Abstract
Graphene and graphene oxide can promote the adhesion, growth and differentiation of mesenchymal stem cells. Further, graphene surface coatings accelerate the differentiation of human mesenchymal stem cells acting as osteogenic inducers. Quantification of the osteogenic induction is conventionally performed with Alizarin Red S (ARS), an anthraquinone derivative used to identify calcium deposits in tissue sections and cell cultures. The ARS staining is quite versatile because the dye forms an Alizarin Red S–calcium complex that can be extracted from the stained monolayer of cells and readily assayed by absorbance measurements. Direct visualization of stained deposits is also feasible; however, an in-situ visualization and quantification of deposits is possible only on transparent supports and not on thick opaque materials like ceramics and graphene composites that are well-known inducers of osteogenesis. In this manuscript, the shape of the 2D-fluorescence spectra of the ARS-calcium complex is used to develop a method to detect and monitor the in-situ differentiation process occurring during the osteogenic induction mediated by opaque graphene oxide surfaces.
Highlights
Bone engineering is aimed at bone repair and regeneration with materials able to restore original tissue properties
Because the 2D spectra shape exhibits specific modifications during Alizarin Red S (ARS)–calcium complex formation, we propose a method for in situ quantification of osteogenic induction on Graphene Oxide (GO), which is feasible for other opaque materials
Among several other advantages, including excellent mechanical properties and good biocompatibility, graphene was demonstrated to be effective in directing stem cell differentiation
Summary
Bone engineering is aimed at bone repair and regeneration with materials able to restore original tissue properties. The osteogenic processes occurring on scaffold and coating materials are usually quantified with Alizarin Red S (ARS) staining. This is the standard method used to evaluate calcium-rich deposits produced by cells of the osteogenic lineage in culture, and is extremely useful for testing matrix mineralization induced by osteo-inductive treatments [3]. ARS staining can be used for a qualitative measurement of calcification with optical microscopy or to quantify the amount of deposits with spectroscopy techniques [3] This method quantifies osteogenesis after solvent extraction of the stained deposits from the material surface [3]. In situ visualization of deposits can be obtained after ARS staining only on transparent materials, such as glass slides or cell culture plastic ware
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