CN110624132B - 一种膀胱修复支架材料及其制备方法和用途 - Google Patents
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Abstract
本发明提供了一种膀胱修复支架材料及其制备方法和用途。该膀胱修复支架材料由原青花素交联小肠黏膜下层膜后而得。本发明膀胱修复支架材料具有良好的生物相容性,更优的力学性能和抗酶解能力。能够有效避免单纯SIS应用于膀胱全层缺损修复过程中容易出现的挛缩、钙化等并发症,同时具有良好的促平滑肌再生效果,克服了现有软组织修复材料成肌困难的问题,具有良好的临床应用前景。
Description
技术领域
本发明属于组织工程学领域,具体涉及一种膀胱修复支架材料及其制备方法和用途。
背景技术
膀胱作为储尿排尿器官具有重要的生理功能,然而许多病理原因导致的膀胱损伤严重影响病人的健康及生活质量(例如:膀胱外翻、神经源性膀胱功能障碍、挛缩性膀胱及尿路上皮癌等)。膀胱损伤大都需要通过手术才能重建其功能。临床上膀胱重建的金标准是膀胱扩大术,常采用患者胃肠道代替膀胱组织重建其结构和功能。但是由于肠段的结构及功能与膀胱有着本质的区别,导致肠段置换术易产生血尿、排尿困难、结石、肿瘤等并发症。近年来,随着组织工程及再生医学的产生及发展,为膀胱缺损修复提供了新的思路和技术手段。
猪小肠黏膜下层(SIS)是一种天然的生物复合材料,被广泛的应用于组织器官修复,其主要由胶原、糖胺聚糖、糖蛋白和多种生长因子构成。由于SIS具有低免疫原性及良好的生物相容性,现已被FDA批准应用于瘘(直肠瘘,尿道瘘、阴道瘘)、疝(腹股沟疝、腹疝)以及硬脑膜等的缺损修复重建。但是,由于特殊的膀胱内环境使SIS在膀胱修复过程中极易导致挛缩、钙化及炎症等并发症的发生,且平滑肌再生效果不理想,极大的限制了其在临床中的应用。因此通过交联手段对SIS进行改性,使其具备一定力学强度,并具备抗炎、抗钙化及促平滑肌再生功能具有重要意义。
生物衍生材料的交联方法可划分为物理交联法和化学交联法,这些方法为材料的交联改性提供更多选择,有助于得到更适于临床的组织工程产品。物理交联的生物衍生材料是指由于盐键、氢键及疏水作用等存在而形成的网络结构,其细胞毒性小,但交联效率低、交联产物不稳定。化学交联法是指在机械力、超声波和交联剂等的作用下,使高分子化合物间通过化学键联结而形成交联网络的方法,其交联效率高、成本低,但交联后材料的细胞相容性下降,免疫反应增强、易钙化。
因此选择一种交联效率高、交联产物稳定、生物相容性好的生物交联剂对SIS进行交联改性,对制备膀胱修复支架材料具有重要意义。
发明内容
本发明的目的是提供一种膀胱修复支架材料及其制备方法和用途。
本发明提供了一种膀胱修复支架材料,它由原青花素交联小肠黏膜下层膜后而得。
进一步地,所述交联指数为0.2%~65%;优选地,所述交联指数为44.2%。
进一步地,所述交联包括如下步骤:将小肠黏膜下层膜浸泡在原青花素溶液中,交联,洗涤,冻干,即可。
进一步地,所述原青花素溶液的浓度范围为0.1~5mg/mL;优选地,所述原青花素溶液的浓度为0.5~1mg/mL;更优选地,所述原青花素溶液的浓度为1mg/mL。
进一步地,所述小肠黏膜下层膜的面积与原青花素溶液的体积比为50~200cm2:100mL;优选地,所述小肠黏膜下层膜的面积与原青花素溶液的体积比为100cm2:100mL。
进一步地,所述交联的温度为35~40℃,时间为24~96h;优选地,所述交联的温度为37℃,时间为48h。
进一步地,所述小肠黏膜下层膜的制备方法如下:
取小肠,去除浆膜层、肌层和黏膜层,保留黏膜下层,脱脂,脱细胞,清洗,冻干,即得。
进一步地,
所述脱脂是用体积比为1:1的甲醇和氯仿的混合溶液浸泡6-24小时;
和/或,所述脱细胞为在4-10℃中,于胰蛋白酶溶液中浸泡6-24小时后,再在十二烷基硫酸钠与氯化钠的混合溶液中处理3~5h;所述胰蛋白酶溶液的浓度为0.01~10%w/v,所述十二烷基硫酸钠的浓度为0.01%-1%w/v,所述氯化钠的浓度范围为0.01%-1%w/v。
进一步地,
所述脱脂是用体积比为1:1的甲醇和氯仿的混合溶液浸泡12小时;
和/或,所述脱细胞为在4℃中,于胰蛋白酶溶液中浸泡12小时后,再在十二烷基硫酸钠与氯化钠的混合溶液中室温处理4h;所述胰蛋白酶溶液的浓度为0.25%w/v,所述十二烷基硫酸钠的浓度为0.5%w/v,所述氯化钠的浓度范围为0.9%w/v。
本发明还提供了一种前述的膀胱修复支架材料的制备方法,它包括如下步骤:
小肠黏膜下层膜浸泡在原青花素溶液中,交联,洗涤,冻干,即可。
进一步地,所述原青花素溶液的浓度范围为0.1~5mg/mL;优选地,所述原青花素溶液的浓度为0.5~1mg/mL;更优选地,所述原青花素溶液的浓度为1mg/mL;
和/或,所述小肠黏膜下层膜的面积与原青花素溶液的体积比为50~200cm2:100mL;优选地,所述小肠黏膜下层膜的面积与原青花素溶液的体积比为100cm2:100mL;
和/或,所述交联的温度为35~40℃,时间为24~96h;优选地,所述交联的温度为37℃,时间为48h。
本发明还提供了前述的膀胱修复支架材料在制备膀胱修复支架中的用途。
进一步地,所述膀胱修复支架材料促进膀胱平滑肌再生。
本发明中,w/v为质量体积比,单位为g/mL。
本发明膀胱修复支架材料具有良好的生物相容性,更优的力学性能和抗酶解能力。能够有效避免单纯SIS应用于膀胱全层缺损修复过程中容易出现的挛缩、钙化等并发症,同时具有良好的促平滑肌再生效果,克服了现有软组织修复材料成肌困难的问题,具有良好的临床应用前景。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为未交联的SIS膜及实施例1~5不同PC浓度交联后的SIS膜(PC-SIS)的大体结构及超微结构图片。
图2为不同浓度的PC与SIS的交联指数(*代表不同交联浓度有统计学差异)。
图3为未交联的SIS膜及实施例1~5不同PC浓度交联后的SIS膜(PC-SIS)体外酶解结果(*代表PC-SIS与SIS组有统计学差异;#代表与SIS无统计学差异)。
图4为原代兔膀胱平滑肌细胞提取后的鉴定结果。
图5为活性染色测定R-B-SMCs接种于未交联的SIS膜及实施例1~5不同PC浓度交联后的SIS膜(PC-SIS)的细胞活性。
图6为CCK8测定未交联的SIS膜及实施例1~5不同PC浓度交联后的SIS膜(PC-SIS)浸提液培养7d以内的原代膀胱平滑肌细胞(R-B-SMCs)的增殖水平(*代表PC-SIS与SIS组有统计学差异;#代表PC-SIS与SIS组无统计学差异)。
图7为不同PC交联浓度的PC-SIS(实施例2制备的0.5mg/ml PC-SIS及实施例3制备的1mg/ml PC-SIS)中PC释放曲线(*代表PC-SIS与SIS组有统计学差异;#代表PC-SIS与SIS组无统计学差异)。
图8为未交联的SIS膜,实施例2制备的0.5mg/ml PC-SIS及实施例3制备的1mg/mlPC-SIS的力学性能分析(*代表PC-SIS与SIS组有统计学差异;#代表PC-SIS与SIS组无统计学差异)。
图9为未交联SIS和不同PC浓度交联的PC-SIS溶血实验结果。
图10为未交联SIS和不同PC浓度交联的PC-SIS体外抗钙化结果。
图11为未交联SIS和不同PC浓度交联的PC-SIS傅里叶红外曲线。
图12为未交联SIS和不同PC浓度交联的PC-SIS接触角结果(*代表不同交联浓度有统计学差异)。
图13为F-actin染色检测R-B-SMCs接种于未交联SIS及不同PC浓度交联的PC-SIS材料细胞粘附及分布状况。
图14为SEM测定未交联SIS和不同PC浓度交联的PC-SIS材料R-B-SMCs表面超微结构。
图15为Q-PCR分析不同交联条件下复合R-B-SMCs平滑肌相关基因表达水平(*代表PC-SIS与SIS组无统计学差异)。
图16为兔膀胱全层缺损模型构建手术流程。
图17为不同材料修复组取材大体观察。
图18为HE染色观察不同材料修复组术后膀胱组织学形态。
图19为Masson染色观察不同材料修复组术后膀胱组织学形态。
具体实施方式
本发明具体实施方式中使用的原料、设备均为已知产品,通过购买市售产品获得。
本发明中涉及的缩略词
SIS:小肠粘膜下层,PC:原青花素,SDS:十二烷基硫酸钠,SEM:扫描电子显微镜,Q-PCR:实时定量聚合酶链反应。
实施例1、本发明膀胱修复支架材料的制备
1、SIS膜的制备
(1)清洁肠段:取新鲜猪小肠一副,清除肠段内多余食糜后,将小肠内外翻转,并用生理盐水反复将内表面清洗干净,接着用剪刀沿小肠纵向剖开,最后剪成长15cm左右的肠段。
(2)物理刮除:使用压舌板刮除肠段上的浆膜层、肌层和黏膜层,保留黏膜下层。该过程需反复用力刮除,直至分离出乳白色半透明状薄膜,并用生理盐水清洗2~3次。
(3)脱脂:用生理盐水反复冲洗干净后,用纱布包裹SIS膜,挤出部分水分,并用滤纸沾除多余水分,将氯仿和甲醇按体积比1:1混合后,得氯仿-甲醇混合液(脱脂溶液),于通风橱内将SIS膜浸泡在脱脂溶液中12h,每小时搅拌一次,使SIS膜与液体充分接触。
(4)冲洗:倒掉脱脂溶液,用大量流动清水反复冲洗脱脂后的膜,直至没有任何有机溶剂气味,去离子水再漂洗3遍,每次5min。
(5)胰酶脱细胞:将SIS膜浸泡于浓度为0.25%w/v的胰蛋白酶溶液中,置于4℃环境中处理12h,大量去离子水反复漂洗,去除胰蛋白酶。
(6)SDS脱细胞:在1000mL去离子水中加入5g SDS粉末和9g NaCl粉末,配制成0.5%w/v的脱细胞溶液,将拧干的膜放入其中,室温处理4h,大量去离子水反复漂洗,去除SDS。
(7)冻干和灭菌:将处理好的膜铺平,真空冷冻干燥机中冻干,密封,环氧乙烷37℃灭菌保存,即得SIS膜。
2、原花青素交联SIS膜
1)交联溶液的配制:称取0.01g的PC粉末溶于100mL PBS溶液中,配制成浓度为0.01%的交联溶液,避光保存;
2)SIS膜的交联:将一张SIS膜(100cm2)放入100mL配制好的交联溶液中,37℃恒温摇床中避光交联48h;
3)交联后SIS膜的清洗:将交联后的SIS膜先用PBS反复洗涤,再用去离子水反复洗涤,直至交联溶液清洗干净;
4)冻干与灭菌:将交联后的SIS膜铺平,黏膜面朝上,真空冷冻干燥机中冻干,密封,环氧乙烷37℃灭菌,即得本发明膀胱修复支架材料(PC-SIS)。
实施例2、本发明膀胱修复支架材料的制备
1、SIS膜的制备
与实施例1相同。
2、原花青素交联SIS膜
1)交联溶液的配制:称取0.05g的PC粉末溶于100mL PBS溶液中,配制成浓度为0.05%的交联溶液,避光保存;
2)SIS膜的交联:将一张SIS膜(100cm2)放入100mL配制好的交联溶液中,37℃恒温摇床中避光交联48h;
3)交联后SIS膜的清洗:将交联后的SIS膜先用PBS反复洗涤,再用去离子水反复洗涤,直至交联溶液清洗干净;
4)冻干与灭菌:将交联后的SIS膜铺平,黏膜面朝上,真空冷冻干燥机中冻干,密封,环氧乙烷37℃灭菌,即得本发明膀胱修复支架材料(PC-SIS)。
实施例3、本发明膀胱修复支架材料的制备
1、SIS膜的制备
与实施例1相同。
2、原花青素交联SIS膜
1)交联溶液的配制:称取0.1g的PC粉末溶于100mL PBS溶液中,配制成浓度为0.1%的交联溶液,避光保存;
2)SIS膜的交联:将一张SIS膜(100cm2)放入100mL配制好的交联溶液中,37℃恒温摇床中避光交联48h;
3)交联后SIS膜的清洗:将交联后的SIS膜先用PBS反复洗涤,再用去离子水反复洗涤,直至交联溶液清洗干净;
4)冻干与灭菌:将交联后的SIS膜铺平,黏膜面朝上,真空冷冻干燥机中冻干,密封,环氧乙烷37℃灭菌,即得本发明膀胱修复支架材料(PC-SIS)。
实施例4、本发明膀胱修复支架材料的制备
1、SIS膜的制备
与实施例1相同。
2、原花青素交联SIS膜
1)交联溶液的配制:称取0.3g的PC粉末溶于100mL PBS溶液中,配制成浓度为0.3%的交联溶液,避光保存;
2)SIS膜的交联:将一张SIS膜(100cm2)放入100mL配制好的交联溶液中,37℃恒温摇床中避光交联48h;
3)交联后SIS膜的清洗:将交联后的SIS膜先用PBS反复洗涤,再用去离子水反复洗涤,直至交联溶液清洗干净;
4)冻干与灭菌:将交联后的SIS膜铺平,黏膜面朝上,真空冷冻干燥机中冻干,密封,环氧乙烷37℃灭菌,即得本发明膀胱修复支架材料(PC-SIS)。
实施例5、本发明膀胱修复支架材料的制备
1、SIS膜的制备
与实施例1相同。
2、原花青素交联SIS膜
1)交联溶液的配制:称取0.5g的PC粉末溶于100mL PBS溶液中,配制成浓度为0.5%的交联溶液,避光保存;
2)SIS膜的交联:将一张SIS膜(100cm2)放入100mL配制好的交联溶液中,37℃恒温摇床中避光交联48h;
3)交联后SIS膜的清洗:将交联后的SIS膜先用PBS反复洗涤,再用去离子水反复洗涤,直至交联溶液清洗干净;
4)冻干与灭菌:将交联后的SIS膜铺平,黏膜面朝上,真空冷冻干燥机中冻干,密封,环氧乙烷37℃灭菌,即得本发明膀胱修复支架材料(PC-SIS)。
以下通过具体的试验例来证明本发明的有益效果。
按照实施例1制备的未交联的SIS膜命名为SIS,实施例1~5制备的膀胱修复支架材料(PC-SIS)按PC交联溶液的浓度分别命名为0.1mg/ml PC、0.5mg/ml PC、1mg/ml PC、3mg/ml PC和5mg/ml PC。
试验例1、本发明膀胱修复支架材料的结构观察
1、试验方法
取按照实施例1SIS膜制备方法制备的未交联的SIS膜(SIS),以及实施例1~5制备的不同PC浓度交联后的SIS膜(PC-SIS),采用数码相机拍摄的SIS及各组PC-SIS的大体结构。并将SIS及各组PC-SIS进行二氧化碳临界点干燥,利用导电胶分别将其粘贴于样品台上,黏膜面向上,进行喷金,利用SEM观察其超微结构。
2、试验结果
本发明膀胱修复支架材料的大体结构和超微结构如图1所示。由图1可知:未交联的SIS膜为白色,经原花青素交联后SIS膜由白色变为红棕色,且交联浓度越高,颜色越深。通过SEM可以观察到SIS黏膜面为多孔结构,且孔径不规则。同时,通过SEM观察到交联SIS膜与未交联SIS膜的黏膜面差异较为显著,未交联的SIS纤维纤细,排列无序,孔径不规则,SIS经PC交联后纤维直径变粗,且排列呈现出一定的方向性,可能与PC与SIS之间形成氢键,发生交联有关。
试验例2、茚三酮比色法检测PC交联指数
1、试验方法
茚三酮溶液的配制:称取1.05g的柠檬酸和0.04g的氯化亚锡溶于10mL(1.0M)的NaOH,加去离子水调至25mL。另将1g茚三酮粉末溶解于25mL的乙二醇甲醚中。再将上述两液体混合搅拌45min,制备成茚三酮溶液,储存于棕色瓶中避光保存备用。
标曲绘制:称取0.1g的甘氨酸溶解于1000mL的去离子水中,制备浓度为0.01%的甘氨酸标准溶液待用。分别吸取50、100、150、200、250、500μL的0.01%的甘氨酸标准溶液,滴加到1mL的茚三酮溶液中,100℃下水浴加热20min,冷却至室温,加5mL 50%的异丙醇混合摇匀,吸取200μL液体加入96孔板中,用酶标仪在波长570nm下测量吸光值,绘制标准曲线。
分别称取实施例1SIS膜制备方法制备的未交联的SIS膜、实施例1~5制备的不同PC浓度交联后的SIS膜(PC-SIS)各1.5mg,将其浸入1mL茚三酮溶液中,100℃下水浴加热20min,冷却至室温后加5mL50%的异丙醇混合摇匀,吸取200μL液体加入96孔板中,采用酶标仪在波长570nm下测量吸光值。根据甘氨酸标准曲线计算SIS和各组PC-SIS中自由氨基的含量,所有反应均在避光条件下进行。交联指数按下述公式计算:
CI(%)=(MS-MG)/MS×100%
其中CI表示交联指数,MS表示未交联的SIS膜中所含有的自由α-氨基酸的摩尔数,MG表示各组PC-SIS中α-氨基酸的摩尔数。
2、试验结果
原花青素通过与SIS中的蛋白肽链发生反应进行交联。如图2所示,原花青素交联后SIS的自由氨基显著下降,未交联的SIS膜吸光值为0.446,计算得到不同浓度交联PC-SIS交联指数范围为0.2%-65,其中,1mg/ml PC的交联指数为44.2%。
试验例3、抗酶解性能评价
1、试验方法
将实施例1SIS膜制备方法制备的未交联的SIS膜(SIS),以及实施例1~5制备的不同PC浓度交联后的SIS膜(PC-SIS)制备成圆形(直径为1.5cm),将样品浸泡在1.8mg/ml的胶原酶溶液中,置于37℃水浴摇床内,分别在1h、2h、4h和24h取出材料,洗涤冻干,用微量天平测定材料的质量。剩余质量百分比按如下公式计算:
W%=Me/Mi×100%
其中Mi为材料的初始质量,Me为不同时间点材料从胶原酶溶液中取出冻干后的质量,W为剩余质量百分比。
2、试验结果
图3为未交联的SIS以及各组PC-SIS在不同时间点酶解统计数据结果。如图3所示,未交联的SIS 24h内在胶原酶溶液中已经基本完全降解,降解率为90%;低浓度PC交联的PC-SIS(0.5mg/ml PC)在24h内降解率为46%,较低浓度PC交联的PC-SIS(1mg/ml PC)和高浓度PC交联的PC-SIS(3mg/ml PC和5mg/ml PC)在胶原酶溶液中均较稳定,24h内降解率均在20%以内。体外降解实验表明交联SIS具有显著的抗酶解能力,且抗酶解能力随着交联浓度的增加而增加。
试验例4、兔膀胱平滑肌细胞(R-B-SMCs)原代提取与鉴定
1、试验方法
首先于无菌条件下获取兔膀胱组织,将膀胱组织浸泡于PBS缓冲液中,运用显微器械在肉眼下剥离膀胱黏膜层和浆膜层,暴露出膀胱平滑肌。将剥离的平滑肌层放入庆大霉素溶液(100U/ml)浸泡10min,再放入D-hank’s液中浸泡5min。将平滑肌组织剪成肉糜样,置于II型胶原酶(1mg/ml)溶液中37℃条件下震荡消化4h后离心(1000r/min)10min,弃上清,再加入0.05%胰酶37℃消化30min,再离心5min,将沉淀细胞移入24孔板内,加入含15%胎牛血清的高糖培养基中,置于5%CO2,37℃培养箱中静置培养,培养液每2-3天更换一次。培养至第七天,细胞拟合度为90%时,行免疫荧光染色和流式细胞表面标记物分析,对原代平滑肌细胞进行生物学鉴定。
2、试验结果
原代细胞提取三天后可见少量细胞贴壁,增殖至第七天细胞拟合度达到90%,免疫荧光及流式分析检测平滑肌相关蛋白α-SMA、Desmin及Myosin均为强阳性表达(图4),细胞纯度≥80%。
试验例5、活性染色评价材料细胞毒性
1、试验方法
用打孔器将实施例1SIS膜制备方法制备的未交联的SIS膜(SIS),以及实施例1~5制备的不同PC浓度交联后的SIS膜(PC-SIS)分别制备成直径16mm的圆形,低温环氧乙烷灭菌待用(材料使用前先用基础培养基在细胞培养箱内水化2h)。以2×104个/每孔的细胞密度将R-B-SMCs接种于24孔板的膜片上,37℃,5%CO2条件下培养5d,PBS洗3遍,加入预先配制好的浓度为1μg/ml的钙黄绿素(AM)和1μg/ml的碘化丙啶(PI),避光孵育30min后在共聚焦显微镜下对材料上的细胞形态进行层扫观察。
2、试验结果
活性染料结合膜脂质在荧光显微镜下激发出绿色荧光标记活细胞,PI只能穿透死细胞的细胞膜,在荧光显微镜下激发出红色荧光。染色结果如图5所示,图5显示:未交联的SIS及低浓度PC交联的PC-SIS(0.1mg/ml PC、0.5mg/ml PC及1mg/ml PC),均未见明显红色荧光,而高浓度PC交联的PC-SIS(3mg/ml PC和5mg/ml PC)有少量死细胞存在。结果表明低浓度PC交联的PC-SIS无明显细胞毒性作用,可以很好的支持R-B-SMCs在材料上生长。
试验例6、CCK-8检测细胞增殖
1、试验方法
将实施例1SIS膜制备方法制备的未交联的SIS膜(SIS),以及实施例1~5制备的不同PC浓度交联后的SIS膜(PC-SIS)裁剪成矩形(2cm×3cm,表面积约为12cm2),37℃环氧乙烷灭菌。按照受试材料表面积/浸提液体积=6:1,将材料浸泡在2mL含10%FBS高糖培养基中,无菌条件下置于37℃恒温培养箱中浸提24h。低温离心机中4℃,12000g,离心5min,吸取上清液制得浸提原液,4℃保存待用。用胰蛋白酶收集对数期生长的兔原代膀胱平滑肌细胞(R-B-SMCs),并用细胞计数板进行计数。将细胞浓度稀释为2×105个细胞/mL,按3000个细胞/孔接种于96孔板内,培养24h使细胞充分贴壁,弃掉原液加入材料浸提液。阴性对照组为正常的DMEM,每组3个副孔,细胞孵箱内进行培养,隔天换液。用无血清培养基按1:10将CCK8原液进行稀释(现用现配)配制成工作液。分别于第1d、3d、5d和7d弃去原有培养基,每孔加入200μL的CCK8工作液,37℃条件下避光孵育2h,采用酶标仪在450nm波长处测定各孔光吸收值。
2、试验结果
CCK8结果(图6)显示高浓度PC交联的PC-SIS(3mg/ml PC和5mg/ml PC)浸提液可显著抑制R-B-SMCs的增殖,表明其具有明显的细胞毒性,而未交联的SIS组与低浓度PC交联的PC-SIS(0.1mg/ml PC、0.5mg/ml PC和1mg/ml PC)OD值无明显差异,表明低浓度PC交联的PC-SIS材料无明显细胞毒性。通过以上交联材料交联度及细胞增殖活性检测,拟采用0.5mg/ml PC及1mg/ml PC进行后续实验。
试验例7、香兰素法检测PC释放
1、试验方法
香兰素法是利用PC在酸性条件下与香兰醛发生显色反应来测定PC的含量。显色液配制:称取2g香草醛溶于甲醇溶液中,并定容至100ml,作为A液,量取8ml浓盐酸溶于甲醇中定容至100ml,作为B液,将A液与B液按1:1(v:v)体积比混合,配成显色液,现用现配。标曲绘制:分别取10μg/ml-500μg/ml的PC标准品甲醇溶液20μl于试管中,依次加入50μl A液和50μl B液,混匀后30℃水浴避光反应20min,以甲醇作为标准品溶液作为空白对照,酶标仪下500nm处测反应液的吸光度值,绘制标准曲线。
PC浸提液:按照ISO推荐的材料与浸提液比例进行浸提液制备。PC-SIS膜片(实施例2制备的0.5mg/ml PC和实施例3制备的1mg/ml PC)分别与DMEM培养液按照6cm2/ml的比例进行浸提,置于37℃水浴摇床中浸提。分别于1d、3d、5d、7d、9d吸取浸提液,用0.22μm滤膜抽滤除菌得到浸提原液。分别取20μl浸提液于试管中,依次加入50μl A液和50μl B液,混匀后30℃水浴避光反应20min,酶标仪下500nm处测反应液的吸光度值,根据标准曲线测出浸提液中PC的含量。
2、试验结果
原花青素的释放柱状图(图7)结果显示:1mg/ml PC浸泡于培养基中初期释放快速,第1d释放量大,为0.142ng/mg,第3d释放量急剧减少,为0.073ng/mg,且3d-9d释放量较稳定,释放量无统计学差异(P>0.05)。而0.5mg/ml PC由于交联浓度较低,从第一天开始释放量就较少,为0.069ng/mg,且1d-9d释放量均较稳定,无统计学差异。
试验例8、力学性能检测
1、试验方法
将实施例1SIS膜制备方法制备的未交联的SIS膜(SIS),以及实施例2制备的0.5mg/ml PC和实施例3制备的1mg/ml PC裁剪成长度为5cm,中间平行部分为4mm的哑铃型,以10mm/min的速率对材料进行单轴拉伸测试,测定材料拉伸应力在最大值时的载荷以及弹性模量。
2、试验结果
如图8所示,交联后SIS的力学性能有显著的提升。未交联的SIS的刚度为9.5±0.7Mpa,随着交联浓度的增加,刚度逐渐增强,其中0.5mg/ml PC的刚度为13.8±1.4Mpa,而1mg/ml PC的刚度为21.8±1.15Mpa(P<0.05)。而最大拉伸应变位移交联前与交联后无统计学差异(P>0.05)。交联后弹性模量变化趋势与刚度一致。由此可见,原花青素交联可显著改善SIS的力学性能。
试验例9、溶血实验检测材料的血液相容性
1、试验方法
健康人静脉抗凝血10ml加入生理盐水100ml,混匀后2000r离心10min,去除上清液,如此方法用生理盐水反复洗3-4次,至上清液无色透明为止。将洗涤好的红细胞用生理盐水配制成2%的细胞悬液备用。将实施例1SIS膜制备方法制备的未交联的SIS膜(SIS),以及实施例2制备的0.5mg/ml PC和实施例3制备的1mg/ml PC分别裁剪为5cm×5cm大小(n=3),置于离心管中并加入1200μl的D-Hank’s溶液,37℃预热30min和300μl红细胞悬液混合,轻轻摇匀,室温静置4h(37℃孵育2h),随后将各组混合物4000r离心2min,然后进行溶血现象拍照;并吸取200μl上清液用酶标仪在545nm波段下进行吸光度值检测。其中阳性对照组与阴性对照组分别取1200μl的蒸馏水和1200μl的0.9%NaCl溶液,操作同上。根据如下公式计算溶血率:
HR(%)=[(ODt-ODn)/(ODp-ODn)]×100%
其中HR表示溶血率,ODt表示待测样本的吸光值,ODn表示阴性对照组的吸光值,ODp表示阳性对照组的吸光值。
2、试验结果
材料溶血情况的肉眼观察结果如图9所示。阳性对照的红细胞全部破裂,试管底部未见红细胞沉淀,发生严重的溶血反应。未交联SIS组和交联SIS组的上清液透明澄清、红细胞沉淀于材料上或试管底部,未见明显破裂,与阴性对照组相似。未交联SIS组和交联SIS组的溶血率均小于5%。
试验例10、PC-SIS抗钙化能力体外评价
1、试验方法
分别将实施例1SIS膜制备方法制备的未交联的SIS膜(SIS),以及实施例2制备的0.5mg/ml PC和实施例3制备的1mg/ml PC按照100cm2/10ml模拟体液(SBF)的比例浸入SBF中,于37℃水浴摇床中以120rpm的速率持续摇动浸泡30d,每5d换液一次。30d后取出材料,用D-Hank’s溶液清洗,冷冻干燥机干燥,喷金后进行SEM-EDS(扫描电镜能谱)分析。模拟体液(SBF)浸泡30d后,将SIS和PC-SIS冷冻干燥,称重(约10-15mg),剪碎成小颗粒(<1mm3),并溶于2ml 0.02mol/L的HCL溶液中搅拌12h以溶解出Ca、P成分,然后上机检测,不同材料的Ca、P定量分析通过电感耦合等离子原子发射(ICP)光谱仪检测得出。
2、试验结果
未交联的SIS及不同PC浓度交联的PC-SIS样品在模拟体液中浸泡30d后,扫描电镜及能谱分析结果直观显示(图10):未交联SIS上有少量钙盐沉积,而PC-SIS材料上钙盐沉积明显减少,且随着交联浓度的增高,抑制钙盐沉积的作用越明显。ICP钙磷含量定量分析结果显示:未交联的SIS的Ca/P在1.6-1.8之间,与羟基磷灰石的Ca/P比1.5十分接近,说明材料发生了钙化,PC-SIS的Ca/P比大于1.9,说明PC交联SIS材料未发生显著钙化。
试验例11、傅里叶红外检测交联化学键变化
1、试验方法
采用衰减全反射(ATR)的方法对实施例1SIS膜制备方法制备的未交联的SIS膜、交联剂PC、实施例2制备的0.5mg/ml PC和实施例3制备的1mg/ml PC进行测试,扫描波数范围为500~4000cm-1。
2、试验结果
1645cm-1为酰胺I带特征吸收峰,1544cm-1为酰胺II带特征吸收峰,1236cm-1为酰胺III带特征吸收峰,3307cm-1为氢键特征吸收峰;图11可知,相比未交联的SIS,不同浓度PC交联得到的PC-SIS氢键吸收峰变宽,证明有氢键形成;此外,酰胺Ⅱ带能灵敏地反映分子间或分子内的氢键缔合作用,当氢键数增加时,谱带会向低波数移动,交联后的PC-SIS酰胺II带所对应的波数都向低波数移动,证明有氢键形成,PC和SIS发生了交联。实验结果表明,PC与SIS之间通过氢键交联。
试验例12、接触角实验检测交联材料亲水性
1、试验方法
将实施例1SIS膜制备方法制备的未交联的SIS膜、实施例2制备的0.5mg/ml PC和实施例3制备的1mg/ml PC剪成1cm×2cm大小,每组3片,用接触角测量仪测量3μL水滴接触材料第1秒时的接触角大小,每片测2个点。
2、试验结果
图12水接触角实验的直观图以及柱状图结果可知两组PC交联的SIS与未交联的SIS相比,接触角均有所减小。其中未交联SIS接触角为85.25±6.11,0.5mg/ml PC和1mg/mlPC接触角显著降低,1mg/ml PC接触角为67.25±7.04,0.5mg/ml PC的接触角最小,为48.75±8.27。结果表明原花青素交联后的SIS亲水性比未交联SIS亲水性显著提高。
试验例13、F-actin染色评价细胞在材料上的黏附分布特点
1、试验方法
将实施例1SIS膜制备方法制备的未交联的SIS膜、实施例2制备的0.5mg/ml PC-SIS和实施例3制备的1mg/ml PC-SIS分别放入24孔板中(水化条件同试验例5活性染色。),以2×104个/每孔的细胞密度将R-B-SMCs分别接种于24孔板的膜片上,37℃,5%CO2条件下培养5d,PBS洗3遍,使用溶于PBS的4%甲醛溶液室温固定30min,用丙酮脱水或者用0.5%TritonX-100溶液透化处理5min,取200μl配置好的TRITC标记鬼笔环肽工作液室温避光孵育30min,使用200μlDAPI(浓度100nm)对细胞核进行复染,约30s。在共聚焦显微镜下对材料上的细胞形态进行观察。
2、试验结果
F-actin细胞骨架染色结果(图13)显示R-B-SMCs在未交联的SIS膜片上细胞排列紊乱,无方向性和规律性,而平滑肌细胞在PC交联后的SIS膜片上呈束装分布,生长有一定方向性。
试验例14、SEM观察细胞在材料上的超微结构
1、试验方法
将实施例1SIS膜制备方法制备的未交联的SIS膜、实施例2制备的0.5mg/ml PC和实施例3制备的1mg/ml PC裁剪成直径为16mm的圆片,37℃环氧乙烷灭菌,于通风橱中放置1周,待用。使用时置于24孔板中,材料水化条件同试验例5活性染色。取对数生长期的R-B-SMCs细胞,胰蛋白酶消化细胞,并用细胞计数板计数,稀释成2×106个/mL的细胞悬液,每孔加入1×104个细胞。隔天换液,于培养第5天收样。将复合细胞的材料于2.5%的戊二醛溶液中固定4h,用蒸馏水清洗3次,注意小心清洗,避免材料上的细胞脱落。经梯度酒精脱水(30%、50%、70%、85%、95%、100%),每次脱水10min。样品进行二氧化碳临界点干燥,贴导电胶,将样品固定于载样台上,喷金和SEM检采图。
2、试验结果
扫描电镜从微观层面展示了R-B-SMCs细胞在材料上的分布,SEM结果(图14)显示平滑肌细胞在未交联的SIS和PC交联的SIS上增殖良好,细胞均有伪足伸出。同时,与未交联SIS相比,交联后SIS的膜片伪足伸展更明显,且能观察到大量分泌的胞外基质因子,说明交联后的SIS膜片更利于细胞的生长。
试验例15、Q-PCR检测R-B-SMCs在不同材料上平滑肌相关基因表达水平
1、试验方法
将实施例1SIS膜制备方法制备的未交联的SIS膜、实施例2制备的0.5mg/ml PC和实施例3制备的1mg/ml PC 37℃环氧乙烷灭菌后水化处理(水化条件同试验例5活性染色),然后取对数生长期的R-B-SMCs细胞,胰蛋白酶消化细胞,并用细胞计数板计数,细胞接种密度同试验例5活性染色。隔天换液,于培养第5天胰蛋白酶消化清洗,溶于Trizol。使用RNA提取试剂盒Foregene对细胞内的总RNA进行抽提,Takara逆转录试剂盒将抽提的RNA反转为cDNA。根据Takara试剂商提供的试剂方法进行聚合酶链反应。
2、试验结果
平滑肌相关基因表达水平如图15所示。由图15可知:与未交联的SIS相比,将平滑肌细胞接种于PC交联后的SIS(即PC-SIS材料)上,平滑肌相关基因(α-SMA、SMHHC、Desmin及Myosin)均有明显上调,其中成熟平滑肌标记物Myosin表达水平升高最为明显,且1mg/mlPC交联的SIS平滑肌相关标记物显著高于未交联的SIS。
试验例16、兔膀胱全层缺损模型构建
1、试验方法
本研究实验动物均为2.5±5kg的雄性新西兰大白兔,实验分为3组,每组有3个取材观察时间点,每个取材时间点有3只实验动物。术前一天对拟实施手术的动物禁食,术前使用固定器将实验动物固定,沿耳缘静脉缓慢注射戊巴比妥钠(2mL/kg)。将麻醉好的实验动物仰卧位摆放于手术板,绳子固定四肢,使用剃毛器将兔胸骨角以下会阴部以上毛发剃除,用碘伏溶液进行消毒处理,铺放治疗巾,留出无菌手术操作区域。以耻骨联合与腹直线交点为基点,纵向切开约4cm皮肤切口,钝性分离皮下组织,切开肌肉层,暴露膀胱。(由于手术时兔子膀胱尿液储量存在个体差异,故可采用插尿管排尿或注射无菌生理盐水等,将膀胱调整至大小合适),在兔膀胱后壁构建一个2cm×2cm的方形全层缺损模型。将环氧乙烷灭菌后2cm×2cm的方形材料(实施例1SIS膜制备方法制备的未交联的SIS膜、实施例2制备的0.5mg/ml PC和实施例3制备的1mg/ml PC)覆盖于缺损处,用5-0可吸收缝线将材料与膀胱全层缝合在一起,缝合结束后用5-0不可吸收线在浆膜层于方形缺损的四角标记。关腹,缝合肌层和皮肤。手术完成后,用碘伏溶液擦拭伤口进行消毒,放回笼中。术后两天常规每天肌肉注射青霉素40万单位预防感染。
2、试验结果
手术具体流程如图16所示。
试验例17、修复膀胱取材大体观
1、试验方法
本试验例在试验例16的基础上进一步完成。
取材:各组(未交联SIS、0.5mg/ml PC、1mg/ml PC)分别在试验例16术后4周、8周和12周取材,每组每个时间点选取3只兔子。大体观察:打开腹腔后,首先观察修复区域的粘连情况,若有粘连,则从粘连组织中游离出膀胱组织,整体取下膀胱后,沿脂肪垫侧剖开,观察膀胱内壁异物结石、材料降解等情况,并拍照记录。
2、试验结果
图17为不同材料修复组取材大体观察,由图17可知,术后4周取材,各组修复区域与肠壁粘连严重,修复区域较正常区域膀胱壁较薄,未交联SIS组较易发生水肿,发生率为60%;术后8周取材,各组未见修复区与肠壁粘连,膀胱内壁可观察到明显的“溃疡”样变化,其中未交联SIS膀胱壁最薄,交联修复组较未交联SIS组大体观察膀胱壁更厚,且交联浓度越高,膀胱修复大体观与正常膀胱越接近;术后12周取材,膀胱壁较8周有明显增厚,1mg/mlPC交联组膀胱内壁修复效果较好,与周围正常组织厚度相近。
试验例18、HE及Masson染色评价膀胱修复效果
1、试验方法
本试验例接着试验例17进一步完成。
将4周、8周及12周膀胱修复区域组织取材后,常规固定,包埋切片,脱蜡。苏木精染色:将复水后的组织切片浸入苏木精染色中,使其对细胞核着色,10min后用流动自来水冲洗5min。将组织切片浸入伊红染色,使其对细胞质着色,5min后流动自来水冲洗5min,脱水,封片。Masson染色:Masson复合液5min,1%盐酸酒精分化,丽春红酸性品红染液染5min、流水冲洗、磷钼酸溶液5min、甲苯胺蓝染色复染5min,1%冰醋酸1min,脱水,封片。
2、试验结果
图18为HE染色观察不同材料修复组术后膀胱组织学形态,图19为Masson染色观察不同材料修复组术后膀胱组织学形态。由图18和图19可知:术后4周,未交联SIS组膀胱壁较薄,新生上皮细胞逐渐迁移至黏膜层,附近有较多新生血管,无明显平滑肌再生。0.5mg/mlPC组黏膜层较为完整、连续,修复区胶原含量显著高于未交联SIS组,1mg/mlPC组可见少量平滑肌纤维再生。术后8周,未交联SIS组上皮结构完整,膀胱壁仍较薄,肌层新生组织中成纤维细胞居多。PC-SIS组膀胱上皮有皱襞样结构形成,膀胱壁厚度与未交联SIS组相比较厚,交联组均可见平滑肌再生,其中0.5mg/ml PC平滑肌呈散在分布,而1mg/mlPC组可见束状平滑肌再生。术后12周,未交联SIS组新生组织主要为胶原纤维;PC-SIS组可观察到明显的平滑肌束,相较于0.5mg/mlPC组,1mg/mlPC组平滑肌更成熟。
综上,本发明膀胱修复支架材料具有良好的生物相容性,更优的力学性能和抗酶解能力。能够有效避免单纯SIS应用于膀胱全层缺损修复过程中容易出现的挛缩、钙化等并发症,同时具有良好的促平滑肌再生效果,克服了现有软组织修复材料成肌困难的问题,具有良好的临床应用前景。
Claims (4)
1.一种膀胱修复支架材料在制备促进膀胱平滑肌再生的膀胱修复支架中的用途,其特征在于:所述膀胱修复支架材料由原青花素交联小肠黏膜下层膜后而得;所述交联包括如下步骤:将小肠黏膜下层膜浸泡在原青花素溶液中,交联,洗涤,冻干,即可;所述原青花素溶液的浓度为1mg/mL;所述小肠黏膜下层膜的面积与原青花素溶液的体积比为50-200cm2:100mL;所述交联的温度为35-40℃,时间为24-96h;交联指数为44.2%;所述小肠黏膜下层膜的制备方法如下:取小肠,去除浆膜层、肌层和黏膜层,保留黏膜下层,脱脂,脱细胞,清洗,冻干,即得;所述脱脂是用体积比为1:1的甲醇和氯仿的混合溶液浸泡6-24小时;所述脱细胞为在4-10℃中,于胰蛋白酶溶液中浸泡6-24小时后,再在十二烷基硫酸钠与氯化钠的混合溶液中处理3-5h;所述胰蛋白酶溶液的浓度为0.01-10%w/v,所述十二烷基硫酸钠的浓度为0.01%-1%w/v,所述氯化钠的浓度范围为0.01%-1%w/v。
2.根据权利要求1所述的用途,其特征在于:所述小肠黏膜下层膜的面积与原青花素溶液的体积比为100cm2:100mL。
3.根据权利要求1所述的用途,其特征在于:所述交联的温度为37℃,时间为48h。
4.根据权利要求1所述的用途,其特征在于:所述脱脂是用体积比为1:1的甲醇和氯仿的混合溶液浸泡12小时;所述脱细胞为在4℃中,于胰蛋白酶溶液中浸泡12小时后,再在十二烷基硫酸钠与氯化钠的混合溶液中室温处理4h;所述胰蛋白酶溶液的浓度为0.25%w/v,所述十二烷基硫酸钠的浓度为0.5%w/v,所述氯化钠的浓度范围为0.9%w/v。
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