CN107149701B - 载碳青霉烯类抗生素的i型胶原/羟基磷灰石钙仿生骨 - Google Patents
载碳青霉烯类抗生素的i型胶原/羟基磷灰石钙仿生骨 Download PDFInfo
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Abstract
本发明公开载碳青霉烯类抗生素的I型胶原/羟基磷灰石钙仿生骨,主要由碳青霉烯类抗生素加载在I型胶原/羟基磷灰石钙仿生骨上构成,其中I型胶原/羟基磷灰石钙仿生骨采用I型胶原蛋白与羟基磷灰石钙生物矿化而成。羟基磷灰石钙晶体嵌入I型胶原蛋白纤维纵向生长,其化学成分和分子结构与人类自身骨组织一致。本发明针对耐药型革兰氏阴性致病菌感染的难治型骨髓炎,将药敏高度敏感的碳青霉烯类抗生素与人工骨结合;载碳青霉烯类抗生素的I型胶原/羟基磷灰石仿生骨,可以缓释出碳青霉烯类抗生素,抑制大肠杆菌等耐药型革兰氏阴性致病菌的感染,从而弥补了目前临床上使用的载万古霉素/庆大霉素硫酸钙人工骨,不能涵盖骨髓炎所有菌谱的缺点。
Description
技术领域
本发明属于医用材料技术领域,涉及一种载碳青霉烯类抗生素的I型胶原/羟基磷灰石钙仿生骨。
背景技术
慢性创伤性骨髓炎多为金黄色葡萄球菌、大肠杆菌等革兰氏阳性、革兰氏阴性菌感染,这些耐药细菌可以在细胞表面产生多糖蛋白复合物,形成生物膜包裹细菌,保护细菌。要杀灭保护在生物蛋白膜内的细菌,到达骨髓炎病灶的抗生素必须对上述致病细菌敏感,而且病灶局部抗生素浓度必须高于致病菌最低抑菌浓度许多倍,全身静脉滴注抗生素难以达到这样的治疗要求。与此同时,骨髓炎病灶清除后,会残留空腔,需要植骨材料填充骨腔。
为了弥补全身用药的不足,以及同时达到植骨的作用。各国学者对载抗生素人工骨进行了多年研究。目前临床应用较多的载抗生素人工骨是医用型CaSO4,它由全球最大的骨科生物材料生产公司Wright研制,其基础晶体是α-半水硫酸钙,水溶性抗生素能够和它结合成固体植入物,而不影响抗菌效果。因此临床上多将万古霉素或庆大霉素与α-半水硫酸钙混合,制成载万古霉素/载庆大霉素硫酸钙人工骨,广泛应用于治疗骨髓炎患者。
但是载万古霉素硫酸钙人工骨只针对金黄色葡萄球等革兰氏阳性菌感染的骨髓炎,载庆大霉素人工骨也只对普通的非耐药革兰氏阴性菌感染骨髓炎有效;对于耐药型大肠杆菌、鲍曼不动杆菌等革兰氏阴性菌感染的骨髓炎,上述两种载抗生素人工骨并不敏感,而上述耐药型革兰氏阴性菌感染的骨髓炎在临床上占了将近42%。所以国际上流行通用的载万古霉素和庆大霉素硫酸钙人工骨已经远远满足不了,临床上治疗耐药型革兰氏阴性菌感染骨髓炎的需要。为了针对大肠杆菌、鲍曼不动杆菌等耐药型革兰氏阴性感染的骨髓炎,国内外曾有学者在硫酸钙人工骨中加入新型抗生素泰能,即亚胺培南/西司他丁;因为亚胺培南/西司他丁是碳青霉烯类抗生素,它对革兰氏阴性菌敏感,尤其是对大肠杆菌、鲍曼不动杆菌等骨髓炎中的耐药菌临床有效率达93%。学者也希望通过在人工骨中加载亚胺培南,达到治疗耐药型革兰氏阴性菌感染骨髓炎的目的。但是,将泰能(亚胺培南/西司他丁)加载至硫酸钙,合成载抗生素硫酸钙人工骨并不简单。因为,亚胺培南与α-半水硫酸钙混合后不能凝固成型,而是变成粉末;而粉末状载亚胺培南硫酸钙人工骨放置于患者骨髓炎病灶中,一经骨髓腔内血液冲刷,立即流失;无法达到缓释抗生素,治疗骨髓炎的效果。
其次,载万古霉素/庆大霉素硫酸钙人工骨还有一个致命缺点就是:其人工骨的化学成分和分子结构与人类天然骨组织并不一致,并不能替代自身骨组织植骨。因为,人类自身天然骨组织由有机大分子I型胶原蛋白和无机矿物质羟基磷灰石钙晶体生物矿化组成;羟基磷灰石钙晶体呈片状镶嵌在胶原纤维中,沿着胶原纤维的长轴纵向生物矿化。而美国Wright公司所研发的载抗生素半水硫酸钙人工骨,其分子方程式为1/2H2O.CaSO4,只是单纯的无机物,不含有有机大分子I型胶原蛋白,更不用说I型胶原蛋白与羟基磷灰石钙生物矿化的分子结构;无论是其化学成分,还是分子结构都与天然骨组织相去甚远。而且当临床应用载万古霉素/庆大霉素硫酸钙人工骨,填充骨髓炎病灶清除后的骨缺损时;半水硫酸钙在人体内水解,1/2H2O.CaSO4=〉1/2H2O+Ca2++SO4 2-,分解为钙离子和硫酸根离子。当抗生素硫酸钙完全水解后,仍然会留下空腔。所以为了填充残留的空腔,又不得不取患者自体髂骨植骨,增加患者创伤和痛苦。
因此,目前临床上所用的载万古霉素/庆大霉素硫酸钙人工骨,既不能对耐药型革兰氏阴性致病菌敏感,又不符合天然骨组织的化学成分和分子结构,故已经满足不了临床上治疗骨髓炎的需要。
发明内容
本发明的目的是针对载万古霉素/庆大霉素硫酸钙人工骨,其化学成分和分子机构与人类天然骨组织不符;而且无法加载针对耐药型革兰氏阴性致病菌敏感的碳青霉烯类抗生素。本发明模拟人体内生物矿化,合成I型胶原/羟基磷灰石仿生骨,并加载碳青霉烯类抗生素,从而形成载碳青霉烯类抗生素的I型胶原/羟基磷灰石仿生骨。其化学成分和分子机构与天然骨组织一致,并能释放出对骨髓炎耐药型革兰氏阴性致病菌敏感的碳青霉烯类抗生素。
为解决上述技术问题,本发明采用的技术方案如下:
本发明载碳青霉烯类抗生素的I型胶原/羟基磷灰石钙仿生骨,主要由碳青霉烯类抗生素加载在I型胶原/羟基磷灰石钙仿生骨上构成,其中I型胶原/羟基磷灰石钙仿生骨采用I型胶原蛋白与羟基磷灰石钙生物矿化而成;
进一步地,碳青霉烯类抗生素包括亚胺培南-西司他丁、美罗培南、厄他培南或比阿培南。
进一步地,碳青霉烯类抗生素与I型胶原/羟基磷灰石钙仿生骨的质量比例是7~10:100。
进一步地,I型胶原蛋白与羟基磷灰石钙的质量比例是0.5~1:4。
上述载碳青霉烯类抗生素的I型胶原/羟基磷灰石钙仿生骨,采用以下方法制备而成:
步骤(1)、I型胶原蛋白的制备
将富含I型胶原蛋白的动物组织破碎后进行酶解或酸解,得到所需的I型胶原蛋白溶液;所述的富含I型胶原蛋白的动物组织可以采用大鼠尾巴肌腱、猪皮、猪骨、跟腱;
酶解的条件是:破碎后的动物组织与胃蛋白酶的质量比为1g:60~200mg;酶解温度为20~32℃,酶解时间为5~8天。
酸解的条件是:破碎后的动物组织与醋酸的质量体积比为1g:50~100ml;酸解温度为20~25℃,醋酸浓度为0.5M~1.5M,酸解时间为7~10天。
步骤(2)、I型胶原蛋白胶体的制备
将步骤(1)获得的I型胶原蛋白溶液,放入戊二醛进行交联,交联1~2小时后,I型胶原蛋白自组装成果冻样胶体;最后将胶体放置在去离子水中浸泡,反复冲洗洗去多余的戊二醛,得到I型胶原蛋白胶体;
I型胶原蛋白与戊二醛的质量体积比:1g:10~20ml。
交联温度为20~25℃。
步骤(3)、生物矿化:
将步骤(2)获得的I型胶原胶体浸泡在矿化液中,进行生物矿化,矿化7~9天后得到I型胶原/羟基磷灰石仿生骨;
矿化液采用以下方法制备:
特制的钙液25ml倒入烧杯中,滴入PAA浓度350ug/ml;滴加1M的HCl,调节PH至7.4。然后缓慢滴入磷液25ml,约16滴/分钟。
25ml钙液由以下组分构成:10mM CaCl2.2H2O、150mM NaCl、50mM Tris、0.02%NaN3;
25ml磷化液由6mM Na2HPO4构成;
步骤(4)、加载抗生素
将I型胶原/羟基磷灰石仿生骨,浸泡在碳青霉烯类抗生素溶液中,30℃~38℃静置24~48h,从而形成载碳青霉烯类抗生素I型胶原/羟基磷灰石仿生骨。
本发明的有益效果是:
1)人类自身骨组织是由有机大分子I型胶原蛋白和无机羟基磷灰石钙组成,在分子结构上,羟基磷灰石钙晶体是以I型胶原蛋白纤维为模板,镶嵌在胶原纤维分子间隙,沿着胶原纤维的长轴纵向矿化生长。而本发明,先是合成与人类自身骨组织中I型胶原蛋白纤维的D-Band结构一致的,I型胶原蛋白胶体。然后,模拟生物矿化,羟基磷灰石晶体沿着胶原蛋白纤维矿化生长,最终形成生物矿化仿生骨。其化学成分和分子结构与人类自身骨组织一致,可以作为植骨材料,填充骨髓病灶清除后残留的空腔。
2)针对耐药型革兰氏阴性致病菌感染的难治型骨髓炎,将药敏高度敏感的碳青霉烯类抗生素与人工骨结合;载碳青霉烯类抗生素的I型胶原/羟基磷灰石仿生骨,可以缓释出碳青霉烯类抗生素,抑制大肠杆菌等耐药型革兰氏阴性致病菌的感染,从而弥补了目前临床上使用的载万古霉素/庆大霉素硫酸钙人工骨,不能涵盖骨髓炎所有菌谱的缺点。
附图说明
图1为胶原蛋白SDS-PAGE图,其中左边:蛋白Marer,中间:新鲜胶原蛋白原液,右边:室温放置过夜样品;
图2为I型胶原蛋白胶体的透射电子显微镜图;
图3为I型胶原/羟基磷灰石仿生骨的电子衍射图;
图4为I型胶原/羟基磷灰石仿生骨的透射电子显微镜图;
图5为加载有亚胺培南的I型胶原/羟基磷灰石仿生骨的抑菌圈。
具体实施方式
下面结合具体实施例对本发明作进一步的分析。
实施例1:
1.I型胶原蛋白的制备:超净台操作取大鼠尾巴洗净,75%的酒精浸泡5分钟。将尾巴剪开,去掉皮毛,并剪成小段,抽出银色的尾巴肌腱。将尾键剪断置于平皿中,灭菌生理盐水浸泡。吸去生理盐水。将尾键置于平皿中剪碎,转移至灭菌过的注射器瓶中。按每克尾键50ml的比例,加入0.1%的醋酸溶液。摇晃,将尾键分散于醋酸溶液中,4℃放置一星期。离心4000r/min,20分钟。在醋酸溶液的酸解下,尾巴肌腱水解成胶冻状凝胶,冰箱4℃保存。将酸解鼠尾胶原蛋白进行SDS-PAGE检测,结果显示:I型胶原蛋白原液在相对分子量130kDa附近有明显条带,另一条带的相对分子量高于170kDa(图1)。
图1所示,I型胶原蛋白原液在相对分子量130kDa附近有明显条带,另一条带的相对分子量高于170kDa。
2.I型胶原蛋白胶体的制备:将I型胶原蛋白溶液,经氨气扩散1小时,然后再放入0.05%戊二醛进行交联;交联1小时后,I型胶原蛋白自组装成半透明果冻样胶体;最后将胶体放置在去离子水中浸泡1小时,接着再反复冲洗3遍,洗去多余的戊二醛,完成I型胶原蛋白胶体的制备。其胶体在透射电子显微镜下显示:胶体内部的胶原纤维具有特征性明暗间隔周期性条纹结构,即D-Band结构。胶原染色D-Band结构周期为67nm,胶原分子长度为300nm;螺纹距长度为40nm,叠长度为27nm。此胶原纤维的D-Band结构与,天然骨组织的I型胶原蛋白纤维的D-Band结构一致(图2)。
图2透射电子显微镜显示:胶体内部的胶原纤维具有特征性明暗间隔周期性条纹结构,即D-Band结构。胶原染色D-Band结构周期为67nm,胶原分子长度为300nm;螺纹距长度为40nm,叠长度为27nm。与天然骨组织的I型胶原蛋白纤维的D-Band结构一致。
3.矿化液的配置:
25ml钙液:10mM CaCl2.2H2O+150mM NaCl+50mM.Tris+0.02%NaN3;25ml磷液:6mMNa2HPO4;钙液25ml倒入烧杯中,滴入PAA浓度350ug/ml;滴加1M的HCl,调节PH至7.4。然后缓慢滴入磷液,约16滴/分钟。
4.生物矿化:吸取已经配好的矿化液倒入小培养皿中,将鼠尾I型胶原胶体浸泡在矿化液中,进行生物矿化;矿化7天,颜色加深至乳白色,从而形成I型胶原/羟基磷灰石仿生骨。电子衍射图像显示:此时的胶原矿化已经完成,羟基磷灰石钙晶体形成,每个亮环对应一个晶面依次是002,211,004(图3)。在透射电子显微镜下显示:黑色羟基磷灰石晶体,嵌入胶原蛋白纤维纵向生长,其分子结构与人类自身骨组织一致(图4)。
5.加载亚胺培南/西司他丁:取亚胺培南/西司他丁70mg,溶于10ml去离子水。将I型胶原/羟基磷灰石仿生骨,浸泡在亚胺培南/西司他丁溶液中,静置在38度恒温箱中一天;亚胺培南/西司他丁加载至I型胶原/羟基磷灰石仿生骨中,从而形成载亚胺培南I型胶原/羟基磷灰石仿生骨。抑菌圈实验显示:将载亚胺培南I型胶原/羟基磷灰石仿生骨放置在耐药型革兰氏阴性菌大肠杆菌的培养基中,在载亚胺培南I型胶原/羟基磷灰石仿生骨周围形成抑菌圈,抑菌圈直径3.6cm(见图5)。在模拟体液缓释实验中:载亚胺培南I型胶原/羟基磷灰石仿生骨,缓释的亚胺培南药物浓度为2.5ug/ml,是亚胺培南/西司他丁对大肠杆菌的最低抑菌浓度0.125ug/ml的20倍,远大于最低抑菌浓度。
实施例2-4
将实施例1中亚胺培南/西司他丁分别更换为美罗培南、厄他培南或比阿培南,最终分别制备得到载美罗培南的I型胶原/羟基磷灰石仿生骨、载厄他培南的I型胶原/羟基磷灰石仿生骨、载比阿培南的I型胶原/羟基磷灰石仿生骨;其抑菌效果如下:
将载美罗培南的I型胶原/羟基磷灰石仿生骨,放置在耐药型革兰氏阴性菌大肠杆菌的培养基中,在载美罗培南I型胶原/羟基磷灰石仿生骨周围形成抑菌圈,抑菌圈直径约2.7cm。
将载厄他培南的I型胶原/羟基磷灰石仿生骨,放置在耐药型革兰氏阴性菌大肠杆菌的培养基中,在载厄他培南I型胶原/羟基磷灰石仿生骨周围形成抑菌圈,抑菌圈直径约1.3cm。
将载比阿培南的I型胶原/羟基磷灰石仿生骨,放置在耐药型革兰氏阴性菌大肠杆菌的培养基中,在载比阿培南I型胶原/羟基磷灰石仿生骨周围形成抑菌圈,抑菌圈直径约3.4cm。
上述实施例并非是对于本发明的限制,本发明并非仅限于上述实施例,只要符合本发明要求,均属于本发明的保护范围。
Claims (9)
1.载碳青霉烯类抗生素的I型胶原/羟基磷灰石钙仿生骨,其特征在于主要由碳青霉烯类抗生素加载在I型胶原/羟基磷灰石钙仿生骨上构成,其中I型胶原/羟基磷灰石钙仿生骨采用I型胶原蛋白与羟基磷灰石钙生物矿化而成;
采用以下方法制备而成,该方法包括以下步骤:
步骤(1)、I型胶原蛋白的制备
将富含I型胶原蛋白的动物组织破碎后进行酶解或酸解,得到所需的I型胶原蛋白溶液;
步骤(2)、I型胶原蛋白胶体的制备
将步骤(1)获得的 I型胶原蛋白溶液,放入0.05%戊二醛进行交联,交联1~2小时后,I型胶原蛋白自组装成果冻样胶体;最后将胶体放置在去离子水中浸泡,反复冲洗洗去多余的戊二醛,得到I型胶原蛋白胶体;
步骤(3)、生物矿化:
将步骤(2)获得的I型胶原蛋白胶体浸泡在矿化液中,进行生物矿化,矿化7~9天后得到I型胶原/羟基磷灰石钙仿生骨;
步骤(4)、加载抗生素
将I型胶原/羟基磷灰石钙仿生骨,浸泡在碳青霉烯类抗生素溶液中,30℃~38℃静置24~48 h,从而形成载碳青霉烯类抗生素I型胶原/羟基磷灰石钙仿生骨。
2.如权利要求1所述的载碳青霉烯类抗生素的I型胶原/羟基磷灰石钙仿生骨,其特征在于羟基磷灰石钙晶体嵌入I型胶原蛋白纤维纵向生长,其分子结构和化学成分与人类自身骨组织一致。
3.如权利要求1所述的载碳青霉烯类抗生素的I型胶原/羟基磷灰石钙仿生骨,其特征在于碳青霉烯类抗生素包括亚胺培南-西司他丁、美罗培南、厄他培南、比阿培南。
4.如权利要求1所述的载碳青霉烯类抗生素的I型胶原/羟基磷灰石钙仿生骨,其特征在于碳青霉烯类抗生素与I型胶原/羟基磷灰石钙仿生骨的质量比为7~10:100。
5.如权利要求1所述的载碳青霉烯类抗生素的I型胶原/羟基磷灰石钙仿生骨,其特征在于I型胶原蛋白与羟基磷灰石钙的质量比为0.5~1:4。
6.如权利要求1所述的载碳青霉烯类抗生素的I型胶原/羟基磷灰石钙仿生骨,其特征在于步骤(1)酶解的条件是:破碎后的动物组织与胃蛋白酶的质量比为1g:60~200mg;酶解温度为20~32℃,酶解时间为5~8天;
酸解的条件是:破碎后的动物组织与醋酸的质量体积比为1g:50~100mL ;酸解温度为20~25℃,醋酸浓度为0.5M~1.5M,酸解时间为7~10天。
7.如权利要求1所述的载碳青霉烯类抗生素的I型胶原/羟基磷灰石钙仿生骨,其特征在于步骤(2)I型胶原蛋白与戊二醛的质量体积比:1g:10~20mL ;交联温度为20~25℃。
8.如权利要求1所述的载碳青霉烯类抗生素的I型胶原/羟基磷灰石钙仿生骨,其特征在于步骤(3)矿化液采用以下方法制备:
在25mL 钙液中滴入350ug/mL PAA,再滴加1M的HCl,调节PH至7.4;然后滴入25mL磷液,滴加速度为16滴/分钟;
25mL 钙液由以下组分构成:10 mM CaCl2.2H2O、150mM NaCl、50mM Tris、0.02%NaN3 ;
25mL 磷液由6mM Na2HPO4构成。
9.如权利要求1所述的载碳青霉烯类抗生素的I型胶原/羟基磷灰石钙仿生骨在制备植骨材料上的应用。
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