Analysis of the Phenotypic Effects of 2D- and 3D-Cultured Jaw Periosteal Cells on THP-1-Derived Macrophages

Abstract

Mesenchymal stem cells (MSCs) can regulate several immune cells and function as sensors or switchers in innate and adaptive immunity. Jaw periosteum-derived mesenchymal stem cells (JPCs) are a promising cell source for bone tissue engineering (BTE) due to their strong osteogenic potential and excellent accessibility. The present study used a direct coculture model and an innovative horizontal coculture system to investigate the phenotypic and underlying paracrine effects of 2D- and 3D-cultured JPCs on macrophage polarization. In the contact coculture model, JPCs and THP-1 macrophages were directly mixed and cocultured using 24 well plates. For horizontal indirect coculture, the 2D-cultured JPCs were cultivated on conventional plastic material of the coculture plates. The 3D-cultured JPCs were constructed by the cell colonization of β-tricalcium phosphate (β-TCP) scaffolds. Untreated or osteogenically induced 2D/3D-JPCs and THP-1 derived M1/M2 macrophages were cocultured in parallel. After five days of horizontal coculture, M1/M2 macrophages related markers were assessed by flow cytometry or quantitative PCR analysis. In addition, human proteome profiler arrays were used to analyze the cytokines/chemokines secretion in the supernatants of cocultured macrophages. The cocultured JPCs decreased the CD80 expression of M1 macrophages in the contact coculture model. In the horizontal coculture system, flow cytometry results showed that untreated and osteogenically induced 2D-JPCs reduced CD80, CD86, HLA-DR and CD197 markers expression, while CD14 expression was increased on the surface of M1 macrophages. Osteogenically induced 2D-JPCs decreased CD86 and CD36 expression whereas increased levels of CD209, CD11b and CD14 on the surface of M2 macrophages. PCR results showed a decrease in human RANTES and an increase in CD163 and CD209 mRNA levels in M1 macrophages after the coculture with untreated or osteogenically induced 2D-JPCs. Gene expression levels of TNF-α and CD163 were shown to be respectively decreased and increased by 3D-JPCs in M1 macrophages. M2 macrophages showed a tendency to increase their CD163 gene levels after coculturing with both osteogenically induced 2D- and 3D-JPCs. CD209 mRNA levels in M2 macrophages were obviously increased under the influence of untreated or osteogenically induced 3D-JPCs. In addition, proteome array analyses showed that both JPCs and macrophages secrete various soluble factors under the different coculture conditions, which might be involved in the process of JPC-mediated macrophage polarization. Therefore, we concluded that 2D- and 3D-cultured JPCs are able to secrete a wide range of factors to convert THP-1 macrophages from the classical M1 towards the alternative M2 phenotype.