CN113024730A - 一种用于骨修复的可注射复合壳聚糖水凝胶的制备及使用方法 - Google Patents

一种用于骨修复的可注射复合壳聚糖水凝胶的制备及使用方法 Download PDF

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CN113024730A
CN113024730A CN202110347214.6A CN202110347214A CN113024730A CN 113024730 A CN113024730 A CN 113024730A CN 202110347214 A CN202110347214 A CN 202110347214A CN 113024730 A CN113024730 A CN 113024730A
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胡巧玲
姜质琦
董晓飞
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Zhejiang University ZJU
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Abstract

本发明涉及一种用于骨修复的可注射复合壳聚糖水凝胶的制备及使用方法，该水凝胶主要由改性壳聚糖、内载细胞以及促细胞生长因子组成。首先通过乳液聚合制备得到水凝胶前体溶液，主要成分为聚N‑异丙基丙烯酰胺接枝改性的壳聚糖大分子，该水凝胶能在室温下呈溶液状态，在人体生理温度环境下实现快速凝胶化。通过与细胞悬液和促细胞生长因子的混合，所得复合溶液可以在骨组织损伤部位实现原位快速凝胶化，实现细胞的原位固定，不仅可以为损伤部位提供良好的支撑，还可以促进其内部细胞的粘附、增殖、分化。由于制备简单、使用方便、响应迅速、生物相容性良好，该水凝胶可用于组织修复、药物控释、细胞培养、癌症治疗等诸多生物医学相关领域。

Description

一种用于骨修复的可注射复合壳聚糖水凝胶的制备及使用 方法
技术领域
本发明涉及一种壳聚糖基可注射水凝胶，尤其涉及一种用于骨修复的可注射复合壳聚糖水凝胶的制备及使用方法。
背景技术
如何高效修复由外伤、感染、肿瘤等因素引起的骨组织损伤或缺损依旧是临床医学的重要挑战。日前，自体移植和异体移植是促进骨缺陷愈合以及骨组织重塑的重要治疗手段，但两者都存在一定的弊端，例如自体移植的细胞活性不理想、迁移率较低，异体移植易引起感染或免疫排斥反应。
理想的骨组织修复再生材料不仅需要合适的力学强度在骨缺损部位提供短期支撑，还应该促进细胞黏附、增殖、分化、矿化。水凝胶材料具有高含水量、与细胞外基质相似的结构、可调控的力学性能，使其在骨修复方面有着良好的应用前景。与传统水凝胶相比，智能水凝胶因其能对外界环境变化产生响应而受到更多关注。温度敏感水凝胶是一种对外界温度变化产生响应的水凝胶，具有可注射性，温敏水凝胶中亲水基团与疏水基团的存在，可以使温敏凝胶在温度改变时发生相转变，由液态转变为固态。具有良好生物相容性的温敏可注射水凝胶可以在组织修复领域发挥极大地作用。
壳聚糖是一种天然氨基多糖，来源于生物体，是虾壳、螃蟹壳的重要组成部分。壳聚糖不仅来源广泛，还具有优异的生物相容性、抑菌性以及良好的生物可降解性。温敏壳聚糖水凝胶兼具水凝胶的特点和优良的本征属性，为骨组织修复提供了新思路和新方法。
发明内容
本发明的目的在于针对现有技术的不足，提供一种用于骨修复的可注射复合壳聚糖水凝胶的制备及使用方法。本发明的可注射复合壳聚糖水凝胶，主要由改性壳聚糖、内载细胞以及促细胞生长因子组成。
所述改性壳聚糖具体为聚N-异丙基丙烯酰胺接枝改性的壳聚糖。
本发明所述的一种用于骨修复的可注射复合壳聚糖水凝胶，其前体的制备方法是：
1)聚N-异丙基丙烯酰胺接枝改性的壳聚糖生物大分子的制备：按照质量计，将1-15份壳聚糖、0.1-5份酸、0.01-10份引发剂、1-20份N-异丙基丙烯酰胺、50-100份溶剂加入烧杯中，搅拌溶解均匀后，加热至40-60℃反应1-12小时，随后将溶液转移入透析袋中，用去离子水反复透析，冷冻干燥得到聚N-异丙基丙烯酰胺接枝改性的壳聚糖粉末；
2)聚N-异丙基丙烯酰胺接枝改性的壳聚糖水溶液的制备：将1-20份1)中的聚N-异丙基丙烯酰胺接枝改性的壳聚糖粉末加入80-99份PBS溶液中，低温环境下搅拌溶解均匀后得到水凝胶前体溶液；
上述技术方案中，进一步的，
所述壳聚糖的分子量为3万-200万，脱乙酰度为65％-99％。
所述酸为盐酸、醋酸、磷酸、柠檬酸、酒石酸、丙烯酸中的一种或几种。
所述引发剂为偶氮二异丁腈、偶氮二异庚腈、偶氮二异丁酸二甲酯、过氧化氢、过硫酸铵、过硫酸钾、叔丁基过氧化氢中的一种或几种。
所述溶剂为去离子水、乙醇、DMSO中的一种或几种。
所述低温环境下为4℃-25℃。
本发明所述的一种用于骨修复的可注射复合壳聚糖水凝胶溶液的使用方法，具体包括如下步骤：
(1)可注射复合壳聚糖水凝胶溶液的制备：将所述步骤2)中的水凝胶前体溶液进行灭菌，将10-50份灭菌后的水凝胶前体溶液、5份内载细胞的细胞悬液与0.1-5份促细胞生长因子低温环境下进行共混，混合均匀得到可注射复合壳聚糖水凝胶溶液。
(2)使用注射器或移液枪缓慢吸取一定量步骤1)中的可注射壳聚糖水凝胶溶液，将其缓慢注射到受损的骨组织创伤部位，使溶液填满缺损部位，静待溶液凝胶化即可。
上述技术方案中，进一步的，
所述内载细胞为胚胎干细胞、间充质干细胞、脂肪干细胞、成骨细胞、软骨细胞中的一种或几种。
所述细胞悬液中内载细胞的浓度为2万-200万/ml，细胞培养基为完全培养基、干细胞生长培养基或成骨分化培养基。
所述低温环境为4℃-25℃。
所述受损的骨组织为硬骨或软骨等，其中所述硬骨为长骨或扁骨，如股骨、胫骨、颅骨等；所述软骨为关节软骨等。
用于硬骨组织损伤修复时所述促细胞生长因子为羟基磷灰石HAP、胶原、骨形成蛋白BMP、RGD分子以及其他氨基酸或多肽中的一种或几种；用于软骨组织损伤修复时所述促细胞生长因子为脱钙骨基质DBM、2-([1,1-联苯]-4-基氨基甲酰)苯甲酸KGN、胶原蛋白、鹿茸多肽PAP、胰岛素样生长因子IGF、转化生长因子TGF、基质细胞衍生因子SDF、骨形成蛋白BMP、RGD分子以及其他氨基酸或多肽中的一种或几种。
本发明的有益效果在于：
本发明首先通过乳液聚合制备得到水凝胶前体溶液，主要成分为聚N-异丙基丙烯酰胺接枝改性的壳聚糖大分子，该水凝胶具有温度敏感特性，能在室温环境下呈溶液状态，在人体生理温度环境下实现快速凝胶化。该温敏水凝胶具有优异的生物相容性，通过与细胞悬液和促细胞生长因子的混合，该水凝胶溶液可以在骨组织损伤部位实现原位快速凝胶化，实现细胞的原位固定，不仅可以为损伤部位提供良好的支撑，还可以促进其内部细胞的粘附、增殖、分化。由于制备简单、使用方便、响应迅速、生物相容性良好等诸多特点，该水凝胶可用于组织修复、药物控释、细胞培养、癌症治疗等诸多生物医学相关领域，特别是应用于骨组织修复再生。
具体实施方式
以下结合具体实例进一步说明本发明。
实施例1：
1)称取1.5g粘均分子量为100W、脱乙酰度为90％的壳聚糖粉末、1.75gN-异丙基丙烯酰胺、0.175g过硫酸铵，分散在95mL去离子水中，然后，加入0.5mL的盐酸，搅拌溶解均匀后，加热至60℃反应8小时，随后将溶液转移入透析袋中，用去离子水反复透析，冷冻干燥得到聚N-异丙基丙烯酰胺接枝改性的壳聚糖粉末；
2)将1-20份步骤1)中的聚N-异丙基丙烯酰胺接枝改性的壳聚糖粉末加入80-99份PBS溶液中，10℃环境下搅拌溶解均匀后得到水凝胶前体溶液；
3)将步骤2)中的水凝胶前体溶液进行灭菌，将30份2)中的水凝胶前体溶液、5份骨髓间充质干细胞细胞悬液与0.1份HAP于10℃下进行共混，混合均匀得到可注射复合壳聚糖水凝胶溶液；
4)使用注射器或移液枪缓慢吸取一定量步骤3)的可注射复合壳聚糖水凝胶溶液，将其缓慢注射到受损的骨组织缺陷部位，使溶液填满缺损部位，静待一定时间后，溶液即可完全凝胶化。
实施例2：
1)称取1.5g粘均分子量为100W、脱乙酰度为90％的壳聚糖粉末、1.75gN-异丙基丙烯酰胺、0.175g过硫酸铵，分散在95mL去离子水中，然后，加入0.5mL的盐酸，搅拌溶解均匀后，加热至60℃反应8小时，随后将溶液转移入透析袋中，用去离子水反复透析，冷冻干燥得到聚N-异丙基丙烯酰胺接枝改性的壳聚糖粉末；
2)将1-20份步骤1)中的聚N-异丙基丙烯酰胺接枝改性的壳聚糖粉末加入80-99份PBS溶液中，10℃环境下搅拌溶解均匀后得到水凝胶前体溶液；
3)将步骤2)中的水凝胶前体溶液进行灭菌，将20份2)中的水凝胶前体溶液、5份骨髓间充质干细胞细胞悬液与0.2份多肽分子与10℃环境下进行共混，混合均匀得到可注射复合壳聚糖水凝胶溶液；
4)使用注射器或移液枪缓慢吸取一定量步骤3)的可注射复合壳聚糖水凝胶溶液，将其缓慢注射到受损的骨组织缺陷部位，使溶液填满缺损部位，静待一定时间后，溶液即可完全凝胶化。
实施例3：
1)称取1.5g粘均分子量为100W、脱乙酰度为90％的壳聚糖粉末、1.75gN-异丙基丙烯酰胺、0.175g过硫酸铵，分散在95mL去离子水中，然后，加入0.5mL的盐酸，搅拌溶解均匀后，加热至60℃反应8小时，随后将溶液转移入透析袋中，用去离子水反复透析，冷冻干燥得到聚N-异丙基丙烯酰胺接枝改性的壳聚糖粉末；
2)将1-20份步骤1)中的聚N-异丙基丙烯酰胺接枝改性的壳聚糖粉末加入80-99份PBS溶液中，10℃环境下搅拌溶解均匀后得到水凝胶前体溶液；
3)将步骤2)中的水凝胶前体溶液进行灭菌，将50份2)中的水凝胶前体溶液、5份成骨细胞细胞悬液与1份HAP于10℃环境下进行共混，混合均匀得到可注射复合壳聚糖水凝胶溶液；
4)使用注射器或移液枪缓慢吸取一定量步骤3)的可注射复合壳聚糖水凝胶溶液，将其缓慢注射到受损的骨组织缺陷部位，使溶液填满缺损部位，静待一定时间后，溶液即可完全凝胶化。

Claims (10)

1.一种用于骨修复的可注射复合壳聚糖水凝胶，其特征在于，该水凝胶主要由改性壳聚糖、内载细胞以及促细胞生长因子组成，所述改性壳聚糖为聚N-异丙基丙烯酰胺接枝改性的壳聚糖，所述水凝胶的前体的制备方法具体包括如下步骤：
1)聚N-异丙基丙烯酰胺接枝改性的壳聚糖生物大分子的制备：按照质量计，将1-15份壳聚糖、0.1-5份酸、0.01-10份引发剂、1-20份N-异丙基丙烯酰胺、50-100份溶剂加入烧杯中，搅拌溶解均匀后，加热至40-80℃反应1-12小时，随后将溶液转移入透析袋中，用去离子水反复透析，冷冻干燥得到聚N-异丙基丙烯酰胺接枝改性的壳聚糖粉末；
2)聚N-异丙基丙烯酰胺接枝改性的壳聚糖水溶液的制备：将1-20份1)中的聚N-异丙基丙烯酰胺接枝改性的壳聚糖粉末加入80-99份PBS溶液中，低温环境下搅拌溶解均匀后得到水凝胶前体溶液。
2.根据权利要求1所述的用于骨修复的可注射复合壳聚糖水凝胶，其特征在于，步骤1)中所述壳聚糖的分子量为3万-200万，脱乙酰度为65％-99％。
3.根据权利要求1所述的用于骨修复的可注射复合壳聚糖水凝胶，其特征在于，步骤1)中所述酸为盐酸、醋酸、磷酸、柠檬酸、酒石酸、丙烯酸中的一种或几种。
4.根据权利要求1所述的用于骨修复的可注射复合壳聚糖水凝胶，其特征在于，步骤1)中所述引发剂为偶氮二异丁腈、偶氮二异庚腈、偶氮二异丁酸二甲酯、过氧化氢、过硫酸铵、过硫酸钾、叔丁基过氧化氢中的一种或几种。
5.根据权利要求1所述的用于骨修复的可注射复合壳聚糖水凝胶，其特征在于，步骤1)中所述溶剂为去离子水、乙醇、DMSO中的一种或几种。
6.根据权利要求1所述的用于骨修复的可注射复合壳聚糖水凝胶，其特征在于，步骤2)中所述低温环境为4℃-25℃。
7.一种如权利要求1所述的用于骨修复的可注射复合壳聚糖水凝胶的使用方法，其特征在于，具体包括如下步骤：
(1)可注射复合壳聚糖水凝胶溶液的制备：将权利要求1所述步骤2)中的水凝胶前体溶液进行灭菌，将10-50份灭菌后的水凝胶前体溶液、5份内载细胞的细胞悬液与0.1-5份促细胞生长因子4℃-25℃下进行共混，混合均匀得到可注射复合壳聚糖水凝胶溶液；
(2)使用注射器或移液枪吸取步骤(1)中的可注射壳聚糖水凝胶溶液，将其注射到受损的骨组织创伤部位，使溶液填满缺损部位，静待溶液凝胶化即可。
8.根据权利要求7所述的一种用于骨修复的可注射复合壳聚糖水凝胶的使用方法，其特征在于，步骤(1)中所述内载细胞为胚胎干细胞、间充质干细胞、脂肪干细胞、成骨细胞、软骨细胞中的一种或几种。
9.根据权利要求7所述的一种用于骨修复的可注射复合壳聚糖水凝胶的使用方法，其特征在于，所述细胞悬液中内载细胞的浓度为2万-200万/ml，细胞培养基为完全培养基、干细胞生长培养基或成骨分化培养基。
10.根据权利要求7所述的一种用于骨修复的可注射复合壳聚糖水凝胶的使用方法，其特征在于，所述受损的骨组织为硬骨或软骨，其中所述硬骨为长骨或扁骨，；所述软骨为关节软骨；用于硬骨组织损伤修复时步骤(1)中所述促细胞生长因子为羟基磷灰石HAP、胶原、骨形成蛋白BMP、RGD分子中的一种或几种；用于软骨组织损伤修复时所述促细胞生长因子为脱钙骨基质DBM、2-([1,1-联苯]-4-基氨基甲酰)苯甲酸KGN、胶原蛋白、鹿茸多肽PAP、胰岛素样生长因子IGF、转化生长因子TGF、基质细胞衍生因子SDF、骨形成蛋白BMP、RGD分子中的一种或几种。
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