Osteogenic Induction of Human Periodontal Ligament Fibroblasts Under Two- and Three-Dimensional Culture Conditions

Abstract

Human periodontal ligament fibroblasts (hPDLF) play a key role in the regeneration of periodontal compartment during guided tissue regeneration procedures. This property is attributed to the progenitor cell subsets residing in the area. The aim of this study was to investigate whether hPDLFs could undergo an osteogenic differentiation under two- and three-dimensional (2D and 3D) culture conditions upon osteogenic induction. hPDLFs were isolated from six healthy donors, cultured, and expanded according to standard protocols. Then, three osteogenic culture conditions (dexamethasone, ascorbic acid, and beta-glycerophosphate) were established: 1) 2D culture as single-cell monolayer, 2) 3D-static culture on mineralized poly(DL-lactic-co-glycolic acid) (PLGA) scaffold, and 3) 3D culture on mineralized PLGA scaffold inside the NASA-approved bioreactor stimulating microgravity conditions. After 21 days of osteogenic induction, the majority of monolayer cultures had undergone differentiation toward osteogenic lineage, as indicated by morphological changes, mineralization assay, and some phenotypical properties. However, immunohistochemistry revealed that the scaffold cultures expressed higher levels of osteogenic marker proteins compared with that of the monolayers. Secondly, hPDLF-PLGA constructs in bioreactor showed an increased expression of osteopontin and osteocalcin compared with that of static 3D culture after 21 days. Results indicate that human periodontal ligament contains a subpopulation of cells capable of undergoing osteogenic differentiation and presumably contributing to regeneration of bone defects in the adjacent area. Human PDLF-seeded mineralized PLGA scaffold in microgravity bioreactor may be used to support osteogenic differentiation in vitro. Thus, this system may offer new potential benefits as a tool for periodontal tissue engineering.