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.

During intervertebral disc (IVD) maturation, notochordal cells (NCs) are replaced by chondrocyte-like cells (CLCs) in the nucleus pulposus, suggesting that NCs play a role in maintaining tissue health. Affirmatively, NC-conditioned medium (NCCM) exerts regenerative effects on CLC proliferation and extracellular matrix (ECM) production. The aim of this study was to identify NC-secreted substances that stimulate IVD regeneration. By mass spectrometry of porcine, canine and human NCCM, 149, 170 and 217 proteins were identified, respectively, with 66 proteins in common. Mainly ECM-related proteins were identified, but also organelle-derived and membranebound vesicle proteins. To determine whether the effect of NCCM was mediated by soluble and/or pelletable factors, porcine and canine NCCM were separated into a soluble (NCCM-S; peptides and proteins) and pelletable (NCCM-P; protein aggregates and extracellular vesicles) fraction by ultracentrifugation, and tested on bovine and canine CLCs in vitro, respectively. In each model, NCCM-S exerted a more pronounced anabolic effect than NCCM-P. However, glycosaminoglycan (GAG) uptake from the medium into the carrier gel prevented more definite conclusions. While the effect of porcine NCCM-P on bovine CLCs was negligible, canine NCCM-P appeared to enhance GAG and collagen type II deposition by canine CLCs. In conclusion, porcine and canine NCCM exerted their anabolic effects mainly through soluble factors, but also the pelletable NCCM factors showed moderate regenerative potential. Although the regenerative potential of NCCM-P should not be overlooked, future studies should focus on unraveling the protein-based regenerative mechanism from NCCM produced from isolated NCs, e.g. by NCCM fractionation and pathway blocking studies.

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For the purpose of obtaining an integral picture of anterior pituitary function in canine pituitary-dependent hyperadrenocorticism (PDH), 47 dogs with PDH and eight control dogs received combined administration of four hypophysiotropic hormones (CRH, GHRH, GnRH and TRH) and measurements were made of ACTH, cortisol, GH, LH, PRL and TSH. Basal plasma levels in 47 dogs with PDH were higher for ACTH, cortisol and PRL, lower for GH, and not different for LH (n = 25 noncastrated dogs) and TSH compared with controls (n = 8). In dogs with PDH the responses to combined hypophysiotropic stimulation, measured as increment and area under the curve (AUC), were not different for ACTH, lower for GH and TSH (increments and AUC) and higher for cortisol (increments), LH (AUC, n = 25 noncastrated dogs) and PRL (increments and AUC) than in controls. We conclude that pituitary function is altered in several respects in dogs with PDH. 1) In spite of persisting hypercortisolemia and the neoplastic transformation of the corticotropic cells, these cells usually remain responsive to combined hypophysiotropic stimulation. 2) Basal plasma GH concentrations and GH responsiveness in the combined stimulation test are decreased, probably as a result of the glucocorticoid-induced increase in somatostatin tone. 3) Plasma PRL concentrations and the PRL response to stimulation are increased, probably as a result of cosecretion with ACTH by the transformed corticotropic cells. 4) Despite the well known effect of glucocorticoids of decreasing circulating concentrations of gonadal steroids and thyroxine, the basal plasma concentrations of LH and TSH remain unchanged and there is a tendency to hyperresponsiveness to stimulation for LH and hyporesponsiveness for TSH. The most likely explanation for these changes is a dual effect of glucocorticoids: a direct effect on the gonads and thyroids and/or the transport and metabolism of their secretory products, and an influence on the sensitivity of the feedback control at the hypothalamic-pituitary level.

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