CN1985778A - 生物型人工角膜 - Google Patents

生物型人工角膜 Download PDF

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CN1985778A
CN1985778A CNA2005101207946A CN200510120794A CN1985778A CN 1985778 A CN1985778 A CN 1985778A CN A2005101207946 A CNA2005101207946 A CN A2005101207946A CN 200510120794 A CN200510120794 A CN 200510120794A CN 1985778 A CN1985778 A CN 1985778A
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cornea
reagent
acid
epoxide
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CN1985778B (zh
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徐国风
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Guan Hao biotech inc
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ZHIGUANG BIOLOGICAL SCI-TECH Co Ltd GUANGZHOU
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Priority to US11/639,828 priority patent/US20070142908A1/en
Priority to EP06828357A priority patent/EP1965733B1/en
Priority to RU2008127980/14A priority patent/RU2421185C2/ru
Priority to JP2008546077A priority patent/JP2009519792A/ja
Priority to AU2006329154A priority patent/AU2006329154B2/en
Priority to PCT/CN2006/003444 priority patent/WO2007071169A1/en
Priority to CA2634323A priority patent/CA2634323C/en
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Abstract

本发明公开了一种生物型人工角膜及其制备方法，它由经非醛类固定剂交联固定和去抗原处理过的动物角膜为基材(1)而构成。本发明的优点是：它的组成和三维结构与人的角膜十分相似，无免疫原性，可诱导和促进角膜再生，可随角膜的再生而作顺应性降解，调控交联固定条件可以控制其降解速度与角膜再生的速度基本同步。物理力学性能与人角膜相近，形态稳定，柔韧性好，可加工成不同曲率，在水中不肿胀，是进行角膜重建的理想载体或支架。

Description

生物型人工角膜
技术领域
本发明涉及一种用于角膜缺损重建的装置，属于植入型医疗器械。
背景技术
角膜损伤或病变引起的失明，是眼科常见病之一，目前靠遗体捐赠的角膜移植来进行治疗。但角膜移植一方面来源困难，另一方面免疫排异问题也常令移植失败。靠人体角膜移植远远不能满足治疗的需要。因此，学者试图开发以动物角膜来治疗人类角膜缺损病，有研究直接用动物角膜来进行角膜移植的，因免疫排异未能克服而失败，也有研究用动物角膜经低温冷冻及简单消毒处理制成人工角膜的，因去抗原不彻底，组织相容性欠佳而未能被人体所接受，用动物角膜制人工角膜的研究至今未获得有效突破。
发明内容
本发明的目的是提供一种生物相容性好、可被降解吸收、可诱导角膜再生的生物型人工角膜及其制备方法。
本发明的技术解决方案是：它由经非醛类固定剂交联固定和去抗原处理过的动物角膜为基材而构成。
动物角膜易被微生物降解或分解，需用固定剂使之交联固定，传统上使用戊二醛作固定剂，有残留毒性，我们选用非醛类固定剂，如环氧化物、二酰二胺、二异氰酸酯、聚乙二醇或碳化二亚胺，就没有这一缺点，以环氧化物为例，当应用环氧化物来代替醛类作固定试剂时，由于环氧化物很不稳定，易发生开环交联反应，控制反应条件可以做到使其交联产物很稳定，不轻易降解，只有在再生组织生长、增殖需对其蚕蚀时，分泌出激肽释放酶、纤溶酶、糖皮质激素协同胶原酶作用下，才能将其缓慢分解为多肽及氨基酸，并吸收利用。这样一种被动式降解与组织的再生是同步的，是最有利于组织的再生性修复的，而且无醛类的残留毒性；根据现代免疫学理论，动物组织的抗原性主要是由蛋白质中某些特殊位置的活性基团及特异构象引起的，这些活性基团主要是-OH，-NH2，-SH等，而特异构象则主要是由于蛋白质分子螺旋链的某些特殊氢键引起，在处理动物角膜时，用一种或多种易与这些基团起反应的活泼试剂(如酸酐、酰氯、酰胺、环氧化物等)与这些基团结合，将其封闭起来，从而有效的去除其抗原，同时，还应用强氢键试剂(如胍类化合物)，置换引起特异构象的氢键，改变其构象，就能有效的消除其抗原性。
作为一种优化方案，在基材表面上偶合有含有可粘附生长因子的多肽或糖胺聚糖类活性组分，形成活性表面层。所述多肽之一是由16个赖氨酸(K16)、甘氨酸(G)、精氨酸(R)、天冬氨酸(D)、丝氨酸(S)、脯氨酸(P)及半胱氨酸(C)缩聚而成的，所述的糖胺聚糖是透明质酸、硫酸软骨素、硫酸皮肤素、肝素、硫酸乙酰肝素或硫酸角质素。这些多肽或糖胺聚糖对生长因子有广谱粘附和富集作用，或能激发未分化细胞定向分化，从而具有诱导机体组织的再生性修复的功能。
本发明的生物型人工角膜的制备方法包括以下步骤：
(1)、选料：收集新鲜动物眼球；
(2)、预处理：摘取动物角膜，并修剪平整；置保存液中在-18～4℃低温下冷冻24～28小时，取出，解冻后，用表面活性剂溶液浸泡16～20小时，或者用蛋白酶溶液浸泡2～4小时，洗净，必要时超声清洗10～20分钟；
(3)、交联固定：使用非醛类固定剂交联固定基材中的胶原蛋白分子；
(4)、去除抗原：使用活泼试剂封闭基材蛋白质中的特异活性基团-OH或-NH2或-SH，并用强氢键试剂置换基材蛋白质分子螺旋链中的特殊氢键，改变特异构象；
(5)、表面活性修饰步骤，应用偶联剂将可粘附生长因子的多肽或糖胺聚糖活性组分偶合到基材表面，形成活性表面层。
生物型人工角膜的制备方法中所述的表面活性剂是指曲拉通(TritonX-100)、胆酸纳、羟甲基氨基甲烷(Tris)、十二烷基璜酸钠(SDS)或CHAPS；所述的蛋白酶是指胃蛋白酶、胰蛋白酶或二者的混合酶。
生物型人工角膜的制备方法中所述的非醛类固定剂为易与蛋白质分子发生交联的试剂如环氧化物、二酰二胺、二异氰酸酯、聚乙二醇或碳化二亚胺试剂中的一种或两种，这里的环氧化物可以是单环氧化物
Figure A20051012079400061
，也可以是双环氧化物 ，这里R＝H，CnH2n+1—，n＝0-10。还可以是低聚环氧化物如聚环氧丙烷。
生物型人工角膜的制备方法中所述的活泼试剂可以是小分子有机酸酐、酰氯、酰胺或环氧化物；强氢键试剂为胍类化合物。
生物型人工角膜的制备方法中偶联剂为二酰二胺、二酸酐、双环氧化物或其它能与-NH2，-OH，-COOH起缩合反应的双官能团试剂。
生物型人工角膜的制备方法中所述的保存液为人工泪液、生理盐水、甘油或甘油和人工泪液的混合液。
本发明的优点是：它的组成和三维结构与人的角膜十分相似，无免疫原性，可诱导和促进角膜再生，可随角膜的再生而作顺应性降解，调控交联固定条件可以控制其降解速度与角膜再生的速度基本同步。物理力学性能与人角膜相近，形态稳定，柔韧性好，可加工成不同曲率，在水中不肿胀，是进行角膜重建的理想载体或支架。
附图说明
附图1为本发明实施例的结构示意图；
附图2为本发明实施例的垂直剖视图；
1、基材，2、活性表面层。
具体实施方式
实施例：
如图1和2所示，生物型人工角膜，由经非醛类固定剂交联固定和去抗原处理过的动物角膜为基材1而构成，在基材1表面上偶合有含有可粘附生长因子的多肽或糖胺聚糖类活性组分，形成活性表面层2。所述多肽之一是由16个赖氨酸(K16)、甘氨酸(G)、精氨酸(R)、天冬氨酸(D)、丝氨酸(S)、脯氨酸(P)及半胱氨酸(C)缩聚而成的，所述的糖胺聚糖是透明质酸、硫酸软骨素、硫酸皮肤素、肝素、硫酸乙酰肝素或硫酸角质素。
本发明的生物型人工角膜的制备方法包括以下步骤：
(1)、选料：收集新鲜健康猪眼球，放入专用保存瓶中冷冻运回；
(2)、预处理：摘取动物角膜，并修剪平整；置入人工泪液或甘油保存液中，在-18℃低温下冷冻24小时，取出，解冻后，用曲拉通(Triton X-100)、胆酸纳、羟甲基氨基甲烷(Tris)、十二烷基璜酸钠(SDS)或CHAPS表面活性剂溶液浸泡16～20小时，或者用胃蛋白酶、胰蛋白酶或二者的混合酶溶液浸泡2～4小时，洗净，必要时超声清洗10～20分钟；
(3)、交联固定：置于环氧化物固定液中，常温下交联固定基材1中的胶原蛋白分子8～48小时；
(4)、去除抗原：使用活泼试剂酸酐或甲基化试剂或环氧化物封闭基材蛋白质中的特异活性基团-OH或-NH2或-SH，并用强氢键试剂盐酸胍溶液置换基材蛋白质分子螺旋链中的特殊氢键，改变特异构象；
(5)、表面活性修饰：在基材1的表面上通过偶联剂偶联16个赖氨酸(K16)、甘氨酸(G)、精氨酸(R)、天冬氨酸(D)、丝氨酸(S)、脯氨酸(P)及半胱氨酸(C)缩聚而成的多肽和糖胺聚糖活性组分，形成活性表面层2。
(6)、包装：用灭菌剂灭菌后，在无菌操作条件下，封装在盛有保存液的小瓶中。

Claims (10)

1、一种生物型人工角膜的制备方法，其特征在于：它包括以下处理步骤：
a)、选料：收集新鲜动物眼球；
b)、预处理：摘取动物角膜，并修剪平整；置保存液中在-18～4℃低温下冷冻24～28小时，取出，解冻后，用表面活性剂溶液浸泡16～20小时，或者用蛋白酶溶液浸泡2～4小时，洗净，必要时超声清洗10～20分钟；
c)、交联固定：使用非醛类固定剂交联固定基材中的胶原蛋白分子；
d)、去除抗原：使用活泼试剂封闭基材蛋白质中的特异活性基团-OH或-NH2或-SH，并用强氢键试剂置换基材蛋白质分子螺旋链中的特殊氢键，改变特异构象；
2、根据权利要求1所述的制备方法，其特征在于：它还设有诱导组织再生的表面活性修饰步骤，即在基材(1)的表面上通过偶联剂偶联可粘附生长因子的多肽或糖胺聚糖活性组分，形成活性表面层(2)。
3、根据权利要求1所述的制备方法，其特征在于：所述的非醛类固定剂为易与蛋白质分子发生交联反应的试剂，如环氧化物、二酰二胺、二异氰酸酯、聚乙二醇或碳化二亚胺试剂中的一种或两种，这里的环氧化物可以是单环氧化物
Figure A2005101207940002C1
，也可以是双环氧化物 ，这里R＝H，CnH2n+1-，n＝0-10，还可以是低聚环氧化物，如聚环氧丙烷。
4、根据权利要求1所述的制备方法，其特征在于：所述的活泼试剂可以是小分子有机酸酐、酰氯、酰胺或环氧化物；强氢键试剂为胍类化合物。
5、根据权利要求1所述的制备方法，其特征在于：所述偶联剂为二酰二胺、二酸酐、双环氧化物或其它能与-NH2，-OH，-COOH起缩合反应的双官能团试剂。
6、根据权利要求1所述的制备方法，其特征在于：所述的保存液为人工泪液、生理盐水、甘油或甘油和人工泪液的混合液。
7、根据权利要求1所述的制备方法，其特征在于：所述的表面活性剂是指曲拉通(Triton X-100)、胆酸纳、羟甲基氨基甲烷(Tris)、十二烷基璜酸钠(SDS)或CHAPS；所述的蛋白酶是指胃蛋白酶、胰蛋白酶或二者的混合酶。
8、一种使用权利要求1所述制备方法得到的生物型人工角膜，其特征在于：它由经非醛类固定剂交联固定和去抗原处理过的动物角膜为基材(1)而构成。
9、一种使用权利要求2所述的制备方法得到的生物型人工角膜，其特征在于：在基材(1)表面上偶合有含有可粘附生长因子的多肽或糖胺聚糖类活性组分，形成活性表面层(2)。
10、根据权利要求8所述的生物型人工角膜，其特征在于：所述多肽之一是由16个赖氨酸(K16)、甘氨酸(G)、精氨酸(R)、天冬氨酸(D)、丝氨酸(S)、脯氨酸(P)及半胱氨酸(C)缩聚而成的，所述的糖胺聚糖是透明质酸、硫酸软骨素、硫酸皮肤素、肝素、硫酸乙酰肝素或硫酸角质素。
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