Impact of 3D collagen-based model and hydrostatic pressure on periodontal ligament fibroblast: A morpho-biochemical analysis

This study developed a customized hydrostatic pressure-based loading environment to investigate the effect of static hydrostatic pressure on the periodontal ligament fibroblasts (PDLf) in a three-dimensional (3D) collagen-based model. The cylindrical tissue constructs were comprised of PDL fibroblast cells seeded in type I collagen matrices and divided into three experimental groups: Control (no load), low-load (∼0.07 kPa), and high-load (∼60 kPa), all subjected to 24 h of experimental duration. Cells in the 3D construct were stained with fluorophore-conjugated antibodies for cytoskeletal protein F-actin and matricellular protein periostin. Cell culture supernatant was assessed for receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) expression. Transmission electron microscopy examined the contact between the cells and the collagen matrix. Ultrastructural changes in the 3D collagen matrix were also analyzed using scanning electron microscopy. Experiments were performed in triplicates, and data was analyzed using one-way ANOVA (p < 0.05). The 3D PDLf constructs from the low-load group demonstrated the highest levels of homogeneous cell distribution and higher expression of F-actin and periostin with enhanced interaction with the matrix. The collagen matrix in this group showed more closely packed fibers forming thicker bundles when compared to the control and the high-load 3D PDLf constructs. Nonuniform cell distribution with decreased expression of F-actin and periostin was observed in the control and high-load PDLf constructs. The high-load group showed the highest RANKL/OPG expression. This study demonstrated low-level hydrostatic pressure's role in regulating PDLf functions and extracellular matrix response, while excessive hydrostatic pressure may be detrimental to PDL fibroblast cell function.