Bone organoids are an emerging novel platform to study human bone biology and bone formation. Biofabrication techniques such as extrusion bioprinting have been used to produce mineralized in vitro bone models. However, cell-cell interactions and mineralization rates are influenced by the initial cell printing density . Here, we investigated the effect of cell density on mineral formation, organoid stiffness, and cell morphology in human 3D bioprinted cell-laden hydrogels under dynamic culture conditions.
Osteoblasts isolated from a femur bone chip of a 15-year-old male were encapsulated in 0.8% (w/v) alginate, 4.1% (w/v) gelatin, 0.1% (w/v) graphene oxide hydrogels . Cells were extrusion bioprinted at 5x106 or 10x106 cells/mL of hydrogel and cultured in compression bioreactors for 10 weeks. Organoids were subjected to uniaxial cyclic compressive loading with 1% strain at 5Hz for 5 minutes 5 times per week. Live/dead assays were performed to determine cell viability after bioprinting (day 1) and after two weeks (day 15) of mechanical loading. F-actin nuclear staining was performed after 30 and 70 days of loading to investigate cell spreading morphology. Time lapsed micro-computed tomography (micro-CT) scans were taken weekly to monitor mineral volume and density. After 10 weeks of loading, organoids were fixed and cryosectioned for histology, immunohistochemistry, and scanning electron microscopy (SEM) to evaluate cellular phenotypes and mineralized matrix formation.
Patient-derived organoids exhibited high cell viabilities after bioprinting (>90%) and after two weeks of daily mechanical loading (>85%). F-actin staining after 30 and 70 days, revealed increased cell spreading and dendrite number in higher cell density organoids. While time lapsed micro-CT images revealed similar endpoint mineral volumes, significant differences were found when comparing mineralization rates and mineral densities between the two cell density groups. Higher cell density organoids exhibited the highest mineral formation rates in the earlier timepoints (28-35 days) while lower cell density organoids reached peak mineral formation after 49-56 days. Notably, a significantly higher average mineral density of 230.8 ± 15 mg HA/cm3 was found in higher cell density organoids compared to 176.9 ± 21.42 mg HA/cm3 in low cell density organoids after 70 days of culture. In line, higher cell density organoids exhibited increased stiffness as compared to lower cell density organoids. A 10-fold increase in stiffness was observed in higher cell density organoids at endpoint compared to day 15 measurements. Meanwhile, lower cell density organoids only showed a 2-fold increase in stiffness during this time. Histology, immunohistochemistry, and SEM imaging revealed distinct cell morphologies in the organoids, including osteoblastic and osteocytic characteristics.
Here, we have established a methodology that better supports the formation of mineralized patient-derived bone organoids resembling native bone tissue. Bioprinting at higher cell densities increased organoid stiffness and mineral density. These clinically relevant bone organoids combine primary patient cells with physiological loading conditions to study mineral formation of healthy bone. In future studies, this platform will be employed to investigate pathological bone and evaluate potential therapies.
 J. Zhang et al., Acta Biomaterialia, 117, 307-322 (2020).