title: Delayed hypertrophic differentiation of epiphyseal chondrocytes contributes to failed secondary ossification in mucopolysaccharidosis VII dogs.
abstract: Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient β-glucuronidase activity, which leads to the accumulation of incompletely degraded glycosaminoglycans (GAGs). MPS VII patients present with severe skeletal abnormalities, which are particularly prevalent in the spine. Incomplete cartilage-to-bone conversion in MPS VII vertebrae during postnatal development is associated with progressive spinal deformity and spinal cord compression. The objectives of this study were to determine the earliest postnatal developmental stage at which vertebral bone disease manifests in MPS VII and to identify the underlying cellular basis of impaired cartilage-to-bone conversion, using the naturally-occurring canine model. Control and MPS VII dogs were euthanized at 9 and 14 days-of-age, and vertebral secondary ossification centers analyzed using micro-computed tomography, histology, qPCR, and protein immunoblotting. Imaging studies and mRNA analysis of bone formation markers established that secondary ossification commences between 9 and 14 days in control animals, but not in MPS VII animals. mRNA analysis of differentiation markers revealed that MPS VII epiphyseal chondrocytes are unable to successfully transition from proliferation to hypertrophy during this critical developmental window. Immunoblotting demonstrated abnormal persistence of Sox9 protein in MPS VII cells between 9 and 14 days-of-age, and biochemical assays revealed abnormally high intra and extracellular GAG content in MPS VII epiphyseal cartilage at as early as 9 days-of-age. In contrast, assessment of vertebral growth plates and primary ossification centers revealed no significant abnormalities at either age. The results of this study establish that failed vertebral bone formation in MPS VII can be traced to the failure of epiphyseal chondrocytes to undergo hypertrophic differentiation at the appropriate developmental stage, and suggest that aberrant processing of Sox9 protein may contribute to this cellular dysfunction. These results also highlight the importance of early diagnosis and therapeutic intervention to prevent the progression of debilitating skeletal disease in MPS patients.
authors.affiliation: Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: Sun H
authors.initials: SH
authors.lastname: Peck
authors.affiliation: Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: Philip J M
authors.initials: PJ
authors.lastname: O'Donnell
authors.affiliation: Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Kang
authors.affiliation: Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: Neil R
authors.initials: NR
authors.lastname: Malhotra
authors.affiliation: Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: George R
authors.initials: GR
authors.lastname: Dodge
authors.affiliation: Division of Orthopedic Surgery, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Pacifici
authors.affiliation: Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: Eileen M
authors.initials: EM
authors.lastname: Shore
authors.affiliation: Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: Mark E
authors.initials: ME
authors.lastname: Haskins
authors.affiliation: Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. Electronic address: lachlans@mail.med.upenn.edu.
authors.forename: Lachlan J
authors.initials: LJ
authors.lastname: Smith
mesh_terms: D000818:Animals; D001847:Bone Diseases / Q000209:etiology / Q000503:physiopathology; D002454:Cell Differentiation; D019902:Chondrocytes / Q000166:cytology*; D004285:Dogs; D004838:Epiphyses / Q000166:cytology*; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D006984:Hypertrophy; D016538:Mucopolysaccharidosis VII / Q000150:complications* / Q000503:physiopathology*; D010012:Osteogenesis; D013131:Spine / Q000502:physiology; D055114:X-Ray Microtomography
chemical_list: D006025:Glycosaminoglycans
keywords: Chondrocyte differentiation; Endochondral ossification; Glycosaminoglycan; Long bones; Lysosomal storage disorder; Vertebra