Study on cell type dependent effects of silver nanoparticles reveals a differential toxicity towards M1 and M2 macrophages : implications on cartilage inflammation and regeneration

Abstract

The wide application of silver nanoparticles (AgNPs) in biomedical applications such as drug delivery and anti-microbial agents raises increasing public concerns on their heath impact. Although there are numerous studies on their cytotoxicity, there is still a lack of consensus due to the variations in the preparation of AgNPs. Therefore, a systematic cytotoxic study on seven well-established mammalian cells lines was conducted by using the same batch of AgNPs. The results demonstrate a cell type-dependent cytotoxicity of AgNPs that is not correlated with tissue origins, the production of reactive oxygen species, p53 status, the extent of DNA double-strand breaks and the uptake of AgNPs.

Next, this cell type-dependent cytotoxic effect was investigated on the cells at two extreme differentiation stages of the same cell line. It shows that AgNPs selectively killed pro-inflammatory classically activated (M1) macrophages whereas the cell viability of anti-inflammatory alternatively activated (M2) macrophages retained significantly high under the same condition. A differential cytotoxicity towards M1 and M2 macrophage has been revealed for the first time. Further in vivo studies are required to investigate the influence of this differential cytotoxicity in inflammation and wound healing.

There are limited applications of AgNPs in cartilage research such as treating arthritis and cartilage tissue engineering. This may be because of the inconsistent findings in the anti-inflammatory effects of AgNPs as well as the lack of sufficient information on the influence of AgNPs on healthy and inflamed cartilage cells. Thus, the effect of AgNPs was investigated with TNFα-treated chondrocytes. The data show that AgNPs significantly attenuated the inflammatory cellular responses in TNFα-treated chondrocytes by suppressing the expressions of pro-inflammatory signaling mediators (NFκB and GADD45β) and cartilage matrix degrading enzymes (ADAMTS4 and MMP9). Moreover, a novel, mild and detergent-free protocol was established for cartilage decellularization.

Scanning electron microscopy and histology imaging demonstrate that the microstructures and biochemical components of native cartilage are largely preserved in decellularized cartilage section (DCS) and DCS was shown to provide cells with a more physiological culture niche. Thus, chondrocytes were seeded on DCS and co-incubated with TNFα and AgNPs. Gene analysis reveals that DCS completely changed the effect of AgNPs on TNFα-treated cells by upregulating the expressions of NFκB, GADD45β, ADAMTS4 and MMP9. The work suggests that the cellular responses to inflammation may be highly influenced by tissue microenvironment.

In summary, this study demonstrated that AgNPs exerted cytotoxicity in a cell type-dependent manner. A selective toxicity of AgNPs towards M1 macrophages has been identified for the first time. AgNPs were shown to attenuate the inflammatory cellular responses in TNFα-induced chondrocytes. However, this anti-inflammatory effect of AgNPs could be reversed by the presence of cartilage matrix, which indicates the importance of tissue microenvironment on regulating cellular responses. Further studies of the correlation between AgNPs, DCS and chondrocytes were needed to further investigate the feasibility of AgNPs as a potential therapeutic agent in cartilage repair. In addition, the decellularization protocol and DCS developed in this study can be further applied to other studies.