The mutual effects between macrophages and cartilage templates in the process of subcutaneous endochondral bone formation

The interplay between implants and the recipient immune environment is key to the long-term effectiveness of bone tissue engineering. In this study, we aimed to investigate the mutual effects between macrophages and cartilage templates in the process of subcutaneous osteogenesis. Primary mice bone marrow derived mesenchymal stem cells (BMSCs) were seeded into gelatin sponge and chondrogenically cultured for 4 weeks in vitro to form cartilage templates. The constructs were then implanted subcutaneously in monocyte-depleted mice or normal C57BL/6 mice. Implants harvested at two months showed inferior osteogenic quality in monocyte-depleted mice compared with that of normal mice. In normal mice, the cartilage templates recruited a high ratio of alternatively activated macrophages (CAM or M2) to classically activated macrophages (AAM or M1), compared with empty sponge. In vitro co-culture assay of macrophages with cartilage templates also showed that the cartilage templates polarized macrophages to the M2 phenotype and that these effects were even stronger than those of primary BMSCs. In turn, the co-culture of polarized macrophages with cartilage templates showed that compared to M0 or M2, M1 significantly increased the expressions of osteogenic and angiogenic markers of cartilage templates. These data suggested that macrophages seem to be indispensable in the osteogenesis of cartilage templates and that cartilage templates have a favorable immunomodulatory ability to polarize macrophages to the M2 phenotype. M1 was the contributing phenotype of macrophages that promoted the osteogenesis and angiogenesis of cartilage templates. Macrophages and cartilage templates cooperate to achieve endochondral bone formation.