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Equilibrative nucleoside transporter 1 (ENT1) mediates passage of adenosine across the plasma membrane. We reported previously that mice lacking ENT1 (ENT1(-/-)) exhibit progressive ectopic mineralization of spinal tissues resembling diffuse idiopathic skeletal hyperostosis (DISH) in humans. Here, we investigated mechanisms underlying aberrant mineralization in ENT1(-/-) mice. Micro-CT revealed ectopic mineralization of spinal tissues in both male and female ENT1(-/-) mice, involving the annulus fibrosus of the intervertebral discs (IVDs) of older mice. IVDs were isolated from wild-type and ENT1(-/-) mice at 2months of age (prior to disc mineralization), 4, and 6months of age (disc mineralization present) and processed for real-time PCR, cell isolation, or histology. Relative to the expression of ENTs in other tissues, ENT1 was the primary nucleoside transporter expressed in wild-type IVDs and mediated the functional uptake of [(3)H]2-chloroadenosine by annulus fibrosus cells. No differences in candidate gene expression were detected in IVDs from ENT1(-/-) and wild-type mice at 2 or 4months of age. However, at 6months of age, expression of genes that inhibit biomineralization Mgp, Enpp1, Ank, and Spp1 were reduced in IVDs from ENT1(-/-) mice. To assess whether changes detected in ENT1(-/-) mice were cell autonomous, annulus fibrosus cell cultures were established. Compared to wild-type cells, cells isolated from ENT1(-/-) IVDs at 2 or 6months of age demonstrated greater activity of alkaline phosphatase, a promoter of biomineralization. Cells from 2-month-old ENT1(-/-) mice also showed greater mineralization than wild-type. Interestingly, altered localization of alkaline phosphatase activity was detected in the inner annulus fibrosus of ENT1(-/-) mice in vivo. Alkaline phosphatase activity, together with the marked reduction in mineralization inhibitors, is consistent with the mineralization of IVDs seen in ENT1(-/-) mice at older ages. These findings establish that both cell-autonomous and systemic mechanisms contribute to ectopic mineralization in ENT1(-/-) mice., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Intervertebral disc (IVD) degeneration is a common problem and treatment options for persistent symptomatic disease are limited. Tissue engineering is being explored for its ability to reconstitute the functional components of the IVD. The purpose of this study was to determine whether it was possible to form in vitro a triphasic construct consisting of nucleus pulposus (NP), cartilage endplate (CEP), and a porous calcium polyphosphate (CPP) bone substitute. Bovine articular chondrocytes were placed on the top surface of a porous CPP construct and allowed to form cartilage in vitro. Nucleus pulposus cells were then placed onto the in vitro-formed hyaline cartilage. At 24 h scanning electron microscopy demonstrated that the NP cells maintained their rounded morphology, similar to NP cells placed directly on porous CPP. At 8 weeks histological examination of the triphasic constructs by light microscopy showed that a continuous layer of NP tissue had formed and was fused to the underlying cartilage tissue, which itself was integrated with the porous CPP. The incorporation of the cartilage layer was beneficial to the construct by improving tissue attachment to the CPP, as demonstrated by increased peak load and increased energy required for failure during shear loading when compared to a biphasic construct composed of nucleus pulposus-bone substitute only. This study demonstrates that it is possible to generate a multi-component construct with the incorporation of a CEP-like layer resulting in improved bone substitute-to-IVD tissue interface characteristics.