Oral Presentation: Promoting intervertebral disc fusion using BMP2, L51P, and the EP4 agonist KMN159: Synergistic osteogenesis in human annulus fibrosus cells

Introduction: Spinal fusion surgery is often performed to restore proper spinal alignment, but it presents notable challenges, particularly in older patients with comorbidities. This study aims to identify factors that promote intervertebral disc fusion, with a focus on minimally invasive approaches to spinal surgery. Specifically, we examine a novel method to induce osteogenic differentiation in annulus fibrosus cells (AFCs) using a combination of bone morphogenetic protein-2 (BMP2), L51P (a BMP2 analog), and KMN159 (a prostaglandin E2 receptor 4 agonist that modulates inflammatory processes). Methods: Primary human AFCs (n=11) were stimulated with BMP2, L51P, and KMN159 and cultured for 21 days. Cell viability was assessed to detect cytotoxic effects following stimulation. At 7, 14, and 21 days, osteogenesis was evaluated by measuring the transcription levels of bone-related genes: alkaline phosphatase (ALP), Runt-related transcription factor 2 (RUNX2), bone gamma-carboxyglutamate protein (BGLAP, also known as osteocalcin), secreted phosphoprotein 1 (SPP1, also known as osteopontin), osterix (SP7), and type I collagen (COL1). In parallel, BMP antagonists such as Noggin, Gremlin 1, and Chordin were quantified using quantitative polymerase chain reaction (qPCR). On Day 14, ALP activity was measured at the protein level, and on Day 21, histological staining with alizarin red (ALZR) was performed to detect calcium deposits, indicating mineralization. Results: KMN159 had no discernible impact on the cell viability of AFCs but demonstrated weaker osteogenic effects on AFCs compared to BMP2. Specifically, fold changes in the expression of osteogenic markers on Day 21 showed BMP2 had a stronger effect than KMN159 in promoting the expression of genes such as ALP (2.828 vs 2.516), RUNX2 (4.199 vs 0.863), BGLAP (2.035 vs 0.7028), SPP1 (6.407 vs 1.078), SP7 (42.20 vs 1.094), and COL1 (1.664 vs 0.9518). When KMN159 was combined with BMP2 and L51P, there was a significant increase in ALP expression in AFCs (P < 0.05), suggesting enhanced osteogenic differentiation. This result was corroborated by increased ALP protein activity on Day 14 and intensified calcium deposition detected by ALZR staining on Day 21. Although other bone-related markers did not show statistically significant changes, an upward trend in expression was observed after 21 days. KMN159 had minimal impact on BMP antagonists (Noggin, Gremlin 1, and Chordin), indicating its minimal influence on their expression. Conclusions: Our results suggest that the combination of KMN159, BMP2, and L51P can successfully induce osteogenic differentiation in human AFCs. This combination may serve as a promising minimally invasive approach to enhance spinal fusion without the need to remove the intervertebral disc, offering potential new treatment options for patients suffering from lower back pain.