To induce osteogenic differentiation, MSC/HUVEC spheroids were bioprinted and cultured in two different strategies in a custom culture media made of 92% osteogenic differentiation media (Cell Applications Inc., San Diego, CA) and 8% HUVEC culture media. To confirm the morphology of bioprinted tissues undergoing osteogenic differentiation, tissues were sectioned and stained with RUNX2, CD31, F-actin, and DAPI. Cross-sectioned samples were washed three times with DPBS, fixed in 4% paraformaldehyde for 60 min, permeabilized in 0.2% Triton X-100 for 30 min, and blocked with 2.5% normal goat serum (NGS) for 60 min at room temperature. To visualize osteogenic and endothelial-specific genes, the samples were incubated with mouse anti-RUNX2 primary antibody (1:100 in 2.5% NGS) and rabbit anti-CD31 primary antibody (1:100 in 2.5% NGS) for 60 min; washed three times with DPBS; and incubated with goat anti-mouse Alexa Fluor 488 secondary antibody (Molecular Probes; 1:250 in 2.5% NGS) to label RUNX2, goat anti-rabbit Alexa Fluor 647 secondary antibody (Molecular Probes; 1:250 in 2.5% NGS) to label CD31, Alexa Fluor 568 phalloidin (Molecular Probes; 1:1000 in 2.5% NGS) to label filamentous actin, and DAPI (1:1000 in 2.5% NGS) to visualize cell nuclei for 60 min. The stained samples were washed three times with DPBS and imaged by an Olympus FV10i-LIV Confocal Laser Scanning Microscope (Olympus America Inc.) and analyzed using ImageJ software. In addition to sectioned samples, bioprinted tissues were also stained, as a whole-mount sample, as explained above, and fluorescent images were taken on the Zeiss Axiozoom microscope.

To confirm the calcium deposition, cross-sectioned samples were fixed in 4% paraformaldehyde, washed three times with distilled water, and incubated with 2% Alizarin red S stain solution for 30 min at room temperature. Stained samples were washed three times with distilled water and imaged using the EVOS microscope.

To quantify the intensity of RUNX2 and CD31, areas of interest on confocal images were selected using ImageJ. Fifty regions of interest were determined at the surface of the assembled tissue, spheroid-spheroid interface, and core of spheroids on fluorescent images. Each box was used for quantification of the fluorescence intensity. The intensity of RUNX2 and CD31 was normalized by the intensity of DAPI. A representative heat map was generated for the RUNX2/DAPI intensity.

Real-time polymerase chain reaction (PCR) was performed to quantify the gene expression levels of BSP, COL1, ALP, RUNX2, and CDH2. The primers of the measured mRNA genes were as follows: BSP (forward, AAC GAA GAA AGC GAA GCA GAA and reverse, TCT GCC TCT GTG CTG TTG), COL1 (forward, ATG ACT ATG AGT ATG GGG AAG CA and reverse, TGG GTC CCT CTG TTA CAC TTT), ALP (forward, AGC TGA ACA GGA ACA ACG TGA and reverse, CTT CAT GGT GCC CGT GGT C), RUNX2 (forward, GGT TAA TCT CCG CAG GTC ACT and reverse, CAC TGT GCT GAA GAG GCT GTT), CDH2 (forward, GAG CAG TGA GCC TGC AGA TTT T and reverse, TGC TCA GAA GAG AGT GGA AAG CT), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (forward, ATG GGG AAG GTG AAG GTC G and reverse, GGG GTC ATT GAT GGC AAC AAT A). Real-time PCR was analyzed by using SsoFast EvaGreen Supermix (Bio-Rad, USA), and all results were normalized using GAPDH.

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