Activation of intervertebral disc cells by co-culture with notochordal cells, conditioned medium and hypoxia.
ABSTRACT: BACKGROUND: Notochordal cells (NC) remain in the focus of research for regenerative therapy for the degenerated intervertebral disc (IVD) due to their progenitor status. Recent findings suggested their regenerative action on more mature disc cells, presumably by the secretion of specific factors, which has been described as notochordal cell conditioned medium (NCCM). The aim of this study was to determine NC culture conditions (2D/3D, fetal calf serum, oxygen level) that lead to significant IVD cell activation in an indirect co-culture system under normoxia and hypoxia (2% oxygen). METHODS: Porcine NC was kept in 2D monolayer and in 3D alginate bead culture to identify a suitable culture system for these cells. To test stimulating effects of NC, co-cultures of NC and bovine derived coccygeal IVD cells were conducted in a 1:1 ratio with no direct cell contact between NC and bovine nucleus pulposus cell (NPC) or annulus fibrosus cells (AFC) in 3D alginate beads under normoxia and hypoxia (2%) for 7 and 14 days. As a positive control, NPC and AFC were stimulated with NC-derived conditioned medium (NCCM). Cell activity, glycosaminoglycan (GAG) content, DNA content and relative gene expression was measured. Mass spectrometry analysis of the NCCM was conducted. RESULTS: We provide evidence by flow cytometry that monolayer culture is not favorable for NC culture with respect to maintaining NC phenotype. In 3D alginate culture, NC activated NPC either in indirect co-culture or by addition of NCCM as indicated by the gene expression ratio of aggrecan/collagen type 2. This effect was strongest with 10% fetal calf serum and under hypoxia. Conversely, AFC seemed unresponsive to co-culture with pNC or to the NCCM. Further, the results showed that hypoxia led to decelerated metabolic activity, but did not lead to a significant change in the GAG/DNA ratio. Mass spectrometry identified connective tissue growth factor (CTGF, syn. CCN2) in the NCCM. CONCLUSIONS: Our results confirm the requirement to culture NC in 3D to best maintain their phenotype, preferentially in hypoxia and with the supplementation of FCS in the culture media. Despite these advancements, the ideal culture condition remains to be identified.
Project description:BACKGROUND:Chronic low back pain can be associated with the pathological ingrowth of blood vessels and nerves into intervertebral discs (IVDs). The notochord patterns the IVD during development and is a source of anti-angiogenic soluble factors such as Noggin and Chondroitin sulfate (CS). These factors may form the basis for a new minimally invasive strategy to target angiogenesis in the IVD. OBJECTIVE:To examine the anti-angiogenic potential of soluble factors from notochordal cells (NCs) and candidates Noggin and CS under healthy culture conditions and in the presence of pro-inflammatory mediators. DESIGN:NC conditioned media (NCCM) was generated from porcine NC-rich nucleus pulposus tissue. To assess the effects of NCCM, CS and Noggin on angiogenesis, cell invasion and tubular formation assays were performed using human umbilical vein endothelial cells (HUVECs) ± tumor necrosis factor alpha (TNF? [10 ng/ml]). vascular endothelial growth factor (VEGF)-A, MMP-7, interleukin-6 (IL-6) and IL-8 mRNA levels were assessed using qRT-PCR. RESULTS:NCCM (10 & 100%), CS (10 and 100 ?g) and Noggin (10 and 100 ng) significantly decreased cell invasion of HUVECs with and without TNF?. NCCM 10% and Noggin 10 ng inhibited tubular formation with and without TNF? and CS 100 ?g inhibited tubules in Basal conditions whereas CS 10 ?g inhibited tubules with TNF?. NCCM significantly decreased VEGF-A, MMP-7 and IL-6 mRNA levels in HUVECs with and without TNF?. CS and Noggin had no effects on gene expression. CONCLUSIONS:We provide the first evidence that soluble factors from NCs can inhibit angiogenesis by suppressing VEGF signaling. Notochordal-derived ligands are a promising minimally invasive strategy targeting neurovascular ingrowth and pain in the degenerated IVD.
Project description: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.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-?1 (TGF-?1) 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.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.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.
Project description:Painful human intervertebral discs (IVDs) exhibit nerve growth deep into the IVD. Current treatments for discogenic back pain do not address the underlying mechanisms propagating pain and are often highly invasive or only offer temporary symptom relief. The notochord produces factors during development that pattern the spine and inhibit the growth of dorsal root ganglion (DRG) axons into the IVD. We hypothesize that notochordal cell (NC)-conditioned medium (NCCM) includes soluble factors capable of inhibiting neurite growth and may represent a future therapeutic target.To test if NCCM can inhibit neurite growth and determine if NC-derived glycosaminoglycans (GAGs) are necessary candidates for this inhibition.Human neuroblastoma (SH-SY5Y) cells and rat DRG cells were treated with NCCM in two-dimensional culture in vitro, and digestion and mechanistic studies determined if specific GAGs were responsible for inhibitory effects.Notochordal cell-conditioned medium was generated from porcine nucleus pulposus tissue that was cultured in Dulbecco's modified eagle's medium for 4 days. A dose study was performed using SH-SY5Y cells that were seeded in basal medium for 24 hours and neurite outgrowth and cell viability were assessed after treatment with basal media or NCCM (10% and 100%) for 48 hours. Glycosaminoglycans from NCCM were characterized using multiple digestions and liquid chromatography mass spectroscopy (LC-MS). Neurite growth was assessed on both SH-SY5Y and DRG cells after treatment with NCCM with and without GAG digestion.Notochordal cell-conditioned medium significantly inhibited the neurite outgrowth from SH-SY5Y cells compared with basal controls without dose or cytotoxic effects; % of neurite expressing cells were 39.0±2.9%, 27.3±3.6%, and 30.2±2.7% and mean neurite length was 60.3±3.5, 50.8±2.4, 53.2±3.7 ?m for basal, 10% NCCM, and 100% NCCM, respectively. Digestions and LC-MS determined that chondroitin-6-sulfate was the major GAG chain in NCCM. Neurite growth from SH-SY5Y and DRG cells was not inhibited when cells were treated with NCCM with digested chondroitin sulfate (CS).Soluble factors derived from NCCM were capable of inhibiting neurite outgrowth in multiple neural cell types without any negative effects on cell viability. Cleavage of GAGs via digestion was necessary to reverse the neurite inhibition capacity of NCCM. We conclude that intact GAGs such as CS secreted from NCs are potential candidates that could be useful to reduce neurite growth in painful IVDs.
Project description:Notochordal cells (NCs) are influential in development of the intervertebral disc (IVD) and species that retain NCs do not degenerate. IVD repair using bone marrow derived mesenchymal stem cells (MSCs) is an attractive approach and the harsh microenvironment of the IVD suggests pre-differentiation is a necessary first step. The goal of this study was to use soluble factors from NCs in alginate and NCs in their native tissue to differentiate human MSCs to a young nucleus pulposus (NP) phenotype.Human MSCs (cultured under micromass conditions for 21 days in hypoxia) were differentiated with conditioned medium derived from porcine notochordal cells in native tissue (NCT) or in alginate beads (NCA), and compared with chondrogenic (TGF?-3) or basal medium. A PCR array of 42 genes was utilized to screen a large number of genes known to be associated with the healthy NP phenotype and pellet cultures were also evaluated for glycosaminoglycan content, histology and viability. Proteomic analysis was used to assess candidate soluble factors in NCA and NCT.Notochordal cell conditioned media had diverse effects on MSC phenotype. NCT resulted in the highest levels of glycosaminoglycan (GAG), as well as up-regulation of SOX9 and Collagen II gene expression. NCA demonstrated effects that were catabolic yet also anti-fibrotic and minimally hypertrophic with down-regulation of Collagens I and III and low levels of Collagen X, respectively. Micromass culture and hypoxic conditions were sufficient to promote chondrogenesis demonstrating that both basal and chondrogenic media produced similar phenotypes. Candidate matricellular proteins, clusterin and tenascin were identified by proteomics in the NCA group.NCs secreted important soluble factors capable of differentiating MSCs to a NP phenotype synthesizing high levels of proteoglycan while also resisting collagen fiber expression and hypertrophy, yet results were sensitive to the conditions in which media was generated (cells in alginate versus cells in their native tissue) so that further mechanistic studies optimizing culture conditions and defining important NC secreted factors are required. Matricellular proteins, such as clusterin and tenascin, are likely to be important to optimize differentiation of MSCs for maximum GAG production and reduced collagen fiber expression.
Project description:During intervertebral disc ageing, chondrocyte-like cells (CLCs) replace notochordal cells (NCs). NCs have been shown to induce regenerative effects in CLCs. Since vesicles released by NCs may be responsible for these effects, we characterized NC-derived extracellular vesicles (EVs) and determined their effect on CLCs. EVs were purified from porcine NC-conditioned medium (NCCM) through size exclusion chromatography, ultracentrifugation or density gradient centrifugation. Additionally, the EVs were quantitatively analyzed by high-resolution flow cytometry. The effect of NCCM-derived EVs was studied on canine and human CLC micro-aggregates in vitro and compared with NCCM-derived proteins and unfractionated NCCM. Porcine NCCM contained a considerable amount of EVs. NCCM-derived EVs induced GAG deposition in canine CLCs to a comparable level as NCCM-derived proteins and unfractionated NCCM, and increased the DNA and glycosaminoglycan (GAG) content of human micro-aggregates, although to a lesser extent than unfractionated NCCM. The biological EV effects were not considerably influenced by ultracentrifugation compared with size exclusion-based purification. Upon ultracentrifugation, interfering GAGs, but not collagens, were lost. Nonetheless, collagen type I or II supplemented to CLCs in a concentration as present in NCCM induced no anabolic effects. Porcine NCCM-derived EVs exerted anabolic effects comparable to NCCM-derived proteins, while unfractionated NCCM was more potent in human CLCs. GAGs and collagens appeared not to mediate the regenerative EV effects. Thus, NC-derived EVs have regenerative potential, and their effects may be influenced by the proteins present in NCCM. The optimal combination of NC-secreted factors needs to be determined to fully exploit the regenerative potential of NC-based technology.
Project description:Introduction Effective therapies that may stop or even reverse disc degeneration remain elusive. A minimally invasive method through which nucleus pulposus (NP) cell viability could be achieved would revolutionize the treatment of degenerative disc disease (DDD). With the presented work, we have investigated if nonchondrodystrophic (NCD) canine intervertebral disc (IVD)-derived notochordal cell conditioned medium (NCCM) and chondrodystrophic (CD) canine IVD-derived conditioned medium (CDCM) are able to protect murine and human NP cells from apoptosis. Materials and Methods We developed NCCM and CDCM from hypoxic culture of freshly isolated NPs from NCD and CD canines, respectively. We obtained murine NP cells from nine different C57BL/6 mice and human NP cells from four patients who underwent surgery for discectomy. The cells were cultured with ADMEM/F-12 (control media), NCCM, or CDCM under hypoxic conditions (3.5% O2) and treated with IL-1β + FasL or Etoposide. All media were supplemented with 2% fetal bovine serum. We then determined the expression of specific apoptotic pathways in the murine and human NP cells by recording activated caspase-8, caspase-9, and caspase-3/7 activity. Results In the murine NP cells, NCCM inhibits IL-1β + FasL- and Etoposide-mediated apoptosis via suppression of activated caspase-9 and caspase-3/7, CDCM demonstrated an inhibitory effect on IL-1β + FasL-mediated apoptosis via caspase-3/7 (Fig. 1A). In the human NP cells, NCCM inhibits Etoposide- mediated apoptosis via suppression of activated caspase-8, caspase-9, and mainly caspase-3/7. CDCM demonstrated an inhibitory effect on Etoposide-mediated apoptosis via suppression of activated caspase-8, caspase-9, and mainly caspase-3/7, though not as effective as NCCM (Fig. 1B). Conclusion IL-1β + FasL are known key molecules in the progression of DDD. Here, we demonstrate that soluble factors secreted by the NCD IVD NP strongly protect murine NP cells not only from IL-1β + FasL but also from Etoposide-induced apoptosis via suppression of activated caspase-9 and caspase-3/7. In the human samples, addition of IL-1β + FasL did not increase cell death. Because the human cell samples were obtained from herniated discs that are probably already undergoing a degenerative process, it is likely that there was already some degree of activation by the endogenously secreted prodegenerative factors such as IL-1β + FasL. It may be that the NP cells, once they have reached a pivotal point of the degenerative cascade, no longer respond to exogenously applied IL-1β + FasL in contrast to the otherwise "healthy" discs obtained from the mice. Interestingly, the rescue effect of NCCM in the etoposide-treated cells (murine and human) suggests that NCCM is capable of influencing the signaling pathways known to be relevant to etoposide-induced cell death. A better understanding and harnessing of the restorative powers of the notochordal cell could lead to novel cellular and molecular strategies for the treatment of DDD.
Project description:Bone marrow stromal cells (BMSCs) have shown promising potential to stop intervertebral disc degeneration in several animal models. In order to restore a healthy state, though, this potential should be further stimulated. Notochordal cells (NCs), influential in disc development, have been shown to stimulate BMSC differentiation, but it is unclear how this effect will translate in an environment where resident disc cells (nucleus pulposus cells [NPCs]) could also influence BMSCs. The goal of this study was, therefore, to evaluate the effects of NCs on BMSCs when cocultured with NPCs, in a simplified 3D in vitro system. Bovine BMSCs and NPCs were mixed (Mix) and seeded into alginate beads. Using culture inserts, the Mix was then cocultured with porcine NCs (alginate beads) and compared to coculture with empty beads or porcine skin fibroblasts (SFs, alginate beads). NPCs alone were also cocultured with NCs, and BMSCs alone cultured under chondrogenic conditions. The effects of coculture conditions on cell viability, matrix production (proteoglycan and collagen), and gene expression of disc markers (aggrecan, type II collagen, and SOX9) were assessed after 4 weeks of culture. The NC phenotype and gene expression profile were also analyzed. Coculture with NCs did not significantly influence cell viability, proteoglycan production, or disc marker gene expression of the Mix. When compared to NPCs, the Mix produced the same amount of proteoglycan and displayed a higher expression of disc marker, indicating a stimulation of the BMSCs (and/or NPCs) in the Mix. Additionally, during the 4 weeks of culture, the NC phenotype changed drastically (morphology, gene expression profile). These results show that NCs might not be as stimulatory for BMSCs in an NPC-rich environment, as believed from individual cultures. This absence of effects could be explained by a mild stimulation provided by (de)differentiating NCs and the costimulation of BMSCs and NPCs by each other.
Project description:The 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.We 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.NCCM 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.Notochordal 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.
Project description:<h4>Background</h4>Because the regenerative ability of intervertebral discs (IVDs) is restricted, defects caused by discectomy may induce insufficient tissue repair leading to further IVD degeneration. An acellular bioresorbable biomaterial based on ultra-purified alginate (UPAL) gel was developed to fill the IVD cavity and prevent IVD degeneration. However, an acellular matrix-based strategy may have limitations, particularly in the elderly population, who exhibit low self-repair capability. Therefore, further translational studies involving product combinations, such as UPAL gel plus bone marrow-derived mesenchymal stem cells (BMSCs), are required to evaluate the regenerative effects of BMSCs embedded in UPAL gel on degenerated IVDs.<h4>Methods</h4>Rabbit BMSCs and nucleus pulposus cells (NPCs) were co-cultured in a three-dimensional (3D) system in UPAL gel. In addition, rabbit or human BMSCs combined with UPAL gel were implanted into IVDs following partial discectomy in rabbits with degenerated IVDs.<h4>Findings</h4>Gene expression of NPC markers, growth factors, and extracellular matrix was significantly increased in the NPC and BMSC 3D co-culture compared to that in each 3D mono-culture. In vivo, whereas UPAL gel alone suppressed IVD degeneration as compared to discectomy, the combination of BMSCs and UPAL gel exerted a more potent effect to induce IVD regeneration. Similar IVD regeneration was observed using human BMSCs.<h4>Interpretation</h4>These findings demonstrate the therapeutic potential of BMSCs combined with UPAL gel as a regenerative strategy following discectomy for degenerated IVDs.<h4>Funding</h4>Ministry of Education, Culture, Sports, Science, and Technology of Japan, Japan Agency for Medical Research and Development, and the Mochida Pharmaceutical Co., Ltd.