Periodontal Fibroblasts Research Articles

Engineered Immunomodulatory Nanoparticles Inhibit Root Resorption and Ankylosis

IntroductionExternal root resorption following avulsion injury is a complex process wherein differentiation of macrophages (Mϕ) to multinucleated osteoclasts is temporally regulated by resident periodontal fibroblasts (PDLF). The current study aims to assess the effect of engineered bioactive chitosan nanoparticles (CSNP), sustained released dexamethasone conjugated CSNP (CS-DEX) and CSNP functionalized with photosensitizer Rose Bengal (CSRB) for application in root resorption using an in-vitro PDLF-Mϕ direct coculture model and in-vivo delayed reimplantation model. MethodsPDLF-Mϕ direct coculture system was exposed to lipopolysaccharide (LPS), macrophage colony stimulating factor, receptor activator of nuclear factor kappa β ligand with or without CSNP/CS-DEX for 7 days. Clastic differentiation was assessed by tartrate resistant acid phosphatase (TRAP) staining on day 7. On day 2 and 7, immunofluorescence analysis was conducted to assess the expression of Mϕ polarization markers (CD80, CD206), multinucleation markers (NFATc1, STAT6) in Mϕ and matricellular protein periostin in PDLF and cytokine profiling in cell culture supernatants. Delayed replantation model with extraoral air dry/LPS exposure for 1h followed by root surface treatment with CS-DEX/CSRB was used in Wistar rats. After 21 days, rats were euthanized for histologic and immunofluorescence analysis. Statistical analysis one-way ANOVA with Tukey's multiple comparisons was used to analyze the data (P < .05). ResultsCS-DEX significantly reduced TRAP+ multinucleated cells and CSNP treatment showed no TRAP+ cells. Immunofluorescence analysis showed that CSNP/CS-DEX reduced CD80, NFATc1 and STAT6 expression and increased periostin as expressed by fluorescence intensity. CSNP/CS-DEX significantly reduced TNFα, MMP9 and increased IL10, TGFβ1. Osteoprotegerin was upregulated only by CSNP. Root surface treatment in delayed replantation model showed that CS-DEX and CSRB substantially reduced the degree of resorption and ankylosis. Further, CD80, CD206, and MMP2 expression in groups with root surface treatment with CS-DEX and CSRB was lower than airdry/LPS group and similar to healthy control and NFATc1, STAT6, and MMP9 expressions were lower than healthy control. ConclusionThe engineered nanosized immunomodulatory bioactive materials chitosan nanoparticles functionalized with photosensitizer and dexamethasone effectively reduced the clastic differentiation of Mϕ in in-vitro coculture and minimized the resorption and ankylosis in a delayed reimplantation model. These biomaterials have the potential to serve as root modification agents, promoting favorable healing outcomes in cases of dental avulsion.

Deciphering 3D periodontal fibroblast-macrophage crosstalk in bioactive nanoparticle-guided immunomodulation for treating traumatic dental avulsion

Prolonged extra-oral period in dental avulsion is often associated with loss of viability of Periodontal fibroblasts (PDLF) and increased risk of ankylosis. Root surface treatment with bioactive agents to reduce the risk of ankylosis can be a potential strategy. The objective of the study was to investigate the impact of an engineered chitosan nanoparticles (CSNP), photosensitizer Rose Bengal (RB) functionalized CSNP (CSRB) and sustained dexamethasone (CSDEX) releasing CSNP for application in management of delayed replantation of avulsed teeth. The 3D PDLF-macrophage (Mϕ) collagen model was developed and exposed to LPS, MCSF, RANKL with and without CSDEX/CSNP. Immunofluorescence and cytokine analysis was done at 2 and 7 days to assess cellular interactions. Maxillary right incisors in male Wistar rats were extracted, exposed to extraoral dry or LPS for 1 h and treated with or without CSDEX/CSRB for 1 min before replantation. Rats were euthanized after 21 days for micro-CT, TRAP, and immunofluorescence analysis. CSDEX/CSNP treatment in 3D model significantly reduced CD80, NFATc1, STAT6 and increased CD206 and periostin expression (p < 0.05). TNFα, MMP9 was downregulated and IL10, TGFβ1, MMP2 upregulated with CSDEX/CSNP (p < 0.05). CSDEX/CSRB in animal study significantly reduced resorption, ankylosis, TRAP activity and osteocalcin expression and increased periostin (p<0.05). CSDEX demonstrated higher anti-inflammatory activity by downregulating TNFα, while CSNP upregulated TGFβ1, periostin, and downregulated MMP9. The combination of matrix stabilization with CSRB with periostin upregulation and sustained releasing CSDEX showed potential for hampering root resorption and ankylosis in dental avulsion.

An in vitro assessment of cytotoxicity and genotoxicity of root repair materials.

The success of the root-end procedure depends on the regeneration of the functional periodontal attachment system, including the cementum on the resected root-end surface, periodontal ligament (PDL), and alveolar bone. As root end filling materials remain in close contact with live periapical tissues, they may influence the endodontic treatment outcome. To assess and compare the cytotoxicity and genotoxicity of three root repair materials, mineral trioxide aggregate (MTA), endosequence, and geristore in human-cultured periodontal ligament fibroblasts. Cultured human periodontal ligament fibroblasts of the third passage were used in the study. They were placed in contact with the root repair materials. The cytotoxic effect on PDL fibroblasts was determined by (3-(4,5-dimethylthiazol-2-yl)-2,5-tetrazolium bromide) assay after 24 hours and 48 hours intervals. Cell viability was determined using an inverted phase contrast microscope. The genotoxic effect on the periodontal fibroblast cells was determined by comet assay using imaging software. Data were analyzed using Tukey's multiple comparison test and Dunnett's multiple test. All the test materials showed higher cytotoxicity and genotoxicity at the 48th hour interval with a statistically significant difference from the control group (P < 0.05). MTA was shown to be least cytotoxic and genotoxic to PDL fibroblasts, followed by endosequence root repair material and geristore at 24 hour and 48 hour intervals. The cytotoxicity and genotoxicity of MTA were the least compared to endosequence and geristore on human-cultured PDL fibroblasts.

Association between androgenetic alopecia and periodontitis.

Androgenic alopecia (AGA) is men's most common form of hair loss. It is affected by changes in the expression and activity of 5αR and the metabolism of testosterone and DHT. There is an association between AGA and systemic inflammatory diseases. We hypothesized that there is an association between AGA and periodontal disease, as inflamed gingiva and periodontal fibroblasts have been shown to express more 5αR. Thus, this study aimed to evaluate the relationship between periodontal disease and AGA and the potential effect of aging on this association. Out of a cohort of 1088 individuals, 385 white males aged 25-65 with similar socioeconomic levels and without systemic disease were included. Periodontitis was defined using NHANES data. AGA was evaluated using the Norwood-Hamilton scale. The relationship between AGA, periodontal disease severity, and age was assessed. There was a correlation between age and baldness (r = .421, p < .001). There was a significant correlation between AGA and periodontal disease in younger patients aged 25-34 and 35-44. (p < .042 and p < .036, respectively). There was no significant correlation between AGA and periodontal disease in the 45-54 and 55-65 age groups (p > .05). There may be a relationship between periodontal disease and AGA in the 25-44 age range, suggesting that this association starts at an early age in adulthood.

Periodontal Fibroblasts—Macrophage Crosstalk in External Inflammatory Root Resorption

IntroductionThis study aimed to understand the influence of periodontal fibroblasts (PDLFs) on clastic differentiation of macrophages (Mφ) in different resorptive environments. MethodsPDLF-Mφ direct coculture (juxtacrine) was seeded on dentin, cementum, and polystyrene with/without lipopolysaccharide, macrophage colony-stimulating factor, and receptor activator of nuclear factor kappa beta ligand for 7 and 14 days and stained for tartrate-resistant acid phosphatase (TRAP) activity. PDLF-Mφ cocultured on polystyrene were immunostained for CD80, CD206, NFATc1, STAT6, and periostin, and cell culture supernatants were assessed for cytokines on days 2 and 7. Mφ grown in conditioned media of PDLFs (paracrine) and Mφ monoculture were used as controls. Data was analyzed using Student t test and one-way analysis of variance with the Tukey multiple comparisons test (P < .05). ResultsPDLF-Mφ coculture showed a higher number of TRAP-positive multinucleated cells than Mφ monoculture on dentin and polystyrene. No TRAP-positive multinucleated cells were observed in paracrine and cementum. The expression of CD80 and CD206 in PDLF-Mφ was similar at day 2, whereas CD206 was greater than CD80 at day 7. The expression of STAT6 was greater than NFATc1 at both days 2 and 7 (P < .05). Periostin expression in the presence of the lipopolysaccharide, macrophage colony-stimulating factor, and receptor activator of nuclear factor kappa beta ligand combination was down-regulated in PDLF monoculture, whereas it was up-regulated in PDLF-Mφ coculture. The cytokine profile of PDLF-Mφ on day 2 was predominated by interleukin (IL)-1β, tumor necrosis factor alpha, and MMP9 and MMP2 on day 7. IL-6 and IL-8 showed steady expression at both days 2 and 7. ConclusionsThe study highlights the juxtacrine effect of PDLFs on the clastic differentiation of Mφ with a difference in clastic activity between dentin and cementum. The study also emphasizes the temporal effect of tumor necrosis factor alpha, MMP2, MMP9, and IL-1β on intercellular crosstalk in resorptive environments.

SARS-CoV-2 infection causes periodontal fibrotic pathogenesis through deregulating mitochondrial beta-oxidation

The global high prevalence of COVID-19 is a major challenge for health professionals and patients. SARS-CoV-2 virus has four structural protein components: the spike protein, envelope protein, membrane protein, and nucleocapsid protein. The SARS-CoV-2 virus mutates predominantly in the spike proteins, whilst the other key viral components usually remain stable. Essentially the pathological functions of the SARS-CoV-2 virus on different cell types are still largely unknown. Previous studies have shown that the human oral cavity can potentially act as reservoir of the SARS-CoV-2 virus. However, the consequence of SARS-CoV-2 viral infection on human oral health has not been systematically examined. COVID-19 can cause severe oral mucosa lesions and is likely to be connected with poor periodontal conditions. Fibroblasts are the major cell type inside periodontal ligament (PDL) and express the SARS-CoV-2 receptor: Angiotensin-converting enzyme 2 (ACE2), whose expression level can increase upon bacterial infection hence potentially provide a direct route of SARS-CoV-2 infection to PDL fibroblasts. In this research, we aimed to study the pathogenicity of SARS-CoV-2 viral components on human fibroblasts. We found that by exposing to SARS-CoV-2, especially to the viral envelope and membrane proteins, the human periodontal fibroblasts could develop fibrotic pathogenic phenotypes, including hyperproliferation that was simultaneously induced with increased apoptosis and senescence. The fibrotic degeneration was mediated by a down-regulation of mitochondrial β-oxidation in the fibroblasts. Fatty acid β-oxidation inhibitor, etomoxir treatment could mirror the same pathological consequence on the cells, similar to SARS-CoV-2 infection. Our results therefore provide novel mechanistic insights into how SARS-CoV-2 infection can affect human periodontal health at the cell and molecular level with potential new therapeutic targets for COVID-19 induced fibrosis.

Regulation of TGF-β1 on human periodontal fibroblasts in inflammatory state

Regulation of TGF-β1 on human periodontal fibroblasts in inflammatory state

Isoimperatorin alleviates lipopolysaccharide-induced periodontitis by downregulating ERK1/2 and NF-κB pathways.

Chronic periodontitis is an inflammatory disease characterized by inflammation of the soft tissues of the gums. To combat this disease, more effective drugs are still needed to identify and develop. Isoimperatorin is a kind of a natural compound, which has anti-inflammatory, analgesic, antitumor, antivirus, and other pharmacological effects. However, its possible effects on the progression of chronic periodontitis are still unclear. In this study, we used human periodontal membrane fibroblasts (hPDLCs), human bone marrow-derived macrophages, and found that isoimperatorin reduced hPDLCs viability. In addition, isoimperatorin alleviated the oxidative stress of periodontal membrane cells. Isoimperatorin reduced proinflammatory factor secretion and receptor activator for nuclear factor-κB ligand-induced osteoclast differentiation in periodontal membrane cells. Further, isoimperatorin inhibited the activation of ERK1/2 and nuclear factor-κB pathways. We, therefore, thought isoimperatorin could serve as a promising drug for the treatment of this disease.

Effect of LASER photobiomodulation on the cell viabilities of periodontal ligament fibroblasts of older and younger individuals - An in vitro study.

Fibroblasts form the major cell type of the periodontal ligament and most often studied for periodontal regeneration. The aim of the present study was to investigate the effects of photobiomodulation (PBM) on aged periodontal fibroblasts and compare the viability of periodontal fibroblasts of older and younger individuals. A total of 32 patients were divided into four groups: A, B, C, and D. Groups A and C and B and D comprised of extracted teeth of older (>60 year) and younger individuals (<25 year), respectively. Extracted teeth from Groups A and B received PBM, (diode laser, 660 nm, 100 mW at 4J) whereas Groups C and D did not. Tissue from extracted teeth of all groups was processed and cultured and subjected to 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay to assess their viability. The difference between the groups in terms of viability was significant (P < 0.0001). The mean viability of A and B (PBM) was 1.04 and 1.19, respectively. The mean viability for C and D (non-PBM) was 0.95 and 0.85, respectively. Older fibroblasts have lower viability than younger fibroblasts. PBM improves viability in both older and younger fibroblasts and more so in younger fibroblasts. However, more than one PBM would be required to sustain the effect longer.

"Efficacy of cytotoxic effect of green tea catechins on the human periodontal fibroblasts and human dental pulp fibroblasts -An in vitro study".

Inflammation of tooth-supporting tissue and the pulp tissue is followed by wound healing and regeneration process that involves the specific type of connective tissue cells, the fibroblasts. During periodontitis and pulpitis, the inflammation of the tissue causes damage to the fibroblasts. These fibroblasts secrete collagen proteins and maintain the structural framework; along with this the inflammatory process moves toward healing where in the specific cells such as the fibroblast cells play important roles. Green tea catechins epigallocatechin-3-gallate (EGCG) being one of the major catechins is known to have multiple beneficial effects on human fibroblasts. To assess the in vitro cytotoxicity of green tea catechins on the human periodontal ligament (PDL) fibroblasts and human dental pulp fibroblasts. Human PDL fibroblasts (hPDLFs) and human dental pulp fibroblasts were isolated from the two extracted premolar teeth that were indicated for orthodontic treatment. The fibroblasts were then seeded in 96 well tissue culture plate for cell viability study. EGCG was used at different concentration to treat the cells. After 48 h; (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (MTT) assay was performed to determine the cell viability. The vitality of hPDLFs and human dental pulp fibroblasts was found to be inversely proportional to EGCG concentrations. hPDLFs have shown 37% proliferation at lowest concentration of EGCG used and human dental pulp fibroblasts have shown 99% viability at lowest concentration of EGCG used.

Plap-1 lineage tracing and single-cell transcriptomics reveal cellular dynamics in the periodontal ligament.

Periodontal tissue supports teeth in the alveolar bone socket via fibrous attachment of the periodontal ligament (PDL). The PDL contains periodontal fibroblasts and stem/progenitor cells, collectively known as PDL cells (PDLCs), on top of osteoblasts and cementoblasts on the surface of alveolar bone and cementum, respectively. However, the characteristics and lineage hierarchy of each cell type remain poorly defined. This study identified periodontal ligament associated protein-1 (Plap-1) as a PDL-specific extracellular matrix protein. We generated knock-in mice expressing CreERT2 and GFP specifically in Plap-1-positive PDLCs. Genetic lineage tracing confirmed the long-standing hypothesis that PDLCs differentiate into osteoblasts and cementoblasts. A PDL single-cell atlas defined cementoblasts and osteoblasts as Plap-1-Ibsp+Sparcl1+ and Plap-1-Ibsp+Col11a2+, respectively. Other populations, such as Nes+ mural cells, S100B+ Schwann cells, and other non-stromal cells, were also identified. RNA velocity analysis suggested that a Plap-1highLy6a+ cell population was the source of PDLCs. Lineage tracing of Plap-1+ PDLCs during periodontal injury showed periodontal tissue regeneration by PDLCs. Our study defines diverse cell populations in PDL and clarifies the role of PDLCs in periodontal tissue homeostasis and repair.

Comparison of a new thermosensitive rhAm carrier versus traditional PGA carrier for in vitro antibacterial activity and biocompatibility

To compare a new thermosensitive recombinant human amelogenin (rhAm) carrier and traditional propylene glycol alginate (PGA) carrier for their characteristics, antibacterial activity, and biocompatibility with human periodontal membrane fibroblasts. PGA-rhAm was prepared by mixing 3.3% PGA and rhAm, and CS-βGP-rhAm was prepared by mixing 2% chitosan (CS) with rhAm and then with 60% β-sodium glycerophosphate solution (βGP) as the crosslinking agent. The biophysical properties of the prepared carriers were characterized, and their antibacterial activity was assessed by observing Staphylococcus aureus growth. The biocompatibility of the carriers was evaluated in human periodontal membrane fibroblasts (hPDLFs) using CCK8 assay and scratch test, and mRNA and protein expressions of osteogenic genes of the cells incubated with the carriers were detected using RT-qPCR and Western blotting; osteogenic differentiation of the cells was detected using alkaline phosphatase staining. PGA-rhAm had a viscosity value of 3.262±0.055 Pa.s. CS-βGP-rhAm had a solidification capacity of 6 min at 37 ℃ with a pH value close to that of the oral cavity and a swelling rate of about 90%. CS-β GP-rhAm maintained sustained release of rhAm for over 2 weeks with a self-degradation time over 3 weeks. CS-βGPrhAm more effectively inhibited the growth of S. aureus than rhAm-loaded PGA. While PGA did not obviously affect the proliferation of hPDLFs, both CS-βGP and CS-βGP-rhAm significantly promoted the cell proliferation(P < 0.001). Scratch test showed that after rhAm loading, both CS-βGP and PGA promoted cell migration (P < 0.01). CS-βGP-rhAm significantly enhanced the mRNA expressions of RUNX2 and OCN mRNA level and the protein expressions of Ki67, RUNX2, collagen I, and β-catenin (P < 0.05); PGA-rhAm only enhanced RUNX2 (P < 0.05) and OCN (P < 0.01) mRNA expressions without significant effects on the protein expressions. Alkaline phosphatase staining results showed that CS-βGP, but not PGA, promoted osteogenic differentiation of hPDLFs. CS-βGP carrier is capable of sustained release of rhAm, inhibiting the growth of S. aureus, and improving the biological activity of hPDLFs without affecting the bioactivity of rhAm after drug loading.

Morphological and Biological Evaluations of Human Periodontal Ligament Fibroblasts in Contact with Different Bovine Bone Grafts Treated with Low-Temperature Deproteinisation Protocol.

Several types of deproteinised bovine bone mineral (DBBM) are available on the market, and each one is obtained with a thermic and chemical process that can differ, achieving different results. Currently, several protocols using low temperature are suggested to reduce the possible particle crystallisation during the production process. This study aimed to evaluate the biomorphological reaction of periodontal fibroblast cultures in contact with different DBBM particles treated with a low-temperature protocol (Thermagen®) and without exposure to sodium hydroxide (NaOH). Morphological evaluation was performed using light, confocal laser, and scanning electron microscopy, and the biological reaction in terms of proliferation was performed using an XTT proliferation assay at 24 h (T1), 72 h (T2), and 7 days (T3). The morphological analysis highlighted how the presence of the materials stimulated a change in the morphology of the cells into a polygonal shape, surface reactions with the thickening of the membrane, and expression of actin. In particular, the morphological changes were appreciable from T1, with a progressive increase in the considered morphological characteristics at T2 and T3 follow-ups. The proliferation assay showed a statistical significance between the different experimental materials and the negative control in T2 and T3 follow-ups. The post hoc analysis did not reveal any differences between the materials. In conclusion, the grafts obtained with the low-temperature extractions protocol and not exposed to NaOH solution showed positive morphological reactions with no differences in the sizes of particles.

Influence of Titanium Surface Treatments on Viability of Periodontal Fibroblasts Grown in an Osteogenic Culture Medium.

Background:The integrity of the protective seal provided by the gingiva in direct contact with the implant surface is one of the main factors involved in the prevention of peri-implantitis.Aim:The aim of this study was to assess the viability of periodontal fibroblasts grown in an osteogenic culture medium in contact with titanium surfaces treated either with acid etching alone or with acid etching + anodizing.Materials and Methods:Periodontal fibroblasts grown in an osteogenic culture medium were distributed in a control group, with cells grown in culture bottles, and two experimental groups, with cells grown in contact with titanium disks measuring 6 mm in diameter. The surface of the disks was subjected to acid etching alone (AEG, n = 25) or to acid etching + anodizing (ANG, n = 25), and then evaluated using scanning electron microscopy (SEM). Cell viability was assessed by the [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium] bromide test on days 1, 2, 3, 7, and 14 of the cell culture. The Mann–Whitney test was used for the statistical analysis (P < 0.05).Results:The SEM assessment revealed that the surface of AEG specimens had micrometric characteristics, whereas the surface of ANG specimens had nanometric characteristics. No significant difference was observed among the groups regarding cell viability at any of the evaluation time points.Conclusion:The titanium surface treatments tested did not affect the viability of periodontal fibroblasts in an osteogenic culture medium.

Platelet-derived growth factor-BB regenerates functional periodontal ligament in the tooth replantation

Tooth ankylosis is a pathological condition of periodontal ligament (PDL) restoration after tooth replantation. Platelet-derived growth factor-BB (PDGF-BB) has been proposed as a promising factor for preventing tooth ankylosis. Using rat tooth replantation model, we investigated whether PDGF-BB accelerates the repair of PDL after tooth replantation without ankylosis, and its molecular mechanisms. In PDGF-BB pretreated replanted teeth (PDGF-BB group), ankylosis was markedly reduced and functionally organized PDL collagen fibers were restored; the mechanical strength of the healing PDL was restored to an average of 76% of that in non-replanted normal teeth at 21 days. The numbers of PDGF-Rβ- and BrdU-positive cells in the periodontal tissues of the PDGF-BB group were greater than those of atelocollagen pretreated replanted teeth (AC group). Moreover, in the PDGF-BB group, the periodontal tissues had fewer osteocalcin-positive cells and decreased number of nuclear β-catenin-positive cells compared to those in the AC group. In vitro analyses showed that PDGF-BB increased the proliferation and migration of human periodontal fibroblasts. PDGF-BB downregulated mRNA expressions of RUNX2 and ALP, and inhibited upregulatory effects of Wnt3a on β-catenin, AXIN2, RUNX2, COL1A1, and ALP mRNA expressions. These findings indicate that in tooth replantation, topical PDGF-BB treatment enhances cell proliferation and migration, and inhibits canonical Wnt signaling activation in bone-tooth ankylosis, leading to occlusal loading of the PDL tissues and subsequent functional restoration of the healing PDL. This suggests a possible clinical application of PDGF-BB to reduce ankylosis after tooth replantation and promote proper regeneration of PDL.

CTHRC1 expressed in periodontitis and human periodontal fibroblasts exposed to inflammatory stimuli.

Collagen triple helix repeat containing-1 (CTHRC1) is a glycoprotein that can be secreted extracellularly and is involved in the regulation of collagen matrix in a variety of diseases. The expression level of CTHRC1 in periodontitis was detected in this study. The gingival tissues from clinically healthy subjects (15 cases) and those with periodontitis (30 cases) were taken for immunohistochemical staining. Lipopolysaccharide of the Porphyromonas gingivalis was added in the periodontal ligament fibroblast culture in vitro. Cells were collected, and the mRNA levels of the intracellular CTHRC1 and protein expression of the extracellular CTHRC1 were detected. The protein expression of CTHRC1 in the periodontitis group was higher than that of the clinically healthy group. The in vitro cell experiments showed that 10μg/ml of P.g LPS could induce a significant increase in protein secretion of CTHRC1, and 5μg/ml P.g LPS had a significant effect on promoting the mRNA expression of CTHRC1. Collagen triple helix repeat containing-1might be involved in the development of periodontitis, and the expression level might be significantly correlated with the stimulation of P.g LPS on fibroblasts. Different stimulation intensities of P.g LPS might result in different expression patterns of CTHRC1.

Role of cytochrome P450 2C8 genetic polymorphism and epoxygenase uncoupling in periodontal remodelling affecting orthodontic treatment.

In genome-wide association studies, the CYP2C8 gene locus has been reported to be associated with bisphosphonate-related osteonecrosis of the jaw, a severe devastating side effect of antiresorptive bone treatment. The aim of this study was to elucidate the putative pathomechanism explaining the association between the genetic polymorphism with the alleles CYP2C8*2 and *3 causing low CYP2C8 activity, and disturbed periodontal remodelling in periodontal fibroblasts cultured from patients undergoing orthodontic treatment. CYP2C8 activity, enzyme expression and substrate metabolism were detected in human periodontal fibroblast cultures. Zoledronic acid caused enhanced reactive oxygen species (ROS) production in periodontal fibroblasts, which was enhanced by arachidonic acid as inflammatory signal. Enhanced bisphosphonate-induced uncoupling of the CYP2C8 enzyme was detected in the variant allele (CYP2C8*3) with the result of increased H2 O2 production and lowered substrate oxidation. Conversely, substrate (amodiaquine) addition led to decreased H2 O2 production in isolated CYP2C8 enzymes, but in CYP2C8*3 enzyme, increased H2 O2 was still detected, especially in presence of arachidonic acid. CYP2C8 variants leading to decreased enzyme activity in substrate oxidation may enhance ROS production by reaction uncoupling, and thus, contribute to difficulties in orthodontic treatment and the risk of side effects of antiresorptive drugs.

Comparative Evaluation of Cytotoxic and Genotoxic Effects of Three Resin-Based Sealers by 3,(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide Assay and Comet Assay - An In Vitro Study.

Background and Aim:The complete sealing of the pulpal space contributes to the better healing potential after an root canal treatment, and root canal sealers are an integral part of this treatment which comes in contact with the periodontal tissue. Biocompatibility of an endodontic sealer plays an important role in the root canal treatment procedure. Hence, the aim of the study was to compare and evaluate the cytotoxicity and genotoxicity effect of three resin-based sealers on human periodontal fibroblast cells.Settings and Design:This in vitro study was conducted in Dr. Prabhakar Kore's Basic Science and Research Center and K. L. E V. K. Institute of Dental Sciences, Belagavi.Materials and Methods:Human periodontal fibroblasts were incubated with 15 specimens of Group 1 – AH Plus Group 2 – MTA Fillapex, and Group 3 - GuttaFlow 2, respectively. Cytotoxicity was assessed by 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and genotoxicity was assessed by Comet assay at time intervals at 24 h and 48 h.Statistical Analysis:Data were analyzed by the Kruskal–Wallis and Mann–Whitney U-test.Results:AH Plus and GuttaFlow 2 did not show any cytotoxicity or genotoxicity. MTA Fillapex was slightly cytotoxic throughout the time interval, and it also showed low genotoxicity throughout the time periods for which it was tested.Conclusion:AH Plus and GuttaFlow 2 did not show any cytotoxic and genotoxic effect; however, MTA Fillapex showed cytotoxic and genotoxic effect throughout the time interval.

Gene Expression of the RANK/RANKL/OPG System on a Three-Dimensional Culture of Human Periodontal Fibroblasts Under Continuous Compression

Gene Expression of the RANK/RANKL/OPG System on a Three-Dimensional Culture of Human Periodontal Fibroblasts Under Continuous Compression

A Potential Role of Semaphorin 3A during Orthodontic Tooth Movement.

Background: Induced tooth movement during orthodontic therapy requires mechano-induced bone remodeling. Besides various cytokines and growth-factors, neuronal guidance molecules gained attention for their roles in bone homeostasis and thus, potential roles during tooth movement. Several neuronal guidance molecules have been implicated in the regulation of bone remodeling. Amongst them, Semaphorin 3A is particular interesting as it concurrently induces osteoblast differentiation and disturbs osteoclast differentiation. Methods: Mechano-regulation of Sema3A and its receptors PlexinA1 and Neuropilin (RT-qPCR, WB) was evaluated by applying compressive and tension forces to primary human periodontal fibroblasts (hPDLF) and alveolar bone osteoblasts (hOB). The association of the transcription factor Osterix (SP7) and SEMA3A was studied by RT-qPCR. Mechanisms involved in SEMA3A-mediated osteoblast differentiation were assessed by Rac1GTPase pull-downs, β-catenin expression analyses (RT-qPCR) and nuclear translocation assays (IF). Osteogenic markers were analyzed by RT-qPCR. Results: SEMA3A, PLXNA1 and NRP1 were differentially regulated by tension or compressive forces in hPDLF. Osterix (SP7) displayed the same pattern of regulation. Recombinant Sema3A induced the activation of Rac1GTPase, the nuclear translocation of β-catenin and the expression of osteogenic marker genes. Conclusion: Sema3A, its receptors and Osterix are regulated by mechanical forces in hPDLF. SEMA3A upregulation was associated with Osterix (SP7) modulation. Sema3A-enhanced osteogenic marker gene expression in hOB might be dependent on a pathway involving Rac1GTPase and β-catenin. Thus, Semaphorin 3A might contribute to bone remodeling during induced tooth movement.