Synthesis of scaffold-free, three dimensional, osteogenic constructs following culture of skeletal osteoprogenitor cells on glass surfaces

Abstract

Background: Efficient differentiation of stem cells into three-dimensional (3D) osteogenic construct is still an unmet challenge. These constructs can be crucial for patients with bone defects due to congenital or traumatic reasons. The modulation of cell fate and function as a consequence of interaction with the physical and chemical properties of materials is well known.

Methods: The current study has examined the osteogenic differentiation potential of human skeletal populations following culture on glass surfaces, as a monolayer, or in glass tubes as a pellet culture. The 3D prosperities were assessed morphometrically and the differentiation was evaluated through molecular characterization as well as matrix formation.

Results: Early temporal expression of alkaline phosphatase expression of skeletal populations was observed following culture on glass surfaces. Skeletal populations seeded on glass tubes, adhered as a monolayer to the tube base and subsequently formed 3D pellets at the air -media interface. The pellets cultured on glass displayed 4.9 ± 1.3 times the weight and 2.9 ± 0.1 the diameter of their counterpart cultured in plastic tubes and displayed enhanced production of osteogenic matrix proteins, such a collagen I and osteonectin. The size and weight of the pellets correlated with surface area in contrast to cell numbers seeded. Global DNA methylation level was decreased in pellets cultured on glass. In contrast, gene expression analysis confirmed upregulation extracellular matrix proteins and osteogenesis-related growth factors.

Conclusion: This simple approach to the culture of skeletal cells on glass tubes provides a scaffold-free, 3D construct platform for generating pellets enabling analysis and evaluation of tissue development and integration of multiple constructs with implications for tissue repair and regenerative application on scale-up.

Keywords: 3D culture; 3D, three-dimensional; A/S, Alcian blue/Sirius red/Weigert's haematoxylin; ALP, Alkaline Phosphatase; BMP, bone morphogenic protein; BMSC, human bone marrow stromal; CSF, colony stimulating factor; Ct, Cycle threshold; Differentiation; EGF, epidermal growth factor; FC, Fetal bone cells; FCS, Fetal Calf Serum; FGF, fibroblast growth factor; FN1, fibronectin; GLI, GLI family zinc finger 1; HIPPIE, Human Integrated Protein Interaction Reference; ITGA3, integrin A3; MMP, matrix metalloprotease; Osteogenesis; Osteoprogenitor cells; P/S, penicillin and streptomycin; Pellets; R, receptor; TGF, β transforming growth factor beta; TGFBR2 transforming growth factor beta receptor 2 VDR, vitamin D receptor; gDNA, genomic DNA; iMSC, immortalized human bone marrow derived, mesenchymal stem cells; vWF, von Willebrand factor.

Graphical abstract

Fig. 1

The activity of ALP commenced on day 3 for MG63 cells cultured on glass (F) with increasing intensity until day 15 (G-J). For the cells cultured on plastic, the activity commenced on day 9 (C) with increasing intensity on day 12 (D) and day 15 (E). The ALP intensity was enhanced on glass compared to plastic over all time points examined. Scale bars = 100 μm.

Fig. 2

A/S staining of FC pellets showed negligible matrix formation when cultured in plastic tubes (A), in contrast, marked enhancement of osteogenic matrix was obtained in self-assembled pellets cultured in glass tubes (E). Immunostaining for collagen I, osteonectin and vWF showed enhancement of the studied markers for the pellets cultured in glass tubes (F, G and H) in comparison to plastic (B, C and D). Interestingly, while collagen I followed the Sirius red staining, vWF seemed to be overlaid on the collagen level (H). Scale bars = 100 μm.

Fig. 3

After three weeks in culture, the pellet diameter and weight, were positively correlated with the surface area of the glass tube base (A and B). Pellet diameter and weight did not correlate with MG63 seeding density (C and D). The pellet weight and diameter were significantly higher on glass in comparison to plastic, for both MG63 and iMSCs (E and F).

Fig. 4

(A) Change in gene expression in pellets cultured on glass and plastic, with the latter as the reference. Out of the 84 markers studied in the PCR arrays, 21 genes were significantly upregulated including; 1) cell surface receptors, such as vitamin D receptor (VDR), transforming growth factor beta receptor 2 (TGFBR2), fibroblast growth factor receptor 1, epidermal growth factor receptor and bone morphogenic protein receptor 1A; b) extracellular matrix proteins, including collagen type Iα2, IIα1, IIIα1, fibronectin, their cell mediator protein, Integrin subunit α3 and the modulating enzyme matrix metalloprotease (MMP) 2; c) signalling molecules including, osterix, growth differentiation factor 10, fibroblast growth factor 2, colony Stimulating factor 1, chordin, SMAD5, SEPRINH1, somatomedin A, GLI family zinc finger 1 and alpha 2 HS glycoprotein. Five genes were downregulated, including collagen type XV α1, colony stimulating factor 2, MMP-9, twist1 and vascular endothelial growth factor. (B) Global DNA methylation level showed decreased levels of DNA methylation with MG63 seeded in glass tubes and a comparable observation in iMSCs samples.

Fig. 5

(A) The interaction between the upregulated genes showed an central role for fibronectin with direct connections to the matrix. (B) The relation between downregulated genes (with green background) showed connection with expected intermediates (white background), according to HIPPIE – Human Scored Interactions. The figures were prepared at STRING website (https://string-db.org/).

Fig. S1

Fetal femur derived cells, seeded in glass tubes, adhered to the tube base as a monolayer for 7 days, and subsequently peeled and rolled to form a pellet by day 13. On day 15, the pellet migrated up the tube sidewall, a process that was noted to continue until day 17, when a band of cells emerged from the pellet, adhered to about two thirds of the inner tube circumference to end in a thick band at the media-air interface. On day 19, the whole pellet was observed at the position of the thick band, covered with thin film of media. Side-view on day 21 demonstrated the 3D configuration of the pellet, with position relative to the tube base and size relative to the tube. Cells cultured in plastic tubes retained their size and configuration throughout the culture (n = 5).

Fig. S2

Characterisation of fetal femur derived cells seeded in a 90mm diameter bottle (A-D). The final pellet is shown in (A). The pellet reached 9 mm in length (B). The matrix displayed intense Sirius red (C) and collagen I staining (D). Characterisation of MG63 pellets on plastic (E) and glass (F) stained with Alcian blue and Sirius red, as well as the relative gene expression of pellets cultured on glass, in comparison to pellets cultured on plastic. * p<0.05.