Evaluation of Tendon and Ligament Microstructure and Mechanical Properties in a Canine Model of Mucopolysaccharidosis I

Abstract

Mucopolysaccharidosis (MPS) I is a lysosomal storage disorder characterized by deficient alpha-L-iduronidase activity, leading to abnormal accumulation of glycosaminoglycans (GAGs) in cells and tissues. Synovial joint disease is prevalent and significantly reduces patient quality of life. There is a strong clinical need for improved treatment approaches that specifically target joint tissues; however, their development is hampered by poor understanding of underlying disease pathophysiology, including how pathological changes to component tissues contribute to overall joint dysfunction. Ligaments and tendons, in particular, have received very little attention, despite the critical roles of these tissues in joint stability and biomechanical function. The goal of this study was to leverage thenaturally canine model to undertake functional and structural assessments of the anterior (cranial) cruciate ligament (CCL) and Achilles tendon in MPS I. Tissues were obtained postmortem from 12-month-old MPS I and control dogs and tested to failure in uniaxial tension. Both CCLs and Achilles tendons from MPS I animals exhibited significantly lower stiffness and failure properties compared to those from healthy controls. Histological examination revealed multiple pathological abnormalities, including collagen fiber disorganization, increased cellularity and vascularity, and elevated GAG content in both tissues. Clinically, animals exhibited mobility deficits, including abnormal gait, which was associated with hyperextensibility of the stifle and hock joints. These findings demonstratethat pathological changes to both ligaments and tendons contribute to abnormal joint function in MPS I and suggest that effective clinical management of joint disease in patients should incorporate treatments targeting these tissues.

Introduction

The mucopolysaccharidoses (MPS) are a family of eleven inherited lysosomal storage disorders characterized by deficient activity of enzymes that degrade glycosaminoglycans (GAGs) due to mutations in associated genes.¹The incidence of MPS across all subtypes is estimated to be approximately 1 in 100,000 births, with MPS I, II and III having the highest incidence overall.²MPS I, also known as Hurler Syndrome, or Hurler-Scheie or Scheie in its attenuated forms, is characterized by deficient alpha-L-iduronidase (IDUA) activity, resulting in the accumulation of heparan and dermatan sulfate GAGs in cells and tissues.³ MPS I patients exhibit disease manifestations in multiple organ systems, including the musculoskeletal, central nervous and cardiopulmonary systems.³ Amongst the musculoskeletal manifestations, collectively termed dysostosis multiplex, synovial joint disease is prevalent.^4–7All joints can be affected, including large joints such as hips, shoulders, knees, wrists, elbows and ankles, as well as the small joints of the hands and feet.⁸Clinically, joint disease manifests as pain, stiffness and diminished range of motion, which negatively impact patient mobility and independence.^{6; 7; 9}While not a direct source of patient mortality, joint manifestations therefore come at an immense personal cost to the quality of life of both patients and their carers.While systemic treatments such as enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT) have been shown to increase patient longevity, the response of joint disease, particularly in the lower extremities, is mixed, and surgical interventionis often indicated.^10–13Furthermore, these therapies typically only prevent disease progression and do not reverse existing manifestations.^{14; 15}There is therefore a strong clinical need for improved treatment approaches that specifically target joint tissues; however, their development is hampered by poor understanding of underlying disease pathophysiology, including how pathological changes to component tissues contribute to overall joint dysfunction.

Ligaments and tendons, in particular, have received very little attention, despite the critical roles of these tissues in joint stability and biomechanical function.In the knee joints, the anterior cruciate ligament (ACL) is located within the intra-articular space, and functions to stabilize the joint by limiting rotation and forward movement of the tibia relative to the femur.¹⁶ Additionally, the ACL contributes to load transmission during weight-bearing activities,¹⁷ and performs important roles in proprioception and balance.¹⁸The ACL is the most frequently damaged ligament in the general population, and is associated clinically with pain, swelling and impaired mobility.¹⁹In the ankle joints, the Achilles (or common calcaneal) tendon connects the calf (soleus and gastrocnemius) muscles to the heel (calcaneal) bone, and is the strongest tendon in the human body.^{20; 21} The Achilles tendon functions to transmit forces from the calf muscles to the foot, facilitating plantar flexion, and thus performs a critical role in mobility, including walking, running and jumping.²⁰ Damage and inflammation to the Achilles tendon in the general population is also relatively common, and like ACL damage, can result in pain and mobility deficits.²¹Whilst structurally and functionally distinct, both the ACL and the Achilles tendon are composed of a fibrous extracellular matrix containing collagen type I, elastin, small leucine-rich repeat proteoglycans and other minor components,are hypocellular, and have limitedendogenous repair capacity.^22–25Few studies have examined pathological changes to the ACL and Achilles tendon in MPS I, and the contributions of these tissues to progressive joint disease and decline in mobility are poorly understood.

Our laboratory studies skeletal disease in MPS I using a naturally occurring canine model.First identified in plot hounds and subsequently outbred into a research colony, MPS I dogs have a homozygous donor splice site mutation in intron 1 of the IDUA gene²⁶and are considered to align most closely with the intermediate severity Hurler-Scheie phenotype found in human patients, based primarily on observed pathological manifestations in the central nervous system, skeleton and corneas.^{27; 28} Similar to humans, MPS I dogs exhibit progressive joint disease, which manifests clinicallyas reduced mobility with increasing age. More broadly, dogs are a well-accepted model for studying tendon and ligament dysfunction, as, similar to humans, theyfrequently exhibit injury and age-related damage to these tissues and associated disability.^{29; 30}The goal of this study was to leveragethe MPS I canine model toadvance understanding of synovial joint pathophysiology in MPS I by undertaking a comprehensivebiomechanical and histologicalcharacterization of the ACL and Achilles tendon.

2. Materials and Methods

2.1. Animals and Study Design

Animal work was approved by the University of Pennsylvania Institutional Animal Care and Use Committee. For this study, we used thenaturally-occurring canine model of MPS I,^26–28with animals bred from our existing MPS I colony.Animals included MPS I-affected dogs (N=6; 3 males and 3 females) and healthy controls (N=5; 2 males and 3 females).All animals were littermates or had at least one parent in common.As not all tissues were available for all animals for all study outcomes, sample sizes for specific experiments are stated in the following sections. MPS I-affected animals (homozygous for the IDUA mutation) were diagnosed at birth by DNA mutation analysis. Healthy controls were heterozygous for the IDUA mutation and phenotypically normal. Animals were raised and housed at the University of Pennsylvania School of Veterinary Medicine under NIH and USDA guidelines for the care and use of animals in research. Animals were housed in kennel runs in groups of 2 or 3, with a light cycle of 12 hours per day and an ambient temperature of 21°C, with food and water provided ad libitum.

Physical examinations, including general clinical evaluations and specific assessments of mobility, gait, joint swelling and muscle tone were performed monthly. At 12 months-of-age, all animals were euthanized via an overdose of sodium pentobarbital consistent with the recommendations of the American Veterinary Medical Association. Following euthanasia, the left and right stifle (knee) and hock (ankle) joints were excised and frozen at −20°C until required for experiments. From the stifle joint, we evaluated the cranial cruciate ligament (CCL), which is the equivalent of the ACL in humans and performs a similar function. From the hock joint we evaluate the Achilles tendon. Tissues were obtained from left and right joints for mechanical and histological evaluations, respectively as described below.

2.2. Biomechanical Testing

Biomechanical testing of both CCLs and Achilles tendons was performed on a servo-hydraulic load frame (Instron 8874; Norwood, MA) equipped with a 10 kN load cell. Samples were kept hydrated in phosphate-buffered saline (PBS)-soaked gauze throughout the course of potting in testing fixtures andtesting was performed at room temperature in air.While testing protocols were similar for both tissues, there were some minor but important distinctions, and as such detailed methods for both are included in the sections below. The individual performing the testing was blinded to the study groups.

2.2.1. Cranial Cruciate Ligament Mechanical Testing

Cranial cruciate ligaments (N=5 MPS I and N=4 controls) were tested to failure in uniaxial tension. Left stifle joints were dissected free of soft tissues between the distal femur and proximal tibia. Prior to mechanical testing, the cross-sectional area of the mid- substance of each CCL was measured with a custom laser-based measurement system.³¹ Samples were then potted in polymethyl methacrylate (PMMA) such that the distal third and proximal third of the femora and tibiae remained exposed (Figure 1A). Mechanical testing of CCLs was based on previously published protocols.^32–34 Custom aluminum fixtures were used to securely fix the stifle joint at 45 degrees of flexion. An X-Y linear stage was used to ensure the CCL was vertical under tensile loads less than 10 N. Prior to the onset of the tensile loading protocol, gauge length measurements were taken with Vernier calipers. Samples were subjected to a pre-load of 10N, followed by 15 cycles of preconditioning (1mm peak-to-peak amplitude at 1Hz). Finally, a ramp-to-failure was performed under displacement control at a rate of 0.01mm/s. Measures of time, force and displacement were analyzed with custom software (MATLAB R2020a, Mathworks; Natick, MA). The following parameters were calculated for each sample: stiffness (N/mm), elastic modulus (MPa), mechanical toughness (MJ.m⁻³), failure load (N), failure stress (MPa) and failure strain (mm/mm).

Figure 1.

Representative images illustrating uniaxial tensile mechanical testing configurations. A. Cranial cruciate ligament (CCL). and B.Achilles tendon. Arrows show loading direction.

2.2.2. Achilles Tendon Mechanical Properties

Achilles tendons (N=5 MPS I and N=5 controls) were tested to failure in uniaxial tension. Hock joints were dissected free of soft tissues except for the calcaneal tendon. Prior to mechanical testing, the cross-sectional areas of the insertion, mid-substance, and myotendinous junction regions of each tendon were measured with a custom laser-based measurement system and averaged.³¹ Next, intact calcaneal bones were potted in Field’s Metal. Mechanical testing was based on several previously published protocols.^{35; 36} Proximal ends of the tendon were affixed to 50-grit sandpaper with cyanoacrylate glue. Custom aluminum fixtures were then used to securely fix the tendon complex orthogonal to the calcaneus, simulating a typical loading angle (Figure1B). A linear stage was used to ensure the tendon was aligned vertically prior to the onset of load application. Specimens were photographed to determine gauge length. Samples were subjected to a pre-load of 10N, followed by 15 cycles of preconditioning (1mm peak-to-peak amplitude at 1Hz). Finally, a ramp-to-failure was performed under displacement control at a rate of 1.5 mm/s. Measures of time, force, and displacement were analyzed with custom software (MATLAB) to determine stiffness (N/mm), elastic modulus (MPa), mechanical toughness (MJ.m⁻³), failure load (N),failure stress (MPa) and failure strain (mm/mm).

2.3. Histological Evaluation of Tendon Structure and Composition

Cranial cruciate ligaments (N=6 MPS I and N=5 controls) and Achilles tendons (N=2 MPS I and N=4 controls), were dissected free of bone and muscle attachments from right stifle and hock joints, respectively, fixed for 1 week in 10% neutral buffered formalin, and processed into paraffin. Sections 8 μm thick were obtained from the sample mid-substance, with the sectioning orientation parallel to the fiber direction. Sections were stained with hematoxylin and eosin for cellularity, Alcian blue for GAGs, or picrosirius red for collagens, according to established protocols.³⁷ Semi-quantitative grading of CCL and Achilles tendon condition was undertaken using an adaptation of previously published schemes,^{38; 39} inclusive of the following parameters: fiber fragmentation, fiber arrangement, rounding of cell nuclei, cell density, vascularity, and GAG staining. Each parameter was graded on a scale of 0 to 3, with 0 being completely normal and 3 being severely abnormal. Detailed assessment criteria for each parameter are shown in Table 1.Overall grade was calculated as the sum of these individual parameters. Average grades for three regions of interest per sample were determined. Grading was performed by three individuals blinded to the study groups, with results averaged prior to statistics.

Table 1.

Summary of assessment criteria used for histological grading of CCLs and Achilles tendons.

Parameter	Grade 0	Grade 1	Grade 2	Grade 3
Fiber fragmentation	Continuous, long fibers	Slightly fragmented	Moderately fragmented	Severely fragmented
Fiber arrangement	Parallel arrangement, well ordered and regular fibers	Slightly loose and wavy	Moderately loose, wavy	Loss of parallel arrangement and deterioration of fibers, no pattern identified
Rounding of tenocyte/ligamentocyte nuclei	Flattened/fusiform nuclei	Slightly rounded	Moderately rounded	Severely rounded
Overall cellularity	Normal	Slightly increased	Moderately increased	Severely increased
Increased vascularity	<10% of field	10-20%	20-30%	>30%
Glycosaminoglycan content	Normal	Slightly increased	Moderately increased	Severely increased

Relative GAG content in both CCLs and Achilles tendons was quantified by measuring staining intensity on Alcian blue-stained sections. Briefly, images were first converted to 8-bit gray scale using ImageJ software (National Institutes of Health; Bethesda, USA). They were then inverted in order to provide an intensity of 0 for white pixels and 255 for black pixels, ensuring that positive Alcian blue staining corresponded to higher pixel intensity. Images were uniformly thresholded to eliminate artifacts and the mean gray intensity calculated.

Degree of collagen fiber disorganization for both CCLs and Achilles tendons was quantified as circular standard deviation (CSD) as previously reported.^{40; 41} Briefly, picrosirius red-stained sections were imaged using a polarizing microscope equipped with a rotating stage, and CSD calculated using a custom Matlab program (Mathworks, Natick, MA) by fitting a sin² function topixel intensity-polarizer angle data to determine the angle corresponding to the minimum pixel intensity, which represents the average direction of fiber alignment.

2.4. Statistical Evaluations

Statistical evaluations were performed using Graphpad Prism v10 (Graphpad Software; Boston, USA).Sample sizes were selected based on prior studies of MPS joint disease pathophysiology.^{37; 42}Statistically-significant differences between MPS I and control samples for biomechanical and histological parameters were established using Mann-Whitney U tests. All results are reported as median and interquartile range, and p<0.05 was considered significant. Due to the small number of MPS I Achilles tendon samples available (N=2), statistical evaluations for histological parameters were not performed for these samples.

3. Results

3.1. Clinical Findings

Allanimals reached the study endpoint of 12 months-of-age without any unexpected pathologies. Median animal weights were 20.00 and 17.25 kg for control and MPS I animals, respectively (not significantly different).Comprehensive clinical findings for these animals were reported previously.⁴² Briefly, all MPS I animals were ambulatory for the study duration. Five out of the 6 of these animals developed mild to moderate joint effusions between 2 and 7 months-of-age, but joints were neither painful nor warm to the touch. By 12 months-of-age, MPS I joint abnormalities also included hyperextended carpi, stifles and tarsi, and widely splayed digits. Throughout the study, no abnormalities were noted in healthy control dogs.

3.2. Cranial Cruciate Ligament Properties

Cranial cruciate ligaments were tested in uniaxial tension to failure (Figure 1A). Median stress versus strain curves for each study group are shown in Figure 2A. Of the mechanical properties assessed (Figures 2B–H),stiffness (65% of control, p=0.016), failure load (54% of control, p=0.016) and failure stress (72% of control, p=0.016)were significantly lower for CCLs from MPS I animals compared to those from healthy controls. Representative bright field hematoxylin and eosin and Alcian blue stained histological sectionsof CCLs, and picrosirius red-stained sections under polarized light, from control and MPS I animals are shown in Figures3A–C, respectively. Subjectively, MPS I CCLs exhibited elevated fiber fragmentation, fiber arrangement, rounding of cell nuclei, cell density, vascularity and GAG staining, compared to controls. These observations were confirmed through semi-quantitative grading, with all parameters, in addition to overall grade, significantly worse from MPS I CCLs compared to controls (Figure 3D–J; all p<0.05). Quantitative assessment of Alcian blue staining intensity revealed significantly higher GAG content for MPS I CCLs compared to controls (Figure 3K).Quantitative assessment of picrosirius red-stained sections under polarized light revealed significantly higher CSD for MPS I CCLs compared to controls (Figure 3L), indicating a higher degree of collagen fiber disorganization in these samples.

Figure 2.

Bulk tensile mechanical properties of cranial cruciate ligaments from control and MPS I dogs. A. Stress-strain plot (median of all samples). B. Cross-sectional area. C. Stiffness. D. Modulus. E. Toughness. F. Failure Load. G. Failure Stress. H. Failure Strain. N=4-5; *p<0.05, MPS I versus control. All data presented as median and interquartile range.

Figure 3.

Histological analysis ofcranial cruciate ligaments (CCLs)from control and MPS I dogs. Representative sections stained with: A.Hematoxylin and eosin, illustrating increased cell density, rounding of cell nuclei (inset), vascularity (arrow), and collagen fiber disorganization and fragmentation in MPS I CCLs compared to controls.B. Alcian blue staining, illustrating elevated glycosaminoglycan deposition in MPS I CCLs compared to controls. C. Picrosirius red staining viewed under polarized light, illustrating increased collagen fiber disorganization in MPS I CCLs compared to controls. D-J. Results of semi-quantitative histological grading of CCL condition confirmed subjective findings. K.Higher GAG staining intensity and L. Circular standard deviation in MPS I CCLs compared to controls. N=5-6; *p<0.05, MPS I versus control. All data presented as median and interquartile range.Scale = 100μm (inset = 20μm).

3.3. Achilles Tendon Properties

Achilles tendons were tested in uniaxial tension to failure (Figure 1B). Median stress versus strain curves for each study group are shown in Figure 4A.Of the mechanical propertiesassessed (Figures 4B–H), stiffness (60% of control, p=0.016) and failure load (64% of control, p=0.008) were found to be significantly lower for Achilles tendons from MPS I animals compared to those fromhealthy controls, while failure strain (129% of control, p=0.032) was significantly higher. Representative bright field hematoxylin and eosin, and Alcian blue stained histological sectionsof Achilles tendons, and picrosirius red-stained sections under polarized light, from control and MPS I animals are shown in Figures5A–C, respectively. Similar to CCLs, Achilles tendons from MPS I animals exhibited subjectively increased fiber fragmentation, fiber arrangement, rounding of cell nuclei, cell densityand GAG staining compared to controls. With respect to semi-quantitative grading, while the small sample size precluded statistical tests, all parameters assessed with the exception of vascularity were higher for MPS I Achilles tendons compared to controls (Figures 5D–J), as were GAG staining intensity (Figure 5K) and CSD (Figure 5L).

Figure 4.

Bulk tensile mechanical properties of Achilles tendons from control and MPS I dogs. A. Stress-strain plot (median of all samples). B. Cross-sectional area. C. Stiffness. D. Modulus. E. Toughness. F. Failure Load. G. Failure Stress. H. Failure Strain. N=5; *p<0.05, MPS I versus control. All data presented as median and interquartile range.

Figure 5.

Histological analysis of Achilles tendons from control and MPS I dogs. Representative sections stained with: A. Hematoxylin and eosin, illustrating increased cell density, rounding of cell nuclei (inset), and collagen fiber disorganization and fragmentation in MPS I tendons compared to controls. B. Alcian blue staining, illustrating elevated glycosaminoglycan deposition in MPS I tendons compared to controls. C. Picrosirius red staining viewed under polarized light, illustrating increased collagen fiber disorganization in MPS I tendons compared to controls. D-J. Results of semi-quantitative histological grading of tendon condition confirmed subjective findings. K. Higher GAG staining intensity and L.Circular standard deviation in MPS I tendons compared to controls. N=2-4. All data presented as median and interquartile range. Note that these results were not tested for statistical significance due to the small sample size.Scale = 100μm (inset = 20μm).

4. Discussion

Progressive synovial joint disease is pervasive in MPS I patients, where it reduces mobility, ability to independently undertake daily living activities and overall quality of life. Almost all joints can be affected, with reported clinical manifestations that includejoint contractures, carpal tunnel syndrome, trigger digits, acetabular dysplasia, coxa valga, and genu valgum,^{7; 43–47} with surgical intervention often required from an early age to reduce pain and restore function.⁴⁷With respect to tendons and ligaments, few studies have examined the functional or morphological properties of these tissues, and relatively little is known about their specific contributions to progressive joint disease. A very recent study using ultrasound imaging reported pathological changes in the ligaments and tendons of the fingers, wrists and knees of MPS I patients, including abnormal thickening.⁴⁸ Notably, the nature and extent of these abnormalities were found to vary considerably between joint types and patients, potentially due to the large variation in subject age and treatment status.

Animal models have proved extremely valuable for advancing understanding of joint disease pathophysiology in MPS I.^{42; 49}Previously, we showed that MPS I dog stifle joints develop synovitis and pathological changes to articular cartilage.⁴² At the molecular level, joint diseaseis mediated in part by local inflammation.^{42; 50}In the current study we build on this prior work to provide insights into altered microstructure and mechanical function of both the CCL and the Achilles tendon in MPS I, two tissues that are critical for healthy joint function. Pathological changes in each of these tissues were broadly similar.With respect to biomechanical properties, both stiffness and failure properties were significantly lower for both CCLs and Achilles tendons. With respect to microstructure and composition, elevated GAG content is consistent with MPS I pathophysiology, which leads to accumulation of undegraded GAGs in cells and tissues across multiple organ systems. Microarchitectural, compositionaland cellular abnormalities are broadly similar to those seen in injured and pathologic tendons in the general population.³⁹For example, tendons from patients with chronic Achilles tendinopathy due to mechanical overloadalso exhibit hypercellularity, vascular proliferation, abnormal fiber structure and focal increases in GAG content.^{39; 51}Similarly, ACLs have been reported to exhibit pathological changes with aging and osteoarthritis that include abnormal GAG deposition and collagen fiber disorganization.⁵²In this respect, the MPS I canine model may serve as serve as anaturally occurringand clinically-relevant platform for evaluating treatments that are also suitable for patients in the general population with tendon and ligament degeneration.In MPS I, pathological changes to these tissues occur secondary to GAG accumulation and are potentially driven by elevated local inflammation and collagen breakdown.^{42; 50; 53}Microstructural abnormalities and associated diminished tensile mechanical properties potentially place these tissues at heighted risk of further damage and failure during the course of daily activity.

Diminishedtensile properties of CCLs are similar in nature to those we reported previously for dogs with MPS VII (beta-glucuronidase deficiency) at 6 months-of-age,³⁷ although the magnitude of the changes is not as severe, which is consistent with the slower progression of joint disease in MPS I compared to MPS VII. Notably, unlike MPS VII dogs at 6 months-of-age, MPS I dogs at 12 months-if-age are still physically active and able to ambulate independently, suggesting that the functional abnormalities observed are not due to disuse.Interestingly, MPS I dogs in this study exhibited hyperextension of joints, including stifles (knees), whereas human MPS I patients typically exhibit joint contractures. Joint laxity is commonly reported in patients with MPS IVA (N-acetyl-galactosamine-6-sulfatase deficiency), but not other subtypes. Whether this is due to cross-species differences in joint anatomy and function, or differences in the underlying pathophysiology of component tissues requires further study.

While systemic treatments such as ERT and HSCT are increasing MPS I patient longevity, musculoskeletal manifestations, including synovial joint disease, generally persist, with patient quality of life significantly impacted.^{12; 54–57} Emerging systemic therapies, such as those targeting inflammation, have shown some promise. These include pentosan polysulfate (PPS, a semi-syntheticsulfatedpolysaccharidepolymer), which improved joint range of motion and reduced pain in MPS I patients.⁵⁸Treatment with adalimumab, a tumor necrosis factor-alpha (TNF-α) inhibitor, was also found to improve joint range-of-motion in MPS I patients in both upper and lower extremities.⁵⁹Oral lithium carbonate treatment was reported to reduce joint swelling and improve mobility in MPS I dogs.⁶⁰Preclinical studies suggest that intra-articular delivery of therapeutic agents may be a promising alternative to systemic treatments due to increased local bioavailability of drugs to joint tissues.^{61; 62}Notably, none of these studies specifically evaluated effects on ligament and tendon function; however, our group recently reported dose-dependent improvements in CCL mechanical properties in response to systemic ERT in MPS VII dogs, and these were associated with improvements in mobility.³⁷

Current treatment strategies for ligament and tendon pathologies in the general population include non-surgical approaches such as physical therapy and ultrasound, surgical approaches focused on repair or reconstruction of the damaged tissue, or a combination of both surgical and non-surgical approaches (e.g. surgery followed by physical therapy).^{19; 21; 63}Notably, due to low cell densities, the endogenous repair capacities of these tissues are typically poor, and would likely be further diminished in MPS I patients due to cellular dysfunction secondary to lysosomal GAG accumulation. Pharmacological approaches to improving tendon and ligament healing, for example through immunomodulation or delivery of growth factors, are actively being explored.^{21; 64; 65}

A limitation of this study was the relatively small sample size; however, the magnitude and consistency of the differences observed gives us a high degree of confidence in the biological significance of our findings.Additionally, we only examined one ligament and one tendon type, and it is likely that there are pathological changes that are distinct to these tissues in other joints, commensurate with their unique mechanical contributions.

Ongoing work is focused on elucidating the underlying molecular pathology of ligament and tendon abnormalities in MPS I dogs, and establishing the extent to which similar structural and function changes occur in human patients through non-invasive imaging and functional assays. In conclusion, our findings demonstrate that pathological changes to both ligaments and tendons contribute to abnormal joint function in MPS I, and suggest that effective clinical management of joint disease in patients should incorporate conservative or surgical treatments targeting these tissues.

Data Availability Statement

Study data will be made available upon reasonable request to the corresponding author.