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

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

Identification and Characterization of Intraoral and Dermal Fibroblasts Revisited

Abstract: 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. Keywords: Dermal fibroblast, differences, gingival fibroblast, oral mucosa fibroblast, periodontal ligament fibroblast, similaritie

Growth factor cocktail to facilitate epithelial differentiation of exfoliated deciduous teeth stem cells

Epithelial cells are important in the regeneration of oral mucosal tissue. The cell source is commonly derived from tissue biopsy, which is obtained through surgery. Stem cells from human exfoliated deciduous teeth (SHED) were demonstrated to differentiate into multiple cell types. As it can be readily available from exfoliated deciduous teeth, it can be induced and become a potential source of epithelial cell for oral tissue study. This study aims to examine a mixture of growth factors in the differentiation of epithelial-like cells obtained from human exfoliated deciduous teeth (SHED) stem cells. This growth factor cocktail constitutes hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), insulin-like growth factor 2 (IGF-II), and epidermal growth factor (EGF). After introducing the cocktail, the treated SHED were assessed for epithelial characteristics and markers using cell proliferation test, morphological transformation, protein and gene expression by immunofluorescence staining and cytometry assessment, respectively. The proliferation rate was analysed statistically using Analysis of Variance (ANOVA) with Repeated Measures (p<0.05). SHED cells demonstrated morphological changes on the 7th day after using the cocktail. These transformations are in alignment with the identification of genes associated with epithelial cells and positive stain outcomes for pan-cytokeratin, E-cadherin, and p63. The cell proliferation test indicated that proliferation of cells and growth factor introduction were significantly correlated. The growth factor cocktail used for this research facilitated SHED differentiation for epithelial-like cells. The outcomes validate the production of epithelial cells using SHED; tissue production studies focus on these aspects immensely

Effect of FGF-2 and PDGF-BB on a co-culture of human gingival fibroblasts and umbilical vein endothelial cells

Gingival recession can be treated by root coverage procedure with tissue graft. The ideal gingiva graft should mimic the properties of the native gingiva. Gingival fibroblasts are main cells that reside in human gingiva, while the endothelial cells are the basis for blood vessel formation. The co-culture of these cells, will help in better understanding of gingival tissue regeneration. This study was aimed to determine the effects of fibroblast growth factor-2 (FGF-2) and plateletderived growth factor-BB (PDGF-BB) on a co-culture of human gingival fibroblasts (HGFs) and human umbilical vein endothelial cells (HUVECs). In this in vitro experimental study, the medium for the establishment of monolayer and coculture of these cells were first optimised. Then, the optimal concentrations of these growth factors were determined by assessing the cell viability using MTT assay. Next, to study the stimulatory effect of these growth factors, both HGF and HUVECs were co-cultured and gene expression analysis for fibroblast and angiogenic biomarkers was assessed using Real-Time RT-PCR. Cell viability assay showed that the effect of FGF-2 on HGF was dose-dependent and was optimum at a concentration of 5 ng mL-1, while that of PDGF-BB on HUVEC was optimum at a concentration of 20 ng mL-1. The stimulatory effect of FGF-2 and PDGF-BB was further supported by the Real-Time PCR results which showed that there is a significant expression of VIM, COL1A1, FN, CD31, VE-Cadherin, and vWF in the treatment group of both cells after three days of co-culture experiment, compared to control group. This study indicates a possible synergistic effect of FGF-2 and PDGF-BB growth factors in a co-culture of HGF and HUVEC leading to proangiogenic activity

Amniotic Membrane as a Scaffold for Periodontal Tissue Engineering

Periodontal disease is characterized by the destruction of tooth supporting structures, and continuous destruction of these structures may lead to alveolar bone defects and tooth loss. Periodontal therapy aims to arrest the disease progression as well as to regenerate the loss of structures. Since, the regeneration of these structures is a complex process, cell-based tissue engineering has become one of the methods for periodontal tissue regeneration. In order to give mechanical support to the cells, an amniotic membrane has been proposed as one type of periodontal scaffold, due to its predictable properties. In this review, the integral structure, properties and the recent research in application of amniotic membranes, basically in medical and dental surgeries, along with its potential as a scaffold in periodontal regeneration are highlighted. Amniotic membranes have shown great potential as a suitable substrate/scaffold in in vitro and animal studies; thus provide an alternative for scaffolds materials nowadays. Nevertheless, further studies are required to establish its role and efficacy in periodontal tissue engineering

The potential of periodontal spheroid for periodontal regeneration

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Characterization of periodontal fibroblast spheroids in vitro, interaction with 3d membranes and potential for tissue regeneration

Autologous transplantation of periodontal fibroblasts may be a promising technique to induce tissue regeneration In the treatment of periodontal disease. Spheroid culture is a form of three dimensional cell culture that promotes cellmatrix interaction and cellular differentiation without recourse to exogenous growth factors. The aim of this study was to develop 3D spheroidal cultures of periodontal fibroblasts In vitro and characterize them with respect to their potential use in periodontal tissue repair and rege09!"8tion. In this study commercial normal periodontal fibroblasts were grown in spheroidal form In vitro using the liquid overlay technique. The fibroblast spheroids were characterized by histology, DNA quantification, scanning electron microscopy and immunohistochemistry. Over 14 days, periodontal fibroblasts formed viable spheroids which Incorporated an extracellular matrix rich in collagen type I and periostln. Production of cementum attachment protein which is a marker of dental hard tissue formation, was not detected. This study shows that periodontal fibroblast spheroids may have the potential to be used as an adjunct for periodontal regeneration

Evaluation of the human amniotic membrane as a scaffold for periodontal ligament fibroblast attachment and proliferation

This study was aimed at evaluating the ability of the human amniotic membrane (HAM) to act as a scaffold for the growth of the main cells in periodontal regeneration, human periodontal ligament fibroblasts (HPDLFs). The HAM has many biological properties that are suitable for periodontal tissue regeneration such as low immunogenicity, anti-fibrosis, anti-inflammation, and a rich extracellular matrix component. Commercially available HPDLFs were seeded onto the HAM, and the attachment and proliferation of the cells were observed through scanning electron microscopy (SEM) and histological analysis. Cell viability was assessed using the alamarBlue® proliferation assay at days 1, 3, 7, 14 and 21. Histologically, the HPDLFs showed a monolayer to multilayer attachment onto the HAM from day 1 to day 7. The SEM analysis demonstrated that the HPDLFs had attached appropriately onto the HAM surface at day 1 to day 3, and began overlapping at day 7, while maintaining their flat shape. However, by days 14 and 21, there was an alteration in the morphology of the cells, where they later became rounded. The proliferation assay showed that the viability of the HPDLFs on the HAM had increased significantly from day 1 to day 7 (p=0.012), but later showed significant reduction at day 14 (p=0.002) and day 21 (p=0.005). In conclusion, this study showed that the HAM was able to function well as a scaffold for HPDLFs within 7 days, and thus, it can be a promising scaffold for periodontal regeneration. However, the behaviour of the cells in relation to the membrane over longer culture duration warrants further investigation

Application of neurotransmitters and dental stem cells for pulp regeneration: A review

Introduction: Although there have been many studies on stem cells, few have investigated how neurotransmitters and stem cell proliferation interact to regenerate dental pulp. Dental pulp regeneration is an innovative procedure for reviving dental pulp, if feasible for the entire tooth. Upon tooth injury, activated platelets release serotonin and dopamine in bulk to mobilize dental pulp stem cells to mediate natural dental repair. This has induced research on the role of neurotransmitters in increasing the proliferation rate of stem cells. This review also covers prospective future treatments for dental pulp regeneration. Methods: A literature search was performed via PubMed and ScienceDirect from 2001 to 2022, using the keywords “neurotransmitter,” “stem cell,” “tooth regeneration,” “tooth repair,” “regenerative dentistry,” and “dental pulp.” Different inclusion/exclusion criteria were used, and the search was restricted to English articles. Results: Nine publications reporting neurotransmitter interactions with stem cells for tooth and pulp regeneration were selected. Conclusion: Neurotransmitters were found to interact with dental stem cells. Evidence pointing to neurotransmitters as a factor in the increased proliferation of stem cells was found. This review thus gives hope for tooth pulp regeneration and repair

TGFβ-1 and ALK5 inhibitor treatment affects expressions of TGF β signalling pathway associated molecules in SHED cultured in keratinocyte growth medium

Stem cells from human exfoliated deciduous teeth (SHED) can be induced to differentiate into epithelial-like cells when cultured in a differentiation medium. Transforming growth factor beta (TGF-β) signalling pathway plays an important regulatory function in epithelial proliferation and differentiation.TGF-β1 functions through a complex mechanism consisting of TGFβ type II and type I (ALK5) receptor to initiate cellular processes which could be inhibited by SB431542 molecule (ALK5 inhibitor). This study aims to analyse the expression of TGF-β signalling pathway associated molecules, ALK5 and Smad4, in SHED cultured in keratinocyte growth medium (KGM), treated with exogenous TGF-β1 and ALK5 inhibitor. SHED was cultured in KGM treated with TGF-β1 or ALK-5 inhibitor. RNA was extracted on cells harvested on day 1, 3, 7, 14 and 21 and subjected to reverse transcriptase-PCR using specific primers for ALK5, Smad4 and stem cell marker (Nanog). PCR products were electrophoresed on a 2% agarose gel stained with SYBR green. Nanog expression was maintained from day1 to 14 in all samples but was down regulated at day 21. ALK5 was expressed in most of the samples analysed from day 1 to 14 and down regulated at day 21. Smad4 expression was up regulated at day 1 to 3, down regulated from day 7 to 14 and lost its expression at day 21. TGFβ-1 and ALK5 inhibitor treatment affects the expressions of Nanog, ALK5 and Smad4 in SHED cultured in KGM. The involvement of these molecules during the differentiation process of SHED into epithelial-like cells will contribute further in the fundamental aspects of tissue regeneration research and highlights the importance of dental pulp stem cell technology