A 3D OsteoblastIn VitroModel for the Evaluation of Biomedical Materials

Abstract

Biomedical materials for bone therapy are usually assessed for their biocompatibility and safety employing animal models orin vitromonolayer cell culture assays. However, alternativein vitromodels may offer controlled conditions closer to physiological responses and reduce animal testing. In this work, we developed a 3D spheroidal cell culture with potential to evaluate simultaneously material-cell and cell-cell interactions. Different cell densities of murine MC3T3-E1 preosteoblasts or human primary osteoblasts (HOb) were used to determine the ideal procedure of spheroidal cultures and their adequacy to material testing. Cells were seeded on 96-well plates coated with agar and incubated in agitation from 1 to 7 days. Aggregate morphology was qualitatively evaluated considering the shape, size, repeatability, handling, and stability of spheroids. Higher cell densities induced more stable spheroids, and handling was considered appropriate starting from 2 × 104cells. Confocal microscopy and Scanning Electron Microscopy indicate that most cells within the aggregate core are viable. Exposure to positive controls has shown a dose dependent cell death as measured by XTT assay. Aggregates were stable and presented good viability when employed on standardized testing of metallic and polymer-based biomaterials. Therefore, osteoblast spheroids may provide a promising tool for material screening and biocompatibility testing.

The two faces of titanium dioxide nanoparticles bio-camouflage in 3D bone spheroids

“…Three-dimensional (3D) models have been used in the area of cancer and bioengineering, however, for nanoparticle safety assessment, they are still unexplored 21–25,36–39 . Although 3D cell culture systems do not replace in vivo studies, they can bridge the gap between traditional 2D in vitro studies and in vivo models 18,27,37,40–43 . The main limitations of 2D models for NPs safety assessment are: absence of tissue architecture, cell polarization and spatial organization of surface receptors, cell communication (most of in vitro studies are single cell type), differences in cell stages (3D cultures usually are a mixture of cells at different stages) and sedimentation effect of NPs on top of the cells that limit their accessibility and diffusion 20,44,45 .…”

“…The role of nanoparticles released by the degradation of an implant system in macrophages/monocytes (2D cell models) is already very well described, however, their direct effect on bone cells in a 3D microenvironment and its contribution to osteolysis is still a mystery. We believe that osteoblast spheroids can mimic the complex cell-cell interaction, and cell-ECM interaction making it an attractive new and promising approach to study the complex interactions of nanometric debris with the biological system 27,28 .…”

Cell Condensation Triggers the Differentiation of Osteoblast Precursor Cells to Osteocyte-Like Cells

“…Here, we first established a novel method to fabricate 3D bone spheroids using mouse precursor osteoblast cells under rotatory culture conditions, by changing several parameters, including the cell density or rotational speed, and the time set for the rotatory shaker, based on the parameters described in the previous study (Furukawa et al, 2001). Although several studies attempted to fabricate the 3D in vitro structure for generating the bone model that enhanced osteogenic differentiation, they utilized scaffold-based techniques, and used biomaterials, such as collagen or agar-agar gel (Restle et al, 2015; Zujur et al, 2017). Moreover, these models could slightly enhance osteocyte differentiation with the accompanying process of osteoblast differentiation, because of which the factor triggering independent osteocyte differentiation remained unknown.…”

Scalable biofabrication and morphology evaluation of tissue spheroids

“…Данные по жизнеспособности клеток помогают выбрать оптимальный срок использования сфероида, который для большинства клеток составляет 3-4 дня. Существуют и исключения из этого правила: например, для сфероидов, приготовленных из первичных хондроцитов барана или первичных остеобластов человека, оптимальным возрастом считается 7-14 дней, когда внеклеточный матрикс накапливается в достаточном количестве и значительно возрастает прочность сфероида, что особенно важно для хрящевой и костной ткани [27].…”