Regenerative potential of TGFβ3 + Dex and notochordal cell conditioned media on degenerated human intervertebral disc cells

Abstract

Injection of soluble cell signaling factors into degenerated intervertebral discs (IVDs) offers a minimally invasive treatment that could limit the processes of degeneration by stimulating native matrix repair. This study evaluated the regenerative capacity of degenerated nucleus pulposus (NP) cells obtained from patients undergoing anterior interbody fusions by measuring metabolic activity, DNA content, glycosaminoglycan (GAG) content, and cellular phenotype using qRT-PCR profiling with a custom array of 42 genes. NP cells were cultured in alginate for 7 days with 4 treatment groups: transforming growth factor beta 3 (TGFβ3) + dexamethasone (Dex), soluble factors released from notochordal cells (NCs) cultured in alginate (NCA), soluble factors released from NCs in their native tissue environment (NCT), and basal media. TGFβ3 + Dex stimulated degenerated human NP cells to proliferate and exhibit an anti-catabolic gene expression profile (with a decrease in ADAMTS5 and MMP1 compared to basal, and an increase in SOX9, decrease in ADAMTS5, MMP1, collagen I and collagen III compared to day 0), while NCA stimulated the greatest GAG per cell. We conclude that degenerated human NP cells exhibit regenerative potential, and that an optimal treatment will likely require treatments, such as TGFβ3 + Dex, which were able to increase cell metabolism and reduce catabolism, as well as treatments with factors found in NC conditioned medium, that were able to produce high amounts of GAG per cell. Additional studies to optimize NC culture conditions are required to determine if NC conditioned medium can be made with the capacity to enhance NP cell proliferation and metabolism.

Potential application of notochordal cells for intervertebral disc regeneration: an in vitro assessment

“…Contrary to the present study, however, NCCM appears to be more potent than chondrogenic medium (TGFβ 3 and dexamethasone) for human cells (Abbott et al, 2012). The lack of sensitivity of human NPCs to chondrogenic stimulation may explain this discrepancy, as the GAG/DNA was lower for TGFβ stimulation than for basal conditions (Abbott et al, 2012). The positive effect of NCCM observed here was not reflected at the gene level, as aggrecan and type II collagen expression remained the same for all medium conditions, which was also reported in the other studies (Abbott et al, 2012;Gantenbein-Ritter and Chan, 2012).…”

“…Our data confirm, in a long term study (4 weeks), that bioactive factors secreted by NCs stimulate proteoglycan synthesis by NPCs (GAG/DNA: 1.8 x Basal), and to levels comparable to those reported in short term (1-2 weeks) studies with porcine NCs and human or bovine NPCs [2.0 x (Abbott et al, 2012) and 1.4 x (Gantenbein-Ritter and Chan, 2012) Basal, respectively]. Contrary to the present study, however, NCCM appears to be more potent than chondrogenic medium (TGFβ 3 and dexamethasone) for human cells (Abbott et al, 2012). The lack of sensitivity of human NPCs to chondrogenic stimulation may explain this discrepancy, as the GAG/DNA was lower for TGFβ stimulation than for basal conditions (Abbott et al, 2012).…”

“…These findings were in line with the reported cell composition of young (< 8 months old) pig NPs (Chen et al, 2006;Purmessur et al, 2011). NC conditioned medium preparation NCCM was produced as described before (Abbott et al, 2012;Purmessur et al, 2011) from lumbar and thoracic intervertebral discs harvested from 4 young pigs (< 10 weeks). NP fragments were obtained following the dissecting protocol previously explained and cultured directly (without tissue digestion) in plain medium, using 30 mL/g of tissue, for 4 d under 5 % O 2 , 5 % CO 2 , 90 % N 2 .…”

“…The lack of sensitivity of human NPCs to chondrogenic stimulation may explain this discrepancy, as the GAG/DNA was lower for TGFβ stimulation than for basal conditions (Abbott et al, 2012). The positive effect of NCCM observed here was not reflected at the gene level, as aggrecan and type II collagen expression remained the same for all medium conditions, which was also reported in the other studies (Abbott et al, 2012;Gantenbein-Ritter and Chan, 2012). The same discrepancy was observed for other cell culture conditions and cell types, and could be explained by the fact that NCCM or TGFβ provide an early stimulation of aggrecan that is no more observed at the time of analysis (day 28).…”

“…Based on the observation that degeneration begins to occur soon after the disappearance (Smolders et al, 2013), or change of phenotype, of another cell type in the NP, the notochordal cells (NCs), it is now hypothesised that disc homeostasis may be thrown out of balance by this event. In vitro experiments with human (Abbott et al, 2012) and bovine (Aguiar et al, 1999;Gantenbein-Ritter and Chan, 2012) NPCs encapsulated in 3D hydrogels, indeed, suggest that NCs could act as stimulators, controlling NPC synthetic www.ecmjournal.org E Potier et al Notochordal cells for IVD regeneration activity of proteoglycans (a main component of disc matrix) through secreted factors. Other studies have also shown that NCs could constitute a source of NPC-like cells, as they can produce proteoglycans and express type II collagen and aggrecan (Kim et al, 2009;Cappello et al, 2006) (main constituents of NP matrix) at levels similar to NPCs.…”