Effects of Hindlimb Suspension on the Development of Hip Bone Morphologies in Growing Rats

Abstract

Abnormal hip bone morphologies are associated with various diseases of the hip joint. Weight bearing, especially during growth, may be important to achieve normal acetabulum development. This study aimed to investigate whether hip bone morphologies were affected by hindlimb suspension (HS) in 4 week-old rats. In HS groups, tail suspension was applied for 0, 2, 4, and 8 weeks. Age-matched rats were used as controls. The complex of hip bones with lumbar and sacral vertebrae were assessed based on morphological indexes using three-dimensional reconstructed images from X-ray computed tomography. Acetabular widths (measured from cranial to caudal) unchanged and depths became larger in both groups with age. Acetabular lengths (from the ventral side to the dorsal side) became larger in control groups but unchanged in HS groups with age. In HS groups, acetabular width, length, and depths were smaller than the control groups at 4 and/or 8 weeks. Acetabular versions became enlarged (rotated inwards) with age in both groups, although this was particularly pronounced in HS groups. Histologically, triradiate cartilage layers in the acetabulum were thinner with age and almost disappeared at 8 weeks in both groups. However, HS decreased Safranin O staining and prolonged the presence of hypertrophic chondrocyte indicating alterations in the chondral ossification processes. Iliac wing angles remained unchanged and anterior superior iliac crest (ASIC) distances increased with age in controls. In contrast, HS groups showed narrowed iliac wing angles with small ASIC distances. These results suggest that reduced mechanical loading during growth can interfere with hip joint formation. Keywords Hindlimb suspension, Hip joint, Acetabular morphology, Triradiate cartilage.

Fig. 1

Schematic representation describing how we measured morphological parameters of hip bone. The upper panel shows the way in which acetabular width, length, and depth were measured. Acetabular width: distance between points (b) and (c) on plane II. Acetabular length: distance between points (d) and (e) on plane III. Acetabular depth: distance between point (a) and the nearest point on the line connecting points (b) and (c). Plane I: a midsagittal plane at the central position of the spine-pelvis complex. Plane II: a plane sharing points (a) and (b) that is orthogonal to plane I. Plane III: a plane crossing the point (a) that is orthogonal to plane II. (a): the deepest point of the acetabulum that is the closest point to plane I. (b): the highest point on the margin of the acetabular (cranial side) that is the farthest point to plane I. (c): the point on the acetabular margin on the opposite (caudal) side of point (b) on plane II. (d): the point on the acetabular margin on the dorsal side on plane III. (e): the point on the acetabular margin on the ventral side on plane III. The lower panel shows the measured portions of the acetabular version, Iliac wing angle, and anterior superior iliac crest (ASIC) distance. Acetabular version: the angle between the line connecting the acetabular margins and plane I on plane IV (θ). Iliac wing angle: bisection of the angle formed by tangents of medial surfaces of the iliac wings on both sides (ϕ) on plane V. ASIC distance: distance between the left and right ASIC sides (B). Plane IV: a plane perpendicular to the line between the promontory and the centre of the dorsal aspect of the pubic symphysis that crosses point (a). Plane V: a transverse section perpendicular to the line between the promontory and the centre of the dorsal aspect of the pubic symphysis at the middle level of the sixth lumber vertebra.

Fig. 2

Light micrographs of triradiate cartilage illustrating the histomorphometric measurements. (A) Histological section in the plane parallel to the dorsal surface of acetabular floor stained with Safranin O fast green was used to analyze the triradiate cartilage. (B) The triradiate cartilage between the ischium-ilium, enlarged and boxed in A. h_TC: Height of the triradiate cartilage. h_Hz1: Height of the hypertrophic zone of the ischium side. h_Hz2: Height of the hypertrophic zone of the ilium side. The height of hypertrophic zone was calculated as h_HZ1 + h_HZ2.

Fig. 3

Histological changes in the triradiate cartilage of hip bones. Representative images of triradiate cartilage stained with safranin O at baseline (A and H), 2 weeks (B, E, I, K), 4 weeks (C, F, J, L), and 8 weeks (D and G) of hindlimb suspension (E, F, G, K, L) and age-matched controls (B, C, D, I, and J). Higher magnification of squared areas in A, B, C, E, and F (upper section) are shown in H, I, J, K, and L, respectively (lower section). Scale bars are 200 μm in A–G and 50 μm in H–L.

Fig. 4

Histomorphometric analysis of the triradiate cartilage. (A) Height of the triradiate cartilage. (B) Height of the hypertrophic zone. ^† indicates a significant main effect for age (P < 0.05). Different letters (a, b, and c) indicate statistically significant differences among HS or control groups (P < 0.05). Values are mean ± standard deviation. BL, baseline; Cont, control; HS, hindlimb suspension.