INTRODUCTION: Chronic inflammation is a major feature of intervertebral disc degeneration (IVDD)¹ that contributes to back pain. TNFα, a key regulator of pro-inflammatory cytokines is increased in IVDD and often used to initiate the painful IVDD degenerative conditions in various models.^1,2 However, TNFα alone underrepresents the cocktail of cytokines released from degenerated IVDs which are not well-characterized.^1,3,4 This study quantified inflammatory cytokines in IVD tissues isolated from surgical patients, and then developed an in-vitro human AF cell culture model that recapitulates the clinical IVDD conditions by treating target AF cells with conditioned media (CM) generated from anterior discectomy and fusion (ADF) surgical IVD tissues as compared to TNFα. We hypothesized that CM is comprised of multiple pro-inflammatory cytokines that will initiate a robust IVDD phenotype in-vitro.

METHODS: IVD specimen collected from ADF patients were cultured to generate conditioned media (ADF-CM), which was analyzed using a 48-Plex inflammatory cytokine array. Human AF cells isolated from these subjects were divided into groups with the following conditions: i) Basal ii) TNFα supplementation (TNF) and iii) CM and analyzed for cell proliferation/morphology, metabolic activity and gene expression. Statistical differences were analyzed using ONE-WAY ANOVA with Tukey’s post-hoc test (p≤0.5).

RESULTS: CM had significantly higher levels of several IVDD and pain related cytokines including TNFα, RANTES, IL-6 and the angiogenic marker VEGF) (Fig.1) as well as 26 additional inflammation-related cytokines (not shown). CM challenged AF cells showed substantially reduced cell numbers and formation of long stress fibers and debris from dead cells within the media (Fig. 2A). MTT quantified these findings by showing a significantly reduced optical density (OD) of AF cells in CM indicating greatly reduced cellular metabolic activity, and consistent with cell damage and death observed on imaging (Fig. 2B). AF cells cultured in CM showed a significant downregulated expression of Col1a1 and an upregulation of Acan (Fig 3A) and somewhat increased pro-inflammatory cytokines IL-1β (p=0.0872), IL-6 (p=0.1160) and TNFα (p=0.1065) compared to basal (Fig 3B).

DISCUSSION: This study demonstrates that ADF-CM contains a cocktail of cytokines related to IVDD and pain. ADF-CM induces significant changes in cell proliferation, metabolic activity and gene expression on challenged AF cells that were substantially larger than TNFα alone and may more closely mimics IVDD microenvironment conditions. Therefore, many more factors than TNFα need to be considered when trying to mimic IVDD. The significantly reduced relative OD, elongated stress fibers and cellular debris demonstrates cell death from ADF-CM indicated that target AF cells are unlikely to recover after 3 days of CM treatment and suggests this human IVDD culture model can be titrated down to have lower duration and/or concentration. Importantly, AF cells from ADF tissue, once isolated were able to recover in basal conditions, suggesting rescue of these native AF cells may be possible if the challenge of these multiple cytokines can be managed. In conclusion, we established an in-vitro model using surgical ADF-CM that promotes a robust IVDD phenotype on target cells for prevention screening or treatment screening after optimizing severity of the model