Notochordal cells protect nucleus pulposus cells from degradation and apoptosis: implications for the mechanisms of intervertebral disc degeneration

Abstract

IntroductionThe relative resistance of non-chondrodystrophic (NCD) canines to degenerative disc disease (DDD) may be due to a combination of anabolic and anti-catabolic factors secreted by notochordal cells within the intervertebral disc (IVD) nucleus pulposus (NP). Factors known to induce DDD include interleukin-1 beta (IL-1ß) and/or Fas-Ligand (Fas-L). Therefore we evaluated the ability of notochordal cell conditioned medium (NCCM) to protect NP cells from IL-1ß and IL-1ß +FasL-mediated cell death and degeneration.MethodsWe cultured bovine NP cells with IL-1ß or IL-1ß+FasL under hypoxic serum-free conditions (3.5% O2) and treated the cells with either serum-free NCCM or basal medium (Advanced DMEM/F-12). We used flow cytometry to evaluate cell death and real-time (RT-)PCR to determine the gene expression of aggrecan, collagen 2, and link protein, mediators of matrix degradation ADAMTS-4 and MMP3, the matrix protection molecule TIMP1, the cluster of differentiation (CD)44 receptor, the inflammatory cytokine IL-6 and Ank. We then determined the expression of specific apoptotic pathways in bovine NP cells by characterizing the expression of activated caspases-3, -8 and -9 in the presence of IL-1ß+FasL when cultured with NCCM, conditioned medium obtained using bovine NP cells (BCCM), and basal medium all supplemented with 2% FBS.ResultsNCCM inhibits bovine NP cell death and apoptosis via suppression of activated caspase-9 and caspase-3/7. Furthermore, NCCM protects NP cells from the degradative effects of IL-1ß and IL-1ß+Fas-L by up-regulating the expression of anabolic/matrix protective genes (aggrecan, collagen type 2, CD44, link protein and TIMP-1) and down-regulating matrix degrading genes such as MMP-3. Expression of ADAMTS-4, which encodes a protein for aggrecan remodeling, is increased. NCCM also protects against IL-1+FasL-mediated down-regulation of Ank expression. Furthermore, NP cells treated with NCCM in the presence of IL-1ß+Fas-L down-regulate the expression of IL-6 by almost 50%. BCCM does not mediate cell death/apoptosis in target bovine NP cells.ConclusionsNotochordal cell-secreted factors suppress NP cell death by inhibition of activated caspase-9 and -3/7 activity and by up-regulating genes contributing anabolic activity and matrix protection of the IVD NP. Harnessing the restorative powers of the notochordal cell could lead to novel cellular and molecular strategies in the treatment of DDD.

The species-specific regenerative effects of notochordal cell-conditioned medium on chondrocyte-like cells derived from degenerated human intervertebral discs

“…Unpublished work by our group indicates that also no difference in biologic activity was established between canine NCCM that was generated under NX or HX conditions. In agreement with our studies, previous work demonstrating the regenerative potential of porcine and canine NC-secreted factors have used NCCM generated under HX (Abbott et al, 2012;Purmessur et al, 2011) and NX (Aguiar et al, 1999;Boyd et al, 2004;Erwin et al, 2011;Korecki et al, 2010) conditions. Altogether, this may imply that oxygen status during NCCM generation does not result in a differential biologic activity of NC-secreted factors on human CLCs.…”

“…NCCM of all species, generated under identical conditions on the basis of wet NP weight, increased CLC cell numbers and GAG deposition, but canine and porcine NCCM appeared more potent than human NCCM. Although the IVD is an avascular hypoxic structure and several studies have demonstrated that NCs respond superior to hypoxic than normoxic culture conditions (Erwin et al, 2009;Omlor et al, 2014), no difference in regenerative potential was found between human NCCM generated under HX or NX conditions. Unpublished work by our group indicates that also no difference in biologic activity was established between canine NCCM that was generated under NX or HX conditions.…”

“…chondrodystrophic dogs) coincides with the onset of IVD degeneration. NC-conditioned medium (NCCM, containing factors secreted by NCs) and NC:CLC co-culture stimulate in vitro differentiation of MSCs into a NP-like phenotype Korecki et al, 2010;Purmessur et al, 2011), protect the NP from apoptosis (Erwin et al, 2011;Mehrkens et al, 2013), and/or increase CLC proliferation and GAG production (Abbott et al, 2012;Aguiar et al, 1999;Gantenbein et al, 2014;Potier et al, 2014). In addition, NCs significantly stimulate CLC activity and GAG production (Gantenbein-Ritter et al, 2012) and produce more GAGs than CLCs (Saggese et al, 2014).…”

An understanding of intervertebral disc development, maturation and cell phenotype provides clues to direct cell-based tissue regeneration therapies for disc degeneration

“…Notochordal cells have been shown to produce more proteoglycans than smaller NP cells [69] and, when in coculture, notochordal cells interact with small NP cells stimulating them to produce a healthier extracellular matrix [70,71]. Recent findings also show that notochordal cells can prevent Il-1-induced cell death in small NP cells [72]. Therefore, defining the notochordal cell phenotype and tracking these cells in human discs would also allow clarifying their role in the disc's physiology and disease.…”

Determination of the potential of induced pluripotent stem cells to differentiate into mouse nucleus pulposus cells in vitro

“…The NP is surrounded by the AF, and is a gel-like structure that contains chondrocyte-like cells (Bowles et al, 2011;Dahia et al, 2012). During IVD, NP cells decrease in number and the remaining cells have an altered phenotype that is associated with an increased breakdown of the extracellular matrix and altered matrix synthesis (Urban et al, 2004;Skubutyte et al, 2010;Erwin et al, 2011). Traditional treatment options for managing IVD mainly include conservative and surgical treatments.…”