Progression of Vertebral Bone Disease in Mucopolysaccharidosis VII Dogs from Birth to Skeletal Maturity

Abstract

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient β-glucuronidase activity, leading to accumulation of incompletely degraded heparan, dermatan and chondroitin sulfate glycosaminoglycans. Patients with MPS VII exhibit progressive spinal deformity, which decreases quality of life. Previously, we demonstrated that MPS VII dogs exhibit impaired initiation of secondary ossification in the vertebrae and long bones. The objective of this study was to build on these findings and comprehensively characterize how vertebral bone disease manifests progressively in MPS VII dogs throughout postnatal growth. Vertebrae were collected postmortem from MPS VII and healthy control dogs at seven ages ranging from 9 to 365 days. Microcomputed tomography and histology were used to characterize bone properties in primary and secondary ossification centers. Serum was analyzed for bone turnover biomarkers. Results demonstrated that not only was secondary ossification delayed in MPS VII vertebrae, but that it progressed aberrantly and was markedly diminished even at 365 days-of-age. Within primary ossification centers, bone volume fraction and bone mineral density were significantly lower in MPS VII at 180 and 365 days-of-age. MPS VII growth plates exhibited significantly lower proliferative and hypertrophic zone cellularity at 90 days-of-age, while serum bone-specific alkaline phosphatase (BAP) was significantly lower in MPS VII dogs at 180 days-of-age. Overall, these findings establish that vertebral bone formation is significantly diminished in MPS VII dogs in both primary and secondary ossification centers during postnatal growth.

1. Introduction

The mucopolysaccharidoses (MPS) are a family of inherited, lysosomal storage disorders characterized by deficient activity of enzymes that degrade glycosaminoglycans (GAGs) due to mutations in associated genes [1]. MPS VII, also called Sly Syndrome, is characterized by deficient beta-glucuronidase (GUSB) activity, leading to incomplete degradation of heparan, dermatan and chondroitin sulfate GAGs [2]. These GAGs accumulate progressively in cells and tissues resulting in multi-organ system disease manifestations. MPS VII patients present with a spectrum of disease severities [3]. Progressive skeletal abnormalities, commonly present in patients with more severe disease and termed “dysostosis multiplex”, include short stature, spinal deformity and joint dysplasia, which result in pain, impaired mobility, and an overall diminished quality of life [2-4].

Abnormal bone growth contributes to the progression of skeletal disease in MPS VII due to failures of endochondral ossification during postnatal growth [5]. Endochondral ossification is the biological process underlying the development and growth of vertebrae and long bones, beginning with formation of a cartilaginous template that is then replaced by mineralized bone [6, 7]. This processes occurs first in primary ossification centers (POCs) and subsequently in secondary ossification centers (SOCs), and also in growth plates, facilitating longitudinal bone growth [7, 8]. Using the naturally-occurring canine model, we previously demonstrated that bone disease in MPS VII first manifests at the tissue level as delayed conversion of epiphyseal cartilage to mineralized bone in the SOCs of both vertebrae and long bones [9]. During later stages of postnatal growth, persistent cartilaginous lesions in vertebral SOCs resulted in reduced stiffness and increased range-of-motion of the intervertebral joints, implicating them as a contributing factor in the progression of spinal deformity [10, 11]. These lesions were found to persist for the lifetime of the animals [12]. Impaired hypertrophic differentiation of epiphyseal and growth plate chondrocytes has been shown to contribute to failed cartilage-to-bone conversion in SOCs, and reduced longitudinal bone growth, respectively [9, 13, 14]. Recent work in MPS VII dogs showed that these cells exhibit significantly elevated lysosomal GAG storage from an early age, together with elevated apoptosis and impaired autophagy [15]. Additionally, studies have demonstrated impaired activation of key osteogenic signaling pathways required to initiate and sustain chondrocyte differentiation, and reduced expression of enzymes required for extracellular matrix remodeling and mineralization [16, 17]. In the POCs of MPS VII dog vertebrae, despite bone cells (osteoblasts and osteocytes) exhibiting significantly elevated storage [15], bone content is normal during early postnatal development [9], and to date, no studies have examined how bone disease progresses in the POCs of these animals at later stages of growth. Studies in dogs with MPS I (α-L-iduronidase deficiency), however, have shown that POCs exhibit significantly lower bone volume fraction and mineral density from 6 months-of-age onwards [18]. A more comprehensive understanding of the temporal progression of abnormal ossification in both POCs and SOCs in MPS VII dogs throughout postnatal growth may provide new clinically-relevant insights into disease etiology and inform optimal timing for therapies specifically targeting skeletal disease manifestations in both canine and human patients.

With the advent of new, clinically-approved treatments for MPS VII such as enzyme replacement therapy (ERT), there is a pressing need for non-invasive biomarkers to monitor bone disease progression and response to treatment. There is evidence that serum levels of markers of bone turnover may be abnormal in MPS patients [19]. These biomarkers, when coupled with diagnostic imaging, can be used to monitor progression of other skeletal diseases characterized by altered bone turnover, including osteoporosis, osteoarthritis and rheumatoid arthritis [20-22]. To date, there have been no controlled, preclinical studies examining whether serum biomarkers can be used as surrogates for abnormal bone turnover in MPS VII.

The objectives of this study were to firstly characterize progression of vertebral bone disease in both POCs and SOCs from birth to skeletal maturity in MPS VII dogs, and secondly, to establish the efficacy of two serum biomarkers as non-invasive indicators of abnormal bone formation in this preclinical large animal model.

2. Materials and Methods

2.1. Animals and Tissue Collection

For this study, we used the naturally-occurring canine model of MPS VII. MPS VII dogs have a missense mutation (R166H) in the GUSB gene [23] and exhibit a similar skeletal phenotype to human patients [10, 23, 24]. Animals were raised at the University of Pennsylvania School of Veterinary Medicine under NIH and USDA guidelines for the care and use of animals in research, and all studies were carried out with IACUC approval. Control (heterozygous) and MPS VII (homozygous) dogs were identified via variant-specific real time PCR analysis at birth. All animals were littermates, or had at least one parent in common. Animals received monthly physical examinations from a veterinarian, which included assessments of body weight and condition score, and ability to ambulate. Body condition scores (BSCs) are subjective assessments based on fat covering the ribs by palpation, the presence of a waist when viewed from above, and an abdominal tuck when viewed from the side, and range from 1/9 (emaciated) to 9/9 (morbidly obese), with 4-5/9 being ideal [25]. Animals (n = 2-5 per time point; 35 animals total; 19 female and 16 male) were euthanized at 9, 14, 30, 42, 90, 180 and 365 days-of-age, using 80 mg/kg of sodium pentobarbital in accordance with the American Veterinary Medical Association guidelines. By 365 days these dogs are considered skeletally mature and growth plates have closed. Thoracic spines were dissected out immediately following euthanasia, and T13 vertebral bodies were isolated by cutting through the intervening intervertebral discs and fixed in neutral buffered 10% formalin for 1 week.

2.2. Microcomputed Tomography

Whole, fixed T13 vertebrae were scanned using high-resolution microcomputed tomography (MicroCT; VivaCT40; Scanco Medical AG, Brüttisellen, Switzerland) using the following imaging parameters: an isotropic voxel size of 19 μm, an integration time of 380 ms, peak tube voltage of 70 kV, current of 0.114 mA, and an acquisition of 1000 projections per 180°. A three-dimensional Gaussian filter of 1.2 with a limited, finite filter support of 2 was used for noise suppression, and mineralized tissue was segmented from air or soft tissue using a threshold of 158. Primary and secondary ossification centers were individually segmented and colorized, and three-dimensional (3D) reconstructions generated to qualitatively assess progression of ossification in each region. For vertebrae from 90 (5 MPS VII and 5 controls), 180 (5 MPS and 5 controls) and 365 (4 MPS VII and 3 controls) -day-old animals, the entire region of trabecular bone in the POCs, excluding the primary spongiosa adjacent to open growth plates, was segmented out and standard 3D morphometric analyses were performed using Scanco software. The following parameters were determined: bone volume fraction (bone volume/total volume, BV/TV), apparent bone mineral density (BMD) calibrated against hydroxyapatite (HA) standards (0-784 mg HA/cm3), trabecular thickness (Tb.Th), trabecular spacing (Tb.Sp), trabecular number (Tb.N), connectivity density (Conn.Dens) and structural model index (SMI).

2.3. Histology

Following microCT, vertebrae from 90, 180 and 365-day-old animals (sample groups as above) were decalcified in formic/ethylenediaminetetraacetic acids (Formical 2000; Statlab; Louisville, USA) and processed into paraffin. Mid-sagittal, 7μm-thick sections were cut and double stained with either Alcian blue and picrosirius red (ABPR) to demonstrate GAGs and collagen, respectively, or hematoxylin and eosin (H&E) to demonstrate cellularity [9]. Sections were imaged under bright field microscopy (Eclipse 90i; Nikon; Tokyo, Japan). ABPR staining was used to quantitatively assess relative cartilage and bone content in primary and secondary centers of ossification. H&E staining was used to assess growth plate morphology for vertebrae from 90- and 180-day old animals as described previously [9]. Vertebrae from 365-day-old animals were not assessed, as growth plates in control animals were closed by this age. Total numbers of proliferating and hypertrophic chondrocytes were counted manually in a standardized 1 mm-wide region in the center of the growth plate and normalized to total area (NIS-Elements software; Nikon; Tokyo, Japan). The mean heights of the proliferating and hypertrophic zones were determined from the same standardized regions.

2.4. Serum Biomarkers

Blood samples were collected from animal at 90, 180 and 365 days-of-age (samples groups as above) using a catheter placed in the cephalic vein immediately prior to euthanasia. Collected blood was placed into serum-separating tubes and let sit for 30 minutes at room temperature until blood clots formed, then placed into a centrifuge and spun at 5000 rpm for 5 minutes. Serum was collected and aliquots were snap frozen in liquid nitrogen, and stored at −80°C. For assessment of bone turnover markers, serum aliquots were thawed on ice, and a 20 μL undiluted volume was assayed for either bone-specific alkaline phosphatase (BAP, a marker of bone formation, expressed as U/L serum) or pyridinoline/deoxypyridinoline (PYD, a marker of bone resorption, expressed as nmol/L serum) using commercial enzyme immunoassay kits (MicroVue; Quidel; San Diego USA). For assessment of GAG levels, 25-50 μL starting volume of serum was assayed using the Blyscan assay kit (Bicolor Ltd; Carrickfergus, United Kingdom), with chondroitin sulfate as the standard and results expressed as μg/mL.

2.5. Statistical Analyses

Statistical analyses for quantitative outcome measures (bone morphometry, growth plate morphology, and serum biomarker expression) were performed using GraphPad Prism version 9.0 (GraphPad Software; San Diego, USA). Significant differences between MPS VII and controls at each age were established using two-way analyses of variance followed by post-hoc Sidak tests. Significance was defined as p≤0.05, and all data are presented as mean ± standard deviation.

3. Results

3.1. Clinical Findings

The mobility of MPS VII animals progressively declined beginning between 56 and 84 days-of-age, and these animals were unable to rise or walk by 180 days-of-age. Body weights in MPS VII and control animals were compared to each other within litters, because of the heterogeneity between litters. While birth weights within litters were similar at birth, MPS VII animals weighed around 25% less by 28 days-of-age, with the decreased trend in weight gain continuing until study termination. Weight gain in MPS VII animals that remained in the study for 180 days plateaued between 140 and 180 days, with 30% of these animals losing weight beginning at 140 days-of-age. Control animals continued to gain weight during this time. While all control animals maintained a BCS between 4.5 and 5/9, MPS VII animals had a BCS of 4/9 at 30 days-of-age and continued to decline to 2.5-3/9 by the end of the study. Loss of muscle was evident in all MPS VII animals by 30 days-of-age and was profound 90 days-of-age.

3.2. MicroCT Analysis of Vertebral Bone Growth

Representative 3D reconstructions of control and MPS VII T13 vertebrae at each age are shown in Figure 1. As expected, based on our previous findings [9], secondary ossification (purple) commenced between 9 and 14 days-of-age in control animals, but was markedly delayed in MPS VII vertebrae and not evident until between 14 and 30 days-of-age. Furthermore, secondary ossification in MPS VII vertebrae exhibited an aberrant distribution pattern and remained incomplete even at 365 days-of-age. Vertebral body lengths were markedly shorter in MPS VII animals compared to controls from 90 days-of-age onwards. In POCs (gray), lower trabecular bone content was evident from 90 days-of-age onwards. Qualitative observations were supported by quantitative morphometric analyses (Figure 2). Trabecular bone volume fraction (BV/TV) was significantly lower in MPS VII at 180 and 365 days-of-age (61% and 59% of control, respectively, both p<0.001). Apparent BMD was also significantly lower in MPS VII at 180 and 365 days-of-age (67% (p=0.001) and 64% (p<0.001) of control, respectively). Trabecular thickness (Tb.Th) was significant lower in MPS VII at 180 days-of-age (78% of control, p=0.008), and trabecular number (Tb.N) was significantly lower in MPS VII at 365 days-of-age (67% of control, p=0.002). Determination of SMI resulted in negative values for 8 samples, likely indicating the presence of concave surfaces [26]. Therefore, SMI results are not reported.

Figure 1.

Representative microcomputed tomography reconstructions of T13 vertebrae from control and MPS VII dogs at 9, 14, 30, 42, 90, 180 and 365 days-of-age, illustrating delayed, aberrant and incomplete secondary ossification (purple), reduced bone content in primary ossification centers (gray), and diminished longitudinal growth in MPS VII vertebrae compared to controls. A. Axial (end) view. Scale = 5 mm B. Mid-sagittal view. Scale = 5 mm. Orientation: d = dorsal and v = ventral.

Figure 2.

Microcomputed tomography analysis of trabecular bone content and architecture in the primary ossification centers of control and MPS VII T13 vertebrae at 90, 180 and 365 days-of-age. A. Bone volume fraction (BV/TV). B. Bone mineral density (BMD). C. Trabecular thickness (Tb.Th). D. Trabecular spacing (Tb.Sp). E. Connectivity density (Conn.Dens). F. Trabecular number (Tb.N). *p≤0.05 vs control; n = 3-5.

3.3. Histological Assessment of Primary and Secondary Ossification Centers, and Growth Plates

Representative, mid-sagittal sections from control and MPS VII vertebrae from 90, 180 and 365-day-old dogs are shown in Figure 3. Vertebrae from MPS VII dogs were markedly shorter at all three of these ages (Figure 3A). Examining SOCs (Figure 3B), at 90 days-of-age, some epiphyseal cartilage was still evident in control vertebrae at the vertebral margins, adjacent to the growth plates. By 180 days-of age, SOCs in controls were completely ossified, though growth plates were still open. By 365 days-of-age, growth plates in controls had closed. For MPS VII vertebrae, at 90 and 180 days-of-age cartilage occupied most of the SOC area. At 365 days-of-age there was some evidence of secondary ossification in MPS VII, but significant epiphyseal cartilage persisted, and growth plates remained open. Examining POCs, consistent with microCT findings, lower trabecular bone content was evident in MPS VII vertebrae at 90, 180 and 365 days-of-age compared to controls (Figure 3C).

Figure 3.

A. Representative histology of T13 vertebrae control and MPS VII dogs at 90, 180 and 365 days-of-age. Scale = 5 mm; mid-sagittal sections; poc = primary ossification center; soc = secondary ossification center; Alcian blue and picrosirius red staining. B. Higher magnification view of secondary ossification centers (corresponding to region depicted by upper right inset in panel A) showing abnormal persistence of cartilage in MPS VII vertebrae. Scale = 1 mm. C. Higher magnification view of primary ossification centers (corresponding to region depicted by central inset in panel A) showing reduced trabecular bone content in MPS VII vertebrae. Scale = 1 mm.

With respect to growth plates, at 90 days-of-age the morphology of the proliferative and hypertrophic regions in MPS VII vertebrae appeared similar to controls; however, at 180 days-of-age, columns of proliferating chondrocytes in MPS VII vertebrae exhibited an abnormal, undulating morphology (Figure 4A, inset). Quantitative assessments revealed significantly lower numbers of proliferating and hypertrophic chondrocytes at 90 days-of-age in MPS VII vertebrae (71% (p=0.02) and 75% (p=0.05) of control, Figures 4B and C, respectively), while proliferating and hypertrophic zone thicknesses were not significant different at either age (Figures 4D and E).

Figure 4.

A. Representative histology of growth plates from T13 vertebrae control and MPS VII dogs at 90 and 180 days-of-age. Scale =100 μm; mid-sagittal sections; hematoxylin and eosin staining. Inset shows abnormal growth plate morphology in 6-month-old MPS VII vertebrae. Quantification of B. Proliferative zone (pz) cellularity; C. Hypertrophic zone (hz) cellularity; D. Proliferative zone thickness; and E. Hypertrophic zone thickness. *p≤0.05 vs control; n = 3-5.

3.4. Serum Biomarkers

Concentrations of BAP, PYD and GAG were measured in serum collected from control and MPS VII dogs at 90, 180 and 365 days-of-age (Figure 5). BAP was significantly lower for MPS VII animals at 180 days-of-age (44% of control, p=0.02, Figure 5A), while PYD was not significantly different at any age (Figure 5B). GAG levels were significantly elevated for MPS VII animals at 90 and 180 days-of-age (212% (p=0.002) and 202% (p=0.013) of control, respectively, Figure 5C).

Figure 5.

Serum biomarkers of bone turnover. A. Bone-specific alkaline phosphatase (BAP). B. Pyridinoline/deoxypyridinoline (PYD). C. Glycosaminoglycan (GAG) content. *p≤0.05 vs control; n = 3-5.

4. Discussion

Bone disease is prevalent in MPS VII patients due to multiple failures of endochondral ossification during postnatal growth. Patients exhibit progressive kyphotic and scoliotic spinal deformity, due in part to abnormal development of the vertebrae. Until recently, treatment options for MPS VII were extremely limited. Enzyme replacement therapy (ERT) recently became FDA approved for clinical use [27, 28]; however, as of yet there is little clinical data on its efficacy specifically for bone disease. Studies in mice suggested that ERT for MPS VII may at best be partially effective in this respect [29, 30], and this is supported by the results of a long-term gene therapy study in MPS VII dogs, where despite very high levels of circulating enzyme, cartilaginous vertebral bone lesions persisted through the lifetime of the animals [12]. The reason for the apparent limited efficacy of ERT for MPS VII cartilage and bone disease is unclear, but may be in part due to the large enzyme size, which likely limits diffusion into these dense and poorly-vascularized tissues, where resident cells exhibit elevated GAG storage from an early age [9, 15]. Therefore, there is an urgent clinical need for therapies that specifically and effectively target bone manifestations in MPS VII patients.

In this study, we leveraged the naturally-occurring MPS VII canine model to comprehensively characterize progression of vertebral bone disease in the primary and secondary ossification centers, and growth plates, from birth to skeletal maturity. In a previous study, we examined vertebral bone morphology in MPS VII dogs at 9 and 14 days-of-age, which is the window across which secondary ossification commences in healthy animals, but fails to initiate in MPS VII animals. At these early ages, POC bone volume fraction and BMD, and growth plate morphology, were found to be normal in MPS VII. Here we build on those findings and examine additional postnatal ages through to skeletal maturity (365 days-of-age). Qualitative examination of 3D microCT images revealed that not only is secondary ossification markedly delayed in MPS VII, but that it also progresses aberrantly, and remains diminished relative to controls at 365 days-of-age. Diminished secondary ossification is attributable to failed conversion of cartilage to bone, which is in turn a consequence of the impaired ability of resident epiphyseal chondrocytes to undergo hypertrophic differentiation [9]. This is unsurprising given that these cells exhibit significantly elevated lysosomal GAG storage, as well as elevated levels of apoptosis and impaired autophagy, from an early age [15]. Extracellular GAG levels are also elevated in MPS VII epiphyseal cartilage from an early age [9], and it has been suggested that this extracellular GAG may bind and sequester secreted signaling molecules required to drive cell differentiation during endochondral ossification [5]. Indeed, molecular profiling has shown that there are lower endogenous expression levels of key osteogenic factors, including elements of the BMP and Wnt/beta-catenin pathways, at the onset of secondary ossification [17]. The temporally and spatially aberrant nature of cartilage-to-bone conversion illustrated in the current study may reflect the aberrant distribution and activity of secreted factors in these pathways. The fact that secondary ossification does ultimately commence, albeit in a dysregulated manner, suggests that at least some of these cells maintain differentiation potential, and that ossification can potentially be rescued through a combination of substrate reduction and exogenous administration of osteogenic factors.

In POCs, bone volume fraction and BMD were found to be significantly lower in MPS VII vertebrae at 180 and 365 days-of-age. These findings are similar to those described previously for MPS I dogs [18]. To the best of our knowledge, there have not been any clinical reports of bone quality in MPS VII patients; however, there have been several studies examining BMD in patients with other MPS subtypes using either dual energy x-ray absorptiometry or quantitative computed tomography [31-36]. While conducting clinical studies poses challenges due to the diverse patient cohort with respect to age, sex and treatment status, in general, MPS patients are reported to have lower BMD compared to age and sex-matched controls [32, 33, 35, 36].

Another common characteristic of skeletal disease in MPS VII is diminished height due to impaired longitudinal bone growth [3, 37, 38]. Here, microCT reconstructions revealed impaired longitudinal growth of canine MPS VII vertebrae that was evident from 90 days-of-age. To establish the underlying cellular basis, we examined vertebral growth plates and found significantly lower numbers of both proliferating and hypertrophic chondrocytes at this same age. Previously, we showed that MPS VII dogs exhibit significantly elevated GAG storage in proliferating and hypertrophic growth plate chondrocytes from early in postnatal growth, and studies in mice have shown that the transition from proliferation to hypertrophic differentiation is delayed in MPS VII growth plates, potentially due to reduced Runt-related transcription factor 2 (RUNX2) expression, persistent SOX9 expression, altered parathyroid hormone-related protein (PTHrP) and WNT5A signaling, and disrupted cell cycle progression [14].

Systemic molecular biomarkers of bone turnover represent a potentially non-invasive means of assessing bone disease progression and response to therapy in MPS patients. Few prior studies have examined serum biomarkers of bone turnover in MPS VII, in either patients or animal models. Yamashita et al found elevated osteocalcin and diminished tartrate-specific acid phosphatase serum levels in MPS VII mice [39]. With respect to other MPS subtypes, Stevenson et al assessed bone turnover biomarkers in MPS I, II and VI patients [19]. This study found significantly higher serum levels of osteocalcin, and a trend towards higher levels of BAP, in MPS patients compared to age, sex and tanner stage-matched controls. In the current study, we found lower levels of BAP throughout postnatal growth (significant at 180 days-of-age) in MPS VII dogs, suggesting reduced osteoblast activity and diminished bone formation, which was supported by microCT findings showing lower bone content in both primary and secondary ossification centers. This disparity with the above clinical and murine findings may reflect subtype, age, or species-specific differences in bone pathophysiology. Additionally, the majority of human patients in this clinical study had or were receiving treatment with ERT or hematopoietic stem cell transplantation (HSCT), which may have impacted bone turnover, although findings appeared to be independent of HSCT status [19]. Importantly, BAP values for control dogs were similar to those reported previously, and showed a similar decline with advancing age [40]. A previous study reported that chondroitin sulfate, one of the GAG types that accumulates in MPS VII, exhibits an inhibitory effect on alkaline phosphatase, although it is unclear if this extends to the bone-specific isoform [41].

Our study had several limitations. Only a limited number of animals were available at 30 and 42 days-of-age, precluding quantitative assessments at these ages. Additionally, the study was not sufficiently powered to robustly examine sex dependent differences within each study group. We only examined two serum biomarkers of bone turnover (BAP and PYD), in part due to limited availability of commercial kits that were validated to cross-react with canine serum. We hope to validate and investigate additional biomarkers in the future. While we tried to collect serum from animals early in the day after 8 hours of fasting where possible, this was not strictly controlled, potentially adding variability to biomarker results. Finally, MPS VII animals exhibited impaired mobility and muscle atrophy due to disuse, which may have resulted in altered bone remodeling.

In conclusion, this study provides new information regarding the nature and progression of vertebral bone disease in the clinically-relevant canine model of MPS VII. Our results highlight the need for improved therapeutic approaches that specifically target bone and cartilage cells in both primary and secondary ossification centers, in order to normalize bone formation and prevent the onset of debilitating skeletal deformity in MPS VII patients.