THE EFFECT OF MECHANICAL LOADING ON THE mRNA EXPRESSION OFGROWTH-PLATE CELLS FOR FOUR DIFFERENT RAT DEVELOPMENTAL STAGES

Introduction: Longitudinal bone growth is a complex process involving temporo-spatial progression of chondrocytes throughout the reserve zone, the proliferative zone and the hypertrophic zone of the growth-plate [1]. The growth rate of a bone is a complex interplay between: 1) cell replication in the proliferative zone; 2) cell enlargement in the hypertrophic zone; and 3) matrix synthesis as well as controlled matrix degradation [1,2]. Static forces alter longitudinal growth; increased pressure on the plates retards growth and reduced pressure accelerates growth [2,3], which has key implications for scoliosis and other deformities. However, the mechanisms by which chondrocytes modify the growth rate of a bone are not well understood and very little is known about the biological response of these cells to load. Our aim was to establish the effects of loading on the mRNA expression of key molecular components of the growth-plate at four developmental stages. Methods: Tibiae were isolated from 6 female Sprague-Dawley rats at each age group: 21 (weaning), 35 (pubertal growth spurt), 56 (midpuberty) and 80 (mature stage) days old. Epiphyseal bone/growthplate/metaphyseal bone plugs were sectioned parallel to the growth plates from the proximal tibial ends. A loaded explant from each rat was paired to its contralateral unloaded control. Plugs were confined along the bone longitudinal axis between two ceramic porous plates. For loaded explants, a cylindrical aluminum bucket filled with copper bullets served as static loading, with magnitude fixed at 55% of the rat body weight. Control and loaded explants were incubated for 24 hours. In situ hybridization techniques were used to assess the mRNA expression of the cells in different zones of the growth-plate. Four key components were investigated: 18s (marker of basic cell metabolism), type II collagen (marker of major extracellular matrix component), type X collagen (marker of hypertrophic chondrocytes) and PTH-PTHrP receptors (marker of pre-hypertrophic chondrocytes). In situ hybridization was performed as described in Alvarez et al. [4]. Normal explants (no incubation step) were used to assess the effects of the incubation procedure. The spatial variation in the mRNA expression between loaded explants and their contralateral unloaded controls was compared to establish the sensitivity of: 1) the different growth-plate zones to loading; 2) the different developmental stages to loading. Results: In normal explants (NN), 18s signal was distributed homogeneously throughout the growth plate cells, indicating normal cell metabolism (Figs.1,2). Type II collagen signal of variable intensity was observed in all zones and type X collagen was primarily confined to the hypertrophic zone (Figs.1,2). Staining of PTH-PTHrP receptors was mainly positioned in the pre-hypertrophic zone, indicating cellular activity at the junction of proliferative and hypertrophic zones (Figs.1,2). Comparison of 80 d.o. culture control and normal explants confirmed that the incubation procedure did not affect normal basic cell metabolism (18s), type II and X collagen syntheses as well as PTHPTHrP receptor activity of the growth plates (Fig.1). However, an increasing culture effect was observed in the 56, 35 and 21 d.o. control explants. It manifested as a diminished signal for cell metabolism (18s), as can be seen in the 35 d.o. rat growth plates (Fig.2). In 80 d.o. loaded explants, 18s signal was expressed throughout the growth plate zones similarly to normal and control explants, indicating that loading did not perturb the basic metabolism of chondrocytes (Fig.1). The expression pattern of type II collagen showed a reduced staining band compared to controls (Fig.1). Type X collagen mRNA also indicated a thinning of the expression band in the hypertrophic zone (Fig.1). No difference was observed between loaded and culture control explants for PTH-PTHrP receptor mRNA (Fig.1). In 35 d.o. loaded explants, a deficit of 18s staining was observed in the central portion of growth plates, suggesting a disruption of normal basic cellular metabolism and cell death due to loading. This 18s deficit expression was also observed in 21 d.o. loaded explants and, less significantly, in 56 d.o. loaded explants. Nevertheless, the relative comparison of control and loaded explants for 21, 35 and 56 d.o. growth-plates showed reduced staining of collagens type II and X (Fig.2, for 35 d.o.). No relative difference could be detected for PTH-PTHrP receptor mRNA in 21, 35 and 56 d.o. explants (Fig.2, for 35 d.o.). Figure 1 In situ hybridization for 80 d.o. rat growth plates.