The effects of immunomodulation by macrophage subsets on osteogenesis in vitro

Abstract

BackgroundBone formation and remodeling are influenced by the inflammatory state of the local microenvironment. In this regard, macrophages are postulated to play a crucial role in modulating osteogenesis. However, the differential effects of macrophage subsets and their plasticity on bone formation are currently unknown.MethodsPolarized primary murine macrophages and preosteoblastic MC3T3 cells were co-cultured to investigate the effect of non-activated M0, pro-inflammatory M1, and tissue-regenerative M2 macrophages on the osteogenic ability of MC3T3-E1 cells in vitro. Furthermore, to model the physiological transition from inflammation to tissue regeneration, M1-MC3T3 co-cultures were treated with interleukin-4 (IL-4) at different time points to modulate the M1 phenotype towards M2. Macrophage phenotypic markers were assessed by flow cytometry and enzyme-linked immunosorbent assay. A time course study of osteogenic markers at different time points was conducted: alkaline phosphatase (ALP) mRNA levels were evaluated at week 1, ALP activity and osteocalcin and osteopontin mRNA levels at week 2, and matrix mineralization and osteocalcin and osteopontin protein concentrations at week 3. Supernatant collected 72 hours after seeding or IL-4 treatment, whichever was later, was analyzed for oncostatin M, a cytokine released by macrophages that has been recognized to enhance osteogenesis. Unpaired t test or one-way ANOVA with Tukey’s or Dunnett’s post hoc tests were used for statistical comparison of the groups.ResultsCo-culture with any of the macrophage subtypes increased the osteogenic ability of MC3T3 cells as indicated by increases in ALP activity and matrix mineralization. Increased ALP activity, osteocalcin concentration, and matrix mineralization demonstrated that osteogenesis by M1-MC3T3 co-cultures was further enhanced by macrophage phenotype modulation to M2 via IL-4 treatment 72 hours after seeding. Increased oncostatin M protein concentration in untreated M1-MC3T3 co-cultures and M1-MC3T3 co-cultures treated with IL-4 at 72 hours correlated with greater ALP activity and matrix mineralization.ConclusionsThese results suggest that a transient inflammatory phase is crucial for enhanced bone formation. Macrophage plasticity may offer new strategies for modulating the local inflammatory microenvironment with the aim of potentially enhancing bone repair.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0276-5) contains supplementary material, which is available to authorized users.

Aging Affects Bone Marrow Macrophage Polarization: Relevance to Bone Healing

“…These findings suggest that aged MΦ remain in a preactivated resting state that enhances their responsiveness to exposure of pro-inflammatory stimuli [29]. Loi et al [30] have shown that modulation of macrophage phenotype from M1 to M2 enhances osteogenic ability of MC3T3 cells in an in vitro coculture model. The present study showed that the majority of the aged M0s are both iNOS+ and CD206+.…”

Exendin-4 Induces Bone Marrow Stromal Cells Migration Through Bone Marrow-Derived Macrophages Polarization via PKA-STAT3 Signaling Pathway

“…Bone fracture is the latent threat associated with osteoporosis [24,25]. The first stage of healing after fracture is inflammation, which leads to granulation tissue formation and disrupts the balance between bone loss and formation [26,27].…”

Dissecting the Role of Sphingosine 1-Phosphate - Sphingosine 1-Phosphate Receptor 1 in Inflammatory Bone Remodelling

“…A possible explanation for this is that during the early stages of bone healing, which is characterized by an acute inflammatory phase, the M1 macrophages act as a key factor in the differentiation of osteoblasts, whereas M2 macrophages take part in the later stage of osteogenesis by inducing the maturation and terminal mineralization of osteoblasts. The transition from the M1 to M2 phenotype is, therefore, considered to be crucial aspect of bone healing and de novo bone formation (Loi, et al, 2016). In the preceding chapter, it was found that the macrophages were capable of inducing the SPHK1 activity in the co-cultured BMSCs, thereby promoting the S1P production.…”

“…Furthermore, the conversion of M1 to M2 macrophages significantly improves mineralization in the co-culture system (Loi et al, 2016 MSCs to the injury site and induce bone formation (Takayanagi, 2007). The two major players in bone remodelling -osteoclasts and osteoblasts are coupled through the RANKL-RANK-OPG axis, that osteoblastsderived RANKL combines with its ligand RANK in osteoclasts and plays an indispensable role in osteoclastogenesis; OPG (which is also derived from osteoblasts) reduces osteoclastogenesis by impairing the RANKL-RANK signalling.…”

“…M1 macrophage activation, it has been suggested, is indispensable during the early stages of bone repair. M2 macrophages, on the other hand, plays a key role in the later stage of bone formation, suggesting the shift from M1 to M2 phenotype is a crucial process in the regulation of osteogenesis (Guihard, et al, 2012;Loi, et al, 2016).…”