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It is well established that mechanical loading plays an important role in skeletal development (Carter,1987; Martin et al.,1998; Frost,2003; Pearson and Lieberman,2004). However, little is know about the effects of in vivo mechanical loading on chondral and osseous histomorphology in limb joints. Mechanically induced deformation of cartilage in vitro stimulates Indian hedgehog (Ihh) expression by prehypertrophic mature chondrocytes, which stimulates chondrocyte proliferation and differentiation (St-Jacques et al.,1999; Wu et al.,2001). Mechanical deformation also stimulates extracellular matrix synthesis by mature chondrocytes (Urban,1994). Specifically, moderate levels of hydrostatic compressive stress (< 10 MPa) stimulate chondrocyte differentiation, proliferation, and extracellular matrix synthesis, while excessive levels suppress biosynthesis (Smith et al.,2000; Liu et al.,2001; Ikenoue et al.,2003; Elder et al.,2005). In this manner, chondral growth and limb bone formation are hypothesized to be regulated by mechanical stresses.
Mechanical stresses in cartilage appear to be necessary for joint space development and maintenance during embryonic development as demonstrated in in vivo experiments on chicks. In the absence of stress from muscular contractions, embryonic joints may become fused or joint cavities may fail to form (Murray and Drachman,1969; Mitrovic,1982). Likewise, increased muscular activity and joint motility lead to increased embryonic joint cavity size (Ruano et al.,1985). These modifications in embryonic joints appear to be the result of mechanically induced chondrogenesis (Osborne et al.,2002). Stresses within cartilage anlagen regulate chondrogenesis during joint morphogenesis to ensure joint congruence as articular surfaces are formed (Heegaard et al.,1999).
Stresses in articular cartilage also appear to regulate postnatal limb joint development through chondrogenic events (Carter and Wong,1990; Frost,1999). In vivo studies using animal models support the hypothesis that mechanical loading is necessary for normal joint conformation. Insufficient mechanical loading of limb joints leads to irregular joint form and retarded joint growth (Steinberg and Trueta,1981; van de Lest et al.,2002). Conversely, increased mechanical loading of limb joints leads to increases in joint diameter and subchondral bone thickness (Radin et al.,1982; Bouvier and Zimny,1987; Murray et al.,2001). Larger articular dimensions would provide greater resistance, and hence greater protection against tissue damage, from large joint loads (Frost,1999). Thus, mechanically regulated cartilage proliferation may adapt joint form to joint loads during postnatal ontogeny to maintain a normal kinetic pathway and minimize the risk of joint damage from high contact strains (Hamrick,1999).
The relationship between joint size and joint loads, however, is not clearly defined. Lieberman et al. (2001) report finding no difference in articular surface area in control and exercise-treated sheep. Across species, articular surface area appears to be more closely related to joint mobility, while underlying trabecular bone mass and structure may correspond more strongly with joint loads (Rafferty and Ruff,1994). Articular responses to loading during periods of skeletal growth may be more constrained than trabecular responses to ensure proper joint function (Lieberman et al.,2001). Additionally, theoretical and computational models of joint growth predict that changes in joint curvature may play an important role in the functional adaptation of joints (Frost,1999; Hamrick,1999; Plochocki,2003). Joints may respond to increased mechanical loading through either changes in size or shape, or changes in both. Examining the relationship between joint size, shape, and loading, as well as comparing subchondral and trabecular responses to loading, may improve our understanding of joint conformation.
Investigations into the effects of mechanical loading on in vivo chondrogenesis have also proven inconclusive. Computational models and in vitro experiments support the hypothesis that mechanical deformation of cartilage tissue leads to cartilage tissue proliferation (Urban,1994; Beaupré et al.,2000; Grodzinsky et al.,2000). Several studies also show an increase in chondrocyte proliferation in response to loading as indicated by differences in cellularity (Wu and Chen,2000; Wu et al.,2001; Wang and Mao,2002). However, several human and animal studies have found little or no change in cartilage thickness in response to mechanical loading (Kiviranta et al.,1992; Eckstein et al.,2002) or immobilization (LeRoux et al.,2001), while others have found evidence of chondrogenesis (Kiviranta et al.,1988; Muhlbauer et al.,2000).
In this study, we make histomorphometric comparisons of cartilage and osseous tissue of the proximal femur of control and exercise-treated subadult mice. The objective of this research is to better define the relationship between mechanical loading and joint tissue form. We test three hypotheses. First, that the osteogenic response to loading will be greater in trabecular bone in comparison with subchondral bone. Second, that the functional adaptation of osseous joint tissue to joint loads includes modifications of both joint size and shape. Third, that cartilage tissue responds to increased loading through significant chondrogenesis as indicated by differences in articular cartilage tissue area, thickness, and cellularity. We also examine differences in curvature of the articular cartilage to determine if chondral shape changes occur in response to loading.
MATERIALS AND METHODS
Fifty virgin female mice of the biological strain C57BL/6J were used in this experiment (000664; Jackson Laboratory, Bar Harbor, ME). Each mouse was housed in 153 square inch cages and provided with mouse chow and water ad lib. All mice were housed in the same room with a 12:12-hr light/dark cycle. After a 7-day acclimation period, the mice were randomly assigned to an exercised experimental group (n = 25) that had continuous voluntary access to an activity wheel (Bio-Serv, Frenchtown, NJ) and a sedentary control group (n = 25). At the commencement of the study, mice in the control group had an average body mass of 15.34 g, while mice in the exercise group had a body mass of 15.25 g. Average daily running distances were used to calculate the total amount of exercise the mice in the experimental group received. Distance was measured using a digital magnetic counter and was further monitored with periods of regular observation. All mice were born on the same day and were 7 weeks old at the beginning of the experiment. Skeletal growth in this strain of mouse gradually slows to a halt between 16 to 24 weeks of age; thus, the mice in the experiment were skeletally immature throughout the duration of the investigation. The mice were sacrificed at an age of 11 weeks on day 29 of the experiment using compressed CO2 in accordance with the Institutional Animal Care and Use Committee. Immediately following sacrifice, left femora were dissected out and cleaned of soft tissue.
Femora were fixed in 10% neutral-buffered formalin, dehydrated in alcohol, and embedded in methyl methacrylate (Polysciences, Warrington, PA). A low-speed saw (Isomet; Buehler, Lake Bluff, IL) was used to cut one 80 μm thin section through the midline of each femoral head in the coronal plane. The sections were then ground to a final thickness of ∼ 20 μm, stained with toluidine blue, and digitally captured (Micropublisher 5.0 RTV camera; QImaging, Canada) under light microscopy (Zeiss Axiostar, Carl Zeiss, Germany).
Image analysis software (ImageJ, NIH) was used to collect histomorphometric data. Osseous measurements included bone tissue area taken as the area of bone tissue in the femoral head, and the subchondral articular perimeter taken as the length along the bony articular surface of the femoral head. Observations of the cartilage tissue included maximum cartilage thickness taken as the maximum perpendicular distance between the bony articular surface and chondral articular surface, articular cartilage tissue area, and articular cartilage perimeter taken as the length along the articular surface of the articular cartilage.
Joint curvature values were also measured from the sections to quantify general shape changes of the articular arcs of the femoral head and articular cartilage. Curvature was calculated after Biewener (1983) and Hamrick (1996) as the ratio of the articular arc breadth to the articular arc height, where arc breadth is the chord across the base of the articular arc and arc height is the perpendicular distance from the arc breadth to the arc of the articular surface. Curvatures of both the articular cartilage and subchondral bone articular arcs of the femoral head were measured.
Lastly, chondrocyte cell counts were taken as an indirect measure of differences in chondrocyte proliferation between the exercised and control groups. Increased cellularity is also associated with the longitudinal expansion of cartilage tissue and extracellular matrix volume (Vanky et al.,1998). Cell count was recorded for the sections stained with toluidine blue on digitally captured images using a 40× objective. The color balance of each image was modified such that chondrocyte nuclei appeared as dark circles on a lighter background. A rectangular area 200 μm wide was then outlined through the full depth of the articular cartilage in the midline of the joint. The area of the rectangle was noted and the number of cells in the rectangle was counted. Cell count is reported as the average number of chondrocytes per 200 μm2 for each joint. Toluidine blue metachromasia in the selected area was also noted for qualitative comparisons in matrix composition between groups. Increased metachromasia would be indicative of increased glycosaminoglycan synthesis in response to stresses in the articular cartilage (Schneiderman et al.,1986).
Observations from the dependent variables were compared using an independent samples t-test with treatment group as the grouping variable. A Levene test of the homogeneity of variances and a Kolmogorov-Smirnoff test were employed to verify that the assumptions of homogeneity of the variances and normality were not violated. Comparisons between variables failing to meet the assumptions were analyzed using a nonparametric Wilcoxon signed-rank test. Statistical significance was set as P < 0.05.
The number of kilometers the mice ran per day on the activity wheels ranged from 5.5 to 9.6 km (mean = 7.6 km/day; standard error = 1.24; range = 4.1 km/day). There was no significant body mass difference between the groups at the start of the experiment (−0.6% difference; t = −0.43; P > 0.05), although mice in the exercised group were 0.91 g larger on average at the end of the study (5.24% difference; t = 3.56; P < 0.01). Mice in the control group gained an average of 1.2 g, while those in the exercised group gained 2.0 g.
Histomorphological properties of the osseous tissue of the femoral head differed significantly between treatment groups (Fig. 1, Table 1). Mice in the exercised group had significantly greater bone area in the femoral head in comparison to control mice (P < 0.01). Mice in the exercised group also had significantly longer subchondral articular perimeters (P < 0.05). Subchondral articular arc breadth and arc height dimensions were significantly longer in the exercised mice (P < 0.05), although the exercised mice also exhibited significantly reduced subchondral articular arc curvature (P < 0.01). Of the variables tested, bone area exhibited the greatest difference between groups (11.6%), while subchondral arc breadth exhibited the smallest difference (3.7%).
Table 1. Histomorphometric data of articular and trabeculae bone of the proximal femur in control and exercised mice*
Means are shown with standard errors.
Bone tissue area (mm2)
0.57 ± 0.016
0.64 ± 0.013
Subchondral articular perimeter (mm)
2.50 ± 0.059
2.74 ± 0.098
Subchondral arc breadth (mm)
1.32 ± 0.018
1.37 ± 0.014
Subchondral arc height (mm)
0.49 ± 0.011
0.55 ± 0.012
Subchondral articular arc curvature
2.73 ± 0.098
2.52 ± 0.060
Histomorphological properties of the articular cartilage of the proximal femur also differed between treatment groups (Table 2). Mice in the exercised group exhibited thicker articular cartilage and greater cartilage tissue area at the proximal femur than control mice (P < 0.001). The exercised mice had a greater chondral articular arc height than the control mice on average (P < 0.05), but similar chondral arc breadths (P > 0.05). The differences in curvature and perimeter of the chondral articular arc between groups were not significant (Fig. 1).
Table 2. Histomorphometric data of articular cartilage of the proximal femur in control and exercised mice*
Means are shown with standard errors.
Maximum cartilage thickness (mm)
0.38 ± 0.011
0.45 ± 0.011
Cartilage tissue area (mm2)
0.54 ± 0.015
0.62 ± 0.014
Cartilage articular perimeter (mm)
2.11 ± 0.107
2.26 ± 0.096
Chondral arc breadth (mm)
1.46 ± 0.015
1.50 ± 0.013
Chondral arc height (mm)
0.84 ± 0.018
0.90 ± 0.017
Chondral articular arc curvature
1.75 ± 0.010
1.67 ± 0.031
A Wilcoxon signed-rank test was used to compare chondrocyte cell counts because the data were not normally distributed. Observations for the exercised mice had a greater variance and were skewed to the right. Cell counts were significantly higher in the exercised group compared to the control group (Fig. 2). In general, exercised mice exhibited noticeably greater toluidine blue metachromasia in the articular cartilage.
The results from this study suggest that the mechanical environment plays an important role in joint development. Voluntary exercise has been shown to influence growth plate morphology (Saino et al.,2003; Niehoff et al.,2004). We show that mechanical loading from voluntary exercise also affects the growth of chondral and osseous tissue of the proximal femur. Exercise-induced mechanical loading affected the size, shape, and distribution of joint tissue, as well as cartilage cellularity, in our study. Therefore, we are unable to reject the hypothesis that joint growth is regulated by mechanical stimulation.
Histomorphometric dimensions of the osseous tissue of the femoral head were greater in the exercised mice compared to the control mice. The exercised mice in our experiment had greater bone tissue area, subchondral articular arc perimeter, and joint diameter (articular arc breadth). This increase in joint size would allow for a reduction in tissue strains by reducing the amount of force per unit of bone the joint must resist (Frost,1999). It is likely that the functional adaptation of articular bone through increases in joint size provides an important mechanism for reducing cartilage strains that can lead to irreparable cartilage (Eckstein et al.,2002).
Our data failed to support conclusively the hypothesis of Rafferty and Ruff (1994) that trabecular bone is more responsive to loading than subchondral bone. Bone area in our study exhibited an 11.6% difference between treatment groups while subchondral articular perimeter, breadth, and height showed differences of 9.2%, 3.7%, and 11.5%, respectively. Although bone area differed most between groups of the variables in our analysis, the difference between the subchondral and trabecular variables was not substantial. Although the molecular pathways by which subchondral and trabecular bone responds to loading differ, the level of response appears to be similar for both locations. We also fail to reject the hypothesis of Lieberman et al. (2001) that articular responses to loading are ontogenetically constrained. All of the articular dimensions used in our analysis differed significantly between treatment groups.
The difference in subchondral bone curvature between groups was significant. Mice in the exercised group had significantly flatter subchondral articular arcs. Decreased subchondral curvature in response to increased mechanical stress in articular cartilage is consistent with clinical observations and computational models of joint growth (Frost,1999; Plochocki,2003). It is also consistent with observations that joints become flatter throughout postnatal ontogeny as joint loads increase (Hamrick,1999). Because stresses at the central portion of a joint are more extreme than those at the peripheries, which are more likely to be in the growth-stimulating range, joint growth occurs in a manner that increases the contact area of the joint to decrease tissue strains (Frost,1999; Hamrick,1999). The flatter joint surfaces found in the exercised mice may be a mechanically induced adaptation that increases bony joint contact area to distribute contact stresses over a larger surface. Our research supports existing models of joint growth and indicates that both shape and size changes occur in response to loading. However, more research is needed to elucidate the precise relationship between joint stress and joint curvature.
Previous investigations into the effects of in vivo mechanical loading on cartilage thickness have been conflicting (Kiviranta et al.,1988,1992; Muhlbauer et al.,2000; LeRoux et al.,2001; Eckstein et al.,2002). Our findings are consistent with in vitro studies that show an increase in chondrocyte proliferation and expansion of chondral tissue in response to loading (Grodzinsky et al.,2000; Wu and Chen,2000; Wu et al.,2001). Our data support the hypothesis that articular cartilage is adapted to the mechanical environment. We find that exercise-treated mice have significantly greater cartilage thickness, area, and cellularity compared to controls, although we find no difference in the length or amount of curvature of the chondral articular arc. The absence of significant differences in the length or curvature of the chondral articular arc in our data indicates that the articular surface of the cartilage layer experiences little change as tissue proliferation occurs. However, the chondral arc height and cartilage tissue thickness are significantly greater in the exercised group compared to the control group, suggesting that cartilage thickness is increasing not from a change in chondral curvature, but as the result of a relative decrease in subchondral curvature. The similarities in chondral curvature between groups may play a role in the maintenance of joint congruence as subchondral adaptations occur, but this is unclear given our data.
The results of our research support the hypothesis that the mechanical regulation of articular cartilage proliferation allows for the continual adaptation of joint form. Larger, flatter subchondral surfaces and greater chondral and osseous tissue areas appear to be in vivo adaptations that provide greater resistance against larger loads. However, the functional adaptation of limb articulations to mechanical loading is complex, involving a combination of changes in tissue size and shape that significantly influence joint conformation. Nonetheless, the histomorphometric differences found in limb joint tissue between control mice and those treated with voluntary exercise support the hypothesis that joint growth is mechanically regulated.