During intervertebral disc (IVD) maturation, the main cell type shifts from notochordal cells (NCs) to chondrocytelike cells (CLCs). NCs secrete factors with regenerative potential, making them an interesting focus for regenerative treatments. During initial development, these strategies preferably employ non-human donors due to easy availability of their NC-rich nucleus pulposus (NP) tissue. To increase the success of translating these strategies for clinical application, this study aimed to delineate whether NC-secreted factors of different species have a regenerative effect on human CLCs. Human, canine and porcine NC-rich NP tissue and NC-conditioned medium (NCCM) were analysed biochemically and histologically. Human CLC micro-aggregates from degenerated IVDs were cultured in human, canine or porcine NCCM. Collagen, glycosaminoglycan (GAG) and DNA content was determined and histology was performed. Canine and porcine NPs were richer in NCs than human NPs. Human NPs contained the highest collagen content, whereas the DNA and GAG content of canine NPs was significantly higher than that of human or porcine NPs. NCCM from all species significantly increased the DNA and GAG content of the human CLC micro-aggregates. Porcine and canine NCCM were significantly more potent than human NCCM in inducing GAG deposition, whereas only human NCCM induced collagen type II production. Secreted factors from human, canine and porcine NC-rich NPs exerted regenerative effects on human CLCs, indicating a cross-species effect. Bioactive compound(s) are present in NCCM of different species that may reverse human IVD degeneration, supporting further research into strategies based on NC-technology employing canine or porcine models for their translation into humans.

Introduction Intervertebral disc (IVD) degeneration, a major cause for low back pain, is characterized by a decreasing number of nucleus pulposus cells (NPCs),1 which are then unable to maintain a healthy disc matrix. Bone marrow-derived stromal stem cells (MSCs) are attractive cells for IVD regeneration because they can acquire a phenotype consistent with NPCs, and injection of MSCs into the IVD has been shown to inhibit the progression of degenerative changes.2 Current regenerative strategies concentrate on augmenting the effect of the transplanted MSCs or promoting the phenotype of the resident cells (NPCs). In this respect, conditioned medium from notochordal cells, present in the immature IVD (NCCM), has been shown to stimulate NPC metabolism,3 and modulate differentiation of MSCs toward a NP-like phenotype.4 The goal of this study was to assess, in a canine in vitro model, the stimulatory effects of NCCM on NPCs, MSCs and their mixture (NPCs + MSCs), as well as the effect of MSCs on NPCs alone. Materials and Methods Canine MSCs and NPCs were harvested from mature chondrodystrophic dogs (Beagles) and cryopreserved until further use. NCCM was produced from 1- to 1.5-year-old nonchondrodystrophic dogs; healthy canine nucleus pulposus tissue was cultured for 4 days in high glucose (hg)DMEM + 1% pen/strep. MSCs, NPCs, and the combination of both cell types (1:1) were then cultured for 4 weeks in 1.2% alginate beads (MSCs, NPCs alone: 3 million cells/mL; NPC + MSC: 6 million cells/mL) at 37°C, 5% O2, and 5% CO2. The beads received either base medium (BM: hgDMEM + 5% stripped FCS + 1% pen/strep), NCCM (supplemented with 5% stripped FCS + 1% pen/strep), or positive control medium (TGF: BM + 10 ng/mL TGF-β1). A crossover group (Cr) was given NCCM for 1 week and BM for the remainder of the culture duration to investigate whether short-term exposure to NCCM was enough to promote matrix production. At days 1 and 28, beads were analyzed for cell viability by confocal microscopy (Calcein AM/Propidium iodide staining), for glycosaminoglycan (GAG) and DNA contents by DMMB assay and Qubit Quantification Platform, respectively, and for GAG deposition by Alcian blue staining. Gene expression (Collagen 2, Aggrecan, and SOX9) was determined by RT-qPCR. Statistical analysis was done with a Kruskal-Wallis test followed by a Mann-Whitney U-test with Bonferroni correction for post hoc testing. Statistical significance was assumed for p < 0.05 Results At days 1 and 28, no notable cell death was observed for any group. When cultured in NCCM, GAG content increased for both NPCs and MSCs, compared with BM (Fig. 1A). Addition of MSCs to NPCs did not increase GAG content. These findings were confirmed by Alcian blue staining. DNA content decreased compared with day 0 for both cell types and NPC + MSC cultured in BM (Fig. 1B). DNA content, however, increased for NPC in NCCM compared with BM. TGF-β1 increased GAG (90 µg/bead) and DNA (1.4 µg/bead) content for NPCs and NPCs + MSCs. In contrast, TGF-β1 did not have an effect on MSC GAG ...