Genesis of osteoclasts on calcium phosphate ceramics and their role in material-induced bone formation.

Innate immune responses play important roles in material-induced bone formation and such roles were further explored in the current study with an emphasis on M2 macrophages and osteoclastogenesis. With the presence of M-CSF and RANKL, M0 macrophages from FVB mouse bone marrow-derived monocytes (BMMs) fused to osteoclasts with both M2 marker and osteoclast marker at day 5, and such osteoclast formation at day 5 was enhanced when the cells were treated with IL-4 at day 3. With IL-4 treatment alone for 24 h, M0 polarized into M2 macrophages. Conditioned medium of M2 macrophages enhanced osteogenic differentiation of MC3T3-E1 (pre-osteoblasts) while osteoclast conditioned medium enhanced osteogenic differentiation of CRL-12424 (osteogenic precursors). TCPs (a typical osteoinductive material) supported M2 macrophage polarization at day 4 and osteoclast formation at day 5, while TCPb (a typical non-osteoinductive material) was less effective. Moreover, osteoclasts formed on TCPs produced osteogenic factors including S1P, Wnt10B and BMP-6, resulting osteogenic differentiation of CRL-12424 cells. Similar to in vitro testing, TCPs favored M2 macrophage polarization followed by the formation of osteoclasts in vivo, as compared to TCPb. The overall data provided evidence of a coupling between M2 macrophages, osteoclasts and material-induced bone formation: osteoclasts formed from M2 macrophages secrete osteogenic cytokines to induce osteogenic differentiation of osteogenic precursor cells to finally form bone. The current findings outlined a biological mechanism of material-induced bone formation and further rationalized the use of osteoinductive materials for bone regeneration. STATEMENT OF SIGNIFICANCE: This paper provides evidence for finding out the relationship between M2 macrophages, osteoclasts and osteogenesis in material-induced bone formation. It suggested that osteoinductive materials enhanced macrophage polarization to M2 macrophages which fuses to osteoclasts, osteoclasts subsequently secret osteogenic cytokines to differentiate finally osteogenic precursors to form bone in osteoinductive materials. The data supports scientifically the superiority of osteoinductive materials for bone regeneration in clinics.

Serial cellular events in bone formation initiated by calcium phosphate ceramics.

To better understand the biological mechanisms triggered by osteoinductive materials in vivo, we evaluated the timeline of cellular responses to osteoinductive materials subcutaneously implanted in FVB mice. More F4/80-positive macrophages were present in osteoinductive tri-CaP ceramic (TCP) with submicron surface topography (TCPs) than non-osteoinductive TCP with micron surface topography (TCPb) at week 1. Moreover, TCPs (but not TCPb) significantly enhanced osteoclastogenesis, and induced macrophages to polarize from M1 to M2 in the first week. The time sequence and relevance of macrophages and osteoclasts responses involved in bone formation was then evaluated through peri-implant injection of specific chemicals in mice implanted with osteoinductive TCPs. Day-1 injection of clodronate liposomes (LipClod) depleted macrophages, inhibited macrophage polarization to M2, blocked osteoclastogenesis and bone formation, while the day-6 injection was less effective. Anti-RANKL antibody (aRANKL) did not affect macrophage colonization but inhibited osteoclastogenesis. Injection of aRANKL before week 2 aborted bone formation in TCPs, while injection at week 4 partially inhibited bone formation. The overall data show that following ectopic implantation, osteoinductive materials allow macrophage colonization in hours to days, macrophage polarization to M2 in days (within 7 days), osteoclastogenesis in weeks (e.g. in 2 weeks) and bone formation thereafter (after 4 weeks). The serial cellular events verified herein bring a new insight on material-induced bone formation and pave the way to further explore the mechanisms triggered by osteoinductive materials. STATEMENT OF SIGNIFICANCE: A series of key cellular events triggered by osteoinductive calcium phosphate ceramic was revealed: macrophages colonized within hours to days, polarization of M2 macrophages occurred within 7 days, osteoclastogenesis mainly occurred in weeks (e.g. in 2 weeks) and bone formation finally arose thereafter (after 4 weeks). Moreover, such time sequence of cellular events was confirmed with specific chemicals (clodronate liposomes and anti-RANKL antibody). The findings verified herein bring a new insight on material-induced bone formation and pave the way to further explore the mechanisms triggered by osteoinductive materials.