In vitro cell-based tests are an important preclinical step for the safety assessment of biomaterials and drugs. Three-dimensional cell culture models (3D) may improve the limitations of the usual 2D models, as they better simulate a physiological environment. This work describes the characterization of a 3D spheroid model of MC3T3-E1 murine preosteoblasts for the testing of bone-substitute materials and investigates its adequacy to some of the most employed cell viability tests. The spheroids presented structural stability for 28 days in culture, with a regular spheroidal aspect, compact surface, and dense inner structure, with high potential for mineralization, but a time-dependent reduction in size. The use of colorimetric tests (MTT, XTT, and NRU) did not achieve satisfactory optical densities and did not correlate with cell density in the 3D model, as the aggregates remain strongly stained even after dye extraction steps. On the other hand, the LDH test achieved appropriate optical density and a high correlation with cell density (r2 = 0.77) and identified a dose–response for a well-known cytotoxic polymer (latex), while no toxicity was identified for biocompatible PLA wires. These results indicate that material testing with 3D bone cell models requires a careful choice of test methods and parameters.

The strategies used to associate peptide arginylglycylaspartic acid (RGD) with calcium phosphate grafts to enhance cell–biomaterial interactions have been controversial in the literature. Several works have demonstrated that RGD-functionalized hydroxyapatite (HA) surfaces improve cell adhesion, whereas others claim that RGD-loaded HA has an inhibitory effect when serum is present in the biological medium. To investigate such contradictory results, we associated RGD with the HA surface using physical adsorption and chemical bonding methods and evaluated the cell adhesion and spreading in pre-osteoblasts culture with and without fetal bovine serum (FBS). The effect of functionalization methods on the physicochemical characteristics of both surfaces was analyzed using multiscale techniques. Adsorption assays of serum allowed us to estimate the impact of the association method on the HA surface’s reactivity. Physically adsorbed RGD did not increase the number of adhered cells due to the weak interactions between the peptide and the surface. Although chemical binding stabilizes RGD on the HA, the functionalization procedure covered the surface with molecules such as (3-aminopropyl)triethoxysilane (APTEs) and carbodiimide, changing the surface’s chemical activity. Serum protein adsorption decreased by 90%, revealing a significant reduction in the surface interactions with molecules of the biological medium. The present study’s findings showed that the RGD’s physical association with HA did not improve cell adhesion and that this phenomenon is highly dependent on the presence of serum proteins.

Platelet-rich Fibrin (PRF), a second-generation blood concentrate, offers a versatile structure for bone regeneration due to its composition of fibrin, growth factors, and cytokines, with adaptations like denatured albumin-enriched with liquid PRF (Alb-PRF), showing potential for enhanced stability and growth factor dynamics. Researchers have also explored the combination of PRF with other biomaterials, aiming to create a three-dimensional framework for enhanced cell recruitment, proliferation, and differentiation in bone repair studies. This study aimed to evaluate a combination of Alb-PRF with nanostructured carbonated hydroxyapatite microspheres (Alb-ncHA-PRF), and how this association affects the release capacity of growth factors and immunomodulatory molecules, and its impact on the behavior of MG63 human osteoblast-like cells. Alb-PRF membranes were prepared and associated with nanocarboapatite (ncHA) microspheres during polymerization. MG63 cells were exposed to eluates of both membranes to assess cell viability, proliferation, mineralization, and alkaline phosphatase (ALP) activity. The ultrastructural analysis has shown that the spheres were shattered, and fragments were incorporated into both the fibrin mesh and the albumin gel of Alb-PRF. Alb-ncHA-PRF presented a reduced release of growth factors and cytokines when compared to Alb-PRF (p < 0.05). Alb-ncHA-PRF was able to stimulate osteoblast proliferation and ALP activity at lower levels than those observed by Alb-PRF and was unable to positively affect in vitro mineralization by MG63 cells. These findings indicate that the addition of ncHA spheres reduces the biological activity of Alb-PRF, impairing its initial effects on osteoblast behavior.