Nucleus pulposus notochord cells secrete connective tissue growth factor and Up‐regulate proteoglycan expression by intervertebral disc chondrocytes

Abstract

Objective. To identify the components of conditioned medium obtained from intervertebral disc nucleus pulposus-derived canine notochord cells, and to evaluate the capacity of such factors to affect discderived chondrocyte gene expression of aggrecan, versican, and hyaluronic acid synthase 2 (HAS-2) as a function of culture conditions.Methods. Canine notochord cells obtained from nonchondrodystrophic dogs were cultured within alginate beads under conditions of serum deficiency (Dulbecco's modified Eagle's medium [DMEM]) to produce notochord cell-conditioned medium (NCCM). NCCM was evaluated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectroscopy. Bovine disc-derived chondrocytes were cultured with serumdeficient medium (DMEM) and NCCM and assayed for the effect of tissue culture conditions on aggrecan, versican, and HAS-2 gene expression. Next, chondrocyte gene expression for aggrecan was evaluated using DMEM containing recombinant connective tissue growth factor (rCTGF), and the results compared with those obtained using NCCM and DMEM.Results. NCCM contained aggrecan, Cu/Zn superoxide dismutase, fibronectin, and CTGF precursor. Culture with NCCM caused an up-regulation of aggrecan, versican, and HAS-2 gene expression. NCCM induced aggrecan gene expression in chondrocytes at a level similar to that induced by 100-200 ng/ml rCTGF. Nonchondrodystrophic and chondrodystrophic canine notochord cells exhibited similar levels of CTGF gene expression.Conclusion. Nucleus pulposus-derived notochord cells secrete CTGF (CCN2), a recently discovered multifunctional growth factor. There is no difference between CTGF gene expression in nonchondrodystrophic and chondrodystrophic canine notochord cells, suggesting a possible role of CTGF as an anabolic factor within the disc nucleus that is, to at least some degree, dependent on the population of notochord cells within the disc nucleus.More than half of all musculoskeletal disability is associated with degenerative disc disease, making this one of the most common and expensive medical conditions in the population; low back pain represents the leading cause of disability in persons under the age of 45 years in the US (1-3). The factors that account for the vulnerability of the disc to degeneration and the limited capacity of the disc for repair remain largely unknown. Moreover, there is currently no biologically explained distinction between individuals who do and those do not develop degenerative disc disease with age. Studies in twins have suggested that genetic susceptibility plays a critical role in the development of degenerative disc disease (4,5).Some animal species, such as dogs, include naturally occurring strains that rarely develop degenerative disc disease. These degenerative disc disease-resistant strains are distinctive in that they maintain a resident population of notochord cells within the nucleus pulposus into adult life. In nonchondrodystrophic dogs, notochord cells in the intervertebral disc are preserved, ...

Soluble and pelletable factors in porcine, canine and human notochordal cell-conditioned medium: implications for IVD regeneration

“…proteoglycans, collagens and keratins, and organelle-derived proteins. However, specific growth factors, potentially responsible for NCCM's regenerative effect, were not identified, which is in contrast with previous studies in which CTGF was identified in canine (Erwin et al, 2006) and porcine (Gantenbein et al, 2014) NCCM. This discrepancy may be due to the method of NCCM generation.…”

“…Up to date, only few studies reported on the potential biologically active NCCM factors. Connective tissue growth factor (CTGF) was found in canine (Erwin et al, 2006) and porcine (Gantenbein et al, 2014) NCCM and alpha-2-macroglobulin, clusterin and tenascin were detected in porcine NCCM (Purmessur et al, 2011).…”

“…In addition, a dimethyl methylene blue (DMMB) assay (Farndale et al, 1982) was performed to determine the glycosaminoglycan (GAG) concentration.…”

The evolutionary importance of cell ratio between notochordal and nucleus pulposus cells: an experimental 3-D co-culture study

“…Future experiments will test the influence of cell-cell contact by seeding pNCs and bNPCs into alginate beads enabling cell-cell interaction and also the importance of hypoxic conditions. Gene expression revealed a significant up-regulation of Col 1 and Col 2 by the pNCs in the presence of bNPCs, which confirms previous findings of Boyd [3] and Aguiar et al [1] that NCs express certain stimulating cytokines, which seems evident and has been demonstrated using conditioning media [3,9,22]. Korecki et al also detected that SOX-9 (transcription factor for chondrogenesis) was down-regulated if human mesenchymal stem cells (hMSCs) are exposed to notochordal conditioned medium (NCM) as compared to a chondrogenic medium containing TGF-b.…”

Médecine régénératrice du disque intervertébral

“…Ainsi, l'hypothèse d'une différenciation progressive des cellules notochordales en nucléopul-pocytes fait actuellement l'objet d'investigations. De plus, de récentes études ont démontré que les cellules notochordales stimulent la synthèse de protéoglycanes (PG) par les nucléopul-pocytes ainsi que leur prolifération, grâce à la sécrétion notamment du connective tissue growth factor (CTGF/CCN2) (Figure 2) [11]. Ces protéoglycanes retiennent les molécules d'eau et offrent au NP des propriétés de déformabilité et de résistance aux contraintes méca-niques incomparables et indispensables au maintien de la dynamique rachidienne [2].…”