Aggregation of human osteoblasts unlocks self-reliant differentiation and constitutes a microenvironment for 3D-co-cultivation with other bone marrow cells

Abstract

Skeletal bone function relies on both cells and cellular niches, which, when combined, provide guiding cues for the control of differentiation and remodeling processes. Here, we propose an in vitro 3D model based on human fetal osteoblasts, which eases the study of osteocyte commitment in vitro and thus provides a means to examine the influences of biomaterials, substances or cells on the regulation of these processes. Aggregates were formed from human fetal osteoblasts (hFOB1.19) and cultivated under proliferative, adipo- and osteoinductive conditions. When cultivated under osteoinductive conditions, the vitality of the aggregates was compromised, the expression levels of the mineralization-related gene DMP1 and the amount of calcification and matrix deposition were lower, and the growth of the spheroids stalled. However, within spheres under growth conditions without specific supplements, self-organization processes occur, which promote extracellular calcium deposition, and osteocyte-like cells develop. Long-term cultivated hFOB aggregates were free of necrotic areas. Moreover, hFOB aggregates cultivated under standard proliferative conditions supported the co-cultivation of human monocytes, microvascular endothelial cells and stromal cells. Overall, the model presented here comprises a self-organizing and easily accessible 3D osteoblast model for studying bone marrow formation and in vitro remodeling and thus provides a means to test druggable molecular pathways with the potential to promote life-long bone formation and remodeling.

Keywords: Calcification; Endothelial cells; Mesenchymal stroma cells; Osteocyte; Osteogenesis; Spheroid.

Figure 1

hFOB spheroid development under proliferative, osteo- and adipo-inductive conditions. (A) Representative hematoxylin/eosin (H&E) histological images of hFOB cell aggregates grown under proliferative condition (growth medium, GM) over 34 days. Aggregates grown in spheroids over the timespan exhibited no necrotic onset. Scale bar 100 µm. (B) Decalcified 6-days-old spheroids grown under proliferative (GM), osteo- (OM) and adipo-inductive (AM) conditions were stained with H&E. Under proliferative condition, spheroids showed a high density of nuclei in the core and no prominent signs of necrosis. In osteo-and adipo-inductive conditions, despite the absence of visible necrosis, more nuclear fragmentation is present. Spheroids in growth medium displayed larger eosinophilic regions. (C) Sirius Red staining of spheroids: red stain indicates a high degree of collagen content in spheroids in growth and, to less extent, in osteogenic media. Scale bar 100 µm.

Figure 2

Calcification in the core of hFOB spheroids. (A) Microscopic evaluation of core mineralization detected by Xylenol Orange (XO). Representative epifluorescence and brightfield (BF) images of 6-days old spheroids cultivated in the presence of 20 µM XO. Control spheroids were cultured in growth medium in the absence of XO. Spheroids in growth and osteogenic media showed a positive signal for XO (red) in the core, while in adipogenic medium the intensity of the signal was comparable to controls. Scale bar, 50 µm (B) Quantitative estimation of XO signal intensity by means of digital image analysis (ImageJ/FIJI). Data represent mean ± s.d of three independent experiments in triplicates. Statistical significance was evaluated by a two-tailed unpaired Student T-test. *P = 0.035; **P = 0.0039; ***P = 0.0006.

Figure 3

Core calcification and apoptosis. (A) TUNEL analysis: representative deconvoluted images of 4-days-old spheroids under different growth conditions containing apoptotic cells (green). Nuclei were counterstained with Hoechst 33342 (blue); scale bar, 50 µM. (B) Spheroids were cultured in growth medium (CTR), or in growth medium containing 50 µm of the caspase inhibitor Z-VAD-FMK. Propidium Iodide (PI) and Hoechst 33342 (Hoechst) stain were performed 5 days post-treatment. Scale bar, 50 µm. (C) Mineralization was visualized under the same conditions with Xylenol Orange (XO) and brightfield microscopy (BF). Scale bar, 50 µm. Statistical significance was evaluated by a two-tailed unpaired Student T-test. Data represent mean ± s.d of at least two independent experiments in triplicates. * P > 0.05.

Figure 4

Osteocalcin expression in spheroids under proliferative conditions. Cells expressing eGFP under the control of human osteocalcin promoter (hFOB hOC_eGFP), were used to form spheroids. Displayed are representative, deconvoluted images of spheroids cultivated for 3 and 6 days at different culture conditions at 34 °C. (A) eGFP expression in hFOB hOC_eGFP spheroids in growth medium was compared to hFOB wild-type spheroids (autofluorescence). (B) Reporter activity of hFOB OC_eGFP spheroids under adipogenic medium was significantly downregulated when compared to osteogenic medium. A semi-quantitative analysis of osteocalcin promotor activity was referred to expression in 2D sub-confluent cultures shortly before spheroid formation. Nuclei were counterstained with Hoechst. For image deconvolution, z-stacks of 40 µm spheroid central-sections were processed with Huygens Essential Software. eGFP intensity estimation was performed by means of ImageJ software on deconvoluted sections. GFP-intensity was normalized to the estimated volume of the spheroids and compared to the value of 1-day-old spheroids. Statistical significance was evaluated by a two-tailed unpaired Student T-test of at least three experiments in triplicate. *P < 0.05; ***P < 0.001; ****P < 0.0001 (C) Gene expression analysis of eGFP and osteogenic marker, osteocalcin. Relative expression was based on gene expression of the 2D sub-confluent cultures used to produce spheroids. Two-tailed unpaired Student T-test were applied for determination of statistical significance ****P < 0.0001.

Figure 5

Osteoblast-osteocytes transition markers. Detection of markers indicative for osteocyte differentiation in spheroids formed under proliferative culture condition. (A) Deconvoluted images of Osteocalcin and DMP1 (Alexa555 in red) together with nuclear counterstaining with Hoechst 33342 (blue). Before image acquisition, spheroids were cleared with 88% glycerol. Control spheroids were assessed with secondary antibody only. Scale bar, 50 µm (B) Expression of DMP1 at higher magnification. DMP1 expression showed a cytoplasmatic localization. The signal is particularly strong in proximity to the plasma membrane, which indicates nascent DMP1 being secreted as component of the osteoid matrix (C) Gene expression analysis of DMP1, Osterix and Osteocalcin in 3D cultures in growth medium. Relative expression for each gene was assessed as fold-induction based on the expression of the corresponding gene in 2D sub-confluent cultures used to produce spheroids. Significance was determined by comparing baseline expression of each gene in 2D with the corresponding expression in spheroids. Two-tailed unpaired Student T-test were applied for statistical analysis. *P < 0.05.

Figure 6

hFOB aggregates constitute a living substrate and 3D-scaffold for different bone marrow cell types. Various cell types were presented to hFOB spheroids in 3D co-culture and could be maintained in hFOB growth medium at 34 °C. Scale bars indicate 100 µm. (A) Leukemia monocytes THP1 were labelled with the lipophilic fluorescent dye DiD (red) and added to 3-days old hFOB aggregates. Cultures were kept in hFOB growth medium for 12 days. Deconvolution microscopy revealed the monocytes being present in spheroid cores. Nuclei were counterstained with Hoechst (blue). (B) Human microvascular endothelial cells (hMEC1) and hFOB expressing eGFP (hFOB_eGFP; green fluorescence) were mixed at a ratio of 3:1 for spheroid formation. Images were taken one (1d) and six (6d) days after joint spheroid formation with the endothelial cells covering the surface of the aggregate. Nuclei were counterstained with Hoechst (blue) (C) Also, wild type hFOB together with human mesenchymal stem cells (hMSC), in the presented case pre-stained with CFSE (green fluorescence), jointly formed stable aggregates. MSCs were found to be located within the spheroid core. Joint aggregates were imaged in bright field illumination; MSC were localized by means of epifluorescence.