CN118105543A - 一种治疗感染性骨缺损的载万古霉素水凝胶多孔手术植入物及其制备方法 - Google Patents
一种治疗感染性骨缺损的载万古霉素水凝胶多孔手术植入物及其制备方法 Download PDFInfo
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Abstract
本发明提供了一种治疗感染性骨缺损的载万古霉素水凝胶多孔手术植入物,所述手术植入物包括多孔钛合金假体、钙磷涂层和载万古霉素水凝胶;所述钙磷涂层涂覆于所述多孔钛合金假体,所述载万古霉素水凝胶填充于所述多孔钛合金假体的孔隙内。本发明手术植入物可以实现载万古霉素水凝胶与3D打印多孔钛合金假体的术中即刻组装使用。本发明手术植入物可实现细菌浓度响应性万古霉素智能释放。本发明通过微弧氧化技术使得3D打印多孔钛合金假体内外表面均匀分布钙磷涂层,在重建骨缺损及控制骨感染后,加速骨整合,加快感染性骨缺损病人的术后康复进程。
Description
技术领域
本发明涉及医学精技术领域,具体是一种治疗感染性骨缺损的载万古霉素水凝胶多孔手术植入物及其制备方法。
背景技术
目前临床上对于感染性骨缺损的治疗原则为通过手术清除死骨后对遗留的缺损进行修复重建,术后给予患者全身用抗生素继续抗感染治疗。但是由于部分感染性骨缺损的形状较为复杂,目前已有的重建方法如自体骨移植、外固定架、Masquelet膜诱导辅助自体骨移植以及利用生物材料重建等都存有无法精准匹配缺损、术后重建区稳定性差的问题。此外,全身用抗生素治疗感染不但效果欠佳,而且副作用大、容易引发耐药产生。而现有针对手术局部的载药方法(如传统的骨水泥载抗生素)由于无法实现对抗生素释放的有效调控,更无法实现针对细菌的响应释放,容易前期大量爆释,所以抗菌效果不理想。更关键的是,感染会严重损害宿主骨生长能力,导致骨缺损重建时间和病人术后康复时间漫长。综上所述,在实现骨缺损精准重建后进行抗菌、促成骨的研究,具有重要的现实意义,这将缩短感染性骨缺损患者的康复进程,减轻患者痛苦以及其家庭和社会的负担。
目前,针对感染性骨缺损的主流治疗方法有膜诱导辅助自体骨移植技术(Masquelet技术)、外固定架为基础的骨搬运技术(Ilizarov技术)以及近几年出现的各种以抗菌促成骨生物材料为基础的新治疗方式。这些技术取得了一定的临床疗效,但也存在着一些问题。
Ilizarov技术核心是利用外固定架缓慢牵张来诱导成骨,其方法是彻底手术清创后,用外固定架将伤肢固定住,在远离缺损区的正常骨结构区截断骨质,每日牵张0.75-1.0毫米以实现骨生长。该技术用在治疗感染性骨缺损时存在以下缺点:首先,无法实现术后即刻稳定重建,外固定架需要等骨缺损区完全愈合后才能摘除,在此之前患者需长时间持续卧床,丧失工作甚至自理能力。其次,有些骨缺损由于结构的异形性,还需要进行植骨处理,治疗成本增加的同时,也带来了植骨相关并发症的风险。另外,由于骨缺损术区局部无抗菌素存在,需要在术后长时间进行抗生素全身用药继续治疗和预防感染,副作用大的同时也增加了耐药产生的风险。最后,该方法完全靠缺损区两端的宿主骨生长实现骨与骨的融合而完成骨缺损重建,无促成骨作用,治疗时间漫长。
Masquelet膜诱导辅助自体骨移植技术允许患者术后部分负重,但仍存在以下缺点:首先,无法根据骨缺损的大小形状实现个性化定制,且骨水泥机械强度低,无法实现术后即刻稳定重建,患者一期手术后仍不能下地活动。已有文献报道接受该方法治疗后早期下地负重导致畸形愈合的案例,也有文献报导随着缺损区的延长而治疗效果不良;其次,仅适用于四肢骨缺损的治疗,无法应用于脊柱缺损治疗;另外,需要二期手术植骨,增加治疗成本的同时,也带来了植骨相关并发症的风险;还有,由于骨水泥抗生素释放行为的不可控,前期爆释抗生素,中后期抗生素释放水平很低,远期抗感染能力差。最后,完全靠宿主骨与所移植的自体骨或者异体骨融合进行缺损重建,无促成骨作用。
针对脊柱的骨缺损治疗,目前应用较多的是的钛网植骨,即在骨缺损区植入圆柱体钛合金网格,网格内需植入大量自体骨或者人工骨,也是依靠宿主骨与移植骨的融合实现脊柱骨缺损的重建,无需二次手术取出。但该方法除了植骨相关并发症,还容易出现钛网松动、下沉、移位甚至融合失败等并发症,并且钛网形状单一,根本不能满足形态各异的脊柱骨缺损,无法实现骨缺损区形状的精准匹配,再加上钛网无自稳或和宿主骨局部的固定作用,患者术后需要长时间戴外固定装置,等缺损局部骨愈合之后才能摘除,给患者的生活带来很大不便。并且在治疗脊柱感染性骨缺损时,该钛网植骨重建方法由于局部无任何抗菌性能,也需全身用抗生素进行感染的治疗或预防。
国内外最新研究探索了利用生物材料重建感染性骨缺损的新治疗方式,比如一些有促成骨功能的生物材料的出现,其优点是可以体内降解,生物相容性较好,比如搭载生物因子甚至干细胞等具有促成骨效应的纳米纤维支架、生物玻璃支架等。此外,抗菌促成骨复合功能生物材料的研究也成为热点,比如壳聚糖季铵盐/聚甲基丙烯酸甲酯/羟基磷灰石复合物,含纳米银/纳米二氧化硅复合水凝胶,羟基磷灰石支架载万古霉素等。这些方法为感染性骨缺损的治疗提供了新思路,但其同样存在上述一种或多种缺点,比如无法实现抗生素的细菌响应性释放等。更重要的是,这些方法最大的缺点是其非金属材料力学强度低、术后稳定性差,对于较大的骨缺损无法提供足够的力学支撑,无法实际应用于临床。
感染性骨缺损的成功治疗除了在于重建缺损、控制感染,另外的关键因素是缺损重建及感染控制后局部的骨生长愈合,然而,由于骨细胞与细菌对假体表面的黏附机制类似,不适合细菌定殖和菌膜形成的材料表面环境往往也不适合骨细胞长入与整合。因此,寻求一种能精准重建骨缺损前提下,在防治感染的同时又能很好地促进骨整合的理想假体成为本领域技术人员亟待解决的问题。
发明内容
针对现有技术的不足,本发明的目的在于提供一种治疗感染性骨缺损的载万古霉素水凝胶多孔手术植入物。本发明将载有万古霉素水凝胶的智能细菌响应性药物控释体系与具有促成骨效应的3D打印多孔钛合金假体微弧氧化钙磷涂层进行有机结合,实现骨缺损精准修复、抗感染及促成骨三重功效,缩短了感染性骨缺损患者术后康复时间。
为达到此发明目的,本发明采用以下技术方案:
第一个方面,本发明提供了一种治疗感染性骨缺损的载万古霉素水凝胶多孔手术植入物,所述手术植入物包括多孔钛合金假体、钙磷涂层和载万古霉素水凝胶;所述钙磷涂层涂覆于所述多孔钛合金假体,所述载万古霉素水凝胶填充于所述多孔钛合金假体的孔隙内。
优选地,所述载万古霉素水凝胶包括:
1)万古霉素;
2)聚乙二醇;
3)四臂聚乙二醇氨基;
4)四臂聚乙二醇琥珀酰亚胺碳酸酯、四臂聚乙二醇琥珀酰亚胺基戊二酸酯或四臂聚乙二醇琥珀酰亚胺酯中的任一种。
优选地,所述3)四臂聚乙二醇氨基与所述4)四臂聚乙二醇琥珀酰亚胺碳酸酯、四臂聚乙二醇琥珀酰亚胺基戊二酸酯或四臂聚乙二醇琥珀酰亚胺酯中的任一种的质量之比为:1:3~3:1。
优选地,所述多孔钛合金假体为圆柱体,其直径为0.5~0.8厘米,高度为0.3~0.7厘米,孔径为500~700μm。
第二个方面,本发明提供了一种制备如上所述的多孔手术植入物的方法,包括以下步骤:
(1)采用电子束熔融成型法制备多孔钛合金假体;
(2)利用微弧氧化电源方法在假体表面制备多孔钙磷涂层;
(3)制备多乙烯基聚乙二醇高分子前体溶液;
(4)将聚乙二醇万古霉素水凝胶与多乙烯基聚乙二醇高分子前体溶液混合后,注入所述多孔钛合金假体的孔隙,制得所述多孔手术植入物。
优选地,所述多乙烯基聚乙二醇高分子包括:四臂聚乙二醇氨基、四臂聚乙二醇琥珀酰亚胺碳酸酯、四臂聚乙二醇琥珀酰亚胺基戊二酸酯和四臂聚乙二醇琥珀酰亚胺酯。
优选地,所述步骤(3)具体为:使用磷酸缓冲溶液溶解四臂聚乙二醇氨基得到前体溶液1;使用磷酸缓冲溶液溶解四臂聚乙二醇琥珀酰亚胺碳酸酯、四臂聚乙二醇琥珀酰亚胺基戊二酸酯或四臂聚乙二醇琥珀酰亚胺酯中的任一种得到前体溶液2。
优选地,所述步骤(4)具体为:
A将聚乙二醇万古霉素水凝胶与所述前体溶液1混合得混合液1;
b将所述混合1与所述前体溶液2混合得混合液2;
c将所述混合液2注入所述多孔钛合金假体的孔隙。
优选地,所述前体溶液1中四臂聚乙二醇氨基的质量浓度为:10~50%;所述前体溶液2中四臂聚乙二醇琥珀酰亚胺碳酸酯、四臂聚乙二醇琥珀酰亚胺基戊二酸酯或四臂聚乙二醇琥珀酰亚胺酯中的任一种的质量浓度为:10%~50%。
第三个方面,本发明提供了如上所述的载万古霉素水凝胶多孔手术植入物在制备治疗感染性骨缺损的组织材料中的应用。
本发明的有益效果在于:
(1)多孔钛合金假体微孔与载万古霉素水凝胶结合界面控制技术是本发明治疗骨感染的关键技术,本发明通过它来实现载万古霉素水凝胶与3D打印多孔钛合金假体的术中即刻组装使用。
(2)细菌浓度响应性万古霉素智能释放技术。本发明通过席夫碱反应修饰万古霉素的方法使该体系释放万古霉素具有酸响应性特点,具体如下:在中性环境,PEG-Van随着凝胶降解缓慢往外渗透,速率由凝胶的降解时间控制,期间伴随着万古霉素从PEG上缓慢地脱落;而在细菌增殖产生乳酸导致的酸性环境中,pH值的降低使席夫碱键迅速断裂,从而加速释放万古霉素。通过克服此关键技术难题实现如下智能化抗菌模式:术后如果没有出现感染,PEG-Van会缓缓从凝胶释放直至消失;一旦发生感染或者细菌产生酸性环境,万古霉素会响应性迅速释放进行杀菌。
(3)针对3D打印多孔钛合金假体促成骨改性的微弧氧化技术。通过该技术使得3D打印多孔钛合金假体内外表面均匀分布钙磷涂层,在重建骨缺损及控制骨感染后,加速骨整合,加快感染性骨缺损病人的术后康复进程。
附图说明
附图用来提供对本发明的进一步理解,并且构成说明书的一部分,与本发明的实施例一起用于解释本发明,并不构成对本发明的限制。在附图中:
图1为样品制备与表征技术路线图;
图2为本发明智能化微弧氧化3D打印多孔钛合金载万古霉素水凝胶假体的(TS-M/H/V组)表征结果示意图(其中图(A)为实物图片(TS-M/H/V);图(B)为微弧氧化假体表面SEM图像(TS-M/H/V组);图(C)为水凝胶表面SEM图像;图(D)为各组假体的弹性模量图;(E)为在应力扫描模式下的流变分析结果图(TS-M/H/V组);图(F)为在频率扫描模式下的流变分析结果图(TS-M/H/V组);图(G)为水凝胶TS-M组、TS-M/H组和TS-M/H/V组分别植入物的FTIR光谱图;图(H)为水凝胶TS-M/H/V组在pH 5.0和pH 7.4的PBS中万古霉素的释放曲线(以均数±标准差表示,n=6));
图3为细胞实验技术路线图;
图4为抗菌与成骨性能评价技术路线图;
图5为建立兔股骨感染性骨缺损模型示意图;
图6为TS-M组、TS-M/H组、TS-M/H/V组假体与大鼠骨髓间充质干细胞共培养第1、3、5天,大鼠骨髓间充质干细胞在各组假体上的细胞死活染色结果示意图;
图7为TS-M组(图A、B、C)、TS-M/H组(图D、E、F)和TS-M/H/V组(G、H、I)假体与大鼠骨髓间充质干细胞共培养第14天后的电子扫描电镜(SEM)结果示意图;
图8为TS-M组、TS-M/H组、TS-M/H/V组假体与大鼠骨髓间充质干细胞共培养7天及14天,各组假体表面细胞的ALP活性比较结果示意图(“*”表示P<0.05);
图9为各组假体与金黄色葡萄球菌共培养1天后,SEM(放大3000倍与25000倍)观察各组假体表面的细菌粘附情况结果示意图(TS-M组(A,a)、TS-M/H组(B,b)、TS-M/H/V组(C,c));
图10为微弧氧化假体组(TS-M组);微弧氧化假体载水凝胶组(TS-M/H组);微弧氧化假体载万古霉素水凝胶组(TS-M/H/V组)兔子在术后42天的白细胞(A)、体温(B)及体重(C)变化趋势对比结果示意图(“**”表示P<0.01);
图11为微弧氧化假体组(TS-M组);微弧氧化假体载水凝胶组(TS-M/H组);微弧氧化假体载万古霉素水凝胶组(TS-M/H/V组)兔子在术后第42天取材后标本大体像、X线摄像及感染评分示意图(“**”P<0.01);
图12为TS-M(图A,B,C),TS-M/H(图D,E,F)和TS-M/H/V(图G,H,I)组的micro-CT图像以及三维重建图(红色箭头指的是TS-M和TS-M/H组兔股骨骨髓炎表现.*p<0.05.**p<0.01(n=6));
图13为微弧氧化假体组(TS-M组)、微弧氧化假体载水凝胶组(TS-M/H组)、微弧氧化假体载万古霉素水凝胶组(TS-M/H/V组)假体周围骨组织黏附细菌数量示意图(图A);微弧氧化假体组(TS-M组)、微弧氧化假体载水凝胶组(TS-M/H组)、微弧氧化假体载万古霉素水凝胶组(TS-M/H/V组)假体黏附细菌数量示意图(图B)(“**”表示P<0.01);
图14为TS-M图(A)、TS-M/H(图B)和TS-M/H/V(图C)组支架病理切片H&E染色图,炎性浸润(黑色箭头示意),含单核细胞和粒细胞,炎性浸润导致骨坏死、骨侵蚀;图D为TS-M/H/V组的组织感染病理分数示意图(*p<0.05.**p<0.01(n=6));
图15为TS-M(图A)、TS-M/H(图B)和TS-M/H/V(图C)组假体的硬组织切片染色图(星号所标为钛合金);图D为TS-M、TS-M/H和TS-M/H/V组的BI和BICR示意图(*p<0.05.**p<0.01(n=6));
图16为本发明微弧氧化假体载万古霉素水凝胶制备过程以及应用示意图。
具体实施方式
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解,下面的实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体技术或条件的,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1智能化微弧氧化3D打印多孔钛合金载万古霉素水凝胶假体的制备(图1)
1、制备多孔钙磷涂层钛合金假体
1)制备多孔钛合金假体:
采用电子束熔融成型法(ElectronBeam Melting,EBM)制备多孔钛合金假体,其规格如下:具有钻石晶格孔隙结构的多孔钛合金圆柱体,其直径为0.7厘米,高度为0.5厘米,孔径为640μm,支柱直径400μm。
2)在假体表面制备多孔钙磷涂层:
利用微弧氧化电源在多孔钛合金假体表面制备多孔钙磷涂层,将制备的多孔钙磷涂层钛合金假体命名为TS-M(Titanium Scaffolds-MAO)。
2、载万古霉素水凝胶的制备:
1)制备成胶前驱体溶液1:
使用pH值为7.4的磷酸盐缓冲溶液溶解四臂PEG氨基(Tetra-PEG-NH2)于一个样品瓶中制备成胶前驱体溶液1;其中,Tetra-PEG-NH2的质量浓度为10~50%,也可以是10%、20%、30%、40%、50%等。
2)制备成胶前驱体溶液2:
使用pH值为7.4的磷酸盐缓冲溶液溶解四臂PEG琥珀酰亚胺碳酸酯(Tetra-PEG-SC)、四臂聚乙二醇琥珀酰亚胺基戊二酸酯(Tetra-PEG-SG)或四臂-聚乙二醇-琥珀酰亚胺酯(Tetra-PEG-SS)其中任一种于另一个样品瓶中制备成胶前驱体溶液2;其中,Tetra-PEG-SC、Tetra-PEG-SG或Tetra-PEG-SS的质量浓度为10~50%,也可以是10%、20%、30%、40%、50%等。
根据Tetra-PEG-SC、Tetra-PEG-SG和Tetra-PEG-SS种类和Tetra-PEG-NH2的配比选择来调控凝胶的降解时间,进而大体控制万古霉素的释放速率。其中,根据骨科术后感染特点,为了保证足够时长,本发明将选用降解时间为1个月的Tetra-PEG-SC水凝胶。
表1:种类和配比
3)制备聚乙二醇-万古霉素水凝胶(PEG-Van)
将万古霉素与单臂聚乙二醇反应生成聚乙二醇-万古霉素水凝胶(PEG-Van);
3、制备多孔钛合金载万古霉素水凝胶假体
将PEG-Van融入胶前驱体溶液1后,使用双通注射器与成胶前驱体溶液2混合,在凝固前注入假体孔隙,制得智能化微弧氧化3D打印多孔钛合金载万古霉素水凝胶假体,并将其命名为TS-M/H/V(Titanium Scaffolds-MAO-Hydrogel-Vancomycin)。
在该体系中,席夫碱键可以在pH值7.4保持稳定,时间超过1个月;但是如果在酸性环境中就可以在短时间内发生断裂,从而释放万古霉素。而细菌在增殖过程中会产生乳酸,进而导致周围产生酸性环境促进万古霉素的释放。最终,通过这种机制实现细菌浓度响应性的智能药物释放功能。
4、将步骤3制备的假体置于含0.5mg/mL碳化二亚胺(EDC),0.5mg/mL琥珀酰亚胺(NHS)的PBS液中反应20min,反应30min之后用PBS清洗,再将其置于含有浓度为0.5mg/mL的短肽的PBS液中,常温孵育2~4h后,用纯水漂洗去除多余短肽。
本实施例中,所使用的短肽是组氨酸、甘氨酸、苯丙氨酸、丝氨酸、谷氨酸、精氨酸组成的六肽(His-Gly-Phe-Ser-Glu-Arg,HGFSER),本实施例短肽可通过化学合成的方法制备获得,将其制备成冻干粉后,配制成浓度为0.5mg/mL的蛋白溶液。经实验证实,该短肽具有不仅具有细胞黏附作用,还具有良好的耐酸性,在弱酸环境(pH值4~6)中稳定性好,不易发生降解,将纯度为95%的纯品溶解制备成蛋白溶液,在室温弱酸环境(pH值4~6)放置18个月后检测其纯度仍能保持在95%以上。
由于细菌释放的乳酸使得假体所处环境呈乳酸性,上述短肽可在弱酸环境中促使细胞生长,有助于伤口的愈合。
实施例2智能化微弧氧化3D打印多孔钛合金载万古霉素水凝胶假体的表征与性能验证
根据上述假体样品的制作过程,将制备的样品进行分组:微弧氧化假体组(TS-M组);微弧氧化假体载水凝胶组(TS-M/H组);微弧氧化假体载万古霉素水凝胶组(TS-M/H/V组),然后检测各组样品的性能。
一、实验方法与步骤:
1、表征实验(图2)
(1)扫描电子显微镜
用扫描电子显微镜观察各组假体孔隙内外部表面的涂层形态。
(2)微弧氧化3D打印多孔钛合金假体的力学表征
用通用力学测试机对微弧氧化处理前后的多孔钛合金支架进行静态压缩测试,得到各组假体的弹性模量。
(3)X射线光电子谱
用X-射线光电子谱分析各组假体表面涂层的化学组成。
(4)载万古霉素水凝胶的表征
用傅里叶变换红外光谱和布鲁克核磁共振等方法对载万古霉素水凝胶进行材料结构的表征。
(5)万古霉素体外释放检测
将TS-M/H/V支架浸入2ml pH值分别为5和7.4磷酸盐缓冲盐水,然后采用高效液相色谱法检测万古霉素释放速率,用于验证智能化微弧氧化3D打印多孔钛合金载万古霉素水凝胶假体的细菌浓度响应性药物释放功能。
2、细胞实验(图3)
(1)样品的细胞相容性检测
将各组实验样品(n=3)置于基础培养基中浸泡1小时,然后滴加50μl含1×105个hMSCs细胞悬液。在接种后3天、7天和14天,用细胞计数试剂盒(CCK-8)检测细胞的增殖活性,用扫描电镜观察hMSCs在各组假体上的生长情况。
(2)样品的促成骨活性检测(图4)
除了培养基换成成骨培养基外,细胞培养和接种方法同前。接种培养7天后,用碱性磷酸酶试剂盒检测各组假体上细胞的碱性磷酸酶活性。
3、体外抗菌性能检测(图4)
(1)SEM观察样品表面细菌。将各组灭菌样品与菌液共培养后用SEM观察各组假体表面细菌形貌及数量。
(2)死菌/活菌荧光染色检测。将各组灭菌样品与菌液共培养后用LIVE/DEADBacLight细菌死活染试剂盒对各组样品进行染色,最后采用激光扫描共聚焦显微镜观察各组假体表面死菌/活菌。
(3)细菌代谢活性检测。将各组灭菌样品与菌液共培养后,放入1ml PB S用超声波清洗仪清洗20min,然后用WST微生物活性试剂盒(Microbial Via bilityAssay Kit-WST,Dojindo)检测细菌线粒体代谢活性,以反映假体的抗菌性能。
(4)抗菌膜形成性能检测。将各组灭菌样品与菌液共培养后用结晶紫对菌膜进行染色,检测菌膜量。
4、动物实验(图5)
(1)建立兔股骨感染性骨缺损模型。
(2)一般指标检测。所有动物均于手术当天(0天),术后第1、3、7、14、28、35及42天进行体温、体重、白细胞检查。
(3)序贯荧光素标记。为了动态观察多孔钛合金支架骨整合情况,分别采用钙黄绿素和四环素序贯荧光标记3周及5周的骨生成情况。
(4)标本大体评估。6周后取材,根据如下评分方法对大体所见进行评分:0分,局部无脓肿、死骨形成,无反应骨形成,肤色不红;1分,肤色红,但局部无脓肿、死骨形成,无反应骨形成;2分,反应新骨形成;3分,局部形成脓肿、骨膜反应、脓性分泌物,甚至窦道形成;4分,严重的骨吸收、脓肿形成并延及骨干。
(5)X线评估。在术后第21天和第42天处死取材后拍摄右膝关节侧位片。根据如下评分标准对各组动物的感染进行量化评分,具体的评分内容为:(1)骨膜反应,(2)骨溶解,(3)软组织肿胀,(4)畸形,(5)整体印象,(6)自发性骨折,(7)死骨形成。其中1-5项评分为:无,0分;轻度,1分;中度,2分。重度,3分。6-7项评分:无,0分;有,1分。
(6)Micro-CT分析。将各组标本进行Micro-CT扫描,对感兴趣区域(假体及周围骨)进行感染状况评估和骨定量组织学分析。感染的评估主要根据下表的骨髓炎的Micro-CT评分体系。
表2骨髓炎评分系统(micro-CT)
(7)微生物学检测。6周后取材,采用涂板计数定量分析方法分别对假体和感染的股骨进行微生物学检测,检测假体黏附的细菌数量。
(8)组织病理学分析。6周后取材置于4%多聚甲醛进行组织固定。24h后对标本进行脱钙并石蜡包埋,包埋后切片,进行HE(Hematoxylin-eosin)染色并在光学显微镜下观察。通过如下方法对骨组织感染的病理改变进行定量评分直接评估感染情况,包括:急性骨感染的评价(Intraosseous acute inflammation,IAI)、慢性骨感染的评价(Intraosseouschronic inflammation,ICI)、骨膜炎症(Periosteal inflammation,PI)及骨坏死(Bonenecrosis,BN)。此外,利用BIOQUANT图像分析系统分析各组假体的骨长入率和骨与假体接触率以间接反映感染情况。
二、实验结果
1、表征结果如图2所示:
(1)扫描电子显微镜
(2)微弧氧化3D打印多孔钛合金假体的力学表征各组假体的弹性模量如下表:
表3:各组弹性模量
组别 | 弹性模量(GPa) |
TS-M | 2.217±0.33 |
TS-M/H | 2.676±0.27 |
TS-M/H/V | 2.572±0.48 |
由上表可知,本发明制备的多孔钛合金假体其弹性模量远低于实体假体的弹性模量(90–115GPa),更接近于人体松质骨的弹性模量(1.37GPa)。
(3)X射线光电子谱
(4)傅立叶红外线光谱仪
(5)万古霉素释放动力学
2、细胞实验结果
(1)大鼠骨髓间充质干细胞(MSCs)与假体的细胞相容性
在假体与大鼠骨髓间充质干细胞共培养第1、3、5天,我们采用荧光显微镜初步观察细胞在假体上的增殖状态,如图6、7所示,绿色为生存状态良好的大鼠骨髓间充质干细胞,红色为死亡的大鼠骨髓间充质干细胞,可见大鼠骨髓间充质干细胞在各组假体上都能良好地生存,尤其在TS-M组,细胞生长状态最佳。
(2)促大鼠骨髓间充质干细胞成骨分化作用
进一步,利用ALP作为早期阶段的成骨标记,我们检测了我们的假体在促大鼠骨髓间充质干细胞成骨分化方面的作用。图8显示了将细胞与3种假体共培养14天的ALP活性结果,TS-M/H组的ALP活性显著优于TS-M组和TS-M/H/V组,差异有统计学意义(P<0.05),TS-M组和TS-M/H/V组之间无显著差异。
3、体外抗菌实验结果
抑制细菌在表面粘附是阻止菌膜及假体相关感染发生的关键。所以,我们将金葡菌与假体共培养来评价假体的抗细菌粘附性能。首先,我们先观察细菌的形貌,如图9中A、B中红色箭头所指,共培养1天后,大量球状细菌在TS-M组、TS-M/H组假体表面粘附聚集成簇。而TS-M/H/V组假体表面无聚集成簇的细菌粘附,只可见少量破裂死亡细菌,如图9C中蓝色箭头所指。
4、动物实验结果
(1)一般指标观察
术后1周,TS-M组和TS-M/H组的所有兔子均表现出局部感染症状,如膝关节肿胀和伤口渗液。而TS-M/H/V组的兔子,局部感染征象明显较轻。如图10(A)所示,所有兔子的白细胞在术后1周内急剧升高。然而,TS-M/H/V组兔子的白细胞在1周以后开始呈现明显降低趋势,显著低于其他组(P<0.01)。如图10(B)所示,TS-M组和TS-M/H组的白细胞计数在术后急剧升高,显著高于TS-M/H/V组(P<0.01);如图10(C)所示,术后所有兔子的体重总体呈现降低趋势,4周内体重无明显差异,但在第4周开始,而TS-M/H/V组兔子的平均体重明显大于其他组(P<0.01)。
(2)取材后大体标本评估
TS-M和TS-M/H组的所有兔子胫骨段均表现出化脓性感染病灶,包括髓内脓液的形成、溶骨性病变、骨膜反应和骨结构畸形等;而TS-M/H/V组兔子无明显表现。如图11所示,术后6周TS-M和TS-M/H组的所有兔子胫骨段,尤其是膝关节处X线表现出骨膜反应、新骨和死骨的形成。而TS-M/H/V组兔子胫骨段几乎没有发现明显的骨感染影像学征象。对于动物标本的感染评分为:TS-M、TS-M/H和TS-M/H/V组依次为8.5±0.43、7.1±0.56、2.3±0.27,与TS-M和TS-M/H组相比,TS-M/H/V组的得分显著降低(P<0.01)。
(3)micro-CT分析
兔子在术后第42天取材后,结果如图12所示我们利用micro-CT对假体及其周围骨进行分析。此外,我们对其感染情况进行双盲评分。骨体积分数(BVF)指的是假体内的骨量比上总骨量,我们用起来反映骨生长.最后,我们对其进行三维重建来获得整体的骨生长状况.
(4)微生物学评价
兔子在术后第42天取材后进行微生物学评价,结果如图13所示。对于假体黏附的细菌数量,TS-M/H/V组显著低于TS-M组和TS-M/H组(P<0.05),TS-M组和TS-M/H组之间无显著差异(P>0.05)。对于假体周围骨内的细菌数量,TS-M/H/V组显著低于TS-M组和TS-M/H组(P<0.05),TS-M组和TS-M/H组之间无显著差异(P>0.05)。
(5)感染的病理学评估
TS-M组和TS-M/H组感染部位的组织学切片(HE染色)显示慢性骨感染的典型表现(图14),包括炎症细胞(含单核细胞和粒细胞)浸润(星号),骨坏死(黑箭头)(H&E染色)和骨侵蚀。
(6)成骨评价
我们利用马森戈德纳三色染色法对硬组织切片进行染色,可将骨组织染为绿色,类骨组织染为红色/橙色,种植体为黑色。支架骨整合的代表性组织学图像如图15A-C所示。图15D显示了三组支架的BI和BICR的定量分析。与TS-M和TS-M/H组相比,TS-M/H/V组的BI和BICR显著高于TS-M/H组,且骨组织量的比例更高(p<0.05)。
综上,本发明实现了万古霉素控释和万古霉素细菌浓度响应性智能化释放,精准有效的抗感染。通过载万古霉素体系中席夫碱键在细菌产生乳酸(pH值降低)时发生断裂释放万古霉素,而实现细菌浓度响应性的智能药物控制释放功能。
本发明研发了既能精准有效的重建骨缺损及控制感染,又能促成骨的智能化3D打印多孔钛合金假体,为感染性骨缺损的治疗和加速康复提供了新思路。该研究巧妙地将将水凝胶的智能药物控释与具有促成骨效应的3D打印多孔钛合金假体微弧氧化涂层有机结合,实现骨修复、抗感染及促成骨三重功效。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (10)
1.一种治疗感染性骨缺损的载万古霉素水凝胶多孔手术植入物,其特征在于,所述手术植入物包括多孔钛合金假体、钙磷涂层和载万古霉素水凝胶;所述钙磷涂层涂覆于所述多孔钛合金假体,所述载万古霉素水凝胶填充于所述多孔钛合金假体的孔隙内。
2.如权利要求1所述的多孔手术植入物,其特征在于,所述载万古霉素水凝胶包括:
1)万古霉素;
2)聚乙二醇;
3)四臂聚乙二醇氨基;
4)四臂聚乙二醇琥珀酰亚胺碳酸酯、四臂聚乙二醇琥珀酰亚胺基戊二酸酯或四臂聚乙二醇琥珀酰亚胺酯中的任一种。
3.如权利要求2所述的多孔手术植入物,其特征在于,所述3)四臂聚乙二醇氨基与所述4)四臂聚乙二醇琥珀酰亚胺碳酸酯、四臂聚乙二醇琥珀酰亚胺基戊二酸酯或四臂聚乙二醇琥珀酰亚胺酯中的任一种的质量之比为:1:3~3:1。
4.如权利要求1所述的多孔手术植入物,其特征在于,所述多孔钛合金假体为圆柱体,其直径为0.5~0.8厘米,高度为0.3~0.7厘米,孔径为500~700μm。
5.一种制备如权利要求1~4任一项所述的多孔手术植入物的方法,其特征在于,包括以下步骤:
(1)采用电子束熔融成型法制备多孔钛合金假体;
(2)利用微弧氧化电源方法在假体表面制备多孔钙磷涂层;
(3)制备多乙烯基聚乙二醇高分子前体溶液;
(4)将聚乙二醇万古霉素水凝胶与多乙烯基聚乙二醇高分子前体溶液混合后,注入所述多孔钛合金假体的孔隙,制得所述多孔手术植入物。
6.如权利要求5所述的方法,其特征在于,所述多乙烯基聚乙二醇高分子包括:四臂聚乙二醇氨基、四臂聚乙二醇琥珀酰亚胺碳酸酯、四臂聚乙二醇琥珀酰亚胺基戊二酸酯和四臂聚乙二醇琥珀酰亚胺酯。
7.如权利要求6所述的方法,其特征在于,所述步骤(3)具体为:使用磷酸缓冲溶液溶解四臂聚乙二醇氨基得到前体溶液1;使用磷酸缓冲溶液溶解四臂聚乙二醇琥珀酰亚胺碳酸酯、四臂聚乙二醇琥珀酰亚胺基戊二酸酯或四臂聚乙二醇琥珀酰亚胺酯中的任一种得到前体溶液2。
8.如权利要求7所述的方法,其特征在于,所述步骤(4)具体为:
a将聚乙二醇万古霉素水凝胶与所述前体溶液1混合得混合液1;
b将所述混合1与所述前体溶液2混合得混合液2;
c将所述混合液2注入所述多孔钛合金假体的孔隙。
9.如权利要求7所述的方法,其特征在于,所述前体溶液1中四臂聚乙二醇氨基的质量浓度为:10~50%;所述前体溶液2中四臂聚乙二醇琥珀酰亚胺碳酸酯、四臂聚乙二醇琥珀酰亚胺基戊二酸酯或四臂聚乙二醇琥珀酰亚胺酯中的任一种的质量浓度为:10~50%。
10.如权利要求1~4任一项所述的载万古霉素水凝胶多孔手术植入物在制备治疗感染性骨缺损的组织材料中的应用。
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