载万古霉素缓释微球纳米羟基磷灰石/壳聚糖支架联合自体红骨髓可修复慢性骨髓炎兔的骨缺损
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摘要
背景:前期研究证实,万古霉素缓释微球对兔慢性骨髓炎具有良好的治疗效果,纳米羟基磷灰石/壳聚糖具有良好的生物相容性、降解性及成骨诱导性,自体红骨髓联合纳米羟基磷灰石/壳聚糖支架可用于修复兔桡骨骨缺损。因此,3者联合修复兔慢性骨髓炎骨缺损的效果有待验证。目的:探讨载万古霉素/聚乳酸-羟基乙酸共聚物(polyactic-co-glycolicacid,PLGA)缓释微球的纳米羟基磷灰石/壳聚糖支架,联合自体红骨髓修复兔慢性骨髓炎骨缺损的治疗效果及可行性。方法:采用水-油-水乳相法制备万古霉素/PLGA缓释微球,将缓释微球与纳米羟基磷灰石/壳聚糖混合液充分混匀,采用真空冷冻干造法制备载万古霉素/PLGA缓释微球的纳米羟基磷灰石/壳聚糖支架,对支架进行力学性能测试及万古霉素释放量检测。在20只5月龄新西兰大白兔(解放军第三军医大学大坪医院实验动物中心提供)左侧胫骨髓腔内注射金黄色葡萄球菌,构建慢性骨髓炎骨缺损模型,随机分4组干预,A组仅作清创处理,B组清创后植入纳米羟基磷灰石/壳聚糖支架,C组清创后植入载万古霉素/PLGA缓释微球纳米羟基磷灰石/壳聚糖支架,D组清创后植入载万古霉素/PLGA缓释微球纳米羟基磷灰石/壳聚糖支架及自体红骨髓。治疗3个月后,通过X射线、病理苏木精-伊红染色评价骨缺损修复效果。结果与结论:(1)载万古霉素/PLGA缓释微球的纳米羟基磷灰石/壳聚糖支架,具有良好的生物力学性能及缓慢平稳释放万古霉素的能力;(2)治疗3个月后的X射线显示,A、B组胫骨近端仍见明显的缺损区、骨质破坏及骨膜反应;C组可见新生骨形成,缺损区稍模糊,但仍有缺损存在;D组可见新骨形成,缺损区模糊,密度稍增高,骨膜反应消失;(3)治疗3个月后的病理苏木精-伊红染色显示,A、B组骨小梁紊乱、破坏较多;C、D组骨小梁排列较为整齐,破坏较少,且D组骨小梁粗大,排列有序;(4)结果表明,载万古霉素/PLGA缓释微球的纳米羟基磷灰石/壳聚糖支架联合自体红骨髓修复兔慢性骨髓炎骨缺损效果良好。
BACKGROUND: Preliminary studies have confirmed that vancomycin sustained-release microspheres exert satisfactory therapeutic effect on chronic osteomyelitis in rabbits. Nano-hydroxyapatite/chitosan(nHA/CS) has good biocompatibility, degradability and osteogenic induction. Autologous red bone marrow can be used as a seed cell source combined with nHA/CS scaffold to repair rabbit tibia bone defects. Therefore, the combined effects of the three materials in the repair of rabbit chronic osteomyelitis bone defects need a further investigation. OBJECTIVE: To explore the efficacy and feasibility of nHA/CS scaffold carrying vancomycin/polyactic-co-glycolic acid(PLGA) sustained-release microspheres combined with autologous red bone marrow in the repair of chronic osteomyelitis bone defects in rabbits. METHODS: Vancomycin/PLGA sustained-release microspheres were prepared by water oil and water emulsion method. The nHA/CS scaffolds carrying vancomycin/PLGA sustained-release microspheres were prepared by vacuum freeze drying, and mechanical properties and vancomycin release of the scaffold were determined. Twenty 5-month-old New Zealand white rabbits provided by Laboratory Animal Centre of Daping Hospital of Third Military Medical University were selected for establishing the chronic osteomyelitis bone defect model by intramedullary infection of Staphylococcus aureus into the left tibia. The model rabbits were randomly divided into four groups: group A was only given only for debridement treatment; in group B, the nHA/CS scaffold was implanted after debridement; in group C, nHA/CS scaffolds carrying vancomycin/PLGA sustained-release microspheres were implanted after debridement; in group D, nHA/CS scaffolds carrying vancomycin/PLGA microspheres and autologous red bone marrow were implanted after debridement. At 3 months after implantation, the repair effect was observed by X-ray and hematoxylin-eosin staining. RESULTS AND CONCLUSION: The nHA/CS scaffolds carrying vancomycin/PLGA sustained-release microspheres held good biomechanical properties and drug sustained release. At 3 months after implantation, X-ray revealed that the defect region in the groups A and B had no obvious improvement, and there were still bone destruction and periosteal reaction. The group C showed the formation of the new bone, the defect area was slightly blurred, but the defect still existed. In the group D, new bone formed, the defect area became blurred, the density was slightly high, and the periosteum reaction disappeared. Hematoxylin-eosin staining showed the bone trabecular disorder and more damage in the groups A and B. The bone trabecula was neatly arranged and less damaged in the groups C and D, and especially the group D had thickened bone trabecula arranged regularly. These results indicate that nHA/CS scaffolds carrying vancomycin/PLGA sustained-release microspheres combined with autologous red bone marrow exhibit good outcomes in repairing the bone defect due to chronic osteomyelitis in rabbits.
BACKGROUND: Preliminary studies have confirmed that vancomycin sustained-release microspheres exert satisfactory therapeutic effect on chronic osteomyelitis in rabbits. Nano-hydroxyapatite/chitosan(nHA/CS) has good biocompatibility, degradability and osteogenic induction. Autologous red bone marrow can be used as a seed cell source combined with nHA/CS scaffold to repair rabbit tibia bone defects. Therefore, the combined effects of the three materials in the repair of rabbit chronic osteomyelitis bone defects need a further investigation. OBJECTIVE: To explore the efficacy and feasibility of nHA/CS scaffold carrying vancomycin/polyactic-co-glycolic acid(PLGA) sustained-release microspheres combined with autologous red bone marrow in the repair of chronic osteomyelitis bone defects in rabbits. METHODS: Vancomycin/PLGA sustained-release microspheres were prepared by water oil and water emulsion method. The nHA/CS scaffolds carrying vancomycin/PLGA sustained-release microspheres were prepared by vacuum freeze drying, and mechanical properties and vancomycin release of the scaffold were determined. Twenty 5-month-old New Zealand white rabbits provided by Laboratory Animal Centre of Daping Hospital of Third Military Medical University were selected for establishing the chronic osteomyelitis bone defect model by intramedullary infection of Staphylococcus aureus into the left tibia. The model rabbits were randomly divided into four groups: group A was only given only for debridement treatment; in group B, the nHA/CS scaffold was implanted after debridement; in group C, nHA/CS scaffolds carrying vancomycin/PLGA sustained-release microspheres were implanted after debridement; in group D, nHA/CS scaffolds carrying vancomycin/PLGA microspheres and autologous red bone marrow were implanted after debridement. At 3 months after implantation, the repair effect was observed by X-ray and hematoxylin-eosin staining. RESULTS AND CONCLUSION: The nHA/CS scaffolds carrying vancomycin/PLGA sustained-release microspheres held good biomechanical properties and drug sustained release. At 3 months after implantation, X-ray revealed that the defect region in the groups A and B had no obvious improvement, and there were still bone destruction and periosteal reaction. The group C showed the formation of the new bone, the defect area was slightly blurred, but the defect still existed. In the group D, new bone formed, the defect area became blurred, the density was slightly high, and the periosteum reaction disappeared. Hematoxylin-eosin staining showed the bone trabecular disorder and more damage in the groups A and B. The bone trabecula was neatly arranged and less damaged in the groups C and D, and especially the group D had thickened bone trabecula arranged regularly. These results indicate that nHA/CS scaffolds carrying vancomycin/PLGA sustained-release microspheres combined with autologous red bone marrow exhibit good outcomes in repairing the bone defect due to chronic osteomyelitis in rabbits.
引文
[1]Dhanireddy S,Neme S.44-Acute and Chronic Osteomyelitis.Infect Dis.2017;1:388-398.
[2]Winkler H,Haiden P.Treatment of Chronic Bone Infection.Oper Tech Orthop.2016;26(1):2-11.
[3]Ning L,Malmstrom H,Ren YF.Porous Collagen-Hydroxyapatite Scaffolds With Mesenchymal Stem Cells for Bone Regeneration.JOral Implantol.2015;41(1):45-49.
[4]David F,Levingstone TJ,Schneeweiss W,et al.Enhanced bone healing using collagen-hydroxyapatite scaffold implantation in the treatment of a large multiloculated mandibular aneurysmal bone cyst in a thoroughbred filly.J Tissue Eng Regen Med.2015;9(10):1193-1199.
[5]Gomes D,Pereira M,Bettencourt AF.Osteomyelitis:an overview of antimicrobial therapy.Braz J Pharm Sci.2013;49(1):13-27.
[6]Inzana JA,Schwarz EM,Kates SL,et al.Biomaterials approaches to treating implant-associated osteomyelitis.Biomaterials.2016;81:58-71.
[7]Kaplan SL.Staphylococcus aureus Infections in Children:The Implications of Changing Trends.Pediatrics.2016;137(4):e20160101.
[8]Yang D,Wijenayaka AR,Solomon LB,et al.Novel Insights into Staphylococcus aureusDeep Bone Infections:the Involvement of Osteocytes.MBio.2018;9(2).pii:e00415-18.doi:10.1128/mBio.00415-18.
[9]Duszynska W,Taccone FS,Hurkacz M,et al.Continuous vs.intermittent vancomycin therapy for Gram-positive infections not caused by methicillin-resistant Staphylococcus aureus.Minerva Anestesiol.2016;82(3):284-293.
[10]Cao G,Liang X,Zhang J,et al.Vancomycin serum trough concentration vs.clinical outcome in patients with gram-positive infection:a retrospective analysis.J Clin Pharm Ther.2016;40(6):640-644.
[11]Kadry AA,Al-Suwayeh SA,Abd-Allah AR,et al.Treatment of experimental osteomyelitis by liposomal antibiotics.J Antimicrob Chemother.2004;54(6):1103-1108.
[12]Broussou DC,Lacroix MZ,Toutain PL,et al.Differential Activity of the Combination of Vancomycin and Amikacin on Planktonic vs.Biofilm-Growing Staphylococcus aureus Bacteria in a Hollow Fiber Infection Model.Front Microbiol.2018;9:572.
[13]Marrelli M,Tatullo M.Influence of PRF in the healing of bone and gingival tissues.Clinical and histological evaluations.Eur Rev Med Pharmacol Sci.2013;17(14):1958-1962.
[14]Marrelli M,Falisi G,Apicella A,et al.Behaviour of dental pulp stem cells on different types of innovative mesoporous and nanoporous silicon scaffolds with different functionalizations of the surfaces.J Biol Regul Homeost Agents.2015;29(4):991-997.
[15]Paduano F,Marrelli M,Alom N,et al.Decellularized bone extracellular matrix and human dental pulp stem cells as a construct for bone regeneration.J Biomater Sci Polym Ed.2017;28(8):730-748.
[16]胡荣康,吴林秀,陈明军,等.不同分子量壳聚糖的制备及其应用研究进展[J].食品工业科技,2017,38(15):324-328.
[17]Boukari Y,Qutachi O,Scurr DJ,et al.A dual-application poly(dl-lactic-co-glycolic)acid(PLGA)-chitosan composite scaffold for potential use in bone tissue engineering.J Biomater Sci Polym Ed.2017;28(16):1966-1983.
[18]Shamekhi MA,Rabiee A,Mirzadeh H,et al.Fabrication and characterization of hydrothermal cross-linked chitosan porous scaffolds for cartilage tissue engineering applications.Mater Sci Eng CMater Biol Appl.2017;80:532-542.
[19]Uswatta SP,Okeke IU,Jayasuriya AC.Injectable porous nano-hydroxyapatite/chitosan/tripolyphosphate scaffolds with improved compressive strength for bone regeneration.Mater Sci Eng C Mater Biol Appl.2016;69:505-512.
[20]Wang XF,Lu PJ,Song Y,et al.Nano hydroxyapatite particles promote osteogenesis in a three-dimensional bio-printing construct consisting of alginate/gelatin/hASCs.Rsc Adv.2016;6(8):6832-6842.
[21]Medeiros JS,Oliveira AM,de Carvalho JO,et al.Nanohydroxyapatite/Graphene Nanoribbons Nanocomposites Induce in Vitro Osteogenesis and Promote in Vivo Bone Neoformation.Acs Biomater Sci Eng.2018;4(5).DOI:10.1021/acsbiomaterials.7b01032
[22]Ge S,Zhao N,Wang L,et al.Bone repair by periodontal ligament stem cellseeded nanohydroxyapatite-chitosan scaffold.Int J Nanomedicine.2012;7:5405-5414.
[23]Biazar E,Heidari KS,Tavirani MR,et al.Bone reconstruction in rat calvarial defects by chitosan/hydroxyapatite nanoparticles scaffold loaded with unrestricted somatic stem cells.Artif Cells Nanomed Biotechnol.2015;43(2):112-116.
[24]杨新明,孟宪勇,张瑛,等.带蒂筋膜瓣包裹自体红骨髓复合体修复骨缺损的疗效[J].中华创伤骨科杂志,2012,14(9):741-747.
[25]邵擎东,李宇飞,许天明,等.自体红骨髓移植治疗鼠胫骨骨不连[J].中华实验外科杂志,2015,32(6):1375-1377.
[26]向柄彦,郭元.载药微球联合BMP-2治疗兔慢性骨髓炎的实验研究[J].重庆医学,2015,44(8):1032-1034.
[27]向柄彦,郭元.载药组织工程支架的构建及其性能研究[J].现代医药卫生,2015,31(9):1281-1282.
[28]向柄彦,韩小松,陈炎城,等.不同浓度富血小板血浆复合自体红骨髓及纳米骨治疗兔桡骨骨缺损的实验研究[J].现代预防医学,2012,39(19):5126-5131.
[29]Li Y,Zhang Z,Zhang Z.Porous Chitosan/Nano-Hydroxyapatite Composite Scaffolds Incorporating Simvastatin-Loaded PLGAMicrospheres for Bone Repair.Cells Tissues Organs.2018;205(1):20-31.
[30]Norden CW,Myerowitz RL,Keleti E.Experimental osteomyelitis due to Staphylococcus aureus or Pseudomonas aeruginosa:a radiographicpathological correlative analysis.Br J Exp Pathol.1980;61(4):451-460.
[31]Hatzenbuehler J,Pulling TJ.Diagnosis and management of osteomyelitis.Am Fam Physician.2011;84(9):1027-1033.
[32]Bibbo C,Stough JD.Reduction calcaneoplasty and local muscle rotation flap as a salvage option for calcaneal osteomyelitis with soft tissue defect.J Foot Ankle Surg.2012;51(3):375-378.
[33]万古霉素临床应用剂量专家组.万古霉素临床应用剂量中国专家共识[J].中华传染病杂志,2012,30(11):641-646.
[34]龙亚周,朱泽兴,于燕,等.万古霉素不同给药途径治疗慢性骨髓炎的研究进展[J].中华外科杂志,2016,54(9):716-720.
[2]Winkler H,Haiden P.Treatment of Chronic Bone Infection.Oper Tech Orthop.2016;26(1):2-11.
[3]Ning L,Malmstrom H,Ren YF.Porous Collagen-Hydroxyapatite Scaffolds With Mesenchymal Stem Cells for Bone Regeneration.JOral Implantol.2015;41(1):45-49.
[4]David F,Levingstone TJ,Schneeweiss W,et al.Enhanced bone healing using collagen-hydroxyapatite scaffold implantation in the treatment of a large multiloculated mandibular aneurysmal bone cyst in a thoroughbred filly.J Tissue Eng Regen Med.2015;9(10):1193-1199.
[5]Gomes D,Pereira M,Bettencourt AF.Osteomyelitis:an overview of antimicrobial therapy.Braz J Pharm Sci.2013;49(1):13-27.
[6]Inzana JA,Schwarz EM,Kates SL,et al.Biomaterials approaches to treating implant-associated osteomyelitis.Biomaterials.2016;81:58-71.
[7]Kaplan SL.Staphylococcus aureus Infections in Children:The Implications of Changing Trends.Pediatrics.2016;137(4):e20160101.
[8]Yang D,Wijenayaka AR,Solomon LB,et al.Novel Insights into Staphylococcus aureusDeep Bone Infections:the Involvement of Osteocytes.MBio.2018;9(2).pii:e00415-18.doi:10.1128/mBio.00415-18.
[9]Duszynska W,Taccone FS,Hurkacz M,et al.Continuous vs.intermittent vancomycin therapy for Gram-positive infections not caused by methicillin-resistant Staphylococcus aureus.Minerva Anestesiol.2016;82(3):284-293.
[10]Cao G,Liang X,Zhang J,et al.Vancomycin serum trough concentration vs.clinical outcome in patients with gram-positive infection:a retrospective analysis.J Clin Pharm Ther.2016;40(6):640-644.
[11]Kadry AA,Al-Suwayeh SA,Abd-Allah AR,et al.Treatment of experimental osteomyelitis by liposomal antibiotics.J Antimicrob Chemother.2004;54(6):1103-1108.
[12]Broussou DC,Lacroix MZ,Toutain PL,et al.Differential Activity of the Combination of Vancomycin and Amikacin on Planktonic vs.Biofilm-Growing Staphylococcus aureus Bacteria in a Hollow Fiber Infection Model.Front Microbiol.2018;9:572.
[13]Marrelli M,Tatullo M.Influence of PRF in the healing of bone and gingival tissues.Clinical and histological evaluations.Eur Rev Med Pharmacol Sci.2013;17(14):1958-1962.
[14]Marrelli M,Falisi G,Apicella A,et al.Behaviour of dental pulp stem cells on different types of innovative mesoporous and nanoporous silicon scaffolds with different functionalizations of the surfaces.J Biol Regul Homeost Agents.2015;29(4):991-997.
[15]Paduano F,Marrelli M,Alom N,et al.Decellularized bone extracellular matrix and human dental pulp stem cells as a construct for bone regeneration.J Biomater Sci Polym Ed.2017;28(8):730-748.
[16]胡荣康,吴林秀,陈明军,等.不同分子量壳聚糖的制备及其应用研究进展[J].食品工业科技,2017,38(15):324-328.
[17]Boukari Y,Qutachi O,Scurr DJ,et al.A dual-application poly(dl-lactic-co-glycolic)acid(PLGA)-chitosan composite scaffold for potential use in bone tissue engineering.J Biomater Sci Polym Ed.2017;28(16):1966-1983.
[18]Shamekhi MA,Rabiee A,Mirzadeh H,et al.Fabrication and characterization of hydrothermal cross-linked chitosan porous scaffolds for cartilage tissue engineering applications.Mater Sci Eng CMater Biol Appl.2017;80:532-542.
[19]Uswatta SP,Okeke IU,Jayasuriya AC.Injectable porous nano-hydroxyapatite/chitosan/tripolyphosphate scaffolds with improved compressive strength for bone regeneration.Mater Sci Eng C Mater Biol Appl.2016;69:505-512.
[20]Wang XF,Lu PJ,Song Y,et al.Nano hydroxyapatite particles promote osteogenesis in a three-dimensional bio-printing construct consisting of alginate/gelatin/hASCs.Rsc Adv.2016;6(8):6832-6842.
[21]Medeiros JS,Oliveira AM,de Carvalho JO,et al.Nanohydroxyapatite/Graphene Nanoribbons Nanocomposites Induce in Vitro Osteogenesis and Promote in Vivo Bone Neoformation.Acs Biomater Sci Eng.2018;4(5).DOI:10.1021/acsbiomaterials.7b01032
[22]Ge S,Zhao N,Wang L,et al.Bone repair by periodontal ligament stem cellseeded nanohydroxyapatite-chitosan scaffold.Int J Nanomedicine.2012;7:5405-5414.
[23]Biazar E,Heidari KS,Tavirani MR,et al.Bone reconstruction in rat calvarial defects by chitosan/hydroxyapatite nanoparticles scaffold loaded with unrestricted somatic stem cells.Artif Cells Nanomed Biotechnol.2015;43(2):112-116.
[24]杨新明,孟宪勇,张瑛,等.带蒂筋膜瓣包裹自体红骨髓复合体修复骨缺损的疗效[J].中华创伤骨科杂志,2012,14(9):741-747.
[25]邵擎东,李宇飞,许天明,等.自体红骨髓移植治疗鼠胫骨骨不连[J].中华实验外科杂志,2015,32(6):1375-1377.
[26]向柄彦,郭元.载药微球联合BMP-2治疗兔慢性骨髓炎的实验研究[J].重庆医学,2015,44(8):1032-1034.
[27]向柄彦,郭元.载药组织工程支架的构建及其性能研究[J].现代医药卫生,2015,31(9):1281-1282.
[28]向柄彦,韩小松,陈炎城,等.不同浓度富血小板血浆复合自体红骨髓及纳米骨治疗兔桡骨骨缺损的实验研究[J].现代预防医学,2012,39(19):5126-5131.
[29]Li Y,Zhang Z,Zhang Z.Porous Chitosan/Nano-Hydroxyapatite Composite Scaffolds Incorporating Simvastatin-Loaded PLGAMicrospheres for Bone Repair.Cells Tissues Organs.2018;205(1):20-31.
[30]Norden CW,Myerowitz RL,Keleti E.Experimental osteomyelitis due to Staphylococcus aureus or Pseudomonas aeruginosa:a radiographicpathological correlative analysis.Br J Exp Pathol.1980;61(4):451-460.
[31]Hatzenbuehler J,Pulling TJ.Diagnosis and management of osteomyelitis.Am Fam Physician.2011;84(9):1027-1033.
[32]Bibbo C,Stough JD.Reduction calcaneoplasty and local muscle rotation flap as a salvage option for calcaneal osteomyelitis with soft tissue defect.J Foot Ankle Surg.2012;51(3):375-378.
[33]万古霉素临床应用剂量专家组.万古霉素临床应用剂量中国专家共识[J].中华传染病杂志,2012,30(11):641-646.
[34]龙亚周,朱泽兴,于燕,等.万古霉素不同给药途径治疗慢性骨髓炎的研究进展[J].中华外科杂志,2016,54(9):716-720.