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.

Inflammation is a defensive mechanism for pathogen clearance and maintaining tissue homeostasis. In the skeletal system, inflammation is closely associated with many bone disorders including fractures, nonunions, periprosthetic osteolysis (bone loss around orthopedic implants), and osteoporosis. Acute inflammation is a critical step for proper bone-healing and bone-remodeling processes. On the other hand, chronic inflammation with excessive proinflammatory cytokines disrupts the balance of skeletal homeostasis involving osteoblastic (bone formation) and osteoclastic (bone resorption) activities. NF-κB is a transcriptional factor that regulates the inflammatory response and bone-remodeling processes in both bone-forming and bone-resorption cells. In vitro and in vivo evidences suggest that NF-κB is an important potential therapeutic target for inflammation-associated bone disorders by modulating inflammation and bone-remodeling process simultaneously. The challenges of NF-κB-targeting therapy in bone disorders include: (1) the complexity of canonical and noncanonical NF-κB pathways; (2) the fundamental roles of NF-κB-mediated signaling for bone regeneration at earlier phases of tissue damage and acute inflammation; and (3) the potential toxic effects on nontargeted cells such as lymphocytes. Recent developments of novel inhibitors with differential approaches to modulate NF-κB activity, and the controlled release (local) or bone-targeting drug delivery (systemic) strategies, have largely increased the translational application of NF-κB therapy in bone disorders. Taken together, temporal modulation of NF-κB pathways with the combination of recent advanced bone-targeting drug delivery techniques is a highly translational strategy to reestablish homeostasis in the skeletal system.

Osteonecrosis of the femoral head (ONFH) is a debilitating disease that may progress to femoral head collapse and subsequently, degenerative arthritis. Although injection of bone marrow-derived mononuclear cells (BMMCs) is often performed with core decompression (CD) in the early stage of ONFH, these treatments are not always effective in prevention of disease progression and femoral head collapse. We previously described a novel 3D printed, customized functionally-graded scaffold (FGS) that improved bone growth in the femoral head after CD in a normal healthy rabbit, by providing structural and mechanical guidance. The present study demonstrates similar results of the FGS in a rabbit steroid-induced osteonecrosis model. Furthermore, the injection of BMMCs into the CD decreased the osteonecrotic area in the femoral head. Thus, the combination of FGS and BMMC provides a new therapy modality that may improve the outcome of CD for early stage of ONFH by providing both enhanced biological and biomechanical cues to promote bone regeneration in the osteonecrotic area.