BMP-2/氰基丙烯酸复合生物胶修复大鼠下颌骨缺损的实验研究
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摘要
目的
1.以壳聚糖为载体,将BMP-2、α-氰基丙烯酸酯正丁酯、β-甘油磷酸钠等复合成生物胶,研究观察其稳定性。制作Wistar大鼠下颌骨骨缺损动物模型,将复合生物胶植入大鼠下颌骨缺损动物模型中观察修复缺损的效果。
2.研究探讨BMP-2/氰基丙烯酸酯复合生物胶对大鼠下颌骨骨缺损的止血、黏结固定、诱导成骨修复颌骨缺损的可行性,为临床应用提供实验依据。
方法
1.BMP-2/氰基丙烯酸复合生物胶的制备
2.壳聚糖温敏性水凝胶的制备。
3.大鼠下颌骨矩形骨缺损动物模型的制备。
4.将大鼠随机分为实验组、对照组、空白组。实验组植入BMP-2/氰基丙烯酸复合生物胶,对照组植入壳聚糖温敏性水凝胶,空白组不植入任何材料。创口分层缝合。术中观察BMP-2/氰基丙烯酸复合生物胶止血、黏结固定等。
5.下颌骨标本处理及检测：术后3、6、9周每组各处死6只动物。标本大体观察,各组下颌骨标本拍摄软X线片,观察其缺损骨修复情况。组织学观察：不同时间的标本固定后,用EDTA液脱钙4周,HE染色、石蜡包埋做连续切片,厚度为4μm,光学显微镜观察。
6.统计学分析：测量软x线片,配合使用Image Pro Plus6.0软件计算各组大鼠骨缺损面积(A),进行数据分析。用SPSS17.0软件对各组间数据进行处理,采用单因素方差分析(One Way ANOVE)进行统计学分析。
结果
1.以壳聚糖为载体,将BMP-2、α-氰基丙烯酸酯正丁酯、甘油磷酸钠等复合成生物胶为半固态状胶冻状、流动性差、具有弹性的凝胶。
2.壳聚糖温敏性水凝胶为淡黄色清亮液体,流动性差可,在37℃水浴,时长约1min可凝固,呈胶冻状。
3.术中实验组植入的BMP-2/氰基丙烯酸复合生物胶表面形成膜状结构覆盖创伤及周围部位,止血及固定效果良好。
4.试验过程中各组大鼠均未出现排异反应,成骨活跃,术后3、6、9周软X线大体观察可见实验组下颌骨骨缺损处密度大于其他两组,其缺损区域缩小。6、9周后实验组下颌骨骨缺损区域小于对照组。统计学分析植入后3、6、9周时,实验组骨缺损面积均低于对照组及空白对照组(P<0.05),说明BMP-2/氰基丙烯酸复合生物胶对颌骨缺损修复有显著作用。组织学观察：术后3周时实验组骨缺损周围除有新生毛细血管、成骨细胞、破骨细胞还可发现有少量的淋巴细胞；术后6周时实验组骨缺损区毛细血管和胶原纤维减少,周围有新骨长入且新生骨量明显高于同期对照组和空白对照组；术后9周时实验组骨缺损区可见大量新骨形成。
结论
BMP-2/氰基丙烯酸复合生物胶具有良好的生物相容性及骨诱导性,对大鼠颌骨缺损修复再生具有显著的促进作用,并且生物安全性好,修复颌骨缺损的效果好,具有良好的临床应用前景。
Object
1. Using the bone morphogenetic protein-2, cyanoacrylate and chitosan and others to made the compound biological glue. To Study the physical and biological properties of this compound. To prepare the model of a Wistar rat mandibular defect by remove part of bone of mandibular of rat.
2. Putting the compound biological glue into the model of the mandibular defect of rats, to research the ability of hemostasis, adhesive and bone regeneration of the compound biological glue.
Materials and methods
1. The production of BMP-2with cyanoacrylate biological glue.
2. The compound of temperature responsive chitosan hydrogel.
3. To prepare the model of mandibular defect of Wistar rat.
4.54Wistar rats were randomly divided into3groups, experimental group, control group and blank control group. The experimental group was implanted by the compound BMP-2with cyanoacrylate biological glue, while the control group was implanted with temperature responsive chitosan hydrogel, and the blank control group was implanted into nothing. The wounds were sutured layered and perfectly. To observe the ability of the hemostasis and adhesive of the compound BMP-2with cyanoacrylate biological glue during and after surgery.
5. The disposal and detection of samples:The rats were anesthetized to death after the operation of implantation for3,6,9weeks. The mandibular samples of rats were examined by soft X-ray and processed by Image-Pro Plus6.0software. Routine histological observation:The joint specimens were exposed; fixed by neutral formalin, colored by HE staining, wrapped and cut into slices by4μm, study and observed them under a optical microscope.
6. Statistical analysis:All the data were processed by SPSS17.0software, one-way ANOVE analysis was used to analysis. There was a statistically significant difference between experimental group and other groups.
Results
1. The compound BMP-2with cyanoacrylate biological glue was compounded successfully. This biological glue is semi-solid, illiquid, and elastic.
2.The temperature responsive chitosan hydrogel is pale yellow, liquid and it may be become solidification glue in the37℃water for about1min.
3. The mandibular defects model of rats were implanted by the compound BMP-2with cyanoacrylate biological glue, the experimental group was repaired with the wound bed and showed excellent hemostasis result.
4. All the rats in the study were healthy without immunological reaction after implantation. The bone regenerated active were very obvious after operation. Soft X ray films confirmed that the defect area in the experimental group was narrower than other groups, after3,6,9weeks of implantation. No inflammation or rejection response was detected during the experimental in54rats, and the wound was healed well. The bone defect area of residual mandibular defects in the experimental group were all smaller than the other two groups after6,9weeks of operation. And the control group was smaller than the blank control group. There was a statistically significant difference between the experimental and the other two groups (P<0.05). Histology observation: There was a lot of newborn capillaries, osteoblasts, and a few lymphocytes are discovered after3weeks; Capillaries and collagenous fiber reduce, while there was a few bone tissue than the other groups after6weeks; There was a mass of bone tissue in the defect area after9weeks.
Conclusions
The compound BMP-2with biological glue has good biocompatibility, safe, stability and ability of osteoinduction. It can be used to regeneration and restruction of rat mandibular bone defect and have a good future in clinical applications.
1.以壳聚糖为载体,将BMP-2、α-氰基丙烯酸酯正丁酯、β-甘油磷酸钠等复合成生物胶,研究观察其稳定性。制作Wistar大鼠下颌骨骨缺损动物模型,将复合生物胶植入大鼠下颌骨缺损动物模型中观察修复缺损的效果。
2.研究探讨BMP-2/氰基丙烯酸酯复合生物胶对大鼠下颌骨骨缺损的止血、黏结固定、诱导成骨修复颌骨缺损的可行性,为临床应用提供实验依据。
方法
1.BMP-2/氰基丙烯酸复合生物胶的制备
2.壳聚糖温敏性水凝胶的制备。
3.大鼠下颌骨矩形骨缺损动物模型的制备。
4.将大鼠随机分为实验组、对照组、空白组。实验组植入BMP-2/氰基丙烯酸复合生物胶,对照组植入壳聚糖温敏性水凝胶,空白组不植入任何材料。创口分层缝合。术中观察BMP-2/氰基丙烯酸复合生物胶止血、黏结固定等。
5.下颌骨标本处理及检测：术后3、6、9周每组各处死6只动物。标本大体观察,各组下颌骨标本拍摄软X线片,观察其缺损骨修复情况。组织学观察：不同时间的标本固定后,用EDTA液脱钙4周,HE染色、石蜡包埋做连续切片,厚度为4μm,光学显微镜观察。
6.统计学分析：测量软x线片,配合使用Image Pro Plus6.0软件计算各组大鼠骨缺损面积(A),进行数据分析。用SPSS17.0软件对各组间数据进行处理,采用单因素方差分析(One Way ANOVE)进行统计学分析。
结果
1.以壳聚糖为载体,将BMP-2、α-氰基丙烯酸酯正丁酯、甘油磷酸钠等复合成生物胶为半固态状胶冻状、流动性差、具有弹性的凝胶。
2.壳聚糖温敏性水凝胶为淡黄色清亮液体,流动性差可,在37℃水浴,时长约1min可凝固,呈胶冻状。
3.术中实验组植入的BMP-2/氰基丙烯酸复合生物胶表面形成膜状结构覆盖创伤及周围部位,止血及固定效果良好。
4.试验过程中各组大鼠均未出现排异反应,成骨活跃,术后3、6、9周软X线大体观察可见实验组下颌骨骨缺损处密度大于其他两组,其缺损区域缩小。6、9周后实验组下颌骨骨缺损区域小于对照组。统计学分析植入后3、6、9周时,实验组骨缺损面积均低于对照组及空白对照组(P<0.05),说明BMP-2/氰基丙烯酸复合生物胶对颌骨缺损修复有显著作用。组织学观察：术后3周时实验组骨缺损周围除有新生毛细血管、成骨细胞、破骨细胞还可发现有少量的淋巴细胞；术后6周时实验组骨缺损区毛细血管和胶原纤维减少,周围有新骨长入且新生骨量明显高于同期对照组和空白对照组；术后9周时实验组骨缺损区可见大量新骨形成。
结论
BMP-2/氰基丙烯酸复合生物胶具有良好的生物相容性及骨诱导性,对大鼠颌骨缺损修复再生具有显著的促进作用,并且生物安全性好,修复颌骨缺损的效果好,具有良好的临床应用前景。
Object
1. Using the bone morphogenetic protein-2, cyanoacrylate and chitosan and others to made the compound biological glue. To Study the physical and biological properties of this compound. To prepare the model of a Wistar rat mandibular defect by remove part of bone of mandibular of rat.
2. Putting the compound biological glue into the model of the mandibular defect of rats, to research the ability of hemostasis, adhesive and bone regeneration of the compound biological glue.
Materials and methods
1. The production of BMP-2with cyanoacrylate biological glue.
2. The compound of temperature responsive chitosan hydrogel.
3. To prepare the model of mandibular defect of Wistar rat.
4.54Wistar rats were randomly divided into3groups, experimental group, control group and blank control group. The experimental group was implanted by the compound BMP-2with cyanoacrylate biological glue, while the control group was implanted with temperature responsive chitosan hydrogel, and the blank control group was implanted into nothing. The wounds were sutured layered and perfectly. To observe the ability of the hemostasis and adhesive of the compound BMP-2with cyanoacrylate biological glue during and after surgery.
5. The disposal and detection of samples:The rats were anesthetized to death after the operation of implantation for3,6,9weeks. The mandibular samples of rats were examined by soft X-ray and processed by Image-Pro Plus6.0software. Routine histological observation:The joint specimens were exposed; fixed by neutral formalin, colored by HE staining, wrapped and cut into slices by4μm, study and observed them under a optical microscope.
6. Statistical analysis:All the data were processed by SPSS17.0software, one-way ANOVE analysis was used to analysis. There was a statistically significant difference between experimental group and other groups.
Results
1. The compound BMP-2with cyanoacrylate biological glue was compounded successfully. This biological glue is semi-solid, illiquid, and elastic.
2.The temperature responsive chitosan hydrogel is pale yellow, liquid and it may be become solidification glue in the37℃water for about1min.
3. The mandibular defects model of rats were implanted by the compound BMP-2with cyanoacrylate biological glue, the experimental group was repaired with the wound bed and showed excellent hemostasis result.
4. All the rats in the study were healthy without immunological reaction after implantation. The bone regenerated active were very obvious after operation. Soft X ray films confirmed that the defect area in the experimental group was narrower than other groups, after3,6,9weeks of implantation. No inflammation or rejection response was detected during the experimental in54rats, and the wound was healed well. The bone defect area of residual mandibular defects in the experimental group were all smaller than the other two groups after6,9weeks of operation. And the control group was smaller than the blank control group. There was a statistically significant difference between the experimental and the other two groups (P<0.05). Histology observation: There was a lot of newborn capillaries, osteoblasts, and a few lymphocytes are discovered after3weeks; Capillaries and collagenous fiber reduce, while there was a few bone tissue than the other groups after6weeks; There was a mass of bone tissue in the defect area after9weeks.
Conclusions
The compound BMP-2with biological glue has good biocompatibility, safe, stability and ability of osteoinduction. It can be used to regeneration and restruction of rat mandibular bone defect and have a good future in clinical applications.
引文
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