CN105903081A - 一种新型双层蛋白多糖基修复材料的制备方法 - Google Patents
一种新型双层蛋白多糖基修复材料的制备方法 Download PDFInfo
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Abstract
本发明涉及一种新型双层蛋白多糖基修复材料的制备方法,该方法采用Ⅰ型胶原蛋白及小分子寡聚透明质酸钠为原材料,二者经过电荷作用、氢键等方式发生交联作用,形成致密有序的三维网状结构,同时通过热交联处理使三维网状结构得以进一步强化,即提高了材料的机械强度又充分保留了Ⅰ型胶原蛋白固有的止血及创面愈合作用。此外,小分子寡聚透明质酸钠的添加,不仅在胶原蛋白海绵固有韧性的基础上进一步提高了其机械强度,同时又可以促进患者抗微生物感染和自身防护能力,小分子生物活性透明质酸钠通过粘膜吸收发挥其生理效应,包括抗炎、促进细胞生长和分化、促进伤口修复等等。
Description
技术领域
本发明涉及医用生物材料技术领域,是一种新型双层蛋白多糖基修复材料的制备方法,该修复材料新型双层胶原蛋白材料用于硬脑膜、硬脊膜及周围神经的修复。
背景技术
Ⅰ型胶原蛋白是哺乳类动物组织中最常见的结构性蛋白之一,具有良好的机械性,生物降解性以及生物相容性,被广泛地用于生物医用高分子材料领域。然而,单独使用胶原蛋白制备的高分子材料力学性能和抗水性能较差,且在体内容易降解,在实际应用过程中常采用其它高分子材料与胶原蛋白共混改性制得性能更优越的复合高分子材料。
透明质酸是人和动物皮肤、玻璃体、关节润滑液和软骨组织的重要成分,它由(1-β-4) D-葡糖醛酸和(1-β-3)N-乙酰基-D-氨基葡糖双糖单位重复连接而成,是脑组织中含量最为丰富的小分子聚糖之一。透明质酸具有良好的理化性能及生物相容性。此外,透明质酸作为一种重要的天然高分子材料,具有超强的保水性和低免疫原性,常常应用于和胶原蛋白共混构建组织工程。
低分子量透明质酸或寡聚透明质酸具有促血管生成、促进创伤愈合、抗肿瘤及免疫调节等生物活性。低分子量透明质酸能够保护肉芽组织免受氧自由基的破坏,并且能够促进伤口愈合。实验证明,相对分子质量较高的透明质酸不能透过表皮层进入真皮层,而低分子量透明质酸能够渗透到真皮层,清除氧自由基。寡聚透明质酸可刺激血管内皮细胞的增生和迁移,从而促进新生血管的生成,同时寡聚透明质酸可以促进内皮细胞合成Ⅰ型和Ⅷ型胶原,这两种胶原在血管形成过程中有重要作用。
胶原-小分子聚糖支架通常应用于皮肤再生,并已在这一领域中取得了较大的成功。近年来,胶原-小分子聚糖支架也广泛应用于软骨、神经等组织工程领域。胶原蛋白良好的生物相容性,生物可降解性等特性,使其在组织工程领域中备受青睐。仅由胶原蛋白单一物质组成的生物材料具有机械性能差,在液体环境中不能保持一定的韧性强度等缺点,从而限制了其在某些医疗领域的应用。
因此,本发明在常规胶原蛋白制备的基础上,引入具有生物学活性的小分子寡聚透明质酸钠,利用其本身携带的负离子与胶原蛋白分子中的正离子发生物理交联反应得到具有三维网状结构的凝胶,然后通过冷冻干燥技术得到复合胶原海绵,同时辅助加热脱氢交联处理进一步强化三维网状结构,即提高了材料的机械强度又充分保留了Ⅰ型胶原蛋白固有的止血及创面愈合作用。此外,小分子寡聚透明质酸钠的添加,不仅在胶原蛋白海绵固有韧性的基础上进一步提高了其机械强度,同时又可以促进患者抗微生物感染和自身防护能力,小分子生物活性透明质酸钠通过粘膜吸收发挥其生理效应,包括抗炎、促进细胞生长和分化、促进伤口修复等。本发明制备的蛋白多糖基修复材料具有相对较高机械性能、利于细胞附着再生的胶原海绵产品,以满足于临床上对硬脑膜、硬脊膜及周围神经的修复材料的需求。
发明内容
本发明的优点:
1.交联反应无需引入任何化学交联剂;
2.本产品具有双层结构,上层由Ⅰ型胶原蛋白构成,下层为胶原蛋白与小分子寡聚透明质酸钠复合物;
3.经本方法制得的修复材料在机械性能上优于传统的胶原蛋白产品,因而更适于临床中的穿刺或缝合处理;
4.本产品为可降解生物高分子材料,使用后无需二次手术取出。
附图说明
图1拉力测试对比结果。
具体实施方式
现结合实施例,对本发明作详细描述,但本发明的实施不仅限于此。
实施例一
取1%的胶原蛋白溶液100g,在低温条件下向其中加入事先预冷好的1%寡聚HA溶液0.5ml,边搅拌边缓慢滴加,滴加完毕后继续搅拌10min使二者充分混匀,搅拌完成后转移至4℃放置过夜。第二天,称取一定量的胶原-寡聚HA溶液放入特定模具中,铺平,进行冷冻干燥。
将冻干后的复合胶原海绵置于真空箱内,真空度0.01Mpa,温度105℃进行热交联,热交联时间为48h,热交联结束后得到胶原-寡聚HA海绵。
实施例二
取1%的胶原蛋白溶液100g,在低温条件下向其中加入事先预冷好的1%寡聚HA溶液1ml,边搅拌边缓慢滴加,滴加完毕后继续搅拌10min使二者充分混匀,搅拌完成后转移至4℃放置过夜。第二天,称取一定量的胶原-寡聚HA溶液放入特定模具中,铺平,进行冷冻干燥。
将冻干后的复合胶原海绵置于真空箱内,真空度0.01Mpa,温度105℃进行热交联,热交联时间为48h,热交联结束后得到胶原-寡聚HA海绵。
实施例三
取1%的胶原蛋白溶液100g,在低温条件下向其中加入事先预冷好的1%寡聚HA溶液2ml,边搅拌边缓慢滴加,滴加完毕后继续搅拌10min使二者充分混匀,搅拌完成后转移至4℃放置过夜。第二天,称取一定量的胶原-寡聚HA溶液放入特定模具中,铺平,进行冷冻干燥。
将冻干后的复合胶原海绵置于真空箱内,真空度0.01Mpa,温度105℃进行热交联,热交联时间为48h,热交联结束后得到胶原-寡聚HA海绵。
实施例四
取1%的胶原蛋白溶液100g,在低温条件下向其中加入事先预冷好的1%寡聚HA溶液5ml,边搅拌边缓慢滴加,滴加完毕后继续搅拌10min使二者充分混匀,搅拌完成后转移至4℃放置过夜。第二天,称取一定量的胶原-寡聚HA溶液放入特定模具中,铺平,进行冷冻干燥。
将冻干后的复合胶原海绵置于真空箱内,真空度0.01Mpa,温度105℃进行热交联,热交联时间为48h,热交联结束后得到胶原-寡聚HA海绵。
实施例五
取1%的胶原蛋白溶液100g,在低温条件下向其中加入事先预冷好的1%寡聚HA溶液2ml,边搅拌边缓慢滴加,滴加完毕后继续搅拌10min使二者充分混匀,搅拌完成后转移至4℃放置过夜。第二天,称取一半质量的未经混合的胶原溶液放入特定模具中,铺平,再称取一半质量的胶原-寡聚HA混合溶液置于上层铺平,然后进行冷冻干燥。
将冻干后的双层胶原海绵置于真空箱内,真空度0.01Mpa,温度105℃进行热交联,热交联时间为24h,热交联结束后得到胶原-胶原与寡聚HA混合双层海绵。
实施例六
取1%的胶原蛋白溶液100g,在低温条件下向其中加入事先预冷好的1%寡聚HA溶液2ml,边搅拌边缓慢滴加,滴加完毕后继续搅拌10min使二者充分混匀,搅拌完成后转移至4℃放置过夜。第二天,称取一半质量的未经混合的胶原溶液放入特定模具中,铺平,再称取一半质量的胶原-寡聚HA混合溶液置于上层铺平,然后进行冷冻干燥。
将冻干后的双层胶原海绵置于真空箱内,真空度0.01Mpa,温度105℃进行热交联,热交联时间为48h,热交联结束后得到胶原-胶原与寡聚HA混合双层海绵。
实施例七
取1%的胶原蛋白溶液100g,在低温条件下向其中加入事先预冷好的1%寡聚HA溶液2ml,边搅拌边缓慢滴加,滴加完毕后继续搅拌10min使二者充分混匀,搅拌完成后转移至4℃放置过夜。第二天,称取一半质量的未经混合的胶原溶液放入特定模具中,铺平,再称取一半质量的胶原-寡聚HA混合溶液置于上层铺平,然后进行冷冻干燥。
将冻干后的双层胶原海绵置于真空箱内,真空度0.01Mpa,温度105℃进行热交联,热交联时间为72h,热交联结束后得到胶原-胶原与寡聚HA混合双层海绵。
实施例八
取1%的胶原蛋白溶液100g,在低温条件下向其中加入事先预冷好的1%寡聚HA溶液2ml,边搅拌边缓慢滴加,滴加完毕后继续搅拌10min使二者充分混匀,搅拌完成后转移至4℃放置过夜。第二天,将未添加寡聚HA的胶原与上述混合均匀的产品按照质量比为1:1的比例进行混合,充分混合均匀后称取一定质量的混合物溶液放入特定模具中,铺平,然后进行冷冻干燥。
将冻干后的双层胶原海绵置于真空箱内,真空度0.01Mpa,温度105℃进行热交联,热交联时间为48h,热交联结束后得到胶原-胶原与寡聚HA混合双层海绵。
实施例九
取1%的胶原蛋白溶液100g,在低温条件下向其中加入事先预冷好的1%寡聚HA溶液2ml,边搅拌边缓慢滴加,滴加完毕后继续搅拌10min使二者充分混匀,搅拌完成后转移至4℃放置过夜。第二天,称取一半质量的胶原-寡聚HA混合溶液置于模具底层,铺平,再称取一半质量的胶原-寡聚HA混合溶液置于上层铺平,然后进行冷冻干燥。
将冻干后的双层胶原海绵置于真空箱内,真空度0.01Mpa,温度105℃进行热交联,热交联时间为72h,热交联结束后得到胶原-胶原与寡聚HA混合双层海绵。
实施例十
将由实施例所得的双层胶原海绵样品于万能拉力机上进行拉伸性能测试。测试条件:拉伸速率10mm/min,断裂敏感度为35%,即当拉力降为最大拉力的75%时即视为产品断裂。测试结果见图1,并与市售天义福硬脑膜产品拉伸性能进行了对比。由对比结果可知,所得产品在拉伸强度和伸展性能上优于传统胶原海绵及天义福产品。
Claims (7)
1.一种新型双层蛋白多糖基修复材料的制备方法,可用于硬脑膜及硬脊膜的修复:其主要特征为以适宜的胶原蛋白溶液为原料,通过添加一定量的小分子聚糖,与胶原蛋白分子进行交联,然后控制胶原蛋白与胶原蛋白-小分子聚糖复合物之间的比例进行铺模,最后通过冷冻干燥和热交联处理制得最终的产品。
2.权利要求1所述的小分子聚糖是指天然粘多糖及其衍生物,包括小分子寡聚透明质酸及其盐、壳寡糖、硫酸软骨素、硫酸皮肤素、硫酸角质素、硫酸乙酰肝素和肝素等。
3.权利要求2中所述的小分子寡聚透明质酸及其盐的分子量为1000-100,000道尔顿。
4.权利要求1所述的一定量的小分子聚糖是指胶原蛋白与小分子聚糖的比例为1/1~1000/1(m/m)。
5.权利要求1所述的适宜的胶原蛋白溶液的浓度为0.1~10%(m/m%)。
6.权利要求1所述的热交联处理的温度范围为90~130℃,处理时间范围为24~120h。
7.权利要求1中的胶原蛋白与胶原蛋白-小分子聚糖复合物之间的比例为1:3-3:1(m/m),优先选择1:1。
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