注射型抗感染骨的制备及治疗兔感染性骨缺损的实验研究
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摘要
目的：评价抗感染活性成分妥布霉素对成骨细胞的影响，探索注射型抗感染骨的制备方式，并检测其理化性质和生物相容性，验证其治疗感染性骨缺损的有效性。
方法：
1、妥布霉素对MC3T3-E1细胞的影响：应用含妥布霉素培养液分别培养细胞1d、3d、6d，采用下述方法检测妥布霉素对细胞的影响：应用台盼蓝拒染计数存活细胞数；MTT检测细胞代谢活力；碱性磷酸酶活性检测细胞主要功能；乳酸脱氢酶活性检测膜损伤程度；RT-PCR检测ALP、COLⅠ和Smad1的表达；碱性磷酸酶染色和茜素红钙结节染色。同时应用上述方法检测妥布霉素作用3d后，无抗生素培养3d、6d后细胞的恢复情况。
2、妥布霉素含量对注射型抗感染骨缓释效果的影响：制备妥布霉素含量分别为25mg/ml、50mg/ml、75mg/ml和100mg/ml的注射型抗感染骨，应用PBS浸泡评价其体外缓释效果、溶胀率和降解率，应用体外细菌吸附试验评价其抗菌能力，应用小鼠肌袋植入实验评价其体内缓释效果。
3、注射型抗感染骨的性质：应用倒置法观察壳聚糖水凝胶的凝胶时间，扫描电镜观察注射型抗感染骨三维结构，浸提液法评价其体外细胞毒性，小鼠肌袋植入试验评价其体内生物相容性。
4、注射型抗感染骨修复兔感染性骨缺损：应用兔股骨髁感染性骨缺损模型，采用大体观察、X线摄片、药物浓度检测、细菌含量测定、显微CT扫描、荧光双标和组织切片观察，评价其防止感染、修复骨缺损的有效性。
结果：
1、高浓度的妥布霉素抑制MC3T3-E1细胞的增殖、代谢、碱性磷酸酶活性和功能基因的表达，增加乳酸脱氢酶的释放。去妥布霉素作用后，较低浓度妥布霉素作用的细胞增殖、代谢、碱性磷酸酶活性和功能基因的表达均较较高浓度组高。
2、50mg/ml组形态维持较好，缓释效果好，溶胀率、体外细菌吸附试验、局部和外周药物浓度检测方面不低于其他实验组。
3、注射型抗感染骨为三维多孔结构，细胞毒性为0~Ⅰ级，体内生物相容性好。
4、植入注射型抗感染骨组动物全部存活，体重无明显减低，外周血药浓度低于安全浓度，局部药物4w仍保存52.6%，局部无细菌检出，骨形态学分析及矿化沉积率均显著优于无植入物组，组织学见新骨生成和材料降解相匹配。
结论：
1、妥布霉素缓释应避免局部过高药物浓度。
2、注射型抗感染骨妥布霉素适宜浓度为50mg/ml。
3、制备的注射型抗感染骨有较好的理化性质和生物相容性。
4、注射型抗感染骨能有效防治感染，并且利于骨缺损修复，是较为理想的防治感染性骨缺损的支架材料。
Objective: To evaluate the effects of tobramycin on MC3T3-E1, develop injectableantibiotic DBM, and evaluate the constructive characteristics, biocompatibility andvalidity of preventing bone infection and repairing bone defects of the injectableantibiotic DBM.
Methods:
1. Evaluate the effect of tobramycin on MC3T3-E1cell viability and function.MC3T3-E1cells were treated with varying concentrations of tobramycin over a periodof1,3,6d, harvesting them after each time period and performing assays to test theirviability(trypan blue exclusion test), metabolic function(MTT assay), Alkalinephosphatase (ALP) activity, expression of ALP, COL Ⅰ and Smad1and cellmembrane viability (LDH). Alkaline phosphatase staining and calcium staining werealso assessed. Cell recovery in the absence of tobramycin was evaluated.
2. Construction of injectable antibiotic DBM with varying concentrations of tobramycin.To construct injectable antibiotic DBM with varying concentrations of tobramycin(25mg/ml、50mg/ml、75mg/ml and100mg/ml) and investigate the difference oftobramycin release rate, swelling ratio, degradation rate, and antibacterial activity invitro, and tobramycin release rate in muscle bag of mouse between groups.
3. Characteristics of injectable antibiotic DBM. Gelation time of thermogelling chitosanwas tested by tube inverting method. SEM observation, cytotoxicity test, andintramusculary implanting test of injectable antibiotic DBM were performed.
4. Treatment of experimental infected bone defects with injectable antibiotic DBM.Injectable antibiotic DBM were implanted in contaminated unicortical defect of rabbits.The validity of preventing bone infection and repairing bone defects were assessed byclinical, radiological, histological, gross examination, bacterial load assay, and highperformance liquid chromatography(HPLC) analysis of tobramycin concentration.
Results:
1.Tobramycin of high concentration inhibit MC3T3-E1cell viability and metabolicfunction, ALP activity, expression of ALP, COLⅠ and Smad1and cell membrane viability. Cells, which had been treated with lower concentration of tobramycin, showedbetter viability and function in the absence of tobramycin.
2. The injectable antibiotic DBM with50mg/ml tobramycin performed better in gelstate maintain and controlled-release, and showed average function of antibacterialactivity in vitro, and tobramycin release rate in vivo.
3. Injectable antibiotic DBM showed a3D structure with varying pores, grade0toⅠcytotoxicity, and good biocompatibility in vivo.
4. Animals implanted with injectable antibiotic DBM all survived without weight lossand high serum concentrations. At4weeks,52.6%tobramycin were conserved in theinjectable antibiotic DBM. Bacterial load assay and radiological analysis showed bestresults in all groups. The degradation of implants matched with new bone formation.
Conclusion:
1. Tobramycin delivery should avoid local excessive drug concentrations.
2. Suitable tobramycin concentration for injectable antibiotic DBM is50mg/ml.
3. Injectable antibiotic DBM had good biocompatibility and physical characteristic.
4. Injectable antibiotic DBM is a good local antibiotic delivery system, which couldprevent bone infection and repair bone defects.
方法：
1、妥布霉素对MC3T3-E1细胞的影响：应用含妥布霉素培养液分别培养细胞1d、3d、6d，采用下述方法检测妥布霉素对细胞的影响：应用台盼蓝拒染计数存活细胞数；MTT检测细胞代谢活力；碱性磷酸酶活性检测细胞主要功能；乳酸脱氢酶活性检测膜损伤程度；RT-PCR检测ALP、COLⅠ和Smad1的表达；碱性磷酸酶染色和茜素红钙结节染色。同时应用上述方法检测妥布霉素作用3d后，无抗生素培养3d、6d后细胞的恢复情况。
2、妥布霉素含量对注射型抗感染骨缓释效果的影响：制备妥布霉素含量分别为25mg/ml、50mg/ml、75mg/ml和100mg/ml的注射型抗感染骨，应用PBS浸泡评价其体外缓释效果、溶胀率和降解率，应用体外细菌吸附试验评价其抗菌能力，应用小鼠肌袋植入实验评价其体内缓释效果。
3、注射型抗感染骨的性质：应用倒置法观察壳聚糖水凝胶的凝胶时间，扫描电镜观察注射型抗感染骨三维结构，浸提液法评价其体外细胞毒性，小鼠肌袋植入试验评价其体内生物相容性。
4、注射型抗感染骨修复兔感染性骨缺损：应用兔股骨髁感染性骨缺损模型，采用大体观察、X线摄片、药物浓度检测、细菌含量测定、显微CT扫描、荧光双标和组织切片观察，评价其防止感染、修复骨缺损的有效性。
结果：
1、高浓度的妥布霉素抑制MC3T3-E1细胞的增殖、代谢、碱性磷酸酶活性和功能基因的表达，增加乳酸脱氢酶的释放。去妥布霉素作用后，较低浓度妥布霉素作用的细胞增殖、代谢、碱性磷酸酶活性和功能基因的表达均较较高浓度组高。
2、50mg/ml组形态维持较好，缓释效果好，溶胀率、体外细菌吸附试验、局部和外周药物浓度检测方面不低于其他实验组。
3、注射型抗感染骨为三维多孔结构，细胞毒性为0~Ⅰ级，体内生物相容性好。
4、植入注射型抗感染骨组动物全部存活，体重无明显减低，外周血药浓度低于安全浓度，局部药物4w仍保存52.6%，局部无细菌检出，骨形态学分析及矿化沉积率均显著优于无植入物组，组织学见新骨生成和材料降解相匹配。
结论：
1、妥布霉素缓释应避免局部过高药物浓度。
2、注射型抗感染骨妥布霉素适宜浓度为50mg/ml。
3、制备的注射型抗感染骨有较好的理化性质和生物相容性。
4、注射型抗感染骨能有效防治感染，并且利于骨缺损修复，是较为理想的防治感染性骨缺损的支架材料。
Objective: To evaluate the effects of tobramycin on MC3T3-E1, develop injectableantibiotic DBM, and evaluate the constructive characteristics, biocompatibility andvalidity of preventing bone infection and repairing bone defects of the injectableantibiotic DBM.
Methods:
1. Evaluate the effect of tobramycin on MC3T3-E1cell viability and function.MC3T3-E1cells were treated with varying concentrations of tobramycin over a periodof1,3,6d, harvesting them after each time period and performing assays to test theirviability(trypan blue exclusion test), metabolic function(MTT assay), Alkalinephosphatase (ALP) activity, expression of ALP, COL Ⅰ and Smad1and cellmembrane viability (LDH). Alkaline phosphatase staining and calcium staining werealso assessed. Cell recovery in the absence of tobramycin was evaluated.
2. Construction of injectable antibiotic DBM with varying concentrations of tobramycin.To construct injectable antibiotic DBM with varying concentrations of tobramycin(25mg/ml、50mg/ml、75mg/ml and100mg/ml) and investigate the difference oftobramycin release rate, swelling ratio, degradation rate, and antibacterial activity invitro, and tobramycin release rate in muscle bag of mouse between groups.
3. Characteristics of injectable antibiotic DBM. Gelation time of thermogelling chitosanwas tested by tube inverting method. SEM observation, cytotoxicity test, andintramusculary implanting test of injectable antibiotic DBM were performed.
4. Treatment of experimental infected bone defects with injectable antibiotic DBM.Injectable antibiotic DBM were implanted in contaminated unicortical defect of rabbits.The validity of preventing bone infection and repairing bone defects were assessed byclinical, radiological, histological, gross examination, bacterial load assay, and highperformance liquid chromatography(HPLC) analysis of tobramycin concentration.
Results:
1.Tobramycin of high concentration inhibit MC3T3-E1cell viability and metabolicfunction, ALP activity, expression of ALP, COLⅠ and Smad1and cell membrane viability. Cells, which had been treated with lower concentration of tobramycin, showedbetter viability and function in the absence of tobramycin.
2. The injectable antibiotic DBM with50mg/ml tobramycin performed better in gelstate maintain and controlled-release, and showed average function of antibacterialactivity in vitro, and tobramycin release rate in vivo.
3. Injectable antibiotic DBM showed a3D structure with varying pores, grade0toⅠcytotoxicity, and good biocompatibility in vivo.
4. Animals implanted with injectable antibiotic DBM all survived without weight lossand high serum concentrations. At4weeks,52.6%tobramycin were conserved in theinjectable antibiotic DBM. Bacterial load assay and radiological analysis showed bestresults in all groups. The degradation of implants matched with new bone formation.
Conclusion:
1. Tobramycin delivery should avoid local excessive drug concentrations.
2. Suitable tobramycin concentration for injectable antibiotic DBM is50mg/ml.
3. Injectable antibiotic DBM had good biocompatibility and physical characteristic.
4. Injectable antibiotic DBM is a good local antibiotic delivery system, which couldprevent bone infection and repair bone defects.
引文
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