Degenerative Grade Affects the Responses of Human Nucleus Pulposus Cells to Link-N, CTGF, and TGFβ3

Abstract

Study Design Cells isolated from moderately and severely degenerated human intervertebral discs (IVDs) cultured in an alginate scaffold. Objective To compare the regenerative potential of moderately vs. severely degenerated cells using three pro-anabolic stimulants. Summary of Background Data Injection of soluble cell signaling factors has potential to slow the progression of IVD degeneration. While degenerative grade is thought to be an important factor in targeting therapeutic interventions it remains unknown whether cells in severely degenerated IVDs have impaired metabolic functions compared to lesser degenerative levels or if they are primarily influenced by the altered microenvironment. Methods NP cells were cultured in alginate for 21 days and treated with three different pro-anabolic stimulants: a growth factor/anti-inflammatory combination of TGFβ3+Dex, or matricellular proteins CTGF or Link-N. They were assayed for metabolic activity, DNA content, glycosaminoglycan (GAG), and qRT-PCR gene profiling. Results Moderately degenerated cells responded to stimulation with increased proliferation, decreased IL-1β, MMP9 and COL1A1 expression, and upregulated HAS1 as compared to severely degenerated cells. TGFβR1 (ALK5) receptors were expressed at greater levels in moderately than severely degenerated cells. TGFβ3+Dex had a notable stimulatory effect on moderately degenerated NP cells with increased anabolic gene expression, and decreased COL1A1 and ADAMTS5 gene expression. Link-N and CTGF had similar responses in all assays, and both treatments up-regulated IL-1β expression and had a more catabolic response than TGFβ3+Dex, particularly in the more severely degenerated group. All groups, including different degenerative grades, produced similar amounts of GAG. Conclusion Pro-anabolic stimulants alone had limited capacity to overcome the catabolic and pro-inflammatory cytokine expression of severely degenerated NP cells and likely require additional anti-inflammatory treatments. Moderately degenerated NP cells had greater TGFβ receptor 1 expression and better responded to anabolic stimulation.

Best Paper NASS 2013: Link-N can stimulate proteoglycan synthesis in the degenerated human intervertebral discs

“…The regenerative potential of Link-N has been studied in many systems (Mwale et al, 2003;Mwale et al, 2011;Antoniou et al, 2012;Abbott et al, 2013), and in the present study human disc cells from NP and AF in their microenvironment, under the influence of Link-N, showed increase in aggrecan synthesis and downregulation of proteases by NP cells. In contrast, recent work by Abbott et al (Abbott et al, 2013) evaluated the effect of Link-N, CTGF and TGF-β + Dex on human disc cells in alginate.…”

“…In contrast, recent work by Abbott et al (Abbott et al, 2013) evaluated the effect of Link-N, CTGF and TGF-β + Dex on human disc cells in alginate. The study did not show much upregulation of aggrecan by disc cells exposed to Link-N compared to other experimental groups.…”

Changes and significance of inflammatory cytokines in a rat model of cervical spondylosis

“…TGF-β has been reported to increase proteoglycan production to adjust the proliferation of type II collagen intervertebral disk cells and reduce matrix degradation to thereby regulate the metabolism of intervertebral disc cells (33,34). While certain studies found that proteoglycan production in nucleus pulposus cells was increased through gene therapy with TGF-β, it is inferred that TGF-β is a protective cytokine in the intervertebral disc (35)(36)(37). In the present study, an increasing trend of TGF-β1 at 1 month post-operation was identified, while it had significantly decreased at 3 months.…”

CCN2 Suppresses Catabolic Effects of Interleukin-1β through α5β1 and αVβ3 Integrins in Nucleus Pulposus Cells

“…Additionally, considering that mechanotransduction through ␣5␤1 integrin is altered in degenerate NP cells, it is also likely that binding of CCN2 to this cognate integrin fails to have the same anabolic/anti-catabolic effect that it does in healthy discs. In fact, in terms of catabolic gene expression and matrix production, recent studies confirm that NP cells from severely degenerated discs respond less robustly to anabolic factors, including CCN2 and TGF-␤, than healthy or moderately degenerated cells (49,50). Further experiments measuring catabolic gene expression after treatment of degenerate or nondegenerate human NP cells with IL-1␤, CCN2, or a Healthy NP Degenerate NP FIGURE 8.…”