Intermittent compressive force regulates matrix metalloproteinases and tissue inhibitors of metalloproteinases expression in human periodontal ligament cells.

OBJECTIVE: This study aims to evaluate the effects of intermittent compressive force (ICF) on the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) by human periodontal ligament cells (hPDLCs). DESIGN: hPDLCs were subjected to ICF with a magnitude of 1.5 g/cm2 and loaded for 24 h. mRNA and protein expression of several MMPs and TIMPs were assessed using RT-PCR and ELISA analyses. An inhibitor of TGF-ß (SB431542) was used to assess a possible role of TGF-ß in the expression of MMPs and TIMPs under ICF. RESULTS: mRNA and protein analyses showed that ICF significantly induced expression of TIMP1 and TIMP3, but decreased expression of MMP1. Incubation with the TGF-ß inhibitor and applied to ICF showed a downregulation of TIMP3, but expression of MMP1 was not affected. CONCLUSION: ICF is likely to affect ECM homeostasis by hPDLCs by regulating the expression of MMP1 and TIMPs. Moreover, TGF-ß1 regulated expression of TIMP3. These findings suggest ICF may decrease the degradation of ECM and may thus be essential for maintaining PDL homeostasis.

Effects of lactalbumin enzymatic hydrolysate on human squamous cell carcinoma cells-an in vitro study.

Objectives: Alpha-lactalbumin, the protein from human and bovine milk has been found to be promising as an alternative of anticancer agent. This study was aimed to investigate the effects of lactalbumin enzymatic hydrolysate (LAH) on cell proliferation, migration, and mRNA expression of matrix metalloproteinase (MMP) on human squamous cell carcinoma (hSCC) cell lines, in vitro. Methods: Tongue (HSC-4 and 7) and pharyngeal (HN-30 and 31) hSCC cell lines were treated with a two-fold dilution of LAH (0.39-100 mg/ml). Cell viability and cell proliferation were examined by MTT assay. Colony forming unit (CFU) was assessed by crystal violet blue staining. Cell migration was investigated by scratch wound healing assay. Gene expression of metastasis-associated MMPs was assessed by RT-qPCR. Statistical analyses were evaluated at p value = 0.05. Results: LAH at concentration of 50 and 100 mg/ml exhibited cytotoxicity on hSCC cells. The proliferation and CFU ability of hSCC cells were significantly attenuated after LAH treatment. The mRNA expression of MMP2, MMP9, and MMP14 was reduced in HN-30 and HN-31 cells while expression of MMP2 and MMP14 was downregulated in HSC-7 cells. Only MMP1 mRNA level was reduced in HSC-4 cells. However, cell migration of all hSCC cell lines did not alter after LAH treatment. Conclusion: LAH treatment exhibits inhibitory effects on hSCC cell growth, proliferation and MMPs gene expression. Thus, LAH should be the promising alternative agent to develop the prospective anti-cancer drug.

Augmented effect of fibroblast growth factor 18 in bone morphogenetic protein 2-induced calvarial bone healing by activation of CCL2/CCR2 axis on M2 macrophage polarization.

Fibroblast growth factor (FGF) signaling plays essential roles in various biological events. FGF18 is one of the ligands to be associated with osteogenesis, chondrogenesis and bone healing. The mouse critical-sized calvarial defect healing induced by the bone morphogenetic protein 2 (BMP2)-hydrogel is stabilized when FGF18 is added. Here, we aimed to investigate the role of FGF18 in the calvarial bone healing model. We first found that FGF18 + BMP2 hydrogel application to the calvarial bone defect increased the expression of anti-inflammatory markers, including those related to tissue healing M2 macrophage (M2-Mø) prior to mineralized bone formation. The depletion of macrophages with clodronate liposome hindered the FGF18 effect. We then examined how FGF18 induces M2-Mø polarization by using mouse primary bone marrow (BM) cells composed of macrophage precursors and BM stromal cells (BMSCs). In vitro studies demonstrated that FGF18 indirectly induces M2-Mø polarization by affecting BMSCs. Whole transcriptome analysis and neutralizing antibody treatment of BMSC cultured with FGF18 revealed that chemoattractant chemokine (c-c motif) ligand 2 (CCL2) is the major mediator for M2-Mø polarization. Finally, FGF18-augmented activity toward favorable bone healing with BMP2 was diminished in the calvarial defect in Ccr2-deleted mice. Altogether, we suggest a novel role of FGF18 in M2-Mø modulation via stimulation of CCL2 production in calvarial bone healing.

Roles of extracellular adenosine triphosphate on the functions of periodontal ligament cells.

OBJECTIVE: Adenosine triphosphate (ATP) is an essential nucleotide that is normally present in both intracellular and extracellular compartments. Extracellular ATP (eATP) has a pivotal role in both physiological and pathological processes of periodontal ligament tissues. Here, this review aimed to explore the various functions of eATP that are involved in the control of behaviours and functions of periodontal ligament cells. METHODS: To identify the included publications for review, the articles were searched in PubMed (MEDLINE) and SCOPUS with the keywords of adenosine triphosphate and periodontal ligament cells. Thirteen publications were used as the main publications for discussion in the present review. RESULTS: eATP has been implicated as a potent stimulator for inflammation initiation in periodontal tissues. It also plays a role in proliferation, differentiation, remodelling, and immunosuppressive functions of periodontal ligament cells. Yet, eATP has diverse functions in regulating periodontal tissue homeostasis and regeneration. CONCLUSION: eATP may provide a new prospect for periodontal tissue healing as well as treatment of periodontal disease especially periodontitis. It may be utilized as a useful therapeutic tool for future periodontal regeneration therapy.

Betaine promotes osteogenic differentiation in immortalized human dental pulp-derived cells.

OBJECTIVES: This study aimed to evaluate the effect of betaine (BET) on immortalized human dental pulp stem cell (ihDP) osteogenic differentiation. MATERIALS AND METHODS: hDPs were immortalized using SV40 T-antigen transfection. Characterization, multilineage differentiation, proliferation, cell cycle, colony-forming unit, and cellular senescence were evaluated (n = 4). The effect of BET on ihDP response was assessed (n = 4). Osteogenic differentiation was detected using ALP, ARS staining, and RT-qPCR (n = 4). To investigate the involvement of calcium signaling, the cells were pretreated with either 8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) or thapsigargin before BET treatment (n = 6). RESULTS: ihDPs retained similar phenotypic characteristics presented in hDPs but exhibited an increase in cell proliferation and extended culture to passage 25. An increased proportion of cells in S and G2/M phases without senescence was observed in ihDPs. BET (50 mM) treatment significantly increased mineral deposition at 14 days and upregulated ALP, MSX2, BMP2, and RUNX2 expression. TMB-8 pretreatment reduced the effect of BET-induced ihDP osteogenic differentiation, whereas thapsigargin promoted osteogenic differentiation in ihDPs synergistically with BET. CONCLUSION: ihDPs showed superior proliferation ability and a longer life span, which could serve as a promising cell for regenerative dentistry. BET promoted odonto/osteogenic differentiation via intracellular calcium regulation.

Effects of decellularized extracellular matrix derived from Jagged1-treated human dental pulp stem cells on biological responses of stem cells isolated from apical papilla.

Objective: Indirect Jagged1 immobilization efficiently activates canonical Notch signaling in human dental pulp stem cells (hDPSCs). This study aimed to investigate the characteristics of the Jagged1-treated hDPSC-derived decellularized extracellular matrix (dECM) and its biological activity on odonto/osteogenic differentiation of stem cells isolated from apical papilla (SCAPs). Methods: Bioinformatic database of Jagged1-treated hDPSCs was analyzed using NetworkAnalyst. hDPSCs seeded on the Jagged1 immobilized surface were maintained with normal or osteogenic induction medium (OM) followed by decellularization procedure, dECM-N, or dECM-OM, respectively. SCAPs were reseeded on each dECM with either the normal medium or the OM. Cell viability was determined by MTT assay. Characteristics of dECMs and SCAPs were evaluated by SEM, EDX, immunofluorescent staining, and alcian blue staining. Mineralization was assessed by alizarin red S, Von Kossa, and alkaline phosphatase staining. Statistical significance was considered at p < 0.05. Results: RNA-seq database revealed upregulation of several genes involved in ECM organization, ECM-receptor interaction, and focal adhesion in Jagged1-treated hDPSCs. Immobilized Jagged1 increased the osteogenesis of the hDPSC culture with OM. dECMs showed fibrillar-like network structure and maintained major ECM proteins, fibronectin, type I-collagen, and glycosaminoglycans. A decrease in calcium and phosphate components was observed in dECMs after the decellularized process. Cell viability on dECMs did not alter by 7 days. Cell attachment and f-actin cytoskeletal organization of SCAPs proliferated on Jagged1-treated dECMs were comparable to those of the control dECMs. SCAPs exhibited significantly higher mineralization on dECM-N in OM and markedly enhanced on dECM-OM with normal medium or OM conditions. Conclusion: Jagged1-treated hDPSC-derived dECMs are biocompatible and increase odonto/osteogenic differentiation of SCAPs. The results suggested the potential of Jagged1 dECMs, which could be further developed into ECM scaffolds for application in regenerative medicine.

Shear Stress Enhances the Paracrine-Mediated Immunoregulatory Function of Human Periodontal Ligament Stem Cells via the ERK Signalling Pathway.

Relevant immunomodulatory effects have been proposed following allogeneic cell-based therapy with human periodontal ligament stem cells (hPDLSCs). This study aimed to examine the influence of shear stress on the immunosuppressive capacity of hPDLSCs. Cells were subjected to shear stress at different magnitudes (0.5, 5 and 10 dyn/cm2). The expression of immunosuppressive markers was evaluated in shear stress-induced hPDLSCs using qRT-PCR, western blot, enzyme activity and enzyme-linked immunosorbent assays. The effects of a shear stress-derived condition medium (SS-CM) on T cell proliferation were examined using a resazurin assay. Treg differentiation was investigated using qRT-PCR and flow cytometry analysis. Our results revealed that shear stress increased mRNA expression of IDO and COX2 but not TGF-ß1 and IFN-γ. IDO activity, kynurenine and active TGF-ß1 increased in SS-CM when compared to the non-shear stress-derived conditioned medium (CTL-CM). The amount of kynurenine in SS-CM was reduced in the presence of cycloheximide and ERK inhibitor. Subsequently, T cell proliferation decreased in SS-CM compared to CTL-CM. Treg differentiation was promoted in SS-CM, indicated by FOXP3, IL-10 expression and CD4+CD25hiCD127lo/- subpopulation. In conclusion, shear stress promotes kynurenine production through ERK signalling in hPDLSC, leading to the inhibition of T cell proliferation and the promotion of Treg cell differentiation.

Cell lineage- and expression-based inference of the roles of forkhead box transcription factor Foxc2 in craniofacial development.

BACKGROUND: Foxc2 is a member of the winged helix/forkhead (Fox) box family of transcription factors. Loss of function of Foxc2 causes craniofacial abnormalities such as cleft palate and deformed cranial base, but its role during craniofacial development remains to be elucidated. RESULTS: The contributions of Foxc2-positive and its descendant cells to the craniofacial structure at E18.5 were examined using a tamoxifen-inducible Cre driver mouse (Foxc2-CreERT2) crossed with the R26R-LacZ reporter mouse. Foxc2 expression at E8.5 is restricted to the cranial mesenchyme, contributing to specific components including the cranial base, sensory capsule, tongue, upper incisor, and middle ear. Expression at E10.5 was still positively regulated in most of those regions. In situ hybridization analysis of Foxc2 and its closely related gene, Foxc1, revealed that expression domains of these genes largely overlap in the cephalic mesenchyme. Meanwhile, the tongue expressed Foxc2 but not Foxc1, and its development was affected by the neural crest-specific deletion of Foxc2 in mice (Wnt1-Cre; Foxc2fl/fl ). CONCLUSIONS: Foxc2 is expressed in cranial mesenchyme that contributes to specific craniofacial tissue components from an early stage, and it seems to be involved in their development in cooperation with Foxc1. Foxc2 also has its own role in tongue development.

Activity of chitosan antifungal denture adhesive against common Candida species and Candida albicans adherence on denture base acrylic resin.

STATEMENT OF PROBLEM: Candida adherence to the denture base is an important cause of denture stomatitis. In addition, infections with drug-resistant Candida have become more prevalent, especially among elderly and immunocompromised patients. Thus, alternative safe antifungal agents for oral applications are needed. PURPOSE: The purpose of this in vitro study was to investigate the activity of chitosan, a natural biopolymer, against common oral Candida species and its efficacy in inhibiting C albicans adherence to denture-base acrylic resin. MATERIAL AND METHODS: The minimum fungicidal concentrations (MFCs) of 5 types of chitosan against 6 species of Candida and 10 C albicans clinical isolates were determined by broth and agar dilution, respectively. N-succinyl chitosan (NSC), low- and high-molecular-weight water-soluble chitosan (LMWC and HMWC), and oligomer and polymer shrimp-chitosan were examined. NSC and HMWC, as pure gel and as a mixture with carboxymethylcellulose (CMC), were applied to acrylic resin disks, incubated with C albicans for 24 hours, and washed, and adherent cells were collected for colony count. The effects of HMWC on human gingival fibroblasts after 1 and 24 hours of treatment were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The retention force of HMWC gel was measured by using a universal testing machine. The Kruskal-Wallis and Mann-Whitney U tests were used to compare the antiadherence activity (α=.05). RESULTS: HMWC had the highest antifungal activity against most Candida species tested and C albicans clinical isolates. HMWC gel completely inhibited C albicans adherence to denture base acrylic resin (P<.001). CMC denture adhesive significantly increased C albicans adherence (P<.001), but adding 2×MFC HMWC into CMC reduced the adherence, although this was not statistically significant (P=.06). HMWC at 1×MFC and 2×MFC showed no toxic effect on gingival fibroblast viability and proliferation. Moreover, the retention force provided by HMWC gel was sufficient for use as a denture adhesive (>5000 Pa). CONCLUSIONS: High-molecular-weight, water-soluble chitosan is a biocompatible biopolymer that could inhibit C albicans adherence and that showed properties suitable for development into an antifungal denture adhesive.