CN117582548A - 一种具有原位修复前交叉韧带功能的可注射多交联复合水凝胶支架及其制备方法 - Google Patents
一种具有原位修复前交叉韧带功能的可注射多交联复合水凝胶支架及其制备方法 Download PDFInfo
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- CN117582548A CN117582548A CN202311701874.5A CN202311701874A CN117582548A CN 117582548 A CN117582548 A CN 117582548A CN 202311701874 A CN202311701874 A CN 202311701874A CN 117582548 A CN117582548 A CN 117582548A
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- chondroitin sulfate
- solution
- anterior cruciate
- cruciate ligament
- hydrogel
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Abstract
本发明公开一种具有原位修复前交叉韧带功能的可注射多交联复合水凝胶支架及其制备方法。利用多巴胺接枝的氧化硫酸软骨素还原镁离子获得复合溶液,并利用席夫碱反应和金属螯合作用与聚赖氨酸和氯化铁共混合后,经多层交联反应快速形成稳定的水凝胶;在水凝胶中混入干细胞、PRP、生长因子、氨甲环酸等生物活性物质,促进前交叉韧带的修复。本发明制得的水凝胶支架可原位成胶,适用于不规则的前交叉韧带创面修复并可一体化修复韧带与骨;同时,该水凝胶支架具备良好抗菌性和湿粘附性;此外,多交联的网络使其降解性能和力学性能适用于前交叉韧带的复杂生长环境及修复周期长等问题,从而有效提高前交叉韧带修复效果。
Description
技术领域
本发明属于生物医学领域,具体涉及一种具有原位修复前交叉韧带功能的可注射多交联复合水凝胶支架及其制备方法。
背景技术
全世界每年前交叉韧带损伤的人数大约有200万人。随着人们对运动需求的增多,前交叉韧带损伤数量在逐年增加。目前临床治疗方法主要包括前交叉韧带重建与前交叉韧带修复。其中,利用肌腱移植或人工韧带替代物的前交叉韧带重建方法,通常伴随着30%的韧带再断裂、术后骨髓道的难以愈合以及骨关节炎严重等问题。
前交叉韧带修复是采用缝线原位缝合的方法,侵入性小且保留了天然前交叉韧带及其本体感受器,有助于反馈膝关节的位置和动态稳定性,有望成为一种更有优势和前途的治疗手段。但面对近端撕裂和不规则创面,缝线缝合难度急剧增加。同时,关节腔中大量的滑液和酶会侵蚀韧带断面不利于韧带愈合。因此,仅靠缝合手术难以有效修复韧带。此外,缝合过程中构建的骨隧道会引起骨损伤,对机体造成破坏,不利于功能恢复。
前交叉韧带与骨骼的天然界面是一种称为“附着点”的特殊组织,包含四种不同类型的组织:韧带、未钙化纤维软骨、钙化纤维软骨和骨。据统计,骨隧道愈合不足是前交叉韧带临床治疗结果不理想的主要原因之一。研究表明镁离子(Mg2+)可以调控新生骨中矿物质的结晶和形成,参与骨衍生干细胞成骨分化的相关细胞因子的表达进而促进骨的修复,同时,镁离子还可以促进血管内皮生成因子表达量升高,促进局部血液灌注。但以往人们只单一的使用镁离子促进骨的愈合,或者单一注重韧带愈合而忽略骨的修复。因此前交叉韧带的修复不仅仅是单一韧带的愈合更需要促进骨的愈合。
有鉴于此,本发明设计了一款能够同时修复韧带与骨的一体化的水凝胶支架,其原位成胶性能可适应各种不规则的前交叉韧带损伤创面;负载的镁离子能够促进骨的愈合;水凝胶支架保护韧带抵御关节腔内滑液和酶的侵袭;复合的生物活性物质进一步增强细胞迁移粘附和血管再生。总之,本发明的水凝胶支架能够促进韧带组织和骨界面再生,最大限度的恢复患者的原本的肢体功能,从而提高的韧带修复的临床疗效,具有重大临床需求。
发明内容
本发明的目的在于提供一种具有原位修复前交叉韧带功能的可注射多交联复合水凝胶支架及其制备方法。所制得的水凝胶支架通过席夫碱反应、金属螯合作用、静电作用、自聚作用等形成多网络结构,具有自愈合性,有效提高水凝胶力学性能;所制得的水凝胶支架具有良好的湿粘附性能;同时,加入的镁离子可以促进一体化韧带与骨的再生与修复;所制得的水凝胶支架还具有良好的抗降解能力和一定的抗菌性能,并且可进行原位成胶,适用于不规则伤口;此外,水凝胶支架修复前交叉韧带的速度快且能够降低骨关节炎。
为实现上述目的,本发明采用如下技术方案:
一种具有原位修复前交叉韧带功能的可注射多交联复合水凝胶支架的制备方法,其包括以下步骤:
(1)将硫酸软骨素用高碘酸钠氧化,得到氧化硫酸软骨素;在氮气保护下,利用1-乙基-3-(3-二甲氨丙基)碳二亚胺盐酸盐和N-羟基琥珀酰亚胺将多巴胺接枝到氧化硫酸软骨素上,得到多巴胺接枝氧化硫酸软骨素;将氯化镁溶液与多巴胺接枝氧化硫酸软骨素溶液混合,25℃反应1~5h,冷冻干燥,得到多巴胺接枝氧化硫酸软骨素-镁;
(2)将ε-聚赖氨酸溶液与多巴胺接枝氧化硫酸软骨素-镁溶液、氯化铁溶液混合,加入生物活性物质,调节pH值为8.0,静置成胶,得到具有原位修复前交叉韧带功能的可注射多交联复合水凝胶支架。
所述步骤(1)中,硫酸软骨素与高碘酸钠的质量比为1:0.5~1。
所述步骤(1)中,多巴胺与氧化硫酸软骨素的质量比为1:0.5~1。
所述步骤(1)中,氯化镁溶液与多巴胺接枝氧化硫酸软骨素溶液的体积比为0.01~0.05:1,氯化镁溶液的浓度为20wt%~30wt%,多巴胺接枝氧化硫酸软骨素溶液的浓度为1wt%~3wt%。
所述步骤(2)中,ε-聚赖氨酸溶液与多巴胺接枝氧化硫酸软骨素-镁溶液、氯化铁溶液的体积比为1~1.5:1~1.5:0.1~0.5,ε-聚赖氨酸溶液的浓度为5wt%~30wt%,多巴胺接枝氧化硫酸软骨素-镁溶液的浓度为5wt%~20wt%,氯化铁溶液的浓度为1wt%~10wt%。
所述步骤(2)中,静置成胶的温度为25~30℃、时间为10~40s。
所述步骤(2)中,生物活性物质选自表皮生长因子、成纤维细胞生长因子、生长分化因子、胰岛素样生长因子、血小板衍生生长因子、或转化生长因子-β、力学生长因子E肽、骨髓间充质干细胞、脂肪间充质干细胞、神经干细胞、氨甲环酸、塞来昔布、氨基葡萄糖、抗生素类药物中的任意一种或多种。
所诉生长因子的溶液浓度在0.001~1mg/L之间,干细胞溶液的浓度在50~200w/mL,富血小板血浆的血小板富集浓度在4~8倍之间,抗生素类药物的溶液的浓度0.5~1mmol/L。
一种由上述制备方法制得的可注射多交联复合水凝胶支架。
上述一种可注射多交联复合水凝胶在制备前交叉韧带修复用制品中的应用。
本发明的显著优点在于:
(1)本发明选用硫酸软骨素制备水凝胶支架,硫酸软骨素能够减少骨关节炎患者疼痛,改善关节功能、减少关节肿胀;
(2)本发明所选用材料ε-聚赖氨酸能够去除湿组织上的水合阳离子,促进儿茶酚基团与组织之间通过氢键、π-π相互作用、静电吸引紧密结合,提供优异湿粘附力,并且有一定的抗菌性能;
(3)本发明形成的水凝胶支架具有多网络结构,提升了水凝胶的机械性能,并且有自愈合能力,降解缓慢;
(4)水凝胶中释放的Mg2+可以促进降钙素基因相关多肽-α(CGRP)介导的成骨分化,并且可促进骨髓干细胞(BMSCs)的成骨分化,从而促进OCN、COL和ALP基因表达,Mg2+还具有促进矿化骨基质形成的积极作用,可以一体化的促进韧带修复;
(5)本发明的水凝胶可原位注射成胶用来针对不同程度的前交叉韧带撕裂类型,具有方便手术的操作、针对不同人群和不同前交叉韧带断裂程度的优点,进而有效的促进伤口的愈合;
(6)本发明的水凝胶支架保护韧带抵御关节腔内滑液和酶的侵袭,复合的生物活性物质进一步增强细胞迁移粘附和血管再生,促进韧带组织再生,最大限度的恢复患者的原本的肢体功能,从而提高的韧带修复的临床疗效,具有非常大的临床需求。
附图说明
图1为损伤的前交叉韧带示意图(左图部分损伤、右图完全断裂损伤)。
图2为前交叉韧带部分损伤注射图。
图3为前交叉韧带部分损伤注射图(放大图)。
图4为前交叉韧带完全损伤注射图。
图5为前交叉韧带完全损伤注射图(放大图)。
图6为实施例1所制得的可注射水凝胶支架产品图。
图7实施例3所制得的可注射水凝胶支架72h 3T3死活荧光图。
图8为实施例3所制得的可注射水凝胶支架对兔子前交叉韧带4周的修复图。
图9为实施例3所制得的可注射水凝胶支架对兔子膝盖12周的核磁图。
具体实施方式
为了使本发明所述的内容更加便于理解,下面结合具体实施方式对本发明所述的技术方案做进一步的说明,但是本发明不仅限于此。
下述实施例中,所述硫酸软骨素平均分子量为20~30kDa,购自阿拉丁试剂(上海)有限公司,货号C832332。
下述实施例中,所述多巴胺购自上海麦克林生化科技股份有限公司,货号A902400。
下述实施例中,所述ε-聚赖氨酸平均分子量为70-150kDa,购自上海源叶生物科技有限公司,货号S20058。
下述实施例中,所述氨基葡萄糖为D-氨基葡萄糖酸,购自上海麦克林生化科技股份有限公司,货号D838500。
实施例1
一种具有原位修复前交叉韧带功能的可注射水凝胶支架的制备方法,包括以下步骤:
S1:制备氧化硫酸软骨素、多巴胺接枝氧化硫酸软骨素-镁:
S1-1:制备氧化硫酸软骨素:
将硫酸软骨素溶解于去离子水中配置为浓度为2wt%的溶液,将高碘酸钠溶解于去离子水中配置为浓度为18wt%的溶液;在搅拌状态下,向100mL 2wt%的硫酸软骨素溶液中滴加10mL 18wt%的高碘酸钠溶液,25℃避光反应2小时,然后加入2mL乙二醇搅拌1小时以终止反应;所得到的溶液倒入截留分子量为3500MW的透析袋中,在室温条件下用去离子水透析3天,每隔8小时换一次水;将所截留透析袋内的溶液全部倒入聚四氟乙烯板中,于-80℃预冻12小时,再在冷冻温度为-55℃、真空度为0.01Pa的条件下冷冻干燥48小时,得到氧化硫酸软骨素;
S1-2:制备多巴胺接枝氧化硫酸软骨素-镁:
将1.0g氧化硫酸软骨素溶解于100mL蒸馏水中,加入0.96g 1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(EDC)和0.288g N-羟基硫代琥珀酰亚胺(NHS),用HCl将体系的pH调节至4.8,室温搅拌1小时后加入1.0g多巴胺,在室温、氮气保护环境下搅拌过夜;所得到的溶液倒入截留分子量为3000MW的透析袋中,在室温条件下用去离子水透析3天,每隔8小时换一次水;将所截留透析袋内的溶液全部倒入聚四氟乙烯板中,于-80℃预冻12小时,再在冷冻温度为-55℃、真空度为0.01Pa的条件下冷冻干燥48小时,得到多巴胺接枝氧化硫酸软骨素;
将多巴胺接枝氧化硫酸软骨素溶解于去离子水中配置为浓度为2wt%的溶液,将氯化镁溶解于去离子水中配置为浓度为20wt%的溶液;将100μL 20wt%的氯化镁溶液加入到10mL2wt%的多巴胺接枝氧化硫酸软骨素溶液中,25℃反应3小时;反应结束后于-80℃预冻12小时,再在冷冻温度为-55℃、真空度为0.01Pa的条件下冷冻干燥48小时,得到多巴胺接枝氧化硫酸软骨素-镁。
S2:多网络交联反应:
将ε-聚赖氨酸溶解于去离子水中配置为浓度为20wt%的溶液,将多巴胺接枝氧化硫酸软骨素-镁溶解于去离子水中配置为浓度为15wt%的溶液,将氯化铁溶解于去离子水中配置为浓度为10wt%的溶液;将10mL 20wt%的ε-聚赖氨酸溶液与10mL 15wt%的多巴胺接枝氧化硫酸软骨素-镁溶液快速混合,在搅拌状态下滴加3mL 10wt%的氯化铁溶液,用NaOH调节pH值为8.0,得到水凝胶前驱体溶液,随后25℃静置一段时间成胶,即得到具有原位修复前交叉韧带功能的可注射水凝胶支架。
实施例2
一种具有原位修复前交叉韧带功能的可注射水凝胶支架的制备方法,包括以下步骤:
S1:制备氧化硫酸软骨素、多巴胺接枝氧化硫酸软骨素-镁:
与实施例1步骤S1相同。
S2:多网络交联反应:
将ε-聚赖氨酸溶解于去离子水中配置为浓度为20wt%的溶液,将多巴胺接枝氧化硫酸软骨素-镁溶解于去离子水中配置为浓度为15wt%的溶液,将氯化铁溶解于去离子水中配置为浓度为10wt%的溶液;将10mL 20wt%的ε-聚赖氨酸溶液与10mL 15wt%的多巴胺接枝氧化硫酸软骨素-镁溶液快速混合,在搅拌状态下滴加4mL 10wt%的氯化铁溶液,用NaOH调节pH值为8.0,得到水凝胶前驱体溶液,随后25℃静置一段时间成胶,即得到具有原位修复前交叉韧带功能的可注射水凝胶支架。
实施例3
一种具有原位修复前交叉韧带功能的可注射水凝胶支架的制备方法,包括以下步骤:
S1:制备氧化硫酸软骨素、多巴胺接枝氧化硫酸软骨素-镁:
与实施例1步骤S1相同。
S2:多网络交联反应:
将ε-聚赖氨酸溶解于去离子水中配置为浓度为20wt%的溶液,将多巴胺接枝氧化硫酸软骨素-镁溶解于去离子水中配置为浓度为15wt%的溶液,将氯化铁溶解于去离子水中配置为浓度为10wt%的溶液;将15mL 20wt%的ε-聚赖氨酸溶液与10mL 15wt%的多巴胺接枝氧化硫酸软骨素-镁溶液快速混合,在搅拌状态下滴加4mL 10wt%的氯化铁溶液,用NaOH调节pH值为8.0,得到水凝胶前驱体溶液,随后25℃静置一段时间成胶,即得到具有原位修复前交叉韧带功能的可注射水凝胶支架。
实施例4
一种具有原位修复前交叉韧带功能的可注射水凝胶支架的制备方法,包括以下步骤:
S1:制备氧化硫酸软骨素、多巴胺接枝氧化硫酸软骨素-镁:
与实施例1步骤S1相同。
S2:多网络交联反应:
将ε-聚赖氨酸溶解于去离子水中配置为浓度为20wt%的溶液,将多巴胺接枝氧化硫酸软骨素-镁溶解于去离子水中配置为浓度为15wt%的溶液,将氯化铁溶解于去离子水中配置为浓度为10wt%的溶液;将10mL 20wt%的ε-聚赖氨酸溶液与15mL 15wt%的多巴胺接枝氧化硫酸软骨素-镁溶液快速混合,在搅拌状态下滴加4mL 10wt%的氯化铁溶液和5mL富血小板血浆,用NaOH调节pH值为8.0,得到水凝胶前驱体溶液,随后25℃静置一段时间成胶,即得到具有原位修复前交叉韧带功能的可注射水凝胶支架。
其中,所述富血小板血浆的制备方法为:采集5mL新鲜兔全血,进行第一次离心(4℃,350g,10min),离心后的血液应分为三层,其中:最上层为血浆层、体积占离心全血体积的40%、内含大量血小板,中间层为较薄的一层白膜层、含大量白细胞和血小板及少量红细胞,最下层为红细胞层,含大量红细胞;将最上层和大部分的中间层抽出并混合均匀,进行第二次离心(4℃,2000g,10min),离心后形成血小板沉淀及血浆上清液,弃去多余的血浆上清液,保留500μL的血浆上清液重悬血小板沉淀,吹打混匀,即制备得到富血小板血浆。经计算,制得的富血小板血浆的血小板富集倍数为5倍。
实施例5
一种具有原位修复前交叉韧带功能的可注射水凝胶支架的制备方法,包括以下步骤:
S1:制备氧化硫酸软骨素、多巴胺接枝氧化硫酸软骨素-镁:
与实施例1步骤S1相同。
S2:多网络交联反应:
将ε-聚赖氨酸溶解于去离子水中配置为浓度为20wt%的溶液,将多巴胺接枝氧化硫酸软骨素-镁溶解于去离子水中配置为浓度为15wt%的溶液,将氯化铁溶解于去离子水中配置为浓度为10wt%的溶液;将10mL 20wt%的ε-聚赖氨酸溶液与10mL 15wt%的多巴胺接枝氧化硫酸软骨素-镁溶液快速混合,在搅拌状态下滴加5mL 10wt%的氯化铁溶液和2mL浓度为0.01mg/L的表皮生长因子(EGF),用NaOH调节pH值为8.0,得到水凝胶前驱体溶液,随后25℃静置一段时间成胶,即得到具有原位修复前交叉韧带功能的可注射水凝胶支架。
实施例6
一种具有原位修复前交叉韧带功能的可注射水凝胶支架的制备方法,包括以下步骤:
S1:制备氧化硫酸软骨素、多巴胺接枝氧化硫酸软骨素-镁:
与实施例1步骤S1相同。
S2:多网络交联反应:
将ε-聚赖氨酸溶解于去离子水中配置为浓度为20wt%的溶液,将多巴胺接枝氧化硫酸软骨素-镁溶解于去离子水中配置为浓度为15wt%的溶液,将氯化铁溶解于去离子水中配置为浓度为10wt%的溶液;将10mL 20wt%的ε-聚赖氨酸溶液与10mL 15wt%的多巴胺接枝氧化硫酸软骨素-镁溶液快速混合,在搅拌状态下滴加4mL 10wt%的氯化铁溶液和2mL浓度为1mmol/L的氨基葡萄糖,用NaOH调节pH值为8.0,得到水凝胶前驱体溶液,随后25℃静置一段时间成胶,即得到具有原位修复前交叉韧带功能的可注射水凝胶支架。
对比例1
一种具有原位修复前交叉韧带功能的可注射水凝胶支架的制备方法,包括以下步骤:
S1:制备氧化硫酸软骨素、多巴胺接枝氧化硫酸软骨素-镁:
与实施例1步骤S1相同。
S2:交联反应:
将ε-聚赖氨酸溶解于去离子水中配置为浓度为20wt%的溶液,将多巴胺接枝氧化硫酸软骨素-镁溶解于去离子水中配置为浓度为15wt%的溶液;将10mL 20wt%的ε-聚赖氨酸溶液与10mL 15wt%的多巴胺接枝氧化硫酸软骨素-镁溶液快速混合,用NaOH调节pH值为8.0,得到水凝胶前驱体溶液,随后25℃静置一段时间成胶,即得到具有原位修复前交叉韧带功能的可注射水凝胶支架。
对比例2
一种具有原位修复前交叉韧带功能的可注射水凝胶支架的制备方法,包括以下步骤:
S1:制备氧化硫酸软骨素:
与实施例1步骤S1相同。
S2:交联反应:
将ε-聚赖氨酸溶解于去离子水中配置为浓度为10wt%的溶液,将氧化硫酸软骨素溶解于去离子水中配置为浓度为10wt%的溶液;将10mL 10wt%的ε-聚赖氨酸溶液与10mL10wt%的氧化硫酸软骨素溶液快速混合,用NaOH调节pH值为8.0,得到水凝胶前驱体溶液,随后25℃静置一段时间成胶,即得到具有原位修复前交叉韧带功能的可注射水凝胶支架。
用流变仪对所制得的原位修复前交叉韧带可注射水凝胶支架进行流变学测试,测试参数如下:在25℃下,通过流变仪(MCR 302,奥地利)使圆柱体(高5mm×直径10mm)在间隙距离为0.8mm下进行流变学研究,频率扫描是在1%应变和0.1至100rad/s的振荡频率下进行的。
对所制得的原位修复前交叉韧带可注射水凝胶支架进行降解能力测试,具体步骤为:将水凝胶支架浸泡在含有0.4U/ml胶原酶及0.4U/ml溶菌酶的PBS缓冲液中,在温度37℃转速120rpm恒温震荡培养,每天换液1次,30天后取出水凝胶支架冻干称重,并计算水凝胶支架的降解率。
采用万能测试机(LLOYD LR100K,CN)对所制得的原位修复前交叉韧带可注射水凝胶支架进行拉伸力学性能测试,两端夹具固定后,以15mm/min的速度进行拉伸,得到水凝胶支架的最大断裂载荷和拉伸位移,实验重复进行5次,其结果表示为(平均值±标准偏差)。
用Stable Micro Systems纹理分析仪(TA-XT plus,UK)对所制得的原位修复前交叉韧带可注射水凝胶支架进行压缩试验,转切质构仪的测量模式为压缩,以20mm/min的速度在80%的应变下进行测试。
通过猪皮肤搭接剪切试验对所制得的原位修复前交叉韧带可注射水凝胶支架进行粘附强度测试。将一块新鲜的猪皮经过去除脂肪和剃毛处理,之后将处理过的猪皮切成10毫米×30毫米的矩形,用水冲洗,将皮肤从水中取出并直接使用而不干燥,将水凝胶前驱体溶液注射在两张粘合面积为10mm×15mm的猪皮之间,然后夹紧粘合剂区域并在室温下放置2小时,使用通用材料试验机(英斯特朗5567)以10mm/min的速度进行拉伸测试,粘合强度(Pa=N/m2)的计算方法是将最大力(N)除以粘合面积(m2)。
对所制得的原位修复前交叉韧带可注射水凝胶支架进行爆破压力测试。将新鲜的猪皮切成宽20毫米×长50毫米的矩形,并粘附在带有氰基丙烯酸酯胶的塑料软管上,之后使用针在猪皮和软管上制作直径为3mm的圆形凹口,然后将水凝胶前驱体溶液(300μL)注入缺口区域并放置2小时,软管的一端连接到注射泵,另一端连接到数字压力表,随后将PBS填充到注射器中并以150mL/h的速度泵送,记录多网络水凝胶的爆破压力。
对所制得的原位修复前交叉韧带可注射水凝胶支架进行抗菌测试,具体为:将活化好的107cfu细菌培养液(E.coil和MRSA)与水凝胶支架共培养12h,然后取细菌培养液在酶标仪OD600nm测定吸光值和涂布平板,计算出杀菌率。
对所制得的原位修复前交叉韧带可注射水凝胶支架进行生物相容性测试:(1)细胞死活染色:制备直径为10mm的圆柱体水凝胶支架待用,实验前将水凝胶支架浸泡在PBS缓冲液中并于紫外下照射24h,而后将水凝胶放入24孔板中,并将50μL密度为4×105cell/mL的3T3细胞悬浮液接种于水凝胶支架上,共培养72h后使用活/死细胞染色液对3T3细胞进行染色,在荧光显微镜下观察细胞的生长情况(图7)。(2)细胞毒性实验:吸出孔板中培养72h后的培养液和水凝胶支架,每孔加入200μL 0.05mg/mL的MTT溶液,于含5%CO2的37℃恒温培养箱中培养4h。吸出MTT溶液,加入200μLDMSO,在37℃恒温震荡培养箱孵育15min,分别分装入96孔板中,使用酶标仪于490nm波长下测试吸光值,计算出细胞相对增殖率。
由表1试验数据可知,相较于对比例1~2,实施例1~6制得的原位修复前交叉韧带可注射水凝胶支架具有良好的湿粘附性能,更优异的力学性能和生物相容性,降解缓慢,因此在韧带的长期愈合中更易保持较完整的形态,以防止滑液对前交叉韧带残端的侵蚀;同时,实施例1~6制得的原位修复前交叉韧带可注射水凝胶支架有一定的抗菌性能,通过复合生物活性物质可以进一步促进细胞的增殖,对前交叉韧带的愈合起促进效果。
表1原位修复前交叉韧带可注射水凝胶支架的性能测试结果
将实施例1~6以及对比例1~2制得的原位修复前交叉韧带可注射水凝胶支架进行动物实验研究。将体重为2.5kg~3kg的新西兰雄性白兔右膝关节的前交叉韧带分别部分断裂和完全断裂,(实施例1所用到的支架用于部分断裂模型为实施例1-1,用于完全断裂模型为实施例1-2;实施例2所用到的支架用于部分断裂模型为实施例2-1,用于完全断裂模型为实施例2-2;实施例3所用到的支架用于部分断裂模型为实施例3-1,用于完全断裂模型为实施例3-2;实施例4所用到的支架用于部分断裂模型为实施例4-1,用于完全断裂模型为实施例4-2;实施例5所用到的支架用于部分断裂模型为实施例5-1,用于完全断裂模型为实施例5-2;实施例6所用到的支架用于部分断裂模型为实施例6-1,用于完全断裂模型为实施例6-2;对比例1所用到的支架用于部分断裂模型为对比例1-1,用于完全断裂模型为对比例1-2;对比例2所用到的支架用于部分断裂模型为对比例2-1,用于完全断裂模型为对比例2-2)通过手术将断端缝合后,在缝合处注入水凝胶前驱体溶液并静置成胶,并设置部分断裂仅缝合韧带无支架的对照组1-1和完全断裂仅缝合韧带无支架的对照组1-2,以及部分断裂韧带未处理的对照组2-1和完全断裂韧带未处理的对照组2-2,处理好伤口后,用支具固定兔右膝关节,两周后拆除。在支具固定4周、8周、12周后观察韧带修复状况以及通过病理染色观察韧带及周围软骨组织,并用Markin进行评分,评分标准如表2。韧带修复结果如表3-1和表3-2所示。
表2评分标准
评分指标 | 分值 |
Ⅰ软骨结构 | |
正常 | 0 |
表面不规整 | 1 |
血管翳的形成和表面不规整 | 2 |
裂隙进入过渡层 | 3 |
裂隙进入辐射层 | 4 |
裂隙进入钙化层 | 5 |
结构完全破坏 | 6 |
Ⅱ软骨细胞 | |
正常 | 0 |
弥漫性细胞增加 | 1 |
局部细胞增加 | 2 |
细胞数目明显减少 | 3 |
Ⅲ软骨基质染色(番红O) | |
正常 | 0 |
轻微减少 | 1 |
中度减少 | 2 |
重度减少 | 3 |
未着色 | 4 |
Ⅳ潮线完整性 | |
完整 | 0 |
被破坏 | 1 |
由表3-1和表3-2试验数据可知,本发明实施例1~6的原位修复前交叉韧带可注射水凝胶支架更能够促进前交叉韧带的快速修复,并促进新生血管的生成,加速前交叉韧带修复速度与质量并且减少骨关节炎症的产生。
表3-1部分断裂模型测试数据
表3-2完全断裂模型测试数据
此外,取实施例3第四周完全损伤模型韧带观测(图8),前交叉韧带处可看见明显的新生韧带样组织,韧带连接情况良好,表明支架能够有效促进损伤韧带的修复。同时,取实施例3第12周完全损伤模型韧带模型膝盖进行核磁观测(图9),结果发现膝盖位置为连续性存在的低信号形态,关节积液较少,修复后的前交叉韧带形态较为完整,表明支架均可以促进前交叉韧带的修复,并减少关节内炎症的产生,从而有利于运动功能恢复。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (9)
1.一种具有原位修复前交叉韧带功能的可注射多交联复合水凝胶支架的制备方法,其特征在于:包括以下步骤:
(1)将硫酸软骨素用高碘酸钠氧化,得到氧化硫酸软骨素;在氮气保护下,利用1-乙基-3-(3-二甲氨丙基)碳二亚胺盐酸盐和N-羟基琥珀酰亚胺将多巴胺接枝到氧化硫酸软骨素上,得到多巴胺接枝氧化硫酸软骨素;将氯化镁溶液与多巴胺接枝氧化硫酸软骨素溶液混合,25℃反应1~5h,冷冻干燥,得到多巴胺接枝氧化硫酸软骨素-镁;
(2)将ε-聚赖氨酸溶液与多巴胺接枝氧化硫酸软骨素-镁溶液、氯化铁溶液混合,加入生物活性物质,调节pH值为8.0,静置成胶,得到具有原位修复前交叉韧带功能的可注射多交联复合水凝胶支架。
2.根据权利要求1所述的制备方法,其特征在于:所述步骤(1)中,硫酸软骨素与高碘酸钠的质量比为1:0.5~1。
3.根据权利要求1所述的制备方法,其特征在于:所述步骤(1)中,多巴胺与氧化硫酸软骨素的质量比为1:0.5~1 。
4.根据权利要求1所述的制备方法,其特征在于:所述步骤(1)中,氯化镁溶液与多巴胺接枝氧化硫酸软骨素溶液的体积比为0.01~0.05:1,氯化镁溶液的质量浓度为20%~30%,多巴胺接枝氧化硫酸软骨素溶液的质量浓度为1%~3%。
5.根据权利要求1所述的制备方法,其特征在于:所述步骤(2)中,ε-聚赖氨酸溶液与多巴胺接枝氧化硫酸软骨素-镁溶液、氯化铁溶液的体积比为1~1.5:1~1.5:0.1~0.5,ε-聚赖氨酸溶液的质量浓度为5%~30%,多巴胺接枝氧化硫酸软骨素-镁溶液的质量浓度为5%~20%,氯化铁溶液的质量浓度为1%~10%。
6.根据权利要求1所述的制备方法,其特征在于:所述步骤(2)中,静置成胶的温度为25~30℃、时间为10~40s。
7.根据权利要求1所述的制备方法,其特征在于:所述步骤(2)中,生物活性物质选自表皮生长因子、成纤维细胞生长因子、生长分化因子、胰岛素样生长因子、血小板衍生生长因子、或转化生长因子-β、力学生长因子E肽、骨髓间充质干细胞、脂肪间充质干细胞、神经干细胞、氨甲环酸、塞来昔布、氨基葡萄糖、抗生素类药物中的任意一种或多种。
8.一种由权利要求1所述制备方法制得的可注射多交联复合水凝胶支架。
9.如权利要求8所述的可注射多交联复合水凝胶在制备前交叉韧带修复用制品中的应用。
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