Genistein loaded into microporous surface of nano tantalum/PEEK composite with antibacterial effect regulating cellular response in vitro, and promoting osseointegration in vivo

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 ].…”

“…Fluorescent live/dead assays are able to discriminate between live and dead cells by evaluating plasma membrane integrity and the activity of the esterase enzyme, both maintained only in viable cells [ 54 , 55 , 60 , 62 , 70 , 74 , 75 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 ]. 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.…”

“…The analysis of alkaline phosphatase (ALPL) activity is one of the most common tests used to evaluate the osteogenic process and numerous easy-to-use and highly sensitive assays to measure ALPL activity are available on the market [ 14 , 53 , 54 , 55 , 58 , 61 , 65 , 66 , 67 , 68 , 70 , 71 , 73 , 76 , 79 , 80 , 83 , 85 , 86 , 88 , 90 , 91 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , 107 , 114 , 115 ].…”