14156

18698

19156

19781

MINAMIZONO, Wataru

OHSAKO, Masafumi

XU, Siqin

YASHIMA, Nao

八嶋, 奈央

南園, 航

大迫, 正文

徐, 思琴

東洋大学学術情報リポジトリ

Study of structural and functional changes during development of the anteriorcortical bone in the rat lumbar vertebral body ― 381 ―ABSTRACTThe purpose of this study was to investigate the structural and functional changes of vertebral cortical bone during its development as part of a basic study to further our knowledge of vertebral cortical bone. Male Wistar rats of 3, 7 and 13W (each n=13) were used as material. After delivery, the rats were quickly euthanized using carbon dioxide gas, and the vertebral cortical bone was removed and used for various analyses. First, bone strength of vertebral cortical bone at each week of age was measured, indicating that bone strength increased significantly with development. Observations of the vertebral cortical bone surface indicated that the vascular space within the vertebral cortical bone decreased with development. Cross-sectional observation of the vertebral cortical bone showed that the 3w vertebral cortical bone exhibited a very coarse structure, while it was understood that it developed a laminar structure toward the 7 and 13w vertebral bodies. In addition, polychrome staining showed that the thickness of the fiber bundles of the matrix fibers within the vertebral cortical bone increased with increasing age. These findings suggest that vertebral cortical bone densifies from a coarse structure during development, which Study of structural and functional changes during development of the anterior cortical bone in the rat lumbar vertebral bodyYASHIMA, Nao*MINAMIZONO, Wataru*Xu, Siqin*OHSAKO, Masafumi**＊Doctoral Program in Graduate School of Health and Sports Sciences, Course of Health and Sports Sciences, First Year Student, Toyo University. ＊Doctoral Program in Graduate School of Human Life Design, Course of Human Life Studies, Second Year Student, Toyo University. ＊Master’s Program in Graduate School of Human Life Design, Course of Health care and Sports, Second Year Student, Toyo University. ＊＊Professor of the Faculty of Health and Sports Sciences, Toyo University.― 382 ―contributes to increased bone strength. In conclusion, the vertebral cortical bone of rats densifies with development, suggesting that this structural change is an adaptation to the increase in body weight and activity with age.Keywords：spine, cortical bone, developingINTRODUCTIONIt is known that vertebral body compression fractures are triggered in many osteoporotic patients and that many vertebral body compression fractures are accompanied by wedge-shaped deformities1). However, previous studies have shown the importance of cortical bone as a factor determining vertebral body strength2-4). Based on this, we hypothesized that among the cortical bones constituting the vertebral body, the anterior cortical bone (vertebral body cortical bone), which provides anterior support, plays a major role in the development of wedge-shaped deformities, and thus revealed the intrinsic vulnerability of the cortical bone of the vertebral body. However, academic findings on vertebral cortical bone are still scarce. Cortical bone is formed by membranous ossification. Normally, cortical bone formation begins with membranous bone, also called woven bone5-7), and matures into lamellar bone8, 9). Cortical bone is formed by the ossification of the laminae of the vertebral column. Reports examining the process of developmental changes in rat tibia have shown that 3-week-old tibial cortical bone is characterized by porosity due to abundant vascular spaces, but becomes denser as it matures. It has also been suggested that these structural changes in the developing cortical bone are a strength adaptation to the increase in body weight and activity that follows aging. However, based on this perspective, no findings have yet been presented on developmental changes in the vertebral cortical bone, the bone of the trunk. Therefore, the purpose of this study was to examine the structural and functional changes that ac-company the development of vertebral cortical bone as part of a basic study to further our knowledge of vertebral cortical bone.METHODSAnimalMale rats [Wistar strain, 39 (13 each), Nippon Bio Supp Center, TYO, JPN] aged 3, 7 and 13 weeks (3W, 7W, 13W) were used in this study. All rats were euthanized by carbon dioxide aspiration after delivery. The vertebral cortical bone of the first lumbar vertebra was then removed from the rats. Some of the extracted vertebrae were used for mechanical analysis, Study of structural and functional changes during development of the anteriorcortical bone in the rat lumbar vertebral body ― 383 ―while others were immersed and fixed in 4% paraformaldehyde solution and then made into various histological specimens.Compressive Strength TestVertebral cortical bone from each week-old vertebra was trimmed into squares (4 mm x 4 mm) and the periosteum and trabecular bone were removed with tweezers. They were then placed in a compression testing machine (MUROMACHI, TYO, JPN) and vertical compression force was applied at a compression speed of 10 mm/min based on the study in the preliminary experiments. Specimens for scanning electron microscopy observationVertebral cortical bone was treated with 6% sodium hypochlorite, carbon and platinum deposited, and observed by Scanning Electron Microscope (SEM, HITACHI, TYO, JPN).Non-decalcified rigolac resin polished specimen (polished specimen)Vertebral cortical bone was encapsulated in rigolac resin. Specimens were polished and stained with toluidine blue.Decalcified paraffin sectionsVertebral cortical bone was demineralized with 8% EDTA for 3 weeks and then embedded in paraffin. The paraffin-embedded samples were sectioned into sections approximately 4㎛ thick. The sections were then deparaffinized with lemosol, hydrated with alcohol, and polychrome stained.Morphometry and Statistical ProcessingImages of the cortical bone surface taken by SEM were converted to binary format with reference to a previous study10). The percentage of vascular spaces in the cortical bone was then measured as porosity (black area/total area). Bone density of vertebral cortical bone was also measured using polished specimens. All statistical data in this study were analyzed by turkey HSD test after one-way analysis of variance (ANOVA) using SPSS ver. 26. The significance level was set at less than 5% (p〈0.05).RESULTSThe strength of the vertebral cortical bone at each week of age was measured to be 9.25 N at 3w, 28.75 N at 7w, and 70.25 N at 13w. Significant differences were shown between the values at each week of age (Figure 1).― 384 ―First, the surface structure of the vertebral cortical bone at each week of age was observed by SEM. The results showed that the vertebral cortical bone of the 3w exhibited an extremely fibrous bone structure. In addition, such fibrous bone structure was formed from thin fibrous bundles. Furthermore, the thin fibrous bundles were irregularly oriented in the direction of the long axis of the bone. On the other hand, the vertebral cortical bone of 7w exhibited the same fibrous bone structure as that of 3w. However, such fibrous bone structure was formed by relatively thick fibrous bundles, unlike 3w. The thick fibrous bundles were relatively more regularly oriented in the longitudinal direction of the bone than in 3w. On the other hand, unlike 3w and 7w, the cortical bone of the vertebral body of 13w did not exhibit a fibrous bone structure, but formed a dense laminar plate structure (Figure 2). On the other hand, numerous vascular cavities were observed in the vertebral cortical bone of both weeks. The number of vascular cavities per unit area was 158.65 for 3w, 119.58 for 7w, and 81.39 for 13w, indicating a decrease with increasing age (Figure 2). The percentage of vascular cavities in the cortical bone was 20.58% for 3w, 4.56% for 7w, and 2.98% for 13w, indicating that, like the number of vascular cavities, the percentage decreased with increasing age (Figure 2). Figure 1. Comparison of bone strength (n=4, *: p<0.05)   Study of structural and functional changes during development of the anteriorcortical bone in the rat lumbar vertebral body ― 385 ―The cross-sectional structure of the vertebral cortical bone at each week of age was then observed under an optical microscope. As a result, the vertebral cortical bone at 3w showed an extremely rough structure. On the other hand, the vertebral cortical bone at 7w showed a relatively laminar structure, with a less coarse structure than at 3w. On the other hand, the 13w vertebral cortical bone formed an even denser laminae structure than the 7w (Figure 3). At each week of age, the bone volume of vertebral cortical bone per unit area was 77.29%, 90.76%, and 96.70% for 3w, 7w, and 13w, respectively, indicating an increase in bone volume with development (Figure 3). Figure 2. Comparison of bone surface structures (upper: specimen for SEM, lower: binary format image, bar=100㎛, n=4, *: p<0.05)   ― 386 ―Polychrome staining revealed that the 3w vertebral cortical bone had a coarse structure formed by thin fibrous bundles, and the fibrous bundles were mainly light red and light blue in color. In contrast, the 7w vertebral cortex was composed of thicker fibrous bundles and the fibrous bundles were predominantly light red in color. 13w vertebral cortex was composed of thicker fibrous bundles that were predominantly light red and light orange in color than 3w and 7w (Figure 4). Figure 3. Comparison of bone cross-sectional structures (resin specimens, bar=100 ㎛, n=4, *: p<0.05)   Study of structural and functional changes during development of the anteriorcortical bone in the rat lumbar vertebral body ― 387 ―DISCUSSIONThe purpose of this study was to examine the changes in structure and function of vertebral cortical bone with development as part of a basic study to further our knowledge of vertebral cortical bone. The results showed that the strength of vertebral cortical bone at each week of age increased significantly with development. On the one hand, the strength of vertebral cortical bone is determined by diverse structural factors11). Therefore, the increase in vertebral cortical bone strength with development was considered to be significantly related to the structural changes associated with vertebral cortical bone development. Cortical bone, on the other hand, forms in a mode of ossification called membranous ossification. In the early stages of cortical bone formation, collagen fibers, the main component of the matrix, are irregularly and intricately arranged, a characteristic of membranous bone, also called woven bone5-7). This irregular morphology of woven bone also results in irregular vascularization within the bone. As the bone matures, the woven bone gradually thickens, narrowing the space for blood vessels to pass through. The bone matrix thus formed has a regularized arrangement of collagen fibers, forming a laminae-like structure with blood vessels arranged in concentric circles around the vessels. The mature bone formed in this way is called lamellar bone and is distinguished from fibrous bone8, 9). In the present study, 3w vertebral cortical bone showed an extremely fibrous bone structure, and such fibrous bone structure was formed from thin fibrous bundles. In addition, such thin fibrous bundles were irregularly oriented in the longitudinal direction of the bone.  Figure. 4 Comparison of reactions in immunohistochemistry in each group (Demineralized paraffin specimen, bar=50 ㎛) ― 388 ―On the other hand, the vertebral cortical bone of 7w had a fibrous bone structure similar to that of 3w, but such fibrous bone structure, unlike that of 3w, was formed from relatively thick fibrous bundles and was oriented relatively regularly in the long axial direction of the bone. On the other hand, the vertebral cortical bone of 13w, unlike that of 3w and 7w, did not exhibit fibrous bone structures and formed a dense laminar plate structure. On the other hand, numerous vascular cavities were observed in the vertebral cortical bone at each week of age, indicating that they decreased with increasing age. The vertebral cortical bone at 3w showed a very coarse structure, while that at 7w was less coarse than at 3w and formed a relatively laminar structure. 13w vertebral cortical bone formed a denser laminar structure than at 7w. Bone volume of vertebral cortical bone at each week of age was shown to increase with development. Polychrome staining revealed that the 3w vertebral cortical bone showed a coarse structure formed by thin fibrous bundles, and the fibrous bundles were predominantly light red and light blue in color. In contrast, the vertebral cortical bone of 7w was composed of thick fibrous bundles, and the fibrous bundles were predominantly light red; the vertebral cortical bone of 13w was formed of thicker fibrous bundles that were predominantly light red and light orange than those of 3w and 7w. These results suggest that vertebral cortical bone undergoes structural changes during development, such as narrowing of the vascular space, increase in bone volume, thickening of the matrix fibers, increase in density and regularization of orientation, and densification according to the typical ossification pattern by membranous ossification. This densification of vertebral cortical bone was considered to be a structural factor contributing to the increase in bone strength with increasing age. In general, rats are considered to correspond to weaning at 3w, puberty at 7w, and late maturity at 13w, as applied to humans. Therefore, it is thought that porosity occurs during the juvenile stage, when maturity is poor, because external growth is prioritized over the acquisition of bone strength due to low body weight and activity. On the other hand, as maturity progresses and body weight and activity increase, such porosity is suggested to become denser and acquire strength to cope with mechanical stresses. The above findings on developmental changes in vertebral cortical bone are expected to serve as a basis for future applied research as an indicator role related to the strength and structure of vertebral cortical bone.COCLUSIONRat vertebral cortical bone densifies with development, suggesting that this is a structural change adapted to the increase in body weight and activity with increasing age.Study of structural and functional changes during development of the anteriorcortical bone in the rat lumbar vertebral body ― 389 ―REFERENCE1.　  Néville D. O. F., et al.: Prevalence of vertebral fractures and quality of life in a sample of postmenopausal Brazilian women with osteoporosis. Arch osteo, 7.1: 101-106. 2012.2.　  Kinney J. H., et al. : Three-dimensional morphometry of the L6 vertebra in the ovariectomized rat model of osteoporosis: biomechanical implications. JBMR, 15:1981–91. 20003.　  Eswaran S. K., et al.: The micro-mechanics of cortical shell removal in the human vertebral body. Compu Method Appl Mech Eng, 196:3025–32. 2007.4.　  Augat P., et al.: The role of cortical bone and its microstructure in bone strength. Age and ageing, 2006, 35. suppl_2: ii27-ii31.5.　  Turner C. H., et al.: Mechanical loading thresholds for lamellar and woven bone formation. Journal of bone and mineral research, 1994, 9.1: 87-97.6.　  Hernandez C. J., et al.: Osteocyte density in woven bone. Bone, 2004, 35.5: 1095-1099.7.　  Akiva A., et al.: An organoid for woven bone. Advanced Functional Materials, 2021, 31.17: 2010524.8.　  Weiner S., et al.: Lamellar bone: structure–function relations. Journal of structural biology, 1999, 126.3: 241-255.9.　  Nyman J. S., et al.: The influence of water removal on the strength and toughness of cortical bone. Journal of biomechanics, 39: 931-938. 2006.10.   Wendeberg B., et al.: Mineral metabolism of fractures of the tibia in man studied with external counting of Sr85. Acta Ortho Scandi, 32. sup52: 3-81. 1961.11.   Ammann P., et al.: Bone strength and its determinants. Osteo. Int, 14: 13-18. 2003.― 390 ―ラット腰椎体における前方皮質骨の発育に伴う構造と 機能の変化に関する研究健康スポーツ科学研究科健康スポーツ科学専攻博士後期課程1年八嶋　奈央ライフデザイン学研究科ヒューマンライフ学専攻博士後期課程2年南園　　航ライフデザイン学研究科健康スポーツ学専攻修士課程2年徐　　思琴健康スポーツ科学部健康スポーツ科学科教授大迫　正文要旨　本研究では、椎体皮質骨の発育に伴う構造的および機能的変化を検討した。材料として3、7および13週齢のWistar系雄性ラットを用いた。椎体皮質骨は発育過程において、血管腔の狭小化、骨量の増加、基質線維の肥厚、密度の増加および配向の規則化などの構造的変化を遂げ、膜性骨化による典型的な骨化様式に従い緻密化が図られていると考えられた。このような椎体皮質骨の緻密化は、増齢に伴う骨強度の増加に寄与する構造的要因であると考えられた。結論として、ラット椎体皮質骨は発育とともに緻密化し、これは増齢による体重および活動性の増加に適応した構造的変化であることが示唆された。キーワード：椎骨、皮質骨、発育

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ラット腰椎体における前方皮質骨の発育に伴う構造と機能の変化に関する研究

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ラット腰椎体における前方皮質骨の発育に伴う構造と機能の変化に関する研究

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