肥大型骨不连断端组织成骨潜能的实验研究
摘要
目的观察肥大型骨不连断端及其周围区域组织的结构特点，评估其成骨潜能。
方法收集2009年至2012年因肥大型骨不连在我院行手术治疗患者的骨不连断端标本共25例，将切取的标本按距离骨不连断端中心的远近分为中心区、周围区及骨痂区三组，通过光学显微镜及透射电镜观察不同区域的组织结构特点和微观特征。采用免疫组织化学方法检测这些组织内BMP-2、DCN及VEGF的表达，在高倍镜下观察阳性细胞并计数，进而作统计分析。
结果肥大型骨不连断端区域呈现出不同的组织类型，中心区主要是纤维化组织，其内可见大量的成纤维样细胞；周围区可见大量的软骨细胞，可见少量的新生血管形成；骨痂区见有一定量的成骨细胞及丰富的新生血管。免疫组化染色显示肥大型骨不连断端中心区、周围区及骨痂区组织BMP-2、DCN及VEGF的表达明显不同（P<0.05）。骨痂区BMP-2、VEGF的表达明显高于中心区和周围区（P<0.05），周围区组织DCN的含量则明显高于中心区及贴骨瘢痕区（P<0.05）。
结论肥大型骨不连在形成的过程中组织结构发生变化，其断端区域的组织类型、细胞类型不同，BMP-2、DCN的含量减少，断端组织的抗纤维化和成骨能力下降，断端区域不同部位组织的成骨潜能不同。
Objective To observe the structure of the intervening and surrounding tissue at thehypertrophic nonunion site, to assess the potential of bone formation.
Methods Tissue from the nonunion site and its surroundings was extracted from25patients with hypertrophic nonunion within2009to2012. Each sample was classified bythe distance to the center of the nonunion site as three teams:central area, surrounding areaand callus area. Tissue structure and microscopic characteristics of the three regions wereinvestigated by using optical microscope, transmission electron micros-cope. Detect theexpression of BMP-2、DCN and VEGF by immunohistochemistry. Assess the potential ofbone formation by analyzing the differences of each group.
Results The area of hypertrophic nonunion ends displayed different non-uniformhistological features. The central area mainly displayed fibrotic tissue which contained alarge number of fibroblast-like cells; The surrounding area contained masses ofchondrocytes without neovascularization; The scar area contained a certain amount ofosteoblasts with rich neovascularization. The results of immunohistochemistry showeddiscrepant expression of BMP-2、DCN and VEGF. The expression of BMP-2and VEGFwas more intense in the scar area than in the center area or in the surrounding area(P<0.05).However, The content of DCN in the surrounding area was significantly higherthan the central area or the scar area.
Conclusion The tissue structure of hypertrophic nonunion changed in the process ofits formation. The stump region contained different tissue types and cell types, the contentof BMP-2, DCN decreased. The anti-fibrosis and ossification ability of the nonunion tissuedecreased. The different parts of stump region had different osteogenic potential.
方法收集2009年至2012年因肥大型骨不连在我院行手术治疗患者的骨不连断端标本共25例，将切取的标本按距离骨不连断端中心的远近分为中心区、周围区及骨痂区三组，通过光学显微镜及透射电镜观察不同区域的组织结构特点和微观特征。采用免疫组织化学方法检测这些组织内BMP-2、DCN及VEGF的表达，在高倍镜下观察阳性细胞并计数，进而作统计分析。
结果肥大型骨不连断端区域呈现出不同的组织类型，中心区主要是纤维化组织，其内可见大量的成纤维样细胞；周围区可见大量的软骨细胞，可见少量的新生血管形成；骨痂区见有一定量的成骨细胞及丰富的新生血管。免疫组化染色显示肥大型骨不连断端中心区、周围区及骨痂区组织BMP-2、DCN及VEGF的表达明显不同（P<0.05）。骨痂区BMP-2、VEGF的表达明显高于中心区和周围区（P<0.05），周围区组织DCN的含量则明显高于中心区及贴骨瘢痕区（P<0.05）。
结论肥大型骨不连在形成的过程中组织结构发生变化，其断端区域的组织类型、细胞类型不同，BMP-2、DCN的含量减少，断端组织的抗纤维化和成骨能力下降，断端区域不同部位组织的成骨潜能不同。
Objective To observe the structure of the intervening and surrounding tissue at thehypertrophic nonunion site, to assess the potential of bone formation.
Methods Tissue from the nonunion site and its surroundings was extracted from25patients with hypertrophic nonunion within2009to2012. Each sample was classified bythe distance to the center of the nonunion site as three teams:central area, surrounding areaand callus area. Tissue structure and microscopic characteristics of the three regions wereinvestigated by using optical microscope, transmission electron micros-cope. Detect theexpression of BMP-2、DCN and VEGF by immunohistochemistry. Assess the potential ofbone formation by analyzing the differences of each group.
Results The area of hypertrophic nonunion ends displayed different non-uniformhistological features. The central area mainly displayed fibrotic tissue which contained alarge number of fibroblast-like cells; The surrounding area contained masses ofchondrocytes without neovascularization; The scar area contained a certain amount ofosteoblasts with rich neovascularization. The results of immunohistochemistry showeddiscrepant expression of BMP-2、DCN and VEGF. The expression of BMP-2and VEGFwas more intense in the scar area than in the center area or in the surrounding area(P<0.05).However, The content of DCN in the surrounding area was significantly higherthan the central area or the scar area.
Conclusion The tissue structure of hypertrophic nonunion changed in the process ofits formation. The stump region contained different tissue types and cell types, the contentof BMP-2, DCN decreased. The anti-fibrosis and ossification ability of the nonunion tissuedecreased. The different parts of stump region had different osteogenic potential.
引文
[1] Nakamura A, Akahane M, Shigematsu H, et al. Cell sheet transplantation of culturedmesenchymal stem cells enhances bone formation in a rat nonunion model. Bone,2010,46(2):418-424
[2]刘傥，曹轶伦.骨不连的病理学研究进展.中国矫形外科杂志，2010,18（12）：1006-1007
[3] Nakamura A, Akahane M, Shigematsu H, et al. Cell sheet transplantation of culturedmesenchymal stem cells enhances bone formation in a rat nonunion model. Bone,2010,46(2):418-424
[4] Hietaniemi K, Peltonen J, Paavolainen P. An experimental model for non-union in rats.Injury,1995,26:681-681
[5]郭征,郭霞,郑振耀.组织隔离法与机械活动法在兔胫骨骨不连模型建立中的作用．中华创伤骨科杂志,2004,11(6):1261-1264
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[7] Santos Neto FLD, Volpon JB. Experimental nonunion in dogs. ClinOrthop,1984,(187):260-271
[8] Dhammi I, Jain A, Singh A, et al. Primary nonunion of intertrochanteric fractures offemur: An analysis of results of valgization and bone grafting. Indian J Orthop,2011,45(6):514-519
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[11] Clases L, Echert-Hubner K, Augat P. The affect of mechanical stability on localvascularization and tissue diffenentiation in callus healing. J Orthop Res,2002,5：1099-1105;
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[14] Boyd HB, Lipinski SW. Nonunion of trochanteric and subtrochanteric fractures. SurgGynecol Obstet,1957,104(4):463-470
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[16] Boyan BD, Schwartz Z, Swain LD, et al. Initial effects of partially purified bonemorphogenetic protein on the expression of glycosaminoglycan, collagen, andalkaline phosphatase in nonunion cell cultures. Clin Orthop RelatRe,.1992,278:286-304
[17] Guerkov HH, Lohmann CH, Liu Y, et al. Pulsed electromagnetic felds increasegrowth factor release by nonunion cells. Clin Orthop Relat Res,2001,384:265–279
[18] Okamoto M, Murai J, Yoshikawa H, et al. Bone morphogenetic proteins in bonestimulate osteoclasts and osteoblasts during bone development. J Bone MinerRes,2006,21:1022-1033
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[20]张华,马维,范志勇.骨折愈合机制的研究进展[J]，河北医药,2009,13:1644-1666
[21]林坚平,宋世锋,姚伦龙.骨折愈合及早期诊断的研究进展[J].中国矫形外科杂志,2009,24:1876-1878
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[23]邱南海，郗根旺.骨不连常见病因分析与治疗进展.中国矫形外科杂志,2009,17(10):762-765
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[27] Nussenbaum B，Teknos TN，Chepeha DB．Tissue engineering：the current status ofthis futuristic modality in headneck reconstruction．Curr OpinOtolaryngol HeadNeckSurg，2004,12:311-315
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[1] Nakamura A, Akahane M, Shigematsu H, et al. Cell sheet transplantation of cultured mesenchymalstem cells enhances bone formation in a rat nonunion model. Bone,2010,46(2):418-424
[2] Hietaniemi K, Peltonen J, Paavolainen P. An experimental model for non-union in rats. Injury,1995,26:681-681
[3]郭征,郭霞,郑振耀.组织隔离法与机械活动法在兔胫骨骨不连模型建立中的作用．中华创伤骨科杂志,2004,11(6):1261-126
[4] Volpon JB. Nonunion using a canie model. Arch Orthop Trauma Surg,1994,113:312-317
[5] Santos Neto FLD, Volpon JB. Experimental nonunion in dogs. Clin Orthop,1984,(187):260-271
[6] Dhammi I, Jain A, Singh A, et al. Primary nonunion of intertrochanteric fractures of femur: Ananalysis of results of valgization and bone grafting. Indian J Orthop,2011,45(6):514-519
[7] STCanale.坎贝尔骨科手术学[M].王岩,主译.11版.北京:人民军医出版社,2009:2768-2783
[8] Paley D, Catagni MA, Argnani F, et al. Ilizarov treatment of tibial nonunions with bone loss. ClinOrthop,1989,241:146–165
[9] Clases L, Echert-Hubner K, Augat P. The affect of mechanical stability on local vascularization andtissue diffenentiation in callus healing. J Orthop Res,2002,5‖1099-1105;
[10] Wen HB, Cui FZ, Zhu XD. Microstructural features of non-union of human humeral shaft fracture.J Struct Biol,1997,119(3):239-246
[11] Qu G, von Schroeder HP, Trabecular microstructure at the human scaphoid nonunion. J Hand SurgAm,2008,33(5):650-655
[12] Boyd HB, Lipinski SW. Nonunion of trochanteric and subtrochanteric fractures. Surg GynecolObstet,1957,104(4):463-470
[13] Iwakura T, Miwa M, Sakai Y, et al. Human hypertrophic nonunion tissue contains mesenchymalprogenit or cells with multilineage capacity in vitro. J Orthop Res,2009,2:208-215
[14] Boyan BD, Schwartz Z, Swain LD, et al. Initial effects of partially purified bone morphogeneticprotein on the expression of glycosaminoglycan, collagen, and alkaline phosphatase in nonunion cellcultures. Clin Orthop Relat Re,.1992,278:286-304
[15] Guerkov HH, Lohmann CH, Liu Y, et al. Pulsed electromagnetic felds increase growth factorrelease by nonunion cells. Clin Orthop Relat Res,2001,384:265–279
[16] Okamoto M, Murai J, Yoshikawa H, et al. Bone morphogenetic proteins in bone stimulateosteoclasts and osteoblasts during bone development. J Bone Miner Res,2006,21:1022-1033
[17]韩昕光,毕郑钢，王东奎等.骨不连接区成骨活性的实验研究.中华刨伤骨科杂志,2009,11（1）：45-50
[18]郭征，郭霞，郑振耀.组织隔离法与机械活动法在兔胫骨骨不连模型建立中的作用.中华创伤骨科杂志,2004,6(11):1261-1264
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[2]刘傥，曹轶伦.骨不连的病理学研究进展.中国矫形外科杂志，2010,18（12）：1006-1007
[3] Nakamura A, Akahane M, Shigematsu H, et al. Cell sheet transplantation of culturedmesenchymal stem cells enhances bone formation in a rat nonunion model. Bone,2010,46(2):418-424
[4] Hietaniemi K, Peltonen J, Paavolainen P. An experimental model for non-union in rats.Injury,1995,26:681-681
[5]郭征,郭霞,郑振耀.组织隔离法与机械活动法在兔胫骨骨不连模型建立中的作用．中华创伤骨科杂志,2004,11(6):1261-1264
[6] Volpon JB. Nonunion using a canie model. Arch Orthop TraumaSurg,1994,113:312-317
[7] Santos Neto FLD, Volpon JB. Experimental nonunion in dogs. ClinOrthop,1984,(187):260-271
[8] Dhammi I, Jain A, Singh A, et al. Primary nonunion of intertrochanteric fractures offemur: An analysis of results of valgization and bone grafting. Indian J Orthop,2011,45(6):514-519
[9] STCanale.坎贝尔骨科手术学[M].王岩,主译.11版.北京:人民军医出版社,2009:2768-2783
[10] Paley D, Catagni MA, Argnani F, et al. Ilizarov treatment of tibial nonunions withbone loss. Clin Orthop,1989,241:146–165
[11] Clases L, Echert-Hubner K, Augat P. The affect of mechanical stability on localvascularization and tissue diffenentiation in callus healing. J Orthop Res,2002,5：1099-1105;
[12] Wen HB, Cui FZ, Zhu XD. Microstructural features of non-union of human humeralshaft fracture. J Struct Biol,1997,119(3):239-246
[13] Qu G, von Schroeder HP, Trabecular microstructure at the human scaphoid nonunion.J Hand Surg Am,2008,33(5):650-655
[14] Boyd HB, Lipinski SW. Nonunion of trochanteric and subtrochanteric fractures. SurgGynecol Obstet,1957,104(4):463-470
[15] Iwakura T, Miwa M, Sakai Y, et al. Human hypertrophic nonunion tissue containsmesenchymal progenit or cells with multilineage capacity in vitro. J Orthop Res,2009,2:208-215
[16] Boyan BD, Schwartz Z, Swain LD, et al. Initial effects of partially purified bonemorphogenetic protein on the expression of glycosaminoglycan, collagen, andalkaline phosphatase in nonunion cell cultures. Clin Orthop RelatRe,.1992,278:286-304
[17] Guerkov HH, Lohmann CH, Liu Y, et al. Pulsed electromagnetic felds increasegrowth factor release by nonunion cells. Clin Orthop Relat Res,2001,384:265–279
[18] Okamoto M, Murai J, Yoshikawa H, et al. Bone morphogenetic proteins in bonestimulate osteoclasts and osteoblasts during bone development. J Bone MinerRes,2006,21:1022-1033
[19]韩昕光,毕郑钢，王东奎等.骨不连接区成骨活性的实验研究.中华刨伤骨科杂志,2009,11（1）：45-50
[20]张华,马维,范志勇.骨折愈合机制的研究进展[J]，河北医药,2009,13:1644-1666
[21]林坚平,宋世锋,姚伦龙.骨折愈合及早期诊断的研究进展[J].中国矫形外科杂志,2009,24:1876-1878
[22] CALDWELL GA. Cause and treatment of nonunion of tibial fractures. J Fla MedAssoc，1953，40(2):98-100
[23]邱南海，郗根旺.骨不连常见病因分析与治疗进展.中国矫形外科杂志,2009,17(10):762-765
[24]林博文,徐忠世,肖德明，实验性骨不连模型的制作[J],中山医科大学学报，2002，25(5)：332-333
[25] Harada S， Rodan GA． Contmlofosteoblast function and regulation of Bonemass．Nature，2003，423：349-355
[26] Boyle WJ．Simonet WS，Laeey DL．Osteoclast differentiation and activation．Nature，2003，423：337-342
[27] Nussenbaum B，Teknos TN，Chepeha DB．Tissue engineering：the current status ofthis futuristic modality in headneck reconstruction．Curr OpinOtolaryngol HeadNeckSurg，2004,12:311-315
[28] Chen D, Harris MA，Rossini G, et a1.Bone morphogenetie protein2(BMP-2)enhances BMP-3,BMP-4,and bone cell differentiation marker geneexpression duringthe induction of mineralized bone matrix formation in cultures of fetal rat calvarialosteoblasts．Calcif Tissue Int,1997,60：283-290
[29] Okamoto M，MumiJ，YoshikawaH，et a1．Bone morphogenetic proteins in bonestimulate osteoclasts and osteoblasts during bone development． J Bone MinerRes,2006,21:1022-1033
[30] Hosseinkhani H, Hosseinkhani M，Khademhosseini A，et a1．Bone regenerationthrough controlled release of bone morphogenetic protein-2from3-D tissueengineered nano-scaffold. J Contml Release,2007,117:380-386
[31] Niikura T, Hak DJ, Reddi AH. Global gene profiling reveals a downregulation of BMPgene expression in experimental atrophic nonunions compared to standard healingfractures. J Orthop Res.2006,24(7):1463-1471
[32] Laine P, Reunanen N, Ravanti L, et al. Activation of extracellular signal-regulatedprotein kinase1,2results in down-regulation of decorin expression in fibroblasts.Biochem J.2000,349(Pt1):19-25
[33] Douglas T，HeinemannS，Bierbaum S，et a1．Fibrillogenesis of collagen types I, II,and Ⅲ with small1eucine-rich proteoglycans decorin and biglycan.Biomacromolecules,2006,7:2388-2393
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