Poor solute transport through the cartilage endplate (CEP) impairs disc nutrition and could be a key factor that limits the success of intradiscal biologic therapies. Here we demonstrate that treating the CEP with matrix metalloproteinase-8 (MMP-8) reduces the matrix constituents that impede solute uptake and thereby improves nutrient diffusion. Human CEP tissues harvested from four fresh cadaveric lumbar spines (age range: 38-66 years old) were treated with MMP-8. Treatment caused a dose-dependent reduction in sGAG, localized reductions to the amount of collagen, and alterations to collagen structure. These matrix modifications corresponded with 16-24% increases in the uptake of a small solute (376 Da). Interestingly, the effects of MMP-8 treatment depended on the extent of non-enzymatic glycation: treated CEPs with high concentrations of advanced glycation end products (AGEs) exhibited the lowest uptake compared to treated CEPs with low concentrations of AGEs. Moreover, AGE concentrations were donor-specific, and the donor tissues with the highest AGE concentrations appeared to have lower uptake than would be expected based on the initial amounts of collagen and sGAG. Finally, increasing solute uptake in the CEP improved cell viability inside diffusion chambers, which supports the nutritional relevance of enhancing the transport properties of the CEP. Taken together, our results provide new insights and in vitro proof-of-concept for a treatment approach that could improve disc nutrition for biologic therapy: specifically, matrix reduction by MMP-8 can enhance solute uptake and nutrient diffusion through the CEP, and AGE concentration appears to be an important, patient-specific factor that influences the efficacy of this approach.

Intervertebral disc degeneration is strongly associated with chronic low back pain, a leading cause of disability worldwide. Current back pain treatment approaches (both surgical and conservative) are limited to addressing symptoms, not necessarily the root cause. Not surprisingly therefore, long-term efficacy of most approaches is poor. Cell-based disc regeneration strategies have shown promise in preclinical studies, and represent a relatively low-risk, low-cost, and durable therapeutic approach suitable for a potentially large patient population, thus making them attractive from both clinical and commercial standpoints. Despite such promise, no such therapies have been broadly adopted clinically. In this perspective we highlight primary obstacles and provide recommendations to help accelerate successful clinical translation of cell-based disc regeneration therapies. The key areas addressed include: (a) Optimizing cell sources and delivery techniques; (b) Minimizing potential risks to patients; (c) Selecting physiologically and clinically relevant efficacy metrics; (d) Maximizing commercial potential; and (e) Recognizing the importance of multidisciplinary collaborations and engaging with clinicians from inception through to clinical trials.

Background

Cardiovascular disease, a progressive manifestation of α-L-iduronidase deficiency or mucopolysaccharidosis type I, continues in patients both untreated and treated with hematopoietic stem cell transplantation or intravenous enzyme replacement. Few studies have examined the effects of α-L-iduronidase deficiency and subsequent glycosaminoglycan storage upon arterial gene expression to understand the pathogenesis of cardiovascular disease.

Methods

Gene expression in carotid artery, ascending, and descending aortas from four non-tolerized, non-enzyme treated 19 month-old mucopolysaccharidosis type I dogs was compared with expression in corresponding vascular segments from three normal, age-matched dogs. Data were analyzed using R and whole genome network correlation analysis, a bias-free method of categorizing expression level and significance into discrete modules. Genes were further categorized based on module-trait relationships. Expression of clusterin, a protein implicated in other etiologies of cardiovascular disease, was assessed in canine and murine mucopolysaccharidosis type I aortas via Western blot and in situ immunohistochemistry.

Results

Gene families with more than two-fold, significant increased expression involved lysosomal function, proteasome function, and immune regulation. Significantly downregulated genes were related to cellular adhesion, cytoskeletal elements, and calcium regulation. Clusterin gene overexpression (9-fold) and protein overexpression (1.3 to 1.62-fold) was confirmed and located specifically in arterial plaques of mucopolysaccharidosis-affected dogs and mice.

Conclusions

Overexpression of lysosomal and proteasomal-related genes are expected responses to cellular stress induced by lysosomal storage in mucopolysaccharidosis type I. Upregulation of immunity-related genes implicates the potential involvement of glycosaminoglycan-induced inflammation in the pathogenesis of mucopolysaccharidosis-related arterial disease, for which clusterin represents a potential biomarker.

Background

We previously demonstrated the therapeutic benefits of pentosan polysulfate (PPS) in a rat model of mucopolysaccharidosis (MPS) type VI. Reduction of inflammation, reduction of glycosaminoglycan (GAG) storage, and improvement in the skeletal phenotype were shown. Herein, we evaluate the long-term safety and therapeutic effects of PPS in a large animal model of a different MPS type, MPS I dogs. We focused on the arterial phenotype since this is one of the most consistent and clinically significant features of the model.

Methodology/principal findings

MPS I dogs were treated with daily oral or biweekly subcutaneous (subQ) PPS at a human equivalent dose of 1.6 mg/kg for 17 and 12 months, respectively. Safety parameters were assessed at 6 months and at the end of the study. Following treatment, cytokine and GAG levels were determined in fluids and tissues. Assessments of the aorta and carotid arteries also were performed. No drug-related increases in liver enzymes, coagulation factors, or other adverse effects were observed. Significantly reduced IL-8 and TNF-alpha were found in urine and cerebrospinal fluid (CSF). GAG reduction was observed in urine and tissues. Increases in the luminal openings and reduction of the intimal media thickening occurred in the carotids and aortas of PPS-treated animals, along with a reduction of storage vacuoles. These results were correlated with a reduction of GAG storage, reduction of clusterin 1 staining, and improved elastin integrity. No significant changes in the spines of the treated animals were observed.

Conclusions

PPS treatment led to reductions of pro-inflammatory cytokines and GAG storage in urine and tissues of MPS I dogs, which were most evident after subQ administration. SubQ administration also led to significant cytokine reductions in the CSF. Both treatment groups exhibited markedly reduced carotid and aortic inflammation, increased vessel integrity, and improved histopathology. We conclude that PPS may be a safe and useful therapy for MPS I, either as an adjunct or as a stand-alone treatment that reduces inflammation and GAG storage.