Degenerative disc disease (DDD) is associated with spinal pain often leading to long-term disability. However, the non-chondrodystrophic canine intervertebral disc is protected from the development of DDD, ostensibly due to its retention of notochordal cells (NC) in the nucleus pulposus (NP). In this study, we hypothesized that secretome analysis of the NC-rich NP will lead to the identification of key proteins that delay the onset of DDD. Using mass-spectrometry, we identified 303 proteins including components of TGFβ- and Wnt-signaling, anti-angiogeneic factors and proteins that inhibit axonal ingrowth in the bioactive fractions of serum free, notochordal cell derived conditioned medium (NCCM). Ingenuity Pathway Analysis revealed TGFβ1 and CTGF as major hubs in protein interaction networks. In vitro treatment with TGFβ1 and CTGF promoted the synthesis of healthy extra-cellular matrix proteins, increased cell proliferation and reduced cell death in human degenerative disc NP cells. A single intra-discal injection of recombinant TGFβ1 and CTGF proteins in a pre-clinical rat-tail disc injury model restored the NC and stem cell rich NP. In conclusion, we demonstrate the potential of TGFβ1 and CTGF to mitigate the progression of disc degeneration and the potential use of these molecules in a molecular therapy to treat the degenerative disc.

The tremendous cost, pain and disability associated with degenerative disc disease (DDD) makes the development of a biological agent that can mitigate the course of DDD, a critical unmet need. We have identified and reported that a single injection of a combination of recombinant human (rh) Transforming growth factor beta 1 (TGF-β1) and Connective tissue growth factor (CTGF) proteins into the injured intervertebral disc (IVD) nucleus pulposus (NP) can mediate DDD in a pre-clinical rodent model. In this study, we developed and evaluated the efficacy of a novel molecular therapy (NTG-101) containing rhTGF-β1 and rhCTGF proteins suspended in an excipient solution using in vivo models of DDD including rat-tail and chondrodystrophic (CD) canines. Needle puncture injury in CD-canine NPs resulted in loss of hydration, disc height and showed radiographic evidence of DDD like humans. However, NTG-101-injected IVDs maintained disc height and demonstrated retention of viscoelastic properties as compared to IVDs injected with phosphate buffer saline (PBS, 1X, pH = 7.2). In addition, a single intra-discal injection of NTG-101 into the injured IVD-NPs resulted in sustained expression of healthy extra-cellular matrix (ECM) proteins (aggrecan, collagen 2A1) and reduced expression of inflammation associated proteins and molecules (IL-1β, IL-6, IL-8, MMP-13, Cox-2 and PGE2) as compared to vehicle controls. In conclusion, we demonstrated that a single intra-discal injection of the novel formulation, NTG-101 confers a robust anti-inflammatory, anti-catabolic and pro-anabolic effects in pre-clinical models of DDD thereby restoring homeostasis. These findings suggest the therapeutic potential of NTG-101 for clinical use.

We propose that the IVD NP represents a regenerative niche suggesting that the IVD could represent a readily accessible source of precursor cells for neural repair and regeneration.

IntroductionIn the present study, we sought to quantify and contrast the secretome and biomechanical properties of the non-chondrodystrophic (NCD) and chondrodystrophic (CD) canine intervertebral disc (IVD) nucleus pulposus (NP).MethodsWe used iTRAQ proteomic methods to quantify the secretome of both CD and NCD NP. Differential levels of proteins detected were further verified using immunohistochemistry, Western blotting, and proteoglycan extraction in order to evaluate the integrity of the small leucine-rich proteoglycans (SLRPs) decorin and biglycan. Additionally, we used robotic biomechanical testing to evaluate the biomechanical properties of spinal motion segments from both CD and NCD canines.ResultsWe detected differential levels of decorin, biglycan, and fibronectin, as well as of other important extracellular matrix (ECM)-related proteins, such as fibromodulin and HAPLN1 in the IVD NP obtained from CD canines compared with NCD canines. The core proteins of the vital SLRPs decorin and biglycan were fragmented in CD NP but were intact in the NP of the NCD animals. CD and NCD vertebral motion segments demonstrated significant differences, with the CD segments having less stiffness and a more varied range of motion.ConclusionsThe CD NP recapitulates key elements of human degenerative disc disease. Our data suggest that at least some of the compromised biomechanical properties of the degenerative disc arise from fibrocartilaginous metaplasia of the NP secondary to fragmentation of SLRP core proteins and associated degenerative changes affecting the ECM. This study demonstrates that the degenerative changes that naturally occur within the CD NP make this animal a valuable animal model with which to study IVD degeneration and potential biological therapeutics.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0733-z) contains supplementary material, which is available to authorized users.

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Equilibrative Nucleoside Transporters (SLC29) are a family of proteins that transport nucleosides, nucleobases and nucleoside analogue drugs across cellular membranes. ENT1 is expressed ubiquitously in mammalian tissues and responsible for a significant portion of nucleoside analog drug uptake in humans. Despite the important clinical role of ENT1, many aspects of the regulation of this protein remain unknown. A major outstanding question in this field is the whether ENT1 is phosphorylated directly. To answer this question, we overexpressed tagged human (h) and mouse (m) ENT1, affinity purified protein using the tag, conducted phosphoamino acid analysis and found that m/hENT1 is predominantly phosphorylated at serine residues. The large intracellular loop of ENT1, between transmembrane domains 6 and 7, has been suggested to be a site of regulation by phosphorylation, therefore we generated His/Ubiquitin tagged peptides of this region and used them for in vitro kinase assays to identify target serines. Our data support a role for PKA and PKC in the phosphorylation of ENT1 within the intracellular loop and show that PKA can phosphorylate multiple sites within this loop while PKC specifically targets serines 279 and 286 and threonine 274. These data demonstrate, for the first time, that ENT1 is a phosphoprotein that can be directly phosphorylated at several sites by more than one kinase. The presence of multiple kinase targets within the loop suggests that ENT1 phosphorylation is considerably more complex than previously thought and thus ENT1 may be subject to phosphorylation by multiple pathways.

BackgroundNotochordal cell conditioned medium (NCCM) derived from non-chondrodystrophic dogs has pro-anabolic and anti-catabolic effects upon nucleus pulposus (NP) cells. Here, for the first time, we assessed the ability of NCCM to influence the production of extracellular matrix and inflammatory proteins by healthy and osteoarthritic human chondrocytes within engineered cartilage tissues. We hypothesized that, similar to its action on NP cells, NCCM exerts metabolic and anti-catabolic effects on human articular chondrocytes and has the potential to significantly counteract inflammatory mediators.MethodsChondrocytes from nine non-osteoarthritic patients and from six osteoarthritic (OA) donors at the time of total knee arthroplasty were chondro-differentiated in pellets for 2 weeks. Non-OA pellets were exposed for 72 hours to IL-1β/TNF-α and then cultured up to 14 days in 2 % FBS-supplemented NCCM or 2 % FBS-supplemented medium (control (ctr)). OA pellets were cultured in NCCM or ctr medium without pro-inflammatory treatment. Tissues after each culture phase were analyzed biochemically (GAG/DNA), (immuno-) histologically (collagen I, II and GAG) and by Western blotting. Supernatants were analyzed by ELISA.ResultsResponse to NCCM was age and disease dependent with healthy chondrocyte pellets (from donors >55 years of age) recovering their glycosaminoglycan (GAG) contents to baseline levels only with NCCM. OA pellets treated with NCCM significantly increased GAG content (1.8-fold) and levels of hyaluronic acid link protein (HAPLN), fibromodulin and SOX-9. The catabolic proteins (matrix metalloproteinase (MMP)-3 and MMP-13) and pro-inflammatory enzyme levels (cyclooxygenase-2 (COX-2)) were markedly reduced and there was significantly reduced secretion of pro-inflammatory chemokines (IL-6 and IL-8).ConclusionsNCCM restores cartilage matrix production of end-stage human OA chondrocytes towards a healthy phenotype and suppresses the production of inflammatory mediators. Harnessing the necessary and sufficient factors within NCCM that confers chondroprotection and regenerative effects could lead to a minimally invasive agent for treatment of degenerative and inflammatory joint diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1026-x) contains supplementary material, which is available to authorized users.

Cellular replacement therapy using mesenchymal stem cells (MSCs) and/or the delivery of growth factors are at the forefront of minimally invasive biological treatment options for Degenerative Disc Disease (DDD). In this study, we compared the therapeutic potential of a novel drug candidate, NTG-101 to MSCs, including rat cartilage derived stem cells (rCDSCs), bone marrow stem cells (rBMSCs) and human Umbilical Cord Derived Mesenchymal Stem Cells (hUCMSCs) for the treatment of DDD. We induced DDD using a validated image-guided needle puncture injury in rat-tail IVDs. Ten weeks post-injury, animals were randomized and injected with MSCs, NTG-101 or vehicle. At the end of the study, histological analysis of the IVD-Nucleus Pulposus (NPs) injected with NTG-101 or rCDSCs showed a healthy or mild degenerative phenotype in comparison to vehicle controls. Immunohistochemical analysis revealed strong expression of aggrecan, collagen 2, brachyury and Oct4 in IVD-NPs injected with NTG-101. Our results also demonstrated suppression of inflammation induced p38 and NFκB resulting in inhibition of catabolic genes, but activation of Smad-2/3, Erk-1/2 and Akt-dependent signaling inducing anabolic genes in IVD-NP on treatment with NTG-101. In conclusion, a single injection of NTG-101 into the degenerative disc demonstrated superior benefits compared to stem cell transplantation.