三种染色方法观察骨骺损伤后骨桥形成过程的组织形态学特征
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摘要
背景:国内外的研究表明,多种染色法联合应用有利于探究骨与软骨组织疾病的形态学表现。目前对于多种染色法探究骨骺损伤后骨桥形成过程的组织形态学表现研究较少。目的:通过苏木精-伊红染色、番红O-固绿染色、Masson染色3种染色方法观察骨骺损伤后骨桥形成过程的组织形态表现。方法:SD大鼠40只由福建中医药大学实验动物中心提供,实验方案经福建中医药大学动物实验伦理委员会批准。随机将40只SD大鼠分为2组:对照组不做处理;模型组建立胫骨近端骨骺损伤模型。分别于造模后1,7,28 d拍摄X射线片和7,28 d行MRI扫描;于造模后1,7,14,28 d麻醉大鼠后取胫骨损伤区组织,行苏木精-伊红染色、番红O-固绿染色、Masson染色,观察术后不同时间点生长板损伤区的组织形态表现。结果与结论:①X射线片显示:造模后1 d,胫骨干骺端骨骺损伤,软骨膜周围生长板间隙增宽;第7天,骨骺损伤处骨膜反应明显;第28天,骨膜反应消失,干骺端与生长板间隙关系基本恢复正常;MRI显示:造模后第28天MRI T2WI示骺板模糊,并可见一黑色线条形低信号影,提示骨桥形成;②造模后第1天,3种染色均可显示入侵的血管,Masson染色更为清晰;第7天:苏木精-伊红染色较番红固绿染色及Masson染色显示了损伤区软骨细胞的形态变化更加明显;第14天,番红O-固绿染色生长板与骨桥对比鲜明,Masson染色可观察到骨桥内尚未骨化的纤维血管组织;第28天,苏木精-伊红染色软骨细胞的周期性形态变化较明显,番红O-固绿染色骨桥和生长板形态分明,可清晰的表现损伤区生长板的压缩性改变;③结果说明,3种染色方法的联合应用能够全面、客观地探究骨骺损伤后骨桥形成过程的组织形态学表现。
BACKGROUND: Existing evidence has shown that combined usage of various staining methods contributes to exploring the morphological performance of bone and cartilage diseases. Histomorphology of bone bridge formation after epiphyseal injury using various staining methods is rarely reported.OBJECTIVE: To observe the histomorphology of bone bridge formation after epiphyseal injury by hematoxylin-eosin staining, safranin O-fast green staining and Masson staining.METHODS: Forty Sprague-Dawley rats were provided by the Laboratory Animal Center of Fujian University of Traditional Chinese Medicine,and the study was approved by the Laboratory Ethics Committee of Fujian University of Traditional Chinese Medicine. The rats were randomly divided into model(proximal tibia epiphyseal injury model) and control groups(n=20/group). X-ray was conducted at 1, 7 and 28 days after modeling, and MRI was scanned at 7 and 28 days after modeling. The tibia damage tissues were removed under anesthesia at 1, 7, 14, and28 days after modeling. Hematoxylin-eosin staining, safranin O-fast green staining and Masson staining were performed to observe the histomorphology of the growth plate damage area at postoperative different time points.RESULTS AND CONCLUSION:(1) X-ray films: at 1 day after modeling, the tibial metaphysis injury and widening growth plate space surrounding the perichondrium were observed. On day 7, periosteal reaction in the injured metaphysis was obvious. On day 28, periosteal reaction disappeared,and the space between metaphysis and growth plate returned to be normal. MRI T2WI at 28 days after modeling showed that epiphyseal plate became obscure and a blank line low signal was observed, suggesting bone bridge formation.(2) At 1 day after modeling, all staining methods revealed blood vessels, especially Masson staining. On day 7, the morphological changes of chondrocytes in the injury region were more obvious in hematoxylin-eosin staining compared with the safranin O-fast green staining and Masson staining. On day 14, under safranin O-fast green staining,the growth plate was clearly contrasted with the bone bridge, while unossified fibrous vascular tissue in the bone bridge can be observed by Masson staining. On day 28, hematoxylin-eosin staining showed obvious periodic morphological changes of chondrocytes. Safranin O-fast green staining showed clear morphology of bone bridge and growth plate, and the compressive change of the growth plate in the damaged area clearly.(3) These results indicate that the combined application of the three staining methods can comprehensively and objectively explore the histomorphological manifestations of the bone bridge formation after epiphyseal injury.
BACKGROUND: Existing evidence has shown that combined usage of various staining methods contributes to exploring the morphological performance of bone and cartilage diseases. Histomorphology of bone bridge formation after epiphyseal injury using various staining methods is rarely reported.OBJECTIVE: To observe the histomorphology of bone bridge formation after epiphyseal injury by hematoxylin-eosin staining, safranin O-fast green staining and Masson staining.METHODS: Forty Sprague-Dawley rats were provided by the Laboratory Animal Center of Fujian University of Traditional Chinese Medicine,and the study was approved by the Laboratory Ethics Committee of Fujian University of Traditional Chinese Medicine. The rats were randomly divided into model(proximal tibia epiphyseal injury model) and control groups(n=20/group). X-ray was conducted at 1, 7 and 28 days after modeling, and MRI was scanned at 7 and 28 days after modeling. The tibia damage tissues were removed under anesthesia at 1, 7, 14, and28 days after modeling. Hematoxylin-eosin staining, safranin O-fast green staining and Masson staining were performed to observe the histomorphology of the growth plate damage area at postoperative different time points.RESULTS AND CONCLUSION:(1) X-ray films: at 1 day after modeling, the tibial metaphysis injury and widening growth plate space surrounding the perichondrium were observed. On day 7, periosteal reaction in the injured metaphysis was obvious. On day 28, periosteal reaction disappeared,and the space between metaphysis and growth plate returned to be normal. MRI T2WI at 28 days after modeling showed that epiphyseal plate became obscure and a blank line low signal was observed, suggesting bone bridge formation.(2) At 1 day after modeling, all staining methods revealed blood vessels, especially Masson staining. On day 7, the morphological changes of chondrocytes in the injury region were more obvious in hematoxylin-eosin staining compared with the safranin O-fast green staining and Masson staining. On day 14, under safranin O-fast green staining,the growth plate was clearly contrasted with the bone bridge, while unossified fibrous vascular tissue in the bone bridge can be observed by Masson staining. On day 28, hematoxylin-eosin staining showed obvious periodic morphological changes of chondrocytes. Safranin O-fast green staining showed clear morphology of bone bridge and growth plate, and the compressive change of the growth plate in the damaged area clearly.(3) These results indicate that the combined application of the three staining methods can comprehensively and objectively explore the histomorphological manifestations of the bone bridge formation after epiphyseal injury.
引文
[1]Chung R, Foster BK, Xian CJ. Injury responses and repair mechanisms of the injured growth plate. Front Biosci(Schol Ed). 2011;3:117-125.
[2]Li W,Xu R,Huang J,et al. Treatment of rabbit growth plate injuries with oriented ECM scaffold and autologous BMSCs. Sci Rep.2017;7:44140.
[3]Brown JH,Deluca SA.Growth plate injuries:Salter-Harris classification.American Family Physician. 1992;46(4):1180.
[4]Basener CJ,Mehlman CT,Dipasquale TG. Growth disturbance after distal femoral growth plate fractures in children:a meta-analysis. J Orthop Trauma. 2009;23(9):663-667.
[5]Chung R,Foster B K, Xian CJ.The potential role of VEGF-induced vascularisation in the bony repair of injured growth plate. J Endocrinol.2014;221(1):63-75.
[6]颉强,胡蕴玉,杨柳,等.大鼠胫骨近端骨骺损伤后骨桥形成分子机制的研究[J].生物化学与生物物理进展, 2008, 35(9):1039-1045.
[7]Lee MA, Nissen TP, Otsuka NY.Utilization of a Murine Model to Investigate the Molecular Process of Transphyseal Bone Formation. J Pediatr Orthop. 2000;20(6):802-806.
[8]Macsai CE,Hopwood B, Chung R, et al. Structural and molecular analyses of bone bridge formation within the growth plate injury site and cartilage degeneration at the adjacent uninjured area. Bone. 2011;49(4):904-912 Bone.
[9]Musumeci, Giuseppe, Castrogiovanni, et al. Post-Traumatic Caspase-3Expression in the Adjacent Areas of Growth Plate Injury Site:A Morphological Study. Int J Mol Sci. 2013;14(8):15767-15784.
[10] Xian CJ, Zhou FH, Mccarty RC, et al. Intramembranous ossification mechanism for bone bridge formation at the growth plate cartilage injury site. J Orthop Res. 2004 Mar;22(2):417-426.
[11] Chung R, Xian CJ. Recent research on the growth plate:Mechanisms for growth plate injury repair and potential cell-based therapies for regeneration. J Mol Endocrinol. 2014;53(1):T45-61.
[12] Isaksson H,Wilson W,Donkelaar CC, et al. Comparison of biophysical stimuli for mechano-regulation of tissue differentiation during fracture healing. J Biomech. 2006;39(8):1507-1516.
[13] Cardiff RD, Miller CH, Munn RJ. Manual hematoxylin and eosin staining of mouse tissue sections. Cold Spring Harbor Protocols.2014;2014(6):655.
[14] Feldman AT, Wolfe D. Tissue processing and hematoxylin and eosin staining. Methods Mol Biol. 2014;1180:31-43.
[15]黄云梅,陈文列,黄美雅,等.多种特殊染色法在骨关节炎组织形态学研究中的应用比较[J].中国比较医学杂志, 2011, 21(5):45-48.
[16] Rosenberg L.Chemical basis for the histological use of safranin O in the study of articular cartilage. J Bone Joint Surg Am. 1971;53(1):69-82.
[17] Eggert FM,Linder JE,Jubb RW.Staining of demineralized cartilage.Histochemistry.1981; 73(3):391-396.
[18] Ji H,Li J,Shao J.Histopathologic comparison of condylar hyperplasia and condylar osteochondroma by using different staining methods. Oral Surg Oral Med Oral Pathol Oral Radiol. 2017;123(3):320-329.
[19] Garvey W. Modified Elastic Tissue-Masson Trichrome Stain. Stain Technology.1984;59(4):4.
[20] Suvik A, Effendy AWM.The use of modified Masson’s trichrome staining in collagen evaluation in wound healing study. Journal of Neurosurgery.2012; 3(1):39-47.
[21]吕学敏,胡浩,鲁明,等.骨骺损伤修复过程中骺板形态及VEGF表达的变化[J].中华骨科杂志,2012, 32(6):570-575.
[22]贺今,纪春岩.NOTCH、VEGF信号通路在急性髓性白血病发生及血管新生中的作用[J].中国临床研究, 2016,29(4):558-559.
[23] Hernandez SL,Banerjee D, Garcia A,et al. Notch and VEGF pathways play distinct but complementary roles in tumor angiogenesis.Vascular Cell.2013;5(1):17.
[2]Li W,Xu R,Huang J,et al. Treatment of rabbit growth plate injuries with oriented ECM scaffold and autologous BMSCs. Sci Rep.2017;7:44140.
[3]Brown JH,Deluca SA.Growth plate injuries:Salter-Harris classification.American Family Physician. 1992;46(4):1180.
[4]Basener CJ,Mehlman CT,Dipasquale TG. Growth disturbance after distal femoral growth plate fractures in children:a meta-analysis. J Orthop Trauma. 2009;23(9):663-667.
[5]Chung R,Foster B K, Xian CJ.The potential role of VEGF-induced vascularisation in the bony repair of injured growth plate. J Endocrinol.2014;221(1):63-75.
[6]颉强,胡蕴玉,杨柳,等.大鼠胫骨近端骨骺损伤后骨桥形成分子机制的研究[J].生物化学与生物物理进展, 2008, 35(9):1039-1045.
[7]Lee MA, Nissen TP, Otsuka NY.Utilization of a Murine Model to Investigate the Molecular Process of Transphyseal Bone Formation. J Pediatr Orthop. 2000;20(6):802-806.
[8]Macsai CE,Hopwood B, Chung R, et al. Structural and molecular analyses of bone bridge formation within the growth plate injury site and cartilage degeneration at the adjacent uninjured area. Bone. 2011;49(4):904-912 Bone.
[9]Musumeci, Giuseppe, Castrogiovanni, et al. Post-Traumatic Caspase-3Expression in the Adjacent Areas of Growth Plate Injury Site:A Morphological Study. Int J Mol Sci. 2013;14(8):15767-15784.
[10] Xian CJ, Zhou FH, Mccarty RC, et al. Intramembranous ossification mechanism for bone bridge formation at the growth plate cartilage injury site. J Orthop Res. 2004 Mar;22(2):417-426.
[11] Chung R, Xian CJ. Recent research on the growth plate:Mechanisms for growth plate injury repair and potential cell-based therapies for regeneration. J Mol Endocrinol. 2014;53(1):T45-61.
[12] Isaksson H,Wilson W,Donkelaar CC, et al. Comparison of biophysical stimuli for mechano-regulation of tissue differentiation during fracture healing. J Biomech. 2006;39(8):1507-1516.
[13] Cardiff RD, Miller CH, Munn RJ. Manual hematoxylin and eosin staining of mouse tissue sections. Cold Spring Harbor Protocols.2014;2014(6):655.
[14] Feldman AT, Wolfe D. Tissue processing and hematoxylin and eosin staining. Methods Mol Biol. 2014;1180:31-43.
[15]黄云梅,陈文列,黄美雅,等.多种特殊染色法在骨关节炎组织形态学研究中的应用比较[J].中国比较医学杂志, 2011, 21(5):45-48.
[16] Rosenberg L.Chemical basis for the histological use of safranin O in the study of articular cartilage. J Bone Joint Surg Am. 1971;53(1):69-82.
[17] Eggert FM,Linder JE,Jubb RW.Staining of demineralized cartilage.Histochemistry.1981; 73(3):391-396.
[18] Ji H,Li J,Shao J.Histopathologic comparison of condylar hyperplasia and condylar osteochondroma by using different staining methods. Oral Surg Oral Med Oral Pathol Oral Radiol. 2017;123(3):320-329.
[19] Garvey W. Modified Elastic Tissue-Masson Trichrome Stain. Stain Technology.1984;59(4):4.
[20] Suvik A, Effendy AWM.The use of modified Masson’s trichrome staining in collagen evaluation in wound healing study. Journal of Neurosurgery.2012; 3(1):39-47.
[21]吕学敏,胡浩,鲁明,等.骨骺损伤修复过程中骺板形态及VEGF表达的变化[J].中华骨科杂志,2012, 32(6):570-575.
[22]贺今,纪春岩.NOTCH、VEGF信号通路在急性髓性白血病发生及血管新生中的作用[J].中国临床研究, 2016,29(4):558-559.
[23] Hernandez SL,Banerjee D, Garcia A,et al. Notch and VEGF pathways play distinct but complementary roles in tumor angiogenesis.Vascular Cell.2013;5(1):17.