Fe0/cells interfacial processes evaluated by multidimensional and epifluorescence microscopies

GRILLO CA; PUNTARULO S; LOZANO PUERTO RM; FERNANDEZ LORENZO DE MELE, M.; FAGALI NS; PÉREZ MACEDA BT

La Plata

Workshop; Imaging techniques for biotechnology and biomedical applications; 2016

1.Degradable metal/cells interfaceMetallic degradable biomaterials such as iron, magnesium and their alloys have been proposed as suitable materials for temporal cardiac and orthopaedic applications. However, in vivo assays have shown that the surrounding tissues are rich in debris particles, ions and insoluble corrosion products, due to degradation processes. Close to orthopaedic implants highly vascularised fibrous membrane, macrophages, multinucleated giant cells and fibroblasts can be found [1]. Macrophages are the cells that are mainly involved in phagocytosis and inflammation processes[2,3]. The debris particles enter into the periprosthetic tissue and macrophages interact with them and, depending on their size, may be phagocytised inducing a cascade of events. Dead cells have been found at the metal/tissue interface. Stea et al. [4] found that the apoptotic cells in the interface membrane collected from revision surgery for aseptic loosening of hip joint prostheses were mainly macrophages, and cell death was related with metal debris. The formation of particles or debris due to breakage or wear of Fe0 biomaterials, generation of soluble and insoluble corrosion products and pH changes may produce side effects such as oxidative stress and inflammation in tissues adjacent to the implant [5]. Considering the complexity of the biomaterial/tissue system the use of cell cultures is a useful model to evaluate such processes. We show here results obtained by the application of two microscopic techniques to follow the changes in this interface: Time-lapse multidimensional microscopy (MM) and epifluorescence microscopy (EM). MM is suitable to follow the alterations of cells during their exposure to the corrosion products and EMis appropriate to detect reactive species (RS) by using DCFH2-DA. As examples we show here the study of the interactions between degradable iron rings (99.9% SpecPure) with fibroblast cells (3T3 Balbc) and murine macrophages (J774-A1).2.Time-lapse multidimensional microscopy: Evaluation of cell behaviourMany cellular and physicochemical processes could be followed by MM. A great number of reports dedicated to introduce faster, more robust and more sensitive equipments and algorithms for image and time-lapse processing were published in recent years [6]. 3.Epifluorescence microscopy: Evaluation of Reactive Species generation To complement the MM information, RS formation was followed in the Feº surrounding area. The formation of reactive oxygen and nitrogen species (reactive species, RS) is commonly detected using the fluorescein derivative DCFH2-DA. This compound is able to penetrate cell membranes, hydrolyzed by cellular esterases and converted via RS to the fluorescent oxidation product DCF. The figure 2 shows the RS production in three areas at different distances from the center of the Feº ring (Near: 1.7 mm (N), Medium (M): 5.0 mm, Far (F): 10.0 mm). A significant increase of RS production in the nearest zone to the ring compared to the control cells (without ring) can be seen. AcknowledgementsAuthors acknowledge UNLP, CONICET, ANPCyT and MAT2011-29152-C02-02 for the financial support.References[1] Yang F, Wu W, Cao L, Huang Y, Zhu Z, Tang T, et al. Pathways of macrophage apoptosis within the interface membrane in aseptic loosening of prostheses. Biomaterials 2011;32:9159-67.[2] Huk OL, Zukor DJ, Ralston W, Lisbona A, Petit A. Apoptosis in interface membranes of aseptically loose total hip arthroplasty. Journal of Materials Science: Materials in Medicine 2001;12:653-8.[3] Landgraeber S, von Knoch M, Löer F, Wegner A, Tsokos M, Hußmann B, et al. Extrinsic and intrinsic pathways of apoptosis in aseptic loosening after total hip replacement. Biomaterials 2008;29:3444-50.[4] Stea S, Visentin M, Granchi D, Cenni E, Ciapetti G, Sudanese A, et al. Apoptosis in peri-implant tissue. Biomaterials 2000;21:1393-8.[5] Held M, Schmitz MHA, Fischer B, Walter T, Neumann B, Olma MH, et al. CellCognition: Time-resolved phenotype annotation in high-throughput live cell imaging. Nature Methods 2010;7:747-54.[6] Huth J, Buchholz M, Kraus JM, Mølhave K, Gradinaru C, Wichert GV, et al. TimeLapseAnalyzer: Multi-target analysis for live-cell imaging and time-lapse microscopy. Computer Methods and Programs in Biomedicine 2011;104:227-34.