BackgroundPentosan 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 FindingsTreatment 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.ConclusionsBased 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.

Deletion of connexin (Cx) 43 from osteoblasts and osteocytes (OCN-Cre;Cx43fl/− mice) or from osteocytes only (DMP1-8kb-Cre;Cx43fl/fl mice) results in increased cortical, but not cancellous, osteocyte apoptosis and widening of the femoral midshaft without changes in cortical thickness. Despite the consequent larger moment of inertia, stiffness and ultimate load, measures of mechanical strength assessed by 3-point bending, are not higher in either model of Cx43 deficiency, due to reduced Young's modulus, a measure of the stiffness of the material per unit of area. In OCN-Cre;Cx43fl/− mice, this was accompanied by a reduced ratio of nonreducible/reducible collagen cross-links as assessed by Fourier Transformed Infrared Imaging (FTIRI) in the femoral diaphysis. On the other hand, DMP1-8kb-Cre;Cx43fl/fl mice did not show a significant reduction in collagen maturation in the same skeletal site, but a small decrease in mineralization was detected by FTIRI. Remarkably, both osteoblastic and osteocytic cells lacking Cx43 expressed lower mRNA levels of lysyl oxidase, a crucial enzyme involved in collagen maturation. These findings suggest that Cx43 expression in osteoblasts is involved in maintaining the quality of the bone matrix in cortical bone through the maturation of collagen cross-links. Osteocytic Cx43 expression is important also to maintain the stiffness of the bone material, where a Cx43 deficiency results in a local reduction in mineralization, possibly due to osteocyte apoptosis.

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Functional adaptation may complicate the choice of phenotype used in genetic studies that seek to identify genes contributing to fracture susceptibility. Often, genetic variants affecting one trait are compensated by coordinated changes in other traits. Bone fracture is a prototypic example because mechanical function of long bones (stiffness and strength) depends on how the system coordinately adjusts the amount (cortical area) and quality (tissue-mineral density, TMD) of bone tissue to mechanically offset the natural variation in bone robustness (total area/length). We propose that efforts aimed at identifying genes regulating fracture resistance will benefit from better understanding how functional adaptation contributes to the genotype-phenotype relationship. We analyzed the femurs of C57BL/6J – ChrA/J/NaJ Chromosome Substitution Strains (CSSs) to systemically interrogate the mouse genome for chromosomes harboring genes that regulate mechanical function. These CSSs (CSS-i, i = the substituted chromosome) showed changes in mechanical function on the order of -26.6 to 11.5% relative to the B6 reference strain after adjusting for body size. Seven substitutions showed altered robustness, cortical area, or TMD, but no effect on mechanical function (CSS-4, 5, 8, 9, 17, 18, 19); six substitutions showed altered robustness, cortical area, or TMD, and reduced mechanical function (CSS-1, 2, 6, 10, 12, 15); and one substitution also showed reduced mechanical function but exhibited no significant changes in the three physical traits analyzed in this study (CSS-3). A key feature that distinguished CSSs that maintained function from those with reduced function was whether the system adjusted cortical area and TMD to the levels needed to compensate for the natural variation in bone robustness. These results provide a novel biomechanical mechanism linking genotype with phenotype, indicating that genes control function not only by regulating individual traits, but also by regulating how the system coordinately adjusts multiple traits to establish function.