Postnatal progression of bone disease in the cervical spines of mucopolysaccharidosis I dogs

Abstract

Introduction: Mucopolysaccharidosis I (MPS I) is a lysosomal storage disorder characterized by deficient α-l-iduronidase activity leading to accumulation of poorly degraded dermatan and heparan sulfate glycosaminoglycans (GAGs). MPS I is associated with significant cervical spine disease, including vertebral dysplasia, odontoid hypoplasia, and accelerated disk degeneration, leading to spinal cord compression and kypho-scoliosis. The objective of this study was to establish the nature and rate of progression of cervical vertebral bone disease in MPS I using a canine model.

Methods: C2 vertebrae were obtained post-mortem from normal and MPS I dogs at 3, 6 and 12 months-of-age. Morphometric parameters and mineral density for the vertebral trabecular bone and odontoid process were determined using micro-computed tomography. Vertebrae were then processed for paraffin histology, and cartilage area in both the vertebral epiphyses and odontoid process were quantified.

Results: Vertebral bodies of MPS I dogs had lower trabecular bone volume/total volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and bone mineral density (BMD) than normals at all ages. For MPS I dogs, BV/TV, Tb.Th and BMD plateaued after 6 months-of-age. The odontoid process appeared morphologically abnormal for MPS I dogs at 6 and 12 months-of-age, although BV/TV and BMD were not significantly different from normals. MPS I dogs had significantly more cartilage in the vertebral epiphyses at both 3 and 6 months-of-age. At 12 months-of-age, epiphyseal growth plates in normal dogs were absent, but in MPS I dogs they persisted.

Conclusions: In this study we report reduced trabecular bone content and mineralization, and delayed cartilage to bone conversion in MPS I dogs from 3 months-of-age, which may increase vertebral fracture risk and contribute to progressive deformity. The abnormalities of the odontoid process we describe likely contribute to increased incidence of atlanto-axial subluxation observed clinically. Therapeutic strategies that enhance bone formation may decrease incidence of spine disease in MPS I patients.

Figure 1. Histology and microCT reconstructions of C2 vertebral bone

Mid-sagittal, histological sections, double-stained with Alcian blue (glycosaminoglycans) and picrosirius red (collagen), from normal and MPS I dogs aged 3, 6 and 12 months (A, B, E, F, I and G), with 3D microCT reconstructions of trabecular bone for the same samples (C, D, G, H, K and L). Lower trabecular bone volume is apparent for MPS I samples at all ages, but is most striking at 12 months-of-age. All histological images are oriented with the ventral (anterior) side at the bottom; ep = caudal vertebral end plate. Scale bars = 2 mm (histology) or 1 mm (microCT). Dashed lines indicate approximate location of odontoid process attachment.

Figure 2. MicroCT analysis of C2 vertebral trabecular bone

A. Bone volume/total volume (BV/TV). B. Trabecular thickness (Tb.Th). C. Trabecular spacing (Tb.Sp). D. Trabecular number (Tb.N). E. Bone mineral density (apparent density, BMD). F Tissue. mineral density (material density, TMD). *p<0.05 vs normal; +p<0.05 vs 3 months; and ◆p<0.05 vs 6 months.

Figure 3. Histology and microCT reconstructionsf theo odontoid process

Mid-sagittal, histological sections, double-stained with Alcian blue (glycosaminoglycans) and picrosirius red (collagen), from normal and MPS I dogs aged 3, 6 and 12 months (A, B, E, F, I and G), with microCT reconstructions of the same samples (C, D, G, H, K and L). MPS I samples appear smaller and narrower, and had irregular surface morphology at 6 and 12 months-of-age. Decreased cartilage on the ventral articulating surface (arrows) was also evident at these ages for MPS I samples. At 3 months-of-age, delayed calcification was apparent towards the tip of the MPS I samples. For all images, ventral (anterior) is towards the bottom and caudal (posterior) is towards the right. Dashed lines indicate approximate location of vertebral body attachment. All scale bars = 2 mm.

Figure 4. MicroCT analysis of the odontoid process

A. Bone volume/total volume (BVTV). B. Bone mineral density (apparent density, BMD). C. Tissue mineral density (material density, TMD). *p<0.05 vs normal; +p<0.05 vs 3 months; and ◆p<0.05 vs 6 months.

Figure 5. Cartilage to bone conversion in the C2 vertebral epiphysis

Mid-sagittal histological sections double-stained with Alcian blue (glycosaminoglycans) and picrosirius red (collagen) illustrating increased cartilage in the ventral epiphyses (*) at 3 months-of-age (A and B) and at 6 months-of-age (C and D). At 12 months-of age, the epiphyseal growth plate in normal animals was absent (E); however, in MPS I animals, this growth plate persisted (F). Scale bars = 500 μm (A–D) and 1 mm (E and F); gp = growth plate and ep = caudal vertebral endplate. All images are oriented with the ventral side at the bottom.

Figure 6. Quantification of cartilage area in the C2 vertebral epiphysis and odontoid process

A. Cartilage area (inclusive of the growth plate) as a percentage of the total epiphysis area was determined from mid-sagittal Alcian blue/picrosirius red-stained histological sections, for 3 and 6 month-old samples. B. Cartilage area in the odontoid process as a percentage of total tissue area was determined from mid-sagittal Alcian blue/picrosirius red-stained histological sections, for 3 month-old samples. *p<0.05 vs normal; +p<0.05 vs 3 months.