Periodontal tissue engineering is expected to overcome the limitations associated with the existing regenerative techniques for the treatment of periodontal defects involving alveolar bone, cementum, and periodontal ligament. Cell-based tissue engineering approaches involve the utilization of in vitro expanded cells with regenerative capacity and their delivery to the appropriate sites via biomaterial scaffolds. The aim of this study was to establish living periodontal ligament cell-containing structures on electrospun poly(DL-lactic-co-glycolic acid) (PLGA) nanofiber membrane scaffolds, assess their viability and characteristics, and engineer multilayered structures amenable to easy handling. Human periodontal ligament (hPDL) cells were expanded in explant culture and then characterized morphologically and immunohistochemically. PLGA nanofiber membranes were prepared by the electrospinning process; mechanical tensile properties were determined, surface topography, nanofiber size, and porosity status were investigated with SEM. Cells were seeded on the membranes at approximately 50,000 cell/cm(2) and cultured for 21 days either in expansion or in osteogenic induction medium. Cell adhesion and viability were demonstrated using SEM and MTT, respectively, and osteogenic differentiation was determined with IHC and immunohistomorphometric evaluation of osteopontin, osteocalcin, and bone sialoprotein marker expression. At days 3, 6, 9, and 12 additional cell/membrane layers were deposited on the existing ones and multilayered hybrid structures were established. Results indicate the feasibility of periodontal ligament cell-containing tissue-like structures engineering with PDL cells and electrospun nanofiber PLGA scaffolds supporting cell adhesion, viability and osteogenic differentiation properties of cells in hybrid structures amenable to macroscopic handling.

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.

Abstract:Osteogenesis is one of the principal components of periodontal tissue development as well as regeneration. As pluripotent cells with unlimited proliferative potential and differentiation ability to all germ layer representatives, embryonic stem cells also hold the promise to become a cell source in bone tissue engineering. Our aim was to investigate osteogenic differentiation potential of human embryonic stem cells (hESCs) under the inductive influence of human periodontal ligament fibroblast (hPDLF) monolayers. After being expanded and characterized morphologically and immunohistochemically, hESCs (HUES-9) were cocultured with hPDLFs for 28 days. Two groups were established: (i) osteogenic induction group with ascorbic acid, b-glycerophosphate, and dexamethasone containing hESC differentiation medium; and (ii) spontaneous differentiation group cultured in hESC differentiation medium. Morphological shift in cells was analyzed under an inverted microscope, and immunohistochemistry was performed on fixed specimens at days 1 and 28 using antibodies against alkaline phosphatase, osteonectin, osteopontin, bone sialoprotein (BSP), and osteocalcin (OSC). Reverse transcription-polymerase chain reaction was utilized for the detection of octameric binding protein-4, BSP, and OSC expression at mRNA level. Mineralization was assessed using alizarin red, and the surface topology shift in colonies was demonstrated with scanning electron microscopy. Results indicate the feasibility of osteogenic differentiation of hESCs in coculture, and suggest a role of periodontal ligament fibroblasts in their differentiation patterns. Advances in the field could allow for potential utilization of hESCs in periodontal tissue engineering applications involving regeneration of bone in periodontal compartment lost as a result of destructive periodontal diseases.