Towards application as reinforcing agents in bone tissue engineering: cytocompatibility of inorganic dichalcogenides molybdenum disulfide nanoplatelets and tungsten disulfide nanotubes

摘要

Introduction: Molybdenum disulfide nanoplatelets (MSNPs) and tungsten disulfide nanotubes (WSNTs) are layered inorganic compounds whose interesting physiochemical properties have been investigated for tribological and electronic applications.Biomedical applications of MSNPs and WSNTs include investigation as lubricants for orthodontic wires and catheters.Recently, we have found that these inorganic nanoparticles are able to reinforce polymeric nanocomposites for tissue engineering better than carbon nanotubes and graphene. A previous study of MSNPs and WSNTs investigated the cytotoxicity of these nanoparticles to cell types that would receive environmental exposure such as lung fibroblasts. This study found no significant effect on cell viability when treated with concentrations up to 100 μg/ml MSNPs and WSNTs. However, as tissue engineering reinforcing agents,the cell types that would be exposed to these particles as the scaffolds degrade would be fibroblastic cell types, including mesenchymal stem cells (MSCs), necessitating investigation into the cytotoxicity of these particles to these cell types. This study investigates the cytotoxicity of MSNPs and WSNTs to fibroblasts and human MSCs, the particles effect on differentiation potential of MSCs, and the uptake of these nanoparticles to determine potentially safe doses for biomedical applications. Materials and Methods: MSNPs were synthesized as described previously, using a high temperature reaction of MoO3 and sulfur powder.WSNTs were purchased from APNano (NY, USA). The nanoparticles were water-solubilized with 1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine conjugated polyethylene glycol (DSPE-PEG). To investigate cytotoxicity, cells were treated with 5,10, SO, 100, or 300 μg/ml concentrations of MSNP or WSNT for 6,12, or 24 hours followed by Presto Blue and lactate dehydrogenase (LDH) assays. Untreated cells or cells treated with DSPE-PEG were used as positive controls while cells treated with 70% ethanol were used as negative control. For differentiation studies, cells were treated with a low (10 μg/ml) or high (50 μg/ml) concentration of nanoparticles for 24 hours and then osteo- or adipogenic differentiation media was used to induce differentiation. Osteogenesis was assessed using Alizarin Red S staining, alkaline phosphatase activity and calcium assays while adipogenesis was assessed using Oil Red O staining and elution against a control of DSPE-PEG treated cells. A sample size of four was used for all groups and Kruskal-Wallis with a Dunn post hoc was used to compare within the groups and with the controls. Results and Discussion: Presto Blue assay indicated that only treatment of NIH-3T3 cells with MSNPs showed a decrease in viability with increasing concentration as compared to DSPE-PEG. Treatment of fibroblasts with MSNPs and treatment of MSCs with MSNPs or WSNTs showed little to no cytotoxicity at any of the concentrations (Figure 1). This trend was confirmed by LDH assay results. An increase in MSC proliferation was observed for all WSNT treatment concentrations compared to the DSPE-PEG control with the highest increase of 45% at 24 hours. MSCs treated with low (10 μg/ml) and high (50 μg/ml) concentrations of MSNPs and WSNTs for 24 hours maintained their differentiation potential to adipocytes and osteoblasts. MSNPs are internalized in vesicles in the cells while WSNTs are internalized in vesicles as well as cytoplasmic matrix. Conclusions: The results indicate that treatment with MSNPs at concentrations up to 10 μg/ml does not significantly affect viability of NIH-3T3 cells. No dose or time dependent increase in cytotoxicity was observed for NIH-3T3 cells treated with WSNTs or MSCs treated with MSNPs or WSNTs. Additionally, treatment with low (10 μg/ml) or high (50 μg/ml) concentrations of the nanoparticles do not affect the differentiation potential of MSCs. The results provide preliminary safety guidelines to further explore the potential of these nanoparti