Compatible Evaluation Of Silver Nanoparticles And Silver Based Coatings With Human Bone Marrow-Derived Mesenchymal Stem Cells

Silver nanoparticles(Ag NPs)are one of the most widely used products in nano-medicine due to their broad-spectrum antimicrobial activity.They are used in many medical products such as wound dressings,catheters,bone cement and artificial cardiac valves.In tissue engineering,Ag NPs are often incorporated as antibacterial agents in scaffolds,which are subsequently loaded with human bone marrow-derived mesenchymal stem cells(hMSCs).Thus,possible adverse effects of silver nanoparticles on hMSCs should be investigated carefully to ensure a safe usage.In this study,Ag NPs with a mean diameter of ~30 nm were prepared and their biocompability with hMSCss was investigated.Ag NPs could be uptaken by hMSCs and localized in the cytoplasm.Ag NPs at high concentrations caused a reduction in both cell viability and mitochondrial membrane potential in a dose-dependent manner.Furthermore,they led an increase in the release of lactate dehydrogenase and the production of reactive oxygen species(ROS).Annexin V-FITC/PI staining implied that Ag NPs did not only induce apoptosis but also cause necrosis.Based on cell cycle analysis,G2/M arrest was detected in cells treated with Ag NPs,implicating DNA damage.The in vitro effect of Ag NPs at low concentrations on hMSCs differentiation was investigated.Studies on osteogenic differentiation demonstrated that Ag NPs generally upregulated osteogenesis-related protein expression,including alkaline phosphatase(ALP),type I collagen(COLI),osteopontin(OPN)and osteocalcin(OCN).Moreover,Ag NPs enhacnced the matrix mineralization,indicating that Ag NPs stimulate osteogenic differentiation of hMSCs.For adipogenesis,Ag NPs showed no influence on adiponectin secretion,lipid droplet formation and the expression of adipogenic marker genes.It is concluded that Ag NPs do not influence the adipogenic differentiation of hMSCs in vitro.As for chondrogenesis,results showed that long-term exposure to Ag NPs at low concentrations led to an increased expression of Sry-related high-mobility-group box 9(SOX9),cartilage oligomeric matrix protein(COMP),glycosaminoglycan(GAG)and aggrecan,while type II collagen expression was unaffected.Short-term exposure to Ag NPs at relatively high concentrations resulted in a slight increase in SOX9 expression,while no change in GAG,aggrecan,type IIcollagen or COMP content was found.Expression of type X collagen,a marker for hypertrophic chondrocytes,was reduced after both long-and short-term exposure.In conclusion,30 nm Ag NPs have positive effects on chondrogenesis since they can promote the expression of chondrogenic markers while reducing hypertrophy of hMSCs.Ag NPs based antibacterial coatings were prepared by the electrostatic immobilization of the 2-mercaptosuccinic acid(MSA)functionalized Ag NPs to an interlayer film rich in amine groups fabricated by plasma deposition from a vapor of allylamine(AA).The coatings show good anti-bacterial properties and did not affect the initial adhesion,spreading and proliferation of hMSCs.Furthermore,silver coated surface did not compromise the osteogenic differentiation capacity but promoted adipogenic differentiation of hMSCs as demonstrated by more accumulation of lipid droplets and upregulation of adipogenesis-related genes and proteins.In addition,silver incorporation activated the expression of antioxidant enzymes as a consequence of accumulation of intracellular reactive oxygen species(ROS)in adipogenic induced cells,which was correlated with the enhanced adipogenic capacity of hMSCs.