CN106581772B - 携带G-CSF缓释***的Fibrin-HA水凝胶及其制备方法和用途 - Google Patents
携带G-CSF缓释***的Fibrin-HA水凝胶及其制备方法和用途 Download PDFInfo
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Abstract
本发明公开了一种软骨修复材料,它含有粒细胞集落刺激因子以及药学上可接受的辅料或者辅助性成分。本发明还公开了前述修复材料的制备方法以及粒细胞集落刺激因子的新用途。本发明携带G‑CSF缓释明胶微球的Fibrin‑HA水凝胶可以有效诱导内源性干细胞/前体细胞归巢,软骨修复效果优良。
Description
技术领域
本发明涉及一种携带G-CSF缓释***的Fibrin-HA水凝胶及其制备方法和用途。
背景技术
关节软骨属于透明软骨,它覆盖于关节的表面,由软骨细胞和基质构成,软骨细胞占软骨组织的5%或更少,基质主要有蛋白多糖凝胶以及II型胶原构成,蛋白多糖约占软骨干重的35%,胶原约占软骨干重的60%,起着缓冲应力、吸收震荡、润滑关节表面、防止磨损等重要作用。造成关节软骨损伤的原因多种多样,包括关节受到暴力挤压或撕裂受到的急性损伤以及长期大运动量、高负荷运动对关节造成的慢性磨损。也有研究表明,关节长期缺乏活动也会造成关节软骨退行性变。根据软骨损伤的深度可以分为以下两种类型:部分厚度的软骨损伤,即缺损深度不超过软骨钙化层和全层关节软骨损伤,即缺损超过软骨钙化层。临床上常根据国际软骨修复协会(ICRS)分级法:0级为正常的关节软骨;I级为软骨肿胀、软化;II级为软骨表面缺损<软骨全层的50%;III级为软骨表面缺损>软骨全层的50%,但软骨下骨未暴露;IV级为全层软骨缺损,软骨下骨裸露或缺损。软骨损伤后最终会导致软骨退变从而发生骨关节炎,出现严重的疼痛并致残、致畸,最终需要行人工关节置换。
软骨损伤修复是目前医学研究尚未攻克的难题之一,主要原因在于其缺少血管、***及神经的组织,难以再生,一经损伤,修复起来十分困难。
目前常用的关节软骨损伤修复方案包括自体软骨移植、微骨折以及组织工程软骨等,但存在自体软骨块或软骨细胞来源受限、移植软骨退变为瘢痕组织、软骨细胞***培养增加疾病传播以及肿瘤风险等,治理效果不佳。
发明内容
为了解决上述问题,本发明提供了一种新的软骨修复材料,及其制备方法和用途。
本发明提供了粒细胞集落刺激因子在制备软骨修复材料中的用途。
其中,所述修复材料是诱导软骨干细胞/前体细胞归巢的材料。
本发明还提供了前述软骨修复材料,它含有粒细胞集落刺激因子以及药学上可接受的辅料或者辅助性成分。
其中,所述修复材料是携带粒细胞集落刺激因子的纤维蛋白/透明质酸水凝胶。
其中,所述修复材料是携带粒细胞集落刺激因子明胶微球的纤维蛋白/透明质酸水凝胶。
其中,所述修复材料,明胶微球直径<50um。
本发明还提供了一种制备前述材料的方法,步骤如下:
a、制备制纤维蛋白原溶液、凝血酶溶液、透明质酸溶液、粒细胞集落刺激因子溶液、明胶微球;
b、将明胶微球加入粒细胞集落刺激因子溶液中,获得携带粒细胞集落刺激因子的明胶微球;
c、将凝血酶溶液、透明质酸溶液和明胶微球混合,得混合溶液,再加入纤维蛋白原溶液,混合,即可。
其中,步骤a中,所述纤维蛋白原溶液中纤维蛋白原的质量/体积分数为10~100mg/ml,优选50mg/ml;
所述所述透明质酸溶液中透明质酸的质量/体积分数为5~10mg/ml,优选10mg/ml;
所述凝血酶溶液中凝血酶的质量/体积分数的50~100units/ml,优选100units/ml;
所述粒细胞集落刺激因子溶液的质量/体积分数为100~4000ng/ml,优选2000ng/ml;
所述明胶微球直径<50um。
其中,所述溶液和微球按照如下方法制备:
(1)配制纤维蛋白原溶液:将0.9%生理盐水加入一定质量的纤维蛋白原粉末中,37℃水浴10分钟,形成纤维蛋白原溶液;
(2)配制凝血酶溶液:将0.9%生理盐水加入一定质量的重组凝血酶粉末中,吸管反复吹吸30秒使其均匀溶解;
(3)配制透明质酸溶液:将0.9%生理盐水加入一定质量的透明质酸粉末中,搅拌5min使其均匀溶解备用;
(4)制备G-CSF溶液:将0.9%生理盐水加入一定质量的重组G-CSF蛋白中,吸管反复吹吸30秒使其均匀溶解备用;
(5)制备明胶微球:将明胶粉末加入生理盐水中,配制成质量/体积分数为10%的明胶溶液,然后将明胶溶液逐滴加入30倍体积预热到50℃的橄榄油中,搅拌形成均匀的乳剂,并迅速将乳剂放入冰水中使明胶微粒凝结;再将100ml冰浴处理过的丙酮加入乳剂中使明胶微粒脱水,收获小于50um明胶微粒,再将其加入10.6mmol/L戊二醛溶液中形成共价交联;然后用100mmol/L氨基乙酸溶液洗涤三次去除残留的醛基,并用去粒子水反复洗涤三次,冻干,即可;
(6)制备携带G-CSF蛋白的明胶缓释微球:称取明胶微球,将其加入1~10倍体积/质量的G-CSF溶液中,优选5倍,使溶液完全吸附于明胶微球,冻干,即可。
其中,步骤b中,所述凝血酶溶液、透明质酸溶液和明胶微球的比例为5:7:(2~8),优选为5:7:3;混合溶液与纤维蛋白原溶液的比例为1:1。
其中,步骤c中,混合溶液与纤维蛋白原溶液混合时的水浴温度控制在30-40℃,持续5min以上。
携带G-CSF缓释明胶微球的Fibrin-HA水凝胶具有以下优点:
1、取材来源广泛,各原材料储存条件要求不高,配制简单,并且价格不高;
2、无细胞毒性,具有良好的生物相容性和可降解性;
3、能够缓慢释放G-CSF,后者能够诱导内源性干细胞/前体细胞归巢,从而修复骨骼肌肉***损伤;
4、操作简单,并且具有非常良好的可塑形性,能够很好填充不同深度、形态的组织缺损,使再生组织在形态和组成结构上接近正常组织。
本发明粒细胞可以诱导软骨干细胞/前体细胞归巢,进而促进软骨缺损处的修复,本发明携带G-CSF缓释明胶微粒Fibrin-HA水凝胶可以用于修复软骨缺损,为临床软骨缺损的治疗提供了一种新的选择,临床应用前景优良。
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 Fibrin-HA水凝胶形态学特征以及生物相容性评价;
图2缓慢释放的G-CSF能够诱导软骨内源性干细胞/前体细胞归巢;
图3体内实验证实释放G-CSF的Fibrin-HA水凝胶能够修复软骨缺损;
图4组织学评估携带G-CSF的Fibrin-HA水凝胶修复软骨缺损。
具体实施方式
mM:mmol/L。
实施例1本发明携带G-CSF缓释***的Fibrin-HA水凝胶的制备方法
(1)配制纤维蛋白原溶液:将0.9%生理盐水加入一定质量的纤维蛋白原粉末中,37℃水浴10分钟,形成质量/体积分数为50mg/ml纤维蛋白原溶液;
(2)配制凝血酶溶液:将0.9%生理盐水加入一定质量的重组凝血酶粉末中,吸管反复吹吸30秒使其均匀溶解,形成质量/体积分数的100units/ml的凝血酶溶液;
(3)配制透明质酸溶液:将0.9%生理盐水加入一定质量的透明质酸粉末中,搅拌5min使其均匀溶解备用,形成质量/体积分数为10mg/ml的透明质酸溶液;
(4)制备G-CSF溶液:将0.9%生理盐水加入一定质量的重组G-CSF蛋白粉末中,吸管反复吹吸30秒使其均匀溶解备用,形成质量/体积分数为2000ng/ml的G-CSF溶液;
(5)制备明胶微球:将明胶粉末加入生理盐水中,震荡2分钟配制成质量/体积分数为10%的明胶溶液,然后将10ml明胶溶液逐滴加入300ml预热到50℃的橄榄油,持续搅拌15分钟(500rpm)形成均匀的乳剂,并迅速将乳剂放入冰水中使明胶微粒凝结;其次将100ml冰浴处理过的丙酮加入乳剂中使明胶微粒脱水,并用孔隙直径为20-50um的滤网过滤收获明胶微粒,再将其加入10.6mM戊二醛溶液中过夜形成共价交联;然后用100mM氨基乙酸溶液洗涤三次去除残留的醛基,并用去粒子水反复洗涤三次,冻干并储存在4℃真空容器中备用;
(6)制备携带G-CSF蛋白的明胶缓释微球:称取明胶微球1g,将其加5mlG-CSF溶液,使溶液完全吸附于明胶微球,冻干获得携带G-CSF蛋白的明胶缓释微球并储存在4℃真空容器中备用;
使用方法:步骤(2)、步骤(3)和步骤(6)配制溶液按体积比5:7:3均匀混合,然后与步骤(1)配制溶液按体积比1:1混合,在凝固之前迅速将混合液注射到缺损部位。
实施例2本发明携带G-CSF缓释***的Fibrin-HA水凝胶的制备方法
(1)配制纤维蛋白原溶液:将0.9%生理盐水加入一定质量的纤维蛋白原粉末中,37℃水浴10分钟,形成质量/体积分数为10mg/ml纤维蛋白原溶液;
(2)配制凝血酶溶液:将0.9%生理盐水加入一定质量的重组凝血酶粉末中,吸管反复吹吸30秒使其均匀溶解,形成质量/体积分数的50units/ml的凝血酶溶液;
(3)配制透明质酸溶液:将0.9%生理盐水加入一定质量的透明质酸粉末中,搅拌5min使其均匀溶解备用,形成质量/体积分数为5mg/ml的透明质酸溶液;
(4)制备G-CSF溶液:将0.9%生理盐水加入一定质量的重组G-CSF蛋白粉末中,吸管反复吹吸30秒使其均匀溶解备用,形成质量/体积分数为100ng/ml的G-CSF溶液;
(5)制备明胶微球:将明胶粉末加入生理盐水中,震荡2分钟配制成质量/体积分数为10%的明胶溶液,然后将10ml明胶溶液逐滴加入300ml预热到50℃的橄榄油,持续搅拌15分钟(500rpm)形成均匀的乳剂,并迅速将乳剂放入冰水中使明胶微粒凝结;其次将100ml冰浴处理过的丙酮加入乳剂中使明胶微粒脱水,并用孔隙直径为20-50um的滤网过滤收获明胶微粒,再将其加入10.6mM戊二醛溶液中过夜形成共价交联;然后用100mM氨基乙酸溶液洗涤三次去除残留的醛基,并用去粒子水反复洗涤三次,冻干并储存在4℃真空容器中备用;
(6)制备携带G-CSF蛋白的明胶缓释微球:称取明胶微球1g,将其加入1ml的G-CSF溶液,使溶液完全吸附于明胶微球,冻干获得携带G-CSF蛋白的明胶缓释微球并储存在4℃真空容器中备用;
使用方法:步骤(2)、步骤(3)和步骤(6)配制溶液按体积比5:7:2均匀混合,然后与步骤(1)配制溶液按体积比1:1混合,在凝固之前迅速将
实施例3本发明携带G-CSF缓释***的Fibrin-HA水凝胶的制备方法
(1)配制纤维蛋白原溶液:将0.9%生理盐水加入一定质量的纤维蛋白原粉末中,37℃水浴10分钟,形成质量/体积分数为100mg/ml纤维蛋白原溶液;
(2)配制凝血酶溶液:将0.9%生理盐水加入一定质量的重组凝血酶粉末中,吸管反复吹吸30秒使其均匀溶解,形成质量/体积分数的80units/ml的凝血酶溶液;
(3)配制透明质酸溶液:将0.9%生理盐水加入一定质量的透明质酸粉末中,搅拌5min使其均匀溶解备用,形成质量/体积分数为8mg/ml的透明质酸溶液;
(4)制备G-CSF溶液:将0.9%生理盐水加入一定质量的重组G-CSF蛋白粉末中,吸管反复吹吸30秒使其均匀溶解备用,形成质量/体积分数为4000ng/ml的G-CSF溶液;
(5)制备明胶微球:将明胶粉末加入生理盐水中,震荡2分钟配制成质量/体积分数为10%的明胶溶液,然后将10ml明胶溶液逐滴加入300ml预热到50℃的橄榄油,持续搅拌15分钟(500rpm)形成均匀的乳剂,并迅速将乳剂放入冰水中使明胶微粒凝结;其次将100ml冰浴处理过的丙酮加入乳剂中使明胶微粒脱水,并用孔隙直径为20-50um的滤网过滤收获明胶微粒,再将其加入10.6mM戊二醛溶液中过夜形成共价交联;然后用100mM氨基乙酸溶液洗涤三次去除残留的醛基,并用去粒子水反复洗涤三次,冻干并储存在4℃真空容器中备用;
(6)制备携带G-CSF蛋白的明胶缓释微球:称取明胶微球1g,将其加入10ml的G-CSF溶液,使溶液完全吸附于明胶微球,冻干获得携带G-CSF蛋白的明胶缓释微球并储存在4℃真空容器中备用;
使用方法:步骤(2)、步骤(3)和步骤(6)配制溶液按体积比5:7:8均匀混合,然后与步骤(1)配制溶液按体积比1:1混合,在凝固之前迅速将混合液注射到缺损部位。
以下用实验例的方式来说明本发明的有益效果:
实验例1本发明水凝胶修复软骨的作用
1、制备方法
(1)配制纤维蛋白原溶液:将0.9%生理盐水加入一定质量的纤维蛋白原粉末中,37℃水浴10分钟,形成质量/体积分数为50mg/ml纤维蛋白原溶液;
(2)配制凝血酶溶液:将0.9%生理盐水加入一定质量的重组凝血酶粉末中,吸管反复吹吸30秒使其均匀溶解,形成质量/体积分数的100units/ml的凝血酶溶液;
(3)配制透明质酸溶液:将0.9%生理盐水加入一定质量的透明质酸粉末中,搅拌5min使其均匀溶解备用,形成质量/体积分数为10mg/ml的透明质酸溶液;
(4)制备G-CSF溶液:将0.9%生理盐水加入一定质量的重组G-CSF蛋白粉末中,吸管反复吹吸30秒使其均匀溶解备用,形成质量/体积分数为2000ng/ml的G-CSF溶液;
(5)制备明胶微球:将明胶粉末加入生理盐水中,震荡2分钟配制成质量/体积分数为10%的明胶溶液,然后将10ml明胶溶液逐滴加入300ml预热到50℃的橄榄油,持续搅拌15分钟(500rpm)形成均匀的乳剂,并迅速将乳剂放入冰水中使明胶微粒凝结;其次将100ml冰浴处理过的丙酮加入乳剂中使明胶微粒脱水,并用孔隙直径为20-50um的滤网过滤收获明胶微粒,再将其加入10.6mM戊二醛溶液中过夜形成共价交联;然后用100mM氨基乙酸溶液洗涤三次去除残留的醛基,并用去粒子水反复洗涤三次,冻干并储存在4℃真空容器中备用;
(6)制备携带G-CSF蛋白的明胶缓释微球:称取明胶微球1g,将其加入5mlG-CSF溶液,使溶液完全吸附于明胶微球,冻干获得携带G-CSF蛋白的明胶缓释微球并储存在4℃真空容器中备用。
3、体外实验证实携带G-CSF明胶缓释微球的Fibrin/HA水凝胶诱导软骨干细胞/前体细胞归巢
(1)取山羊膝关节骨软骨块进行体外培养,在软骨上制造直径4mm软骨缺损;
(2)组1的软骨缺损,不做修复处理;
(3)组2的软骨缺损,修复方法是:将第2节步骤(2)、步骤(3)配制溶液按5:7混合,然后与第2节步骤(1)配制溶液按1:1均匀混合,在凝固前迅速将混合液注射到缺损部位;
(4)组3的兔软骨缺损,修复方法是:将第2节步骤(2)、步骤(3)和步骤(6)配制溶液按5:7:3均匀混合,然后与第2节步骤(1)配制溶液按1:1混合,在凝固之前迅速将混合液注射到缺损部位;
(5)在软骨缺损修复后的第1天、第7天和第14天,通过激光共聚焦显微镜观察是否有细胞迁移到软骨缺损修复区域;
(6)将有细胞迁移到软骨缺损修复区域的水凝胶取出,用胶原酶消化后进行多向分化能力鉴定、流式细胞分析表面标记物,确定细胞种类;
4、兔膝关节股骨髁负重区制造软骨缺损模型
(1)在兔耳缘静脉注射浓度为20%氨基甲酸乙酯的麻醉剂,然后膝关节周围备皮,酒精和碘伏先后擦洗膝关节周围皮肤,铺无菌桌布;
(2)膝关节屈曲90°,于膝关节正前方纵行切开长约3cm皮肤,分离皮下组织并显露关节囊和股四头肌腱,沿股四头肌腱及髌骨内侧缘切开关节囊,髌骨向外侧推移完整显露股骨内侧髁;
(3)用直径5mm的磨钻在股骨内侧髁负重区制造软骨缺损,深度约2mm。
5、不同治疗方案修复软骨缺损
(1)组1的兔软骨缺损,不做修复处理,制造软骨缺损后直接缝合关节囊和皮肤;
(2)组2的兔软骨缺损,修复方法是:将第2节步骤(2)、步骤(3)配制溶液按5:7混合,然后与第2节步骤(1)配制溶液按1:1均匀混合,在凝固前迅速将混合液注射到缺损部位,待凝固后反复屈伸膝关节,检查Fibrin-HA水凝胶固定良好后逐层缝合关节囊和皮肤;
(3)组3的兔软骨缺损,修复方法是:将第2节步骤(2)、步骤(3)和步骤(6)配制溶液按5:7:3均匀混合,然后与第2节步骤(1)配制溶液按1:1混合,在凝固之前迅速将混合液注射到缺损部位,待凝固后反复屈伸膝关节,检查携带G-CSF缓释明胶微球的Fibrin-HA水凝胶固定良好后逐层缝合关节囊和皮肤。
6、术后处理
所有大白兔术后连续3天肌肉注射青霉素预防感染,标准饲养程序,不限制其活动。
7、标本采集与处理
术后6周和12周分别采集膝关节标本,每组每次采集10只。首先是于耳缘静脉推注大量空气处死兔子,然后沿原手术切口切开显露膝关节股骨髁,对软骨缺损修复区域进行肉眼评分,拍照,并使用摆锯截取缺损修复组织,放入10%甲醛溶液固定1周,然后脱钙1月。
8、组织切片染色
将经过脱钙处理膝关节标本适当修整后切片,厚度5um,分别行HE染色、阿尔新蓝染色、番红-固绿染色和II型胶原免疫组化染色,进行组织学评价软骨缺损修复效果。
二、实验结果
实验结果如图1~图4所示:
从图1可以看出,Fibrin-HA水凝胶具有良好的可塑形性和降解特性,能够非常良好地充填不同深度、不同形态组织缺损,并且具有良好的生物相容性(图1)。
体外实验结果如图2(对应实验步骤3)所示,仅缓慢释放G-CSF的Fibrin-HA水凝胶能够诱导软骨干细胞/前体细胞(细胞分化鉴定、流式细胞鉴定确定)归巢,而空白缺损和单纯的Fibrin-HA水凝胶对照组则未发现细胞归巢现象(图2),说明G-CSF能诱导软骨干细胞/前体细胞归巢;
体内实验进一步证实了***研究结果如图3软骨大体形态和图4软骨组织学形态所示,在空白对照组和不能释放G-CSF的Fibrin-HA阴性对照组,缺损处在术后6周和12周均清晰可见,而在释放G-CSF的Fibrin-HA实验组在术后6周有软骨样组织完全充填缺损,并且在术后12周完全修复缺损,再生软骨形态学评分接近正常软骨。实验结果说明空白对照和单纯Fibrin-HA水凝胶不能修复软骨缺损,而本发明携带G-CSF缓释明胶微粒Fibrin-HA水凝胶则能够诱导体内内源性干细胞/前体细胞,进而修复兔软骨缺损,进一步说明G-CSF能够诱导体内内源性干细胞/前体细胞,进而修复兔软骨缺损。
实验结果说明,本发明G-CSF可以诱导软骨干细胞/前体细胞归巢,进而修复软骨,本发明携带G-CSF缓释明胶微粒Fibrin-HA水凝胶诱导软骨干细胞/前体细胞归巢并修复软骨的性能优良。
Claims (10)
1.一种软骨修复材料,其特征在于:所述修复材料是携带粒细胞集落刺激因子明胶微球的纤维蛋白/透明质酸水凝胶。
2.根据权利要求1所述的修复材料,其特征在于:所述修复材料中明胶微球直径<50μm。
3.一种制备权利要求1或2所述材料的方法,其特征在于:步骤如下:
a、制备纤维蛋白原溶液、凝血酶溶液、透明质酸溶液、粒细胞集落刺激因子溶液、明胶微球;
b、将明胶微球加入粒细胞集落刺激因子溶液中,获得携带粒细胞集落刺激因子的明胶微球;
c、将凝血酶溶液、透明质酸溶液和明胶微球混合,得混合溶液,再加入纤维蛋白原溶液,混合,即可。
4.根据权利要求3所述的方法,其特征在于:步骤a中,
所述纤维蛋白原溶液中纤维蛋白原的质量体积比为10~100mg/ml;
所述透明质酸溶液中透明质酸的质量体积比为5~10mg/ml;
所述凝血酶溶液中凝血酶的浓度为50~100units/ml;
所述粒细胞集落刺激因子溶液的质量体积比为100~4000ng/ml;
所述明胶微球直径<50μm。
5.根据权利要求4所述的方法,其特征在于:步骤a中,
所述纤维蛋白原溶液中纤维蛋白原的质量体积比为50mg/ml;
所述透明质酸溶液中透明质酸的质量体积比为10mg/ml;
所述凝血酶溶液中凝血酶的浓度为100units/ml;
所述粒细胞集落刺激因子溶液的质量体积比为2000ng/ml;
所述明胶微球直径<50μm。
6.根据权利要求4所述的方法,其特征在于:所述溶液和微球按照如下方法制备:
(1)配制纤维蛋白原溶液:将0.9%生理盐水加入一定质量的纤维蛋白原粉末中,37℃水浴10分钟,形成纤维蛋白原溶液;
(2)配制凝血酶溶液:将0.9%生理盐水加入一定质量的重组凝血酶粉末中,吸管反复吹吸30秒使其均匀溶解;
(3)配制透明质酸溶液:将0.9%生理盐水加入一定质量的透明质酸粉末中,搅拌5min使其均匀溶解备用;
(4)制备G-CSF溶液:将0.9%生理盐水加入一定质量的重组G-CSF蛋白中,吸管反复吹吸30秒使其均匀溶解备用;
(5)制备明胶微球:将明胶粉末加入生理盐水中,配制成质量/体积分数为10%的明胶溶液,然后将明胶溶液逐滴加入30倍体积预热到50℃的橄榄油中,搅拌形成均匀的乳剂,并迅速将乳剂放入冰水中使明胶微粒凝结;再将100ml冰浴处理过的丙酮加入乳剂中使明胶微粒脱水,收获小于50μm明胶微粒,再将其加入10.6mmol/L戊二醛溶液中形成共价交联;然后用100mmol/L氨基乙酸溶液洗涤三次去除残留的醛基,并用去离子水反复洗涤三次,冻干,即可;
(6)制备携带G-CSF蛋白的明胶缓释微球:称取明胶微球,将其加入1~10倍体积/质量的G-CSF溶液中,使溶液完全吸附于明胶微球,冻干,即可。
7.根据权利要求6所述的方法,其特征在于:步骤(6)中,所述制备携带G-CSF蛋白的明胶缓释微球:称取明胶微球,将其加入5倍体积/质量的G-CSF溶液中,使溶液完全吸附于明胶微球,冻干,即可。
8.根据权利要求3所述的方法,其特征在于:步骤c中,所述凝血酶溶液、透明质酸溶液和明胶微球的体积比为5:7:(2~8);混合溶液与纤维蛋白原溶液的体积比为1:1。
9.根据权利要求8所述的方法,其特征在于:所述凝血酶溶液、透明质酸溶液和明胶微球的体积比为5:7:3。
10.根据权利要求3所述的方法,其特征在于:步骤c中,混合溶液与纤维蛋白原溶液混合时的水浴温度控制在30-40℃,持续5min以上。
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