Poster Presentation 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

Introduction: Intervertebral disc (IVD) degeneration is associated with reduced active nucleus pulposus (NP) cells and chronic inflammation¹. The platelet-derived growth factor (PDGF)-BB has mitogenic² and anti-inflammatory³ effects in various cell types. However, the effects of PDGF-AB (AB) and -BB (BB) on IVD degeneration have not been fully described. The aim of this study was to 1) characterize the time-course effects of AB and BB on cell proliferation and inflammatory responses in human IVD cells and 2) investigate if PDGF administration suppressed IVD degeneration in inflamed IVDs utilizing a needle puncture induced IVD degeneration model.

Methods: Following informed consent in accordance with the institutional review board approval, human degenerated IVD tissues were obtained from patients diagnosed with IVD degeneration (n=6, Pfirrmann grade 4-5). NP and AF cells were extracted from the samples. For monolayer cultures (n=6) and pellet cultures (n=5), NP cells were treated with recombinant human AB or BB. For inflammatory responses, the NP and AF cells were serum-deprived for 1 day, followed by stimulating with 20ng/ml TNFa and 20ng/ml AB or BB. MTT assay (n=4) was performed to examine cell proliferation and gene expression was evaluated. Mouse experiments were performed with IACUC approval. Coccygeal IVDs (CC5-10) from 4-month-old wildtype mice (n=3/each timepoint) were punctured with 30-gauge needles and AB (100ng/ml) or BB (40ng/ml) was injected into punctured levels after 1 week of puncture. The IVDs were collected for Safranin O/fast green staining after 1 or 2 weeks of treatment. Data were analyzed using one-way or two-way ANOVA with a statistical significance of p<0.05.   

Results: MTT assay showed that AB and BB stimulated cell proliferation (Fig. 1A). Meanwhile, the expression of cell proliferation marker MKI67 and cell cycle regulator CCNB1 was significantly increased after AB and BB treatment for 5 days while RUNX1 gene expression was inhibited at day1 (Fig. 1B-D). In pellet cultures, AB treatment significantly increased MKI67 expression while reducing RUNX1 expression in a dose-dependent manner at day 1 (Fig. 1E-F). However, there was a trend of increased MKI67expression and decreased RUNX1 expression in BB treatment groups (Fig. 1E-F). TNFa treatment increased the gene expression levels of IL6 and END1 in human NP and AF cells while AB or BB treatment inhibited TNFa-induced IL6 and END1 gene expression (Fig. 2). In punctured IVDs, most of NP cells were lost and the boundary between NP and AF was indistinguishable compared to control levels while the NP tissues were partially restored with increased cell number and proteoglycans in AB or BB treated IVDs (Fig. 3).

Discussion: Our study reveals that AB and BB promoted mitogenesis and inhibited inflammatory responses, leading to attenuated degenerative changes. The anti-inflammatory effects of AB and BB might contribute to enhance the production of extracellular matrix and cell proliferation, and eventually IVD restoration. The concurrent reduction in RUNX1 expression with increased cell proliferation suggest that RUNX1 is possibly involved in regulating cell proliferation in human NP cells. Taken together, PDGF -AB and -BB might be promising candidates for treating IVD degeneration.