Zurairah Berahim

Biologic Interaction of Three-Dimensional Periodontal Fibroblast Spheroids With Collagen-Based and Synthetic Membranes

BACKGROUND Cell-based therapy using autologous cells has been suggested as a potential approach for periodontal tissue regeneration. Spheroid systems are a form of three-dimensional cell culture that promotes cell matrix interaction, which could recapitulate the aspect of cell homeostasis in vivo. The aim of this study is to assess the interaction of periodontal fibroblast spheroids with synthetic and collagen-based membranes that have been used in guided tissue regeneration. METHODS Commercially available normal human periodontal ligament fibroblasts were grown in spheroid forms using a liquid overlay technique and then transplanted onto a collagen-based and a polyglycolic acid-based membrane. The biologic interaction of the spheroids with the membranes was assessed using basic histology, Alamar blue tissue viability assay, scanning electron microscopy, and immunohistochemical analysis. RESULTS Periodontal fibroblast spheroids adhered to both membranes, and the cells were able to proliferate and migrate from the spheroids both horizontally and vertically into the membrane scaffolds. Immunohistochemical analysis showed expression of collagen type I, periostin, and Runx2 by the periodontal fibroblasts. CONCLUSION Periodontal fibroblast spheroids were able to grow three-dimensionally on the biologic membranes and may have the potential to be used together with guided tissue regeneration approaches as an adjunct for periodontal regeneration.

Synergistic effects of chitosan scaffold and TGFβ1 on the proliferation and osteogenic differentiation of dental pulp stem cells derived from human exfoliated deciduous teeth.

OBJECTIVE Multipotent stem cells derived from human exfoliated deciduous teeth (SHED) represent a promising cell source for tissue regeneration. In the present study we decided to test the inductive effect of chitosan and transforming growth factor-β1 (TGFβ1) as a scaffold/factor combination on SHED proliferation and osteogenic differentiation. DESIGN Cell proliferation was quantitatively assessed by PrestoBlue, live/dead assay was performed and cell attachment to chitosan scaffold was examined by scanning electron microscopy (SEM). For osteogenic differentiation analysis, alkaline phosphatase activity was quantified, cells were stained with Alizarin Red, and the lineage specific genes/proteins ALP, COL I, BSP, and OCN were analysed by real-time PCR and Western blot. RESULTS SHED remained viable and attached well to the chitosan structure. Moreover, TGFβ1 significantly enhanced the proliferative activity of SHED on the chitosan scaffold. Our data further revealed that chitosan and TGFβ1 enhanced the osteogenic differentiation of SHED, as evidenced by high ALP activity, strong mineral deposition, and the up-regulation of ALP, COL I, BSP, and OCN gene/protein expression. CONCLUSION Together, data from our study indicate that the combination of chitosan scaffolds and TGFβ1 enhanced proliferation and osteogenic differentiation of SHED. These findings suggest that the combined application of chitosan scaffold and TGFβ1 in conjunction with SHED might be beneficial for in vivo bone regeneration.

Isolation and Enhancement of a Homogenous in Vitro Human Hertwig’s Epithelial Root Sheath Cell Population

Hertwig’s epithelial root sheath (HERS) cells play a pivotal role during root formation of the tooth and are able to form cementum-like tissue. The aim of the present study was to establish a HERS cell line for molecular and biochemical studies using a selective digestion method. Selective digestion was performed by the application of trypsin-EDTA for 2 min, which led to the detachment of fibroblast-like-cells, with the rounded cells attached to the culture plate. The HERS cells displayed a typical cuboidal/squamous-shaped appearance. Characterization of the HERS cells using immunofluorescence staining and flow cytometry analysis showed that these cells expressed pan-cytokeratin, E-cadherin, and p63 as epithelial markers. Moreover, RT-PCR confirmed that these cells expressed epithelial-related genes, such as cytokeratin 14, E-cadherin, and ΔNp63. Additionally, HERS cells showed low expression of CD44 and CD105 with absence of CD34 and amelogenin expressions. In conclusion, HERS cells have been successfully isolated using a selective digestion method, thus enabling future studies on the roles of these cells in the formation of cementum-like tissue in vitro.

Evaluation of Osteogenic and Cementogenic Potential of Periodontal Ligament Fibroblast Spheroids Using a Three-Dimensional In Vitro Model of Periodontium.

The aim of this study was to develop a three-dimensional in vitro model of periodontium to investigate the osteogenic and cementogenic differentiation potential of the periodontal ligament fibroblast (PDLF) spheroids within a dentin-membrane complex. PDLFs were cultured in both spheroid forms and monolayers and were seeded onto two biological collagen-based and synthetic membranes. Cell-membrane composites were then transferred onto dentin slices with fibroblasts facing the dentin surface and further cultured for 20 days. The composites were then processed for histology and immunohistochemical analyses for osteocalcin, Runx2, periostin, and cementum attachment protein (CAP). Both membranes seeded with PDLF-derived cells adhered to dentin and fibroblasts were present at the dentin interface and spread within both membranes. All membrane-cell-dentine composites showed positive staining for osteocalcin, Runx2, and periostin. However, CAP was not expressed by any of the tissue composites. It can be concluded that PDLFs exhibited some osteogenic potential when cultured in a 3D matrix in the presence of dentin as shown by the expression of osteocalcin. However the interaction of cells and dentin in this study was unable to stimulate cementum formation. The type of membrane did not have a significant effect upon differentiation, but fibroblast seeded-PGA membrane demonstrated better attachment to dentin than the collagen membrane.

Biological Interaction Between Human Gingival Fibroblasts and Vascular Endothelial Cells for Angiogenesis: A Co-culture Perspective

Advancement in cell culture protocols, multidisciplinary research approach, and the need of clinical implication to reconstruct damaged or diseased tissues has led to the establishment of three-dimensional (3D) test systems for regeneration and repair. Regenerative therapies, including dental tissue engineering, have been pursued as a new prospect to repair and rebuild the diseased/lost oral tissues. Interactions between the different cell types, growth factors, and extracellular matrix components involved in angiogenesis are vital in the mechanisms of new vessel formation for tissue regeneration. In vitro pre-vascularization is one of the leading scopes in the tissue-engineering field. Vascularization strategies that are associated with co-culture systems have proved that there is communication between different cell types with mutual beneficial effects in vascularization and tissue regeneration in two-dimensional or 3D cultures. Endothelial cells with different cell populations, including osteoblasts, smooth muscle cells, and fibroblasts in a co-culture have shown their ability to advocate pre-vascularization. In this review, a co-culture perspective of human gingival fibroblasts and vascular endothelial cells is discussed with the main focus on vascularization and future perspective of this model in regeneration and repair.

Identification and Characterization of Intraoral and Dermal Fibroblasts Revisited

BACKGROUND Fibroblasts are the common cells used in clinical regenerative medicine and dentistry. These cells are known to appear heterogeneous in vivo. Previous studies have only investigated the biological properties of these cell subpopulations in vitro. Despite sharing similarity in their spindle-shaped appearance, previous literatures revealed that they play distinguished functional and biological activities in the body. OBJECTIVE This paper highlights the similarities and differences among these cell subpopulations, particularly between intraoral fibroblasts (human periodontal ligament, gingival and oral mucosa fibroblasts) and dermal fibroblasts based on several factors including their morphology, growth and proliferation rate. RESULTS It could be suggested that each subpopulation of fibroblasts demonstrate different positionspecified gene signatures and responses towards extracellular signals. These dissimilarities are crucial to be taken into consideration to employ specific methodologies in stimulating these cells in vivo. CONCLUSION A comparison of the characteristics of these cell subpopulations is desired for identifying appropriate cellular applications.

Cementoblastic lineage formation in the cross-talk between stem cells of human exfoliated deciduous teeth and epithelial rests of Malassez cells.

ObjectivesThe purpose of this study was to evaluate the synergistic effect of epithelial rests of Malassez cells (ERM) and transforming growth factor-β1 (TGF-β1) on proliferation, cementogenic and osteogenic differentiation of stem cells derived from human exfoliated deciduous teeth (SHED).Materials and methodsSHED were co-cultured with ERM with/without TGF-β1. Then, SHED proliferation, morphological appearance, alkaline phosphatase (ALP) activity, mineralization behaviour and gene/protein expression of cemento/osteoblastic phenotype were evaluated.ResultsTGF-β1 enhanced SHED proliferation when either cultured alone or co-cultured with ERM. ERM induced the cementoblastic differentiation of SHED which was significantly accelerated when treated with TGF-β1. This activity was demonstrated by high ALP activity, strong mineral deposition and upregulation of cementum/bone-related gene and protein expressions (i.e. ALP, collagen type I, bone sialoprotein, osteocalcin and cementum attachment protein).ConclusionsERM were able to induce SHED differentiation along the cemento/osteoblastic lineage that was triggered in the presence of TGF-β1.Clinical relevanceThe cemento/osteoblastic differentiation capability of SHED possesses a therapeutic potential in endodontic and periodontal tissue engineering.