Morphologic and gene expression analysis of periodontal ligament fibroblasts subjected to pressure

Force application (FA) during orthodontic tooth movement is mediated through periodontal ligament (PDL) fibroblasts. FA on deciduous teeth has an inherent risk of root resorption, which is less in permanent teeth. Currently, the root resorption mechanism is poorly understood. We hypothesized that FA alters the morphology and gene expression of PDL fibroblasts. This study was designed to achieve homogenous PDL fibroblast cultures, establish an in-vitro FA model, analyze fibroblast morphology after FA, and compare the gene expressions of PDL fibroblasts of deciduous and permanent teeth after FA. Fibroblasts were sorted from primary cultures of deciduous and permanent tooth PDLs. Cell viability was evaluated in the Opticell (Thermo Scientific, Waltham, Mass) FA model. Cellular morphology was analyzed using immunofluorescence staining for actin and focal adhesion complexes. Gene expressions of untreated or pressure-treated PDL fibroblasts of deciduous and permanent teeth were compared by gene array and confirmed by real-time polymerase chain reaction. Cell sorting resulted in cultures containing 98% of PDL fibroblasts. The Opticell model showed 94% cell survival after FA. FA increased fibroblasts' adhesion. Gene arrays and real-time polymerase chain reactions indicated greater up-regulation of DKK2 mRNA in untreated PDL fibroblasts of deciduous teeth and greater up-regulation of ADAMTS1 mRNA in pressurized PDL fibroblasts of deciduous and permanent teeth. Cell sorting is an efficient method to establish homogenous PDL fibroblast cultures. Using the Opticell FA model allows the maintenance of excellent cell viability. FA increased the surface adherence of fibroblasts. Up-regulation of ADAMTS1 after FA may indicate its involvement in the remodeling of the periodontium during orthodontic tooth movement. Understanding root resorption mechanisms under FA will help to prevent it during orthodontic treatment.