The socioeconomic burden of chronic back pain related to intervertebral disc (IVD) disease is high and current treatments are only symptomatic. Minimally invasive strategies that promote biological IVD repair should address this unmet need. Notochordal cells (NCs) are replaced by chondrocyte-like cells (CLCs) during IVD maturation and degeneration. The regenerative potential of NC-secreted substances on CLCs and mesenchymal stromal cells (MSCs) has already been demonstrated. However, identification of these substances remains elusive. Innovatively, this study exploits the regenerative NC potential by using healthy porcine NC-derived matrix (NCM) and employs the dog as a clinically relevant translational model. NCM increased the glycosaminoglycan and DNA content of human and canine CLC aggregates and facilitated chondrogenic differentiation of canine MSCs in vitro. Based on these results, NCM, MSCs and NCM+MSCs were injected in mildly (spontaneously) and moderately (induced) degenerated canine IVDs in vivo and, after six months of treatment, were analyzed. NCM injected in moderately (induced) degenerated canine IVDs exerted beneficial effects at the macroscopic and MRI level, induced collagen type II-rich extracellular matrix production, improved the disc height, and ameliorated local inflammation. MSCs exerted no (additive) effects. In conclusion, NCM induced in vivo regenerative effects on degenerated canine IVDs. NCM may, comparable to demineralized bone matrix in bone regeneration, serve as ‘instructive matrix’, by locally releasing growth factors and facilitating tissue repair. Therefore, intradiscal NCM injection could be a promising regenerative treatment for IVD disease, circumventing the cumbersome identification of bioactive NC-secreted substances.

Objectives: Conditioned medium derived from notochordal cell-rich nucleus pulposus tissue (NCCM) was previously shown to have a stimulatory effect on bone marrow stromal cells (BMSCs) and nucleus pulposus cells (NPCs) individually, in mixed species in vitro cell models. The objective of the current study was to assess the stimulatory effect of NCCM on NPCs in a homologous canine in vitro model and to investigate whether combined stimulation with NCCM and addition of BMSCs provides a synergistic stimulatory effect. Methods: BMSCs and NPCs were harvested from chondrodystrophic dogs with confirmed early intervertebral disc (IVD) degeneration. NCCM was produced from NP tissue of nonchondrodystrophic dogs with healthy IVDs. BMSCs or NPCs alone (3 · 10 6 cells/mL) and NPCs + BMSCs (6 · 10 6 cells/mL; mixed 1:1) were cultured for 4 weeks in 1.2% alginate beads under base medium (BM), NCCM, or with addition of 10 ng/mL transforming growth factor-b1 (TGF-b1) as a positive control. Beads were assessed for glycosaminoglycan (GAG) and DNA contents by biochemical assays, GAG deposition by Alcian blue staining, and gene expression (aggrecan, versican, collagen 1 and 2, SOX9, A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), and matrix metalloproteinase 13 [MMP13]) with real-time quantitative RT-PCR. Results: NCCM increased NPC proliferation, proteoglycan production, and expression of genes associated with a healthy NP-like phenotype. BMSCs also showed increased proteoglycan production under NCCM, but these effects were not observed at the gene level. Combined stimulation of NPCs with NCCM and coculturing with BMSCs did not result in increased proteoglycan content compared to stimulation with NCCM alone. Discussion: NCCM stimulates matrix production by both NPCs and BMSCs and directs NPCs toward a healthier phenotype. NCCM is therefore promising for IVD regeneration and identification of the bioactive components will be helpful to further develop this approach. In the current study, no synergistic effect of adding BMSCs was observed.

This study shows that, indeed, NCCM is capable of stimulating NPC matrix production within the NP environment. The lack of increased DNA content in the BMSC-injected groups indicates that BMSCs have died over time. Identification of the bioactive factors in NCCM is crucial for further development of an NCCM-based treatment for intervertebral disc regeneration.

Notochordal cell derived matrix (NCM) can induce regenerative effects on nucleus pulposus cells and may exert such effects on chondrocytes as well. Furthermore, when dissolved at low concentrations, NCM forms a viscous fluid with potential lubricating properties. Therefore, this study tests the feasibility of the use of NCM as a regenerative lubricant for the osteoarthritic joint. Chondrocyte-seeded alginate beads were cultured in base medium (BM), BM with NCM (NCM), or BM with TGF-β1 (TGF), as well as BM and NCM treated with IL-1β. NCM increased GAG deposition and cell proliferation (stronger than TGF), and GAG/DNA ratio and hydroxyproline content (similar to TGF). These effects were maintained in the presence of IL-1β. Moreover, NCM mitigated expression of IL-1β-induced IL-6, IL-8, ADAMTS-5 and MMP-13. Reciprocating sliding friction tests of cartilage on glass were performed to test NCM’s lubricating properties relative to hyaluronic acid (HA), and showed a dose-dependent reduction in coefficient of friction with NCM, similar to HA. NCM has anabolic and anti-inflammatory effects on chondrocytes, as well as lubricating properties. Therefore, intra-articular NCM injection may have potential as a treatment to minimize pain while restoring the affected cartilage tissue in the osteoarthritic joint.

In cartilage tissue engineering studies, the stimulatory effect of mechanical perturbation declines after the first 2 weeks of culture. Similarly, it is known that chondrocyte-agarose constructs should not be loaded within the first days after seeding, to prevent considerable cell death, suggesting a mechanical threshold. This study aims to establish a relationship between chondrocyte deformation and death, and to evaluate the protective effect of the pericellular matrix (PCM) that is formed in 3D cultures. Chondrocyte viability was monitored every hour for 24 h after applying a strain range of 0% to 25% to agarose constructs containing chondrocytes, cultured for 1, 3, 5, 7, or 10 days. At these culture time points, the PCM thickness and chondrocyte deformation were assessed by means of histology and assayed for biochemical contents. Inverse finite element (FE) simulations were used to evaluate the change of mechanical properties of the chondrocyte and PCM over the 10-day culture duration. Chondrocyte death was demonstrated to be dependent on both the magnitude and duration of straining. The highest cell death was observed at day 1 (43%), reducing over culture duration (15% at day 3 and 2.5% at day 10). Cell deformation at 25% compression decreased significantly over culture duration (aspect ratio of 2.24±0.67 at day 1 and 1.45±0.24 at day 3) and with increased matrix production. Inverse FE simulations showed an increasing PCM Young's modulus of 45 kPa at day 3 to 162 kPa at day 10. The current results provide evidence for a mechanical threshold for chondrocyte death and for the protective effect of the PCM. As such, these insights may help in establishing mechanical loading protocols for cartilage tissue engineering studies.