Investigating the Effect of Mineralized Collagen Scaffolds on the Immunomodulatory Capacity of Human Mesenchymal Stem Cells Under Varying Co-Culture Conditions

Craniomaxillofacial (CMF) bone injuries cannot heal naturally, requiring surgical intervention due to their large size and complex topography. The gold standards for treatment of these wounds are autografts and allografts, which are faced with the respective challenges of limited patient bone availability and risk of disease transfer from external sources. Our lab has previously developed mineralized collagen scaffolds which enhance osteogenic differentiation and bone formation without added exogenous factors, both in vitro and in vivo. At the wound site, human mesenchymal stem cells (hMSCs) play critical roles in osteogenic processes and can differentiate into osteoblasts. Further, when exposed to inflammatory stimuli, hMSCs can adopt an immunomodulatory phenotype and induce anti-inflammatory and regenerative behavior in other cell types, including macrophages, via paracrine factors. Macrophages play a key role in the inflammatory response by polarizing across a gradient of phenotypes, which can support functions ranging from inflammation to wound healing. The interactions of hMSCs and macrophages via paracrine factors have been previously characterized in 2D. Before material properties can be leveraged to exploit them, however, further exploration of their interactions in a 3D environment is needed. Thus, leveraging the interaction of hMSCs with macrophages would be crucial to resolution of the inflammatory response and initiation of healing. We investigated hMSC immunomodulatory potential via culture of hMSCs (exposed either to basal or inflammatory conditions) on scaffolds with varying composition (chondroitin-6-sulfate or heparin) and pore structure (isotropic or anisotropic). In the single culture, basal hMSCs showed significantly higher osteoprotegerin (OPG) elution than licensed hMSCs on all scaffolds. Further, licensed hMSCs showed higher secretion of immunomodulatory proteins like IL-6 and PGE2 compared to basal hMSCs. Cells cultured on the anisotropic scaffold showed the highest cell proliferation and elution of immunomodulatory proteins compared to the isotropic scaffold. The Heparin scaffold arbitrated the highest elution of osteogenic protein OPG compared to chondroitin-6-sulfate scaffolds. Secondly, an indirect co-culture of hMSCs (basal vs inflammatory) with pre-polarized macrophages (unpolarized or M1) was performed using anisotropic scaffolds in a transwell plate. In this study, we observed an increase in both metabolic activity and OPG secretion in the presence of macrophages. Future work will investigate a direct co-culture between hMSCs and macrophages, allowing comparison of the decoupled influence of paracrine factors and direct cell-cell contact. The development of a biomaterial platform capable of mediating interactions between multiple cell types to achieve better healing would be transformative to the field of tissue engineering.