Inflammation is a protective response to damaged tissue and foreign bodies, such as biomaterials, and is usually considered to be negative. More recently, however, the active anti-inflammatory, pro-regenerative role of various mediators and inflammatory cytokines from immune cells have become widely recognised. In particular, macrophages play an essential mediating role in modulating inflammation and thus macrophage phenotype and function has received considerable attention.
Nanoclay has attracted attention in the field of regenerative medicine due to the inherent osteogenic bioactivity and ability of nanoclay to interact with proteins [1,2]. However, to date, no studies have explored how macrophages respond to nanoclay in terms of immunomodulation potential.
In this study, we have evaluated macrophage responses to nanoclay particles. Mouse bone marrow-derived macrophages were isolated from balb/c male mice (4-8 weeks old) and were cultured with various concentrations of nanoclay particles (50, 100, 500, and 1000 µg/ml) for 1 and 3 days. Intracellular and extracellular macrophages were observed by transmission electron microscopy, and the localisation of nanoclay particles in the cells was confirmed by energy-dispersive X-ray spectroscopy (EDX). Macrophage phenotype was evaluated by flow cytometry, and the concentrations of pro-and anti-inflammatory cytokines in culture media were measured by enzyme-linked immunosorbent assay (ELISA). Furthermore, the expression levels of pro-and anti-inflammatory cytokine related genes were assessed by quantitative polymerase chain reaction.
Macrophages actively phagocytosed nanoclay particles, regardless of nanoclay concentration. The nanoclay particles were found in extracellular macrophages, near actin filaments and intracellularly, e.g. within phagosomes and lysosomes. In the presence of 100 µg/ml nanoclay particles, in cell culture media, the population of M1-like macrophages dramatically increased for 24 hours (p<0.034). However, after 3 days, the population of M1-like macrophages decreased, but the number of M2-like macrophages increased. Similarly, significantly higher levels of anti-inflammatory genes, interleukin (IL)-10 and transforming growth factor (TGF)-β1, were observed in macrophages cultured in 100 µg/ml nanoclay particles for 3 days (p<0.017, p<0.032). For Tumour Necrosis Factor (TNF)-α, a pro-inflammatory cytokine, macrophages cultured with nanoclay particles, regardless of their concentrations, showed a significantly lower gene expression level compared to the macrophage without nanoclay (p<0.039). The current studies demonstrate the potential of nanoclay to modulate the phenotype of macrophages in vitro. Nanoclays can promote the development of M2-like macrophages expressing enhanced levels of the inflammatory genes, IL-10 and TGF-β1 with important implications therein for reparative processes in tissue engineering.
1. Shi P. et al. Adv. Healthcare Mater. 7, 1800331 (2018).
2. Dawson J.I. & Oreffo. R.O.C. Adv. Mater 25, 4069-4086 (2013).
Supported by an MRC-AMED Regenerative Medicine and Stem Cell Research Initiative (ref.MR/V00543X/1).