CN109157305B - 复合人工角膜及其制备方法 - Google Patents
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Abstract
本发明公开了一种复合人工角膜及其制备方法，该复合人工角膜包括由聚甲基丙烯酸羟乙酯(PHEMA)制备的中心光学镜柱、由脱细胞基质水凝胶制备的角膜裙边结构、以及连接所述中心光学镜柱和所述角膜裙边结构的中间夹层网格支架，其中所述角膜裙边结构中设置有粘附生长了细胞的神经细胞支架层和角膜基质细胞支架层。该复合人工角膜结构简单，生物相容性好，易加工，并能很好地模拟角膜在体内的环境，且有利于移植。
Description
技术领域
本发明涉及一种复合人工角膜及其制备方法。
背景技术
角膜疾病是导致眼盲的仅次于白内障的第二大原因。角膜混浊是双侧失明的主要原因之一，影响全世界700万人。其中，有285万人因角膜神经功能障碍或变性而减少或没有感觉。角膜移植是使眼盲患者复明的唯一手段，然而，传统的角膜移植手术有其两大弊端，一是角膜供体的来源困难，二是对新生血管多的角膜受体则手术成功率极低，如严重化学和热烧伤的角膜，多次角膜移植失败的角膜，严重干眼症的角膜，天胞疮等，人工角膜的出现正能解决这两个问题，即来源丰富，也可抵抗新生血管。因此开发一种来源方便，可替代供体捐献角膜的人工眼角膜就变得格外重要和迫切。
目前应用最广泛的人工角膜结构为“裙-核”型，即由多孔裙边支架材料和光学中心两部分组成。其中，裙边支架是直接与植床接触的部分，起到与人眼组织细胞融合的作用，但是大部分异质材料与人体组织间没有良好的相容性，导致在人工角膜植入后常常会出现角膜脱落、发炎等并发症。
脱细胞基质是生物新鲜组织经过脱细胞方法处理，脱去组织中的所有细胞抗原、可溶性蛋白质和糖胺多糖等物质，已有的脱细胞基质研究表明经多步骤的脱细胞处理，只保留了原有组织结构中不可溶成分，主要包括胶原、弹性蛋白、氨基多糖和结构蛋白等，为细胞生长和植入体内修复和缺损提供了基础。脱细胞基质的主要特点是良好的生物相容性、柔韧性和低抗原性并含有促进细胞黏附生长、增殖和分化的物质，将脱细胞基质制备成水凝胶，可通过模具很容易制备出角膜裙边结构，且性能可控。
发明内容
本发明的主要目的在于克服现有技术的不足，提供一种复合人工角膜及其制备方法。
为实现上述目的，本发明采用以下技术方案：
一种复合人工角膜，包括由聚甲基丙烯酸羟乙酯(PHEMA)制备的中心光学镜柱、由脱细胞基质水凝胶制备的角膜裙边结构、以及连接所述中心光学镜柱和所述角膜裙边结构的中间夹层网格支架，其中所述角膜裙边结构中设置有粘附生长了细胞的神经细胞支架层和角膜基质细胞支架层。
进一步地：
所述中间夹层网格支架由静电直写打印而成，优选为PCL或PLGA材料。
所述中心光学镜柱由HEMA与水、交联剂、引发剂、催化剂混合制成，其中，交联剂为乙二醇二甲基丙烯酸酯(EDMA)，引发剂为过硫酸铵水溶液(APS)，催化剂为四甲基乙二胺(TEMED)。
所述中心光学镜柱按照浓度为99％无水级的HEMA 4mL，水2mL，EDMA 20μL,质量分数为10％的APS水溶液200μL，TEMED 100μL的配比制备成型。
所述脱细胞基质为脱细胞角膜基质或脱细胞软骨基质。
所述神经细胞支架层为仿神经支架，材料为甲基丙烯酸酯化PDC预聚物(methacrylated PDC(mPDC)prepolymer)，其结构为有规律或杂乱无章。
所述神经细胞支架层通过掩膜光刻的方式加工出来。
所述角膜基质细胞支架层为由PCL材料打印而成的网格支架。
所述复合人工角膜的整体直径为10mm，厚度为500μm,所述中心光学镜柱的直径为5mm，所述中间夹层网格支架的内径和外径分别为4mm、6mm，所述中间夹层网格支架具有一层或者多层网格。
一种所述的复合人工角膜的制备方法，包括：
打印中间夹层网格支架，优选地，通过静电直写打印；
通过模具注塑成型加工出PHEMA材料的中心光学镜柱，所述中间夹层网格支架部分嵌入到成型的所述中心光学镜柱中，形成PHEMA/网格支架复合体；
将所述PHEMA/网格支架复合体以及已经粘附生长有细胞的神经细胞支架层和角膜基质细胞支架层放入模具，通过模具注塑成型的方式浇注脱细胞基质水凝胶到模具中制备角膜裙边结构，得到所述复合人工角膜，其中所述中间夹层网格支架作为所述复合人工角膜的夹层以及所述中心光学镜柱和所述角膜裙边结构的中间连接物。
本发明具有如下有益效果：
本发明提供的复合人工角膜包括由聚甲基丙烯酸羟乙酯(PHEMA)制备的中心光学镜柱、由脱细胞基质水凝胶制备的角膜裙边结构、以及连接所述中心光学镜柱和所述角膜裙边结构的中间夹层网格支架，其中所述角膜裙边结构中设置有粘附生长有细胞的神经细胞支架层和角膜基质细胞支架层两层支架，采用上述结构和材料，形成由中间夹层网格支架介导的PHEMA、脱细胞基质水凝胶的复合人工角膜，结构简单，生物相容性好，易加工，并能很好地模拟角膜在体内的环境，且由于角膜裙边结构中设置已经粘附生长好细胞的神经细胞支架层和角膜基质细胞支架层，神经细胞支架层和角膜基质细胞支架层两层支架更好地模拟角膜在人体内的环境，提供了角膜基质细胞和神经细胞的共培养条件，从而本发明的复合人工角膜可以在体外细胞共培养形成神经和角膜组织，生物相容性好且更有利于移植。
在一些优选的方案中，通过静电直写网格支架作为夹层连接中心透明镜柱和裙边结构制备出复合人工角膜，静电直写网格支架结构均一、生物相容性好、机械性能良好且参数可控，用其作为人工角膜中心镜柱和裙边结构的中间连接物可使人工角膜材料选择更加多样，复合结构更加可靠，且其多孔网格支架结构和脱细胞基质水凝胶裙边结构利于细胞的粘附生长。通过在裙边结构中引入两层支架层以实现角膜基质细胞和神经细胞的共培养，可以更好地模拟角膜在体内的环境。脱细胞基质水凝胶来源于动物的组织或器官，生物相容性极好，为细胞提供良好的生存环境，体内排斥极低。
附图说明
图1a为本发明一种实施例的复合人工角膜的整体示意图；
图1b为本发明一种实施例的复合人工角膜的分解示意图；
图2a为本发明一种实施例中的角膜裙边结构的整体示意图；
图2b为本发明一种实施例中的角膜裙边结构及其中设置的角膜基质细胞支架层和神经细胞支架层的俯视图；
图3为六种不同的神经细胞支架层的结构示意图；
图4为两种不同的角膜基质细胞支架层的结构示意图。
具体实施方式
以下对本发明的实施方式作详细说明。应该强调的是，下述说明仅仅是示例性的，而不是为了限制本发明的范围及其应用。
参阅图1a至图4，在一种实施例中，一种复合人工角膜11，包括由聚甲基丙烯酸羟乙酯(PHEMA)制备的中心光学镜柱12、由脱细胞基质水凝胶制备的角膜裙边结构14、以及连接所述中心光学镜柱12和所述角膜裙边结构14的中间夹层网格支架13，其中所述角膜裙边结构14中设置有粘附生长了细胞的神经细胞支架层15和角膜基质细胞支架层16两层支架。
在优选的实施例中，所述中间夹层网格支架13由静电直写打印而成，例如通过静电直写生物打印机熔融打印而成。在更优选的实施例中，采用PCL或PLGA材料打印得到上述中间夹层网格支架13。中间夹层网格支架13可为一层或者多层网格。
在优选的实施例中，所述中心光学镜柱12由HEMA与水、交联剂、引发剂、催化剂混合制成，其中交联剂为乙二醇二甲基丙烯酸酯(EDMA)，引发剂为过硫酸铵水溶液(APS)，催化剂为四甲基乙二胺(TEMED)。将HEMA与水、交联剂、引发剂、催化剂混合均匀后，可通过模具注塑成型，制备简单。通过调整各成分比例可以得到不同所需性质的PHEMA。
在特别优选的实施例中，所述中心光学镜柱按照浓度为99％无水级的HEMA 4mL，水2mL，EDMA 20μL,质量分数为10％的APS水溶液200μL，TEMED 100μL的配比制备成型。制备时，将以上各成分混合均匀加入到模具中静置3h可得到成型的PHEMA，去离子水清洗待用。
所述脱细胞基质水凝胶来源于动物的组织或器官，生物相容性极好，为细胞提供良好的生存环境，体内排斥极低。在优选的实施例中，所述脱细胞基质为脱细胞角膜基质或脱细胞软骨基质。
参阅图2a至图2b，为了良好地模拟角膜在人体内的环境，所述角膜裙边结构14中设置神经细胞支架层15和角膜基质细胞支架层16两层支架，实现角膜基质细胞和神经细胞的共培养，优选地，两层支架周围轮廓尺寸与角膜裙边结构14相同。
在优选的实施例中，所述神经细胞支架层15为仿神经支架，材料为甲基丙烯酸酯化PDC预聚物(methacrylated PDC(mPDC)prepolymer)。参阅图3，在不同的实施例中，所述神经细胞支架层15的结构可以为有规律的或杂乱无章的。图3所示的几种神经细胞支架层15，其中3-1、3-2、3-3、3-4、3-5为结构有规律的支架，3-6为结构杂乱无章的支架。实际应用中，仿神经支架结构可有更丰富的设计。
所述神经细胞支架层15可以通过掩膜光刻的方式加工出来。
在优选的实施例中，所述角膜基质细胞支架层16为由PCL材料打印而成的网格支架。优选地，角膜基质细胞支架层16采用静电直写设备打印而成。图4中4-1、4-2分别为两种不同尺寸的网格支架结构。实际应用中，角膜基质细胞支架层16的网格支架尺寸可有更多的选择。
在一个具体的优选实施例中，所述复合人工角膜11的整体直径为10mm，厚度为500μm,所述中心光学镜柱12的直径为5mm，所述中间夹层网格支架13的内径和外径分别为4mm、6mm，所述中间夹层网格支架13具有一层或者多层网格，实现有效的连接作用。
参阅图1a至图4，在另一种实施例中，一种所述的复合人工角膜11的制备方法，包括：
打印中间夹层网格支架13，优选地，通过静电直写打印；
通过模具注塑成型加工出PHEMA材料的中心光学镜柱12，所述中间夹层网格支架13部分嵌入到成型的所述中心光学镜柱12中，形成PHEMA/网格支架复合体；
将所述PHEMA/网格支架复合体以及已经粘附生长有细胞的神经细胞支架层15和角膜基质细胞支架层16放入模具，通过模具注塑成型的方式浇注脱细胞基质水凝胶到模具中制备角膜裙边结构14，得到所述复合人工角膜11，其中所述中间夹层网格支架13作为所述复合人工角膜11的夹层以及所述中心光学镜柱12和所述角膜裙边结构14的中间连接物。
以上内容是结合具体/优选的实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，其还可以对这些已描述的实施方式做出若干替代或变型，而这些替代或变型方式都应当视为属于本发明的保护范围。
Claims (5)
1.一种复合人工角膜，其特征在于,包括由PHEMA制备的中心光学镜柱、由脱细胞基质水凝胶制备的角膜裙边结构、以及连接所述中心光学镜柱和所述角膜裙边结构的中间夹层网格支架，其中所述角膜裙边结构中设置有已经粘附生长有细胞的神经细胞支架层和角膜基质细胞支架层；
所述中间夹层网格支架由静电直写打印而成，所述静电直写打印的材料为PCL或PLGA材料；所述中间夹层网格支架呈环形；
所述神经细胞支架层为仿神经支架，材料为甲基丙烯酸酯化PDC预聚物，其结构为有规律或杂乱无章；
所述神经细胞支架层通过掩膜光刻的方式加工出来；所述角膜基质细胞支架层为由PCL材料打印而成的网格支架；
所述复合人工角膜的制备方法，包括：通过静电直写打印中间夹层网格支架；通过模具注塑成型加工出PHEMA材料的中心光学镜柱，所述中间夹层网格支架部分嵌入到成型的所述中心光学镜柱中，形成PHEMA/网格支架复合体；将所述PHEMA/网格支架复合体以及已经粘附生长有细胞的神经细胞支架层和角膜基质细胞支架层放入模具，通过模具注塑成型的方式浇注脱细胞基质水凝胶到模具中制备角膜裙边结构，得到所述复合人工角膜，其中所述中间夹层网格支架作为所述复合人工角膜的夹层以及所述中心光学镜柱和所述角膜裙边结构的中间连接物。
2.如权利要求1所述的复合人工角膜，其特征在于,所述中心光学镜柱由HEMA与水、交联剂、引发剂、催化剂混合制成，其中交联剂为EDMA，引发剂为APS水溶液，催化剂为TEMED。
3.如权利要求2所述的复合人工角膜，其特征在于,所述中心光学镜柱按照浓度为99％无水级的HEMA 4mL，水2mL，EDMA 20μL,质量分数为10％的APS水溶液200μL，TEMED 100μL的配比制备成型。
4.如权利要求1至3任一项所述的复合人工角膜，其特征在于,所述脱细胞基质水凝胶为脱细胞角膜基质水凝胶或脱细胞软骨基质水凝胶。
5.如权利要求1至3任一项所述的复合人工角膜，其特征在于,所述复合人工角膜的整体直径为10mm，厚度为500μm,所述中心光学镜柱的直径为5mm，所述中间夹层网格支架的内径和外径分别为4mm、6mm，所述中间夹层网格支架具有一层或者多层网格。
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Effective date of registration: 20230330 Address after: 510000 No. 54 martyrs South Road, Guangdong, Guangzhou Patentee after: ZHONGSHAN OPHTHALMIC CENTER, SUN YAT-SEN University Address before: 510000 Unit 701, 7th Floor, No. 6 Spiral Third Road, Guangzhou International Biological Island, Guangdong Province (self-declaration) Patentee before: GUANGZHOU RUICHENG MEDICAL TECHNOLOGY Co.,Ltd. |