Background Accelerated proliferation of human periodontal ligament stem cells (PDLSCs) is present in periodontitis. It is known that fibroblast growth factor 2 (FGF2) regulates the proliferation of PDLSCs, while the function of FGF2 in myogenic cell differentiation is mediated by Linc-RNA Activator of Myogenesis (Linc-RAM) lncRNA. Therefore, Linc-RAM lncRNA may also participate in periodontitis. Methods This study included 28 patients with periodontitis (patient group) and 22 patients without periodontitis but received orthodontic treatment (control group) in the stomatological hospital of Sun Yat-Sen university. Gingival biopsies were obtained from participants. RT-qPCR, cell transfection, cell proliferation assay and western blot were carrying out to analyze the samples. Results We found that FGF2 mRNA was upregulated, while Linc-RAM was downregulated in PDLSCs derived from periodontitis-affected teeth than in healthy teeth. FGF2 mRNA and Linc-RAM were inversely correlated in both types of PDLSCs. FGF2 overexpression led to inhibited Linc-RAM expression in PDLSCs derived from periodontitis-affected teeth and promoted the proliferation of PDLSCs. Linc-RAM overexpression failed to significantly affect FGF2 expression but attenuated the enhancing effects of FGF2 overexpression on the proliferation of PDLSCs. Conclusions Therefore, downregulation of Linc-RAM lncRNA may participate in FGF-2 mediated- proliferation of human PDLSCs.
Background Hyperglycemic micro-environment induced by diabetes could regulate the response of periodontal tissues to pathogenic microorganisms in which disruption of autophagy lysosomal pathway (ALP) may participate. This study aimed to explore the mechanisms underlying how high glucose (HG) regulates ALP in gingival epithelial cells (GECs). Methods Human gingival tissues in healthy group (C), periodontitis group (P), diabetes group (DM), and diabetes + periodontitis group (DP) were collected and were applied to observe the fusion of autophagy and lysosome. Diabetic mouse model with periodontitis was established and RNA-seq was applied to investigate the expression of ALP-associated genes in gingival epithelium. To explore the key role of ATPase transmembrane v0 domain, subunit c (ATP6V0C) in the disruption of ALP by HG, human gingival epithelial cells (HGECs) were cultured in 5.5 mM/25 mM glucose medium for 48 hours and followed by Porphyromonas gingivalis stimulation for 0, 6, and 12 hours. HBLV-h-ATP6V0C was transfected in HGECs that were stimulated by 25 mM HG condition. Results Immunofluorescence double staining exhibited the disruption of ALP in human gingival epithelium in diabetes groups and HGECs under 25 mM glucose condition, accompanied with significantly downregulated lysosomal acidity. RNA-seq of mouse gingival epithelium screened out Atp6v0c. Compared with HGECs in normal culture medium, ATP6V0C expression and LC3-II/I expression ratio were significantly downregulated, with an upregulated expression of P62, IL-1 beta in HGECs under HG condition. Over-expression of ATP6V0C rescued HG-induced disruption of ALP in HBLV-h-ATP6V0C transfected HGECs, with significantly upregulation of LC3-II/I and downregulation of P62, IL-1 beta. Conclusion ATP6V0C mediates HG-induced ALP disruption in HGECs, eventually exacerbates periodontal inflammation.
Background The present study aimed to explore the effects of the active form of vitamin D (calcitriol, 1 alpha,25-dihydroxyvitamin D-3, 1 alpha,25 (OH)(2)D-3, 1,25D) on live Porphyromonas gingivalis internalized into KB cells and U937 cells. Methods Quantitative real-time polymerase chain reaction method was used to evaluate the number of surviving P. gingivalis internalized into KB cells and U937 cells. Transmission electron microscopy was used to detect P. gingivalis in cells. A western blot analysis was performed to observe LC3 expressions. Results 1) Treatment with 1,25D decreased the number of live P. gingivalis in KB cells and U937 cells in a dose-dependent manner. 2) Dividing P. gingivalis were found only in KB cells but not in U937 cells. The cell walls of most P. gingivalis in KB cells were intact, while those in U937 cells were disrupted. Treatment with 1,25D promoted the encapsulation of P. gingivalis in autophagosomes in both KB and U937 cells. 3) Both 1,25D treatment and P. gingivalis infection increased the LC3 II/I ratio. Furthermore, 1,25D treatment increased the P. gingivalis-upregulated LC3 II/I ratio. 4) Treatment with 3-methyladenine (3-MA) decreased the number of P. gingivalis by 11.41% in KB cells, while increased that by 121.51% in U937 cells. Under 1,25D treatment conditions, 3-MA treatment increased the number of P. gingivalis by 88.71% in KB cells and by 284.70% in U937 cells. Conclusions Autophagy may facilitate P. gingivalis survival in KB cells and eliminate P. gingivalis in U937 cells. Treatment with 1,25D may help decrease the number of live P. gingivalis in KB cells and U937 cells by promoting functional autophagy.
Background The present study was to determine the role of Toll-like receptor 4 (TLR4) signaling in inflammation and alveolar bone resorption using a murine model of Porphyromonas gingivalis-associated ligature-induced peri-implantitis. Methods Smooth surface titanium implants were placed in the left maxilla alveolar bone 6 weeks after extraction of first and second molars in Wild-type (WT) and TLR4(-/-) (TLR4 KO) mice. Silk ligatures immersed with P. gingivalis were tied around the implants 4 weeks after the implant placement and confirmation of osteointegration. Two weeks after the ligation, bone resorption, osteoclastogenesis, cellular inflammatory responses, and gingival mRNA expression levels of cytokines were assessed by micro-computed tomography, tartrate-resistant acid phosphatase (TRAP) staining, immunobiological examination and Real-time quantitative polymerase chain reaction, respectively. Results In both WT and TLR4 KO mice, the bone resorption around implants was significantly increased in the P. gingivalis/ligation group compared with control group. In P. gingivalis/ligation group, the levels of bone resorption, TRAP+ cell formation, and gingival CD3+ and CD45+ cell infiltration were significantly decreased in TLR4 KO mice compared with that in WT mice. Receptor activator of nuclear factor-kappa B ligand /osteoprotegerin (RANKL/OPG) ratio was significantly increased after P. gingivalis/ligation treatment in WT mice not in TLR4 KO mice. When comparing the P. gingivalis/ligation group with the respective control group, gingival mRNA expressions of IL-1 beta, IFN-gamma, and 1L-17 were significantly increased in TLR4 KO mice. Conclusion This study suggests that TLR4 mediates alveolar bone resorption in P. gingivalis associated ligature-induced peri-implantitis through regulation of immune B cell infiltration, RANKL/OPG expression ratio, and differential inflammatory cytokine production.
Background The development of platelet concentrated biomaterials has gained increasing awareness for regenerative medicine. With different protocol, derivatives such as advanced platelet-rich fibrin (A-PRF), injected platelet-rich fibrin, and concentrated growth factor (CGF) have been demonstrated effectively in preclinical and clinical studies. The aim of this study was to compare the level of growth factors releasing from A-PRF and CGF, and their clinical efficacy in the regenerative management of intrabony defects (IBDs). Methods Thirty-two blood samples were collected from eight healthy donors and assessed for platelet-derived growth factor-alpha beta, vascular endothelial growth factor, bone morphogenetic protein-2, and transforming growth factor-beta 1 release at indicated times. In addition, the clinical records of 45 patients (15 per group) who had undergone guided tissue regeneration (GTR) with or without A-PRF/CGF were retrieved. The probing depth (PD) and clinical attachment level (CAL) were recorded preoperatively and 6 months postoperatively. Intrabony component (IC) depth, radiographic bone level (RBL), and bone defect filling were assessed radiographically. Results A-PRF had a looser fibrin network than the CGF but presented larger amounts of growth factors with a more sustained release period. Although there was no difference in PD reduction, CAL gain, RBL height change and defect filling (%) between A-PRF and CGF group, both achieved a more favorable clinical result in IC height reduction and defect filling (%) than the control. Conclusions A-PRF and CGF have the ability to stimulate a continual and steady release of total growth factors over a 14-day period. A-PRF and CGF show a similar effectiveness in periodontal bone regeneration with a potential benefit of improving GTR outcomes in IBD treatment.
Background To report 4-year natural periodontal progression of mandibular first molars based on radiographic records in 15 to 44-year-old Chinese villagers. Methods In 1992 (N = 486) and 1996 (N = 413), panoramic radiographs were recorded. Tooth loss of mandibular first molars was calculated. Relative bone height (RBH), intrabony defect (IBD) depth, and furcation involvement (FI) were measured on 918 and 755 mandibular first molars in 1992 and 1996, respectively. The progression of the three parameters and their relationship with widened periodontal ligament space (WPDL) were analyzed. Results In 1992, of 31 lost mandibular first molars, 29 belonged to the 35- to 44-year age group. At 4-year follow-up, five of eight lost teeth belonged to the 35- to 44-year age group. RBH decreased from 83% in 1992 to 77% in 1996. RBH progression was significantly faster in the 25- to 34- and 35- to 44-year age groups than in the 15- to 24-year age group. The mean IBD depth was 2.81 +/- 0.55 mm (n = 32) in 1992 and 3.70 +/- 0.73 mm (n = 33) in 1996. Prevalence of FI increased from 20.8% to 27.4%. Teeth with WPDL showed greater RBH and IBD progression than those without WPDL (RBH: 12% +/- 1% versus 6% +/- 0.01%, P < 0.001; IBD depth: 0.31 +/- 0.08 versus 0.01 +/- 0.00 mm, P <0.001). FI-area progression in teeth with WPDL showed a trend of greater expansion than in those without WPDL (0.92 +/- 0.18 versus 0.56 +/- 0.11 mm(2), P = 0.051). Conclusions Tooth loss mainly occurred in the 35- to 44-year age group. RBH progression was faster in the 25- to 44-year age group. WPDL was associated with progression of RBH, IBDs, and FI.
Background Serum amyloid A (SAA) has been identified to trigger inflammation response, and play a crucial role in chronic inflammatory diseases. However, the regulatory mechanism of SAA still remains unclear during the development of periodontitis Methods SAA mRNA and protein expression were detected in healthy and inflammatory gingival tissues using real-time polymerase chain reaction (PCR) and immunohistochemistry. Human recombinant SAA (Apo-SAA), Pam3CSK4 (a Toll-like receptor (TLR) 2 ligand), siRNA-SAA, or TLR2 neutralizing antibody was applied to treat human gingival fibroblasts, respectively, or combined. SAA, TLRs, and inflammatory cytokines interleukin (IL)-6 and IL-8 were analyzed by real-time PCR, western blotting, or enzyme-linked immunosorbent assay. Results SAA expression increased in human inflammatory gingival tissues from patients with periodontitis (P <0.05). Apo-SAA could increase not only the mRNA expression of TLR2 (P <0.05), but also IL-6 and IL-8 mRNA and protein levels (P <0.05) which was suppressed by TLR2 antibody in human gingival fibroblasts. Pam3CSK4 increased SAA, IL-6, and IL-8 levels (P <0.05). However, the expression of SAA, IL-6, and IL-8 decreased after transfection of siRNA-SAA (P <0.05). Conclusion SAA not only increases in inflammatory gingiva, but also triggers inflammatory cytokine secretion via interacting with TLR2 pathway in human gingival fibroblasts, which indicates that SAA is involved in periodontal inflammation.
Background Furcation involvement and attachment loss are major predictors of tooth loss. The aim of this study was to describe specific designs for papilla preservation flaps (PPFs) and minimally invasive surgery to be used in compromised molars and report proof-of-principle data with 3 to 16-year follow-up in severely compromised molars due to the presence of combined furcation and intrabony defects. Methods Forty-nine subjects with furcated molars and deep intrabony defects were treated with PPFs, application of periodontal regenerative devices. Improvement as a consequence of therapy was defined as tooth retention, reduction in horizontal and vertical furcation involvement, decrease in probing depths, and increases in clinical attachment level. Subjects were maintained with regular supportive periodontal care. Results At 1 year, 100% of maxillary and 92% of mandibular molars showed improvements. Improvements were not observed in molars with baseline hypermobility: two mandibular molars with hypermobility were extracted at the 1-year follow-up. Improvement in vertical sub-classification was observed in 87.5% of maxillary and in 84.6% of mandibular molars. One-year improvements could be maintained over the 3 to 16-year follow-up. Conclusions PPFs and periodontal regeneration can be applied and provide clinical benefits to severely compromised molars due to the combined presence of furcation involvement and deep intrabony defects. These results were obtained in cases with an interdental peak of bone and gingival margin coronal to the furcation entrance in well-maintained and compliant subjects. Randomized controlled clinical trials with medium- to long-term follow-up are needed to confirm these findings.
Background The aim of this study was to simultaneously and quantitatively assess the expression levels of 20 periodontal disease-related proteins in gingival crevicular fluid (GCF) from normal controls (NOR) and severe periodontitis (SP) patients with an antibody array. Methods Antibodies against 20 periodontal disease-related proteins were spotted onto a glass slide to create a periodontal disease antibody array (PDD). The array was then incubated with GCF samples collected from 25 NOR and 25 SP patients. Differentially expressed proteins between NOR and SP patients were then used to build receiver operator characteristic (ROC) curves and compare five classification models, including support vector machine, random forest, k nearest neighbor, linear discriminant analysis, and Classification and Regression Trees. Results Seven proteins (C-reactive protein, interleukin [IL]-1 alpha, interleukin-1 beta, interleukin-8, matrix metalloproteinase-13, osteoprotegerin, and osteoactivin) were significantly upregulated in SP patients compared with NOR, while receptor activator of nuclear factor-kappa was significantly downregulated. The highest diagnostic accuracy using a ROC curve was observed for IL-1 beta with an area under the curve of 0.984. Five of the proteins (IL-1 beta, IL-8, MMP-13, osteoprotegerin, and osteoactivin) were identified as important features for classification. Linear discriminant analysis had the highest classification accuracy across the five classification models that were tested. Conclusion This study highlights the potential of antibody arrays to diagnose periodontal disease.
Background Dental implant failures still occur because of peri-implant diseases such as peri-implantitis. The objective of this study was to treat peri-implantitis in beagle dogs by fabricating minocycline hydrochloride (MH)-loaded graphene oxide (GO) films on implant abutment surfaces. Methods Beagle dogs with silk ligature were used to establish the peri-implantitis model. Modified sulcus bleeding index, peri-implant probing pocket depth, radiographic evaluation, micro-CT tomography analysis, and histological evaluation were determined to evaluate the therapeutic effect of MH-loaded GO films on abutment surfaces for peri-implantitis. Results Radiographic and micro-CT analysis showed that lots of marginal bone loss could be found on Ti and MH/Ti groups, especially for Ti group. Little bone less could be seen on GO/Ti group while bone less on MH/GO/Ti group was negligible. Results of the histological analysis presented that lots of neutrophils could be found on Ti and MH/Ti groups. However, almost none of the neutrophils could be observed on GO/Ti and MH/GO/Ti while lots of osteocytes could be found. Conclusion MH-loaded GO films on implant abutment surfaces could prevent the further development of peri-implantitis and exhibit good therapeutic effect for peri-implantitis in beagle dogs.
Background Autophagy has recently emerged as a protective mechanism in response to compressive force and an important process in maintenance of bone homeostasis. It appears to be involved in the degradation of osteoclasts, osteoblasts, and osteocytes. The aim of this study was to investigate the role of compressive force-induced autophagy in periodontal ligament (PDL) cells in regulating osteoclastogenesis of orthodontic tooth movement (OTM). Methods An OTM model and compressive force on PDL cells were employed to investigate the expression of autophagy markers in vivo and in vitro, respectively. Autophagosomes and autolysosomes were observed in PDL cells by transmission electron microscope (TEM) and autophagy LC3 double labelling. 3-Methyladenine (3-MA) and rapamycin were respectively used to inhibit and promote autophagy, and the effect of autophagy on osteoclastogenesis was explored via microcomputed tomography, hematoxylin and eosin (H&E) staining, histochemistry of titrate-resistant acid phosphatase, and real-time polymerase chain reaction (RT-PCR) in vivo. Receptor activator of nuclear factor-kappa B ligand/osteoprotegerin (RANKL/OPG) was investigated by RT-PCR and ELISA in vitro. Results Orthodontic force-induced autophagy was prominent on the pressured side of PDL tissues. Administration of 3-MA downregulated bone density and upregulated osteoclasts, while rapamycin had reverse results in OTM. The autophagy activity increased initially then decreased in PDL cells during compressive force application and responded to light force. In PDL cells, administration of 3-MA upregulated while rapamycin downregulated the RANKL/OPG ratio. Conclusion Autophagy is activated by compressive force in PDL cells. Besides, it could modulate OTM by negatively regulating osteoclastogenesis and keep bone homeostasis via RANKL/OPG signaling.
Background The relationship between chronic periodontitis and pulpal/cemental changes is seldom reported. This study aimed to report on the microstructural changes of cementum and histopathological features of the dental pulp in teeth with severe chronic periodontitis. Methods Eighty molar teeth with severe chronic periodontitis and 50 extracted third molars (as normal controls) were collected. The microstructure of cementum was evaluated by scanning electron microscopy, and the pulp was stained with hematoxylin and eosin. Interleukin (IL)-17 and IL-1 beta levels were examined by immunohistochemistry/western blotting. Reactive oxygen species (ROS) and superoxide dismutase 1 (SOD 1) levels were also checked. Caspase 3 expression and terminal-deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining were used as apoptotic indices, and LC3B and P62 were detected to demonstrate the level of autophagy. Results The surface of cementum showed irregularities; the dental pulp was inflamed and under oxidative stress. IL-17 and IL-1 beta levels were increased in the pulp of teeth with periodontitis. ROS and apoptosis levels were higher than in normal dental pulp, while SOD 1 level was reduced. Intriguingly, autophagy markers LC3B and P62 were upregulated in the pulp obtained from teeth with periodontitis. Conclusions Severe chronic periodontitis influenced the microstructure of both cementum and dental pulp. The dental pulp collected from teeth with periodontitis was inflamed, under oxidative stress, and presented increased levels of apoptosis and autophagy relative to normal dental pulp.
Background Association between BMI and periodontitis were controversial. A study indicated that not only overweight or obesity but also underweight was correlated with generalized aggressive periodontitis (GAgP). However, the exact relationship between BMI and GAgP and the optimal BMI value for the lowest risk of GAgP remain unknown. Objective To explore the exact relationship between BMI and GAgP risk, periodontal status and WBC (white blood cell) count and find the optimal BMI value associated with the lowest risk, periodontal status and lowest WBC of GAgP in Chinese. Methods 300 GAgP patients and 133 healthy controls were recruited. Height and weight of participants were accurately measured to calculate BMI value. Clinical periodontal parameters, including probing depth (PD), attachment loss (AL), and bleeding index (BI) were recorded. WBC was obtained from routine blood examination. Smooth curve fitting and segmented regression model were used to analyze the threshold effect between BMI and variables. The shape of the curve was used to describe the relationships between BMI and GAgP. Results U-shaped relationships between BMI and risk of GAgP, AL, and WBC count in GAgP patients were observed. The optimal value of BMI for the lowest risk of GAgP and lowest WBC count was 22 kg/m(2). The risk of GAgP increased by 39% in patients per unit increase of BMI when BMI ranged from 22 to 28 kg/m(2) (adjusted OR = 1.39, 95% CI: 1.17, 1.67) and increased by 18% per unit decrease of BMI when BMI ranged from 22 to 18 kg/m(2) (adjusted OR = 0.82, 95% CI: 0.69, 0.97). The count of WBC increased by 1.12 x 10(9)/L in patients per unit increase of BMI when BMI ranged from 22 to 28 kg/m(2) (adjusted beta = 0.12, 95% CI: 0.01, 0.23) and increased by 0.2 x 10(9)/L per unit decrease of BMI when BMI ranged from 22 to18 kg/m(2) (adjusted beta = -0.2, 95% CI: -0.35, -0.04). Conclusion U-shaped relationships exist between BMI and risk of GAgP, AL, and WBC count in patients with GAgP among Chinese aged below 36 years old with their BMI range from 18 to 28 kg/m(2); the optimal BMI value for lowest odds ratio and lowest WBC count of GAgP was 22 kg/m(2).
Background Periodontal disease is characterized by alveolar bone destruction and degenerative lesions of the periodontal ligament (PDL); it is initiated by bacterial infection of the oral cavity, but the clinical effects are secondary to an aberrant host immune response. Primary hypertension (PH), which causes significant morbidity and mortality worldwide, has also been shown to be an inflammatory disease characterized by aberrant immune cell infiltration and activation. Clinical retrospective studies have shown a link between PH and periodontitis with PH exacerbating periodontitis and vice versa, but the pathophysiologic mechanisms responsible for this remain unknown. Methods In this study, we investigate the underlying mechanisms behind PH exacerbation of periodontitis by using a bacteria-induced periodontitis model in normotensive and hypertensive (Nos3(-/-)) mice treated with or without an Angiotensin II (Ang II) specific receptor 1 (AT1) antagonist, losartan. The histologic analyses including immunohistochemistry, immunofluorescence were carried out. The qRT-PCR and ELISAs were applied for the target gene and protein detection. Results We find that PH worsens bone resorption and PDL destruction in periodontitis and that treatment with losartan, rescues this. We also show that PH increases dendritic cell (DC) and osteoclast (OC) infiltration in periodontitis, which is also dependent on Ang II. Finally, we show that PH augments the pro-inflammatory state in periodontitis infiltrating DCs in an Ang II-dependent manner and use in vitro studies to show that Ang II directly augments DC Toll-like receptor 4 (TLR4) signaling. Conclusion Our studies show a central role for Ang II as a pro-inflammatory Toll-like receptor mediator in the pathogenesis of PH-exacerbated periodontitis, indicating that Ang II may be a reasonable target in patients with PH and periodontitis comorbidity.
Background The aim of this study was to evaluate the relationship among clinical periodontal, microbiologic parameters and lung function in participants with chronic obstructive pulmonary disease (COPD). Methods A total of 160 participants were recruited, including 80 participants with COPD (COPD group) and 80 participants without COPD (control group). All participants completed questionnaires and underwent clinical periodontal and lung function examinations. Subgingival plaques were obtained to determine the prevalence of selected oral and respiratory bacterial species. Results 1) Significant relationships were noted in the participants among oral hygiene index-simplified (OHI-S), clinical attachment level (CAL) and forced expiratory volume in one second (FEV1%). 2) Porphyromonas gingivalis (Pg), Klebsiella pneumonia (Kp), Pseudomonas aeruginosa (Pa) and Streptococcus pneumonia (Sp) prevalence was increased in participants with COPD compared with control participants. 3) A significant negative association was noted between the relative content of Pg and FEV1% in participants with COPD. Conclusion The results of this study confirm that periodontal destruction and oral pathogens are associated with lung function.