Mucopolysaccharidosis (MPS) VII is an inherited lysosomal storage disorder characterized by deficient activity of the enzyme b-glucuronidase. Skeletal abnormalities are common in patients and result in diminished quality of life. Enzyme replacement therapy (ERT) for MPS VII using recombinant human b-glucuronidase (vestronidase alfa) was recently approved for use in patients; however, to date there have been no studies evaluating therapeutic efficacy in a large animal model of MPS VII. The objective of this study was to establish the effects of intravenous ERT, administered at either the stan-dard clinical dose (4 mg/kg) or a high dose (20 mg/kg), on skel-etal disease progression in MPS VII using the naturally occur-ring canine model. Untreated MPS VII animals exhibited progressive synovial joint and vertebral bone disease and were no longer ambulatory by age 6 months. Standard-dose ERT-treated animals exhibited modest attenuation of joint dis-ease, but by age 6 months were no longer ambulatory. High -dose ERT-treated animals exhibited marked attenuation of joint disease, and all were still ambulatory by age 6 months. Vertebral bone disease was recalcitrant to ERT irrespective of dose. Overall, our findings indicate that ERT administered at higher doses results in significantly improved skeletal disease outcomes in MPS VII dogs.

Mucopolysaccharidosis I is a lysosomal storage disorder characterized by deficient alpha-L-iduronidase activity, leading to abnormal accumulation of glycosaminoglycans in cells and tissues. Synovial joint disease is prevalent and significantly reduces patient quality of life. There is a critical need for improved understanding of joint disease pathophysiology in MPS I, including specific biomarkers to predict and monitor joint disease progression, and response to treatment. The objective of this study was to leverage the naturally-occurring MPS I canine model and undertake an unbiased proteomic screen to identify systemic biomarkers predictive of local joint disease in MPS I. Synovial fluid and serum samples were collected from MPS I and healthy dogs at 12 months-of-age, and protein abundance characterized using liquid chromatography tandem mass spectrometry. Stifle joints were evaluated postmortem using magnetic resonance imaging (MRI) and histology. Proteomics identified 40 proteins for which abundance was significantly correlated between serum and synovial fluid, including markers of inflammatory joint disease and lysosomal dysfunction. Elevated expression of three biomarker candidates, matrix metalloproteinase 19, inter-alpha-trypsin inhibitor heavy-chain 3 and alpha-1-microglobulin, was confirmed in MPS I cartilage, and serum abundance of these molecules was found to correlate with MRI and histological degenerative grades. The candidate biomarkers identified have the potential to improve patient care by facilitating minimally-invasive, specific assessment of joint disease progression and response to therapeutic intervention.

Purpose: Mucopolysaccharidosis (MPS) VII is a genetic, lysosomal storage disease characterized by abnormal accumulation of glycosaminoglycans in cells and tissues. MPS VII patients exhibit multiple failures of endochondral ossification during postnatal growth, including markedly delayed cartilage-to-bone conversion in the vertebrae and long bones. Cartilage canals provide the template for vascularization at the onset of secondary ossification. The objective of this study was to investigate whether abnormal cartilage canal architecture and enzyme-mediated extracellular matrix (ECM) remodeling contribute to delayed cartilage-to-bone conversion in MPS VII. Materials and Methods: The epiphyseal cartilage canal networks of 9-day-old healthy control and MPS VII-affected dog vertebrae were characterized using high-resolution, contrast-free quantitative susceptibility mapping magnetic resonance imaging. Relative expression levels of matrix metalloproteinases (MMPs) 9, 13 and 14 were examined using immunohistochemistry, while tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) were examined using in situ enzyme staining. Results: Interestingly, the density, number, connectivity and thickness of cartilage canals was not significantly different between MPS VII and control vertebrae. Immunohistochemistry revealed diminished MMP-9, but normal MMP-13 and 14 expression by epiphyseal cartilage chondrocytes, while ALP and TRAP enzyme expression by chondrocytes and chondroclasts, respectively, were both diminished in MPS VII. Conclusions: Our findings suggest that while the epiphyseal cartilage canal network in MPS VII is normal at the onset of secondary ossification, expression of enzymes required for cartilage resorption and replacement with mineralized ECM, and initiation of angiogenesis, is impaired.

Intervertebral disc degeneration is implicated as a leading cause of low back pain. Persistent, local inflammation within the disc nucleus pulposus (NP) and annulus fibrosus (AF) is an important mediator of disc degeneration and negatively impacts the performance of therapeutic stem cells. There is a lack of validated large animal models of disc degeneration that recapitulate clinically relevant local inflammation. We recently described a goat model of disc degeneration in which increasing doses of chondroitinase ABC (ChABC) were used to reproducibly induce a spectrum of degenerative changes. The objective of this study was to extend the clinical relevance of this model by establishing whether these degenerative changes are associated with the local expression of inflammatory cytokines and catabolic enzymes. Degeneration was induced in goat lumbar discs using ChABC at different doses. After 12 weeks, degeneration severity was determined histologically and using quantitative magnetic resonance imaging (MRI). Expression levels of inflammatory cytokines (tumor necrosis factor‐α [TNF‐α], interleukin‐1β [IL‐1β], and IL‐6) and catabolic enzymes (matrix metalloproteinases‐1 [MMPs‐1] and 13, and a disintegrin and metalloproteinase with thrombospondin type‐1 motifs‐4 [ADAMTS‐4]) were assessed as the percentage of immunopositive cells in the NP and AF. With the exception of MMP‐1, cytokine, and enzyme expression levels were significantly elevated in ChABC‐treated discs in the NP and AF. Expression levels of TNF‐α, IL1‐β, and ADAMTS‐4 were positively correlated with histological grade, while all cytokines and ADAMTS‐4 were negatively correlated with MRI T2 and T1ρ scores. These results demonstrate that degenerate goat discs exhibit elevated expression of clinically relevant inflammatory mediators, and further validate this animal model as a platform for evaluating new therapeutic approaches for disc degeneration.

Bone elongation is driven by chondrocyte proliferation and hypertrophy in the growth plate. Both processes are modulated by multiple signaling pathways including the Indian Hedgehog (IHH) signaling pathway. Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders characterized by accumulation of glycosaminoglycans (GAGs) in multiple tissues and organs, leading to a range of clinical symptoms including bone shortening through mechanisms that are not fully understood. Using MPS VII mice, we previously observed a reduction in the number of proliferating and hypertrophic chondrocytes and a reduced gene expression of Ihh in the tibial growth plate. We further demonstrate here that IHH secretion by MPS VII chondrocytes was reduced both in vitro and in vivo. While normal chondrocytes showed no response to exogenous IHH, proliferation of MPS VII chondrocytes was stimulated in response to exogenous IHH in vitro. This was accompanied by an elevated gene expression of patched receptor (Ptch1). The results from this study suggested that reduced proliferation in MPS VII growth plate may be partially due to dysfunction of the IHH signaling pathway.

The mucopolysaccharidoses (MPS) are a group of inherited lysosomal storage disorders characterized by abnormal accumulation of glycosaminoglycans (GAGs) in cells and tissues. MPS patients frequently exhibit failures of endochondral ossification during postnatal growth leading to skeletal deformity and short stature. In this review, we outline the current understanding of the cellular and molecular mechanisms underlying failures of endochondral ossification in MPS and discuss associated treatment challenges and opportunities. Studies in MPS patients and animal models have demonstrated that skeletal cells and tissues exhibit significantly elevated GAG storage from early in postnatal life and that this is associated with impaired cartilage-to-bone conversion in primary and secondary ossification centers, and growth plate dysfunction. Recent studies have begun to elucidate the underlying cellular and molecular mechanisms, including impaired chondrocyte proliferation and hypertrophy, diminished growth factor signaling, disrupted cell cycle progression, impaired autophagy, and increased cell stress and apoptosis. Current treatments such as hematopoietic stem cell transplantation and enzyme replacement therapy fail to normalize endochondral ossification in MPS. Emerging treatments including gene therapy and small molecule–based approaches hold significant promise in this regard. Failures of endochondral ossification contribute to skeletal deformity and short stature in MPS patients, increasing mortality and reducing quality of life. Early intervention is crucial for effective treatment, and there is a critical need for new approaches that normalize endochondral ossification by directly targeting affected cells and signaling pathways.

The mucopolysaccharidoses (MPS) are a family of lysosomal storage disorders characterized by deficient activity of enzymes that degrade glycosaminoglycans (GAGs). Abnormal development of the vertebrae and long bones is a hallmark of skeletal disease in several MPS subtypes; however, the underlying cellular mechanisms remain poorly understood. The objective of this study was to conduct an ultrastructural examination of how lysosomal storage differentially affects major skeletal cell types in MPS I and VII using naturally occurring canine disease models. We showed that both bone and cartilage cells from MPS I and VII dog vertebrae exhibit significantly elevated storage from early in postnatal life, with storage generally greater in MPS VII than MPS I. Storage was most striking for vertebral osteocytes, occupying more than forty percent of cell area. Secondary to storage, dilation of the rough endoplasmic reticulum (ER), a marker of ER stress, was observed most markedly in MPS I epiphyseal chondrocytes. Significantly elevated immunostaining of light chain 3B (LC3B) in MPS VII epiphyseal chondrocytes suggested impaired autophagy, while significantly elevated apoptotic cell death in both MPS I and VII chondrocytes was also evident. The results of this study provide insights into how lysosomal storage differentially effects major skeletal cell types in MPS I and VII, and suggests a potential relationship between storage, ER stress, autophagy, and cell death in the pathogenesis of MPS skeletal defects.

Endochondral bone growth is abnormal in 6 of the 11 types of mucopolysaccharidoses (MPS) disorders; resulting in short stature, reduced size of the thoracic cavity and compromised manual dexterity. Current therapies for MPS have had a limited effect on bone growth and to improve these therapies or develop adjunct approaches requires an understanding of the underlying basis of abnormal bone growth in MPS. The MPS VII mouse model replicates the reduction in long bone and vertebral length observed in human MPS. Using this model we have shown that the growth plate is elongated but contains fewer chondrocytes in the proliferative and hypertrophic zones. Endochondral bone growth is in part regulated by entry and exit from the cell cycle by growth plate chondrocytes. More MPS VII chondrocytes were positive for Ki67, a marker for active phases of the cell cycle, suggesting that more MPS VII chondrocytes were in the cell cycle. The number of cells positive for phosphorylated histone H3 was significantly reduced in MPS VII chondrocytes, suggesting fewer MPS VII chondrocytes progressed to mitotic division. While MPS VII HZ chondrocytes continued to express cyclin D1 and more cells were positive for E2F1 and phos pRb than normal, fewer MPS VII HZ chondrocytes were positive for p57kip2 a marker of terminal differentiation, suggesting fewer MPS VII chondrocytes were able to exit the cell cycle. In addition, multiple markers typical of PZ to HZ transition were not downregulated in MPS VII, in particular Sox9, Pthrpr and Wnt5a. These findings are consistent with MPS VII growth plates elongating at a slower rate than normal due to a delay in progression through the cell cycle, in particular the transition between G1 and S phases, leading to both reduced cell division and transition to the hypertrophic phenotype.

To develop a nomogram based on age and disease type for sclerotomy placement in pars plana vitrectomy for infants and children with congenital and acquired vitreoretinopathies.A retrospective, single-center, single-surgeon comparative case series.A total of 171 eyes of 93 patients ranging in age from a postmenstrual age (PMA) of 34 weeks to a chronological age of 23 years with congenital and acquired vitreoretinopathies.The corneal white-to-white diameter and the distance between the ora serrata and surgical limbus (ora-limbus distance) in the nasal, temporal, inferior, and superior quadrants were measured externally in vivo under general anesthesia prior to planned pars plana vitrectomy or laser procedure. The average ora-limbus distance of the quadrants was calculated for each eye (mean ora-limbus distance); and mean ora-limbus distance was evaluated as a function of age and compared among age-matched subjects from control and disease groups. Results were used to create a nomogram for safe sclerotomy placement based on age and disease type.The main outcome measure was distance (millimeters) from the ora serrata to the surgical limbus; a secondary measurement was corneal white-to-white distance (millimeters).Among all subjects, the ora-limbus distance was greatest in the superior quadrant, followed by the temporal, inferior, and nasal quadrants. A positive logarithmic relationship between ora-limbus distance and age was demonstrated in all groups. Whereas age-adjusted mean ora-limbus distances were similar among the familial exudative vitreoretinopathy and control groups, growth trends differed in patients with coloboma, persistent fetal vasculature, Stickler syndrome and/or myopia, and retinopathy of prematurity (ROP). In ROP, when subjects <12 months of age were considered separately, a negative correlation was found between the ora-limbus distance and PMA, with infants of lesser gestational age exhibiting greater restriction in ora-limbus distance.Placement of sclerotomies for pars plana vitrectomy in children requires consideration of ocular development in congenital and acquired vitreoretinopathies; we present an age-based nomogram for sclerotomy placement in infants and children based on analyses of ora-limbus distance in several pediatric ocular conditions.

Short stature is a characteristic feature of most of the mucopolysaccharidoses, a group of inherited lysosomal storage disorders caused by a single enzyme deficiency. MPS patients present with progressive skeletal defects from an early age, including short stature due to impaired cartilage-to-bone conversion (endochondral ossification). The aim of this study was to determine which murine MPS model best reproduces the bone length reduction phenotype of human MPS and use this model to determine the earliest developmental stage when disrupted endochondral ossification first appears. Gus(mps/mps) mice representing severe MPS VII displayed the greatest reduction in bone elongation and were chosen for histopathological analysis. Tibial development was assessed from E12.5 to 6 months of age. Chondrocytes in the region of the future primary ossification center became hypertrophic at a similar age to normal in the MPS VII mouse fetus, but a delay in bone deposition was observed with an approximate 1 day delay in the formation of the primary ossification centre. Likewise, chondrocytes in the region of the future secondary ossification center also became hypertrophic at the same age as normal in the MPS VII early postnatal mouse. Bone deposition in the secondary ossification centre was delayed by two days in the MPS VII proximal tibia (observed at postnatal day 14 (P14) compared to P12 in normal). The thickness of the tibial growth plate was larger in MPS VII mice from P9 onwards. Abnormal endochondral ossification starts in utero in MPS VII and worsens with age. It is characterized by a normal timeframe for chondrocyte hypertrophy but a delay in the subsequent deposition of bone in both the primary and secondary ossification centres, accompanied by an increase in growth plate thickness. This suggests that the signals for vascular invasion and bone deposition, some of which are derived from hypertrophic chondrocytes, are altered in MPS VII.

Successful fracture repair requires restoration of bone morphology and mechanical integrity. Recent evidence shows that fractured bones of neonatal mice undergo spontaneous realignment, dubbed “natural reduction.” Here, we show that natural reduction is regulated by the proprioceptive system and improves with age. Comparison among mice of different ages revealed, surprisingly, that 3-month-old mice exhibited more rapid and effective natural reduction than newborns. Fractured bones of null mutants for transcription factor Runx3, lacking functional proprioceptors, failed to realign properly. Blocking Runx3 expression in the peripheral nervous system, but not in limb mesenchyme, recapitulated the null phenotype, as did inactivation of muscles flanking the fracture site. Egr3 knockout mice, which lack muscle spindles but not Golgi tendon organs, displayed a less severe phenotype, suggesting that both receptor types, as well as muscle contraction, are required for this regulatory mechanism. These findings uncover a physiological role for proprioception in non-autonomous regulation of skeletal integrity.