Differentiation of Human Embryonic Stem Cells on Periodontal Ligament Fibroblasts.

Human embryonic stem cells' (hESCs) unlimited proliferative potential and differentiation capability to all somatic cell types makes them one of the potential cell sources in cell-based tissue engineering strategies as well as various experimental applications in fields such as developmental biology, pharmacokinetics, toxicology, and genetics. Periodontal tissue engineering is an approach to reconstitute the ectomesenchymally derived alveolar bone, periodontal ligament apparatus, and cementum tissues lost as a result of periodontal diseases. Cell-based therapies may offer potential advantage in overcoming the inherent limitations associated with contemporary regenerative procedures, such as dependency on defect type and size and the pool and capacity of progenitor cells resident in the wound area. Further elucidation of developmental mechanisms associated with tooth formation may also contribute to valuable knowledge based upon which the future therapies can be designed. Protocols for the differentiation of pluripotent hESCs into periodontal ligament fibroblastic cells (PDLF) as common progenitors for ligament, cementum, and alveolar bone tissue represent an initial step in developing hESC-based experimental and tissue engineering strategies. The present protocol describes methods associated with the guided differentiation of hESCs by the use of coculture with adult PDLFs and the resulting change of morphotype and phenotype of the pluripotent embryonic stem cells toward fibroblastic and osteoblastic lineages.

Stem cells in tooth tissue regeneration--challenges and limitations.

The accelerated pace of research in the stem cell field in recent decades and the accumulated body of knowledge has spurred the interest in potential clinical applications of stem cells in all branches of medicine including regenerative dentistry. In humans, embryonic and adult stem cells are two major groups of cells that can serve as a donor source in tissue engineering strategies based on ex-vivo cellular expansion. It has been shown that adult stem cell populations are present in all examined living tissues of the organism, thus being a crucial source of tissue homeostasis and regeneration, and offering a target population for in situ stimulation of extensive tissue regeneration. Experimental findings indicate that in the complex structure of the tooth organ, both periodontal and endodontic tissues harbour adult stem cells with characteristics peculiar to early stages of cellular differentiation. Myriad of strategies incorporating both embryonic and adult stem cells for the regeneration of a particular tooth structure or the whole teeth were proposed; however their successful application to solve real problems encountered in the clinical practice of dentistry remains an elusive and challenging objective.

Human embryonic stem cell differentiation on periodontal ligament fibroblasts.

Human embryonic stem cells' (hESCs) unlimited proliferative potential and differentiation capability to all somatic cell types made them potential cell source in different cell-based tissue engineering strategies as well as various experimental applications in fields such as developmental biology, pharmacokinetics, toxicology, and genetics. Periodontal tissue engineering aims to improve the outcome of regenerative therapies which have variable success rates when contemporary techniques are used. Cell-based therapies may offer potential advantage in overcoming the inherent limitations associated with guided tissue-regeneration procedures, such as dependency on defect type and size and the pool and capacity of progenitor cells resident in the wound area. Elucidation of developmental mechanisms of different periodontal tissues may also contribute to valuable knowledge based upon which the future therapies can be designed. Prior to the realization of such a potential, protocols for the differentiation of pluripotent hESCs into periodontal ligament fibroblastic cells (PDLF) as common progenitors for ligament, cementum, and alveolar bone tissue need to be developed. The present protocol describes methods associated with the guided differentiation of hESCs by the use of coculture with adult PDLFs, and the resulting change of morphotype and phenotype of the pluripotent embryonic stem cells toward fibroblastic and osteoblastic lineages.

In vitro differentiation and attachment of human embryonic stem cells on periodontal tooth root surfaces.

Periodontal tissue engineering based on cell replacement therapies is a promising field for improved regeneration of tooth supporting structures lost as a result of destructive periodontal diseases. Human embryonic stem cells (hESCs) could become adequate cell source for tissue engineering because of their unlimited proliferative potential and ability to differentiate to all somatic cell types. The aim of this study was to analyze the differentiation capacity of hESCs toward periodontal compartment cells and their relationship with tooth root surfaces in vitro. Periodontal ligament fibroblastic cell (PDLF) cultures were established and characterized; hESCs (HUES-9 line) were expanded in undifferentiated state and characterized for pluripotency morphologically and immunohistochemically. Extracted tooth root slices (RS) of 300 microm thickness, prepared with both periodontal and endodontic instrumentation, were used. Three different experimental groups were established: (i) undifferentiated hESC colonies cultured on and around the RS; (ii) undifferentiated hESC colonies cultured on and around RS with PDLF coculture, and (iii) undifferentiated hESC colonies cultured on and around RS with PDLF coculture in osteoinductive medium for 3 weeks. The fibrogenic and osteogenic marker expression was assessed with immunohistochemistry; histological staining and scanning electron microscopy were utilized to determine the relationship between differentiating hESCs and mineralized tooth root structures. Results demonstrate that hESC differentiation is influenced by tooth structures, PDLFs, and osteogenic medium, resulting with increased propensity toward mesenchymal lineage commitment, and formation of soft-hard tissue relationship in close contact areas. The proposed experimental system may facilitate further understanding in development of periodontal structures and contribute to realization of hESCs as a cell source in periodontal tissue engineering applications.

Periodontal ligament cellular structures engineered with electrospun poly(DL-lactide-co-glycolide) nanofibrous membrane scaffolds.

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 embryonic stem cell differentiation on tissue engineering scaffolds: effects of NGF and retinoic acid induction.

The indefinite proliferative capacity and ability to differentiate into all somatic cell types can make human embryonic stem cells (hESCs) useful in experimental and applied studies in embryonic development, tissue engineering, genetic engineering, pharmacokinetics, and the like. Cellular differentiation dynamics can be studied in monolayer cell cultures; however, it proceeds in three-dimensional (3D) organization in vivo. The aim of this study was to assess the effects of retinoic acid (RA) and nerve growth factor (NGF) on the differentiation patterns of hESCs in 3D culture environment and to compare it with the monolayer culture. Expanded hESCs (HUES-9) were differentiated in two experimental groups for 21 days: (i) two-dimensional (2D) monolayer cultures of hESC colonies, and (ii) 3D culture of hES single cells in poly(DL-lactic-co-glycolic acid) scaffolds. The media used were embryonic stem cell expansion medium (ES-EM), embryonic stem cell differentiation medium containing fetal calf serum (ES-DM), ES-EM containing either 10 ng/mL NGF or 10(-6) M RA, and their combination. Fixed specimens were analyzed with scanning electron microscopy, and expression of nestin, pan-cytokeratin, troponin, and alpha-fetoprotein at days 7, 14, and 21 was evaluated by immunohistomorphometry and reverse transcriptase--polymerase chain reaction. Results indicate different patterns of ectodermal, mesodermal, and endodermal marker expressions between groups, where NGF and RA preferentially favors the differentiation toward ectodermal and mesodermal lineages. While troponin and nestin expression is significantly elevated in 3D culture environment, pan-cytokeratin expression is favored by 2D culture instead. The effects of 3D scaffold culture imply the usefulness of testing in vitro differentiation properties of hESCs in various culture settings designed as models in prospective tissue engineering applications.

Differentiation of human embryonic stem cells on periodontal ligament fibroblasts in vitro.

Human embryonic stem cells (hESCs) are pluripotent cells with unlimited proliferation potential and differentiation capacity to all types of somatic cells. Periodontal tissue engineering based on in vitro expanded cells holds the promise to overcome the limitations associated with contemporary regenerative techniques. The aim of this study was to investigate the differentiation patterns of hESCs under the influence of periodontal ligament cells in vitro. hESCs (HUES-9) were expanded and characterized for their pluripotency. Then they were transfected with green fluorescent protein-carrying plasmid, and cocultured with human periodontal ligament fibroblastic cells for 21 days. Two experimental groups were established with different medium constituents. Specimens were fixed at days 7, 14, and 21 and were analyzed morphologically under inverted light microscope, and by immunohistochemistry using antibodies against collagen types I and III, fibronectin, fibroblast surface protein, vimentin, and pancytokeratin. Our results demonstrate different patterns of cell differentiation between groups, with about one-fifth of cells in colonies acquiring characteristics similar to periodontal ligament fibroblastic progenitors while others proceed toward distinctive lineages. This indicates the feasibility to direct the differentiation of hESCs toward the periodontal ligament fibroblastic progenitors to some extent. These findings support the notion that hESCs may become a cell source with unlimited supply for periodontal tissue engineering applications.

Encapsulation and osteoinduction of human periodontal ligament fibroblasts in chitosan-hydroxyapatite microspheres.

Periodontal ligament cells play a crucial role in the regeneration of periodontal tissues and an undifferentiated mesenchymal cell subset is thought to exist within this population. The aim of this study was to assess the osteogenic differentiation potential of human periodontal ligament fibroblasts (hPDLFs) in three dimensional (3D)-osteogenic culture environment following encapsulation in chitosan-hydroxyapatite (C/HA) microspheres with the size range of 350-450 microm. Human PDLF cultures were established and three experimental groups were formed: (i) two-dimensional (2D)-culture as single cell monolayer, (ii) 3D-static culture of C/HA encapsulated hPDLFs, and (iii) 3D-dynamic culture of C/HA encapsulated hPDLFs in a rotating wall vessel bioreactor. The cells were cultured in standard culture medium supplemented with beta-glycerophosphate, dexamethasone, and ascorbic acid. After 21 days, immunohistochemistry was performed using antibodies against osteonectin, osteopontin, bone-sialoprotein, and osteocalcin as osteogenic differentiation markers. Phase-contrast and scanning electron microscopy observations were used for histological and morphological evaluation. The combined effects of osteoinductive medium and HA-containing composite microsphere material on encapsulated hPDLFs resulted in the transformation of a considerable portion of the cells into osteoblastic lineage at the end of the experiments. Results demonstrate the ability of hPDLFs to undergo osteogenic differentiation upon induction in vitro, both under 2D and 3D culture conditions. C/HA microspheres in microgravity bioreactor may serve as a suitable 3D environment to support the osteogenic differentiation of human PDLFs, in vitro.

Effect of osteogenic induction on the in vitro differentiation of human embryonic stem cells cocultured with periodontal ligament fibroblasts.

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, beta-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.

Osteogenic induction of human periodontal ligament fibroblasts under two- and three-dimensional culture conditions.

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.