001042A
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- bacterial cellulose
- chitosan
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Abstract
本发明公开了一种可完全降解组织工程皮肤支架材料，包括细菌纤维素/壳聚糖纳米纤维层和大孔径阵列微米纤维支架两部分，细菌纤维素/壳聚糖纳米纤维层为上层，大孔径阵列微米纤维支架上方为下层，层间通过戊二醛交联结合；所述细菌纤维素/壳聚糖纳米纤维层由木葡糖酸醋杆菌原位合成，表面固定有纤维素酶；所述大孔径阵列微米纤维支架为静电纺乳酸/透明质酸/丝素微纳米纤维有序排列的支架，表面修饰有聚乳酸‑羟基乙酸生长因子缓释微球。本发明所用原料均无毒无害，纤维性质稳定，具有良好的生物相容性，能够在体内完全降解，下层支架的孔隙直径与细胞尺寸接近，使细胞能够进入，还能稳定释放生长因子，有利于皮肤组织再生。
Description
技术领域
本发明涉及生物医用材料技术领域，具体涉及一种可完全降解组织工程皮肤支架材料及其制备方法。
背景技术
皮肤作为人体最大的器官，起着保护人体免受外界物体入侵的作用。皮肤的损伤可导致细菌、病原体的入侵，严重者可导致死亡。因此在过去的三十年中，生物医用材料和组织工程领域大量开展了皮肤支架的研究。人体皮肤为双层结构，由表皮和真皮组成。表皮主要由致密的角化细胞组成，起防护作用；真皮主要由成纤细胞及其分泌的胶原蛋白组成，有较好的弹性和机械强度，在皮肤再生过程中起重要的作用。
双层支架材料应用于皮肤的修复，取得了良好的效果。但目前文献和公开专利中采用的双层支架多为微米多孔结构，且采用单一的方法制备。最新的组织工程支架研究表明，纳米纤维结构的支架能提供更多的细胞吸附位点，促进细胞的生长，因此，纳米纤维的双层支架对于皮肤的修复是一种更好的选择。然而采用静电纺丝制备的纳米纤维膜大多不能完全降解，还存在易收缩变形，纤维稳定性不足的现象，作为皮肤组织工程表皮支架时无法满足结构稳定性的要求；真皮支架结构则存在孔径较小，结构致密，不利于细胞进入支架内部形成三维培养的问题。3D打印制备支架时，虽然孔隙率和孔径大小可以调节，解决了静电纺丝纤维结构致密的问题，但是由于打印材料需要达到一定的硬度才能得到设计的形状和结构，这就造成3D打印支架应用于皮肤组织工程时硬度过大，难以彻底降解，不易与创面贴合，不利于细胞粘附和迁移。
因此，寻找可完全降解、纤维稳定性强的纳米纤维膜和大孔径纤维支架材料构建功能稳定、利于细胞粘附生长的组织工程皮肤支架材料意义重大。
发明内容
本发明提供一种上层纤维网络稳定性强、下层孔径大、纤维规则排列的双层结构可完全降解组织工程皮肤支架材料及其制备方法。
本发明的目的通过如下技术方案实现：
一种可完全降解组织工程皮肤支架材料，包括细菌纤维素/壳聚糖纳米纤维层和大孔径阵列微米纤维支架两部分，细菌纤维素/壳聚糖纳米纤维层为上层，大孔径阵列微米纤维支架上方为下层，层间通过戊二醛交联结合；所述细菌纤维素/壳聚糖纳米纤维层由木葡糖酸醋杆菌原位合成，表面固定有纤维素酶；所述大孔径阵列微米纤维支架为静电纺乳酸/透明质酸/丝素微纳米纤维有序排列的支架，表面修饰有聚乳酸-羟基乙酸生长因子缓释微球。
一种所述可完全降解组织工程皮肤支架材料的制备方法，包括以下步骤：
步骤一、细菌纤维素/壳聚糖纤维层的制备：将溶解壳聚糖添加到木葡糖酸醋杆菌培养基中，原位合成细菌纤维素/壳聚糖复合凝胶，复合凝胶经0.5mol/LNaOH溶液常温碱解8-10h纯化后，再采用冻干法将纤维素酶固定在细菌纤维素/壳聚糖纤维的表面，得到细菌纤维素/壳聚糖纳米纤维层；
步骤二、聚乳酸/透明质酸/丝素微纳米纤维的制备：以丝素和透明质酸的混合溶液为纺丝液1，聚乳酸溶液为纺丝液2，采用同轴静电纺丝技术得到以纺丝液1为壳层，纺丝液2为核层的聚乳酸/透明质酸/丝素微纳米纤维；
步骤三、大孔径阵列微米纤维支架的制备：使用探针阵列收集器接收，得到大孔径阵列微米纤维支架材料，再将大孔径阵列微米纤维支架材料浸润在聚乳酸-羟基乙酸生长因子缓释微球溶液中，取出后真空干燥即得；
步骤四、戊二醛交联：在培养皿中加入适量25wt％的戊二醛水溶液，然后将其放入干净密闭的干燥器底部，再将大孔径阵列微米纤维支架放在铝箔上，细菌纤维素/壳聚糖纤维层叠放在大孔径阵列微米纤维支架上方，将叠加后的支架材料置于干燥器孔板中间，盖好盖子并密封后，室温下交联8-12h，取出水洗，充分干燥。
进一步地，步骤二所述聚乳酸/透明质酸/丝素微纳米纤维的制备方法，包括以下步骤：(1)丝素膜的制备：桑蚕丝脱胶后溶于80℃的氯化钙、水和乙醇体积比为1:8:2的混合溶液中，搅拌溶解，透析后得纯丝素溶液，流延成膜备用；(2)纺丝液1的制备：将丝素膜与相对分子质量为150万的透明质酸粉末共同加入甲酸溶液中，得到纺丝液1；(3)纺丝液2的制备：聚乳酸溶于氯仿和丙酮体积比2:1的混合溶剂中，配制成5wt％的聚乳酸溶液，室温搅拌均匀，得到纺丝液2；(4)同轴静电纺丝：取纺丝液1、纺丝液2分别装于10mL的注射器内，将注射器分别安装到两个SN-50微量注射泵上，再用聚四氟乙烯软管将纺丝液2连于内层针管，纺丝液1连于外层嵌套装置，对同轴装置施加相同高压，使纺丝液2核层液体与纺丝液1壳层液体在喷口处汇合而成同心分层流，在高压静电场的静电力牵引下，得到同轴复合纤维。
进一步地，所述同轴静电纺丝的工艺参数为：电压12kV，收集距离15cm，核-壳溶液推进速度为0.1-0.2和0.3-0.4mL/h。
进一步地，所述探针阵列收集器为将探针嵌入平板收集器泡沫板中，探针之间等距形成6×6阵列，探针间距为2-4cm。
进一步地，所述聚乳酸-羟基乙酸生长因子缓释微球的制备方法如下：配制生长因子水溶液，将其加入PLGA二氯甲烷溶液中，用超声细胞破碎仪超声分散10s至液体呈乳白色；然后将乳白色溶液到PVA溶液中，继续超声分散2min，至液体呈淡乳白色后，磁力搅拌2-4h，使二氯甲烷挥发完全；待微球成型后，离心、弃上清，用去离子水清洗沉淀，重复离心、清洗三次，-20℃冷冻，真空干燥后即得聚乳酸-羟基乙酸生长因子缓释微球。
进一步地，聚乳酸-羟基乙酸生长因子缓释微球的制备方法中离心转速为4000rpm，离心时间5min。
大量细胞实验显示细菌纤维素没有细胞毒性，支持角化细胞和成纤维细胞的生长和增殖。本发明可完全降解组织工程皮肤支架材料所用细菌纤维素/壳聚糖纤维层为木葡糖酸醋杆菌原位合成细菌纤维素/壳聚糖纤维，与将细菌纤维素粉碎处理后再制成纤维相比，原位合成能够使复合纤维层保持细菌纤维素原来的网络结构，复合纤维也具有极高的稳定性。由于人体内没有纤维素酶，细菌纤维素无法在体内自行降解，因此通过冻干法将纤维素酶固定在细菌纤维素/壳聚糖纤维的表面实现其在体内的完全降解，并可通过纤维素酶的量来调节其降解周期。
丝素与透明质酸的复合能够良好的模拟细胞外基质的蛋白与多糖结构，对细胞生长起到促进作用，但是二者复合后纤维的稳定性和力学强度较差，本发明通过以聚乳酸为核层，丝素与透明质酸复合为壳层，由于核壳溶液皆不易扩散，两者在喷口处汇合时间较短，故不会导致核壳溶液混合，因而能够采用同轴静电纺丝技术制备得到聚乳酸/透明质酸/丝素微纳米纤维膜，既能够有效调控丝素/透明质酸静电纺纤维的形态，降低纤维的溶失率，又能增强纳米纤维的稳定性和力学性能，显著提高了支架的弹性模量和压缩应力，同时不会影响纤维的生物相容性，壳层较高的孔壁粗糙度还有利于聚乳酸-羟基乙酸生长因子缓释微球的结合。
本发明在大孔径阵列微米纤维支架的制备过程中，使用传统平板接收器时，得到的纤维呈无序排列，而使用探针阵列收集器时，收集到的纤维呈规则有序排列。所得大孔径阵列微米纤维的孔隙率大于92％，比平板接收增加了11％；孔隙直径范围在50-120μm，平均孔径为68μm，是平板接收的四倍左右；在后续实验中大孔径阵列支架与平板接收支架相比，表现出较好的细胞粘附和增殖能力，细胞形态以及三维生长倾向。
本发明与现有技术相比具有以下有益效果：
(1)本发明提供的可完全降解组织工程皮肤支架材料所用原料均无毒无害，具有良好的生物相容性，能够在体内完全降解，不同于现有双层皮肤支架上层采用静电纺丝纳米纤维膜，本发明是通过原位合成的方法，充分利用细菌纤维素自身网络结构的特点，同时细菌纤维素与壳聚糖的复合由木葡糖酸醋杆菌完成，因而复合纤维具有很高的稳定性；
(2)本发明提供的可完全降解组织工程皮肤支架材料上下层通过戊二醛交联作用结合在一起，下层为大孔径阵列微米纤维支架，通过调节探针阵列的排布可以调节纤维支架的孔径大小及孔隙率，制备得到孔径平均值与细胞尺寸接近的支架，使细胞能够进入，有利于形成较厚的细胞组织；
(3)本发明提供的可完全降解组织工程皮肤支架材料机械强度高，有良好的生物相容性、适合于细胞的黏附、迁移和增殖，下层支架材料表面固定有聚乳酸-羟基乙酸生长因子缓释微球，能够稳定释放生长因子，而微球的多孔结构还可以保护生长因子免受外界环境的影响，保持性能稳定，将负载生长因子的多孔PLGA微球修饰在支架材料上，能够避免单纯的微球在组织中容易发生团聚或迁移的现象，得到的支架材料不仅具有传导性和诱导性，便于细胞粘附、增殖和分化，而且能为皮肤组织再生提供必要的力学支持、充足的血供及养分。
具体实施方式
为更进一步阐述本发明所采取的技术手段及其效果，以下结合具体实施例进行详细描述。
实施例1
细菌纤维素/壳聚糖纤维层的制备：将溶解壳聚糖添加到木葡糖酸醋杆菌培养基中，原位合成细菌纤维素/壳聚糖复合凝胶，复合凝胶经0.5mol/L NaOH溶液常温碱解8h纯化后，再采用冻干法将纤维素酶固定在细菌纤维素/壳聚糖纤维的表面，得到细菌纤维素/壳聚糖纳米纤维层。
实施例2
除细菌纤维素/壳聚糖纤维层的制备过程中复合凝胶经碱解10h纯化，其余同实施例1。
实施例3
聚乳酸/透明质酸/丝素微纳米纤维的制备方法，包括以下步骤：(1)丝素膜的制备：桑蚕丝脱胶后溶于80℃的氯化钙、水和乙醇体积比为1:8:2的混合溶液中，搅拌溶解，透析后得纯丝素溶液，流延成膜备用；(2)纺丝液1的制备：将丝素膜与相对分子质量为150万的透明质酸粉末共同加入甲酸溶液中，得到纺丝液1；(3)纺丝液2的制备：聚乳酸溶于氯仿和丙酮体积比2:1的混合溶剂中，配制成5wt％的聚乳酸溶液，室温搅拌均匀，得到纺丝液2；(4)同轴静电纺丝：取纺丝液1、纺丝液2分别装于10mL的注射器内，将注射器分别安装到两个SN-50微量注射泵上，再用聚四氟乙烯软管将纺丝液2连于内层针管，纺丝液1连于外层嵌套装置，对同轴装置施加相同高压，使纺丝液2核层液体与纺丝液1壳层液体在喷口处汇合而成同心分层流，在高压静电场的静电力牵引下，得到同轴复合纤维。
其中，所述同轴静电纺丝的工艺参数为：电压12kV，收集距离15cm，核-壳溶液推进速度分别为0.1和0.3mL/h。
实施例4
除所述同轴静电纺丝的工艺参数为：电压12kV，收集距离15cm，核-壳溶液推进速度分别为0.1和0.4mL/h，其余同实施例1。
实施例5
一种可完全降解组织工程皮肤支架材料，包括细菌纤维素/壳聚糖纳米纤维层和大孔径阵列微米纤维支架两部分，细菌纤维素/壳聚糖纳米纤维层为上层，大孔径阵列微米纤维支架上方为下层，层间通过戊二醛交联结合；所述细菌纤维素/壳聚糖纳米纤维层由木葡糖酸醋杆菌原位合成，表面固定有纤维素酶；所述大孔径阵列微米纤维支架为静电纺乳酸/透明质酸/丝素微纳米纤维有序排列的支架，表面修饰有聚乳酸-羟基乙酸生长因子缓释微球。
一种所述可完全降解组织工程皮肤支架材料的制备方法，包括以下步骤：
步骤一、细菌纤维素/壳聚糖纤维层的制备：将溶解壳聚糖添加到木葡糖酸醋杆菌培养基中，原位合成细菌纤维素/壳聚糖复合凝胶，复合凝胶经0.5mol/LNaOH溶液常温碱解9h纯化后，再采用冻干法将纤维素酶固定在细菌纤维素/壳聚糖纤维的表面，得到细菌纤维素/壳聚糖纳米纤维层；
步骤二、聚乳酸/透明质酸/丝素微纳米纤维的制备：以丝素和透明质酸的混合溶液为纺丝液1，聚乳酸溶液为纺丝液2，采用同轴静电纺丝技术得到以纺丝液1为壳层，纺丝液2为核层的聚乳酸/透明质酸/丝素微纳米纤维；
步骤三、大孔径阵列微米纤维支架的制备：使用探针阵列收集器接收，得到大孔径阵列微米纤维支架材料，再将大孔径阵列微米纤维支架材料浸润在聚乳酸-羟基乙酸生长因子缓释微球溶液中，取出后真空干燥即得；
步骤四、戊二醛交联：在培养皿中加入适量25wt％的戊二醛水溶液，然后将其放入干净密闭的干燥器底部，再将大孔径阵列微米纤维支架放在铝箔上，细菌纤维素/壳聚糖纤维层叠放在大孔径阵列微米纤维支架上方，将叠加后的支架材料置于干燥器孔板中间，盖好盖子并密封后，室温下交联10h，取出水洗，充分干燥。
进一步地，步骤二所述聚乳酸/透明质酸/丝素微纳米纤维的制备方法，包括以下步骤：(1)丝素膜的制备：桑蚕丝脱胶后溶于80℃的氯化钙、水和乙醇体积比为1:8:2的混合溶液中，搅拌溶解，透析后得纯丝素溶液，流延成膜备用；(2)纺丝液1的制备：将丝素膜与相对分子质量为150万的透明质酸粉末共同加入甲酸溶液中，得到纺丝液1；(3)纺丝液2的制备：聚乳酸溶于氯仿和丙酮体积比2:1的混合溶剂中，配制成5wt％的聚乳酸溶液，室温搅拌均匀，得到纺丝液2；(4)同轴静电纺丝：取一定量的纺丝液1、纺丝液2分别装于10mL的注射器内，将注射器分别安装到两个SN-50微量注射泵上，再用聚四氟乙烯软管将纺丝液2连于内层针管，纺丝液1连于外层嵌套装置，对同轴装置施加相同高压，使纺丝液2核层液体与纺丝液1壳层液体在喷口处汇合而成同心分层流，在高压静电场的静电力牵引下，得到同轴复合纤维。
进一步地，所述同轴静电纺丝的工艺参数为：电压12kV，收集距离15cm，核-壳溶液推进速度为0.1和0.3mL/h。
进一步地，所述探针阵列收集器为将探针嵌入平板收集器泡沫板中，探针之间等距形成6×6阵列，探针间距为3cm。
进一步地，所述聚乳酸-羟基乙酸生长因子缓释微球的制备方法如下：配制生长因子水溶液，将其PLGA二氯甲烷溶液中，用超声细胞破碎仪超声分散10s至液体呈乳白色；然后将乳白色溶液到PVA溶液中，继续超声分散2min，至液体呈淡乳白色后，磁力搅拌3h，使二氯甲烷挥发完全；待微球成型后，离心、弃上清，用去离子水清洗沉淀，重复离心、清洗三次，-20℃冷冻，真空干燥后即得聚乳酸-羟基乙酸生长因子缓释微球。
进一步地，聚乳酸-羟基乙酸生长因子缓释微球的制备方法中离心转速为4000rpm，离心时间5min。
对比例1
除可完全降解组织工程皮肤支架材料的制备方法中步骤一、细菌纤维素/壳聚糖纤维层的制备过程中复合凝胶经0.5mol/L NaOH溶液常温碱解7h纯化，其余同实施例1。
对比例2
除可完全降解组织工程皮肤支架材料的制备方法中步骤一、细菌纤维素/壳聚糖纤维层的制备过程中复合凝胶经0.5mol/L NaOH溶液常温碱解11h纯化，其余同实施例1。
对比例3
除可完全降解组织工程皮肤支架材料的制备方法中步骤一、细菌纤维素/壳聚糖纤维层的制备过程中复合凝胶经0.5mol/L NaOH溶液碱煮1h纯化，其余同实施例1。
对比例4
除所述同轴静电纺丝的工艺参数为：电压12kV，收集距离15cm，核-壳溶液推进速度为0.1和0.1mL/h，其余同实施例1。
对比例5
除所述同轴静电纺丝的工艺参数为：电压12kV，收集距离15cm，核-壳溶液推进速度为0.1和0.5mL/h，其余同实施例1。
形态表征
纳米纱层、纳米纤维束、组织工程支架样本表面喷金镀膜后，在扫描电子显微镜加速电压为25kV时，拍摄得到放大5000倍的SEM照片，观察纤维形态、直径、孔隙率，纳米纤维、纳米纱和纳米纤维束的平均直径均通过NanoMeasurer 1.2测量软件测量不同样品20次后统计得到。
吸水性测试
将样本裁剪成相同尺寸大小，充分干燥，用电子天平分别称取各样品的起始重量记为m0，然后分别将各样本放入培养皿中，向培养皿中加入重蒸水，在不同的时间用镊子夹出样品，用吸水纸擦去表面水分，电子天平称重，记录为mt。利用如下公式计算吸水率，吸水率
抗菌性测试
将样本裁剪成相同尺寸大小，用接种环从固体培养基上取一定量的新鲜大肠杆菌、金黄色葡萄球菌、白色念珠菌，加入液体培养基，稀释成相同浓度，即1.5×106cfu/ml；分别取1ml的上述菌液滴加在样品上，用灭菌覆盖膜覆盖在样品上，在37℃，湿度大于90％的条件下培养24h；用洗脱液24ml反复清洗覆盖膜和样品，然后取0.2ml洗脱液滴入固体琼脂培养基上，在37℃条件下培养1～48h，然后进行活菌计数，测定活菌数目，计算杀菌率。
机械性能测试
取纳米纤维层、大孔径阵列微米纤维支架、组织工程支架样本(长×宽：1cm×5cm)，用螺旋测微器测量样品的厚度，将样品两端1cm处固定于材料测试机的夹具上，在室温20℃、相对湿度60％环境下，采用H5K-S型万能材料实验机施50N的力，10mm/min的速度进行拉伸测试。通过应力-应变曲线计算样本的断裂强度、断裂伸长及拉伸模量(每组测量5个标本)。
对实施例1～2及对比例1～3制备得到的细菌纤维素/壳聚糖纤维层进行机械性能测试，结果如表1所示：
表1细菌纤维素/壳聚糖纤维层机械性能测试结果
从表1可以看出，碱解的方式和时间对细菌纤维素/壳聚糖纤维层机械性能影响很大，对比例1为NaOH溶液常温碱解7h，纤维中有成块现象，不连续，纤维平均直径较大，这可能是由于碱解时间不足，纤维中杂质较多；对比例2为NaOH溶液常温碱解11h，纤维均匀、但不连续，断裂伸长率、断裂强度、拉伸模量与对比例2相比大幅下降，这可能是由于碱解时间过长，细菌纤维素纤维长度和直径减小；从实施例1～2和对比例1～2的机械性能测试结果可知，常温碱解时间在8～10h制得的细菌纤维素/壳聚糖纤维层纯度高，且具有最佳的机械性能。对比例3为NaOH溶液碱煮1h，纤维直接成了细碎状态，直径也只有48nm，这可能是由于原位生成的细菌纤维素/壳聚糖凝胶中纤维呈纤维束的状态，碱煮过程在除去杂质的同时，也导致纤维束散开，变得细碎。
通过在金相显微镜下观察实施例3～4和对比例4～5所得聚乳酸/透明质酸/丝素微纳米纤维的形态，可以看到实施例3～4能够形成均匀良好的核-壳结构，核层与壳层之间界限明显；而对比例4纤维表面粗糙，直径不均匀，为非核-壳结构，这可能是由于壳层纺丝液流速较快，突破壳层纺丝液的包覆而造成的；对比例5虽然能够得到核壳结构，但是核层含量降低，造成纤维机械强度减弱。
将实施例5所得细菌纤维素/壳聚糖纤维层、大孔径阵列微米纤维支架和复合的可完全降解组织工程皮肤支架材料分别进行拉伸试验，结果表明复合的可完全降解组织工程皮肤支架材料的拉伸强度高于二者单独的拉伸强度。采用游标卡尺对支架材料的厚度进行测量，本实施例复合的可完全降解组织工程皮肤支架材料厚度为432±14μm，扫描电子显微镜结果显示纳米纤维的直径为190-220nm，微米纤维的直径为2-10μm，作为真皮层的支架孔隙直径范围在50-120μm，孔隙率为92.8％，28天降解率为99.7％。通过NIT/3T3细胞检测实施例5制得的可完全降解组织工程皮肤支架材料，检测结果表明其具有良好的生物相容性、适合于细胞的黏附、迁移和增殖。
在家兔皮肤缺损修复方面的应用试验：将12只月龄、体重相当的健康家兔分为3组，空白对照组、试验组、对比组，每组4只，在其背部相同部位形成相同面积和深度的皮肤缺损，空白对照组不使用组织工程支架材料，试验组使用实施例5制得的可完全降解组织工程皮肤支架材料，对比组使用与实施例5支架材料相同厚度的3D打印聚乳酸组织工程皮肤支架材料，其他喂养条件一致。分别在第3天、9天、15天对家兔背部进行HE染色观察，结果显示：第3天时，空白对照组和对比组的4只家兔背部缺损处表皮结构和孔结构还处于缺失状态，伤口由细胞，大量渗出物和炎症介质组成；而试验组4只家兔的伤口处渗出物和炎症介质极少，伤口深层出现肉芽组织。到第9天时，3组家兔的伤口面积均明显减小，均可以观察到伤口再生区域中出现一些新生毛囊，试验组和对比组还能看到成形的再生组织顶部的表皮，试验组的家兔伤口上的新生乳头状结构与对比组相比更加致密；第14天，能够看到试验组家兔伤口形成完整表皮，ECM的密度与正常皮肤接近，皮下组织丰富，新生毛囊占据再生组织；而空白对照组家兔伤口处还有部分真皮裸露，对比组表皮接近完整，但皮下组织疏松、不规则，毛囊较少。家兔皮肤缺损修复试验表明实施例5制得的可完全降解组织工程皮肤支架材料生物相容性好，无排异反应，能够完全降解，可减轻伤口感染，有效诱导创伤面部位自体细胞生长、促进创面愈合，对皮肤缺损修复效果优于3D聚乳酸打印组织工程皮肤支架。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，本领域普通技术人员对本发明的技术方案所做的其他修改或者等同替换，只要不脱离本发明技术方案的精神和范围，均应涵盖在本发明的权利要求范围当中。
Claims (7)
1.一种可完全降解组织工程皮肤支架材料，其特征在于，包括细菌纤维素/壳聚糖纳米纤维层和大孔径阵列微米纤维支架两部分，细菌纤维素/壳聚糖纳米纤维层为上层，大孔径阵列微米纤维支架上方为下层，层间通过戊二醛交联结合；所述细菌纤维素/壳聚糖纳米纤维层由木葡糖酸醋杆菌原位合成，表面固定有纤维素酶；所述大孔径阵列微米纤维支架为静电纺乳酸/透明质酸/丝素微纳米纤维有序排列的支架，表面修饰有聚乳酸-羟基乙酸生长因子缓释微球。
2.根据权利要求1所述可完全降解组织工程皮肤支架材料，其特征在于，所述聚乳酸-羟基乙酸生长因子缓释微球的制备方法如下：配制生长因子水溶液，取PBS加入PLGA二氯甲烷溶液中，用超声细胞破碎仪超声分散10 s至液体呈乳白色；然后将乳白色溶液到PVA溶液中，继续超声分散2 min，至液体呈淡乳白色后，磁力搅拌2-4 h，使二氯甲烷挥发完全；待微球成型后，离心、弃上清，用去离子水清洗沉淀，重复离心、清洗三次，-20℃冷冻，真空干燥后即得聚乳酸-羟基乙酸生长因子缓释微球。
3.根据权利要求2所述可完全降解组织工程皮肤支架材料，其特征在于，聚乳酸-羟基乙酸生长因子缓释微球的制备方法中离心转速为4000 rpm，离心时间5 min。
4.一种根据权利要求1所述可完全降解组织工程皮肤支架材料的制备方法，其特征在于，包括以下步骤：
步骤一、细菌纤维素/壳聚糖纤维层的制备：将溶解壳聚糖添加到木葡糖酸醋杆菌培养基中，原位合成细菌纤维素/壳聚糖复合凝胶，复合凝胶经0.5 mol/LNaOH溶液常温碱解8-10 h纯化后，再采用冻干法将纤维素酶固定在细菌纤维素/壳聚糖纤维的表面，得到细菌纤维素/壳聚糖纳米纤维层；
步骤二、聚乳酸/透明质酸/丝素微纳米纤维的制备：以丝素和透明质酸的混合溶液为纺丝液1，聚乳酸溶液为纺丝液2，采用同轴静电纺丝技术得到以纺丝液1为壳层，纺丝液2为核层的聚乳酸/透明质酸/丝素微纳米纤维；
步骤三、大孔径阵列微米纤维支架的制备：使用探针阵列收集器接收，得到大孔径阵列微米纤维支架材料，再将大孔径阵列微米纤维支架材料浸润在聚乳酸-羟基乙酸生长因子缓释微球溶液中，取出后真空干燥即得；
步骤四、戊二醛交联：在培养皿中加入适量25wt%的戊二醛水溶液，然后将其放入干净密闭的干燥器底部，再将大孔径阵列微米纤维支架放在铝箔上，细菌纤维素/壳聚糖纤维层叠放在大孔径阵列微米纤维支架上方，将叠加后的支架材料置于干燥器孔板中间，盖好盖子并密封后，室温下交联8-12 h，取出水洗，充分干燥。
5.根据权利要求4所述可完全降解组织工程皮肤支架材料的制备方法，其特征在于，步骤二所述聚乳酸/透明质酸/丝素微纳米纤维的制备方法，包括以下步骤：（1）丝素膜的制备：桑蚕丝脱胶后溶于80 ℃的氯化钙、水和乙醇体积比为1:8:2的混合溶液中，搅拌溶解，透析后得纯丝素溶液，流延成膜备用；（2）纺丝液1的制备：将丝素膜与相对分子质量为150万的透明质酸粉末共同加入甲酸溶液中，得到纺丝液1；（3）纺丝液2的制备：聚乳酸溶于氯仿和丙酮体积比2:1的混合溶剂中，配制成5wt%的聚乳酸溶液，室温搅拌均匀，得到纺丝液2；（4）同轴静电纺丝：取一定量的纺丝液1、纺丝液2分别装于 10 mL的注射器内，将注射器分别安装到两个SN-50微量注射泵上，再用聚四氟乙烯软管将纺丝液2连于内层针管，纺丝液1连于外层嵌套装置，对同轴装置施加相同高压，使纺丝液2核层液体与纺丝液1壳层液体在喷口处汇合而成同心分层流，在高压静电场的静电力牵引下，得到同轴复合纤维。
6.根据权利要求5所述可完全降解组织工程皮肤支架材料的制备方法，其特征在于，所述同轴静电纺丝的工艺参数为：电压12 kV，收集距离15 cm，核-壳溶液推进速度为0.1-0.2和0.3-0.4 mL/h。
7.根据权利要求4~6任一项所述可完全降解组织工程皮肤支架材料的制备方法，其特征在于，所述探针阵列收集器为将探针嵌入平板收集器泡沫板中，探针之间等距形成6×6阵列，探针间距为2-4 cm。
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| CN112138215A (zh) * | 2020-09-26 | 2020-12-29 | 江苏大学 | 基于纳米纤维素的细胞生长因子缓释各向异性支架构建方法和应用 |
| CN112210888A (zh) * | 2020-10-23 | 2021-01-12 | 中原工学院 | 利于组织再生的聚乳酸弹性无纺材料及其制备方法 |
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| CN112138215B (zh) * | 2020-09-26 | 2021-11-05 | 江苏大学 | 基于纳米纤维素的细胞生长因子缓释各向异性支架构建方法和应用 |
| CN112210888A (zh) * | 2020-10-23 | 2021-01-12 | 中原工学院 | 利于组织再生的聚乳酸弹性无纺材料及其制备方法 |
| CN112210888B (zh) * | 2020-10-23 | 2021-06-25 | 中原工学院 | 利于组织再生的聚乳酸弹性无纺材料及其制备方法 |
| CN112704555A (zh) * | 2021-01-26 | 2021-04-27 | 山东建筑大学 | 一种可降解多孔尺骨中端骨折接骨器的制备方法 |
| CN113209373A (zh) * | 2021-04-16 | 2021-08-06 | 华南理工大学 | 一种皮肤组织修复支架及其制备方法与应用 |
| CN115844857A (zh) * | 2022-12-21 | 2023-03-28 | 方达医药技术（苏州）有限公司 | 药物透皮缓释支架 |
| CN116036368A (zh) * | 2023-02-08 | 2023-05-02 | 苏州汇涵医用科技发展有限公司 | 一种简洁高效的细菌纤维素的人工皮肤及其制作工艺 |
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