Trabecular Bone Deficit and Enhanced Anabolic Response to Re-Ambulation after Disuse in Perlecan-Deficient Skeleton

Abstract

Perlecan/Hspg2, a large monomeric heparan sulfate proteoglycan, is found in the basement membrane and extracellular matrix, where it acts as a matrix scaffold, growth factor depot, and tissue barrier. Perlecan deficiency leads to skeletal dysplasia in Schwartz-Jampel Syndrome (SJS) and is a risk factor for osteoporosis. In the SJS-mimicking murine model (Hypo), inferior cortical bone quality and impaired mechanotransduction in osteocytes were reported. This study focused on trabecular bone, where perlecan deficiency was hypothesized to result in structural deficit and altered response to disuse and re-loading. We compared the Hypo versus WT trabecular bone in both axial and appendicular skeletons of 8-38-week-old male mice, and observed severe trabecular deficit in Hypo mice, approximately 50% reduction of Tb.BV/TV regardless of skeletal site and animal age. Defects in endochondral ossification (e.g., accelerated mineralization), increases in osteoclast activity, and altered differentiation of bone progenitor cells in marrow contributed to the Hypo phenotype. The Hypo trabecular bone deteriorated further under three-week hindlimb suspension as did the WT. Re-ambulation partially recovered the lost trabecular bone in Hypo, but not in WT mice. The novel finding that low-impact loading could counter detrimental disuse effects in the perlecan-deficient skeleton suggests a strategy to maintain skeletal health in SJS patients.

Keywords: Schwartz-Jampel syndrome; hindlimb suspension; micro-computed tomography; osteoclastogenesis; osteoporosis; perlecan; re-ambulation; trabecular bone.

Figure 1

Severe trabecular osteoporosis in perlecan-deficient (Hypo) male skeletons. Distal femur (long bone) was examined (A) in various age groups by ex vivo μCT imaging and (B) in a longitudinal study using in vivo μCT imaging. (C) Histomorphometric and histological analysis of osteoblastic bone formation indices and osteoclastic TRAP staining. The axial skeleton (spinal column and/or the vertebral body L4) was examined (D) in various age groups by ex vivo μCT imaging (E) and in a longitudinal study by in vivo μCT imaging. Significance for within-genotype age comparisons is indicated by different letters (uppercase for WT and lowercase for Hypo) and between-genotype comparisons are indicated by * p < 0.05). Scale bar in Figure 1D = 1 mm.

Figure 2

Perlecan deficiency altered MSC differentiation, but not proliferation (A), and increased osteoclastogenesis and resorptive activity (B). Perlecan content was reduced in Hypo marrow (C). Treating the primary MSC and osteoclast progenitor cells with exogenous heparin rescued the effects from the perlecan deficiency (D). Significance for within-genotype dose comparisons are indicated by different letters (uppercase for WT and lowercase for Hypo) and between-genotype comparisons indicated by * (p < 0.05). Open bars: WT; filled bars: Hypo.

Figure 3

Trabecular bone responses to HLS were similar in WT and Hypo mice. (A) Sequential in vivo CT imaging showed bone loss after 3-weeks HLS regardless of genotype. (B) HLS-induced relative changes in the trabecular bone parameters were similar in Hypo and WT mice. (C) Fold-change of bone markers for GND control mice (Hypo-GND/WT-GND) and the fold-changes induced by HLS (HLS/GND) in both WT and Hypo mice (* p < 0.05, Student t test). Significance in multiple group comparisons was indicated by different letters (one-way ANOVA and Tukey post hoc tests).

Figure 4

Different trabecular responses of WT and Hypo mice to re-ambulation following HLS. Tb.BV/TV was decreased by HLS and partially restored by re-ambulation in Hypo, but not WT, groups (A). Age and re-ambulation showed different effects on Tb.Th (B), Tb.N (C), and Tb.Sp (D). Significance in multiple group comparisons was indicated by different letters (uppercase for WT and lowercase for Hypo, one-way ANOVA and Tukey post hoc tests). Percentage of change is shown for the HLS group relative to its GND control ((HLS-GND)/GND, %).