CN106039400B - 冰晶模板法制备规则片层结构三维生物支架的方法和应用 - Google Patents
冰晶模板法制备规则片层结构三维生物支架的方法和应用 Download PDFInfo
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Abstract
本发明公开了一种冰晶模板法制备规则片层结构三维生物支架的方法和应用。将蚕茧脱胶后得到的丝素纤维依次经过溶解、过滤、透析和离心后,浓缩获得丝素蛋白水溶液,采用冰晶模板法制备冰晶/丝素支架,以恒定的降温速度调节进行冷冻处理,通过冷冻干燥去除冰晶/丝素支架中的规则片层冰晶,获得具有规则片层结构的丝素支架,作为规则片层结构三维生物支架。由此,本发明具有优良的生物相容性和力学性能,显著提高了人体细胞的增殖性能和干细胞的定向分化性能,从而提高生物支架的骨组织修复作用,在组织工程、药物缓释、止血材料、过滤膜领域有广泛的应用前景。
Description
技术领域
本发明公开了一种冰晶模板法制备规则片层结构三维生物支架的方法和应用,属于生物医药材料领域。
背景技术
以家蚕丝素蛋白、柞蚕丝素蛋白和蜘蛛丝蛋白为代表的丝蛋白生物材料具有优良的力学性能、生物相容性、无免疫排斥、可降解性以及本质是天然蛋白质的结构特点,在生物材料领域受到高度的关注。丝蛋白生物材料可用作骨组织修复、创面覆盖材料、药物缓释材料等不同形状与功能的生物材料,以满足人类对生物材料的不同需求。
多孔材料可用于组织工程领域创伤组织的再生,所以支架材料的制备除了需要生物相容性之外还需要具有一定的强度,便于成型等性能,是支架材料能否应用于临床的关键。多孔支架的孔结构能够为细胞的粘附和增殖提供一种类似体内的微环境,使得细胞具有了类似在体内时的形态,能够影响细胞的生长、信号传递和分化,在重塑和修复组织的过程中起着至关重要的作用。成纤维细胞、间充质干细胞等人体细胞都可以在丝蛋白生物材料上良好地生长,发挥其特有的生理功能。现有技术制备多孔三维支架方法包括冷冻干燥法、粒子致孔法、气体发泡法等方法。目前,通过冷冻干燥法制备丝蛋白多孔支架的研究最多,可以通过改变丝蛋白溶液的浓度、时间来控制支架材料的孔的尺寸;粒子致孔法是在丝蛋白溶液中加入致孔剂粒子,将两者混合均匀、固化,再利用致孔剂与丝蛋白材料溶解性不同,置换出致孔剂粒子,最后干燥得到丝蛋白多孔支架的方法;气体发泡法一般采用碳酸盐类化合物为化学发泡剂,调节pH产生气体,最后经冷冻干燥制得多孔支架。尽管组织工程丝蛋白多孔支架的制备方法众多,不同方法制备的丝蛋白多孔支架的表面形貌、孔径和孔隙率会有所差异。但是以上制孔方法存在需要加入制孔剂或者需要添加发泡剂的缺点,同时存在力学性能不足,得到的孔径不可控,支架内部表面孔与下部孔会出现重叠及堵塞的缺点。对于微米级的细胞来说,只能在表面孔粘附与增殖,而不能深入到下部孔,这种细胞粘附方式与二维材料一样,使得三维多孔支架空有三维形貌的外壳。以上制孔方法严重阻碍了三维多孔支架在生物医药的应用。
因此,需要克服现有技术中的上述问题,模拟细胞外基质形貌的结构特征,保持三维多孔支架的具有规则形貌和力学强度,使得细胞能够在三维多孔支架内部粘附、增殖与分化,以满足当前组织工程材料的需要。
发明内容
为了解决背景技术中的应用,本发明公开了一种冰晶模板法制备规则片层结构三维生物支架的方法和应用,通过改善丝蛋白多孔支架的形貌特征与力学强度来满足实际的需求。
本发明采用可控梯度降温技术,通过在一定浓度的丝蛋白溶液中恒定降温至冰点以下,获得形貌规则的冰晶结构,冷冻干燥去除冰晶/丝素支架中的冰晶,获得具有规则片层结构的丝蛋白支架。
为达到上述发明目的,本发明采用的技术方案是:
一、一种冰晶模板法制备规则片层结构三维生物支架的方法:
(1)将蚕茧脱胶后得到的丝素纤维依次经过溶解、过滤、透析和离心后,浓缩至获得质量百分数为2%~25%的丝素蛋白水溶液;
(2)采用冰晶模板法制备冰晶/丝素支架:
(3)接着通过冷冻干燥去除冰晶/丝素支架中的规则片层冰晶,获得具有规则片层结构的丝素支架,作为规则片层结构三维生物支架。
所述步骤(2)具体为:将步骤(1)获得的丝素蛋白水溶液注入支架模具中,将支架模具放置降温调节环境中,以恒定的降温速度调节进行冷冻处理,在常温~-150℃低温范围内经过1~240分钟完成降温冷冻处理,使得丝素蛋白溶液中的冰晶以恒定速度由下往上生长,得到冰冻的冰晶/丝素支架。
所述恒定的降温速率为0.1℃/分钟~100℃/分钟。
将所述支架模具在梯度降温装置上面,梯度降温装置包括铜棒、加热线圈、液氮容器和加热器,铜棒上部缠绕加热线圈,加热线圈连接加热器,通过加热器给铜棒加热,铜棒下部置于液氮容器中,所述支架模具放置于铜棒顶端。
所述步骤(2)中梯度降温装置为一种可控降温的装置,可将温度将至-150℃的低温,降温速度可达到0.1℃/分钟~100℃/分钟范围,降温装置与支架模具之间具有很好的热传导。
所述步骤(1)中的丝素原料采用家蚕丝胶、丝腺蛋白、野蚕丝胶、蜘蛛丝蛋白或重组丝蛋白等丝蛋白,但不限于此。
所述丝素原料替换为其它生物大分子,其它生物大分子为壳聚糖、海藻酸钠或者胶原等材料。
二、一种冰晶模板法制备规则片层结构三维生物支架的应用:采用所述规则片层结构三维生物支架用于接种人体纤维细胞、人体充质干细胞中的应用。
所述的规则片层结构丝蛋白支架用于与人体纤维细胞实验:将人体纤维细胞接种到所述规则片层结构丝蛋白支架中,细胞能够在支架表面和支架内部生长,表现出良好的细胞增殖能力。
所述的规则片层结构丝蛋白支架用于与人间充质干细胞实验:将人间充质干细胞接种在所述规则片层结构丝蛋白支架中,一天培养后既表现出高的贴壁效果,两周培养后,能够促进细胞大量分泌胶原蛋白和碱性磷酸酶,表现出良好的促进干细胞定向分化能力。
本发明所使用到的丝蛋白物质为天然活性物质,已普遍用于生物医药行业,对细胞或组织无毒性反应。以冰晶为模板能够快速、准确地构建具有平行排列的片层拓扑结构的丝蛋白多孔材料。片层结构能够为细胞提供单独的储存空间;同时,与普通的网络状多孔支架相比,同方向的片层结构能够在z轴方向提供很好的支撑作用(如骨骼的着力点方向),从而提高支架材料的力学性能。细胞实验表面:具有片层结构的丝蛋白多孔支架表现出较高的细胞粘附率和增殖率;细胞能够在支架内部正常粘附与生长,具有良好的生物相容性,生物安全性高,能满足生物医学中的应用。
因此,本发明充分发挥了多孔结构特点对细胞相容性和力学性能的改善,综合了生物多孔支架材料的优势,属于最为理想的一种三维多孔支架材料,而该发明也为研究生物大分子多孔材料的设计和制备工艺提供参考信息。
由于上述技术方案的运用,本发明与现有技术相比具有以下突出特点:
(1)优良的生物相容性:丝蛋白成分,天然安全,能被生物体吸收再利用,是一种对机体组织无毒副作用;
(2)对环境没有污染:制备过程不使用有机或有毒试剂,制备条件温和,不产生有对人体和环境有毒害的产物;
(3)工艺简单,快速:以冰晶为模板,通过恒速降温能够快速、准确地在丝蛋白支架内部构建具有平行排列的片层拓扑结构;
(4)提高细胞在支架内部的生长:均一形貌的片层支架,能够很好地为细胞粘附提供着力点和生存空间,解决了细胞在三维支架内部中的细胞缺氧气及缺养分的问题,克服现有技术制备的三维多孔支架内部无法使得细胞生存的缺点。
(5)提高支架的力学性能:与普通的网络状多孔支架相比,同方向的片层结构能够在z轴方向提供很好的支撑作用,从而提高支架材料的力学性能。
由此,本发明不另加制孔剂,并具有优良的生物相容性和力学性能,显著提高了人体细胞的增殖性能和干细胞的定向分化性能,从而提高生物支架的骨组织修复作用,在组织工程、药物缓释、止血材料、过滤膜领域有广泛的应用前景。
附图说明
图1为实施例1中所获得的规则片层结构的丝素支架的扫描电镜图(图1A)和传统冷冻干燥法制备的丝素支架的扫描电镜图(图1B)。
图2为人纤维细胞在实施例1中所获得的规则片层结构的丝素支架上的细胞形貌图(图2A)和人纤维细胞在传统冷冻干燥法制备的丝素支架上的细胞形貌图(图2B)。
具体实施方式
下面通过实施例对本发明作进一步的详细说明,以下实施例是对本发明的解释而本发明并不局限于以下实施例。
本发明的实施例如下:
实施例1:
(1)将蚕茧脱胶后得到的丝蛋白纤维依次经过溶解、过滤、透析和离心后,浓缩至质量百分数为2%的丝素蛋白水溶液;
(2)冰晶模板法制备冰晶/丝素支架,将丝素蛋白水溶液注入模具中,将模具放置在梯度降温装置上面,通过恒定速度调节降温速率为0.1℃/分钟,经过240分钟,使得模具内的支架降至-24℃,得到冰冻的冰晶/丝素支架;
(3)通过冷冻干燥去除冰晶/丝素支架中的规则片层冰晶,获得具有规则片层结构的丝素支架,而对照组组支架(冷冻干燥法制备)的形貌则是无规则的多孔状;
(4)实施例1中所获得的规则片层结构的丝素支架的扫描电镜如图1A所示,人纤维细胞在实施例1中所获得的规则片层结构的丝素支架上的细胞形貌如图2A所示。以传统冷冻干燥法作为对照组,传统冷冻干燥法制备的丝素支架的扫描电镜如图1B所示,人纤维细胞在传统冷冻干燥法制备的丝素支架上的细胞形貌如图2B所示。
图中可见,人纤维细胞能够在该本发明的丝素支架内部粘附与增殖,具有良好的生物相容性,而传统冷冻干燥法的对照组中的多孔支架(冷冻干燥法制备)上的细胞数量较少,细胞形貌较小。
实施例2:
(1)将蚕茧脱胶后得到的丝蛋白纤维依次经过溶解、过滤、透析和离心后,浓缩至质量百分数为25%的丝素蛋白水溶液;
(2)冰晶模板法制备冰晶/丝素支架,将丝素蛋白水溶液注入模具中,将模具放置在梯度降温装置上面,通过恒定速度调节降温速率为100℃/分钟,经过1分钟,使得模具内的支架以恒定降温方式降至-100℃,得到冰冻的冰晶/丝素支架;
(3)通过冷冻干燥去除冰晶/丝素支架中的规则片层冰晶,获得具有规则片层结构的丝素支架,75%酒精处理后使得片层结构的丝素支架结构β化;
(5)细胞实验表明该支架能够诱导人间充质干细胞向成骨细胞分化。
实施例3:
(1)将蚕茧脱胶后得到的丝蛋白纤维依次经过溶解、过滤、透析和离心后,浓缩至质量百分数为12%的丝素蛋白水溶液;
(2)冰晶模板法制备冰晶/丝素支架,将丝素蛋白水溶液注入模具中,将模具放置在梯度降温装置上面,通过恒定速度调节降温速率为5℃/分钟,经过20分钟,使得模具内的支架以恒定降温方式降至-100℃,得到冰冻的冰晶/丝素支架;
(3)通过冷冻干燥去除冰晶/丝素支架中的规则片层冰晶,获得具有规则片层结构的丝素支架,100%甲醇处理后使得片层结构的丝素支架结构β化;
(5)细胞实验表明该规则片层结构支架能够诱导人间充质干细胞向成骨细胞分化。
实施例4:
(1)将明胶溶液溶于水溶液中得到质量百分数为10%的明胶溶液;
(2)冰晶模板法制备冰晶/明胶支架,将明胶水溶液注入模具中,将模具放置在梯度降温装置上面,通过恒定速度调节降温速率为5℃/分钟,经过30分钟,使得模具内的支架以恒定降温方式降至-150℃,得到冰冻的冰晶/明胶支架;
(3)通过冷冻干燥去除冰晶/明胶支架中的规则片层冰晶,获得具有规则片层结构的明胶支架;
(5)细胞实验表明该该规则片层结构的明胶支架能够诱导人间充质干细胞向成骨细胞分化。
综上,本发明实施例采用冰晶模板法制备冰晶/丝素支架,以恒定的降温速度调节进行冷冻处理,经冷冻干燥后得到规则片层结构的丝素支架,克服了需要加入制孔剂或者需要添加发泡剂的缺点。细胞能够在该规则片层结构的丝素支架内部粘附、增殖与分化。解决了细胞在三维支架内部中的细胞缺氧气及缺养分的问题,克服现有技术制备的三维多孔支架内部无法使得细胞生存的缺点。同时片层结构能够诱导间充质干细胞的定向分化。该发明不仅可以应用到片层丝蛋白支架的制备,还可以应用到其它生物大分子(如:壳聚糖、海藻酸钠或者胶原等)片层结构生物支架的制备。因此本发明是一种生物相容性好、细胞三维培养效果显著,可以诱导间充质干细胞分化的生物支架。
最后,还需要注意的是,以上列举的仅是本发明的具体实施例子。显然,本发明不限于以上实施例子,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
Claims (5)
1.一种冰晶模板法制备规则片层结构三维生物支架的方法,依次包括如下步骤:
(1)将蚕茧脱胶后得到的丝素纤维依次经过溶解、过滤、透析和离心后,浓缩至获得质量百分数为2%~25%的丝素蛋白水溶液;
(2)采用冰晶模板法制备冰晶/丝素支架;
(3)接着通过冷冻干燥去除冰晶/丝素支架中的规则片层冰晶,获得具有规则片层结构的丝素支架,作为规则片层结构三维生物支架;
所述步骤(2)具体为:将步骤(1)获得的丝素蛋白水溶液注入支架模具中,将支架模具放置降温调节环境中,以恒定的降温速度调节进行冷冻处理,在常温~-150℃低温范围内经过1~240分钟完成降温冷冻处理,使得丝素蛋白溶液中的冰晶以恒定速度由下往上生长,得到冰冻的冰晶/丝素支架;
所述恒定的降温速度为0.1℃/分钟~100℃/分钟。
2.根据权利要求1所述的一种冰晶模板法制备规则片层结构三维生物支架的方法,其特征在于:将所述支架模具在梯度降温装置上面,梯度降温装置包括铜棒、加热线圈、液氮容器和加热器,铜棒上部缠绕加热线圈,加热线圈连接加热器,通过加热器给铜棒加热,铜棒下部置于液氮容器中,所述支架模具放置于铜棒顶端。
3.根据权利要求1所述的一种冰晶模板法制备规则片层结构三维生物支架的方法,其特征在于:所述步骤(1)中的丝素原料采用家蚕丝胶、丝腺蛋白、野蚕丝胶、蜘蛛丝蛋白或重组丝蛋白。
4.根据权利要求1所述的一种冰晶模板法制备规则片层结构三维生物支架的方法,其特征在于:所述丝素原料替换为其它生物大分子,其它生物大分子为壳聚糖、海藻酸钠或者胶原材料。
5.一种冰晶模板法制备规则片层结构三维生物支架的应用,其特征在于:采用权利要求1~4任一所述方法获得的规则片层结构三维生物支架用于接种人体纤维细胞、人体充质干细胞中的应用。
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