Electroless Palladium-Coated Polymer Scaffolds for Electrical Stimulation of Osteoblast-Like Saos-2 Cells

Abstract

Three-dimensional porous scaffolds offer some advantages over conventional treatments for bone tissue engineering. Amongst all non-bioresorbable scaffolds, biocompatible metallic scaffolds are preferred over ceramic and polymeric scaffolds, as they can be used as electrodes with different electric field intensities (or voltages) for electric stimulation (ES). In the present work we have used a palladium-coated polymeric scaffold, generated by electroless deposition, as a bipolar electrode to electrically stimulate human osteoblast-like Saos-2 cells. Cells grown on palladium-coated polyurethane foams under ES presented higher proliferation than cells grown on foams without ES for up to 14 days. In addition, cells grown in both conditions were well adhered, with a flat appearance and a typical actin cytoskeleton distribution. However, after 28 days in culture, cells without ES were filling the entire structure, while cells under ES appeared rounded and not well adhered, a sign of cell death onset. Regarding osteoblast differentiation, ES seems to enhance the expression of early expressed genes. The results suggest that palladium-coated polyurethane foams may be good candidates for osteoblast scaffolds and demonstrate that ES enhances osteoblast proliferation up to 14 days and upregulate expression genes related to extracellular matrix formation.

Translating Material Science into Bone Regenerative Medicine Applications: State-of-The Art Methods and Protocols

“…Cell localization and distribution on the biomaterial can be analyzed by simply labeling the nuclei at different timepoints after cell seeding on the scaffold [ 52 ]. The morphology of the biomaterial-seeded cells can also be examined by scanning electron microscopy (SEM) [ 14 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 ], as well as the intracellular structures by transmission electron microscopy (TEM) [ 65 , 68 ]. Cell morphology and distribution can also be assessed using immunofluorescence staining of actin filaments, by means of anti-phalloidin antibodies among others, which allows for the analysis of the cytoskeleton conformation of cell growth in contact with the biomaterials [ 14 , 52 , 54 , 55 , 57 , 60 , 62 , 64 , 65 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 ].…”

“…Other commercial colorimetric assays for the evaluation of cell viability are available on the market and, among the most widely used are the cell counting kit-8 (CCK-8) assay that allows a sensitive colorimetric measure of viable cells, using a water-soluble tetrazolium salt that produces, in presence of active dehydrogenases in living cells, an orange formazan product, and the amount of formazan produced is directly proportional to the number of viable cells [ 55 , 57 , 58 , 62 , 73 , 76 , 80 , 81 , 85 , 86 , 87 , 88 , 89 ]. Moreover, the Alamar Blue assay method is frequently used to assess the metabolic activity of proliferating cells: the active resazurin compound, upon entering living cells, is reduced to resorufin, a red fluorescent molecule that can be quantified [ 14 , 59 , 64 , 72 , 75 , 90 , 91 ].…”

“…Different cell lines also find an approved use in the field: the most used is the mouse calvarial preosteoblast MC3T3-E1 cell line [ 52 , 54 , 62 , 63 , 71 , 78 , 80 , 82 , 85 , 86 , 87 , 90 , 114 ]. Other reported cell lines are: human osteosarcoma SaOS-2 cells [ 56 , 64 , 88 ], the mouse osteoblastic KUSA-A1 cell line [ 84 ], the human osteoblast NHOst-Osteoblasts OGM cell line [ 66 ], and the human osteosarcoma MG63 osteoblast-like cells [ 58 , 95 , 97 ].…”

“…In the vast majority of papers in the literature, the expression levels of one or more genes associated with osteoblast differentiation are usually evaluated by quantitative real-time PCR (qRT-PCR) and/or Western blot and/or immunofluorescence analysis and/or ELISA assay [ 14 , 55 , 57 , 58 , 60 , 62 , 64 , 65 , 68 , 69 , 70 , 72 , 73 , 76 , 77 , 79 , 80 , 82 , 83 , 84 , 86 , 87 , 89 , 90 , 91 , 94 , 96 , 98 , 100 , 108 , 109 , 113 ]. Table 3 reports the most representative osteogenic genes usually analyzed.…”

“…During osteogenic differentiation, calcium deposition can be measured by Alizarin Red S staining [ 14 , 53 , 54 , 55 , 57 , 61 , 64 , 65 , 67 , 68 , 69 , 71 , 72 , 76 , 83 , 84 , 85 , 86 , 87 , 90 , 91 , 93 , 94 , 96 , 99 , 114 ]. The direct visualization and analysis of the Alizarin Red stained deposits provides a qualitative measure of the mineralized matrix deposition, but it is possible only on transparent supports and not on thick opaque materials like ceramics and graphene composites.…”