Methods: Human PDL cells were embedded in thin-films of Extracel (80–100 µm), a commercially-available hydrogel composed of hyaluronan and gelatin, and grown on type I collagen-coated 6-well plates. To accelerate the growth and direct fibroblast differentiation from PDL progenitor cells, connective tissue growth factor (CTGF; 100 ng/mL) and fibroblast growth factor-2 (FGF-2; 10 ng/mL) were incorporated into the gel matrix and culture media. The following parameters were measured over a 3-week period: (1) cell viability by the fluorescein diacetate (FDA)–propidium iodide (PI) method; (2) Expression of 18 genes using real-time RT-PCR, and (3) MMP and TIMP-1 synthesis by immunoassays.
Results: Addition of CTGF and FGF-2 produced a significant increase (p<0.001) in cell proliferation and viability compared to the controls. Twelve of the 18 genes were expressed at Ct values < 35, and of these RUNX2, COL1A1, COL3A1, MMP1, MMP2 and TGFB1 were upregulated in growth factor-treated cultures. Growth factors also stimulated the synthesis of MMPs and TIMP-1.
Conclusions: In summary, we have demonstrated that the addition of a third dimension provides another level of complexity to the existing two-dimensional culture systems designed to investigate the biology of the PDL. The system is also suitable for investigating the pathogenesis of periodontal diseases, and with minor adaptations, in a mechanically-active environment.