Recent research has brought about a clear understanding that successful fracture healing is based on carefully coordinated cross-talk between inflammatory and bone forming cells. In particular, the key role that macrophages play in the recruitment and regulation of the differentiation of mesenchymal stem cells (MSCs) during bone regeneration has been brought to focus. Indeed, animal studies have comprehensively demonstrated that fractures do not heal without the direct involvement of macrophages. Yet the exact mechanisms by which macrophages contribute to bone regeneration remain to be elucidated. Macrophage-derived paracrine signaling molecules such as Oncostatin M, Prostaglandin E2 (PGE2), and Bone Morphogenetic Protein-2 (BMP2) have been shown to play critical roles; however the relative importance of inflammatory (M1) and tissue regenerative (M2) macrophages in guiding MSC differentiation along the osteogenic pathway remains poorly understood. In this review, we summarize the current understanding of the interaction of macrophages and MSCs during bone regeneration, with the emphasis on the role of macrophages in regulating bone formation. The potential implications of aging to this cellular cross-talk are reviewed. Emerging treatment options to improve facture healing by utilizing or targeting MSC-macrophage crosstalk are also discussed.

The MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) locus is misregulated in many human cancers and produces an abundant long nuclear-retained noncoding RNA. Despite being transcribed by RNA polymerase II, the 39 end of MALAT1 is produced not by canonical cleavage/polyadenylation but instead by recognition and cleavage of a tRNA-like structure by RNase P. Mature MALAT1 thus lacks a poly(A) tail yet is expressed at a level higher than many protein-coding genes in vivo. Here we show that the 39 ends of MALAT1 and the MEN b long noncoding RNAs are protected from 39-59 exonucleases by highly conserved triple helical structures. Surprisingly, when these structures are placed downstream from an ORF, the transcript is efficiently translated in vivo despite the lack of a poly(A) tail. The triple helix therefore also functions as a translational enhancer, and mutations in this region separate this translation activity from simple effects on RNA stability or transport. We further found that a transcript ending in a triple helix is efficiently repressed by microRNAs in vivo, arguing against a major role for the poly(A) tail in microRNA-mediated silencing. These results provide new insights into how transcripts that lack poly(A) tails are stabilized and regulated and suggest that RNA triple-helical structures likely have key regulatory functions in vivo.

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Complex interactions among cells of the monocyte-macrophage-osteoclast lineage and the mesenchymal stem cell-osteoblast lineage play a major role in the pathophysiology of bone healing. Whereas the former lineage directs inflammatory events and bone resorption, the latter represents a source of cells for bone regeneration and immune modulation. Both of these lineages are affected by increasing age, which is associated with higher baseline levels of inflammatory mediators, and a significant reduction in osteogenic capabilities. Given the above, fracture healing, osteoporosis, and other related events in the elderly present numerous challenges, which potentially could be aided by new therapeutic approaches to modulate both inflammation and bone regeneration.

Bone fractures are among the most common orthopaedic problems that affect individuals of all ages. Immediately after injury, activated macrophages dynamically contribute to and regulate an acute inflammatory response that involves other cells at the injury site, including mesenchymal stem cells (MSCs). These macrophages and MSCs work in concert to modulate bone healing. In this study, we co-cultured undifferentiated M0, pro-inflammatory M1, and anti-inflammatory M2 macrophages with primary murine MSCs in vitro to determine the cross-talk between polarized macrophages and MSCs and their effects on osteogenesis. After 4 weeks of co-culture, MSCs grown with macrophages, especially M1 macrophages, had enhanced bone mineralization compared to MSCs grown alone. The level of bone formation after 4 weeks of culture was closely associated with prostaglandin E2 (PGE2) secretion early in osteogenesis. Treatment with celecoxib, a cyclooxygenase-2 (COX-2) selective inhibitor, significantly reduced bone mineralization in all co-cultures but most dramatically in the M1-MSC co-culture. We also found that the presence of macrophages reduced the secretion of osteoprotegerin (OPG), the decoy RANKL receptor, suggesting that macrophages may indirectly modulate osteoclast activity in addition to enhancing bone formation. Taken together, these findings suggest that an initial pro-inflammatory phase modulated by M1 macrophages promotes osteogenesis in MSCs via the COX-2-PGE2 pathway.

BackgroundMesenchymal stem cells (MSCs) are capable of immunomodulation and tissue regeneration, highlighting their potential translational application for treating inflammatory bone disorders. MSC-mediated immunomodulation is regulated by proinflammatory cytokines and pathogen-associated molecular patterns such as lipopolysaccharide (LPS). Previous studies showed that MSCs exposed to interferon gamma (IFN-γ) and the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) synergistically suppressed T-cell activation.MethodsIn the current study, we developed a novel preconditioning strategy for MSCs using LPS plus TNF-α to optimize the immunomodulating ability of MSCs on macrophage polarization.ResultsPreconditioned MSCs enhanced anti-inflammatory M2 macrophage marker expression (Arginase 1 and CD206) and decreased inflammatory M1 macrophage marker (TNF-α/IL-1Ra) expression using an in-vitro coculture model. Immunomodulation of MSCs on macrophages was significantly increased compared to the combination of IFN-γ plus TNF-α or single treatment controls. Increased osteogenic differentiation including alkaline phosphate activity and matrix mineralization was only observed in the LPS plus TNF-α preconditioned MSCs. Mechanistic studies showed that increased prostaglandin E2 (PGE2) production was associated with enhanced Arginase 1 expression. Selective cyclooxygenase-2 inhibition by Celecoxib decreased PGE2 production and Arginase 1 expression in cocultured macrophages.ConclusionsThe novel preconditioned MSCs have increased immunomodulation and bone regeneration potential and could be applied to the treatment of inflammatory bone disorders including periprosthetic osteolysis, fracture healing/nonunions, and osteonecrosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0730-z) contains supplementary material, which is available to authorized users.

Chronic inflammation is associated with upregulation of the transcription factor NF-κB and excessive inflammatory cytokine secretion by M1 macrophages. The anti-inflammatory cytokine IL-4 converts pro-inflammatory M1 macrophages into an anti-inflammatory and tissue regenerative M2 phenotype, thus reducing inflammation and enhancing tissue regeneration. We have generated NF-κB responsive, or constitutively active IL4-expression lentiviral vectors transduced into murine bone marrow-derived mesenchymal stromal cells (MSCs). MSCs with a constitutively active IL-4 expression vector produced large quantities of IL-4 continuously whereas IL-4 secretion was significantly induced by lipopolysaccharide (LPS) in the NF-κB sensing MSCs. In contrast, LPS had no effect on MSCs with IL-4 secretion driven by a constitutively active promoter. We also found that intermittent and continuous LPS treatment displayed distinct NF-κB activation profiles, and this regulation was independent of IL-4 signaling. The supernatant containing IL-4 from the LPS treated MSCs suppressed M1 marker (iNOS and TNFα) expression and enhanced M2 marker (Arginase 1, CD206, and IL1Ra) expression in primary murine macrophages. The IL-4 secretion at the basal, non-LPS induced level was sufficient to suppress TNFα and enhance Arginase 1 at a lower level, but had no significant effects on iNOS, CD206, and IL1Ra expression. Finally, IL-4 secretion at basal or LPS-induced levels significantly suppressed osteogenic differentiation of MSCs. Our findings suggest that the IL-4 secreting MSCs driven by NF-κB sensing or constitutive active promoter have great potential for mitigating the effects of chronic inflammation and promoting earlier tissue regeneration.

SummaryBone healing involves complex biological pathways and interactions among various cell types and microenvironments. Among them, the monocyte–macrophage–osteoclast line-age and the mesenchymal stem cell–osteoblast lineage are critical, in addition to an initial inflammatory microenvironment. These cellular interactions induce the necessary inflammatory milieu and provide the cells for bone regeneration and immune modulation. Increasing age is accompanied with a rise in the basal state of inflammation, potentially impairing osteogenesis.The translational potential of this articleTranslational research has shown multiple interactions between inflammation, ageing, and bone regeneration. This review presents recent, relevant considerations regarding the effects of inflammation and ageing on bone healing.