Human Periodontal Ligament Research Articles

Comparative biological properties of resin-free and resin-based calcium silicate-based endodontic repair materials on human periodontal ligament stem cells

ObjectivesTo investigate the effect of three different calcium silicate-based materials (CSBM) on the biological behavior of human periodontal ligament stem cells (hPDLSCs).MethodsEluates of Biodentine, NeoPutty and TheraCal PT prepared at 1:1, 1:2, and 1:4 ratios were extracted under sterile conditions. The cytotoxicity of the extracts to the hPDLSCs was assessed using the MTT assay. Scratch wound healing assay was utilized for assessing cell migration. Scanning electron microscopy was used to detect cell attachment and morphology. Calcium ion release was measured using inductively coupled plasma-optical emission spectrometry; the pH-value was evaluated with a pH-meter. ANOVA with post hoc Tukey test was used for statistical analysis.ResultsCell viability was significantly higher for Biodentine and NeoPutty at day 1 with all dilutions (p < 0.05), while at day 3 and day 7 with dilutions 1:2 and 1:4; all materials showed similar behavior (p > 0.05). Biodentine had the highest percentage of cell migration into the scratched area at day 1 for all dilutions (p < 0.05). Stem cells were attached favorably on Biodentine and NeoPutty with evident spreading, and intercellular communications; however, this was not shown for TheraCal PT. Biodentine showed the highest pH values and calcium ion release (p < 0.05).ConclusionsThe resin-free CSBM showed better performance and favorable biological effects on hPDLSCs and were therefore considered promising for usage as endodontic repair materials.Clinical significance: Proper selection of materials with favorable impact on the host stem cells is crucial to ensure outcome in different clinical scenarios.

Validation of a physiological type 2 diabetes model in human periodontal ligament stem cells.

Type 2 diabetes (T2DM), a recognized risk factor for periodontitis, is characterized by insulin resistance. However, the molecular mechanisms concerning the role of insulin resistance in linking T2DM and periodontitis remain poorly elucidated due to the absence of an appropriate T2DM cell model. We aimed to explore an appropriate model of T2DM in human periodontal ligament stem cells (hPDLSCs) and uncover the involved mechanisms. hPDLSCs were incubated with common reagents for recapitulating insulin resistance state including high glucose (HG) (15, 25, 35, 45 mM), glucosamine (0.8, 8, 18, 28, 38 mM), or palmitic acid (PA; 100, 200, 400, 800 μM), combined with LPS for 48 h. The insulin signaling pathway, inflammation, and pyroptosis were detected by western blots and quantitative real-time polymerase chain reaction (RT-qPCR). The effects on osteogenesis were evaluated by alkaline phosphatase staining, alizarin red S staining, RT-qPCR, and western blots. HG failed to recapitulate insulin resistance. Glucosamine was sufficient to induce insulin resistance but failed to trigger inflammation. In total, 100 and 200 μM PA exhibited the most proinflammatory, insulin resistance, and pyroptosis induced role, and inhibited the osteogenic differentiation of hPDLSCs. Palmitic acid is a promising candidate for developing T2DM model in hPDLSCs.

Effect of icariin surface treatment on the resorption of denuded roots after replantation in rat.

Limiting the incidence of resorption associated with delayed replantation of avulsed teeth is critical for long-term tooth survival. In this study, we assessed whether icariin, a natural product with anti-osteoclastic properties, could reduce root resorption in a rat model of tooth replantation. Cytocompatibility of icariin (10, 20, 40 and 80 μM) was evaluated by CCK-8 proliferation assay in vitro, and an osteoclastogenesis assay was performed to evaluate the effect of icariin on the differentiation of rat bone marrow macrophages and human peripheral blood monocytes into tartrate-resistant acid phosphatase-stained (TRAP+ ) multinucleated giant cells (MNGCs). Differentiation of human periodontal ligament stem cells (hPDLSCs) treated with icariin (10 μM) was also evaluated at 5, 10 and 21 days of osteogenic induction. The first maxillary molars of five-week-old male Sprague-Dawley rats were extracted, denuded of PDL, then treated either with neutralized collagen solution (Carrier control) or icariin in collagen (3 μg/μL) before replantation into their sockets. The animals were euthanized 2 weeks post-surgery for micro-computed tomography (micro-CT) imaging and histological analyses. Icariin was cytocompatible and significantly reduced the differentiation of TRAP+ MNGCs in a dose-dependent manner compared to the control. Moreover, icariin enhanced alkaline phosphatase activity, expression of osteogenic marker genes and proteins, and calcium deposition in hPDLSCs. Micro-CT imaging of the replanted samples demonstrated a significantly higher volume of remaining roots in the icariin-treated group than in the control group. Histological analysis revealed a marked number of resorptive lacunae with TRAP activity in the control group, whereas icariin-treated samples showed signs of functional healing and reduced osteoclastic activity. Icariin was biocompatible and demonstrated potent anti-osteoclastic and pro-osteogenic properties that reduced resorption and promoted functional healing of denuded roots in a rat maxillary first molar model of replantation. These findings indicate that root surface treatment with icariin may be a clinically relevant and practical method for improving the retention and survival of teeth with compromised PDL after delayed replantation following traumatic 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.

Injectable periodontal ligament stem cell-metformin-calcium phosphate scaffold for bone regeneration and vascularization in rats.

Injectable and self-setting calcium phosphate cement scaffold (CPC) capable of encapsulating and delivering stem cells and bioactive agents would be highly beneficial for dental and craniofacial repairs. The objectives of this study were to: (1) develop a novel injectable CPC scaffold encapsulating human periodontal ligament stem cells (hPDLSCs) and metformin (Met) for bone engineering; (2) test bone regeneration efficacy in vitro and in vivo. hPDLSCs were encapsulated in degradable alginate fibers, which were then mixed into CPC paste. Five groups were tested: (1) CPC control; (2) CPC +hPDLSC-fibers +0% Met (CPC + hPDLSCs + 0%Met); (3) CPC +hPDLSC-fibers +0.1% Met (CPC + hPDLSCs + 0.1%Met); (4) CPC +hPDLSC-fibers +0.2% Met (CPC + hPDLSCs + 0.2%Met); (5) CPC +hPDLSC-fibers +0.4% Met (CPC + hPDLSCs + 0.4%Met). The injectability, mechanical properties, metformin release, and hPDLSC osteogenic differentiation and bone mineral were determined in vitro. A rat cranial defect model was used to evaluate new bone formation. The novel construct had good injectability and physical properties. Alginate fibers degraded in 7 days and released hPDLSCs, with 5-fold increase of proliferation (p＜0.05). The ALP activity and mineral synthesis of hPDLSCs were increased by Met delivery (p＜0.05). Among all groups, CPC+hPDLSCs+0.1%Met showed the greatest cell mineralization and osteogenesis, which were 1.5-10folds those without Met (p＜0.05). Compared to CPC control, CPC+hPDLSCs+0.1%Met enhanced bone regeneration in rats by 9 folds, and increased vascularization by 3 folds (p＜0.05). The novel injectable construct with hPDLSC and Met encapsulation demonstrated excellent efficacy for bone regeneration and vascularization in vivo in an animal model. CPC+hPDLSCs+0.1%Met is highly promising for dental and craniofacial applications.

Isolation and characterization of human periodontal ligament stem cells under the terms of use in clinical application: A pilot study

Isolation and characterization of human periodontal ligament stem cells under the terms of use in clinical application: A pilot study

Effects of low-level laser on the expression of interleukin-6, tumor necrosis factor‑α, osteoprotegerin, and receptor activator of nuclear factor-κB ligand in human periodontal ligament cells.

This study aims to determine the effects of low-level laser (LLL) on the expression of interleukin-6 (IL-6), tumor necrosis factor (TNF)-α, osteoprotegerin (OPG), and receptor activator of nuclear factor-κB ligand (RANKL) in human periodontal ligament cells (HPDLCs) stimulated by high glucose; and identify the molecular mechanism of LLL therapy in the regulation of periodontal inflammation and bone remodeling during orthodontic treatment in diabetic patients. HPDLCs were cultured in vitro to simulate orthodontic after loading and irradiated with LLL therapy. The cultured cells were randomly divided into four groups: low glucose Dulbecco's modification of Eagle's medium (DMEM)+stress stimulation (group A), high glucose DMEM+stress stimulation (group B), hypoglycemic DMEM+LLL therapy+stress stimulation (group C), and hyperglycemic DMEM+LLL therapy+stress stimulation (group D). Groups C and D were further divided into C1 and D1 (energy density: 3.75 J/cm2) and C2 and D2 (energy density: 5.625 J/cm2). Cells in groups A, B, C, and D were irradiated by LLL before irradiation. At 0, 12, 24, 48, and 72 h, the supernatants of the cell cultures were extracted at regular intervals, and the protein expression levels of IL-6, TNF-α, OPG, and RANKL were detected by enzyme-linked immunosorbent assay. 1) The levels of IL-6 and TNF-α secreted by HPDLCs increased gradually with time under static pressure stimulation. After 12 h, the levels of IL-6 and TNF-α secreted by HPDLCs in group A were significantly higher than those in groups B, C1, and C2 (P<0.05), which in group B were significantly higher than those in groups D1, and D2 (P<0.01). 2) The OPG protein concentration showed an upward trend before 24 h and a downward trend thereafter. The RANKL protein concentration increased, whereas the OPG/RANKL ratio decreased with time. Significant differen-ces in OPG, RANKL, and OPG/RANKL ratio were found among group A and groups B, C1, C2 as well as group B and groups D1, D2 (P<0.05). 1) In the high glucose+stress stimulation environment, the concentrations of IL-6 and TNF-α secreted by HPDLCs increased with time, the expression of OPG decreased, the expression of RANKL increased, and the ratio of OPG/RANKL decreased. As such, high glucose environment can promote bone resorption. After LLL therapy, the levels of IL-6 and TNF-α decreased, indicating that LLL therapy could antagonize the increase in the levels of inflammatory factors induced by high glucose environment and upregulate the expression of OPG in human HPDLCs, downregulation of RANKL expression in HPDLCs resulted in the upregulation of the ratio of OPG/RANKL and reversed the imbalance of bone metabolism induced by high glucose levels. 2) The decrease in inflammatory factors and the regulation of bone metabolism in HPDLCs were enhanced with increasing laser energy density within 3.75-5.625 J/cm2. Hence, the ability of LLL therapy to modulate bone remodeling increases with increasing dose.

Mechanical force application and inflammation induce osteoclastogenesis by independent pathways.

To investigate the functional changes of PDL fibroblasts in the presence of mechanical force, inflammation, or a combination of force and inflammation. Inflammatory supernatants were prepared by inoculating human neutrophils with Porphyromonas gingivalis. Primary human PDL fibroblasts (PDLF), gingival fibroblasts (GFs), and osteoblasts (Saos2) were then exposed to the inflammatory supernatants. Orthodontic force on the PDLFs was simulated by centrifugation. Analyses included cell proliferation, cell viability, cell cycle, and collagen expression, as well as osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-Β ligand (RANKL) expression. Mechanical force did not affect PDLF viability, but it increased the metabolic rate compared to resting cells. Force application shifted the PDLF cell cycle to the G0/G1 phase, arresting cell proliferation and leading to elevated collagen production, mild OPG level elevation, and robust RANKL level elevation. Including an inflammatory supernatant in the presence of force did not affect PDLF viability, proliferation, or cytokine expression. By contrast, the inflammatory supernatant increased RANKL expression in GFs, but not in Saos2 cells. Applying mechanical force significantly affects PDLF function. Although inflammation had no effect on PDLF or Saos2 cells, it promoted RANKL expression in GF cells. Within the limitations of the in vitro model, the results suggest that periodontal inflammation and mechanical forces could affect bone catabolism through effects on different cell types, which may culminate in synergistic bone resorption.

CREB1 alleviates the apoptosis and potentiates the osteogenic differentiation of zoledronic acid-treated human periodontal ligament stem cells via up-regulating VEGF

Periodontitis represents a severe inflammatory illness in tooth supporting tissue. It has been supported that cAMP response element binding protein 1 (CREB1), a common transcription factor, extensively participates in osteogenic differentiation. Here, the current study was to look into the impacts of CREB1 on the process of periodontitis and its possible action mechanism. After human periodontal ligament stem cells (PDLSCs) were challenged with zoledronic acid (ZA), CREB1 expression was examined with RT-qPCR and western blotting. CCK-8 assay appraised cell activity. Following CREB1 elevation or/and vascular endothelial growth factor (VEGF) silencing in ZA-treated PDLSCs, CCK-8 and TUNEL assays separately estimated cell viability and apoptosis. Western blotting tested the expression of apoptosis- and osteogenic differentiation-associated proteins. ALP staining measured PDLSCs osteogenic ability and ARS staining estimated mineralized nodule formation. JASPAR predicted the potential binding of CREB1 with VEGF promoter, which was then testified by ChIP and luciferase reporter assays. RT-qPCR and western blotting tested VEGF expression. CREB1 expression was declined in ZA-exposed PDLSCs and CREB1 elevation exacerbated the viability and osteogenic differentiation while obstructed the apoptosis of PDLSCs. Additionally, CREB1 bond to VEGF promoter and transcriptionally activated VEGF expression. Further, VEGF absence partially stimulated the apoptosis while suppressed the osteogenic differentiation of CREB1-overexpressing PDLSCs treated by ZA. To be concluded, CREB1 might activate VEGF transcription to obstruct the apoptosis while contribute to the osteogenic differentiation of ZA-treated PDLSCs.

Effect of intra-alveolar delivery of Frondoside A on inflammatory response of delayed tooth replantation.

Frondoside A is a sea cucumber extract which is well known for its anti-inflammatory and immunomodulatory properties. The purpose of this study was to evaluate the effect of Frondoside A application in the alveolar socket on inflammatory responses after delayed replantation in rat teeth. Human periodontal ligament cells were cultured and exposed to Frondoside A. Cell-counting kit-8 assay was performed to evaluate the cell viability and nitric oxide assay was performed to assess the anti-inflammatory effect of Frondoside A. Molars were extracted from 32 Sprague-Dawley rats and randomly divided into control and Frondoside A groups. After 30 min of extra-oral dry time, molars were replanted. In the Frondoside A group, Frondoside A solution was applied in the alveolar socket before replantation. The animals were sacrificed after 28 days and histologically and immunohistochemically evaluated. 0.5 μM Frondoside A showed higher cellular viability at 6 h and lower production of nitric oxide compared with other Frondoside A solutions (p < .05). The Frondoside A group demonstrated lower inflammatory resorption scores in both middle 1/3 and apical 1/3 of root compared to the control group (p < .05). The Frondoside A group showed lower levels of expression in both cathepsin K and CD45 compared with the control group (p < .05). Within the limits of this study, intra-alveolar delivery of Frondoside A alleviates inflammatory root resorption in delayed replantation of rat teeth.

AFF4 regulates osteogenic potential of human periodontal ligament stem cells via mTOR-ULK1-autophagy axis.

Scaffold protein AF4/FMR2 family member 4 (AFF4) has been found to play a role in osteogenic commitment of stem cells. However, function of AFF4 in human periodontal ligament stem cells (hPDLSCs) has not been studied yet. This present study aims to investigate the biological effect of AFF4 on osteogenic differentiation of hPDLSCs and potential mechanistic pathway. First, AFF4 expression profile was evaluated in conditions of periodontitis and osteogenic differentiation of hPDLSCs by immunohistochemical staining, western blot and qRT-PCR. Next, si-RNA mediated knockdown and lentiviral transduction mediated overexpression of AFF4 were adopted to explore impact of AFF4 on osteogenic capacity of hPDLSCs. Then, possible mechanistic pathway was identified. At last, pharmacological agonist of autophagy, rapamycin, was utilized to affirm the role of autophagy in AFF4-regulated osteogenesis of hPDLSCs. First, AFF4 expressions were significantly lower in inflamed periodontal tissues and lipopolysaccharides-treated hPDLSCs than controls, and were up-regulated during osteogenic differentiation of hPDLSCs. Next, osteogenic potential of hPDLSCs was impaired by AFF4 knockdown and potentiated by AFF4 overexpression. Moreover, AFF4 was found to positively regulate autophagic activity in hPDLSCs. At last, rapamycin treatment was shown to be able to partly restore AFF4 knockdown-suppressed osteogenic differentiation. Our study demonstrates that AFF4 regulates osteogenic potential of hPDLSCs via targeting autophagic activity. The involvement of AFF4 in periodontal homeostasis was identified for the first time.

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.

CD40-CD40 ligand interaction between periodontal ligament cells and cementoblasts enhances periodontal tissue remodeling in response to mechanical stress.

We analyzed the localization and expression of Cluster of differentiation 40 ligand (CD40L) in murine periodontal tissue applied with the orthodontic force to determine the CD40L-expressing cells under mechanical stress. Furthermore, we investigated whether CD40-CD40L interaction played an important role in transducing mechanical stress between periodontal ligament (PDL) cells and cementoblasts and remodeling the periodontal tissue for its homeostasis. PDL is a complex tissue that contains heterogeneous cell populations and is constantly exposed to mechanical stress, such as occlusal force. CD40 is expressed on PDL cells and upregulated under mechanical stress. However, whether its ligand, CD40L, is upregulated in periodontal tissue in response to mechanical stress, and which functions the CD40-CD40L interaction induces by converting the force to biological functions between the cement-PDL complex, are not fully understood. The orthodontic treatment was applied to the first molars at the left side of the upper maxillae of mice using a nickel-titanium closed-coil spring. Immunohistochemistry was performed to analyze the localization of CD40L in the periodontal tissue under the orthodontic force. Human cementoblasts (HCEM) and human PDL cells were stretched in vitro and analyzed CD40L and CD40 protein expression using flow cytometry. A GFP-expressing CD40L plasmid vector was transfected into HCEM (CD40L-HCEM). CD40L-HCEM was co-cultured with human PDL cells with higher alkaline phosphatase (ALP) activity (hPDS) or lower ALP (hPDF). After co-culturing, cell viability and proliferation were analyzed by propidium iodide (PI) staining and bromodeoxyuridine (BrdU) assay. Furthermore, the mRNA expression of cytodifferentiation- and extracellular matrix (ECM)-related genes was analyzed by real-time PCR. Immunohistochemistry demonstrated that CD40L was induced on the cells present at the cementum surface in periodontal tissue at the tension side under the orthodontic treatment in mice. The flow cytometry showed that the in vitro-stretching force upregulated CD40L protein expression on HCEM and CD40 protein expression on human PDL cells. Co-culturing CD40L-HCEM with hPDF enhanced cell viability and proliferation but did not alter the gene expression related to cytodifferentiation and ECM. In contrast, co-culturing CD40L-HCEM with hPDS upregulated cytodifferentiation- and ECM-related genes but did not affect cell viability and proliferation. We revealed that in response to a stretching force, CD40L expression was induced on cementoblasts. CD40L on cementoblasts may interact with CD40 on heterogeneous PDL cells at the necessary time and location, inducing cell viability, proliferation, and cytodifferentiation, maintaining periodontal tissue remodeling and homeostasis.

Antibacterial Activity and Sustained Effectiveness of Calcium Silicate-Based Cement as a Root-End Filling Material against Enterococcus faecalis.

Several calcium silicate cement (CSC) types with improved handling properties have been developed lately for root-end filling applications. While sealing ability is important, a high biocompatibility and antimicrobial effects are critical. This study aimed to conduct a comparative evaluation of the antimicrobial efficacy and sustained antibacterial effectiveness against Enterococcus faecalis (E. faecalis) of commercially available CSCs mixed with distilled water (DW) and chlorhexidine (CHX). Various products, viz., ProRoot mixed with DW (PRW) or with CHX (PRC), Endocem mixed with DW (EW) or with CHX (EC), and Endocem premixed (EP) syringe type, were used. While antibacterial activity against E. faecalis was evaluated using a direct contact method, the specimens were stored in a shaking incubator for 30 d for antibacterial sustainability. The cytotoxicity was evaluated using a cell counting kit-8 assay in human periodontal ligament stem cells. The antibacterial activities of EP, EW, and EC were greater than those of PRC and PRW (p < 0.05). The antibacterial sustainability of EP was the highest without cytotoxicity for up to 30 days (p < 0.05). In conclusion, the pre-mixed injectable type EP was most effective in terms of antibacterial activity and sustained antibacterial effectiveness without cytotoxicity.

Calvaria defect regeneration via human periodontal ligament stem cells and prevascularized scaffolds in athymic rats

BackgroundVascularization plays an important role in dental and craniofacial regenerations. Human periodontal ligament stem cells (hPDLSCs) are a promising cell source and, when co-cultured with human umbilical vein endothelial cells (hUVECs), could promote vascularization. The objectives of this study were to develop a novel prevascularized hPDLSC-hUVEC-calcium phosphate construct, and investigate the osteogenic and angiogenic efficacy of this construct with human platelet lysate (hPL) in cranial defects in rats for the first time. MethodshPDLSCs and hUVECs were co-cultured on calcium phosphate cement (CPC) scaffolds with hPL. Cell proliferation, angiogenic gene expression, angiogenesis, alkaline phosphatase activity, and cell-synthesized minerals were determined. Bone and vascular regenerations were investigated in rat critical-sized cranial defects in vivo. ResultshPDLSC-hUVEC-CPC-hPL group had 2-fold greater angiogenic expressions and cell-synthesized mineral synthesis than hPDLSC-hUVEC-CPC group (p < 0.05). Microcapillary-like structures were formed on scaffolds in vitro. hPDLSC-hUVEC-CPC-hPL group had more vessels than hPDLSC-hUVEC-CPC group (p < 0.05). In cranial defects in rats, hPDLSC-hUVEC-CPC-hPL group regenerated new bone amount that was 2.1 folds and 4.0 folds, respectively, that of hPDLSC-hUVEC-CPC group and CPC control (p < 0.05). New blood vessel density of hPDLSC-hUVEC-CPC-hPL group was 2 folds and 7.9 folds, respectively, that of hPDLSC-hUVEC-CPC group and CPC control (p < 0.05). ConclusionThe hPL pre-culture method is promising to enhance bone regeneration via prevascularized CPC. Novel hPDLSC-hUVEC-CPC-hPL prevascularized construct increased new bone formation and blood vessel density by 4–8 folds over CPC control. Clinical significanceNovel hPDLSC-hUVEC-hPL-CPC prevascularized construct greatly increased bone and vascular regeneration in vivo and hence is promising for a wide range of craniofacial applications.

RETRACTED ARTICLE: Identification and assessment of differentially expressed necroptosis long non-coding RNAs associated with periodontitis in human

BackgroundPeriodontitis is the most common oral disease and is closely related to immune infiltration in the periodontal microenvironment and its poor prognosis is related to the complex immune response. The progression of periodontitis is closely related to necroptosis, but there is still no systematic study of long non-coding RNA (lncRNA) associated with necroptosis for diagnosis and treatment of periodontitis.Material and methodsTranscriptome data and clinical data of periodontitis and healthy populations were obtained from the Gene Expression Omnibus (GEO) database, and necroptosis-related genes were obtained from previously published literature. FactoMineR package in R was used to perform principal component analysis (PCA) for obtaining the necroptosis-related lncRNAs. The core necroptosis-related lncRNAs were screened by the Linear Models for Microarray Data (limma) package in R, PCA principal component analysis and lasso algorithm. These lncRNAs were then used to construct a classifier for periodontitis with logistic regression. The receiver operating characteristic (ROC) curve was used to evaluate the sensitivity and specificity of the model. The CIBERSORT method and ssGSEA algorithm were used to estimate the immune infiltration and immune pathway activation of periodontitis. Spearman’s correlation analysis was used to further verify the correlation between core genes and periodontitis immune microenvironment. The expression level of core genes in human periodontal ligament cells (hPDLCs) was detected by RT-qPCR.ResultsA total of 10 core necroptosis-related lncRNAs (10-lncRNAs) were identified, including EPB41L4A-AS1, FAM30A, LINC01004, MALAT1, MIAT, OSER1-DT, PCOLCE-AS1, RNF144A-AS1, CARMN, and LINC00582. The classifier for periodontitis was successfully constructed. The Area Under the Curve (AUC) was 0.952, which suggested that the model had good predictive performance. The correlation analysis of 10-lncRNAs and periodontitis immune microenvironment showed that 10-lncRNAs had an impact on the immune infiltration of periodontitis. Notably, the RT-qPCR results showed that the expression level of the 10-lncRNAs obtained was consistent with the chip analysis results.ConclusionsThe 10-lncRNAs identified from the GEO dataset had a significant impact on the immune infiltration of periodontitis and the classifier based on 10-lncRNAs had good detection efficiency for periodontitis, which provided a new target for diagnosis and treatment of periodontitis.

TRPC expression in human periodontal ligament cells and the periodontal tissue of periodontitis mice: a preliminary study

BackgroundTransient receptor potential canonical (TRPC) channels are non-selective cationic channels with permeability to Ca2+ and Na+. Despite their importance, there are currently few studies on TRPC in the periodontal ligament (PDL) and bone cells in the dental field. To provide biological information regarding TRPC in PDL cells and periodontal tissue, we evaluated TRPC channels expression in the osteoblast differentiation of PDL cells and periodontitis-induced tissue. Human PDL cells were cultured in osteogenic differentiation media for 28 days, and the expression of Runx2, osteocalcin (OCN), and TRPC1, 3, 4, and 6 was evaluated by real-time PCR. In ligature-induced periodontitis mice, the alveolar bone and osteoid areas, the osteoclast number, and the expression of Runx2, OCN, TRPC3, and TRPC6 was evaluated by H&E staining, TRAP staining, and immunohistochemistry, respectively.ResultsIn the PDL cell differentiation group, TRPC6 expression peaked on day 7 and TRPC3 expression generally increased during differentiation. During the 28 days of periodontitis progression, alveolar bone loss and osteoclast numbers increased compared to the control group during the experimental period and the osteoid area increased from day 14. TRPC6 expression in the periodontitis group increased in the PDL area and in the osteoblasts compared to the control group, whereas TRPC3 expression increased only in the PDL area on days 7 and 28.ConclusionsThese results indicate changes of TRPC3 and TRPC6 expression in PDL cells that were differentiating into osteoblasts and in periodontitis-induced tissue, suggesting the need for research on the role of TRPC in osteoblast differentiation or periodontitis progression.

Local Immunomodulation of Human Periodontal Ligaments Stem Cells (PDLSC) in Periodontitis: A Systematic Review

Background: Human periodontal ligament stem cells (PDLSC) are multipotent cells, derived from periodontal ligament (PDL), that can attain the formation of new bone, cementum, and periodontal ligament. However, the quantity of stem cells found in PDL tissues is deficient. Therefore, in periodontitis, the limited PDLSCs make regeneration of the periodontium challenging and unpredictable. Objectives: To critically appraise the available experimental evidence of the local immunomodulation signaling systems and their surrounding functional molecules for repair and application in periodontal regeneration therapy. Methods: This systematic review was conducted using PRISMA 2020 guidelines and research papers were selected based on strict exclusion and inclusion criteria. PubMed, Cochrane Library, Google Scholar and Web of Science databases were used to collect all the research papers ranging from January 2012 to December 2022. Results: Nine out of 586 research papers that focused on PDLSCs during inflammatory conditions were selected based on the Cochrane Risk of Bias Assessment. Seven articles documented that non-coding microRNAs (ncRNAs) played critical roles in the modification of PDLSC multipotency in periodontitis. An article showed that the post-translational modifications are influenced by histone deacetylases (HDAC) indicating the involvement of novel epigenetic signaling systems. A natural product-derived functional molecule resveratrol is described to have anti-inflammatory properties through the nuclear factor kappa B (NF-kB) pathway. Conclusion: The results of our present systematic review clearly apprise that the ncRNAs signaling system and natural products derived anti-inflammatory functional molecules play a clinically significant therapeutic target in novel periodontitis therapy as preventive, diagnostic, therapeutic, and prognostic markers.

Effect of Polydeoxyribonucleotide on Human Periodontal Ligament Cells as a Storage Medium for Avulsed Tooth

Objective: This study aimed to evaluate the suitability of polydeoxyribonucleotides (PDRN) as a storage medium for avulsed teeth.Materials and Methods: The viability of human periodontal ligament (PDL) cells stored in Hank’s balanced salt solution and PDRN solutions (concentrations, 10, 25, 50, and 100 μg/mL) and tap water was measured using the Cell Counting Kit-8 and Live/Dead assays. In addition, Nitric oxide detection and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to evaluate the anti-inflammatory effect of PDRN.Results: The viability of PDL cells stored in a 100 μg/mL PDRN solution was significantly higher than that of cells stored in the other solutions (&lt;i&gt;p&lt;/i&gt; &lt; 0.01). Furthermore, cells stored in 100 μg/mL PDRN solution demonstrated a significantly reduced NO production (&lt;i&gt;p&lt;/i&gt; &lt; 0.0001), and cells stored in 50 and 100 μg/mL PDRN solutions expressed significantly lower levels of tumor necrosis factor α, interleukin (IL) -4, IL-6, and IL-10 (&lt;i&gt;p&lt;/i&gt; &lt; 0.01) compared to cells stored in HBSS.Conclusion: The PDRN solution exhibited cell-preserving and anti-inflammatory effects on the PDL cells. The findings of this study can serve as a basis for further experiments directed at the development of an effective storage medium for avulsed teeth.

Avenanthramide-C Shows Potential to Alleviate Gingival Inflammation and Alveolar Bone Loss in Experimental Periodontitis.

Periodontal disease is a chronic inflammatory disease that leads to the gradual destruction of the supporting structures of the teeth including gums, periodontal ligaments, alveolar bone, and root cementum. Recently, interests in alleviating symptoms of periodontitis (PD) using natural compounds is increasing. Avenanthramide-C (Avn-C) is a polyphenol found only in oats. It is known to exhibit various biological properties. To date, the effect of Avn-C on PD pathogenesis has not been confirmed. Therefore, this study aimed to verify the protective effects of Avn-C on periodontal inflammation and subsequent alveolar bone erosion in vitro and in vivo. Upregulated expression of catabolic factors, such as matrix metalloproteinase 1 (MMP1), MMP3, interleukin (IL)-6, IL-8, and COX2 induced by lipopolysaccharide and proinflammatory cytokines, IL-1β, and tumor necrosis factor α (TNF-α), was dramatically decreased by Avn-C treatment in human gingival fibroblasts and periodontal ligament cells. Moreover, alveolar bone erosion in the ligature-induced PD mouse model was ameliorated by intra-gingival injection of Avn-C. Molecular mechanism studies revealed that the inhibitory effects of Avn-C on the upregulation of catabolic factors were mediated via ERK (extracellular signal-regulated kinase) and NF-κB pathway that was activated by IL-1β or p38 MAPK and JNK signaling that was activated by TNF-α, respectively. Based on this study, we recommend that Avn-C may be a new natural compound that can be applied to PD treatment.