Progressive Ankylosis Protein Regulates Macrophage Immunological Responses in Bone Healing

Objectives: Biomineralization is regulated by phosphate (P_i), a mineralization promoter, and pyrophosphate (PP_i), a hydroxyapatite crystal growth inhibitor. Progressive ankylosis protein (ANK/Ank) increases extracellular PP_i and inhibits biomineralization. Previously, we demonstrated that ablation of Ank in mice (Ank^-/- mice) promoted cementum regeneration, although bone mineral density (BMD) of the regenerated alveolar bone (AB) is decreased due to increased number of osteoclasts compared to wild-type (WT) mice. Osteal macrophages (OMs) play important roles in immune responses during AB healing; however, the role of ANK in OMs remains unclear. Hypothesizing that ANK modulates immunological responses of OMs in AB healing, here we investigate the role of ANK in OMs using Ank^-/- mice.
Methods: Mandibular fenestration defects were created in WT and Ank^-/- mice. The regenerated AB were analyzed histomorphologically using micro-computed tomography (micro-CT) and immunohistochemical staining at postoperative days (POD) 15 and 30. Putative OMs were isolated from the regenerated AB at POD15 and 30 with fluorescence-activated cell sorting (FACS) using markers such as 7-aminoactinomycin D, Hoechst, and hematopoietic cell lineage markers, including CD45 and CD11b. Gene expression in sorted cells was analyzed with RNA-seq.
Results: Micro-CT analysis revealed that BMD of the regenerated AB was decreased by 7% in Ank^-/- mice compared to WT mice at POD30 (p<0.05). No statistically significant differences were observed in the regenerated AB volume, the number of OMs counted by FACS, or the distribution of OMs labeled with Galectin-3, a macrophage marker. The isolated cells expressed macrophage markers including Lgals3. RNA-seq revealed that expressions of inflammatory cytokines/chemokines such as Tnf-a and Cxcl1, 2 were upregulated in sorted cells from Ank^-/- compared to WT mice (P-adjusted<0.05).
Conclusions: These findings suggest that ANK modulates immunological responses of OMs and, subsequently, AB healing via P_i/PP_i regulation. These results provide new approaches to consider for promoting AB wound healing.