载异烟肼、利福平纳米羟基磷灰石-硫酸钙-壳聚糖人工骨在兔脊柱结核模型中的释药研究
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摘要
目的 :研究载异烟肼(isoniazid,INH)、利福平(rifampicin,RFP)纳米羟基磷灰石(nHA)-硫酸钙(CSH)-壳聚糖(CTS)人工骨在兔脊柱结核模型病灶中的释药特点。方法:用结核分枝杆菌H37Rv标准株,采用侧腹入路的手术方法建立兔脊柱结核模型27只,其中雌性15只,雄性12只,3个月龄,平均体重为2.50±0.25kg,每只模型兔均在行结核病灶清除术后置入载INH、RFP纳米羟基磷灰石-硫酸钙-壳聚糖人工骨,按照时相点分为24h、72h和1周、2周、4周、6周、8周、10周、12周共9组,每组3只。采用高效液相色谱法(high performance liquid chromatography,HPLC)测定各时相点动物模型病灶中椎体及椎旁肌肉中的INH及RFP的药物浓度,并研究其浓度与时间的关系。结果:人工骨中的INH在兔脊柱结核模型的病灶椎体中24h、72h和1周、2周、4周、6周、8周的药物浓度分别为75.66±1.95μg/g、48.46±2.34μg/g、30.69±2.74μg/g、20.34±1.63μg/g、9.36±1.17μg/g、5.19±1.40μg/g、2.73±1.19μg/g,在第10周以后未检测到药物浓度,INH在病灶椎旁肌肉中的各时相点浓度为39.51±2.25μg/g、23.65±1.55μg/g、16.39±2.10μg/g、10.38±1.44μg/g、3.66±0.79μg/g、1.89±0.73μg/g、0.26±0.10μg/g,在第10周以后未检测到药物浓度。对INH在同一时间的椎体和椎旁肌两种不同组织中的药物浓度进行两两比较,均有统计学意义(P<0.05);RFP在动物模型的病灶椎体中24h、72h和1周、2周、4周、6周、8周、10周、12周的药物浓度分别为10.85±2.45μg/g、22.47±1.94μg/g、38.32±1.73μg/g、24.22±1.45μg/g、17.85±1.50μg/g、9.81±1.30μg/g、6.35±1.30μg/g、5.11±0.53μg/g、1.32±0.33μg/g,RFP在病灶椎旁肌肉中的药物浓度分别为5.39±1.50μg/g、20.66±1.29μg/g、48.72±2.24μg/g、32.27±1.63μg/g、15.58±1.88μg/g、8.69±0.79μg/g、3.43±0.39μg/g,在第10周后未检测到药物浓度。RFP在椎体和椎旁肌两个部位的药物浓度除72h、4周和6周外,其余时间点的差异均有统计学意义(P<0.05)。结论:载INH、RFP纳米羟基磷灰石-硫酸钙-壳聚糖人工骨在兔脊柱结核模型病灶中可持续、长效的释放抗结核药物。
Objectives: To study drug release characteristics of isoniazid(INH) and rifampicin(RFP) nano-hydroxyapatite(nHA)-calcium sulfate(CSH)-chitosan(CTS) artificial bone in rabbit spinal tuberculosis model.Methods: By using standard strain of Mycobacterium tuberculosis H37 Rv, 27 rabbit spinal tuberculosis models were established by lateral ventral approach, 15 females and 12 males, 3 months old, with an average body weight of 2.50±0.25 kg, each model rabbit was implanted with artificial bone materials after spinal tuberculosis debridement. According to the time points, the models were divided into 24 h, 72 h and 1, 2, 4, 6, 8,10, 12 weeks, a total of nine groups, with 3 rabbits in each group. High performance liquid chromatography(HPLC) was used to determine the concentration of INH and RFP in focal bone tissue and focal muscle tissue of animal models at different time points, and the relationship between concentration and time was studied. Results: The concentration of INH in focal bone tissue at 24 h, 72 h and 1 week, 2 weeks, 4 weeks,6 weeks and 8 weeks was 75.66±1.95μg/g, 48.46±2.34μg/g, 30.69±2.74μg/g, 20.34±1.63μg/g, 9.36±1.17μg/g,5.19±1.40μg/g and 2.73±1.19μg/g respectively, no drug concentration was detected after 10 weeks, the concentration of INH in focal muscle tissue was 39.51 ±2.25μg/g, 23.65 ±1.55μg/g, 16.39 ±2.10μg/g, 10.38 ±1.44μg/g, 3.66±0.79μg/g, 1.89±0.73μg/g, 0.26±0.10μg/g respectively, no drug concentration was detected after10 weeks. Comparison of drug concentrations of INH in two different tissues at the same time showed statistical significance(P<0.05). At 24 h, 72 h and 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks and 10 weeks, the drug concentration of RFP in focal bone tissue was 10.85±2.45μg/g, 22.47±1.94μg/g, 38.32±1.73μg/g, 24.22±1.45μg/g, 17.85±1.50μg/g, 9.81±1.30μg/g, 6.35±1.30μg/g, 5.11±0.53μg/g, 1.32±0.33μg/g respectively. The drug concentration of RFP in focal muscle tissue was 5.39±1.50μg/g, 20.66±1.29μg/g, 48.72±2.24μg/g, 32.27±1.63μg/g, 15.58±1.88μg/g, 8.69±0.79μg/g, 3.43±0.39μg/g respectively. No drug concentration was detected after 10 weeks. The concentrations of RFP in two different tissues were significantly different at other time points except 72 h, 4 weeks and 6 weeks(P<0.05). Conclusions: INH-RFP nano hydroxyapatite-calcium sulfate-chitosan artificial bone can sustainably and effectively release anti-tuberculosis drugs in the focal tissues of rabbit spinal tuberculosis model.
Objectives: To study drug release characteristics of isoniazid(INH) and rifampicin(RFP) nano-hydroxyapatite(nHA)-calcium sulfate(CSH)-chitosan(CTS) artificial bone in rabbit spinal tuberculosis model.Methods: By using standard strain of Mycobacterium tuberculosis H37 Rv, 27 rabbit spinal tuberculosis models were established by lateral ventral approach, 15 females and 12 males, 3 months old, with an average body weight of 2.50±0.25 kg, each model rabbit was implanted with artificial bone materials after spinal tuberculosis debridement. According to the time points, the models were divided into 24 h, 72 h and 1, 2, 4, 6, 8,10, 12 weeks, a total of nine groups, with 3 rabbits in each group. High performance liquid chromatography(HPLC) was used to determine the concentration of INH and RFP in focal bone tissue and focal muscle tissue of animal models at different time points, and the relationship between concentration and time was studied. Results: The concentration of INH in focal bone tissue at 24 h, 72 h and 1 week, 2 weeks, 4 weeks,6 weeks and 8 weeks was 75.66±1.95μg/g, 48.46±2.34μg/g, 30.69±2.74μg/g, 20.34±1.63μg/g, 9.36±1.17μg/g,5.19±1.40μg/g and 2.73±1.19μg/g respectively, no drug concentration was detected after 10 weeks, the concentration of INH in focal muscle tissue was 39.51 ±2.25μg/g, 23.65 ±1.55μg/g, 16.39 ±2.10μg/g, 10.38 ±1.44μg/g, 3.66±0.79μg/g, 1.89±0.73μg/g, 0.26±0.10μg/g respectively, no drug concentration was detected after10 weeks. Comparison of drug concentrations of INH in two different tissues at the same time showed statistical significance(P<0.05). At 24 h, 72 h and 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks and 10 weeks, the drug concentration of RFP in focal bone tissue was 10.85±2.45μg/g, 22.47±1.94μg/g, 38.32±1.73μg/g, 24.22±1.45μg/g, 17.85±1.50μg/g, 9.81±1.30μg/g, 6.35±1.30μg/g, 5.11±0.53μg/g, 1.32±0.33μg/g respectively. The drug concentration of RFP in focal muscle tissue was 5.39±1.50μg/g, 20.66±1.29μg/g, 48.72±2.24μg/g, 32.27±1.63μg/g, 15.58±1.88μg/g, 8.69±0.79μg/g, 3.43±0.39μg/g respectively. No drug concentration was detected after 10 weeks. The concentrations of RFP in two different tissues were significantly different at other time points except 72 h, 4 weeks and 6 weeks(P<0.05). Conclusions: INH-RFP nano hydroxyapatite-calcium sulfate-chitosan artificial bone can sustainably and effectively release anti-tuberculosis drugs in the focal tissues of rabbit spinal tuberculosis model.
引文
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4.马赫,蔡则成,梁思敏,等.载异烟肼、利福平纳米羟基磷灰石-硫酸钙人工骨体外降解及释药研究[J].中国脊柱脊髓杂志,2018,28(7):641-647.
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18.梁卫寰,谭竹钧,区硕俊,等.羟基磷灰石/壳聚糖-柚皮苷缓释材料的制备及性能初探[J].功能材料,2015,46(19):19131-19135.
19.Shi P,Zuo Y,Li X,et al.Gentamicin-impregnated chitosan/nanohydroxyapatite/ethyl cellulose microspheres granules for chronic osteomyelitis therapy[J].J Biomed Mater Res A,2010,93A(3):1020-1031.
20.Kurz SG,Furin JJ,Bark CM.Drug-resistant tuberculosis:challenges and progress[J].Infect Dis Clin North Am,2016,30(2):509-522.
21.Duan Q,Chen Z,Chen C,et al.The prevalence of drug-resistant tuberculosis in mainland China:an updated systematic review and meta-analysis[J].Plos One,2016,11(2):e0148041.doi:10.1371/journal.pone.0148041.
22.Gao Y,Zuo J,Nadia BC,et al.In vitrorelease kinetics of antituberculosis drugs from nanoparticles assessed using a modified dissolution apparatus[J].Biomed Res Int,2013,7:527-531.
23.Jutte PC,Rutgers SR,Van AR,et al.Penetration of isoniazid,rifampicin and pyrazinamide in tuberculous pleural effusion and psoas abscess[J].Int J Tuberc Lung Dis,2004,8(11):1368-1372.
24.Fan SW,Hu ZA.Emphasis on the importance of chemical drug treatment for spinal tuberculosis[J].China J Orthop Trauma,2017,30(9):783-786.
2.Liu P,Jiang H,Li S,et al.Determination of anti-tuberculosis drug concentration and distribution from sustained release microspheres in the vertebrae of a spinal tuberculosis rabbit model[J].J Orthop Res,2017,35(1):200-208.
3.Huang D,Li D,Wang T,et al.Isoniazid conjugated poly(lactide-co-glycolide):Long-term controlled drug release and tissue regeneration for bone tuberculosis therapy[J].Biomaterials,2015,52(1):417-425.
4.马赫,蔡则成,梁思敏,等.载异烟肼、利福平纳米羟基磷灰石-硫酸钙人工骨体外降解及释药研究[J].中国脊柱脊髓杂志,2018,28(7):641-647.
5.陈振,吴鹏,马荣,等.构建新西兰兔脊柱结核模型的实验研究[J].宁夏医科大学学报,2015,37(10):216-223.
6.Ge Z,Wang Z,Wei M.Measuremen of the concentration of three antituberculosis drugs in the focus of spinal tuberculosis[J].Eur Spine J,2008,17(11):1482-1487.
7.Dong J,Zhang S,Liu H,et al.Novel alternative therapy for spinal tuberculosis during surgery:reconstructing with anti-tuberculosis bioactivity implants[J].Expert Opin Drug Deliv,2014,11(3):299-305.
8.Liu Y,Luo D,Wang T.Hierarchical structures of bone and bioinspired bone tissue engineering[J].Small,2016,12(34):4611-4632.
9.邱耿韬,史占军,赵亮.磷酸钙骨水泥在骨组织再生修复应用中的研究进展[J].中国矫形外科杂志,2013,21(14):1406-1409.
10.叶向阳,甄平,李晓飞,等.新型抗结核多孔磷酸钙骨水泥缓释载体的制备与性能研究[J].中国矫形外科杂志,2010,18(23):1981-1986.
11.Kundu B,Soundrapandian C,Nandi SK,et al.Development of new localized drug delivery system based on ceftriaxone-sulbactam composite drug impregnated porous hydroxyapatite:a systematic approach for in vitro and in vivo animal trial[J].Pharm Res,2010,27(8):1659-1676.
12.Chaudhry AA,Yan H,Gong K,et al.High-strength nanograined and translucent hydroxyapatite monoliths via continuous hydrothermal synthesis and optimized spark plasma sintering[J].Acta Biomaterialia,2011,7(2):791-799.
13.廖建国,李艳群,段星泽,等.纳米羟基磷灰石/聚合物复合骨修复材料[J].化学进展,2015,27(Z1):220-228.
14.葛亮.可注射式硫酸钙/纳米羟基磷灰石人工骨的研制及其生物学性能的初步研究[D].第二军医大学,2007.
15.Loi F,Córdova LA,Pajarinen J,et al.Inflammation,fracture and bone repair[J].Bone,2016,86:119-130.doi:10.1016/j.bone.2016.02.020.
16.Foox M,Zilberman M.Drug delivery from gelatin-based systems[J].Expert Opin Drug Deliv,2015,12(9):1547-1563.
17.Sun Y,Li X,Liang X,et al.Calcium phosphate/octadecyl-quatemized carboxymethyl chitosan nanoparticles:an efficient and promising carrier for gene transfection[J].JNanosci Nanotechnol,2013,13(8):5260-5266.
18.梁卫寰,谭竹钧,区硕俊,等.羟基磷灰石/壳聚糖-柚皮苷缓释材料的制备及性能初探[J].功能材料,2015,46(19):19131-19135.
19.Shi P,Zuo Y,Li X,et al.Gentamicin-impregnated chitosan/nanohydroxyapatite/ethyl cellulose microspheres granules for chronic osteomyelitis therapy[J].J Biomed Mater Res A,2010,93A(3):1020-1031.
20.Kurz SG,Furin JJ,Bark CM.Drug-resistant tuberculosis:challenges and progress[J].Infect Dis Clin North Am,2016,30(2):509-522.
21.Duan Q,Chen Z,Chen C,et al.The prevalence of drug-resistant tuberculosis in mainland China:an updated systematic review and meta-analysis[J].Plos One,2016,11(2):e0148041.doi:10.1371/journal.pone.0148041.
22.Gao Y,Zuo J,Nadia BC,et al.In vitrorelease kinetics of antituberculosis drugs from nanoparticles assessed using a modified dissolution apparatus[J].Biomed Res Int,2013,7:527-531.
23.Jutte PC,Rutgers SR,Van AR,et al.Penetration of isoniazid,rifampicin and pyrazinamide in tuberculous pleural effusion and psoas abscess[J].Int J Tuberc Lung Dis,2004,8(11):1368-1372.
24.Fan SW,Hu ZA.Emphasis on the importance of chemical drug treatment for spinal tuberculosis[J].China J Orthop Trauma,2017,30(9):783-786.