CN107412861A - 重组胶原蛋白复合硫酸软骨素和聚乙二醇的骨修复凝胶 - Google Patents
重组胶原蛋白复合硫酸软骨素和聚乙二醇的骨修复凝胶 Download PDFInfo
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Abstract
本发明公开了重组胶原蛋白复合硫酸软骨素和聚乙二醇的骨修复凝胶,主要通过复合重组胶原蛋白、硫酸软骨素、以及聚乙二醇三种材料,利用三者之间的化学反应交联形成,相比较从动物中提取的天然胶原蛋白,重组胶原蛋白具有低抗原性,彻底杜绝了动物源材料所不可避免的病毒隐患,保证了凝胶材料在临床使用中的安全性,本发明所提供的凝胶的制备过程中没有任何有毒物质的引入,所用的原料均无生物学毒性,并且所加的硫酸软骨素具有止痛,促进软骨再生的功效。本发明制备的复合水凝胶具备良好的生物相容性和可降解性,并对骨缺损起到良好的修复作用,在生物材料领域具有极大的潜力和应用价值。
Description
技术领域
本发明涉及蛋白质工程,医用生物材料制备技术领域,具体为重组胶原蛋白复合硫酸软骨素和聚乙二醇的骨修复凝胶。
背景技术
骨缺损是临床常见病,也是骨科治疗中的一个技术难题。目前,治疗骨缺损的方法主要包括骨移植、组织工程技术和基因治疗法及生长因子、物理治疗法的辅助治疗等,但仍没有彻底的骨缺损修复方法。因此寻找一种理想的骨缺损修复材料具有重要意义。
水凝胶具有良好的生物相容性及生物可降解性,具备类细胞外基质的仿生特性及三维水化网状结构,有利于细胞的迁移和生长,因此是移植细胞或缓释生长因子的适宜载体。因此将水凝胶体系作为一种组织工程骨缺损修复材料具有重要应用前景。
胶原蛋白主要分布在哺乳动物的结缔组织中,对动物和人体皮肤、血管、骨骼、筋腱、牙齿和软骨的形成都十分重要,是这些结缔组织的主要物质基础。畜禽源动物组织是人们获取天然胶原蛋白及其胶原肽的主要途径,但由于相关畜类疾病和某些宗教信仰使得人们对陆生哺乳动物胶原蛋白的获取受到限制。重组胶原蛋白是通过生物基因工程技术在大肠杆菌体系中培养、分离和纯化的一种蛋白。和天然动物胶原蛋白一样,重组胶原蛋白同样有着典型的三股螺旋结构以及(Gly-Xaa-Yaa)n重复序列、良好的生物相容性、生物降解性。自动物体提取的胶原蛋白,存在水溶性不佳,可加工性弱,质量不稳定等缺陷。而且,动物来源的胶原蛋白难以排除疯牛病,口蹄疫等病毒风险。重组胶原蛋白没有这些问题。
发明内容
本发明的目的在于提供重组胶原蛋白复合硫酸软骨素和聚乙二醇的骨修复凝胶,以解决上述背景技术中提出的问题。
为实现上述目的,本发明提供如下技术方案:重组胶原蛋白复合硫酸软骨素和聚乙二醇的骨修复凝胶,主要包括重组胶原蛋白、硫酸软骨素以及聚乙二醇三种材料。
优选的,其制备方法包括以下步骤:
A、利用生物基因工程技术制备重组胶原蛋白,包括以下步骤:
a、合成编码重组胶原蛋白的核酸,构建导入核酸的质粒,将质粒转化大肠杆菌BL21-DE3菌株;
b、在大肠杆菌BL21-DE3菌株中表达重组胶原蛋白,主要包括以下步骤:
(1)将少量菌液加入50ml含氨苄西林钠100μg/ml的LB培养基中,置于37℃摇床过夜培养;
(2)然后将培养基转移至1L含氨苄西林钠100μg/ml的LB培养基中,在37℃环境下继续扩大培养;
(3)紫外分光光度计测量菌液在波长600nm处的OD值,当菌液的OD值在0.6~0.8时,加入1mM IPTG同时降低摇床温度至20℃-25℃继续培养8h-10h诱导蛋白表达,将菌液离心,收集细胞沉淀;
c、重组胶原蛋白的纯化,主要包括以下步骤:
(1)将离心后的菌体用缓冲液A使其溶解,缓冲液A由20mM咪唑、20mM磷酸钠、0.5M氯化钠组成,其pH为7.4;
(2)将细菌悬浊液放入超声波细胞破碎仪中进行细胞破碎,即可释放出蛋白并且蛋白会溶于缓冲液A中,其中细胞破碎条件:用2s超声、2s间歇的条件破碎100分钟,超声时将细菌悬浊液放于冰浴中,将破碎完的悬浊液再次离心,使细胞碎片与蛋白溶液分离,其中离心条件:离心速率10000-15000r/min,离心温度2℃-6℃,离心时间20min-30min;
(3)收集上清液,此即为粗蛋白溶液,通过Ni-NTA琼脂糖亲和层析柱进行纯化,将上清蛋白液加入镍柱,用结合缓冲液洗6~8次后再用高浓度咪唑洗脱液,其中结合缓冲液由30mM咪唑、0.5M NaCl、20mM Na2HPO4,组成,其pH为7.4;高浓度咪唑洗脱液由500mM咪唑、0.5M NaCl、20mM Na2HPO4,组成,其pH为7.4,将蛋白洗脱,收集蛋白洗脱液后用甘氨酸透析液于4℃透析4次;
(4)紫外分光光度计测得蛋白液浓度,计算蛋白含量后用胰蛋白酶按一定质量比酶切蛋白得目标蛋白,蛋白液用20mM pH为7.4PBS透析,收集透析后的蛋白液冷冻干燥后得到蛋白CL冻干粉,冻干粉于-15℃--20℃保存;
B、制备ADH修饰硫酸软骨素,其制备方法包括以下步骤:将硫酸软骨素溶于蒸馏水中,加入1-3-二甲氨基丙基-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺,将pH调至5.5,然后加入己二酸二酰肼并调节pH调至6.0,搅拌反应24h,将产物在蒸馏水中透析3天,冷冻干燥后与室温保存;
C、将一定质量硫酸软骨素和重组胶原蛋白混合配置成溶液,配制活性酯聚乙二醇活性酯溶液;其中硫酸软骨素浓度为100mg/ml、重组胶原蛋白浓度为40mg/ml、活性酯聚乙二醇活性酯浓度为160mg/ml;
D、将步骤C中的两种溶液按体积比1:1混合搅拌均匀后静置于25℃形成凝胶。
与现有技术相比,本发明的有益效果是:本发明所提供的复合重组胶原蛋白、硫酸软骨素、以及聚乙二醇的骨修复凝胶,其中的硫酸软骨素具有止痛,促进软骨再生的功效;胶原蛋白是动物和人体皮肤、血管、骨骼、筋腱、牙齿和软骨等结缔组织的主要成分,重组胶原蛋白具有天然胶原蛋白的优良性质,并完全没有疯牛病等病毒隐患;聚乙二醇作为一种常见的水溶性高分子,是制备功能水凝胶的优良原料。复合这三种材料制备的水凝胶具备生物相容性和可降解性,将该凝胶应用于大鼠骨缺损模型,表明这一新型的重组胶原蛋白复合凝胶对骨缺损起到良好的修复作用,在生物材料领域拥有极大的潜力和应用价值。
附图说明
图1为本发明的重组胶原蛋白、硫酸软骨素以及聚乙二醇复合凝胶的合成流程图;
图2为本发明的重组胶原蛋白复合凝胶的红外光谱图;
图3为本发明的重组胶原蛋白复合凝胶的扫描电镜图;
图4为本发明的重组胶原蛋白复合凝胶修复大鼠骨缺损效果的CT图。
图5为本发明的重组胶原蛋白复合凝胶修复大鼠骨缺损效果的磁共振扫描图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明提供一种技术方案:重组胶原蛋白复合硫酸软骨素和聚乙二醇的骨修复凝胶,主要包括重组胶原蛋白、硫酸软骨素以及聚乙二醇三种材料。硫酸软骨素是一种酸性粘多糖,广泛存在于动物组织的细胞外基质和细胞表面,在医学应用上作为一种治疗关节疾病的药物,具有止痛,促进软骨再生的功效;聚乙二醇是一种常见的水溶性高分子,因其分子链末端为活泼性基团—羟基,很容易发生化学反应得到聚乙二醇功能单体,所以利用这种大分子单体很容易制备出结构和性能各异的水凝胶。聚乙二醇水凝胶毒性低,生物相容性好,广泛应用于生物医学和药学材料。
实施例一:
本发明的制备方法包括以下步骤:
本发明的重组胶原蛋白复合凝胶的制备方法包括以下步骤:
A、利用生物基因工程技术制备重组胶原蛋白,包括以下步骤:
a、合成编码重组胶原蛋白的核酸,构建导入核酸的质粒,将质粒转化大肠杆菌BL21-DE3菌株;
b、在大肠杆菌BL21-DE3菌株中表达重组胶原蛋白,主要包括以下步骤:
(1)将少量菌液加入50ml含氨苄西林钠100μg/ml的LB培养基中,置于37℃摇床过夜培养;
(2)然后将培养基转移至1L含氨苄西林钠100μg/ml的LB培养基中,在37℃环境下继续扩大培养;
(3)紫外分光光度计测量菌液在波长600nm处的OD值,当菌液的OD值在0.8时,加入1mM IPTG同时降低摇床温度至20℃继续培养12h诱导蛋白表达,将菌液离心,收集细胞沉淀;
c、重组胶原蛋白的纯化,主要包括以下步骤:
(1)将离心后的菌体用缓冲液A使其溶解,缓冲液A由20mM咪唑、20mM磷酸钠、0.5M氯化钠组成,其pH为7.4;
(2)将细菌悬浊液放入超声波细胞破碎仪中进行细胞破碎,即可释放出蛋白并且蛋白会溶于缓冲液A中,其中细胞破碎条件:用2s超声、2s间歇的条件破碎100分钟,超声时将细菌悬浊液放于冰浴中,将破碎完的悬浊液再次离心,使细胞碎片与蛋白溶液分离,其中离心条件:离心速率10000r/min,离心温度2℃,离心时间20min;
(3)收集上清液,此即为粗蛋白溶液,通过Ni-NTA琼脂糖亲和层析柱进行纯化,将上清蛋白液加入镍柱,用结合缓冲液洗6次后再用高浓度咪唑洗脱液进行洗脱,其中结合缓冲液由30mM咪唑、0.5M NaCl、20mM Na2HPO4,组成,其pH为7.4;高浓度咪唑洗脱液由500mM咪唑、0.5M NaCl、20mM Na2HPO4,组成,其pH为7.4,收集蛋白洗脱液后用甘氨酸透析液于4℃透析4次;
(4)紫外分光光度计测得蛋白液浓度,计算蛋白含量后用胰蛋白酶按一定质量比酶切蛋白得目标蛋白,蛋白液用20mM pH为7.4的PBS缓冲液透析,收集透析后的蛋白液冷冻干燥后得到重组胶原蛋白(CL)冻干粉,冻干粉于4℃保存;
B、制备ADH修饰硫酸软骨素,其制备方法包括以下步骤:将硫酸软骨素溶于蒸馏水中,加入1-3-二甲氨基丙基-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺,将pH调至5.5,然后加入己二酸二酰肼并调节pH调至6.0,搅拌反应24h,将产物在蒸馏水中透析3天,冷冻干燥后与室温保存;
C、将一定质量硫酸软骨素和重组胶原蛋白混合配置成溶液,配制活性酯聚乙二醇活性酯溶液;其中硫酸软骨素浓度为100mg/ml、重组胶原蛋白浓度为30mg/ml、活性酯聚乙二醇活性酯浓度为160mg/ml;
D、将步骤C中的两种溶液按体积比1:1混合搅拌均匀后静置于25℃形成凝胶。
重组胶原蛋白复合凝胶的制备过程如图1所示。首先制备ADH修饰硫酸软骨素,将它与制备好的重组胶原蛋白和活性酯聚乙二醇活性酯混合,反应即可交联生成凝胶。
如图2的红外光谱图所示,a,b,c线分别为单独的重组胶原蛋白,己二酸二酰肼修饰后的硫酸软骨素,以及活性酯聚乙二醇活性酯的红外光谱,d线为重组胶原蛋白/活性酯聚乙二醇活性酯/己二酸二酰肼修饰硫酸软骨素复合形成的凝胶的红外光谱。凝胶样品真空干燥后研碎与KBr混合均匀后,压片制为红外光谱样品。重组胶原蛋白1664cm-1处的酰胺C=O的伸缩振动吸收峰,己二酸二酰肼修饰硫酸软骨素3415cm-1处O-H的伸缩振动吸收峰,活性酯聚乙二醇活性酯2888cm-1处的C-H伸缩振动吸收峰,均出现在三种材料复合凝胶的红外图谱中;而且,凝胶在1640cm-1处的酰胺吸收峰明显增强,说明活性酯聚乙二醇活性酯与己二酸二酰肼修饰后的硫酸软骨素以及重组胶原蛋白上的氨基发生反应生成了新的酰胺键。
如图3扫描电镜图所示,不同浓度的重组胶原蛋白与硫酸软骨素以及聚乙二醇的复合凝胶,都可形成良好的孔状结构。活性酯聚乙二醇活性酯和己二酸二酰肼修饰硫酸软骨素的浓度保持为80mg/ml和50mg/ml。从A-D图中,重组胶原蛋白的浓度由0mg/ml,增加为10mg/ml,20mg/ml以及40mg/ml。当重组胶原蛋白的浓度增加时,凝胶的孔径变大变厚。所有溶液均用生理盐水配制。将凝胶样品用液氮冷冻,样品切成薄片后冷冻干燥,表面喷金后通过扫描电子显微镜观察形貌。
本发明将该凝胶用于骨缺损动物模型的方法,操作方法包括以下步骤:
1)选SPF级SD大鼠45只,随机分成三组(空白对照组,PEG-ChS(硫酸软骨素(ChS)和聚乙二醇(PEG)的复合凝胶)对照组,PEG-ChS-CL(重组胶原蛋白(CL)复合硫酸软骨素(ChS)和聚乙二醇(PEG)的凝胶)组),每组15只,待大鼠适应环境5日后制备骨缺损模型。
2)使用10%水合氯醛(1ml/100mg大鼠体重)腹腔注射麻醉大鼠,备皮,消毒后切开分离皮肤、黏膜、骨膜,使用牙科钻在大鼠头部制备2个约5mm大小骨缺损面,制备过程使用生理盐水降温。
3)待大鼠骨缺损创面止血后,PEG-ChS-CL组大鼠植入重组胶原蛋白复合凝胶材料(PEG-ChS-CL),PEG-ChS对照组大鼠植入不含重组胶原蛋白的凝胶材料(PEG-ChS),空白对照组大鼠不植入任何材料;缝合皮肤黏膜后将大鼠放入饲养盒中,并用SPF的标准饲料进行喂养。
4)密切观察大鼠生理状况,全部45只大鼠无意外死亡,无感染。分别在凝胶植入大鼠体内1天,7天、14天、28天、以及56天时麻醉处死大鼠。
如图4CT(电子计算机断层扫描)图所示,不同凝胶材料修复大鼠骨缺损的效果存在明显差异。A为PEG-ChS凝胶组,B为PEG-ChS-CL凝胶组,C为空白对照组。这三组实验使用相同的大鼠品系,相同的手术方式和相似大小的颅骨缺损模型。这三组骨缺损动物分别在7、14、28、56天拍摄CT图,观测颅骨缺损变化。当动物实验进行到第56天时,我们发现空白对照组无明显骨形成,PEG-ChS凝胶组有部分骨形成出现,而PEG-ChS-CL凝胶组颅骨缺损区域减小明显,并可见骨组织呈现突出缺损边缘的生长方式。可见,PEG-ChS-CL复合凝胶表现出优良的成骨效果,重组胶原蛋白的加入有利于骨缺损的修复。
如图5磁共振扫描图所示,重组胶原蛋白复合凝胶表现出良好的骨修复效果。术后第一天(A-C)可以看到双侧的颅骨缺损(箭头所示),同时可以看到脑组织的轻度膨出和周围组织的水肿。加入PEG-ChS凝胶和PEG-ChS-CL凝胶的样品,可以清楚的看到在颅骨缺损处植入的凝胶(箭头所示)。术后第56天,空白对照组没有看到颅骨缺损处代表骨组织的低信号,说明该处骨组织的修复能力弱,局部纤维等沉积不多,并且有明显的脑组织膨出(箭头所示),说明存在骨缺损后并发症;对于PEG-ChS凝胶组(E),可以看到原来的缺损处存在明显的连续的低信号,证明该处有新骨形成;在PEG-ChS-CL凝胶组(F),可以看到明显的T2和T2-tirm的低信号,而且此处的低信号比周围颅骨的范围广(箭头所示),强烈提示该处的骨组织修复能力强于PEG-ChS凝胶组。磁共振从组织信号变化水平表明重组胶原蛋白复合凝胶促进新骨的形成。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (2)
1.重组胶原蛋白复合硫酸软骨素和聚乙二醇的骨修复凝胶,其特征在于:主要包括重组胶原蛋白、硫酸软骨素以及聚乙二醇三种材料。
2.实现权利要求1所述的重组胶原蛋白(CL)复合硫酸软骨素(ChS)和聚乙二醇(PEG)的骨修复凝胶(PEG-ChS-CL)的制备方法,其特征在于:其制备方法包括以下步骤:
A、利用生物基因工程技术制备重组胶原蛋白,包括以下步骤:
a、合成编码重组胶原蛋白的核酸,构建导入核酸的质粒,将质粒转化大肠杆菌BL21-DE3菌株;
b、在大肠杆菌BL21-DE3菌株中表达重组胶原蛋白,主要包括以下步骤:
(1)将少量菌液加入50ml含氨苄西林钠100μg/ml的LB培养基中,置于37℃摇床过夜培养;
(2)然后将培养基转移至1L含氨苄西林钠100μg/ml的LB培养基中,在37℃环境下继续扩大培养;
(3)紫外分光光度计测量菌液在波长600nm处的OD值,当菌液的OD值在0.6~0.8时,加入1mM IPTG同时降低摇床温度至20℃-25℃继续培养8h-10h诱导蛋白表达,将菌液离心,收集细胞沉淀;
c、重组胶原蛋白的纯化,主要包括以下步骤:
(1)将离心后的菌体用缓冲液A使其溶解,缓冲液A由20mM咪唑、20mM磷酸钠、0.5M氯化钠组成,其pH为7.4;
(2)将细菌悬浊液放入超声波细胞破碎仪中进行细胞破碎,即可释放出蛋白并且蛋白会溶于缓冲液A中,其中细胞破碎条件:用2s超声、2s间歇的条件破碎100分钟,超声时将细菌悬浊液放于冰浴中,将破碎完的悬浊液再次离心,使细胞碎片与蛋白溶液分离,其中离心条件:离心速率10000-15000r/min,离心温度2℃-6℃,离心时间20min-30min;
(3)收集上清液,此即为粗蛋白溶液,通过Ni-NTA琼脂糖亲和层析柱进行纯化,将上清蛋白液加入镍柱,用结合缓冲液洗6~8次后再用高浓度咪唑洗脱液,其中结合缓冲液由30mM咪唑、0.5M NaCl、20mM Na2HPO4,组成,其pH为7.4;高浓度咪唑洗脱液由500mM咪唑、0.5M NaCl、20mM Na2HPO4,组成,其pH为7.4,将蛋白洗脱,收集蛋白洗脱液后用甘氨酸透析液于4℃透析4次;
(4)紫外分光光度计测得蛋白液浓度,计算蛋白含量后用胰蛋白酶按一定质量比酶切蛋白得目标蛋白,蛋白液用20mM pH为7.4PBS透析,收集透析后的蛋白液冷冻干燥后得到蛋白CL冻干粉,冻干粉于-10℃--20℃保存;
B、制备ADH修饰硫酸软骨素,其制备方法包括以下步骤:将硫酸软骨素溶于蒸馏水中,加入1-3-二甲氨基丙基-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺,将pH调至5.5,然后加入己二酸二酰肼并调节pH调至6.0,搅拌反应24h,将产物在蒸馏水中透析3天,冷冻干燥后与室温保存;
C、将一定质量硫酸软骨素和重组胶原蛋白混合配置成溶液,配制活性酯聚乙二醇活性酯溶液;其中硫酸软骨素浓度为100mg/ml、重组胶原蛋白浓度为40mg/ml、活性酯聚乙二醇活性酯浓度为160mg/ml;
D、将步骤C中的两种溶液按体积比1:1混合搅拌均匀后静置于25℃形成凝胶。
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