Abstract
The development of bioactive glasses (BGs) and ceramics, such as β-Tricalcium phosphate (β-TCP), Hydroxyapatite (HAp), and apatite-wollastonite (A-W), has revolutionized regenerative medicine (RM), offering innovative solutions for bone and tissue repair, due to the ability of these materials to bond with living bone tissue. Despite significant advancements, evaluating the bioactivity and biological responsiveness of these biomaterials remains a critical challenge. This review provides a comprehensive synthesis of the available methodologies, critically analyzing their advantages, disadvantages, and the possible gap between in vitro and in vivo assessments, including false positives and false negatives. Classical immersion tests techniques for bioactivity evaluation in simulated physiological solutions, such as simulated body fluid (SBF), Tris-buffer (TRIS), or phosphate-buffered saline (PBS) solutions, are discussed, along with the more innovative Simulated Wound Fluid (SWF). Additionally, traditional standardized methods, such as MTT, BrdU, EdU, and XTT, as well as emerging methods like qPCR and immunocytochemistry, used to study cellular behavior, proliferation, adhesion, and differentiation, are compared. Staining assays, including crystal violet, neutral red, and alizarin red, have also been investigated for their effectiveness in evaluating cellular adhesion and quantification. Notably, while all techniques have shown promise in studies involving BGs and ceramics, a multi-parametric approach remains the most reliable strategy for assessing bioactivity and biological responsiveness, highlighting the need for comprehensive studies to validate the results. Finally, the choice between static and dynamic approaches represents a further critical issue, as it significantly affects assay outcomes.