0
Research Papers

Local Changes to the Distal Femoral Growth Plate Following Injury in Mice

[+] Author and Article Information
Lauren M. Mangano Drenkard

Biomedical Engineering,
Boston University,
110 Cummington Mall,
Boston, MA 02215
e-mail: lmangano@bu.edu

Meghan E. Kupratis

Biomedical Engineering,
Boston University,
110 Cummington Mall,
Boston, MA 02215
e-mail: mkup@bu.edu

Katie Li

Biomedical Engineering,
Boston University,
110 Cummington Mall,
Boston, MA 02215
e-mail: kli430@bu.edu

Louis C. Gerstenfeld

Biochemistry,
Boston University School of Medicine,
72 East Concord Street,
Boston, MA 02118;
Orthopaedic Surgery,
Boston University School of Medicine,
72 East Concord Street,
Boston, MA 02118
e-mail: lgersten@bu.edu

Elise F. Morgan

Mem. ASME
Biomedical Engineering,
Boston University,
110 Cummington Mall,
Boston, MA 02215;
Mechanical Engineering,
Boston University,
110 Cummington Mall,
Boston, MA 02215;
Orthopaedic Surgery,
Boston University School of Medicine,
72 East Concord Street,
Boston, MA 02118
e-mail: efmorgan@bu.edu

1Corresponding author.

Manuscript received November 29, 2016; final manuscript received April 30, 2017; published online June 6, 2017. Assoc. Editor: Eric A Kennedy.

J Biomech Eng 139(7), 071010 (Jun 06, 2017) (9 pages) Paper No: BIO-16-1485; doi: 10.1115/1.4036686 History: Received November 29, 2016; Revised April 30, 2017

Injury to the growth plate is associated with growth disturbances, most notably premature cessation of growth. The goal of this study was to identify spatial changes in the structure and composition of the growth plate in response to injury to provide a foundation for developing therapies that minimize the consequences for skeletal development. We used contrast-enhanced microcomputed tomography (CECT) and histological analyses of a murine model of growth plate injury to quantify changes in the cartilaginous and osseous tissue of the growth plate. To distinguish between local and global changes, the growth plate was divided into regions of interest near to and far from the injury site. We noted increased thickness and CECT attenuation (a measure correlated with glycosaminoglycan (GAG) content) near the injury, and increased tissue mineral density (TMD) of bone bridges within the injury site, compared to outside the injury site and contralateral growth plates. Furthermore, we noted disruption of the normal zonal organization of the physis. The height of the hypertrophic zone was increased at the injury site, and the relative height of the proliferative zone was decreased across the entire injured growth plate. These results indicate that growth plate injury leads to localized disruption of cellular activity and of endochondral ossification. These local changes in tissue structure and composition may contribute to the observed retardation in femur growth. In particular, the changes in proliferative and hypertrophic zone heights seen following injury may impact growth and could be targeted when developing therapies for growth plate injury.

FIGURES IN THIS ARTICLE
<>
Copyright © 2017 by ASME
Your Session has timed out. Please sign back in to continue.

References

Caine, D. , DiFiori, J. , and Maffulli, N. , 2006, “ Physeal Injuries in Children's and Youth Sports: Reasons for Concern?,” Br. J. Sports Med., 40(9), pp. 749–760. [CrossRef] [PubMed]
Mann, D. C. , and Rajmaira, S. , 1990, “ Distribution of Physeal and Nonphyseal Fractures in 2650 Long-Bone Fractures in Children Aged 0–16 Years,” J. Pediatr. Orthop., 10(6), pp. 713–716. [CrossRef] [PubMed]
Langenskiold, A. , 1967, “ The Possibility of Eliminating Premature Partial Closure of an Epiphyseal Plate Caused by Trauma or Disease,” Acta Orthop. Scand., 38(1–4), pp. 267–269. [CrossRef]
Czitrom, A. , Salter, R. , and Willis, R. , 1981, “ Fractures Involving the Distal Epiphyseal Plate of the Femur,” Int. Orthop., 4(4), pp. 269–277. [PubMed]
Ogden, J. , 1987, “ The Evaluation and Treatment of Partial Physeal Arrest,” J. Bone Jt. Surg., 69(8), pp. 1297–1302. [CrossRef]
Makela, E. A. , Vainionpaa, S. , Vihtonen, K. , Mero, M. , and Rokkanen, P. , 1988, “ The Effect of Trauma to the Lower Femoral Epiphyseal Plate: An Experimental Study in Rabbits,” J. Bone Joint Surg. Br., 70(2), pp. 187–191. [PubMed]
Xian, C. , Zhou, F. , McCarthy, R. , and Foster, B. , 2004, “ Intramembranous Ossification Mechanism for Bone Bridge Formation at the Growth Plate Cartilage Injury Site,” J. Orthop. Res., 22(2), pp. 417–426. [CrossRef] [PubMed]
Chung, R. , and Xian, C. J. , 2014, “ Recent Research on The Growth Plate: Mechanisms for Growth Plate Injury Repair and Potential Cell-Based Therapies for Regeneration,” J. Mol. Endocrinol., 53(1), pp. T45–T61. [CrossRef] [PubMed]
Chung, R. , Cool, J. , Scherer, M. , Foster, B. , and Xian, C. , 2006, “ Roles of Neutrophil-Mediated Inflammatory Response in the Bony Repair of Injured Growth Plate Cartilage in Young Rats,” J. Leukocyte Biol., 80(6), pp. 1272–1280. [CrossRef]
Macsai, C. , Georgiou, K. , Foster, B. , Zannenttino, A. , and Xian, C. , 2012, “ Microarray Expression Analysis of Genes and Pathways Involved in Growth Plate Cartilage Injury Response and Bony Repair,” Bone, 50(5), pp. 1081–1091. [CrossRef] [PubMed]
Coleman, R. , Phillips, J. , Lin, A. , Schwartz, Z. , Boyan, B. , and Guldberg, R. , 2010, “ Characterization of a Small Animal Growth Plate Injury Model Using Microcomputed Tomography,” Bone, 46(6), pp. 1555–1563. [CrossRef] [PubMed]
Macsai, C. , Hopwood, B. , Chung, R. , Foster, B. , and Xian, C. , 2011, “ Structural and Molecular Analyses of Bone Bridge Formation Within the Growth Plate Injury Site and Cartilage Degeneration at the Adjacent Uninjured Area,” Bone, 49(4), pp. 904–912. [CrossRef] [PubMed]
Seil, R. , Pape, D. , and Khon, D. , 2008, “ The Risk of Growth Changes During Transphyseal Drilling in Sheep With Open Physes,” Arthroscopy: J. Arthroscopic Relat. Surg., 24(7), pp. 824–833. [CrossRef]
Lee, M. A. , Nissen, T. P. , and Otsuka, N. Y. , 2000, “ Utilization of a Murine Model to Investigate the Molecular Process of Transphyseal Bone Formation,” J. Pediatr. Orthop., 20(6), pp. 802–806. [CrossRef] [PubMed]
Hajdu, S. , Schwendenwein, E. , Kaltenecker, G. , Laszlo, I. , Lang, S. , Vecsei, V. , and Sarahrudi, K. H. , 2011, “ The Effect of Drilling and Screw Fixation of the Growth Plate—An Experimental Study in Rabbits,” J. Orthop. Res., 29(12), pp. 1834–1839. [CrossRef] [PubMed]
Joshi, N. , Bansal, P. , Stewart, R. , Snyder, B. , and Grinstaff, M. , 2009, “ Effect of Contrast Agent Charge on Visualization of Articular Cartilage Using Computed Tomography: Exploiting Electrostatic Interactions for Improved Sensitivity,” J. Am. Chem. Soc., 131(37), pp. 13234–13235. [CrossRef] [PubMed]
Bansal, P. , Joshi, N. , Entezari, V. , Malone, B. , Stewart, R. , Snyder, B. , and Grinstaff, M. , 2010, “ Cationic Contrast Agents Improve Quantification of Glycosaminoglycan (GAG) Content by Contrast Enhanced CT Imaging of Cartilage,” J. Orthop. Res., 29(5), pp. 704–709. [CrossRef] [PubMed]
Bansal, P. , Joshi, N. , Grinstaff, M. , and Snyder, B. , 2010, “ Contrast Enhanced Computed Tomography Can Predict the Glycosaminoglycan Conent and Biomechanical Properties of Articular Cartilage,” Oseoarhritis Cartilage, 18(2), pp. 184–191. [CrossRef]
Bonnarens, F. , and Einhorn, T. A. , 1984, “ Production of a Standard Closed Fracture in Laboratory Animal Bone,” J. Orthop. Res., 2(1), pp. 97–101. [CrossRef] [PubMed]
Schneider, C. A. , Rasband, W. S. , and Eliceiri, K. W. , 2012, “ NIH Image to ImageJ: 25 Years of Image Analysis,” Nat. Methods, 9(7), pp. 671–675. [CrossRef] [PubMed]
Hayward, L. N. M. , de Bakker, C. M.-J. , Lusic, H. , Gerstenfeld, L. C. , Grinstaff, M. W. , and Morgan, E. F.-I. , 2012, “ MRT Letter: Contrast-Enhanced Computed Tomographic Imaging of Soft Callus Formation in Fracture Healing,” Microsc. Res. Tech., 75(1), pp. 7–14. [CrossRef] [PubMed]
Hayward, L. N. M. , de Bakker, C. M. J. , Gerstenfeld, L. C. , Grinstaff, M. W. , and Morgan, E. F. , 2013, “ Assessment of Contrast-Enhanced Computed Tomography for Imaging of Cartilage During Fracture Healing,” J. Orthop. Res., 31(4), pp. 567–573. [CrossRef] [PubMed]
Ridler, T. , and Calvard, S. , 1978, “ Picture Threshold Using an Iterative Selection Method,” IEEE Syst. Man Cybern., 8(8), pp. 630–632. [CrossRef]
Martin, I. , Obradovic, B. , Freed, L. , and Vunjak-Novakovic, G. , 1999, “ Method for Quantitative Analysis of Glycosaminoglycan Distribution in Cultured Natural and Engineered Cartilage,” Ann. Biomed. Eng., 27(5), pp. 656–662. [CrossRef] [PubMed]
Wikström, B. , Hjerpe, A. , Hultenby, K. , Reinholt, F. P. , and Engfeldt, B. , 1984, “ Stereological Analysis of the Epiphyseal Growth Cartilage in the Brachymorphic (bm/bm) Mouse, With Special Reference to the Distribution of Matrix Vesicles,” Virchows Arch. B, 47(1), pp. 199–210. [CrossRef]
Jaramillo, D. , Hoffer, F. , Shapiro, F. , and Rand, F. , 1990, “ MR Imaging of Fractures of the Growth Plate,” Am. J. Roentgenol., 155(6), pp. 1261–1265. [CrossRef]
Jaramillo, D. , and Hoffer, F. , 1999, “ Cartilaginous Epiphysis and Growth Plate: Normal and Abnormal MR Imaging Findings,” Am. J. Roentgenol., 158(5), pp. 1105–1110. [CrossRef]
Sailhan, F. , Chotel, F. , Guibal, A. , Gollogly, A. , Adam, P. , Berard, J. , and Guibaud, L. , 2004, “ Three-Dimensional MR Imaging in the Assessment of Physeal Growth Arrest,” Eur. Radiol., 14(9), pp. 1600–1608. [CrossRef] [PubMed]
Young, J. , Bright, R. , and Whitely, N. , 1986, “ Computed Tomography in the Evaluation of Partial Growth Plate Arrest in Children,” Skeletal Radiol., 15(7), pp. 530–535. [CrossRef] [PubMed]
Coleman, R. M. , Schwartz, Z. , Boyan, B. D. , and Guldberg, R. E. , 2013, “ The Therapeutic Effect of Bone Marrow-Derived Stem Cell Implantation After Epiphyseal Plate Injury is Abrogated by Chondrogenic Predifferentiation,” Tissue Eng., Part A, 19(3–4), pp. 475–483. [CrossRef]
Larsson, S.-E. , Ray, R. , and Kuettner, K. , 1973, “ Microchemical Studies on Acid Glycosaminoglycans of the Epiphyseal Zones During Endochondral Calcification,” Calcif. Tissue Res., 13(1), pp. 271–285. [CrossRef] [PubMed]
Byers, S. , Rooden, J. C. , and Foster, B. K. , 1997, “ Structural Changes in the Large Proteoglycan, Aggrecan, in Different Zones of the Ovine Growth Plate,” Calcif. Tissue Int., 60(1), pp. 71–78. [CrossRef] [PubMed]
Byers, S. , Caterson, B. , Hopwood, J. , and Foster, B. , 1992, “ Immunolocation Analysis of Glycosaminoglycans in the Human Growth Plate,” J. Histochem. Cytochem., 40(2), pp. 275–282. [CrossRef] [PubMed]
Chen, J. , Lee, C. , Coleman, R. , Yoon, J. , Lohmann, C. , Zustin, J. , Guldberg, R. , Schwartz, Z. , and Boyan, B. , 2009, “ Formation of Tethers Linking the Epiphysis and Metaphysis is Regulated by Vitamin D Receptor-Mediated Signaling,” Calcif. Tissue Int., 85(2), pp. 134–145. [CrossRef] [PubMed]
Fischerauer, E. , Heidari, N. , Neumayer, B. , Deutsch, A. , and Weinberg, A. , 2011, “ The Spatial and Temporal Expression of VEGF and Its Receptors 1 and 2 in Post-Traumatic Bon Bridge Formation of the Growth Plate,” J. Mol. Histol., 42(6), pp. 513–522. [CrossRef] [PubMed]
Chung, R. , Foster, B. K. , and Xian, C. J. , 2014, “ The Potential Role of VEGF-Induced Vascularisation in the Bony Repair of Injured Growth Plate Cartilage,” J. Endocrinol., 221(1), pp. 63–75. [CrossRef] [PubMed]
Gerber, H.-P. , Vu, T. H. , Ryan, A. M. , Kowalski, J. , Werb, Z. , and Ferrara, N. , 1999, “ VEGF Couples Hypertrophic Cartilage Remodeling, Ossification and Angiogenesis During Endochondral Bone Formation,” Nat. Med., 5(6), pp. 623–628. [CrossRef] [PubMed]
Welting, T. , Caron, M. , Emans, P. , Janssen, M. , Sanen, K. , Coolsen, M. , Voss, L. , Surtel, D. , Cremers, A. , Voncken, J. , and van Rhijn, R. , 2011, “ Inhibition of Cyclooxygenase-2 Impacts Chondrocyte Hypertrophic Differentiation During Endochondral Ossification,” Eur. Cells Mater., 22, pp. 420–437. [CrossRef]
Arasapam, G. , Scherer, M. , Cool, J. , Foster, B. , and Xian, C. , 2006, “ Roles of COX-2 and iNOS in the Bony Repair of the Injured Growth Plate Cartilage,” J. Cell. Biochem., 99(2), pp. 450–561. [CrossRef] [PubMed]
Simsa-Maziel, S. , and Monsonego-Ornan, E. , 2012, “ Interleukin-1β Promotes Proliferation and Inhibits Differentiation of Chondrocytes Through a Mechanism Involving Down-Regulation of FGFR-3 and p21,” Endocrinology, 153(5), pp. 2296–2310. [CrossRef] [PubMed]
Zhou, F. , Forster, B. , Sander, G. , and Xian, C. , 2004, “ Expression of Proinflammatory Cytokines and Growth Factors at the Injured Growth Plate Cartilage in Young Rats,” Bone, 35(6), pp. 1307–1315. [CrossRef] [PubMed]
Kato, Y. , and Iwamoto, M. , 1990, “ Fibroblast Growth Factor is an Inhibitor of Chondrocyte Terminal Differentiation,” J. Biol. Chem., 265(10), pp. 5903–5909. [PubMed]
Dailey, L. , Laplantine, E. , Priore, R. , and Basilico, C. , 2003, “ A Network of Transcriptional and Signaling Events is Activated by FGF to Induce Chondrocyte Growth Arrest and Differentiation,” J. Cell Biol., 161(6), pp. 1053–1066. [CrossRef] [PubMed]
Hunziker, E. B. , Wagner, J. , and Zapf, J. , 1994, “ Differential Effects of Insulin-Like Growth Factor I and Growth Hormone on Developmental Stages of Rat Growth Plate Chondrocytes In Vivo,” J. Clin. Invest., 93(3), pp. 1078–1086. [CrossRef] [PubMed]
Enomoto-Iwamoto, M. , Iwamoto, M. , Mukudai, Y. , Kawakami, Y. , Nohno, T. , Higuchi, Y. , Takemoto, S. , Ohuchi, H. , Noji, S. , and Kurisu, K. , 1998, “ Bone Morphogenetic Protein Signaling is Required for Maintenance of Differentiated Phenotype, Control of Proliferation, and Hypertrophy in Chondrocytes,” J. Cell Biol., 140(2), pp. 409–418. [CrossRef] [PubMed]
Ngo, T. Q. , Scherer, M. A. , Zhou, F. H. , Foster, B. K. , and Xian, C. J. , 2006, “ Expression of Bone Morphogenic Proteins and Receptors at the Injured Growth Plate Cartilage in Young Rats,” J. Histochem. Cytochem., 54(8), pp. 945–954. [CrossRef] [PubMed]
Williams, J. L. , Vani, J. N. , Eick, J. D. , Petersen, E. C. , and Schmidt, T. L. , 1999, “ Shear Strength of the Physis Varies With Anatomic Location and is a Function of Modulus, Inclination, and Thickness,” J. Orthop. Res., 17(2), pp. 214–222. [CrossRef] [PubMed]
Zhu, W. , Mow, V. C. , Koob, T. J. , and Eyre, D. R. , 1993, “ Viscoelastic Shear Properties of Articular Cartilage and the Effects of Glycosidase Treatments,” J. Orthop. Res., 11(6), pp. 771–781. [CrossRef] [PubMed]
Kaviani, R. , Londono, I. , Parent, S. , Moldovan, F. , and Villemure, I. , 2016, “ Growth Plate Cartilage Shows Different Strain Patterns in Response to Static Versus Dynamic Mechanical Modulation,” Biomech. Model. Mechanobiol., 15(4), pp. 933–946. [CrossRef] [PubMed]
Shimizu, C. , Coutts, R. D. , Healey, R. M. , Kubo, T. , Hirasawa, Y. , and Amiel, D. , 1997, “ Method of Histomorphometric Assessment of Glycosaminoglycans in Articular Cartilage,” J. Orthop. Res., 15(5), pp. 670–674. [CrossRef] [PubMed]
Király, K. , Lapveteläinen, T. , Arokoski, J. , Törrönen, K. , Módis, L. , Kiviranta, I. , and Helminen, H. J. , 1996, “ Application of Selected Cationic Dyes for the Semiquantitative Estimation of Glycosaminoglycans in Histological Sections of Articular Cartilage by Microspectrophotometry,” Histochem. J., 28(8), pp. 577–590. [CrossRef] [PubMed]
Kiviranta, I. , Jurvelin, J. , Säämänen, A. M. , and Helminen, H. J. , 1985, “ Microspectrophotometric Quantitation of Glycosaminoglycans in Articular Cartilage Sections Stained With Safranin O,” Histochemistry, 82(3), pp. 249–255. [CrossRef] [PubMed]
Camplejohn, K. L. , and Allard, S. A. , 1988, “ Limitations of Safranin ‘O’ Staining in Proteoglycan-Depleted Cartilage Demonstrated With Monoclonal Antibodies,” Histochemistry, 89(2), pp. 185–188. [CrossRef] [PubMed]
Collins, M. J. , Arns, T. A. , Leroux, T. , Black, A. , Mascarenhas, R. , Bach, B. R., Jr. , and Forsythe, B. , 2016, “ Growth Abnormalities Following Anterior Cruciate Ligament Reconstruction in the Skeletally Immature Patient: A Systematic Review,” Arthroscopy, 32(8), pp. 1714–1723. [CrossRef] [PubMed]

Figures

Grahic Jump Location
Fig. 1

(a) Schematic of surgical model. (b) Length of the injured and uninjured (contralateral) femora, measured from the apex of the greater trochanter to apex of the left condyle: The height of each bar represents the group mean, and the error bars represent one standard deviation. *p < 0.01 over all time points.

Grahic Jump Location
Fig. 2

Local thickening of the injured growth plate: (a) Top: Sections of the growth plate stained with safranin O and fast green. Bottom: Matched CECT slices (cartilage is rendered according to the colorbar that shows attenuation in Hounsfield units (HU)). Arrows indicate the injury site. (b) Correlation of histologically and CECT-measured thickness of the growth plate (R2 = 0.94, p < 0.0001). Each point indicates the average thickness of one sample. (c) Maximum thickness projection maps of the growth plate. Arrows indicate the injury site. (d) Average thickness of the growth plate near to (within 0.5 mm) and far from the injury site and at matched locations in the contralateral growth plate. The height of each bar represents the group mean, and the error bars represent one standard deviation. *p < 0.0001 versus injured near (across all time points). The average thickness of the contralateral growth plate was 0.086 mm, 0.064 mm, and 0.042 mm at days 7, 21, and 42, respectively.

Grahic Jump Location
Fig. 3

CECT attenuation remains high at the injury site: (a) Maximum intensity projection maps of CECT attenuation in the growth plate. Arrows indicate the injury site. (b) Average CECT attenuation of the growth plate near to (within 0.5 mm) and far from the injury site and at matched locations in the contralateral growth plate. The height of each bar represents the group mean, and the error bars represent one standard deviation. *p < 0.0001 versus day 7, #p < 0.0001 versus day 21; p < 0.05, ††p < 0.005, and †††p < 0.0001 versus corresponding VOI in contralateral.

Grahic Jump Location
Fig. 4

Bone bridge formation. (a) Histological sections of injured growth plate. Arrows indicate disorganized chondrocyte columns. Asterisks indicate hypertrophic cells. (b) Sagittal μCT sections of the distal femora containing the injured growth plates shown in panel A. Arrows indicate bone bridges at the injury site. Circles indicate bone bridges outside the injury site. (c) Bone volume fraction (BV/TV) and tissue mineral density (TMD) of bone bridges within the growth plate. The height of each bar represents the group mean, and the error bars represent one standard deviation. *p < 0.01 and **p < 0.001. (d) Matched histological section and matched μCT section of the injury site at day-7 in a sample where the bone bridge has not yet formed. Arrows indicate the injury site. (e) Matched histological section and matched μCT section of the injury site at day-42 in a sample where the mineralized bone bridge is stained with safranin O. Arrows indicate the injury site. (f) Histology and matched CECT of a bone bridge formed outside the injury site and matched histological section and matched μCT section of bone bridges formed in the contralateral growth plate and outside the injury site. Stars indicate bone bridges.

Grahic Jump Location
Fig. 5

Zone heights. (a) Height of the resting, proliferative, and hypertrophic zones near to the injury site, far from the injury site, and in the contralateral growth plate. (b) Proportional height of the resting, proliferative, and hypertrophic zones near to the injury site, far from the injury site, and in the contralateral growth plate. In both plots, the height of each bar represents the group mean, and the error bars represent one standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Grahic Jump Location
Fig. 6

Collagen X immunostaining at day 21. (a) Injury site, (b) injured growth plate far from injury site, (c) contralateral growth plate. Arrow indicates the injury site. The scale bar applies to all three images.

Tables

Errata

Discussions

Some tools below are only available to our subscribers or users with an online account.

Related Content

Customize your page view by dragging and repositioning the boxes below.

Related Journal Articles
Related eBook Content
Topic Collections

Sorry! You do not have access to this content. For assistance or to subscribe, please contact us:

  • TELEPHONE: 1-800-843-2763 (Toll-free in the USA)
  • EMAIL: asmedigitalcollection@asme.org
Sign In