gms | German Medical Science

Objectives: The most common operation for osteonecrosis of the femoral head (ONFH) without collapse has been core decompression (CD). More recently, harvested and concentrated bone marrow aspirate has been added to CD to help reconstitute the necrotic segment. Bone healing proceeds through a series of stages, the first of which is the inflammatory stage. During this stage, pro-inflammatory M1 macrophages (mø) orchestrate a series of events including bone degradation by osteoclasts and ingress of osteoprogenitor and vascular progenitor cells. Subsequently, M1 mø are followed by anti-inflammatory and pro-tissue healing M2 mø. Recently it has become apparent that mesenchymal stem cells (MSCs) and mø engage in continuous co-modulation. We hypothesized that this MSC- mø crosstalk could be further modulated to enhance osteogenesis.

Methods: Primary murine bone marrow mø were isolated from 10-12 week old male C57BL-6J mice, and were polarized to M0 (undifferentiated), M1, and M2 phenotypes for 24h using macrophage media alone, macrophage media with 100 ng/mL LPS, and macrophage media with 20 ng/mL IL-4, respectively. Primary murine bone marrow MSCs were isolated from 6-8 week old male C57BL-6J mice and cultured through P5. Macrophage-MSC co-cultures were grown in mixed osteogenic-macrophage media (MM) for 4 weeks at 2 seeding densities: 1:1 Mac-MSC and 5:1 Mac-MSC. Alkaline phosphatase (ALP) activity was measured after 2 weeks, and Alizarin Red staining measured bone mineralization at 4 weeks. ELISAs for prostaglandin E2 (PGE2) and osteoprotegerin (OPG) were performed on supernatants. All groups were conducted with n=4. One-way ANOVA followed by Tukey's Test was performed using GraphPad Prism. The animal protocol was approved by our Animal Care Committee.

Results and Conclusion: When cultured at a 5:1 macrophage-MSC ratio, co-cultures with M1 and M2 mø exhibited higher levels of PGE2 early in osteogenesis and ultimately had increased bone mineralization after 4 weeks. PGE2 activity at 3 weeks closely matched trends seen in ALP activity, in which M1-MSC and M2- MSC groups (5:1) had lower ALP activity despite similar or higher levels of bone mineralization at 4 weeks of culture. Reduced PGE2 expression after 2 weeks may be due to negative feedback regulation by autocrine and/or paracrine mechanisms during dynamic mø -MSC crosstalk. These observations also suggest that PGE2 may additionally activate an ALP-independent pathway to enhance osteogenesis. Furthermore, the presence of mø reduced the expression of OPG, suggesting that enhanced osteogenesis is likely due to an increase in MSC/osteoblast function rather than suppression of osteoclast activity. Inflammation is a key component of bone healing. Modulating mø-MSC interactions may have the potential to enhance bone healing in ON and other diseases.