Elsevier

Journal of Biomechanics

Volume 46, Issue 10, 21 June 2013, Pages 1597-1603
Journal of Biomechanics

Spatial periodicity in growth plate shear mechanical properties is disrupted by vitamin D deficiency

https://doi.org/10.1016/j.jbiomech.2013.04.023Get rights and content

Abstract

The growth plate is a highly organized section of cartilage in the long bones of growing children that is susceptible to mechanical failure as well as structural and functional disruption caused by a dietary deficiency of vitamin D. The shear mechanical properties of the proximal tibial growth plate of rats raised either on normal or vitamin D and calcium deficient diets were measured. A sinusoidal oscillating shear load was applied to small excised growth plate specimens perpendicular to the direction of growth while imaging the deformation in real time with a fast confocal microscope. Local deformations and shear strains were quantified using image correlation. The proliferative zone of the growth plate bores the majority of the shear strain and the resting, hypertrophic and calcification zones deformed less. Surprisingly, we regularly observed discontinuous deformations in the proliferative zone in both groups that resembled cell columns sliding past one another in the direction of growth. These discontinuities manifested as regions of concentrated longitudinal shear strain. Furthermore, these shear strain concentrations were spaced evenly in the proliferative zone and the spacing between them was similar across growth plate regions and across control specimens. In contrast to the healthy controls, the vitamin D deficient growth plate exhibited larger variations in the size and orientation of cellular columns in the proliferative and hypertrophic zones. High strains were observed between columns, much as they were in the controls. However, the regular spacing of shear strain concentrations was not preserved, echoing the observation of decreased structural organization.

Introduction

The growth plate is the collection of tissues located near the ends of long bones which together provide the mechanisms for longitudinal bone growth. Central to its function is a layer of growth plate cartilage, the primary source of new bone scaffold material. Growth plate cartilage is comprised of four distinct zones that lie along the longitudinal axis of the bone: resting, proliferative, hypertrophic, and calcified (Martin et al., 1998). This axis is both spatial and temporal, representing the life cycle of chondrocytes in the growth plate. Cells in the resting zone (closest to the epiphysis) are the smallest by diameter. As these cells age, they enter the proliferative zone, the site of growth plate cell division. Adjacent to time and space is the hypertrophic zone, where cells increase in volume by accumulating glycogen. Finally, cells enter the calcification zone where they die as the extracellular matrix is calcified. During all stages of growth, growth plate cells sit along distinct columnar structures known as chondrons (Bonucci and Motta, 1990). The growth plate also contains longitudinal struts known as tethers (Martin et al., 2003) that connect the epiphysis to the metaphysis. Collectively this cellular and matrix structure imparts the growth plate with a highly organized arrangement that is critical to its function.

Disruption of the organization of the growth plate significantly impedes skeletal growth. Such disruptions can arise from a variety of conditions, including genetic mutations (Oh et al., 2012, Marks et al., 2000, Bonaventure et al., 1998), dietary insufficiencies (Ehrlich et al., 1973, Tardivel et al., 1992), and mechanical trauma (Lee et al., 1985, Revel et al., 1985, Wattenbarger et al., 2002). Of specific interest here are insufficiencies of dietary components necessary for skeletal growth, particularly vitamin D. Insufficient dietary intake of vitamin D is a major cause of rickets, one of the most frequent childhood diseases in the developing world (Allgrove, 2004). Rickets is characterized by bone shortening, bone deformities, and increased susceptibility to fractures (Özkan, 2010). The rachitic growth plate is characterized by decreased synthesis of extracellular matrix (Genever and Dickson, 1996, Takechi and Itakura, 1995), abnormal vascularization (Gay et al., 2007), increased chondrocyte hypertrophy (Sabbagh et al., 2005), and disruption of the cell columns (Sabbagh et al., 2005) as well as the tether network that connects the epiphysis to metaphysis (Lee et al., 1985).

Mechanical trauma impedes skeletal growth through premature closure of the growth plate caused by chondrocyte apoptosis (Gaber et al., 2009, Macsai et al., 2011). Such injuries can result from a single overload event or from repetitive loading and overuse (DiFiori, 2010). Failure of the growth plate under load occurs frequently in shear (van Leeuwen et al., 2004), with local damage occurring most commonly in the hypertrophic zone (Lee et al., 1985). Despite the frequency and potentially devastating consequences of such injuries, relatively little is known about the mechanical properties of the growth plate, particularly in comparison to other cartilaginous tissues.

The bulk compressive mechanical properties of growth plate along and perpendicular to the direction of growth have been studied in vitro (Villemure and Stokes, 2009). While the compressive modulus (E) in the direction of growth is approximately 0.5 MPa (Cohen et al., 1994), E is 10 times greater in the transverse direction (Villemure and Stokes, 2009). Spatial variations in compressive strain along the growth plate have been measured using partial thickness sectioning (Sergerie et al., 2009) and by imaging fluorescently stained growth plate nuclei as the tissue is loaded uniaxially (Villemure et al., 2007). In the latter study, texture correlation was used to quantify patterns in axial strain throughout the growth plate. The growth plate was found to be stiffest under compression in the proliferation zone. In contrast, using atomic force microscopy the indentation modulus of the extracellular matrix increased monotonically from the reserve zone to the calcification zone (Radhakrishnan et al., 2004). Villemure et al. (2007) suggest that these apparent contradictions may be the result of the changing ratio of extracellular matrix volume to cell volume from 1.6 in the proliferative zone to 0.4 in the hypertrophic zone (Farnum and Wilsman, 1998).

These studies collectively suggest that growth plate tensile and compressive properties are spatially heterogeneous in a way that depends on growth plate structure. However, very little is known about the shear properties of the growth plate, which are critical to understand how the tissue fails. Previous studies have established confocal elastography techniques capable of mapping shear strains in articular cartilage with a spatial resolution of approximately 20μm (Buckley et al., 2008, Buckley et al., 2010). The goals of the current study were to apply these confocal elastography techniques to characterize the spatial pattern of shear strains in the normal rat growth plate and to characterize changes in this pattern due to structural disruption of the growth plate induced by dietary vitamin D insufficiency.

Section snippets

Dietary Vitamin D insufficiency model

Vitamin D deficiency in male Sprague-Dawley rats (Charles River, Wilmington, MA) was achieved using a modified dietary intervention based on the methods of Sonnenberg et al. (1984) and Kim et al. (2012). At 3 weeks of age, animals were weaned and started on their respective diets. Controls were fed normal rat chow replete in vitamin D3 (2000 IU/kg) and Ca (0.47%) for the 10 week experimental duration (Harlan Teklad, TD. 08364, Indianapolis, IN). The vitamin D-deficient group was fed rat chow

Results

The shear strain profiles γ0(s) of growth plate were sensitive to distance s from the chondro-osseous junction in controls (Fig. 3c, d). In general, the largest shear strains were observed in the proliferation zone while small strains were observed in the hypertrophic/calcification and resting zones. Storage modulus |G|(s) reached a maximum value of 20–60 kPa near the chondro-osseous junction (Fig. 3b). In all but one sample, the global minimum of |G|(s) was in the range 10–20 kPa and located

Discussion

This study established that growth plate exhibits a complex, heterogeneous response to shear strain transverse to the direction of growth. The proliferation zone experienced larger shear strains than the resting and hypertrophic zones. While all samples were most compliant near the middle of the growth plate, the location-dependent shear strain and modulus demonstrated significant variation between samples. We surmised that these variations were a result of the non-uniform geometry of bone

Conflict of interest statement

The authors have no conflict of interest to disclose.

Acknowledgments

This work was supported by the National Institutes of Health (R01-AR053571, R01-AR053571-S1 and R21-AR054867). We thank Darvin Griffin and Jesse Silverberg for their advice and assistance and Drs. Nelly Farnum and Adele Boskey for their keen insight and helpful conversations.

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