CN111494700A - 一种促进碎骨快愈合的骨胶及制备方法 - Google Patents
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Abstract
本发明属于用于骨科的药物制备技术领域,公开了一种促进碎骨快愈合的骨胶及制备方法,促进碎骨快愈合的骨胶为复合氰基丙烯酸酯‑BMP生物胶;制备方法包括将壳聚糖溶解于乙酸溶液中,在磁力搅拌器下按l:1的比例逐滴加入到β‑甘油磷酸钠溶液中;再持续搅拌0.5h,制备成温敏性胶体;使用95%盐酸溶液将步骤二制备的温敏性胶体调至pH值3.5,边搅拌边滴加适量氰基丙烯酸正丁酯,所得溶液中加入rhBMP,使终溶液中rhBMP为200μg/L,磁力搅拌器常温下持续搅拌3h,密封后4℃冰箱保存。利用本发明提供的骨胶将碎骨片粘在一起,进一步减少利用螺钉固定骨折血运的破坏而影响骨折的愈合。
Description
技术领域
本发明属于用于骨科的药物制备技术领域,尤其涉及一种促进碎骨快愈合的骨胶及制备方法。
背景技术
目前,骨折手术中对游离的粉碎性难复位骨折块的固定一直是难点,采用钢板内固定装置通常不能达到稳定的复位效果,对骨膜也有一定程度的破坏作用,进而影响骨折愈合。a-氰基丙烯酸丁酯(BCA)类医用胶已广泛应用于临床,但用于骨折治疗时会受到一定限制,原因在于其黏合力低、骨传导性能差。
目前,医用胶以丁酯和辛酯为主要成分配制,广泛应用于皮肤缝合、胃肠道手术、颅骨修补等。Brochu等报道在骨折手术治疗中加用自由基改性的a-BCA,结果发现a-BCA抗弯模量及抗疲劳性能均显著提高。
BMP系TGF-β超家族成员,BMP-2、BMP-6、BMP-7等对骨髓间充质干细胞(BMSC)的增殖和向软骨及成骨细胞方向分化均起到重要作用,BMP 2具有促进BMSC向成骨细胞诱导分化的作用,BMP-7则更趋向于使BMSC向骨细胞诱导分化。张赞等为进一步证实BMP-2复合a-BCA促进骨折愈合的效果,构建小鼠下颌骨缺损模型,实验组骨缺损处加入CA-BMP-2复合生物骨胶缓释系统,结果表明术后出血及感染率明显降低,X线片检查提示实验组骨缺损修复面积高于对照组;因此认为,a-BCA利于组织止血,降低术后创面感染,且通过BMP-2持续释放使新骨形成达到良好效果。
通过上述分析,现有技术存在的问题及缺陷为:(1)现有技术提供的仅用于骨折治疗的药物,黏合力低、骨传导性能差,不能进一步减少利用螺钉固定骨折血运的破坏而影响骨折的愈合。
(2)手术操作过程中骨胶可能对骨折周围软组织造成一定程度的损伤。
(3)骨胶在缓慢释放过程中可能对人体产生一定程度的副作用。
解决以上问题及缺陷的难度为:复合氰基丙烯酸酯与BMP的最佳比例难以掌握;生物材料对人体的副作用难以控制。
解决以上问题及缺陷的意义为:通过实验能够找到复合氰基丙烯酸酯与BMP相对较好的比例,来达到更好的效果。手术操作过程中尽量将骨折周围软组织分开,减少骨胶与软组织的接触;骨胶的用量在达到愈合量后不再增加。
发明内容
针对现有技术存在的问题,本发明提供了一种促进碎骨快愈合的骨胶及制备方法。
本发明是这样实现的,一种促进碎骨快愈合的骨胶,为复合氰基丙烯酸酯-BMP生物胶。
本发明的另一目的在于提供一种促进碎骨快愈合的骨胶的制备方法包括:
步骤一,将壳聚糖溶解于浓度为14-16mol/L的乙酸溶液中,在磁力搅拌器下按l:1的比例逐滴加入到1.10-1.30mol/Lβ-甘油磷酸钠溶液中;
步骤二,再持续搅拌0.5h-0.6,制备成温敏性胶体;
步骤三,使用95%盐酸溶液将步骤二制备的温敏性胶体调至pH值3.5-4.5,边搅拌边滴加5-10ml氰基丙烯酸正丁酯,所得溶液中加入rhBMP,使终溶液中rhBMP为180-200μg/L,磁力搅拌器常温下持续搅拌2.5-3h,密封后4℃冰箱保存。
进一步,所述步骤三,制备的复合氰基丙烯酸酯-BMP生物胶为白色、半流动胶体。
进一步,所述促进碎骨快愈合的骨胶的制备方法进一步包括壳聚糖温敏性胶体的制备,所述壳聚糖温敏性胶体的制备包括:
步骤1,将壳聚糖溶解于浓度为14-16mol/L的乙酸溶液中,在磁力搅拌器下按l:1的比例逐滴加入到1.10-1.30mol/Lβ-甘油磷酸钠溶液中;
步骤2,再持续搅拌0.5-0.6h,制备成温敏性胶体;
步骤3,使用95%盐酸溶液将步骤2制备的温敏性胶体调至pH值3.5-4.5;密封后4℃冰箱保存。
本发明的另一目的在于提供一种验证所述促进碎骨快愈合的骨胶药效的动物模型构建方法,所述动物模型构建方法包括:
步骤1,取实验动物54只键康成年大白兔,重量2.0-2.5kg,雌雄各半;
步骤2,壳聚糖温敏性胶体的制备:将壳聚糖溶解于浓度为16mol/L的乙酸溶液中,在磁力搅拌器下按l:1的比例逐滴加入到1.30mol/Lβ-甘油磷酸钠溶液中;再持续搅拌0.5h,制备成温敏性胶体;
步骤3,动物分组,54只新西兰大白兔随机分为3组,复合氰基丙烯酸酯-BMP生物胶组、壳聚糖温敏性胶体组、空白对照组,每组18只,雌雄各半;
步骤4,造模。
进一步,步骤4后,还需进行:
步骤I,复合氰基丙烯酸酯-BMP生物胶组、壳聚糖温敏性胶体组、空白对照组指标检测。
步骤II,X射线检查,分析骨折区域骨痂生长及骨折愈合情况;
步骤III,骨密度测定:对骨折断端包括上下0.5cm骨质的骨密度测量4次,取平均值;
步骤IV,生物力学测定:用Merlin软件进行三点弯曲法测量最大抗折载荷,夹具跨度40mm.以骨折部位为中心在支具上方中点施加载荷,速率5mm/min;
步骤V,组织形态学检查:用0.9%氯化钠溶液冲洗标本后,置入10%甲醛溶液中固定24h;按顺序进行脱钙、脱水、包埋、切片、染色处理;切片制作好后,置采用显微摄像系统摄入计算机,分析成骨细胞、骨小粱及胶原纤维生成情况,并比较各组之间的组织学差异;
步骤VI,统计学分析,实验数据以均数±标准差表示.采用软件包进行分析与处理。
结合上述的所有技术方案,本发明所具备的优点及积极效果为:粉碎性骨折的小碎骨片,利用本发明提供的骨胶将碎骨片粘在一起,进一步减少利用螺钉固定骨折血运的破坏而影响骨折的愈合。BMP是现代医学被证明的对骨折愈合最好的生物材料之一,有了BMP骨折的愈合程度大大得到提高。骨胶对于骨折的小碎片不仅能够让碎骨片解剖复位,又能够让骨折小碎骨片快速愈合。骨胶对粉碎性骨折的社会效益得到大大改善。
本发明相比于现有技术,对比的技术效果或者实验效果有:骨胶在手术过程中操作方便简单,减少骨科医生对粉碎性骨折的手术时间;大大减少骨折愈合时间。提高患者的生活质量。
附图说明
图1是本发明实施例提供的促进碎骨快愈合的骨胶及制备方法流程图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
现有技术提供的但用于骨折治疗的药物,黏合力低、骨传导性能差,不能进一步减少利用螺钉固定骨折血运的破坏而影响骨折的愈合。
针对现有技术存在的问题,本发明提供了一种促进碎骨快愈合的骨胶及制备方法,下面结合附图对本发明作详细的描述。
本发明实施例提供的促进碎骨快愈合的骨胶为复合氰基丙烯酸酯-BMP生物胶(100%)。其中可为:复合氰基丙烯酸酯:BMP=4:5。本发明大大提高了骨折碎骨快的愈合率。改善了患肢的功能;降低了患肢的致残率。
如图1所示,本发明实施例提供的促进碎骨快愈合的骨胶的制备方法包括:
S101,将壳聚糖溶解于浓度为14-16mol/L的乙酸溶液中,在磁力搅拌器下按l:1的比例逐滴加入到1.10-1.30mol/Lβ-甘油磷酸钠溶液中。
S102,再持续搅拌0.5-0.6h,制备成温敏性胶体。
S103,使用95%盐酸溶液将步骤二制备的温敏性胶体调至pH值3.5-4.5,边搅拌边滴加5-10ml氰基丙烯酸正丁酯,所得溶液中加入rhBMP,使终溶液中rhBMP为180-200μg/L,磁力搅拌器常温下持续搅拌2.5-3h,密封后4℃冰箱保存。
本发明中,促进碎骨快愈合的骨胶的制备方法进一步包括壳聚糖温敏性胶体的制备,所述壳聚糖温敏性胶体的制备包括:
步骤1,将壳聚糖溶解于浓度为14-16mol/L的乙酸溶液中,在磁力搅拌器下按l:1的比例逐滴加入到1.10-1.30mol/Lβ-甘油磷酸钠溶液中。
步骤2,再持续搅拌0.5-0.6h,制备成温敏性胶体。
步骤3,使用95%盐酸溶液将步骤二制备的温敏性胶体调至pH值3.5-4.5;密封后4℃冰箱保存。
下面结合具体实施例对本发明作进一步描述。
实施例1
本发明实施例提供的促进碎骨快愈合的骨胶的制备方法包括:
步骤一,将壳聚糖溶解于浓度为14mol/L的乙酸溶液中,在磁力搅拌器下按l:1的比例逐滴加入到1.10mol/Lβ-甘油磷酸钠溶液中。
步骤二,再持续搅拌0.5h,制备成温敏性胶体。
步骤三,使用95%盐酸溶液将步骤二制备的温敏性胶体调至pH值3.5,边搅拌边滴加5ml氰基丙烯酸正丁酯,所得溶液中加入rhBMP,使终溶液中rhBMP为180μg/L,磁力搅拌器常温下持续搅拌2.5h,密封后4℃冰箱保存。
实施例2
本发明实施例提供的促进碎骨快愈合的骨胶的制备方法包括:
步骤一,将壳聚糖溶解于浓度为16mol/L的乙酸溶液中,在磁力搅拌器下按l:1的比例逐滴加入到1.30mol/Lβ-甘油磷酸钠溶液中。
步骤二,再持续搅拌0.6h,制备成温敏性胶体。
步骤三,使用95%盐酸溶液将步骤二制备的温敏性胶体调至pH值4.5,边搅拌边滴加10ml氰基丙烯酸正丁酯,所得溶液中加入rhBMP,使终溶液中rhBMP为1200μg/L,磁力搅拌器常温下持续搅拌3h,密封后4℃冰箱保存。
实施例3
本发明实施例提供的促进碎骨快愈合的骨胶的制备方法包括:
步骤一,将壳聚糖溶解于浓度为15mol/L的乙酸溶液中,在磁力搅拌器下按l:1的比例逐滴加入到1.20mol/Lβ-甘油磷酸钠溶液中。
步骤二,再持续搅拌0.55h,制备成温敏性胶体。
步骤三,使用95%盐酸溶液将步骤二制备的温敏性胶体调至pH值4.0,边搅拌边滴加7.5ml氰基丙烯酸正丁酯,所得溶液中加入rhBMP,使终溶液中rhBMP为190μg/L,磁力搅拌器常温下持续搅拌2.75h,密封后4℃冰箱保存。
下面结合实验对本发明的效果作进一步描述。
1材料
1.1实验动物54只键康成年新西兰大白兔,重量2.0-2.5kg,雌雄各半,由江苏省中医药研究院提供。常规单笼饲养,自由进食和饮水,饲料为全价颗粒料。
1.2试剂及仪器rhBMP,β-甘油磷酸钠(Sigma公司,美国),氰基丙烯酸正丁酯(北京瞬康科技发展有限公司),壳聚糖(青岛海汇生物有限公司),磁力搅拌机(上海大中分析仪器厂),水浴箱(巩义市予华仪器有限责任公司),高频钼靶扫描仪(Planmed SoIphie公司,芬兰)。
2复合氰基丙烯酸酯-BMP生物胶及壳聚糖温敏性胶体的制备
复合氰基丙烯酸酯-BMP生物胶的制备:将壳聚糖溶解于浓度为16mol/L的乙酸溶液中,在磁力搅拌器下按l:1的比例逐滴加入到1.30mol/Lβ-甘油磷酸钠溶液中,再持续搅拌0.5h,制备成温敏性胶体。
在此基础上使用95%盐酸溶液将已经准备好的温敏性胶体调至pH值3.5,边搅拌边滴加适量氰基丙烯酸正丁酯,所得溶液中加入rhBMP,使终溶液中rhBMP为200μg/L,磁力搅拌器常温下持续搅拌3h,密封后4℃冰箱保存备用。制备物最终状态为白色胶体,半流动。
壳聚糖温敏性胶体的制备:方法同复合氰基丙烯酸酯-BMP生物胶,止于温敏性胶体的制备,不放入rhBMP及氰基丙烯酸正丁酯。
3动物分组
54只新西兰大白兔随机分为3组:实验组(复合氰基丙烯酸酯-BMP生物胶组)、对照组(壳聚糖温敏性胶体组)、空白对照组,每组18只,雌雄各半。
4方法
4.1造模:兔适应性饲养1周后,经耳缘静脉注射浓度为2.5%的戊巴比妥钠(1ml/kg),麻醉后,放置经消毒后的外科手术台,在双前肢内侧桡骨中段处剪毛、消毒、备皮,再经活力碘消毒,无菌条件下,通过手术方式切开皮肤,剥离周围软组织和血管,露出桡骨。用小型骨科电锯沿桡骨垂直方向造成中段完全横断骨折模型。
4.2手术过程:复合氰基丙烯酸酯-BMP生物胶组拭干骨折断端血迹后在断端间滴入一两滴复合氰基丙烯酸酯-BMP生物胶,加压并维持1min,以使胶体充分固化;壳聚糖温敏性胶体组拭干骨折断端血迹后在断端间滴入一两滴壳聚糖温敏性胶体,加压并维持1min,以使胶体充分固化;空白对照组不采用内固定,利用桡骨为支架自然固定。三组动物冲洗伤口后逐层缝合切口,术后正常饲养,每天肌肉注射青霉素40万单位,共持续3天。术后2,4,6,8,10,12周分批处死动物,3只/组。
5检测方法和指标
5.1X射线检查:术后2,4,6,8,10,12周每组随机取3只家兔行耳缘静脉空气栓塞处死。按原切口入路切开手术桡侧皮肤及周围结缔组织.将整个融合的尺桡骨游离。彻底剔除尺桡骨周围软组织。采用西门子公司计算机X射线摄像仪,于术后2,4,6,8,10,12周分别摄片各组家兔骨折端。摄侧位片:观察骨折区域骨痂生长及骨折愈合情况。
5.2骨密度测定:采用美国Lunar公司的DPX-IQ型双能X射线骨密度仪.统一选取小动物扫描模式:图像分辨率1.0mm×1.0mm:准确度0.01%:扫描速率60mm/s。于术后2,4,6,8,10,12周分别测定家兔骨折断端包括上下0.5cm骨质的骨密度。测量4次,取平均值。
5.3生物力学测定(仅限第8周):第8周时将各组标本置于英国INSTRON3369电子万能试验机上。用Merlin软件进行三点弯曲法测量其最大抗折载荷.夹具跨度约40mm.以骨折部位为中心在支具上方中点施加载荷,速率约5mm/min。
5.4组织形态学检查:术后2,4,6,8,10,12周,将已完成上述检查的标本予以修整后用于组织形态学观察。用0.9%氯化钠溶液冲洗标本后.迅速置入10%甲醛溶液中固定24h。按顺序进行脱钙、脱水、包埋、切片、染色处理。切片制作好后.将其置于Leica公司kicaDM LB2型双目显微镜低倍镜下(×100),再采用Motic—B5显微摄像系统摄入计算机。观察成骨细胞、骨小粱及胶原纤维生成情况:并比较各组之间的组织学差异。
6统计学方法
实验数据以均数±标准差表示.采用SPSS 17.0统计软件包进行分析与处理。同一时间计量资料组间比较采用单因素方差分析.两两比较采用SNK检验。P<0.05为差异有统计学意义。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,都应涵盖在本发明的保护范围之内。
Claims (6)
1.一种促进碎骨快愈合的骨胶,其特征在于,所述促进碎骨快愈合的骨胶为复合氰基丙烯酸酯-BMP生物胶。
2.一种如权利要求1所述促进碎骨快愈合的骨胶的制备方法,其特征在于,所述促进碎骨快愈合的骨胶的制备方法包括:
步骤一,将壳聚糖溶解于浓度为14-16mol/L的乙酸溶液中,在磁力搅拌器下按l:1的比例逐滴加入到1.10-1.30mol/Lβ-甘油磷酸钠溶液中;
步骤二,再持续搅拌0.5-0.6h,制备成温敏性胶体;
步骤三,使用95%盐酸溶液将步骤二制备的温敏性胶体调至pH值3.5-4.5,边搅拌边滴加5-10ml氰基丙烯酸正丁酯,所得溶液中加入rhBMP,使终溶液中rhBMP为180-200μg/L,磁力搅拌器常温下持续搅拌2.5-3h,密封后4℃冰箱保存。
3.如权利要求2所述促进碎骨快愈合的骨胶的制备方法,其特征在于,所述步骤三中,制备的复合氰基丙烯酸酯-BMP生物胶为白色、半流动胶体。
4.如权利要求2所述促进碎骨快愈合的骨胶的制备方法,其特征在于,所述促进碎骨快愈合的骨胶的制备方法进一步包括壳聚糖温敏性胶体的制备,所述壳聚糖温敏性胶体的制备包括:
步骤1,将壳聚糖溶解于浓度为14-16mol/L的乙酸溶液中,在磁力搅拌器下按l:1的比例逐滴加入到1.10-1.30mol/Lβ-甘油磷酸钠溶液中;
步骤2,再持续搅拌0.5-0.6h,制备成温敏性胶体;
步骤3,使用95%盐酸溶液将步骤2制备的温敏性胶体调至pH值3.5-4.5;密封后4℃冰箱保存。
5.一种验证权利要求1所述促进碎骨快愈合的骨胶药效的动物模型构建方法,其特征在于,所述动物模型构建方法包括:
步骤1,取实验动物54只键康成年大白兔,重量2.0-2.5kg,雌雄各半;
步骤2,壳聚糖温敏性胶体的制备:将壳聚糖溶解于浓度为16mol/L的乙酸溶液中,在磁力搅拌器下按l:1的比例逐滴加入到1.30mol/Lβ-甘油磷酸钠溶液中;再持续搅拌0.5h,制备成温敏性胶体;
步骤3,动物分组,54只新西兰大白兔随机分为3组,复合氰基丙烯酸酯-BMP生物胶组、壳聚糖温敏性胶体组、空白对照组,每组18只,雌雄各半;
步骤4,造模。
6.如权利要求5所述的动物模型构建方法,其特征在于,步骤4后,还需进行:
步骤I,复合氰基丙烯酸酯-BMP生物胶组、壳聚糖温敏性胶体组、空白对照组指标检测。
步骤II,X射线检查;
步骤III,骨密度测定;
步骤IV,生物力学测定:用Merlin软件进行三点弯曲法测量最大抗折载荷,夹具跨度40mm.以骨折部位为中心在支具上方中点施加载荷,速率5mm/min;
步骤V,组织形态学检查:用0.9%氯化钠溶液冲洗标本后,置入10%甲醛溶液中固定24h;按顺序进行脱钙、脱水、包埋、切片、染色处理;切片制作好后,置采用显微摄像系统摄入计算机,分析成骨细胞、骨小粱及胶原纤维生成情况,并比较各组之间的组织学差异;
步骤VI,统计学分析,实验数据以均数±标准差表示.采用软件包进行分析与处理。
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