Human Oral Mucosal Fibroblasts Research Articles

Adiponectin inhibits ROS/NLRP3 inflammatory pathway through FOXO3A to ameliorate oral submucosal fibrosis.

Oral submucous fibrosis (OSF) is an oral condition characterized by chronic progression, which may lead to the development of malignancy. Currently, available treatments for OSF only provide temporary relief of symptoms, and there is a limited availability of effective interventions that can effectively cure this condition. In this study, we aimed to investigate whether adiponectin (APN) could ameliorate OSF and the mechanisms involved in it. First, human oral mucosal fibroblasts (HOMFs) were cultured, an OSF model was established using arecoline, and APN and Imiquimod treatment were administered. Then we overexpressed NLRP3 and knocked down FOXO3A. FOXO3A, fibrosis-related factors (ɑ-SMA, COL1A, CTGF), TGF-β1/Smad3 signaling-related factors (TGF-β1, p-Smad3, Smad3), NLRP3 inflammasome-related factors (NLRP3, Caspase-1, IL-1β), and ROS levels were evaluated. Finally, we explored the effect of APN on OSF in mice by in vivo experiments. We found that arecoline significantly increased ɑ-SMA, COL1A, CTGF, and TGF-β1 expressions and promoted Smad3 phosphorylation, while APN significantly inhibited the elevation of these fibrosis-related factors. ROS production was significantly elevated in HOMFs after arecoline treatment, while APN treatment inhibited ROS production. However, the addition of Imiquimod and overexpression of NLRP3 exhibited a trend of elevated ROS, resisting the inhibitory effect of APN. Furthermore, adding Imiquimod and overexpression of NLRP3 elevated ɑ-SMA, COL1A and CTGF and activated TGF-β1/Smad3 signaling pathway. Additionally, knockdown of FOXO3A enhanced APN-inhibited ɑ-SMA and COL1A. In vivo experiments further confirmed that APN ameliorated OSF in mice by inhibiting ROS/NLRP3 inflammatory pathway. In conclusion, APN ameliorated arecoline-induced OSF by promoting FOXO3A expression and downregulating the ROS/NLRP3 pathway.

MiR-3529-3p from PDGF-BB-induced cancer-associated fibroblast-derived exosomes promotes the malignancy of oral squamous cell carcinoma

AimsThis study aims to explore the role of exosomes from cancer-associated fibroblasts (CAFs) induced by PDGF-BB in promoting the malignancy of oral squamous cell carcinoma (OSCC) and provide new insight into the mechanism of OSCC progression and its treatment.Main methodsExosomes were extracted from human oral mucosa fibroblasts (hOMFs) and CAFs. Differentially expressed miRNAs of exosomes between hOMFs and CAFs were analysed using high-throughput sequencing and self-programmed R software. Cal-27, a human tongue squamous carcinoma cell line, was treated with exosomes. Differentially expressed miRNAs between clinical cancer tissues and adjacent tissues and between hOMF and CAF exosomes were verified by qRT‒PCR. The effect of miR-3529-3p on Cal-27 cells was clarified by overexpressing or knocking down miR-3529-3p in Cal-27 cells. Sample expression and differentially expressed miRNA expression were compared between cancer and paracarcinoma tissues.Key findingsWe found that exosomes from CAFs (CAF-Exos) were internalized by tongue squamous carcinoma cells and promoted their proliferation, migration, invasion, and antiapoptotic effects. MiR-3529-3p was a significant differentially expressed miRNA between CAF-Exos and exosomes from hOMFs (hOMF-Exos). The overexpression of miR-3529-3p promoted proliferation, migration, and invasion and inhibited apoptosis of Cal-27 cells.SignificanceThis study explores the role of PDGF-BB-induced CAFs in promoting malignancy in OSCC. This study will provide new insight into the mechanism of OSCC progression and its treatment.

In Vitro Cell Proliferation and Migration Properties of Oral Mucosal Fibroblasts: A Comparative Study on the Effects of Cord Blood- and Peripheral Blood-Platelet Lysate.

Cord blood-platelet lysate (CB-PL), containing growth factors such as a platelet-derived growth factor, has a similar efficacy to peripheral blood-platelet lysate (PB-PL) in initiating cell growth and differentiation, which makes it a unique alternative to be implemented into oral ulceration healing. This research study aimed to compare the effectiveness of CB-PL and PB-PL in promoting oral wound closure in vitro. Alamar blue assay was used to determine the optimal concentration of CB-PL and PB-PL in enhancing the proliferation of human oral mucosal fibroblasts (HOMF). The percentage of wound closure was measured using the wound-healing assay for CB-PL and PB-PL at the optimal concentration of 1.25% and 0.3125%, respectively. The gene expressions of cell phenotypic makers (Col. I, Col. III, elastin and fibronectin) were determined via qRT-PCR. The concentrations of PDGF-BB were quantified using ELISA. We found that CB-PL was as effective as PB-PL in promoting wound-healing and both PL were more effective compared to the control (CTRL) group in accelerating the cell migration in the wound-healing assay. The gene expressions of Col. III and fibronectin were significantly higher in PB-PL compared to CB-PL. The PDGF-BB concentration of PB-PL was the highest and it decreased after the wound closed on day 3. Therefore, we concluded that PL from both sources can be a beneficial treatment for wound-healing, but PB-PL showed the most promising wound-healing properties in this study.

Arecoline induces activation of human oral fibroblasts by promoting macrophage secretion of exosomes containing miR-155-5p

To investigate the mechanism by which arecoline regulates the level of miR-155-5p in macrophage-secreted exosomes to induce the transformation of human oral mucosal fibroblasts (HOMFs) into fibroblast phenotype. Exosomes were harvested from human monocytic cell line THP-1 with or without arecoline treatment. The effects of arecoline-treated THP-1 cell culture supernatant (CS), THP-1-derived exosomes (EXO), exosome-depleted THP-1 cell supernatant (NES), miR-155-5p overexpression, and miR-155-5p inhibitor on migration ability of arecoline-treated HOMF cells were examined using Transwell migration assay. The polarization of THP-1 cells was detected using flow cytometry. DCFH-DA was used to detect the level of oxidative stress in the cells with different treatments. The mRNA and protein expressions of α- SMA, type I collagen and SOCS1 in the cells were detected with qRT-PCR and Western blotting. Flow cytometry showed that arecoline-treated THP-1 cells exhibited obvious polarization from M0 to M1. Both the supernatant and exosomes from arecoline-treated THP-1 cells significantly enhanced the migration ability of HOMF cells, increased intracellular oxidative stress, up-regulated the expressions of miR-155- 5p and the mRNA and protein levels of α-SMA and type I collagen, and lowered the mRNA and protein expressions of SOCS1. In HOMF cells treated with exosomes from arecoline- treated THP-1 cells, overexpression of miR-155-5p significantly enhanced cell migration ability and increased cellular expressions of α-SMA and type I collagen, and miR-155-5p inhibitor caused the opposite changes. Arecoline can up-regulate miR-155-5p expression in THP-1 cells and inhibit the expression of SOCS1 protein in HOMF cells via the exosome pathway, thus promoting the fibrotic phenotype transformation of HOMF cells.

Human Oral Mucosal Fibroblasts from Limbal Stem Cell Deficient Patients as an Autologous Feeder Layer for Epithelial Cell Culture

Purpose To investigate if human oral mucosal fibroblasts (HOMF) from patients with limbal stem cell deficiency (LSCD) can be used as an autologous feeder layer to support the culture of epithelial cells for potential clinical use. Methods HOMF were isolated from oral mucosal biopsies obtained from the following groups of patients with LSCD: aniridia, mucous membrane pemphigoid (MMP), Stevens–Johnson syndrome (SJS), and ectodermal dysplasia (ED). The ability of these cells to support the culture of human limbal epithelial cells (HLE) was compared to that of HOMF from non-LSCD donors and 3T3s commonly used to culture epithelial cells for use in the clinic to treat LSCD. Results HOMF were successfully obtained by explant culture for 3/3 aniridia patients, 3/3 MMP patients, 1/3 SJS patients, and 1/1 ED patients. All HOMF cultured from these LSCD groups supported the expansion of HLE with epithelial culture times and total colony forming efficiency (CFE) comparable to those achieved on HOMF isolated from donors without LSCD. PCR showed that all HLE cultured on LSCD donor HOMF expressed p63α, CK15, PAX6, CK12, and MUC16 as did HLE cultured on the control non-LSCD donor HOMF and 3T3s. Western blotting detected CK15 and MUC16 protein expression in all groups. Conclusions HOMF from patients with LSCD can be successfully used to support the expansion of epithelial cells. These cells may therefore be useful as autologous feeder fibroblasts for the expansion of epithelial cells for use in the clinic to treat LSCD.

Generation of a novel model of bioengineered human oral mucosa with increased vascularization potential.

ObjectiveThe aim of this study was to generate novel models of bioartificial human oral mucosa with increased vascularization potential for future use as an advanced therapies medicinal product, by using different vascular and mesenchymal stem cell sources.BackgroundOral mucosa substitutes could contribute to the clinical treatment of complex diseases affecting the oral cavity. Although several models of artificial oral mucosa have been described, biointegration is a major issue that could be favored by the generation of novel substitutes with increased vascularization potential once grafted in vivo.MethodsThree types of mesenchymal stem cells (MSCs) were obtained from adipose tissue, bone marrow, and dental pulp, and their in vitro potential was evaluated by inducing differentiation to the endothelial lineage using conditioning media. Then, 3D models of human artificial oral mucosa were generated using biocompatible fibrin‐agarose biomaterials combined with human oral mucosa fibroblasts and each type of MSC before and after induction to the endothelial lineage, using human umbilical vein endothelial cells (HUVEC) as controls. The vascularization potential of each oral mucosa substitute was assessed in vitro and in vivo in nude mice.Results In vitro induction of MSCs kept in culture was able to increase the expression of VEGF, CD31, and vWF endothelial markers, especially in bone marrow and dental pulp‐MSCs, and numerous proteins with a role in vasculogenesis become overexpressed. Then, in vivo grafting resulted in a significant increase in blood vessels formation at the interface area between the graft and the host tissues, with significantly positive expression of VEGF, CD31, vWF, and CD34 as compared to negative controls, especially when pre‐differentiated MSCs derived from bone marrow and dental pulp were used. In addition, a significantly higher number of cells committed to the endothelial lineage expressing the same endothelial markers were found within the bioartificial tissue.ConclusionOur results suggest that the use of pre‐differentiated MSCs could contribute to a rapid generation of a vascular network that may favor in vivo biointegration of bioengineered human oral mucosa substitutes.

Oral Mucosa Tissue Equivalents for the Treatment of Limbal Stem Cell Deficiency

Cultured limbal and oral epithelial cells have been successfully used to treat patients with limbal stem cell deficiency (LSCD). The most common culture method for these cell therapies utilizes amniotic membrane as a cell support and/or murine 3T3s as feeder fibroblasts. The aim of this study is to refine the production of autologous oral mucosal cell therapy for the treatment of LSCD. Real architecture for 3D tissue (RAFT) is used as an alternative cell culture support. In addition, oral mucosal cells (epithelial and fibroblast) are used as autologous alternatives to donor human limbal epithelial cells (HLE) and murine 3T3s. The following tissue equivalents are produced and characterized: first, for patients with bilateral LSCD, an oral mucosa tissue equivalent consisting of human oral mucosal epithelial cells on RAFT supported by human oral mucosal fibroblasts (HOMF). Second, for patients with unilateral LSCD, HLE on RAFT supported by HOMF. For both tissue equivalent types, features of the cornea are observed including a multi-layered epithelium with small cells with a stem cell like phenotype in the basal layer and squamous cells in the top layers, and p63α and PAX6 expression. These tissue equivalents may therefore be useful in the treatment of LSCD.

Effects of mechanical stress on cells derived from human oral mucosa

Abstract Objective When a denture is applied, the oral mucosa is exposed to mechanical stress (MS) including occlusal force via the denture. Clinically, inflammation is occasionally observed in the oral mucosa immediately below the denture despite a proper fit. In this study, we applied MS to cells derived from human oral mucosa and evaluated its effects. Materials and Methods Subconfluent human oral mucosal epithelial cells (HO-1-N-1) and human gingival fibroblasts (HGF) were used to seed 1´105 cells/dish and, after 24 hours, subjected to MS (1 and 3 MPa, 60 min) using a hydrostatic pressurizer. Morphological changes of the cells were examined by Giemsa stain, and changes in cell activity were measured by the WST assay. In addition, mRNA was collected from the cells, and I Inflammatory cytokines and growth factors genes were measured by real-time RT-PCR assay and ELISA. Results HO-1-N-1 showed no morphological change by Giemsa stain but exhibited a significant decrease in cell activity by WST assay. Next, in HO-1-N-1, the expression of Inflammatory cytokines, and ICAM mRNA was significantly increased after MS at 1 MPa but was attenuated at 3 MPa. In HGF, the expression of IL-6 mRNA was enhanced. In HO-1-N-1 and HGF, the IL-6 and IL-8 protein levels increased significantly after MS. Furthermore, in HGF, the EGF and FGF-2 protein levels increased significantly by stimulation at 3 MPa. Discussion MS applied to the oral mucosa immediately below the denture caused a decrease in the activity of oral mucosal epithelial cells and induced enhancement of inflammatory cytokine production. Also, excessive MS was suggested to promote the cure of oral mucosa wounds by promoting the production of growth factors in the oral mucosa.

Inhibition of HIF1A-AS1 impedes the arecoline-induced migration activity of human oral mucosal fibroblasts

Long non-coding RNA hypoxia-inducible factor 1α-antisense RNA 1 (HIF1A-AS1) has been known to participate in various types of malignancies, but its role in the development of precancerous oral submucous fibrosis (OSF) has not been investigated. In the current study, we first observed the aberrant upregulation of HIF1A-AS1 in OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs) isolated from OSF specimens. Next, we demonstrated that administration of arecoline, a natural alkaloid that is found in areca nut, induced the elevation of HIF1A-AS1 in BMFs. This finding showed that the habit of areca nut chewing may lead to an increase of HIF1A-AS1 in oral mucosa. Moreover, we found that knockdown of HIF1A-AS1 hindered the arecoline-stimulated migration capacity in BMFs, suggesting HIF1A-AS1 was critical to the transdifferentiation of BMFs into myofibroblasts. Altogether, our results demonstrated that overexpression of HIF1A-AS1 in OSF tissues may result from the use of areca nut and lead to activation of BMFs, which contribute to the progression of OSF.

Construction of Vascularized Oral Mucosa Equivalents Using a Layer-by-Layer Cell Coating Technology.

There have been many advances in tissue engineering with respect to in vitro and in vivo models of oral mucosa equivalents (OMEs). To apply in vitro reconstructed oral mucosa models to regenerative medicine and alternatives to animal testing, it is necessary to develop the technology of reconstructing different types of oral tissues, such as control of epithelial differentiation and introduction of appendages. We previously reported that functional three-dimensional (3D) tissue models could be quickly constructed by using a layer-by-layer (LbL) cell coating technique that assembles extracellular matrix (ECM) nanofilms to a cell surface. In this study, 3D human OMEs composed of lamina-propria, keratinized or non-keratinized epithelium, and blood capillaries were constructed by using the LbL cell coating technology. Human oral mucosal fibroblasts (HOMFs) were coated with ECM nanofilms and accumulated for the construction of oral mucosal lamina-propria. To construct OMEs with keratinized or non-keratinized epithelium, human oral keratinocytes isolated from gingiva (human oral gingival keratinocytes: HOGKs) or human oral keratinocytes isolated from oral mucosa (human oral mucosal keratinocytes: HOMKs) were used in this study. We further studied the construction of epithelialized OMEs with density- and size-controlled blood capillary networks by using human umbilical vein endothelial cells (HUVECs). It was revealed that these constructions had barrier functions in accordance with their histological characterization. The OMEs with keratinization (K-OMEs) showed higher transepithelial electrical resistance (TEER) values compared with OMEs with non-keratinization (N-OMEs). The constructed epithelialized OMEs with blood capillaries are useful for in vitro/ex vivo research models and regenerative medicine as in oral tissue regeneration. The results suggest that OMEs with oral tissue appendages are more promising alternatives to animal testing and can be applied to the design of in vitro oral models that mimic human tissue organs.

Smokeless tobacco extract inhibits proliferation and promotes apoptosis in oral mucous fibroblasts.

The consumption of smokeless tobacco extract (STE) is growing rapidly, and it has been implicated in several human diseases including diabetes, inflammation and a number of types of cancer. The toxicity of STE requires evaluation, as it is known to induce numerous public health issues. To investigate whether STE serves a role in cultured human oral mucosa fibroblasts (hOMFs), the present study examined HOMF morphology with inverted microscopy and immunofluorescence staining. The cell viability was measured with MTT assays, which detected the cell apoptosis rate via flow cytometry. The activities of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) were measured via flow cytometry and commercial kits, subsequent to exposing the cells to various concentrations of STE. Reverse transcription quantitative polymerase chain reaction and western blot analyses were used to demonstrate that the mRNA and the protein expression levels of cell cycle-associated genes (cyclin-dependent kinase inhibitor 1 and cyclin D1), apoptosis-associated genes [B cell lymphoma 2 (Bcl-2) and Bcl-2-associatied X protein], tumor protein (p53), nuclear factor kappa light chain enhancer of activated B cells (NF-κB)-transcription factor (p65) signaling pathways, NF-E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and NAD(P)H: quinoneoxidoreductase1 (NQO1). The results indicated that the hOMF cells were positive for cytokeratin staining. STE induced G1-S cell cycle progression and cell apoptosis by regulating the cell cycle or apoptosis-associated proteins. STE treatment increased the concentrations of ROS and MDA, and decreased the concentrations of SOD and CAT. STE unregulated phosphorylated-p53, NF-κB p65, Nrf2, HO-1, and NQO1 expression levels in the hOMF cells. The present study demonstrated that STE appears to promote oral disease.

Tanshinone Suppresses Arecoline-Induced Epithelial-Mesenchymal Transition in Oral Submucous Fibrosis by Epigenetically Reactivating the p53 Pathway.

Oral submucous fibrosis (OSF) induced by chewing of the areca nut has been considered to be a precancerous lesion with a high probability of developing oral squamous cell carcinoma. Tanshinone (TSN) is the main component extracted from Salvia miltiorrhiza, a traditional Chinese medicine, which was found to have diverse pharmacological effects, such as anti-inflammatory and antitumor. In the current study, we aimed to identify the inhibitory effects and the underlying mechanism of TSN on OSF progress. We found that treatment with TSN inhibited the arecoline-mediated proliferation of primary human oral mucosal fibroblasts and reversed the promotive effects of arecoline on the EMT process. By RNA deep sequencing, we screened two possible targets for TSN: LSD1 and p53. We confirmed that p53 is much lower in OSF than in normal mucous tissues. In addition, p53 and its downstream molecules were decreased by arecoline treatment in oral mucosal fibroblasts, which was reversed by treatment with TSN in a dose-dependent manner. Our results also revealed that arecoline stimulation resulted in hypermethylation of the promoter of TP53 and subsequent downregulation of p53 levels, which was reversed by TSN. Furthermore, we identified that LSD1 could epigenetically activate TP53 by recruiting H3K27me1 and H3K4m2 to its promoter. Our findings provide new insights into the mechanism by which TSN influences arecoline-induced OSF and rationale for the development of clinical intervention strategies for OSF and even oral squamous cell carcinoma.

Anti-cancer activity of a new dihydropyridine derivative, VdiE-2N, in head and neck squamous cell carcinoma

This study aims to examine the effects of a new 1,4-dihydropyridine derivative, VdiE-2N, on cell signaling pathways and mitochondrial events in head and neck squamous cell carcinoma (HNSCC) cells, and on a mice model of xenograft tumor growth/cell proliferation. Four HNSCC cell lines (HN13, HN12, HN6, and CAL27), HEK293 cells (human embryonic kidney 293 cells), and human oral healthy mucosa fibroblasts (OHMF) were used for in vitro assessment of cell viability (resazurin assay) and invasion capacity (modified Boyden chamber assay), and mitochondrial membrane potential (JC-1 fluorescence assay), morphology (transmission electron microscopy), and number of mitochondria (MitoTracker® imaging). SET and pDRP1 proteins were analyzed by immunofluorescence, and proteins involved in cell death/survival pathways were analyzed by Western blotting. HN12 xenograft tumors were established in the flank of Balb/c nude mice, and their characteristics and sensitivity to VdiE-2N were determined by immunohistochemistry and histology. VdiE-2N decreased cell viability in HNSCC cells (IC50 = 9.56 and 22.45µM for HN13 and HN12 cells, respectively) more strongly than it decreased cell viability in OHMF and HEK293 cells (IC50 = 32.90 and > 50µM, respectively). In HN13 cells, VdiE-2N dissipated mitochondrial membrane potential and altered the mitochondria size, shape, and number in a concentration-dependent manner, as well as it induced apoptosis and reduced their invasion capacity. Treatment of mice bearing xenograft tumors with VdiE-2N significantly diminished proliferation of cancer cells. Therefore, VdiE-2N induces HNSCC cell death in vitro through mitochondria-mediated apoptotic pathways and dampens tumor growth in vivo, thus supporting a potential anti-cancer effect.

Identification and Characterization of Intraoral and Dermal Fibroblasts Revisited.

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. 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. 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. A comparison of the characteristics of these cell subpopulations is desired for identifying appropriate cellular applications.

The characterization of human oral mucosal fibroblasts and their use as feeder cells in cultivated epithelial sheets.

Aim:To characterize human oral mucosa middle interstitial tissue fibroblasts (hOMFs) and their application in the cultivation of epithelial sheets.Methodology:hOMFs were cultured with methylcellulose to form cell clusters. hOMFs amplified in adhesive culture were analyzed by flow cytometry, and were found to differentiate into multiple cell types suitable for the cultivation of human corneal epithelial sheets. hOMFs were expanded from clusters to analyze CD56 and PDGFRα expression.Results:These cells showed similar differentiation patterns as keratocytes, and similar expression patterns as mesenchymal and neural cells. Furthermore, we established human corneal epithelial sheets using hOMFs.Conclusion:hOMFs may be of neural crest origin and possess multipotent differentiation capacity, and are suitable for use as an autologous cell source for corneal regeneration.

Human-derived feeder fibroblasts for the culture of epithelial cells for clinical use.

To investigate human oral mucosal fibroblasts (HOMF) and human limbal fibroblasts (HLF) as alternatives to murine 3T3 feeder fibroblasts currently used to support epithelial cell expansion for the treatment of limbal epithelial stem cell deficiency. HLF and HOMF were compared with 3T3s for their ability to support the culture of human limbal epithelial cells and human oral mucosal epithelial cells. HOMF, but not HLF, were equivalent to 3T3s in terms of the number of epithelial population doublings achieved. Human limbal epithelial cells co-cultured with HOMF or 3T3s had similar expression of corneal and putative stem cell markers. HOMF are a suitable and safer feeder fibroblast alternative to 3T3s for the production of epithelial cells for clinical use.

In vitro and in vivo biocompatibility evaluation of a novobiocin stimulus-responsive poly(ethylene glycol)-based hydrogel designed for soft tissue regeneration

In vitro and in vivo biocompatibility evaluation of medical devices designed for soft tissue healing and regeneration is mandatory for new biomaterials targeted toward human application. To this end, a novel stimulus-responsive eight-arm poly(ethylene glycol) (PEG)-based hydrogel for soft tissue regeneration was subjected to preclinical safety evaluation. While in vitro safety testing included determination of half-maximal inhibitory concentrations by cell index impedance measurements, an in vivo pilot rat study focused on detectable signs of oral mucosal inflammation. In a hydrogel with a total volume of 40 µL, the following concentrations of the constituents were released upon hydrogel dissolution in 1 mL body fluid: PEG, 19 µM; gyrase B, 152 µM; and coumermycin, 76 µM, while the dissolving agent novobiocin was effective in a range from 50 to 100 µM. Concerning the components’ biological effects, half-maximal inhibitory concentrations of 806 and 95 µM for human oral mucosal gingival fibroblasts and gingival keratinocytes were calculated for novobiocin, proving gingival keratinocytes more susceptible to substance effects. Furthermore, with respect to coumermycin concentrations, half-maximal inhibitory concentrations of 10 and 3 µM for gingival fibroblasts and gingival keratinocytes, respectively, were obtained, and thereby revealed coumermycin to be toxic in vitro. In vivo, submucosal hard palatal implantation of standard hydrogels showed no evidence of inflammation at any time point under study, which was supported by immunohistochemistry analysis. In order to realize the potential of such a promising stimulus-responsive hydrogel biomaterial concept for soft tissue wound healing and/or regenerative applications, the crosslinker coumermycin and its competitor novobiocin should be replaced by more innocuous alternative protein–ligand pairs.

Gene Signature of Human Oral Mucosa Fibroblasts: Comparison with Dermal Fibroblasts and Induced Pluripotent Stem Cells.

Oral mucosa is a useful material for regeneration therapy with the advantages of its accessibility and versatility regardless of age and gender. However, little is known about the molecular characteristics of oral mucosa. Here we report the first comparative profiles of the gene signatures of human oral mucosa fibroblasts (hOFs), human dermal fibroblasts (hDFs), and hOF-derived induced pluripotent stem cells (hOF-iPSCs), linking these with biological roles by functional annotation and pathway analyses. As a common feature of fibroblasts, both hOFs and hDFs expressed glycolipid metabolism-related genes at higher levels compared with hOF-iPSCs. Distinct characteristics of hOFs compared with hDFs included a high expression of glycoprotein genes, involved in signaling, extracellular matrix, membrane, and receptor proteins, besides a low expression of HOX genes, the hDFs-markers. The results of the pathway analyses indicated that tissue-reconstructive, proliferative, and signaling pathways are active, whereas senescence-related genes in p53 pathway are inactive in hOFs. Furthermore, more than half of hOF-specific genes were similarly expressed to those of hOF-iPSC genes and might be controlled by WNT signaling. Our findings demonstrated that hOFs have unique cellular characteristics in specificity and plasticity. These data may provide useful insight into application of oral fibroblasts for direct reprograming.

An early and late cytotoxicity evaluation of lidocaine on human oral mucosa fibroblasts

Local anesthetic drugs are extensively used in dentistry. However, the cytotoxic effects of these pharmaceutical compounds remain unclear. In this work, we have evaluated the cell viability and cell function of human oral mucosa fibroblasts exposed to different concentrations of lidocaine for increasing incubation times, using a global screening methods including structural, metabolic and microanalytical analyses. Our results demonstrate that lidocaine is able to alter cell viability and function even at low concentrations and times, although the effect of lidocaine concentration was more important than the incubation time. First, the structural analysis methods revealed that ≥5% concentrations of lidocaine are able to significantly reduce cell viability. Then, the metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and water-soluble tetrazolium salt (WST-1) assays suggest that concentrations starting from 1% were able to significantly hinder cell physiology. Finally, electron-probe X-ray microanalysis confirmed the deleterious effects of lidocaine and allowed us to demonstrate that these effects are associated to an apoptosis process of cell death. Therefore, care should be taken when lidocaine is clinically used, and the lowest efficient concentrations should always be used. Furthermore, these results suggest that the comprehensive evaluation method used in this work is accurate and efficient for screening of local anesthetics.

Single Exposure of Human Oral Mucosa Fibroblasts to Ultraviolet B Radiation Reduces Proliferation and Induces COX-2 Expression and Activation

The lip vermillion constitutes a transition tissue, between oral mucosa and skin, where oral mucosal cells from epithelial and connective tissue compartments are exposed to ultraviolet (UV) sunlight. Fibroblasts are abundant resident cells of the connective tissue which are key regulators of extracellular matrix composition, as well as, epithelial and endothelial cell function. UVB light, an inherent component of sunlight, causes several alterations in skin fibroblasts, including premature senescence and increased cyclooxygenase COX-2 expression. To assess if UVB irradiation had similar effects on fibroblasts derived from human oral mucosa (HOM), primary cultures of HOM fibroblasts were irradiated with a single dose of 30 or 60mJ/cm2 of UVB light or sham-irradiated. Fibroblast proliferation was assessed from 3 to 48hrs after UVB-irradiation utilizing [3H]-thymidine incorporation and MTT assays. In addition, COX-2 mRNA expression was detected by RT-PCR, and PGE2 production was assessed using enzyme immunoassay from 0.5 to 24hrs after UVB-irradiation. The results showed a significant decrease in proliferation of UVB-irradiated HOM fibroblasts as compared to controls as measured by both [3H]-thymidine incorporation and MTT assays (p<0.001). HOM fibroblasts had increased COX-2 mRNA expression at 0.5 and 12hrs after irradiation, and PGE2 production was elevated at 12 and 24hrs post-irradiation as compared to controls (p<0.05). The results showed an inhibitory effect of a single dose of UVB irradiation on HOM fibroblast proliferation with an increase in COX-2 expression and activation. Therefore, photodamaged fibroblasts may play and important role in the pathogenesis of UV-induced lesions of the lip.