Low-intensity pulsed ultrasound promotes spinal fusion by regulating macrophage polarization

Abstract

Background: Spinal fusion is considered the gold standard procedure for treating spinal degeneration, tumors, and trauma. An inflammatory response is an important part of bone repair. We investigated the polarization change of inflammatory macrophages (M1) and resident macrophages (M2) during low-intensity pulsed ultrasound (LIPUS) treatment.

Methods: Thirty male Sprague Dawley rats (age: 12 weeks; weight: 300 g) were used in the study. A rat spinal fusion model was established by surgical procedures. LIPUS treatment (20 min. d, 5 d/wk) was begun 3 days after surgery. The rats were randomly divided into a control group (5 subgroups, 3 rats in each subgroup) and LIPUS group (5 subgroups, 3 rats in each subgroup), and sacrificed on day 3, 5, 7, 10, and 14 after spinal fusion surgery for further evaluation. Bone volume was measured by micro-CT, fusion region was examined by histological analyses, types of macrophages in the fusion area were examined by immunohistochemical staining. Raw264.7 cells and bone marrow-derived macrophages (BMDM) were used in cell experiments. Cells were divided into a control group and LIPUS group. Flow cytometry was used to examine the rate of resident macrophages, and real-time PCR was used to examine the mRNA expression of anti-inflammation genes.

Results: LIPUS promoted spinal fusion and stimulated the transition of F4-80⁺/Mac-2⁺ (M1) to F4-80⁺/Mac-2^- (M2), leading to the early appearance of resident macrophages. Cell experiments showed CD206⁺ macrophages (M2) were significantly increased after LIPUS treatment. M2-related genes and anti-inflammation factors (Arg-1, PPAR-γ, and IL-4) were increased after LIPUS treatment.

Conclusion: The earlier transition from inflammatory to resident macrophage might be one reason for the positive effect of LIPUS on spinal fusion.

Keywords: Low-intensity pulsed ultrasound; Macrophage polarization; Spinal fusion.