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

Study Design: Cells isolated from moderately and severely degenerated human intervertebral disks (IVDs) cultured in an alginate scaffold. Objective: To compare the regenerative potential of moderately versus severely degenerated cells using 3 proanabolic stimulants. Summary of Background Data: Injection of soluble cell signaling factors has potential to slow the progression of IVD degeneration. Although 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: Nucleus pulposus (NP) cells were cultured in alginate for 21 days and treated with 3 different proanabolic stimulants: a growth factor/anti-inflammatory combination of transforming growth factor beta 3 (TGF beta 3)+dexamethasone (Dex), or matricellular proteins connective tissue growth factor (CTGF) or Link-N. They were assayed for metabolic activity, DNA content, glycosaminoglycan, and qRT-PCR gene profiling. Results: Moderately degenerated cells responded to stimulation with increased proliferation, decreased IL-1 beta, MMP9, and COL1A1 expression, and upregulated HAS1 as compared with severely degenerated cells. TGF beta R1 (ALK5) receptors were expressed at greater levels in moderately than severely degenerated cells. TGF beta 3+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 upregulated IL-1 beta expression and had a more catabolic response than TGF beta 3+Dex, particularly in the more severely degenerated group. All groups, including different degenerative grades, produced similar amounts of glycosaminoglycan. Conclusions: Proanabolic stimulants alone had limited capacity to overcome the catabolic and proinflammatory cytokine expression of severely degenerated NP cells and likely require additional anti-inflammatory treatments. Moderately degenerated NP cells had greater TGF beta receptor 1 expression and better responded to anabolic stimulation.