Human Periodontal Ligament Fibroblast Cells Research Articles

Dentin Collagen versus Er:YAG Laser as Surface Biomodifiers for Intact Root Slices Simulating Delayed Replanted Roots.

Objective: To evaluate effects of dentin collagen versus Er:YAG laser application through enhancing human periodontal ligament fibroblast (PDLF) cells to attach to intact root surfaces imitating delayed replanted roots. Background Data: Accidental traumatic injuries with teeth avulsion are managed by replantation. Root resorption, poor conditioning, and non-viable fibroblasts are factors responsible for failure. Methods: Thirty six human healthy single-rooted premolars were collected. Six teeth were used for PDLF, six teeth used for dentin collagen, whereas the remaining 24 teeth (48 root slices) were used for PDLF cell density and morphology. Each root was soaked in 5.25% NaOCl. Three groups (n = 16 slices/each) were planned as follows: I: Control (untreated); II: dentin collagen application; III: Er:YAG laser irradiation (4 mm distance, 40 mJ/pulse, under coolant). Following incubation, cell density and morphology of PDLF were investigated under SEM. Statistical analysis was performed using analysis of variance with Scheffé's test, and p < 0.05 was considered significant. Results: All groups showed increased cultured PDLF following incubation. Regarding cell density, attached PDLFs were significantly lower in untreated controls (36.5 ± 6.36) (p < 0.00001 i.e., <0.05) in negative empty and/or light cellular areas, compared with dentin collagen (65 ± 6) and laser-irradiated (66.75 ± 5.77) groups that did not show significant differences (p = 0.940 i.e., >0.05) and showed intermediate and/or heavy cellular areas. Regarding cell morphology, controls showed round and/or oval appearance with less lamellipodia, whereas dentin collagen and laser groups showed flat morphology with cytoplasmic processes. Conclusions: Both dentin collagen and Er:YAG laser showed comparable effectiveness as biomodification tools with good biocompatibility for human PDLF cell attachment on intact root slices imitating delayed replantation. Dentin collagen as a natural bioactive material is considered an alternative to Er:YAG laser to enhance the regenerative effects.

Postbiotics of the Lactiplantibacillus plantarum EIR/IF-1 Strain Show Antimicrobial Activity against Oral Microorganisms with pH Adaptation Capability

Postbiotics offer better properties than probiotics. This study investigated the antimicrobial activity of Lactiplantibacillus plantarum EIR/IF-1 postbiotics against pH-adaptive bacteria, namely Prevotella denticola, Fusobacterium nucleatum, and Streptococcus sanguinis. Cell-free culture media of L. plantarum EIR/IF-1 were used as postbiotics in either crude (acidic) or neutralized form to also understand non-pH-dependent antimicrobial potential. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and viable cell counts were determined for crude and neutralized postbiotics. Culture media adjusted to different pH values were also compared to adjusted media with postbiotics to understand the strength of organic acids in postbiotics. Antibiofilm activity of postbiotics was determined against polymicrobial biofilm formation. Finally, the toxicity of crude postbiotics was tested on human periodontal ligament fibroblast cells (hPDLFCs). MIC values of crude postbiotics were 12.5 mg/mL for all strains. F. nucleatum and P. denticola strains were sensitive to neutralized postbiotics after 48 h of incubation. Moreover, 12.5 and 25 mg/mL postbiotics inhibited biofilm formation and 2.5 mg/mL and lower concentrations of crude postbiotics showed no cytotoxicity in hPDLFCs. This study showed that postbiotics have antimicrobial activity against pH-adaptive oral bacteria and no cytotoxic effect on hPDLFCs depending on the dose. The non-acidic antimicrobial components of postbiotics could also enable their safe use in the oral cavity.

Synthesis, characterization and anticancer evaluation of novel 4-hydrazinothiazole analogues.

Single-step synthesis of novel 4-hydrazinothiazole derivatives 6a-e was achieved under mild conditions via the sequential four components method using isothiocyanate, aminoguanidine, carbonyl adduct, and α-haloketone derivatives. Deprotection of these hydrazinothiazoles was influenced by acylation, providing a novel group of diacylated molecular structures with a broader scope for the design of thiazolyl-containing drugs 7a and 7b. FTIR, 1 H/13 C NMR, LC-MS spectroscopy, and CHN elemental analyses have been used to study the compounds' chemical structures. Using an MTT assay on Human Periodontal Ligament Fibroblast (HPDLF) cells, the 4-hydrazinothiazole derivatives were screened for cytotoxicity in an in vitro cytotoxicity investigation. The 4-hydrazinothiazole compound 6b bearing an isopropylidene-hydrazino group demonstrated a strongly potent cytotoxicity against CAKI1 (IC50 = 1.65±0.24 μM) and A498 (IC50 of 0.85±0.24 μM). Furthermore, the chloroacetyl-containing thiazole compound 7a has displayed efficient inhibition of growth against the test cell lines CAKI1 and A498 at low micromolar IC50 of 0.78 and 0.74 μM, respectively.

Collagen Scaffolds Laden with Human Periodontal Ligament Fibroblasts Promote Periodontal Regeneration in SD Rat Model.

Periodontitis, a chronic inflammatory disease caused by microbial communities carrying pathogens, leads to the loss of tooth-supporting tissues and is a significant contributor to tooth loss. This study aims to develop a novel injectable cell-laden hydrogel consisted of collagen (COL), riboflavin, and a dental light-emitting diode (LED) photo-cross-linking process for periodontal regeneration. Utilizing α-SMA and ALP immunofluorescence markers, we confirmed the differentiation of human periodontal ligament fibroblasts (HPLFs) into myofibroblasts and preosteoblasts within collagen scaffolds in vitro. Twenty-four rats with three-wall artificial periodontal defects were divided into four groups, Blank, COL_LED, COL_HPLF, and COL_HPLF_LED, and histomorphometrically assessed after 6 weeks. Notably, the COL_HPLF_LED group showed less relative epithelial downgrowth (p < 0.01 for Blank, p < 0.05 for COL_LED and COL_HPLF), and the relative residual bone defect was significantly reduced in the COL_HPLF_LED group compared to the Blank and the COL_LED group (p < 0.05). The results indicated that LED photo-cross-linking collagen scaffolds possess sufficient strength to withstand the forces of surgical process and biting, providing support for HPLF cells embedded within them. The secretion of cells is suggested to promote the repair of adjacent tissues, including well-oriented periodontal ligament and alveolar bone regeneration. The approach developed in this study demonstrates clinical feasibility and holds promise for achieving both functional and structural regeneration of periodontal defects.

Effect of genetic polymorphisms rs2301113 and rs2057482 in the expression of HIF-1α protein in periodontal ligament fibroblasts subjected to compressive force.

Many genes and signaling molecules are involved in orthodontic tooth movement, with mechanically and hypoxically stabilized HIF-1α having been shown to play a decisive role in periodontal ligament signaling during orthodontic tooth movement. Thus, this in vitro study aimed to investigate if genetic polymorphisms in HIF1A (Hypoxia-inducible factor α-subunits) influence the expression pattern of HIF-1α protein during simulated orthodontic compressive pressure. Samples from human periodontal ligament fibroblasts were used and their DNA was genotyped using real time Polymerase chain reaction for the genetic polymorphisms rs2301113 and rs2057482 in HIF1A . For cell culture and protein expression experiments, six human periodontal ligament fibroblast cell lines were selected based on the patients' genotype. To simulate orthodontic compressive pressure in fibroblasts, a 2 g/cm2 force was applied under cell culture conditions for 48 hours. Protein expression was evaluated by Western Blot. Paired t-tests were used to compare HIF-1α expression with and without compressive pressure application and unpaired t-tests were used to compare expression between the genotypes in rs2057482 and rs2301113 (p<0.05). The expression of HIF-1α protein was significantly enhanced by compressive pressure application regardless of the genotype (p<0.0001). The genotypes in the genetic polymorphisms rs2301113 and rs2057482 were not associated with HIF-1α protein expression (p>0.05). Our study confirms that compressive pressure application enhances HIF-1α protein expression. We could not prove that the genetic polymorphisms in HIF1A affect HIF-1α protein expression by periodontal ligament fibroblasts during simulated orthodontic compressive force.

IL-6 Plays a Critical Role in Stromal Fibroblast RANKL Induction and Consequent Osteoclastogenesis in Ameloblastoma Progression

Ameloblastoma (AB) is the most common benign, epithelial odontogenic tumor that occurs in the jawbone. AB is a slow-growing, benign epithelial tumor but shows locally invasive growth, with bone resorption or recurrence if not adequately resected. From these points of view, understanding the mechanism of AB-induced bone resorption is necessary for better clinical therapy and improving patients’ quality of life. In bone resorption, osteoclasts play critical roles, and RANKL is a pivotal regulator of osteoclastogenesis. However, the source of RANKL-expressing cells in the AB tumor microenvironment is controversial, and the mechanism of osteoclastogenesis in AB progression is not fully understood. In this study, we investigated the distribution of the RNA expression of RANKL in AB specimens. We found that PDGFRα- and S100A4-positive stromal fibroblasts expressed RANKL in the AB tumor microenvironment. Moreover, we analyzed the mechanisms of osteoclastogenesis in the AB tumor microenvironment using the human AB cell line AM-1 and a human primary periodontal ligament fibroblast cells. The results of histopathologic and in vitro studies clarified that the interaction between AB cells and stromal fibroblasts upregulated IL-6 expression and that AB cells induced RANKL expression in stromal fibroblasts and consequent osteoclastogenesis in AB progression.

Surface functionalization of gallium nitride for biomedical implant applications

GaN is an aqueous and chemically stable material that has demonstrated biocompatibility with potential in biomedical engineering and offers enormous opportunities to tailor its inherent surface properties and promote efficient biomolecule adhesion with targeted functionalities. In this article, functionalization of GaN surfaces via a three-step process to enhance cell attachment and its effective utilization in in-vivo and in-vitro applications is reported. The morphology and surface chemistry of GaN surfaces were modified using sodium hydroxide and 3-aminopropyltriethoxy silane followed via bioconjugation with type 1-human collagen (T1HC), and the changes in chemical states, morphology, wettability, pH, and aqueous stability were investigated. Further, cell culture studies using human periodontal ligament fibroblast (HPdlF) cells were undertaken. The proliferation and adhesion/survivability studies at different time intervals on functionalized GaN surfaces were performed and analyzed via MTT Assay and SEM evaluation. It has been observed that the hydroxyl species and protonated amines promotes the adhesion of the silane coupling agent and human collagen, which remains stable under standard cell culture media. The chemically functionalized T1HC bio-conjugated GaN surfaces display excellent cell culture properties with complete cell adhesion and viability of ∼ 95 %. The research revealed that chemically functionalized GaN might be useful in various procedures where integrating soft tissues with implant surfaces is crucial.

Effect of curcumin-loaded photoactivatable polymeric nanoparticle on peri-implantitis-related biofilm

Curcumin has been used as a photosensitizer (PS) for antimicrobial photodynamic chemotherapy (PACT). However, its low solubility, instability, and poor bioavailability challenge its in vivo application. This study aimed to synthesize curcumin-loaded polymeric nanoparticles (curcumin-NP) and determine their antimicrobial and cytotoxic effects. Nanoparticles (NP) were synthesized using polycaprolactone (PCL) as a polymer by the nanoprecipitation method. Curcumin-NP was characterized by particle size, polydispersity index and zeta potential, scanning electron microscopy, and curcumin encapsulation efficiency (EE). Curcumin-NP was compared to free curcumin solubilized in 10% DMSO as photosensitizers for PACT in single and multispecies Porphyromonas gingivalis, Fusobacterium nucleatum, and Streptococcus oralis biofilms. Chlorhexidine 0.12% (CHX) and ultrapure water were used as positive and negative controls. The cytotoxic effect of curcumin-NP was evaluated on human periodontal ligament fibroblast cells (HPLF). Data were analyzed by ANOVA (α=0.05). Curcumin-NP exhibited homogeneity and stability in solution, small particle size, and 67.5% EE of curcumin. Curcumin-NP presented reduced antibiofilm activity at 500 µg/ml, although in planktonic cultures it showed inhibitory and bactericidal effect. Curcumin-NP and curcumin with and without photoactivation were not cytotoxic to HPLF cells. Curcumin-NP has antimicrobial and antibiofilm properties, with better effects when associated with blue light, being a promising therapy for preventing and treating peri-implant diseases.

Effects of erythropoietin on osteoblast in the tooth extraction socket in mice periodontitis model.

Periodontitis is an excessive inflammatory event in tooth-supporting tissues and can cause tooth loss. We used erythropoietin (EPO), which has been reported to play an important role in bone healing and modulation of angiogenesis, as a therapeutic agent in vivo and in vitro experimental models to analyze its effect on periodontitis. First, EPO was applied to in vitro MC3T3-E1 cells and human periodontal ligament fibroblast (hPDLF) cells to examine its function in altered cellular events and gene expression patterns. In vitro cultivation of MC3T3-E1 and hPDLF cells with 10 IU/ml EPO at 24 and 48 h showed an obvious increase in cell proliferation. Interestingly, EPO treatment altered the expression of osteogenesis-related molecules, including alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP-2), and osteocalcin (OC) in MC3T3-E1 cells but not in hPDLF cells. In particular, MC3T3-E1 cells showed increased expression of ALP, BMP-2, and OC on day 5, while hPDLF cells showed increased expression of BMP-2, and OC on day 14. Based on the in vitro examination, we evaluated the effect of EPO on bone formation using an experimentally-induced animal periodontitis model. After the induction of periodontitis in the maxillary left second M, 10 IU/ml of EPO was locally applied to the extraction tooth sockets. Histomorphological examination using Masson’s trichrome (MTC) staining showed facilitated bone formation in the EPO-treated groups after 14 days. Similarly, stronger positive reactions against vascular endothelial growth factor (VEGF), cluster of differentiation 31 (CD31), runt-related transcription factor 2 (RUNX2), and osteocalcin (OC) were detected in the EPO-treated group compared to the control. Meanwhile, myeloperoxidase, an inflammatory marker, was decreased in the EPO-treated group on days 1 and 5. Overall, EPO facilitates bone healing and regeneration through altered signaling regulation and modulation of inflammation in the osteoblast cell lineage and to a lesser extent in hPDLF cells.

Molecular mechanism of hyperactive tooth root formation in oculo-facio-cardio-dental syndrome.

Mutations in the B-cell lymphoma 6 (BCL6) interacting corepressor (BCOR) cause oculo-facio-cardio-dental (OFCD) syndrome, a rare X-linked dominant condition that includes dental radiculomegaly among other characteristics. BCOR regulates downstream genes via BCL6 as a transcriptional corepressor. However, the molecular mechanism underlying the occurrence of radiculomegaly is still unknown. Thus, this study was aimed at identifying BCOR-regulated genetic pathways in radiculomegaly. The microarray profile of affected tissues revealed that the gene-specific transcriptional factors group, wherein nucleus factor 1B, distal-less homeobox 5, and zinc finger protein multitype 2 (ZFPM2) were the most upregulated, was significantly expressed in periodontal ligament (PDL) cells of the diseased patient with a frameshift mutation (c.3668delC) in BCOR. Wild-type BCOR overexpression in human periodontal ligament fibroblasts cells significantly hampered cellular proliferation and ZFPM2 mRNA downregulation. Promoter binding assays showed that wild-type BCOR was recruited in the BCL6 binding of the ZFPM2 promoter region after immunoprecipitation, while mutant BCOR, which was the same genotype as of our patient, failed to recruit these promoter regions. Knockdown of ZFPM2 expression in mutant PDL cells significantly reduced cellular proliferation as well as mRNA expression of alkaline phosphatase, an important marker of odontoblasts and cementoblasts. Collectively, our findings suggest that BCOR mutation-induced ZFPM2 regulation via BCL6 possibly contributes to hyperactive root formation in OFCD syndrome. Clinical data from patients with rare genetic diseases may aid in furthering the understanding of the mechanism controlling the final root length.

Drug Release and Cytotoxicity of Hyaluronic Acid and Zinc Oxide Gels, An In-Vitro Study

ABSTRACT Hyaluronic acid (HA) is a naturally occurring biopolymer, with a remarkable wound healing property. Zinc-oxide non-eugenol is a material widely used for periodontal dressing in dentistry. However, it has been reported that zinc oxide non-eugenol is toxic to osteoblasts and fibroblasts. Hence, the present study aimed to evaluate the drug release and cytotoxicity of HA and zinc-oxide gels. Hydrogels of HA and zinc oxide were formulated with carbopol as a carrier. In vitro drug release was performed by UV spectrophotometry, dialysis, and vial bag methods. Cytotoxicity assessment of HA and zinc-oxide gels was performed in human periodontal ligament fibroblasts (HPdLF) and human gingival fibroblasts (hGFs). An inverted phase-contrast microscope was used to assess the morphological changes. At 24 and 48 hr, HPdLF cells showed the highest viability in 0.1% low molecular weight-HA (LMW-HA) with a median value of 131.9, and hGFs showed the highest viability in 5% LMW-HA with a median of 129.56. The highest viability of HPdLF cells was observed in 5% high molecular weight-HA (HMW-HA), with a median value of 127.11. hGFs showed the highest viability in 1% HMW-HA with a median value of 97.99. Within the limitations of the present study, we concluded that LMW-HA is more efficient than HMW-HA. Both HPdLF and hGF cells showed complete cell morbidity with zinc-oxide hydrogels. Therefore, zinc oxide-based gels in concentrations as low as 9% could be toxic intraorally to soft tissues that harbor gingival and periodontal ligament fibroblasts.

The efficacy of salivary Histatin-1 protein in wound closure of nicotine treated human periodontal ligament fibroblast cells – In vitro study

ObjectivesThe aims of this study were to investigate the efficacy of Histatin-1 in wound closure as well as effects on gene expression of nicotine-treated human Periodontal Ligament Fibroblast cells (HPDL) in vitro. DesignHPDL grown in 2.5% culture medium treated with 10 ng/ml Histatin − 1 in the presence/absence of 0.5 µM nicotine were subjected to wound assay and migration was studied at 0 h, 6 h, 12 h and 24 h. Cells grown in 2.5% medium served as control. Cell migration was studied by wound gap and transwell migration assays. The effect of Histatin-1 on expression of matrix metalloproteinase 8 (MMP-8), insulin-like growth factor 1 (IGF-1), transforming growth factor beta (TGF-β), collagen type I (COL1) and plasminogen activator inhibitor 1 (PAI-1) were studied. ResultsHistatin-1 treatment significantly decreased percentage wound gap at 12 h (62.96 ± 3.22 vs 79.23 ± 1.73; p < 0.05) and at 24 h (38.78 ± 7.59 vs 75.21 ± 4.94; p < 0.001) compared with controls. In nicotine+Histatin-1 treated cells, wound gap decreased to 70.2 ± 2.9% (p < 0.01) at 24 h compared to nicotine alone in which 82 ± 1.64% of wound gap was retained. Transwell migration assays showed significant migration of HPDL with Histatin-1 (p < 0.05). Gene expression demonstrated significant upregulation for IGF-1, TGF β, COL1 and PAI-1 with Histatin-1. ConclusionHistatin-1 significantly mitigated the effect of nicotine in wound healing assay involving HPDL fibroblast cells at 24 h. Histatin-1 aided wound closure is attributed to the upregulation of IGF-1, TGF β, COL1, and PAI-1 genes.

Exploring the Therapeutic Potentials of Exopolysaccharides Derived From Lactic Acid Bacteria and Bifidobacteria: Antioxidant, Antitumor, and Periodontal Regeneration.

The metabolites of lactic acid bacteria (LAB) and bifidobacteria (Bb) have recently received a lot of attention due to their ability to protect interactions in blood and tissues, as well as their biodegradability and biocompatibility in human tissue. Exopolysaccharides (EPS) derived from bacteria have a long history of use in therapeutic and other industrial applications with no adverse effects. In this regard, EPSs were isolated and characterized from LAB and Bb culture supernatants to determine their antioxidant, antitumor, and periodontal regeneration properties. The antioxidant capacity of the EPSs varied with concentration (0.625–20 mg/ml). The highest antioxidant activity was found in LAB: Streptococcus thermophiles DSM 24731-EPS1, Lactobacillus delbrueckii ssp. bulgaricus DSM 20081T-EPS5, Limosilactobacillus fermentum DSM 20049-EPS6, and Bb; Bifidobacterium longum ssp. longum DSM 200707-EPS10. Human breast cancer cells (MCF7), human colon cancer cells (CaCo2), human liver cancer cells (HepG2), and human embryonic kidney 293 (HEK 293) cells were used as controls to assess the antitumor properties of the selected EPSs. According to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) assay, EPS5 had the highest cytotoxicity against MCF7, CaCo2, and HepG2, with IC50 values of 7.91, 10.69, and 9.12 mg/ml, respectively. Lactate dehydrogenase (LDH) activity was significantly higher in cell lines treated with EPS5-IC50 values compared to other EPSs-IC50 values (p < 0.05). Real time (RT)-PCR results showed that EPS5 treatment increased Bax, Caspase 8, Caspase 3, and p53 gene expression. The expression of the BCL2, MCL1, and Vimentin genes, on the other hand, was reduced. The MTT test was used to examine the effect of EPS5 on the viability of human periodontal ligament fibroblast cells (hPDLFCs), and it was discovered that EPS5 increased hPDLFC viability. According to high-performance liquid chromatography (HPLC) analysis, galactose made up 12.5% of EPS5. The findings of this study pave the way for the use of EPS, which hold great promise for a variety of therapeutic purposes such as antioxidant, antitumor, and periodontal regeneration.

Uji Sitotoksisitas Hidroksiapatit Cangkang Telur Ayam Ras (Gallus gallus) terhadap Sel Fibroblas Ligamen Periodontal Manusia

Periodontitis is an inflammation disease of the periodontal tissues surrounding the teeth, include periodontal ligament fibroblast cell. The recently researches developed tissue engineering concept to accelerate regeneration and proliferation periodontal ligament cells. One of the major components of tissue engineering is scaffold. The hydroxyapatite is biomaterial that can be used as scaffold. The egg shell has the potential biowaste to be a source of hydroxyapatite biomaterial. Before being applied as a scaffold in the medical field, cytotoxicity testing needs to be done. This study aimed to examine toxicity effect of egg shell hydroxyapatite in various concentration toward human periodontal ligament fibroblast cells. This study used human periodontal ligament fibroblast primary cells (5x103) in microplate 96 wells which divided into 7 groups, control cells, control media without cells and the treatment groups with concentration egg shell hydroxyapatite100 μg/ml, 75 μg/ml, 50 μg/ml, 25 μg/ml dan 25 μg/ml and incubated for 24 hours. Cytotoxicity test was conducted using MTT assay. Optical density values describe the viability of living cells and the readings were done using ELISA readers, subsequently the data were analyzed using One way ANOVA. The number of human periodontal ligament cells at all concentration showed that the percentage of cell life is more than 90%. Egg shell hydroxyapatite at all concentrations tested is not toxic toward human periodontal ligament fibroblas cells.

Cytotoxic Tumour-Selective 1,5-Diaryl-3-Oxo-1,4-Pentadienes Mounted on a Piperidine Ring.

A series of 3,5-bis(benzylidene)-4-piperidones 2a–u were prepared as candidate cytotoxic agents. In general, the compounds are highly toxic to human gingival carcinoma (Ca9-22), human squamous carcinoma-2 (HSC-2) and human squamous carcinoma-4 (HSC-4) neoplasms, but less so towards non-malignant human gingival fibroblast (HGF), human periodontal ligament fibroblast (HPLF) and human pulp cells (HPC), thereby demonstrating tumour-selective toxicity. A further study revealed that most of the compounds in series 2 were more toxic to the human Colo-205 adenocarcinoma cell line (Colo-205), human HT29 colorectal adenocarcinoma cells (HT-29) and human CEM lymphoid cells (CEM) neoplasms than towards non-malignant human foreskin Hs27 fibroblast line (Hs27) cells. The potency of the cytotoxins towards the six malignant cell lines increased as the sigma and sigma star values of the aryl substituents rose. Attempts to condense various aryl aldehydes with 2,2,6,6-tetramethyl-4-piperidone led to the isolation of some 1,5-diaryl-1,4-pentadien-3-ones. The highest specificity for oral cancer cells was displayed by 2e and 2r. In the case of 2r, its selective toxicity exceeded that of doxorubicin and melphalan. The enones 2k, m, o have the highest SI values towards colon cancer and leukemic cells. Both 2e,r inhibited mitosis and increased the subG1 population (with a transient increase in G2/M phase cells). Slight activation of caspase-3, based on the cleavage of poly(ADP-ribose)polymerase (PARP) and procaspase 3, was detected.

Impact of FGF1 on human periodontal ligament fibroblast growth, osteogenic differentiation and inflammatory reaction in vitro.

To investigate in vitro the impact of fibroblast growth factor1 (FGF1) in comparison to ascorbic acid (AscA) on human periodontal ligament fibroblast (HPdLF) growth, their osteogenic differentiation, and modulation of their inflammatory reaction to mechanical stress. The influence of different concentrations of FGF1 (12.5-200 ng/mL) on growth and proliferation of HPdLF cells was analyzed over 20days by counting cell numbers and the percentage of Ki67-positive cells. Quantitative expression analysis of genes encoding the osteogenic markers alkaline phosphatase (ALPL), Runt-related transcription factor2 (RUNX2), osteocalcin (OCN), and osteopontin (OSP), as well as the fibroblast markers vimentin (VIM) and fibroblast-specific protein1 (FSP1), was performed after 2and 20days of cultivation. Metabolic activity was determined by MTT assay. For comparison with AscA, 50 ng/mL FGF1 was used for stimulation for 2and 20days. Cell number, percentage of Ki67-positive cells, and expression of osteoblast- and fibroblast-specific genes were examined. Alkaline phosphatase activity was visualized by NBT/BCIP and calcium deposits were stained with alizarin red. Cytokine (IL‑6, IL‑8, COX2/PGE2) expression and secretion were analyzed by qPCR and ELISA in 6 h mechanically compressed HPdLF cultured for 2days with FGF1 or ascorbic acid. Higher concentrations of FGF1 promoted cell proliferation upon short-term stimulation, whereas prolonged treatment induced the expression of osteogenic markers even with low concentrations. AscA promotes cell growth more markedly than FGF1 in short-term cultures, whereas FGF1 induced osteogenic cell fate more strongly in long-term culture. Both factors induced an increased inflammatory response of HPdLF to mechanical compression. Our data suggest that FGF1 promotes an osteogenic phenotype of HPdLF and limits inflammatory response to mechanical forces compared to AscA.

Bio-Morphological Reaction of Human Periodontal Ligament Fibroblasts to Different Types of Dentinal Derivates: In Vitro Study.

Understanding the biological and morphological reactions of human cells towards different dentinal derivate grafting materials is fundamental for choosing the type of dentin for specific clinical situations. This study aimed to evaluate human periodontal ligament fibroblasts (hPLF) cells exposed to different dentinal derivates particles. The study design included the in vitro evaluation of mineralized dentine (SG), deproteinized and demineralized dentine (DDP), and demineralized dentine (TT) as test materials and of deproteinized bovine bone (BIOS) as the positive control material. The materials were kept with the hPLF cell line, and the evaluations were made after 24 h, 72 h, and 7 days of in vitro culture. The evaluated outcomes were proliferation by using XTT assays, the morphological characteristics by light microscopy (LM) and by the use of scanning electron microscopy (SEM), and adhesion by using confocal microscopy (CLSM). Overall, the experimental materials induced a positive response of the hPLFs in terms of proliferation and adhesion. The XTT assay showed the TT, and the SG induced significant growth compared to the negative control at 7 days follow-up. The morphological data supported the XTT assay: the LM observations showed the presence of densely packed cells with a modified shape; the SEM observations allowed the assessment of how fibroblasts exposed to DDP and TT presented cytoplasmatic extensions; and SG and BIOS also presented the thickening of the cellular membrane. The CLMS observations showed the expression of the proliferative marker, as well as and the expression of cytoskeletal elements involved in the adhesion process. In particular, the vinculin and integrin signals were stronger at 72 h, while the actin signal remained constantly expressed in all the follow-up of the sample exposed to SG material. The integrin signal was stronger at 72 h, and the vinculin and actin signals were stronger at 7 days follow-up in the sample exposed to DDP material. The vinculin and integrin signals were stronger at 72 h follow-up in the sample exposed to TT material; vinculin and integrin signals appear stronger at 24 h follow-up in the sample exposed to BIOS material. These data confirmed how dentinal derivates present satisfying biocompatibility and high conductivity and inductivity properties fundamental in the regenerative processes. Furthermore, the knowledge of the effects of the dentin’s degree of mineralization on cellular behavior will help clinicians choose the type of dentine derivates material according to the required clinical situation.

In Vitro Compression Model for Orthodontic Tooth Movement Modulates Human Periodontal Ligament Fibroblast Proliferation, Apoptosis and Cell Cycle.

Human Periodontal Ligament Fibroblasts (hPDLF), as part of the periodontal apparatus, modulate inflammation, regeneration and bone remodeling. Interferences are clinically manifested as attachment loss, tooth loosening and root resorption. During orthodontic tooth movement (OTM), remodeling and adaptation of the periodontium is required in order to enable tooth movement. hPDLF involvement in the early phase-OTM compression side was investigated for a 72-h period through a well-studied in vitro model. Changes in the morphology, cell proliferation and cell death were analyzed. Specific markers of the cell cycle were investigated by RT-qPCR and Western blot. The study showed that the morphology of hPDLF changes towards more unstructured, unsorted filaments under mechanical compression. The total cell numbers were significantly reduced with a higher cell death rate over the whole observation period. hPDLF started to recover to pretreatment conditions after 48 h. Furthermore, key molecules involved in the cell cycle were significantly reduced under compressive force at the gene expression and protein levels. These findings revealed important information for a better understanding of the preservation and remodeling processes within the periodontium through Periodontal Ligament Fibroblasts during orthodontic tooth movement. OTM initially decelerates the hPDLF cell cycle and proliferation. After adapting to environmental changes, human Periodontal Ligament Fibroblasts can regain homeostasis of the periodontium, affecting its reorganization.

Gene Expression Profile in Immortalized Human Periodontal Ligament Fibroblasts Through hTERT Ectopic Expression: Transcriptome and Bioinformatic Analysis.

Human periodontal ligament fibroblast (hPLF) cells play an important role in maintaining oral cavity homeostasis with special function in tissue regeneration and maintenance of dental alveoli. Although their primary cell cultures are considered a good experimental model with no genetic changes, the finite life span may limit some experimental designs. The immortalization process increases cell life span but may cause genetic changes and chromosomal instability, resulting in direct effects on physiological cell responses. In this way, we aimed to investigate the global gene expression of hPLFs after the immortalization process by the ectopic expression of the catalytic subunit of the enzyme telomerase reverse transcriptase (hTERT) through transcriptome analysis. The embryonic origin of the primary culture of hPLF cells and immortalized hPLF-hTERT was also tested by vimentin staining, hTERT synthesis evaluated by indirect immunocytochemistry, analysis of cell proliferation, and morphology. The results indicated that hPLFs and hPLF-hTERT were positive for vimentin. On the 20th cell passage, hPLFs were in senescence, while hPLF-hTERT maintained their proliferation and morphology characteristics. At the same passage, hPLF-hTERT presented a significant increase in hTERT synthesis, but transcriptome did not reveal overexpression of the hTERT gene. Fifty-eight genes had their expression altered (11 upregulated and 47 downregulated) with the absence of changes in the key genes related to these cell types and in the main cancer-associated genes. In addition, the increase in hTERT protein expression without the overexpression of its gene indicates posttranscriptional level regulation. Successful immortalization of hPLFs through the ectopic expression of hTERT encourages further studies to design experimental protocols to investigate clinical questions from a translational perspective.

Physicochemical properties and cytocompatibility of newly developed calcium silicate-based sealers.

The purpose of this study was to compare the physical properties and cytocompatibility of contemporary calcium silicate-based sealers. Four calcium silicate-based sealers (BrightEndo MTA sealer, CeraSeal, EndoSeal TCS and One-Fil) were compared to an epoxy resin-based sealer (AH Plus). Flow, setting time, radiopacity and dimensional change were evaluated according to ISO 6876 standards. Cytotoxicity on human periodontal ligament fibroblast (hPDLF) cells was compared for biological properties using MTT assay. The surface of the sealer was analysed using scanning electron microscopy to evaluate cell attachment. Flow and radiopacity of all sealers met ISO standards, while setting time and dimensional stability did not meet the ISO standards. Calcium silicate-based sealers showed favourable cytocompatibility, and hPDLF cells were well attached to the calcium silicate-based sealers. Calcium silicate-based sealers have clinically acceptable flow and radiopacity, and cytocompatibility. However, these sealers had longer setting time and higher dimensional change than those required by ISO 6876.