Alon Lai

Pentosan Polysulfate: A Novel Therapy for the Mucopolysaccharidoses

Background Pentosan polysulfate (PPS) is an FDA-approved, oral medication with anti-inflammatory and pro-chondrogenic properties. We have previously shown that animal models of the mucopolysaccharidoses (MPS) exhibit significant inflammatory disease, contributing to cartilage degeneration. Enzyme replacement therapy (ERT) only partly reduced inflammation, and anti-TNF-alpha antibody therapy significantly enhanced clinical and pathological outcomes. Here we describe the use of PPS for the treatment of MPS type VI rats. Methodology/Principal Findings Treatment began during prenatal development and at 1 and 6 months of age. All animals were treated until they were 9 months old. Significant reductions in the serum and tissue levels of several inflammatory markers (e.g., TNF-alpha, MIP-1alpha and RANTES/CCL5) were observed, as was reduced expression of inflammatory markers in cultured articular chondrocytes. ADAMTS-5/aggrecanase-2 levels also were reduced in chondrocytes, consistent with an elevation of serum tissue inhibitor of metalloproteinase 1. Marked improvements in motility and grooming behavior occurred, along with a reduction in eye and nasal secretions and a lessening of the tracheal deformities. MicroCT and radiographic analyses further revealed that the treated MPS skulls were longer and thinner, and that the teeth malocclusions, misalignments and mineral densities were improved. MicroCT analysis of the femurs and vertebrae revealed improvements in trabecular bone mineral densities, number and spacing in a subset of treated MPS animals. Biomechanical assessments of PPS-treated spines showed partially restored torsional behaviors, suggesting increased spinal stability. No improvements were observed in cortical bone or femur length. The positive changes in the PPS-treated MPS VI rats occurred despite glycosaminoglycan accumulation in their tissues. Conclusions Based on these findings we conclude that PPS could be a simple and effective therapy for MPS that might provide significant clinical benefits alone and in combination with other therapies.

Effects of torsion on intervertebral disc gene expression and biomechanics, using a rat tail model.

Study Design. In vitro and in vivo rat tail model to assess effects of torsion on intervertebral disc biomechanics and gene expression. Objective. Investigate effects of torsion on promoting biosynthesis and producing injury in rat caudal intervertebral discs. Summary of Background Data. Torsion is an important loading mode in the disc and increased torsional range of motion is associated with clinical symptoms from disc disruption. Altered elastin content is implicated in disc degeneration, but its effects on torsional loading are unknown. Although effects of compression have been studied, the effect of torsion on intervertebral disc gene expression is unknown. Methods In vitro biomechanical tests were performed in torsion on rat tail motion segments subjected to 4 treatments: elastase, collagenase, genipin, control. In vivo tests were performed on rats with Ilizarov-type fixators implanted to caudal motion segments with five 90 minute loading groups: 1 Hz cyclic torsion to ±5 ±15° and ±30°, static torsion to +30°, and sham. Anulus and nucleus tissues were separately analyzed using qRT-PCR for gene expression of anabolic, catabolic, and proinflammatory cytokine markers. Results In vitro tests showed decreased torsional stiffness following elastase treatment and no changes in stiffness with frequency. In vivo tests showed no significant changes in dynamic stiffness with time. Cyclic torsion upregulated elastin expression in the anulus fibrosus. Up regulation of TNF-&agr;and IL-1&bgr; was measured at ±30°. Conclusion. We conclude that strong differences in the disc response to cyclic torsion and compression are apparent with torsion increasing elastin expression and compression resulting in a more substantial increase in disc metabolism in the nucleus pulposus. Results highlight the importance of elastin in torsional loading and suggest that elastin remodels in response to shearing. Torsional loading can cause injury to the disc at excessive amplitudes that are detectable biologically before they are biomechanically.

Short-term effects of backpack load placement on spine deformation and repositioning error in schoolchildren

Backpack weight of 10–15% has been recommended as an acceptable limit for schoolchildren. However, there is still no clear guideline regarding where the backpack centre of gravity (CG) should be positioned. The changes of spinal curvature and repositioning error when carrying a backpack loaded at 15% of body weight at different CG locations (anterior or posterior at T7, T12 or L3) in schoolchildren were analysed. Both spinal curvature and repositioning error were found to be affected by backpack anterior–posterior position and CG level. A relatively smaller change was observed during anterior carriage with the least change when the backpack CG was positioned at T12. The results also suggested that alternative carriage by changing the backpack position occasionally between anterior and posterior positions might help to relieve the effects of backpack on spine. However, future study is recommended to further substantiate the beneficial effects of alternative carriage on children. Statement of Relevance: Anteriorly carried backpack with centre of gravity positioned at T12 was shown to induce relatively less effect on spinal deformation and repositioning error in schoolchildren. Changing backpack carriage position occasionally may help to relieve its effects on spinal deformation. The findings are important for ergonomic schoolbag design and determining a proper load carriage method.

Dose responsive effects of subcutaneous pentosan polysulfate injection in mucopolysaccharidosis type VI rats and comparison to oral treatment.

Background We previously demonstrated the benefits of daily, oral pentosan polysulfate (PPS) treatment in a rat model of mucopolysaccharidosis (MPS) type VI. Herein we compare these effects to once weekly, subcutaneous (sc) injection. The bioavailability of injected PPS is greater than oral, suggesting better delivery to difficult tissues such as bone and cartilage. Injected PPS also effectively treats osteoarthritis in animals, and has shown success in osteoarthritis patients. Methodology/Principal Findings One-month-old MPS VI rats were given once weekly sc injections of PPS (1, 2 and 4 mg/kg, human equivalent dose (HED)), or daily oral PPS (4 mg/kg HED) for 6 months. Serum inflammatory markers and total glycosaminoglycans (GAGs) were measured, as were several histological, morphological and functional endpoints. Overall, weekly sc PPS injections led to similar or greater therapeutic effects as daily oral administration. Common findings between the two treatment approaches included reduced serum inflammatory markers, improved dentition and skull lengths, reduced tracheal deformities, and improved mobility. Enhanced effects of sc treatment included GAG reduction in urine and tissues, greater endurance on a rotarod, and better improvements in articular cartilage and bone in some dose groups. Optimal therapeutic effects were observed at 2 mg/kg, sc. No drug-related increases in liver enzymes, coagulation factor abnormalities or other adverse effects were identified following 6 months of sc PPS administration. Conclusions Once weekly sc administration of PPS in MPS VI rats led to equal or better therapeutic effects than daily oral administration, including a surprising reduction in urine and tissue GAGs. No adverse effects from sc PPS administration were observed over the 6-month study period.

Effects of static compression with different loading magnitudes and durations on the intervertebral disc : an in vivo rat-tail study

Study Design. An in vivo rat-tail model was used to study the effects of static compression with different loading magnitudes and durations on the intervertebral disc. Objective. To investigate the effects of static compression with different loading magnitudes and durations on the intervertebral disc over a period of time. Summary of Background Data. A disc degeneration model is essential for studying therapeutic effects on degenerated disc. Static compression can induce degenerative-like changes in the intervertebral disc. However, the consequences of the simulation model over a period of resting have not been clearly documented, which may have confounding effects on the experimental outcome. Methods. Thirty-five rats were used. Static compressions with different loads (11 or 17 N) and durations (1 hour daily or continuous) were applied to the rat-tail caudal 8–9 disc for 2 weeks, and followed with 3 weeks of rest. The disc height was quantified in vivo on days 4, 18, and 39. The rats were killed and the discs were harvested for morphologic examination on day 39 after the disc height measurement. Results. Significant decrease in disc height was observed after continuous static compression for both 11 and 17 N, and continued during the resting period. The morphologic evaluation of the continuous compressed disc showed a decreased nuclear size, reduced number of nuclear cells, and irregular nuclear shape with inward bulging of disorganized annular collagen lamellas. Daily compression of 1 hour was found to induce a transient increase in disc height, but restored after the 3-week resting period. Favorable morphologic changes, including vacuolated nuclear cells and oval nuclear shape with well-organized annular collagen lamellas, were seen in the rat disc specimens with daily compression of 1 hour. Conclusion. Disc degenerative-like changes without recovery were demonstrated in the rat caudal disc after continuous compression. The changes in disc height and disc morphology were found to be dependent on the duration of load application and may have clinical implication.

Assessment of Functional and Behavioral Changes Sensitive to Painful Disc Degeneration

The development of an in vivo rodent discogenic pain model can provide insight into mechanisms for painful disc degeneration. Painful disc degeneration in rodents can be inferred by examining responses to external stimuli, observing pain‐related behaviors, and measuring functional performance. This study compared the sensitivity of multiple pain and functional assessment methods to disc disruption for identifying the parameters sensitive to painful disc degeneration in rats. Disc degeneration was induced in rats by annular injury with saline injection. The severity of disc degeneration, pain sensitivity, and functional performance were compared to sham and naïve control rats. Saline injection induced disc degeneration with decreased disc height and MRI signal intensity as well as more fibrous nucleus pulposus, disorganized annular lamellae and decreased proteoglycan. Rats also demonstrated increased painful behaviors including decreased hindpaw mechanical and thermal sensitivities, increased grooming, and altered gait patterns with hindpaw mechanical hyperalgesia and duration of grooming tests being most sensitive. This is the first study to compare sensitivities of different pain assessment methods in an in vivo rat model of disc degeneration. Hindpaw mechanical sensitivity and duration of grooming were the most sensitive parameters to surgically induced degenerative changes and overall results were suggestive of disc degeneration associated pain.

Effects of traction on structural properties of degenerated disc using an in vivo rat-tail model.

Study Design. An in vivo rat-tail model was adopted to study the structural changes of degenerated intervertebral disc after different traction protocols. Objective. To investigate the effects of traction with different modes and magnitudes on disc with simulated degeneration. Summary of Background Data. Traction has been commonly used in clinical practice for treating low back pain. Its effects on disc with degeneration have not been fully investigated. Methods. Forty-seven mature rats were used. Continuous static compression of 11 N was applied to the rat caudal 8–9 disc for 2 weeks to simulate disc degeneration. Tractions with different modes (static or intermittent) and magnitudes (1.4 N or 4.2 N) were applied to the degenerated disc for 3 weeks. The disc height was quantified in vivo on days 4, 18, and 39. The treated discs were then harvested for morphologic analysis. Results. Significant decrease in disc height with degenerative morphologic changes was observed after the application of the static compression. The changes in disc height after the application of traction were found to be magnitude dependent. Continuous decrease in disc height was observed after 4.2-N traction, whereas the disc height maintained after traction of 1.4 N. However, no obvious morphologic change was found in comparison with the degenerated discs without traction. Conclusion. Although traction was not demonstrated to have restored disc with degeneration, traction with relatively low magnitude was found to have significant beneficial effect in maintaining disc height of degenerated disc, and it might be a potential intervention to slow down the process of degeneration. Future studies of the effects of low-magnitude traction on degenerated disc are recommended.

Annular puncture with tumor necrosis factor-alpha injection enhances painful behavior with disc degeneration in vivo

BACKGROUND CONTEXT Painfulintervertebral disc degeneration is extremely common and costly. Effective treatments are lacking because the nature of discogenic pain is complex with limited capacity to distinguish painful conditions from age-related changes in the spine. Hypothesized sources of discogenic pain include chronic inflammation, neurovascular ingrowth, and structural disruption. PURPOSE This study aimed to investigate inflammation, pro-neurovascular growth factors, and structural disruption as sources of painful disc degeneration STUDY DESIGN/SETTING This study used an in vivo study to address these hypothesized mechanisms with anterior intradiscal injections of tumor necrosis factor-alpha (TNFα), pro-neurovascular growth factors: nerve growth factor and vascular endothelial growth factor (NGF and VEGF), and saline with additional sham surgery and naïve controls. Depth of annular puncture was also evaluated for its effects on structural and painful degeneration. METHODS Rat lumbar discs were punctured (shallow or deeper puncture) and intradiscally injected with saline, TNFα, or NGF and VEGF. Structural disc degeneration was assessed using X-ray, magnetic resonance imaging (MRI), and histology. The rat painful condition was evaluated using Von Frey hyperalgesia measurements, and substance P immunostaining in dorsal root ganglion (DRG) was performed to determine the source of pain. RESULTS Saline injection increased painful responses with degenerative changes in disc height, MRI intensity, and morphologies of disc structure and cell. TNFα and NGF/VEGF accelerated painful behavior, and TNFα-injected animals had increased substance P in DRGs. Deeper punctures led to more severe disc degeneration. Multiple regression analysis showed that the painful behavior was correlated with disc height loss. CONCLUSIONS We concluded that rate and severity of structural disc degeneration was associated with the amount of annular disruption and puncture depth. The painful behavior was associated with disc height loss and discal inflammatory state, whereas pro-inflammatory cytokines might play a more important role in the level of pain, which might have resulted from enhanced DRG sensitization. These in vivo painful disc degeneration models with different severities of structural changes may be useful for investigating discogenic pain mechanisms and for screening therapies, although interpretations must note the differences between all surgically induced animal models and the human condition.

Effects of electroacupuncture on a degenerated intervertebral disc using an in-vivo rat-tail model

Abstract Electroacupuncture (EA) has long been used as conservative treatment for low back pain (LBP). Its effect on relief of back pain has been demonstrated in many clinical studies. However, whether it has any effect on the biological properties of an intervertebral disc, which is one of the major causes of LBP, is still unclear. The aim of this study was, therefore, to investigate the effects of EA with different simulation frequencies on an intervertebral disc with simulated degeneration using an in-vivo rat-tail model. In this study, 33 rats were used. Disc degeneration was simulated in the rat caudal 8—9 disc via continuous static compressive loading of 11 N for 2 weeks. EA with a frequency of 2 or 100 Hz was then applied to the degenerated disc for 3 weeks with 3 sessions/week and 20 min/session. The intervertebral disc height was measured before and after compression as well as after EA intervention for 3 weeks. The static compression was found to result in a reduction in the disc height of about 22 per cent. There was no evidence that this change could be reversed after resting or the EA intervention. However, EA at 100 Hz was found to induce a further decrease in disc height, which was not shown for the rats after resting or EA at 2 Hz. The results of this study showed that effects of EA on disc degeneration are frequency dependent and adverse effects could result if EA at a certain frequency was used.

Structural, compositional, and biomechanical alterations of the lumbar spine in rats with mucopolysaccharidosis type VI (Maroteaux–Lamy syndrome)

Mucopolysaccharidosis (MPS) VI is an inherited lysosomal storage disorder resulting from deficiency of N‐acetylgalactosamine‐4‐sulfatase activity and subsequent accumulation of incompletely degraded dermatan sulfate (DS) containing glycosaminoglycans (GAGs). Painful spinal deformities are commonly found in MPS VI patients. We characterized lumbar spine structure, composition, and biomechanics in a naturally occurring rat MPS VI model and evaluated the role of MMP‐13, ADAMTS‐5 and TNF‐α in modulating the observed changes. MPS VI rats had discs with large vacuolated cells and sizable nuclear defects. MPS spine segments also had structural and functional changes suggestive of spinal instability, including decreased nuclear pressurization, increased joint laxity and increased disc height index. These functional changes were at least partly associated with elevated ADAMTS‐5, MMP‐13, and TNF‐α. Vertebral and endplate biomechanics were also affected by MPS VI with decreased failure load and stiffness. The discal and vertebral dysfunctions observed in MPS VI rats are likely to be associated with pathological spinal conditions, similar to those that afflict MPS patients. Our findings also suggest more broadly that abnormal accumulation of GAGs and the associated chronic pro‐inflammatory and catabolic cascade may also be a source of spinal dysfunction.