(H-313) Licensed hMSCs exhibit immunomodulatory capacity enhanced in macrophage co-culture

Introduction:: Bone repair is guided by a series of phases namely inflammatory, remodeling, and regenerative. A key cell type in the inflammatory phase is the macrophage which can take on a gradient of different phenotypes largely categorized as either pro-inflammatory (M1) or pro-healing (M2). Macrophages are responsible for the initiation of the inflammatory response, take on a pro-inflammatory phenotype and clear the site of injury of any debris or pathogens. With time these will polarize towards an M2 phenotype which are responsible for vascular maturation and the recruitment of stem cells to initiate the remodeling phase. However, in critical sized craniomaxillofacial bone defects where surgery is required to implant a graft, the inflammatory phase is reset and any stem cells at the site of injury are exposed to highly inflammatory conditions in the presence of macrophages. MSCs have been shown to elicit immunomodulatory functions when licensed by inflammatory cytokines such as interferon-γ (IFN-γ) and tumor necrosis factor (TNF). Theses immunomodulator functions come in the form of MSC secretion of prostaglandin E2 (PGE2) and interleukin-6 (IL-6) where PGE2 has been shown to include IL-10 production by macrophages, indicative of a pro-healing phenotype. Here we investigate the decoupled effect of macrophage secretome and direct cell-cell contact influences on MSC osteogenic and immunomodulatory expression as a function of licensing in mineralized collagen scaffolds.


Materials and Methods:: Mineralized collagen scaffolds were fabricated by homogenizing and subsequently lyophilizing a collagen-glycosaminoglycan precursor solution with calcium salts into porous scaffolds. MSCs were cultured in basal media or licensed with inflammatory cytokines 24 hours prior to seeding on scaffolds. Scaffolds were then placed in the top compartment of a transwell to allow for an indirect co-culture of THP-1 macrophages in the lower compartment for 14 days. MSC osteogenic and immunomodulatory responses were quantified via protein and gene expression, and metabolic activity as a function of macrophage secretome. Next, MSCs were directly cultured with primary MΦ on anisotropic scaffolds for 14 days of culture to identify influences of prolonged exposure to inflammatory stimuli and direct cell-cell contact. MSC osteogenic and immunomodulatory responses were quantified via similar methods as mentioned above while macrophage polarization was quantified via flow cytometry, protein secretion, and gene expression. Statistical testing was performed using ANOVA with error reported as mean ± standard deviation.


Results, Conclusions, and Discussions:: MSC interactions with macrophages have garnered increasing attention with the realization that these cell types do not act independently in the wound microenvironment but actually work synergistically to lead to bone repair. However, most studies conducted to discern their interactions are done in 2D or in materials that do not mimic the native bone microenvironment. Here we explored the influence of macrophage secretome and direct cell contact on MSC osteogenic and immunomodulatory response under basal and licensed conditions in mineralized collagen scaffolds. Our study demonstrates that macrophage secretome enhances both MSC osteogenesis and immunomodulation in a licensing dependent manner. Specifically, we observed that macrophage secretome enhances MSC mediated osteoprotegerin secretion, an osteoclast inhibitor, while it significantly enhances immunomodulatory factor expression as shown via PGE₂ secretion in all groups with the most significant effect in the licensed group exposed to M0 macrophages (Figure 1). We further demonstrate that the direct co-culture of macrophages with hMSCs not only influences MSC response but also directs macrophage polarization towards an M2 phenotype (Figure 2). This work expands our understanding of MSC-macrophage interactions and illuminated the opportunity to exploit this relationship for improved biomaterial design.


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