Effects of Environmental pH on Macrophage Polarization and Osteoimmunomodulation

Biodegradable bone materials can degrade gradually in response to physiological environment and simultaneously promote bone regeneration, thus avoiding the demand of secondary surgery. With the continuous degradation, the environmental pH around the materials could be significantly changed. In the current work, the effects of environmental pH on macrophage polarization and osteoimmunomodulation were investigated. The results showed that the viability of macrophages was inhibited when pH decreased from 7.4 to 6.2 while remained unchanged when pH increased to 8.6. The morphology of macrophages was sensitive to environmental pH changes. The roundish macrophages (pH 7.4) transformed fusiform and polygon-shaped cells at pH 6.6 and 8.2, respectively. The polarization state of macrophages was also found to be pH-dependent. The acidic environment (pH 6.6) tended to polarize macrophages to M2 phenotype, evidenced by the enhanced secretion of arginase-1 (Arg-1), downregulated expressions of M1-related genes such as CD86, tumor necrosis factor-α (TNF-α), interleukin-27 (IL-27), and IL-1β as well as upregulated gene expression of M2 surface marker CD206. The alkaline environment (pH 8.2) led to M1 polarization of macrophages, evidenced by the enhanced production of inducible nitric oxide synthase and upregulated expressions of M1-related genes such as CD11c, TNF-α, IL-1β, and IL-6. Moreover, the gene expressions of growth factors including bone morphogenetic protein-2 (BMP-2), BMP-6, and oncostatin-M (OSM) as well as toll-like receptor (TLR) pathways (TLR-3, TLR-4, and MyD88) were also upregulated at pH 8.2. In addition, the alkaline environment (pH 8.2) was found to exhibit a positive osteoimmunomodulatory effect because the microenvironment created by culturing macrophages in the culture medium with a pH value of 8.2 promoted the alkaline phosphatase (ALP) activity, collagen (Col) production, mineralization, as well as the gene expressions of ALP, Col-I, and osteocalcin (OCN) in osteoblasts. It is considered to be attributed to the enhanced productions of BMP-6 and OSM by macrophages in response to the alkaline environment (pH 8.2). The osteoimmunomodulatory effect of acidic environment (pH 6.6) was found to be weaker. Together, the results indicate that the behaviors of macrophages such as viability, morphology, and polarization state could be modulated by extracellular pH change. The development of novel biomaterials that can create a proper alkaline microenvironment at the materials/bone interface could be beneficial for enhanced bone regeneration.