• Home
  • Search Results
  • Delayed hypertrophic differentiation of epiphyseal chondrocytes contributes to failed secondary ossification in mucopolysaccharidosis VII dogs.

Delayed hypertrophic differentiation of epiphyseal chondrocytes contributes to failed secondary ossification in mucopolysaccharidosis VII dogs.

Molecular genetics and metabolism (2015-10-01)
Sun H Peck, Philip J M O'Donnell, Jennifer L Kang, Neil R Malhotra, George R Dodge, Maurizio Pacifici, Eileen M Shore, Mark E Haskins, Lachlan J Smith
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
2-Propanol, BioReagent, for molecular biology, ≥99.5%
Sigma-Aldrich
HEPES, ≥99.5% (titration)
Sigma-Aldrich
2-Propanol, ACS reagent, ≥99.5%
Sigma-Aldrich
HEPES, BioPerformance Certified, ≥99.5% (titration), suitable for cell culture
Sigma-Aldrich
2-Propanol, anhydrous, 99.5%
Sigma-Aldrich
Magnesium chloride, anhydrous, ≥98%
Sigma-Aldrich
Magnesium chloride solution, for molecular biology, 1.00 M±0.01 M
Sigma-Aldrich
Ethylenediaminetetraacetic acid, ACS reagent, 99.4-100.6%, powder
Sigma-Aldrich
Isopropyl alcohol, ≥99.7%, FCC, FG
Sigma-Aldrich
Ethylenediaminetetraacetic acid, anhydrous, BioUltra, ≥99% (titration)
Sigma-Aldrich
HEPES buffer solution, 1 M in H2O
Sigma-Aldrich
Isopropyl alcohol, meets USP testing specifications
Sigma-Aldrich
2-Propanol, puriss. p.a., ACS reagent, reag. ISO, reag. Ph. Eur., ≥99.8% (GC)
Sigma-Aldrich
2-Propanol, BioUltra, for molecular biology, ≥99.5% (GC)
Sigma-Aldrich
Ethylenediaminetetraacetic acid, anhydrous, crystalline, BioReagent, suitable for cell culture
Sigma-Aldrich
Ethylenediaminetetraacetic acid solution, 0.02% in DPBS (0.5 mM), sterile-filtered, BioReagent, suitable for cell culture
SAFC
HEPES
Sigma-Aldrich
Ethylenediaminetetraacetic acid, purified grade, ≥98.5%, powder
Sigma-Aldrich
Magnesium chloride, powder, <200 μm
Sigma-Aldrich
Magnesium chloride solution, BioUltra, for molecular biology, ~1 M in H2O
Sigma-Aldrich
HEPES, BioUltra, for molecular biology, ≥99.5% (T)
Sigma-Aldrich
2-Propanol, Laboratory Reagent, ≥99.5%
Sigma-Aldrich
2-Propanol, puriss. p.a., ACS reagent, ≥99.8% (GC)
Sigma-Aldrich
2-Propanol, electronic grade, 99.999% trace metals basis
Sigma-Aldrich
Ethylenediaminetetraacetic acid, 99.995% trace metals basis
Sigma-Aldrich
Magnesium chloride solution, BioUltra, for molecular biology, 2 M in H2O
Sigma-Aldrich
2-Propanol, puriss., meets analytical specification of Ph. Eur., BP, USP, ≥99.5% (GC)
Sigma-Aldrich
Magnesium chloride, BioReagent, suitable for insect cell culture, ≥97.0%
Sigma-Aldrich
Magnesium chloride solution, PCR Reagent, 25 mM MgCI2 solution for PCR
SAFC
HEPES