β-磷酸三钙复合改良型富血小板纤维蛋白促成骨:影像学及免疫组织化学分析
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摘要
背景:目前研究证明多种骨替代材料与血液衍生物联合应用均具有更好的成骨作用。现阶段国内外均未见关于β-磷酸三钙与改良型富血小板纤维蛋白复合应用于骨缺损修复的相关报道。目的:观察β-磷酸三钙复合改良型富血小板纤维蛋白用于骨缺损修复的成骨特点及效果。方法:将39只日本大耳兔随机分为4组,均在兔双侧后肢股骨髁外侧面制备6.0 mm×8.0 mm的柱状临界性骨缺损,改良型富血小板纤维蛋白组(n=12)骨缺损中填入兔自体改良型富血小板纤维蛋白碎屑,β-磷酸三钙组(n=12)填入β-磷酸三钙颗粒,复合组(n=12)填入β-磷酸三钙颗粒与兔自体改良型富血小板纤维蛋白碎屑的等质量混合物,空白组(n=3)不填入任何材料。术后1,2,3个月获取完整股骨段,拍摄X射线片后进行破骨细胞分化因子、骨保护素蛋白免疫组织化学分析。结果与结论:(1)X射线显示随着时间的推移,改良型富血小板纤维蛋白组圆形透射影从边缘向中心密度逐渐增高,β-磷酸三钙组和复合组圆形阻射影从边缘向中心密度逐渐降低,最后密度都接近周围天然骨或者与周围天然骨一致;(2)免疫组织化学显示,4组均有破骨细胞分化因子、骨保护素蛋白阳性表达,相同时间点下破骨细胞分化因子与骨保护素蛋白表达的平均吸光度值比较为:复合组>β-磷酸三钙组>改良型富血小板纤维蛋白组>空白组(P均<0.05);(3)结果表明,β-磷酸三钙与改良型富血小板纤维蛋白复合应用时的成骨效果优于二者单独应用。
BACKGROUND: It has been proved that various bone substitute materials combined with blood derivatives contribute to osteogenesis. At present, no relevant reports have been reported on the combination of β-tricalcium phosphate (β-TCP) combined with advanced platelet-rich fibrin (A-PRF) in the repair of bone defects. OBJECTIVE: To observe the characteristics and effects of β-TCP combined with A-PRF for repair of bone defects. METHODS: Thirty-nine Japanese big ear rabbits (provided by the Chengdu Dashuo Experimental Animal Center in China) were randomly divided into A-PRF group (n=12), β-TCP group (n=12), composite group (n=12), and blank control group (n=3). A 6.0 mm× 8.0 mm cylindrical critical bone defect was made on the lateral femoral condyle of each side of hind legs of each rabbit and filled in with different materials in corresponding groups, respectively. No implantation was done in the blank control group. The whole femur of each rat was taken at 1, 2 and 3 months after implantation, and X-ray films were taken as well as receptor activator nuclear factor kappa B ligand and osteoprotegerin immunohistochemical analysis. RESULTS AND CONCLUSION:(1) Over time, X-ray films showed high-density shadow in the bone defect area in the A-PRF group, and there was a centripetal growth trend from the edge of bone defect to the center, while in the β-TCP group and composite group, there was a centripetal decrease trend from the edge to the center of the bone defect, and finally the density was close to natural bone or consistent to the surrounding bone tissues.(2) Receptor activator nuclear factor kappa B ligand and osteoprotegerin immunohistochemical analysis showed a positive expression in all the groups. The order of the mean absorbance value in the four groups was as follows: the composite group > β-TCP group > A-PRF group > blank group, all of which were statistically significant (P < 0.05). To conclude, β-TCP combined with A-PRF has a better osteogenic effect than individual use.
BACKGROUND: It has been proved that various bone substitute materials combined with blood derivatives contribute to osteogenesis. At present, no relevant reports have been reported on the combination of β-tricalcium phosphate (β-TCP) combined with advanced platelet-rich fibrin (A-PRF) in the repair of bone defects. OBJECTIVE: To observe the characteristics and effects of β-TCP combined with A-PRF for repair of bone defects. METHODS: Thirty-nine Japanese big ear rabbits (provided by the Chengdu Dashuo Experimental Animal Center in China) were randomly divided into A-PRF group (n=12), β-TCP group (n=12), composite group (n=12), and blank control group (n=3). A 6.0 mm× 8.0 mm cylindrical critical bone defect was made on the lateral femoral condyle of each side of hind legs of each rabbit and filled in with different materials in corresponding groups, respectively. No implantation was done in the blank control group. The whole femur of each rat was taken at 1, 2 and 3 months after implantation, and X-ray films were taken as well as receptor activator nuclear factor kappa B ligand and osteoprotegerin immunohistochemical analysis. RESULTS AND CONCLUSION:(1) Over time, X-ray films showed high-density shadow in the bone defect area in the A-PRF group, and there was a centripetal growth trend from the edge of bone defect to the center, while in the β-TCP group and composite group, there was a centripetal decrease trend from the edge to the center of the bone defect, and finally the density was close to natural bone or consistent to the surrounding bone tissues.(2) Receptor activator nuclear factor kappa B ligand and osteoprotegerin immunohistochemical analysis showed a positive expression in all the groups. The order of the mean absorbance value in the four groups was as follows: the composite group > β-TCP group > A-PRF group > blank group, all of which were statistically significant (P < 0.05). To conclude, β-TCP combined with A-PRF has a better osteogenic effect than individual use.
引文
[1]胡文军,朱靖恺,马威.植骨材料在口腔种植中的应用概况及进展[J].中国实用口腔科杂志,2018,11(1):17-23.
[2]Jeon IS,Heo MS,Han KH,et al.Vertical ridge augmentation with simultaneous implant placement usingβ-TCP and PRP:A report of two cases.J Oral Maxillofac Surg Med Pathol.2013;25(3):226-231.
[3]Sager M,Ferrari D,Wieland M,et al.Immunohis-tochemical characterization of wound healing at two different bone graft substitutes.Int J Oral Maxil-lofac Surg.2012;41(5):657-666.
[4]Luo T,Zhang W,Shi B,et al.Enhanced bone regeneration around dental implant with bone mor-phogenetic protein 2 gene and vascular endothelial growth factor protein delivery.Clin Oral Implants Res.2012;23(4):467-473.
[5]宋乔健.口腔颌面部骨组织缺损修复材料的研究进展[J].临床医药文献电子杂志,2017,4(25):175-177.
[6]Walsh W,Vizesi F,Michael D,et al.Beta-TCP bone graft substitutes in a bilateral rabbit tibial defect model.Biomaterials.2008;29(3):266-271.
[7]孙佳佳,王熙.A-PRF诱导软硬组织再生的研究[J].现代口腔医学杂志,2018,32(2):115-117.
[8]毛俊丽,孙勇,赵峰,等.兔PRF、A-PRF制备方法的筛选[J].西南国防医药,2016,26(6):593-596.
[9]Schmitz JP,Hollinger JO.The critical size defect as an experimental model for craniomandibulofacial nonunions.Clin Orthop Relat Res.1986;(205):299-308.
[10]周芳,李静,余磊,等.兔桡骨临界骨缺损模型的制备[J].中国组织工程研究,2011,15(50):9385-9388.
[11]Patel ZS,Young S,Tabata Y,et al.Dual delivery of an angiogenic and an osteogenic growth factor for bone regeneration in a critical size defect model.Bone.2008;43(5):931-940.
[12]Kargozar S,Lotfiakhshaiesh N,Ai J,et al.Osteogenic Evaluation of Stem Cell-Seeded Bioactive Glasses Containing Strontium and Cobalt in a Critical Size Defect in Rabbit Femur:Conference of the European Society for Biomaterials[C].Conference:28th ANNUALCONFERENCE OF THE EUROPEAN SOCIETY FORBIOMATERIALS(ESB),At ATHENS-GREECE Megaron Athens International Conference Centre,2017.
[13]李阳.牙龈成纤维细胞成骨能力的体外及体内研究[D].天津:天津医科大学,2017.
[14]Ghanaati S,Booms P,Orlowska A,et al.Advanced platelet-rich fibrin:a new concept for cell-based tissue engineering by mean of inflammatory cells.J Oral Implantol.2014;40(6):679-689.
[15]Kobayashi E,Flückiger L,Fujiokakobayashi M,et al.Comparative release of growth factors from PRP,PRF,and advanced-PRF.Clin Oral Investig.2016;20(9):2353-2360.
[16]Masuki H,Okudera T,Watanebe T,et al.Growth factor and pro-inflammatory cytokine contents in platelet-rich plasma(PRP),plasma rich in growth factors(PRGF),advanced platelet-rich fibrin(A-PRF),and concentrated growth factors(CGF).Int J Implant Dent.2016;2(1):19.
[17]何婷婷.两种富血小板纤维蛋白的降解特性研究[D].泸州:西南医科大学,2017.
[18]焦志立,谢晓玲,付冬梅,等.改良型富血小板纤维蛋白在兔颅骨诱导成骨中的组织学观察[J].中国组织工程研究,2017,21(14):2208-2214.
[19]Gao P,Zhang H,Liu Y,et al.Beta-tricalcium phosphate granules improve osteogenesis in vitro and establish innovative osteo-regenerators for bone tissue engineering in vivo.Sci Rep.2016;6:23367.
[20]Cornell CN.Osteoconductive materials and their role as substitudes for autogenous bone grafts.Orthop Clin North Am.1999;30(4):591-598.
[21]Tanaka T,Kumagae Y,Chazono M,et al.A novel evaluation system to monitor bone formation andβ-tricalcium phosphate resorption in opening wedge high tibial osteotomy.Knee Surg Sports Traumatol Arthrosc.2015;23(7):2007-2011.
[22]Meijer HJ,Steen WH,Bosman F.Standarized radiograghs of the alveolar crest around implants in the mandible.J Prosthet Dent.1992;68:318-321.
[23]于威,李建军.去抗原牛松质骨支架复合骨形态发生蛋白2基因在骨缺损修复过程中的血管化反应[J].中国组织工程研究与临床康复,2008,12(23):4559-4562.
[24]Grzibovskis M,Pilmane M,Urtane I.Today's under-standing about bone aging.Stomatologija.2010;12(4):99-104.
[25]Yu T,Pan H,Hu Y,et al.Autologous platelet-rich plasma induces bone formation of tissue-engineered bone with bone marrow mesenchymal stem cells on beta-tricalcium phosphate ceramics.J Orthop Surg Res.2017;12(1):178.
[26]Nelson C,Warren J,Wang MH,et al.RANKL Employs Distinct Binding Modes to Engage RANK and the Osteoprotegerin Decoy Receptor.Structure.2012;20(11):1971-1982.
[27]Stein NC,Kreutzmann C,Zimmermann SP,et al.Interleukin-4 and interleukin-13 stimulate the osteoclast inhibitor osteoprotegerin by human endothelial cells through the STAT6 pathway.J Bone Miner Res.2008;23(5):750-758.
[28]Kearns AE,Khosla S,Kostenuik PJ.Receptor activator of nuclear factor kappaB ligand and osteoprotegerin regulation of bone remodeling in health and disease.Endocr Rev.2008;29(2):155-192.
[29]Nakashima T,Takayanagi H.Osteoimmunology:crosstalk between the immune and bone systems.J Clin Immunol.2009;29(5):555-567.
[30]Zhou L,Liu Q,Yang ML,et al.Dihydroartemisinin,an Anti-Malaria drug,suppresses estrogen deficiency-induced osteoporosis,osteoclast formation,and RANKL-Induced signaling pathways.JBone Miner Res.2016;31(5):964-974.
[31]J Rieu J,Goeuriot P.Ceramic composites for Biomedical applations.Clin Mater.1993;12:211-217.
[2]Jeon IS,Heo MS,Han KH,et al.Vertical ridge augmentation with simultaneous implant placement usingβ-TCP and PRP:A report of two cases.J Oral Maxillofac Surg Med Pathol.2013;25(3):226-231.
[3]Sager M,Ferrari D,Wieland M,et al.Immunohis-tochemical characterization of wound healing at two different bone graft substitutes.Int J Oral Maxil-lofac Surg.2012;41(5):657-666.
[4]Luo T,Zhang W,Shi B,et al.Enhanced bone regeneration around dental implant with bone mor-phogenetic protein 2 gene and vascular endothelial growth factor protein delivery.Clin Oral Implants Res.2012;23(4):467-473.
[5]宋乔健.口腔颌面部骨组织缺损修复材料的研究进展[J].临床医药文献电子杂志,2017,4(25):175-177.
[6]Walsh W,Vizesi F,Michael D,et al.Beta-TCP bone graft substitutes in a bilateral rabbit tibial defect model.Biomaterials.2008;29(3):266-271.
[7]孙佳佳,王熙.A-PRF诱导软硬组织再生的研究[J].现代口腔医学杂志,2018,32(2):115-117.
[8]毛俊丽,孙勇,赵峰,等.兔PRF、A-PRF制备方法的筛选[J].西南国防医药,2016,26(6):593-596.
[9]Schmitz JP,Hollinger JO.The critical size defect as an experimental model for craniomandibulofacial nonunions.Clin Orthop Relat Res.1986;(205):299-308.
[10]周芳,李静,余磊,等.兔桡骨临界骨缺损模型的制备[J].中国组织工程研究,2011,15(50):9385-9388.
[11]Patel ZS,Young S,Tabata Y,et al.Dual delivery of an angiogenic and an osteogenic growth factor for bone regeneration in a critical size defect model.Bone.2008;43(5):931-940.
[12]Kargozar S,Lotfiakhshaiesh N,Ai J,et al.Osteogenic Evaluation of Stem Cell-Seeded Bioactive Glasses Containing Strontium and Cobalt in a Critical Size Defect in Rabbit Femur:Conference of the European Society for Biomaterials[C].Conference:28th ANNUALCONFERENCE OF THE EUROPEAN SOCIETY FORBIOMATERIALS(ESB),At ATHENS-GREECE Megaron Athens International Conference Centre,2017.
[13]李阳.牙龈成纤维细胞成骨能力的体外及体内研究[D].天津:天津医科大学,2017.
[14]Ghanaati S,Booms P,Orlowska A,et al.Advanced platelet-rich fibrin:a new concept for cell-based tissue engineering by mean of inflammatory cells.J Oral Implantol.2014;40(6):679-689.
[15]Kobayashi E,Flückiger L,Fujiokakobayashi M,et al.Comparative release of growth factors from PRP,PRF,and advanced-PRF.Clin Oral Investig.2016;20(9):2353-2360.
[16]Masuki H,Okudera T,Watanebe T,et al.Growth factor and pro-inflammatory cytokine contents in platelet-rich plasma(PRP),plasma rich in growth factors(PRGF),advanced platelet-rich fibrin(A-PRF),and concentrated growth factors(CGF).Int J Implant Dent.2016;2(1):19.
[17]何婷婷.两种富血小板纤维蛋白的降解特性研究[D].泸州:西南医科大学,2017.
[18]焦志立,谢晓玲,付冬梅,等.改良型富血小板纤维蛋白在兔颅骨诱导成骨中的组织学观察[J].中国组织工程研究,2017,21(14):2208-2214.
[19]Gao P,Zhang H,Liu Y,et al.Beta-tricalcium phosphate granules improve osteogenesis in vitro and establish innovative osteo-regenerators for bone tissue engineering in vivo.Sci Rep.2016;6:23367.
[20]Cornell CN.Osteoconductive materials and their role as substitudes for autogenous bone grafts.Orthop Clin North Am.1999;30(4):591-598.
[21]Tanaka T,Kumagae Y,Chazono M,et al.A novel evaluation system to monitor bone formation andβ-tricalcium phosphate resorption in opening wedge high tibial osteotomy.Knee Surg Sports Traumatol Arthrosc.2015;23(7):2007-2011.
[22]Meijer HJ,Steen WH,Bosman F.Standarized radiograghs of the alveolar crest around implants in the mandible.J Prosthet Dent.1992;68:318-321.
[23]于威,李建军.去抗原牛松质骨支架复合骨形态发生蛋白2基因在骨缺损修复过程中的血管化反应[J].中国组织工程研究与临床康复,2008,12(23):4559-4562.
[24]Grzibovskis M,Pilmane M,Urtane I.Today's under-standing about bone aging.Stomatologija.2010;12(4):99-104.
[25]Yu T,Pan H,Hu Y,et al.Autologous platelet-rich plasma induces bone formation of tissue-engineered bone with bone marrow mesenchymal stem cells on beta-tricalcium phosphate ceramics.J Orthop Surg Res.2017;12(1):178.
[26]Nelson C,Warren J,Wang MH,et al.RANKL Employs Distinct Binding Modes to Engage RANK and the Osteoprotegerin Decoy Receptor.Structure.2012;20(11):1971-1982.
[27]Stein NC,Kreutzmann C,Zimmermann SP,et al.Interleukin-4 and interleukin-13 stimulate the osteoclast inhibitor osteoprotegerin by human endothelial cells through the STAT6 pathway.J Bone Miner Res.2008;23(5):750-758.
[28]Kearns AE,Khosla S,Kostenuik PJ.Receptor activator of nuclear factor kappaB ligand and osteoprotegerin regulation of bone remodeling in health and disease.Endocr Rev.2008;29(2):155-192.
[29]Nakashima T,Takayanagi H.Osteoimmunology:crosstalk between the immune and bone systems.J Clin Immunol.2009;29(5):555-567.
[30]Zhou L,Liu Q,Yang ML,et al.Dihydroartemisinin,an Anti-Malaria drug,suppresses estrogen deficiency-induced osteoporosis,osteoclast formation,and RANKL-Induced signaling pathways.JBone Miner Res.2016;31(5):964-974.
[31]J Rieu J,Goeuriot P.Ceramic composites for Biomedical applations.Clin Mater.1993;12:211-217.