Characterization of human periodontal ligament fibroblasts in three-dimensional collagen gels

Development of three-dimensional (3-D) models to study orthodontic tooth movement (OTM) is essential to the elucidation of the cellular and molecular basis of tooth movement, much of which remains unknown. Currently, few 3-D in vitro models of OTM exist. Such 3-D models should incorporate analogues of tooth, alveolar bone, and a three-dimensional periodontal ligament (PDL). Objectives: To develop a PDL analogue by evaluating the viability and function of human periodontal ligament fibroblasts (hPDLF) in 3-D collagen gels. Methods: HPDLFs (ScienCell Cat#2630) were expanded in standard culture conditions and seeded (3.33x10⁵cells/mL) into 2mm thick fibrillar collagen gels constrained in 16mm diameter culture wells. The viability, proliferation, and function of the hPDLFs were characterized 1, 3, 7, and 14 days after seeding by Live/Dead assay, DNA quantification, H&E and picrosirius staining, SEM, and gel contraction measurements. Results: Cell viability was uniform throughout the volume of the gel at all time points. The number of cells in the gels increased five-fold by D7 (p<0.05) and remained similar until D14. At later time points, collagen fibres in the constrained gels became radially-oriented. The degree of contraction of the released gels was highest immediately upon release, with the highest contraction observed in the D7 and D14 gels (p<0.05), and no further increase in contraction over time for each gel. Conclusions: These results indicate that the collagen gel supports cell proliferation, viability, and the emergence of a possible contractile phenotype, replicating the constrained condition of the human PDL in vivo. This suggests that hPDLFs maintained over time in 3-D collagen gels can be used as a PDL analogue for studies to better understand the effects of compression and tension forces in 3-D models of OTM.

This study was supported by the Alpha Omega Foundation of Canada and the AAOF.