New Approach Methodologies (NAMs) in Alternative Methods: A Comparative Cytotoxicity Analysis of Nanostructured Hydroxyapatite in Adipose Stem Cells Spheroids

Toxicological assessment is essential in NP approval for health and medical applications. Although 2D cell culture has been widely used, 3D models, especially spheroids, provide better predictive value for toxicological risk assessments since they replicate complex cellular interactions more accurately. In this study, different cytotoxicity assays were used to conduct a comparative nanotoxicological analysis of Adipose Stem Cells (ASC) grown in 2D and 3D (spheroid) systems, based on OECD guidelines. To evaluate the toxicity of nanostructured carbonated hydroxyapatite (nCHA) in ASC spheroids, the Neutral Red Uptake assay, and an ATP quantification assay were used. The results obtained from 2D cell monolayers and 3D spheroids were compared to determine the most suitable assay for spheroid analysis. Moreover, the IC50 for SDS treatment was determined and the spheroid morphology was analyzed after treatment with the nanoparticles. Overall, the three assays confirmed the absence of cytotoxicity of nCHA NPs in the ASC monolayer. In addition, the ATP quantification assay confirmed the absence of cytotoxicity of nCHA NPs in the ASC spheroids. Curiously, SDS cytotoxicity was higher in spheroids than in monolayer cultures, inducing the disaggregation of spheroids in a facile and concentration-dependent manner (IC₅₀ = 9.67 μg/mL in spheroids, approximately eight times lower than the IC₅₀ observed in monolayer cultures, 76 μg/mL). The nCHA NPs were also explored as spheroid functionalization agents. The nanoparticles did not affect ASC spheroids' morphology and diameter, and the nCHA are located mainly in the extracellular matrix of spheroids mimicking the mineral component of bone. Collectively, our findings demonstrated that spheroids are more sensitive than monolayers for evaluating nanoparticle biocompatibility, highlighting the potential of 3D cultures as new advanced models (NAMs) for improved nanotoxicology assessments. Furthermore, the functionalization of ASC spheroids with nCHA NP holds potential both as a 3D osteogenesis model and as a therapeutic product to promote bone regeneration.