Proliferation Of Periodontal Ligament Cells Research Articles

Apelin Counteracts the Effects of Fusobacterium nucleatum on the Migration of Periodontal Ligament Cells In Vitro

To better understand the link between periodontitis and metabolic diseases, our in vitro study aimed to assess the influence of the adipokine apelin and/or the periodontal pathogen Fusobacterium nucleatum on periodontal cells. Periodontal ligament (PDL) cells were exposed to F. nucleatum in the presence and absence of apelin. Scratch assays were used to analyze the in vitro wound healing and velocity of cell migration. To investigate if F. nucleatum and/or apelin have a regulatory effect on cell proliferation and apoptosis, proliferation and viability assays were performed as well as an analysis of caspase 9 expression. Both the in vitro wound closure and the cell migration rate were significantly reduced by F. nucleatum. Simultaneous incubation with apelin counteracted the adverse effects of F. nucleatum. The proliferation assay demonstrated that neither apelin nor F. nucleatum significantly affected PDL cell proliferation. Furthermore, neither apelin nor F. nucleatum was cytotoxic or affected apoptosis after 48 h. Apelin could play a modulatory role in the pathogenesis of periodontitis, as it was able to compensate for the inhibitory effects of the periodontal pathogen F. nucleatum on PDL cell migration in vitro.

Mesenchymal stem cell-derived protein extract induces periodontal regeneration: MSC-protein induces periodontal regeneration

BackgroundPeriodontal disease is characterized by chronic inflammation and destruction of supporting periodontal tissues, ultimately leading to tooth loss. In recent years, “cell-free treatment” without stem cell transplantation has attracted considerable attention for tissue regeneration. This study investigated the effects of extracts of mesenchymal stem cells (MSC-extract) and their protein components (MSC-protein) on the proliferation and migration of periodontal ligament (PDL) cells and whether MSC-protein can induce periodontal regeneration. MethodsMSC-extract and MSC-protein were obtained by subjecting mesenchymal stem cells (MSCs) to freeze–thaw cycles and acetone precipitation. Cell proliferation was examined using a WST-8 assay and Ki67 immunostaining, and cell migration was examined using Boyden chambers. The MSC-protein content was analyzed using liquid chromatography-mass spectrometry, protein arrays, and enzyme-linked immunosorbent assays (ELISAs). Gene expression in MSC-protein-treated PDL cells was examined using RNA-sequencing and Gene Ontology analyses. The regenerative potential of MSC-protein was examined using micro-computer tomography (CT) and histological analyses after transplantation into a rat periodontal defect model. ResultsMSC-extract and MSC-protein promoted the proliferation and migration of PDL cells. Protein array and ELISA revealed that MSC-protein contained high concentrations of basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF). Exogenous bFGF promoted the proliferation and migration of PDL cells. Furthermore, the transplantation of MSC-protein enhanced periodontal tissue regeneration with the formation of new alveolar bone and PDLs. ConclusionsThese results indicate that the MSC-protein promotes the proliferation and migration of PDL cells and induces significant periodontal tissue regeneration, suggesting that the MSC-protein could be used as a new cell-free treatment for periodontal disease.

TNF reduces osteogenic cell fate in PDL cells at transcriptional and functional levels without alteration of periodontal proliferative capacity.

To investigate the effect of tumor necrosis factor (TNF) on the growth of human periodontal ligament (PDL) cells, their osteogenic differentiation and modulation of their matrix secretion in vitro. The influence of 10 ng/ml TNF on proliferation and metabolic activity of PDL cells was analyzed by cell counting (DAPI [4',6-diamidino-2-phenylindole] staining) and the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. In addition, cells were cultured under control conditions and osteogenic conditions (media containing 10 mMβ-glycerophosphate). Quantitative expression analysis of genes encoding the osteogenic markers alkaline phosphatase (ALP), collagen typeI alpha1 chain (COL1A1), osteoprotegerin (OPG), and osteopontin (OPN) was performed after 7 and 14days of cultivation. Calcium deposits were stained with alizarin red. Our studies showed that 10 ng/ml TNF did not affect the survival and metabolic activity of PDL cells. Quantitative expression analysis revealed that long-term cultures with TNF impaired osteogenic cell fate at early and late developmental stages. Furthermore, TNF significantly reduced matrix secretion in PDL cells. The present data confirm TNF as aregulatory factor of proinflammatory remodeling that influences the differentiation behavior but not the metabolism and cell proliferation of the periodontium. Therefore, TNF represents an interesting target for the regulation of orthodontic remodeling processes in the periodontium.

Periodontal tissue regeneration with cementogenesis after application of brain-derived neurotrophic factor in 3-wall inflamed intra-bony defect.

The purpose of this study is to investigate regenerative process by immunohistochemical analysis and evaluate periodontal tissue regeneration following a topical application of BDNF to inflamed 3-wall intra-bony defects. Brain-derived neurotrophic factor (BDNF) plays a role in the survival and differentiation of central and peripheral neurons. BDNF can regulate the functions of non-neural cells, osteoblasts, periodontal ligament cells, endothelial cells, as well as neural cells. Our previous study showed that a topical application of BDNF enhances periodontal tissue regeneration in experimental periodontal defects of dog and that BDNF stimulates the expression of bone (cementum)-related proteins and proliferation of human periodontal ligament cells. Six weeks after extraction of mandibular first and third premolars, 3-wall intra-bony defects were created in mandibular second and fourth premolars of beagle dogs. Impression material was placed in all of the artificial defects to induce inflammation. Two weeks after the first operation, BDNF (25 and 50 μg/mL) immersed into atelocollagen sponge was applied to the defects. As a control, only atelocollagen sponge immersed in saline was applied. Two and four weeks after the BDNF application, morphometric analysis was performed. Localizations of osteopontin (OPN) and proliferating cell nuclear antigen (PCNA)-positive cells were evaluated by immunohistochemical analysis. Two weeks after application of BDNF, periodontal tissue was partially regenerated. Immunohistochemical analyses revealed that cells on the denuded root surface were positive with OPN and PCNA. PCNA-positive cells were also detected in the soft connective tissue of regenerating periodontal tissue. Four weeks after application of BDNF, the periodontal defects were regenerated with cementum, periodontal ligament, and alveolar bone. Along the root surface, abundant OPN-positive cells were observed. Morphometric analyses revealed that percentage of new cementum length and percentage of new bone area of experimental groups were higher than control group and dose-dependently increased. These findings suggest that BDNF could induce cementum regeneration in early regenerative phase by stimulating proliferation of periodontal ligament cells and differentiation into periodontal tissue cells, resulting in enhancement of periodontal tissue regeneration in inflamed 3-wall intra-bony defects.

Periodontal Guided Tissue Regeneration Membranes: Limitations and Possible Solutions for the Bottleneck Analysis.

Guided tissue regeneration (GTR) is an important surgical method for periodontal regeneration. By placing barrier membrane on the root surface of the tooth to guide the adhesion and proliferation of periodontal ligament cells, periodontal tissue regeneration can be achieved. This review intends to analyze the current limitations of GTR membranes and to propose possible solutions for developing new ones. Limitations of current GTR membranes include nonabsorbable membranes and absorbable synthetic polymer membranes exhibit weak biocompatibility; when applying to a large defect wound, the natural collagen membrane with fast degradation rate have limited mechanical strength, and the barrier function may not be maintained well. Although the degradation time can be prolonged after cross-linking, it may cause foreign body reaction and affect tissue integration; The clinical operation of current barrier membranes is inconvenient. In addition, most of the barrier membranes lack bioactivity and will not actively promote periodontal tissue regeneration. Possible solutions include using electrospinning (ELS) techniques, nanofiber scaffolds, or developing functional gradient membranes to improve their biocompatibility; adding Mg, Zn, and/or other metal alloys, or using 3D printing technology to improve their mechanical strength; increasing the concentration of nanoparticles or using directional arrangement of membrane fibers to control the fiber diameter and porosity of the membrane, which can improve their barrier function; mixing natural and synthetic polymers as well as other biomaterials with different degradation rates in proportion to change the degradation rate and maintain barrier function; to improve the convenience of clinical operation, barrier membranes that meets personalized adhesion to the wound defect can be manufactured; developing local controlled release drug delivery systems to improve their bioactivity. Impact statement This review provides an up-to-date summary of commonly commercial periodontal guided tissue regeneration membranes, and analyze their limitations in clinical use. Using studies published recently to explore possible solutions from several perspectives and to raise possible strategies in the future. Several strategies have tested in vivo/in vitro, which will guide the way to propel clinical translation, meeting clinical needs.

Smoking habits do not affect biological responses induced by leucocyte and platelet-rich fibrin in periodontal ligament cells.

Leucocyte- and platelet-rich fibrin has been developed to stimulate wound healing response. However, it is currently unknown whether smoking affects the biological responses elicited by leucocyte- and platelet-rich fibrin on periodontal ligament-derived mesenchymal stromal cells. This study analyzes the kinetics of biomolecule release from leucocyte- and platelet-rich fibrin derived from smokers and nonsmokers and their effect on periodontal ligament cell proliferation and migration as essential biological activities during wound healing. Biomolecules present in leucocyte- and platelet-rich fibrin exudates and conditioned media collected from smokers and nonsmokers were analyzed by Luminex arrays. Periodontal ligament-derived mesenchymal stromal cell obtained from one nonsmoker were treated with leucocyte- and platelet-rich fibrin exudates or leucocyte- and platelet-rich fibrin conditioned media derived from both smokers and nonsmokers. The parameters evaluated included cell proliferation, determined by Ki67 immunostaining and migration assessed using transwell assays. Also, cells were treated with nicotine in the presence of fetal bovine serum 10% or leucocyte- and platelet-rich fibrin conditioned media. A similar biomolecular profile was detected in leucocyte- and platelet-rich fibrin exudates and leucocyte- and platelet-rich fibrin conditioned media from smokers and nonsmokers, stimulating (periodontal ligament-derived mesenchymal stromal cell) proliferation, and migration to a comparable degree. Nicotine reduced cell proliferation and migration of periodontal cells; however, this effect was recovered in the presence of leucocyte- and platelet-rich fibrin conditioned media. Leucocyte- and platelet-rich fibrin derived from smokers could be an autologous source of biomolecules to stimulate cell biological activities involved in wound healing in smokers who have difficulties in ceasing this habit. Clinical trials are required to evaluate the impact of leucocyte- and platelet-rich fibrin on healing responses in smokers.

Fluid shear stress promotes periodontal ligament cells proliferation via p38-AMOT-YAP.

Periodontal ligament (PDL) cells are a promising tool for periodontal regeneration therapy. Achieving a sufficient number of PDL cells is essential to PDL regeneration. In our study, appropriate flow shear stress (FSS, 1-6 dyn/cm2) promotes the proliferation of PDL cells. FSS remodels cytoskeleton and focal adhesion in a duration-dependent manner. FSS induces PDL cells to form the actin cap within 10min, flattens the nuclei, and increases the nuclear pore size, which promotes nuclear translocation of Yes-associated protein (YAP). FSS activates p38, which plays a dual function in YAP regulation. p38 regulates the phosphorylation of Akt and cofilin, as well as induced F-actin polymerization to induce YAP activity. In addition, p38 inhibits pLATS and consecutively regulates angiomotin (AMOT) and YAP phosphorylation. AMOT competitively binds to F-actin and YAP to participate in FSS-mediated YAP nuclear translocation and cell proliferation. Taken collectively, our results provide mechanistic insights into the role of p38-AMOT-YAP in FSS-mediated PDL cells proliferation and indicate potential applications in dental regenerative medicine.

Intermittent compressive force regulates human periodontal ligament cell behavior via yes-associated protein

Intermittent compressive force influences human periodontal ligament (PDL) cell behavior that facilitates periodontal tissue regeneration. In response to mechanical stimuli, Yes-associated protein (YAP) has been recognized as a mechanosensitive transcriptional activator that regulates cell proliferation and cell fate decisions. This study aimed to investigate whether compressive forces influence cell proliferation and cell fate decisions of human PDL cells via YAP signaling. YAP expression was silenced by shRNA. The effect of YAP on cell proliferation, adipogenesis and osteogenesis of PDL cells under ICF loading were determined. Adipogenic differentiation bias upon ICF loading was confirmed by fourier-transform infrared spectroscopy (FTIR). The results revealed that ICF-induced YAP promotes osteogenesis, but it inhibits adipogenesis in PDL cells. Depletion of YAP results in PDL cells that are irresponsive to ICF and, therefore, the failure of the PDL cells to undergo osteogenic differentiation. This was shown by a significant reduction in calcium deposited in the CF-derived osteoblasts of the YAP-knockdown (YAP-KD) PDL cells. As to control treatment, reduction of YAP promoted adipogenesis, whereas ICF-induced YAP inhibited this mechanism. However, the adipocyte differentiation in YAP-KD cells was not affected upon ICF treatment as the YAP-KD cells still exhibited a better adipogenic differentiation that was unrelated to the ICF. This study demonstrated that, in response to ICF treatment, YAP could be a crucial mechanosensitive transcriptional activator for the regulation of PDL cell behavior through a mechanobiological process. Our results may provide the possibility of facilitating PDL tissue regeneration by manipulation of the Hippo-YAP signaling pathway.

Effects of High-Glucose Environment on Periodontal Ligament Cell Proliferation and Apoptosis via NF-κB Signaling Pathway.

The purpose of this study was to discuss the function of the high-glucose environment on the periodontal ligament cell (PDLC) proliferation and apoptosis and the action mechanism of the NF-κB signaling pathway in this process. For this purpose, the human PDLCs were cultured in vitro using 5.5 mM (control group)/24.0 mM glucose (HG group) of glucose and 10 μM of QNZ+24.0 mM of glucose (HG+QNZ), respectively, and the cell proliferation level was checked through CCK-8 assay. TUNEL assay was used to perform cell apoptosis. ELISA was utilized to explore the secretion levels of the proinflammatory factors interleukin (IL)-1β and IL-6 proteins. The p65 and p50 proteins level were tested via the Western blotting (WB) assay. Results showed that in comparison with the control group, 24.0 mM of glucose could significantly decrease the proliferation ability of the PDLCs (p<0.01), cause cell apoptosis (p<0.05) and promote the secretion of IL-6 and IL-1β (p<0.05). The expressions of p65 and p50 proteins were up-regulated obviously in the high-glucose environment (p<0.05). QNZ could exert a specific inhibitory effect on the NF-κB activity to significantly down-regulate the expressions of p65 and p50 proteins (p<0.05) and reverse the effects of the high-glucose environment on the cell apoptosis and proliferation (p<0.05). In conclusion, hyper-glucose may affect PDLC proliferation and apoptosis by suppressing the NF-κB signaling pathway activity.

Nisin probiotic prevents inflammatory bone loss while promoting reparative proliferation and a healthy microbiome

Dysbiosis of the oral microbiome mediates chronic periodontal disease. Realignment of microbial dysbiosis towards health may prevent disease. Treatment with antibiotics and probiotics can modulate the microbial, immunological, and clinical landscape of periodontal disease with some success. Antibacterial peptides or bacteriocins, such as nisin, and a nisin-producing probiotic, Lactococcus lactis, have not been examined in this context, yet warrant examination because of their biomedical benefits in eradicating biofilms and pathogenic bacteria, modulating immune mechanisms, and their safety profile in humans. This study’s goal was to examine the potential for nisin and a nisin-producing probiotic to abrogate periodontal bone loss, the host inflammatory response, and changes in oral microbiome composition in a polymicrobial mouse model of periodontal disease. Nisin and a nisin-producing Lactococcus lactis probiotic significantly decreased the levels of several periodontal pathogens, alveolar bone loss, and the oral and systemic inflammatory host response. Surprisingly, nisin and/or the nisin-producing L. lactis probiotic enhanced the population of fibroblasts and osteoblasts despite the polymicrobial infection. Nisin mediated human periodontal ligament cell proliferation dose-dependently by increasing the proliferation marker, Ki-67. Nisin and probiotic treatment significantly shifted the oral microbiome towards the healthy control state; health was associated with Proteobacteria, whereas 3 retroviruses were associated with disease. Disease-associated microbial species were correlated with IL-6 levels. Nisin or nisin-producing probiotic’s ability to shift the oral microbiome towards health, mitigate periodontal destruction and the host immune response, and promote a novel proliferative phenotype in reparative connective tissue cells, addresses key aspects of the pathogenesis of periodontal disease and reveals a new biomedical application for nisin in treatment of periodontitis and reparative medicine.

The effect of phenytoin on the proliferative ability of periodontal and gingival ligament fibroblasts in cell culture medium.

Periodontal ligament fibroblasts (PDLFs) play a vital role in the period of periodontal regeneration. In addition, studies show that diphenylhydantoin (phenytoin) increases the growth of gingival fibroblasts. If this effect is also present in the periodontal ligament (PDL) fibroblasts, it may be used to regenerate periodontal tissues. Accordingly, this study aimed to compare the effect of phenytoin on the growth rate of gingival fibroblast cells and PDL in the cell culture medium. In this regard, 10 Wistar rats were selected. The gingival specimen was obtained from the area between the upper teeth, and the PDL specimen was obtained from the middle third of the lower teeth root. After transferring the samples to a suitable culture medium for culturing PDL and gingival fibroblasts, each sample was divided into two experimental and control groups. In the experimental group, 20 mg/ml phenytoin dissolved in sodium hydroxide was added to Dulbecco's modified Eagle's medium (DMEM). After 48 hours, fibroblast cell proliferation was assessed through a 1-WST cell proliferation kit by ELISA. The proliferation of gingival fibroblast cells and PDL in both test and control groups were statistically analyzed by the independent t-test. The results showed that the effect of phenytoin on the proliferation of gingival fibroblast cells and PDL fibroblast cells is significant. Also, the proliferation of PDL cells was significantly different from gingival cells in the experimental group (P <0.001) and was higher in PDL cells. In general, in this study, it was found that phenytoin in vitro, like in vivo, is able to increase the proliferation of gingival fibroblast cells, and this phenytoin effect is also present in PDL fibroblast cells.

MicroRNA-28-5p as a potential diagnostic biomarker for chronic periodontitis and its role in cell proliferation and inflammatory response

Background/purposeRecent studies have pointed to the crucial role of microRNAs (miRNAs) in chronic periodontitis (CP). This study investigated the regulation and potential mechanisms of miR-28-5p in CP patients and lipopolysaccharide (LPS)-induced periodontal ligament cells (PDLCs).Materials and methods76 CP patients and 71 periodontally healthy subjects were included. RT-qPCR was employed to examine miR-28-5p and sphingosine kinase −1 (SPHK1) in subjects’ gingival sulcus fluid and PDLCs. The diagnostic performance was evaluated by measuring the area under the curve (AUC) of the receiver operating characteristic (ROC) analysis. Pearson correlation coefficient (r) was adopted to explore the statistical relation between indicators. PDLCs proliferation and inflammation factors were determined by CCK-8 and ELISA assay. The direct target gene was validated by a dual-luciferase reporter assay.ResultsmiR-28-5p was lowly expressed in CP patients and LPS-induced PDLCs (P < 0.05). AUC for miR-28-5p was 0.937, which had certain diagnostic value. Additionally, miR-28-5p was negatively correlated with periodontal clinical indicators and inflammatory factors. Cell proliferation of PDLCs was inhibited and inflammation was promoted under LPS induction, however, elevated miR-28-5p diminished the effect of LPS (P < 0.05). SPHK1 acts as a miR-28-5p target and the elevation of SPHK1 caused by LPS treatment was inhibited by the increased miR-28-5p.ConclusionPresent study revealed that miR-28-5p could be served as a potential diagnostic biomarker for CP. And miR-28-5p may participate in CP progression by targeting SPHK1 to regulate the proliferation and inflammation of PDLCs. This study may offer insights into CP treatment and diagnosis.

Effects of Emodin on Alveolar Bone Resorption via the IL-23/Th17 Inflammatory Axis in Rats with Periodontitis

This study aimed to analyze the effect of emodin on alveolar bone resorption in rats with periodontitis through the interleukin-23 (IL-23)/T helper 17 (Th17) cells inflammatory axis. Twenty-four male Sprague Dawley rats were randomly divided into the following three groups of eight rats each: the control group (group N), periodontitis group (group P), and periodontitis + emodin intervention group (group E). After 2 weeks of continuous emodin intervention, alveolar bone loss (ABL) was evaluated by morphological analysis. Tartrate-resistant acid phosphatase staining was performed to determine the number of osteoclasts (OCs) in periodontal tissue. The ABL and trabecular separation were greater and the number of OCs and the G1/G0 and G2/M phase ratios were higher in group P than those in groups N and E (P < 0.05). In contrast, the trabecular number and thickness were decreased and the S and G2+S phase ratios were lower in group P than those in groups N and E (P < 0.05). However, these indices did not differ significantly between groups E and N. The IL-23, IL-17, RANKL, RANK, RANKL/OPG, IL-2, IL-6, IL-1β, and TNF-α levels across the three groups were in the order of group P > group E > group N and the OPG levels were in the order of group P < group E < group N (P < 0.05). Emodin may decrease the expression of RANKL and RANK proteins and levels of inflammatory factors; increase OPG, PPAR-γ, and NF-κB protein expression levels; and decrease the number of OCs through the IL-23/Th17 inflammatory axis, thus inhibiting ABL and bone resorption in rats with periodontitis and promoting the proliferation of periodontal ligament cells and alveolar bone repair.

Ultrathin 2D Titanium Carbide MXene (Ti3 C2 Tx ) Nanoflakes Activate WNT/HIF-1α-Mediated Metabolism Reprogramming for Periodontal Regeneration.

Periodontal defect regeneration in severe periodontitis relies on the differentiation and proliferation of periodontal ligament cells (PDLCs). Recently, an emerging 2D nanomaterial, MXene (Ti3 C2 Tx ), has gained more and more attention due to the extensive antibacterial and anticancer activity, while its potential biomedical application on tissue regeneration remains unclear. Through a combination of experimental and multiscale simulation schemes, Ti3 C2 Tx has exhibited satisfactory biocompatibility and induced distinguish osteogenic differentiation of human PDLCs (hPDLCs), with upregulated osteogenesis-related genes. Ti3 C2 Tx manages to activate the Wnt/β-catenin signaling pathway by enhancing the Wnt-Frizzled complex binding, thus stabilizing HIF-1α and altering metabolic reprogramming into glycolysis. In vivo, hPDLCs pretreated by Ti3 C2 Tx display excellent performance in new bone formation and osteoclast inhibition with enhanced RUNX2, HIF-1α, and β-catenin in an experimental rat model of periodontal fenestration defects, indicating that this material has high efficiency of periodontal regeneration promotion. It is demonstrated in this work that Ti3 C2 Tx has highly efficient therapeutic effects in osteogenic differentiation and periodontal defect repairment.

Phenytoin Regulates Migration and Osteogenic Differentiation by MAPK Pathway in Human Periodontal Ligament Cells.

Periodontal healing requires an adequate number of periodontal ligament (PDL) cells to rebuild the impaired tissue. Phenytoin (PHT) has been reported to promote wound healing and extracellular matrix deposition, which indicates its promising application of periodontal healing. However, the effects of PHT on PDL cells behavior and the underlying mechanism are still unknown. Human PDL cells were cultured and identified. 20-100 μg/mL PHT were used in our study. The proliferation of PDL cells was determined by the EdU assay. A wound healing assay was used to detect cell migration. Matrix metalloproteinase (MMP)-1, MMP-2, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 expression were analyzed by real time-PCR. The protein expression of MMP-1 and phosphorylated mitogen-activated protein kinases (MAPKs) were detected by western blotting assay. Osteogenic differentiation was assessed by alkaline phosphatase (ALP) staining. We found that 20-100 μg/mL of PHT did not affect PDL cells proliferation, whereas 50-100 μg/mL of PHT inhibited cell migration. The 50 or 100 μg/mL of PHT decreased the gene and protein expression of MMP-1, but increased the gene expression of TIMP-1. MMP-2 and TIMP-2 were not affected by 20-100 μg/mL of PHT. Further, 20-50 μg/mL of PHT increased ALP expression, but 100 μg/mL of PHT depressed ALP expression. The extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 were activated by PHT. JNK and ERK are involved in PHT-regulated migration. JNK plays an essential role in PHT-induced osteogenic differentiation. MAPK pathway involved in PHT-regulated migration and osteogenic differentiation in human PDL cells.

In vitro comparison of nanofibrillar and macroporous-spongious composite tissue scaffolds for periodontal tissue engineering

ABSTRACT Purpose/Aim of the study The ultimate goal of periodontal treatment is to regenerate the lost periodontal tissues. The interest in nanomaterials in dentistry is growing rapidly and has focused on improvements in various biomedical applications, such as periodontal regeneration and periodontal tissue engineering. To enhance periodontal tissue regeneration, hydroxyapatite (HA) was used in conjunction with other scaffold materials, such as Poly lactic-co-glycolic-acid (PLGA) and collagen (C). The main target of this study was to compare the effects of nano and macrostructures of the tissue scaffolds on cell behavior in vitro for periodontal tissue engineering. Materials and Methods Nanofibrillar and macroporous-spongious composite tissue scaffolds were produced using PLGA/C/HA. Subgroups with BMP-2 signal molecule and without HA were also created. The scaffolds were characterized by FTIR, SEM/EDX techniques, and mechanical tests. The scaffolds were compared in the periodontal ligament (PDL) and MCT3-E1 cell cultures. The cell behaviors; adhesions by SEM, proliferation by WST-1, differentiation by ALP and mineralization with Alizarin Red Tests were determined. Results Cell adhesion and mineralization were higher in the nanofibrillar scaffolds compared to the macroporous-spongious scaffolds. Macroporous-spongious scaffolds seemed better for the proliferation of PDL cells and differentiation of MC3T3-E1-preosteoblastic cells, while nanofibrillar scaffolds were more convenient for the differentiation of PDL cells and proliferation of MC3T3-E1-preosteoblastic cells. Conclusions In general, nanofibrillar scaffolds showed more favorable results in cell behaviors, compared to the macroporous-spongious scaffolds, and mostly, BMP-2 and HA promoted the activities of the cells.

LncRNA DCST1-AS1 inhibits PDLCs' proliferation in periodontitis and may bind with miR-21 precursor to upregulate PLAP-1.

This study aimed to investigate the potential interactions among long noncoding RNA domain containing 1-antisense (lncRNA DCST1-AS1), miR-21, and periodontal ligament-associated protein-1 (PLAP-1) in periodontitis. It has been verified that miR-21 can target PLAP-1 to regulate the osteogenic differentiation of periodontal ligament cells (PDLCs). Differential expression of DCST1-AS1 and miR-21 in PDLCs derived from periodontitis patients and healthy controls was determined by qPCR and unpaired t test. QPCR and Western blots were conducted to evaluate the effects of overexpression of DCST1-AS1 and miR-21 on the expression of PLAP-1. CCK-8 assay was applied to evaluate the effect of DCST1-ASI, miR-21, or PLAP-1 on PDLCs' proliferation. Western blotting was conducted to detect the expression levels of CKD family (CDK4, CDK6, and CCND1). DCST1-AS1 was downregulated in PDLCs derived from periodontitis patients, and its expression was inversely correlated with the expression of miR-2 but positively correlated with PLAP-1. Bioinformatics analysis showed that DCST1-AS1 might bind with miR-21 precursor but not mature miR-21. Transfection experiments showed that overexpression of DCST1-AS1 led to decreased expression levels of miR-21 and significantly increased the expression levels of PLAP-1 at both mRNA and protein levels, while overexpression of miR-21 resulted in a dramatic lower level of PLAP-1. CCK-8 assay indicated that overexpression of DCST1-AS1 or PLAP-1 prohibited PDLCs' proliferation. However, elevation of miR-21 had a contrary effect on the proliferation of PDLCs. And increased expression levels of DCST1-AS1 could significantly inhibit the expression of CDK4, CDK6, and CCND-1, while overexpression of miR-21 inversed the effects of DCST1-AS1. Therefore, the expression levels of DCST1-AS1 are much lower in periodontitis patients compared to that in healthy controls, and overexpression of DCST1-AS1 can significantly elevate the expression of PLAP-1 by inhibiting miR-21 in PDLCs.

Small Extracellular Vesicles from Lipopolysaccharide-Preconditioned Dental Follicle Cells Promote Periodontal Regeneration in an Inflammatory Microenvironment.

Lipopolysaccharide (LPS)-induced inflammatory microenvironment can enhance the dental follicle cells (DFCs) proliferation, differentiation, and adhesion abilities beneficial to periodontal regeneration, which possibly attributes the success to exosomes according to recent studies. This study aimed to investigate the therapeutic efficacy and underlying mechanisms of LPS-preconditioned DFC-derived small extracellular vesicles (sEVs), which enriched exosomes for periodontal regeneration in an inflammatory microenvironment. LPS preconditioning could significantly increase the secretion of sEVs derived from DFCs. Both LPS-preconditioned dental follicle cell-derived sEV (L-D-sEV) and DFC-derived sEV (D-sEV) promoted the proliferation of periodontal ligament cells from periodontitis (p-PDLCs) with a dose-dependent and saturable manner and also enhanced the migration and differentiation of p-PDLCs. Furthermore, L-D-sEV showed a modest benefit than D-sEV to promote p-PDLCs differentiation. In vivo, an L-D-sEV-loaded hydrogel applied in the treatment of periodontitis was beneficial to repair lost alveolar bone in the early stage of treatment and to maintain the level of alveolar bone in the late stage of treatment in experimental periodontitis rats, which could partly decrease the expression of the RANKL/OPG ratio. In conclusion, L-D-sEV was beneficial to p-PDLCs forming an integrity periodontal tissue. The biological injectable L-D-sEV-loaded hydrogel could be used as a treatment method for experimental periodontitis in rats, promoting periodontal tissue regeneration and providing a new alternative cell therapy method for periodontal tissue regeneration.

Preparing polycaprolactone scaffolds using electrospinning technique for construction of artificial periodontal ligament tissue

ObjectivesThe strategies of tissue-engineering led to the development of living cell-based therapies to repair lost or damaged tissues, including periodontal ligament and to construct biohybrid implant. This work aimed to isolate human periodontal ligament stem cells (hPDLSCs) and implant them on fabricated polycaprolactone (PCL) for the regeneration of natural periodontal ligament (PDL) tissues. MethodshPDLSCs were harvested from extracted human premolars, cultured, and expanded to obtain PDL cells. A PDL-specific marker (periostin) was detected using an immunofluorescent assay. Electrospinning was applied to fabricate PCL at three concentrations (13%, 16%, and 20% weight/volume) in two forms, which were examined through field emission scanning electron microscopy (FESEM). The isolated hPDLSCs were implanted on the fabricated PCL. After 21 days, FESEM was conducted to evaluate the implanted scaffolds, and an MTT assay was performed to characterize the biological response of the PCL scaffold at different cell exposure durations (24, 48, and 72 h). ResultsPeriostin was expressed in the expanded PDL cells, and this result revealed that 20% weight/volume PCL scaffold with a pore size of more than 10 μm was the best. The growth rates of PDLSCs were high. Cytotoxicity test of fabricated PCL scaffold demonstrated no significant change in the cell viability when compared with the negative control and no deteriorating or inhibitory effect on growth after different durations. ConclusionsA cell sheet was successfully formed by using PCL as a scaffold to cover dental implants and promote PDL cell attachment, proliferation, and growth for biohybrid implant construction.

Fully amino acid-based hydrogel as potential scaffold for cell culturing and drug delivery.

Polymer hydrogels are ideal scaffolds for both tissue engineering and drug delivery. A great advantage of poly(amino acid)-based hydrogels is their high similarity to natural proteins. However, their expensive and complicated synthesis often limits their application. The use of poly(aspartic acid) (PASP) seems an appropriate solution for this problem due to the relatively cheap and simple synthesis of PASP. Using amino acids not only as building blocks in the polymer backbone but also as cross-linkers can improve the biocompatibility and the biodegradability of the hydrogel. In this paper, PASP cross-linked with cystamine (CYS) and lysine-methylester (LYS) was introduced as fully amino acid-based polymer hydrogel. Gels were synthesized employing six different ratios of CYS and LYS. The pH dependent swelling degree and the concentration of the elastically active chain were determined. After reduction of the disulfide bonds of CYS, the presence of thiol side groups was also detected. To determine the concentration of the reactive cross-linkers in the hydrogels, a new method based on the examination of the swelling behavior was established. Using metoprolol as a model drug, cell proliferation and drug release kinetics were studied at different LYS contents and in the presence of thiol groups. The optimal ratio of cross-linkers for the proliferation of periodontal ligament cells was found to be 60−80% LYS and 20−40% CYS. The reductive conditions resulted in an increased drug release due to the cleavage of disulfide bridges in the hydrogels. Consequently, these hydrogels provide new possibilities in the fields of both tissue engineering and controlled drug delivery.