Denosumab alleviates intervertebral disc degeneration adjacent to lumbar fusion by inhibiting endplate osteochondral remodeling and vertebral osteoporosis in ovariectomized rats

Abstract

Abstract Background Although adjacent segmental intervertebral disc degeneration (ASDD) is one of the most common complications after lumbar fusion, its exact mechanism remains unclear. As an antibody to RANKL, denosumab (Dmab) effectively reduces bone resorption and stimulates bone formation, which can increase bone mineral density (BMD) and improve osteoporosis. However, it has not been confirmed whether Dmab has a reversing or retarding effect on ASDD. Methods Three-month-old female Sprague-Dawley rats that underwent L4–L5 posterolateral lumbar fusion (PLF) with spinous-process wire fixation 4 weeks after bilateral ovariectomy (OVX) surgery were given Dmab 4 weeks after PLF surgery (OVX+PLF+Dmab group). In addition, the following control groups were defined: Sham, OVX, PLF, and OVX+PLF (n=12 each). Next, manual palpation and X-ray were used to evaluate the state of lumbar fusion. The bone microstructure in the lumbar vertebra and endplate as well as the disc height index (DHI) of L5/6 was evaluated by microcomputed tomography (μCT). The characteristic alterations of ASDD were identified via Safranin-O green staining. Osteoclasts were detected using tartrate-resistant acid phosphatase (TRAP) staining, and the biomechanical properties of vertebrae were evaluated. Aggrecan (Agg), metalloproteinase-13 (MMP-13), and a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4) expression in the intervertebral disc were detected by immunohistochemistry and real-time polymerase chain reaction (RT-PCR) analysis. In addition, the expression of CD24 and Sox-9 was assessed by immunohistochemistry. Results Manual palpation showed clear evidence of the fused segment’s immobility. Compared to the OVX+PLF group, more new bone formation was observed by X-ray examination in the OVX+PLF+Dmab group. Dmab significantly alleviated ASDD by retaining disc height index (DHI), decreasing endplate porosity, and increasing vertebral biomechanical properties and BMD. TRAP staining results showed a significantly decreased number of active osteoclasts after Dmab treatment, especially in subchondral bone and cartilaginous endplates. Moreover, the protein and mRNA expression results in discs (IVDs) showed that Dmab not only inhibited matrix degradation by decreasing MMP-13 and ADAMTS-4 but also promoted matrix synthesis by increasing Agg. Dmab maintained the number of notochord cells by increasing CD24 but reducing Sox-9. Conclusions These results suggest that Dmab may be a novel therapeutic target for ASDD treatment.

Keywords

• Osteoporosis
• Ovariectomy
• Vertebral
• Endplate
• Osteochondral remodeling