Promoting intervertebral disc fusion using BMP2, L51P, and the EP4 agonist KMN-159: synergistic osteogenesis in human annulus fibrosus cells.

Purpose Spinal fusion surgery is widely used to restore spinal stability but remains challenging in elderly patients with comorbidities. This study aimed to explore whether combining bone morphogenetic protein-2 (BMP2), its analogue L51P, and a prostaglandin E2 receptor 4 agonist KMN159 could induce osteogenic differentiation of human annulus fibrosus cells (AFCs), thereby providing a new biological strategy for spinal fusion. Methods Primary human AFCs (n = 11) were cultured under osteogenic conditions and treated with BMP2, L51P, and KMN159 individually or in combination. Cell viability was evaluated using the tetrazolium salt (XTT) assay, osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity, bone-related gene expression (ALP, SP7, COL1), and calcium deposition by alizarin red staining. Expression of BMP antagonists (Noggin, Gremlin1, Chordin) was also quantified by qPCR. Results KMN159 alone showed no cytotoxicity but weaker osteogenic induction than BMP2. When combined with BMP2 and L51P, KMN159 significantly enhanced ALP activity, bone-related gene expression trend, and calcium deposition, with the triple combination showing the strongest effect. Synergistic osteogenesis was particularly evident in AFCs from elderly patients, females, degeneration-derived discs, and lower lumbar segments. Conclusion The combination of BMP2, L51P, and KMN159 effectively promotes osteogenic differentiation of human AFCs in vitro. This minimally invasive strategy may enable disc-preserving spinal fusion, offering potential therapeutic benefits for patients with degenerative disc disease and low back pain.