Erythropoietin receptor signal is crucial for periodontal ligament stem cell-based tissue reconstruction in periodontal disease.

Alveolar bone loss caused by periodontal disease eventually leads to tooth loss. Periodontal ligament stem cells (PDLSCs) are the tissue-specific cells for maintaining and repairing the periodontal ligament, cementum, and alveolar bone. Here, we investigated the role of erythropoietin receptor (EPOR), which regulates the microenvironment-modulating function of mesenchymal stem cells, in PDLSC-based periodontal therapy. We isolated PDLSCs from patients with chronic periodontal disease and healthy donors, referred to as PD-PDLSCs and Cont-PDLSCs, respectively. PD-PDLSCs exhibited reduced potency of periodontal tissue regeneration and lower expression of EPOR compared to Cont-PDLSCs. EPOR-silencing suppressed the potency of Cont-PDLSCs mimicking PD-PDLSCs, whereas EPO-mediated EPOR activation rejuvenated the reduced potency of PD-PDLSCs. Furthermore, we locally transplanted EPOR-silenced and EPOR-activated PDLSCs into the gingiva around the teeth of ligament-induced periodontitis model mice and demonstrated that EPOR in PDLSCs participated in the regeneration of the periodontal ligament, cementum, and alveolar bone in the ligated teeth. The EPOR-mediated paracrine function of PDLSCs maintains periodontal immune suppression and bone metabolic balance via osteoclasts and osteoblasts in the periodontitis model mice. Taken together, these results suggest that EPOR signaling is crucial for PDLSC-based periodontal regeneration and paves the way for the development of novel options for periodontal therapy.

Rational design of viscoelastic hydrogels for periodontal ligament remodeling and repair.

The periodontal ligament (PDL) is a distinctive yet critical connective tissue vital for maintaining the integrity and functionality of tooth-supporting structures. However, PDL repair poses significant challenges due to the complexity of its mechanical microenvironment encompassing hard-soft-hard tissues, with the viscoelastic properties of the PDL being of particular interest. This review delves into the significant role of viscoelastic hydrogels in PDL regeneration, underscoring their utility in simulating biomimetic three-dimensional microenvironments. We review the intricate relationship between PDL and viscoelastic mechanical properties, emphasizing the role of tissue viscoelasticity in maintaining mechanical functionality. Moreover, we summarize the techniques for characterizing PDL's viscoelastic behavior. From a chemical bonding perspective, we explore various crosslinking methods and characteristics of viscoelastic hydrogels, along with engineering strategies to construct viscoelastic cell microenvironments. We present a detailed analysis of the influence of the viscoelastic microenvironment on cellular mechanobiological behavior and fate. Furthermore, we review the applications of diverse viscoelastic hydrogels in PDL repair and address current challenges in the field of viscoelastic tissue repair. Lastly, we propose future directions for the development of innovative hydrogels that will facilitate not only PDL but also systemic ligament tissue repair. STATEMENT OF SIGNIFICANCE.

Construction of hyperelastic model of human periodontal ligament based on collagen fibers distribution.

OBJECTIVE: The human periodontal ligament (PDL) dominated by collagen fibers showed hyperelastic mechanical behavior under orthodontic force. Despite previous researches on the hyperelastic model of PDL, there were certain limitations because of the types of samples and the ignorance of distribution of collagen fibers. Therefore, the aim of this study was to quantify the effect of collagen fibers distribution of human PDL on its hyperelastic behavior. METHODS: A total of 6 human PDL samples of root neck, root middle and root apex were cut from human maxillary central incisor and lateral incisor. The spatial angles of collagen fibers were observed by optical microscope, the hyperelastic model was constructed combined with the observation results. The quasi-static uniaxial tensile tests with displacement load 0.05 mm/min were carried out, and the test data were used to identify the parameters of model. RESULTS: The mechanical behavior of human PDL conformed to the trend of hyperelastic materials, and greatly depended on the spatial angles of internal collagen fibers. The R2 value statistical fit of the constitutive model to the data is excellent (R2 > 0.98). This model could excellently describe the hyperelastic properties of human PDL. SIGNIFICANCE: In this study, we quantitatively described the effect of spatial distribution of collagen fibers on the mechanical properties of human PDL. The accuracy of this model was verified by the uniaxial test data, and the relevant model parameters were acquired, which have certain reference value in subsequent researches on hyperelasticity of human PDL and clinical treatment.

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.

Fibre-guiding biphasic scaffold for perpendicular periodontal ligament attachment.

Periodontal regeneration is characterized by the attachment of oblique periodontal ligament fibres on the tooth root surface. To facilitate periodontal ligament attachment, a fibre-guiding tissue engineered biphasic construct was manufactured by melt electrowriting (MEW) for influencing reproducible cell guidance and tissue orientation. The biphasic scaffold contained fibre-guiding features in the periodontal ligament component comprising of 100 µm spaced channels (100CH), a pore size gradient in the bone component and maintained a highly porous and fully interconnected interface between the compartments. The efficacy of the fibre-guiding channels was assessed in an ectopic periodontal attachment model in immunocompromised rats. This demonstrated an unprecedented and systematic tissue alignment perpendicular to the dentin in the 100CH group, resulting in the close mimicry of native periodontal ligament architecture. In addition, the histology revealed high levels of tissue integration between the two compartments as observed by the perpendicular collagen attachment on the dentin surface, which also extended and infiltrated the scaffold's bone compartment. In conclusion, the 100 µm fibre-guiding scaffold induced a systematic tissue orientation at the dentin-ligament interface, resembling the native periodontium and thus resulting in enhanced alignment mimicking periodontal ligament regeneration. STATEMENT OF SIGNIFICANCE: Periodontitis is a prevalent inflammatory disease affecting a large portion of the adult population and leading to the destruction of the tooth-supporting structures (alveolar bone, periodontal ligament, and cementum). Current surgical treatments are unpredictable and generally result in repair rather than functional regeneration. A key feature of functional regeneration is the re-insertion of the oblique or perpendicularly orientated periodontal ligament fibre in both the alveolar bone and root surface. This study demonstrates that a highly porous scaffold featuring 100 µm width channels manufactured by the stacking of melt electrospun fibres, induced perpendicular alignment and attachment of the neo-ligament onto a dentine surface. The fibre guiding micro-architecture may pave the way for enhanced and more functional regeneration of the periodontium.

Transcriptome Profile of Membrane and Extracellular Matrix Components in Ligament-Fibroblastic Progenitors and Cementoblasts Differentiated from Human Periodontal Ligament Cells.

Ligament-fibroblastic cells and cementoblasts, two types of progenitor cells that differentiate from periodontal ligament stem cells (hPDLSCs), are responsible for the formation of the adhesive tissues in the tooth root. Since one of the factors that determines the fate of stem cell differentiation is the change in the microenvironment of the stem/progenitor cells, this study attempted to compare and analyze the molecular differences in the membrane and ECM of the two progenitor cells. Single cells derived from hPDLSCs were treated with TGF-ß1 and BMP7 to obtain ligament-fibroblastic and cementoblastic cells, respectively. The transcriptome profiles of three independent replicates of each progenitor were evaluated using next-generation sequencing. The representative differentially expressed genes (DEGs) were verified by qRT-PCR, Western blot analysis, and immunohistochemistry. Among a total of 2245 DEGs identified, 142 and 114 DEGs related to ECM and cell membrane molecules were upregulated in ligament-fibroblastic and cementoblast-like cells, respectively. The major types of integrin and cadherin were found to be different between the two progenitor cells. In addition, the representative core proteins for each glycosaminoglycan-specific proteoglycan class were different between the two progenitors. This study provides a detailed understanding of cell-cell and cell-ECM interactions through the specific components of the membrane and ECM for ligament-fibroblastic and cementoblastic differentiation of hPDLSCs.

BMP-2 and asporin expression regulate 5-aza-dC-mediated osteoblast/cementoblast differentiation of periodontal dental ligament mesenchymal progenitor cells.

Periodontal dental ligament (PDL) is composed of heterogeneous population of mesenchymal progenitor cells. The mechanisms that regulate the differentiation of these cells towards osteoblast/cementoblast phenotype are not fully understood. Some studies have demonstrated that is possible to change the pattern of cell differentiation via epigenetic mechanisms. The proposal of this study was to investigate whether 5-aza-2'-deoxycytidine (5-aza-dC) treatment would stimulate the osteoblast/cementoblast differentiation of periodontal ligament mesenchymal progenitor cells (PDL-CD105+ enriched cells), characterized as low osteoblast potential, through bone morphogenetic protein-2 (BMP-2) modulation. PDL-CD105+ cells from a single donor were cloned and characterized in two populations as high osteoblast/cementoblast potential (HOP) and low osteoblast/cementoblast potential (LOP) by mineralization in vitro and expression of osteogenic gene markers, such as runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN), bone morphogenetic protein 2 (BMP-2) and asporin (ASPN). Next, two LOP clones (L1 and L2) were pretreated with 5-aza-dC (10 µM) for 48 h, cultured under osteogenic condition and evaluated for mineralized matrix in vitro, transcription modulation of osteogenic gene markers, methylated and hydroxymethylated DNA levels of BMP-2 and ASPN and intracellular/extracellular expression of BMP-2 protein. LOP clones showed high expression of ASPN transcripts associated with low mRNA levels of BMP-2, RUNX2, ALP, and OCN. 5-aza-dC treatment raised hydroxymethylated DNA levels of BMP-2 and increased the expression of BMP-2 transcripts in both LOP clones. However, BMP-2 protein (intracellular and secreted forms) was detected only in L1 cell clones, in which it was observed an increased expression of osteoblast/cementoblast markers (RUNX2, ALP, OCN) associated with higher mineralization in vitro. In L2 cell clones, 5-aza-dC increased gene expression of ASPN, with no great change in for osteoblast/cementoblast differentiation potential. These data show that 5-aza-dC improves osteoblast/cementoblast differentiation of PDL-CD105+ cells via BMP-2 secretion, and this effect depends on low levels of ASPN expression.

Scleraxis upregulated by transforming growth factor-ß1 signaling inhibits tension-induced osteoblast differentiation of priodontal ligament cells via ephrin A2.

During tooth movement in orthodontic treatment, bone formation and resorption occur on the tension and compression sides of the alveolar bone, respectively. Although the bone formation activity increases in the periodontal ligament (PDL) on the tension side, the PDL itself is not ossified and maintains its homeostasis, indicating that there are negative regulators of bone formation in the PDL. Our previous report suggested that scleraxis (Scx) has an inhibitory effect on ossification of the PDL on the tension side through the suppression of calcified extracellular matrix formation. However, the molecular biological mechanisms of Scx-modulated inhibition of ossification in the tensioned PDL are not fully understood. The aim of the present study is to clarify the inhibitory role of Scx in osteoblast differentiation of PDL cells and its underlying mechanism. Our in vivo experiment using a mouse experimental tooth movement model showed that Scx expression was increased during early response of the PDL to tensile force. Scx knockdown upregulated expression of alkaline phosphatase, an early osteoblast differentiation marker, in the tensile force-loaded PDL cells in vitro. Transforming growth factor (TGF)-ß1-Smad3 signaling in the PDL was activated by tensile force and inhibitors of TGF-ß receptor and Smad3 suppressed the tensile force-induced Scx expression in PDL cells. Tensile force induced ephrin A2 (Efna2) expression in the PDL and Efna2 knockdown upregulated alkaline phosphatase expression in PDL cells under tensile force loading. Scx knockdown eliminated the tensile force-induced Efna2 expression in PDL cells. These findings suggest that the TGF-ß1-Scx-Efna2 axis is a novel molecular mechanism that negatively regulates the tensile force-induced osteoblast differentiation of PDL cells.

Curcumin reduces apoptosis and promotes osteogenesis of human periodontal ligament stem cells under oxidative stress in vitro and in vivo.

AIMS: Human periodontal ligament stem cells (hPDLSCs) tether the teeth to the surrounding bone and are considered as major functional stem cells responsible for regeneration of the alveolar bone and periodontal ligament tissue. However, the outcome of stem cell regenerative therapy is affected by the survival rate and their differentiation potential of transplanted cells. This is primarily because of local oxidative stress and chronic inflammation at the transplantation site. Therefore, our study aimed to explore whether a natural antioxidant, curcumin could increase the tissue regeneration ability of transplanted hPDLSCs. MAIN METHODS: A hydrogen peroxide environment and a rat cranial bone defect model were built to mimic the oxidative stress conditions in vitro and in vivo, respectively. We evaluated the effect of curcumin on oxidative status, apoptosis, mitochondrial function and osteogenic differentiation of H2O2-stimulated hPDLSCs in vitro. We also measured the effect of curcumin on cell viability and bone repair ability of transplanted hPDLSCs in vivo. KEY FINDINGS: Our data showed that curcumin enhanced cell proliferation, reduced the reactive oxygen species (ROS) levels and apoptosis, maintained the standard mitochondrial structure and function, and promoted osteogenic differentiation of H2O2-stimulated hPDLSCs. The extracellular regulated protein kinases 1/2 (Erk1/2) signaling pathway was determined to be involved in the osteogenic differentiation of the H2O2-stimulated hPDLSCs. Moreover, curcumin enhanced the viability and the bone repair ability of hPDLSCs in vivo. SIGNIFICANCE: Curcumin reduced apoptosis and promoted osteogenesis of the hPDLSCs under oxidative stress, and might therefore have a potential clinical use with respect to tissue regeneration.

Estrogen Enhances Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Activating the Wnt/ß-Catenin Signaling Pathway.

OBJECTIVES: This study was designed to investigate the role of the Wnt/ß-catenin signaling pathway in estrogen-enhanced osteogenic differentiation of human peridontal ligament stem cells (hPLSCs). METHODS: The limiting dilution technique was used for cloning and purification of hPLSCs. Flow cytometric analysis of STRO-1, CD146, and CD45 was conducted to identify hPLSCs. The P3 hPDLSCs were divided into 4 groups: Control, 10M E2, 10M E2+100 ng/mL Wnt3a, 10M E2+5 × 10M Xav939. After 7 days of osteogenic induction, qRT-PCR was used to detect the mRNA expression of ß-catenin, CyclinD1, alkaline phosphatase, Runx2, and OCN; Western blot was used to detect the protein expression of ß-catenin, GSK3ß, P-GSK3ß, CyclinD1, Runx2, and OCN; After 1, 3, 5, 7 days of osteogenic induction, the activity of alkaline phosphatase was detected. RESULTS: The authors' results showed that E2 was able to enhance the osteogenic differentiation of hPDLSCs and Wnt/ß-catenin signaling pathway was involved. Wnt3a activated the signaling pathway of Wnt/ß-catenin and further enhanced the osteogenesis of hPDLSCs. Xav939 inhibited the Wnt/ß-catenin signaling pathway in estrogen-mediated environment, but did not obviously inhibit the osteogenic differentiation of hPDLSCs. CONCLUSIONS: E2 enhanced osteogenic differentiation of hPDLSCs through the activation of the Wnt/ß-catenin signaling pathway.

Elevated activity levels do not influence extrinsic fiber attachment morphology on the surface of muscle-attachment sites.

Extrinsic fibers (EFs) are a type of penetrating collagenous fiber, closely related to the periodontal ligament, which help anchor soft tissue into bone. These fibers are associated with muscle attachment sites (entheses). Their size and grouping patterns are thought to be indicative of the loading history of the muscle. EFs are of particular significance in anthropology as potential tools for the reconstruction of behavior from skeletal remains and, specifically, entheses. In this study, we used a mouse model to experimentally test how activity level alters the morphology of EF insertion sites on the bone surface of a fibrocartilaginous enthesis, the biceps brachii insertion. Further, we adapted surface metrological techniques from studies of dental wear to perform automated, quantitative and non-destructive analysis of bone surface histology. Our results show that experimentally increased activity had no significant effect on the quantity or density of EF insertions at the enthesis, nor on the size of those insertions. Although EF presence does indicate muscle attachment, activity did not have an observable effect on EF morphology.

Salvianolic Acid C Attenuates LPS-Induced Inflammation and Apoptosis in Human Periodontal Ligament Stem Cells via Toll-Like Receptors 4 (TLR4)/Nuclear Factor kappa B (NF-κB) Pathway.

BACKGROUND Periodontitis is a chronic inflammatory disease that causes gingival detachment and disintegration of alveolar bone. Salvianolic acid C (SAC) is a polyphenol compound with anti-inflammatory and antioxidant activities that is isolated from Danshen, a traditional Chinese medicine made from the roots of Salvia miltiorrhiza Bunge. The aim of this study was to investigate the mechanisms of underlying its protective effects and its inhibition effect on inflammation and apoptosis in human periodontal ligament stem cells (hPDLSCs). MATERIAL AND METHODS LPS-induced hPDLSCs, as a model mimicking an inflammatory process of periodontitis in vivo, were established to investigate the therapeutic effect of SAC in periodontitis. The inflammatory cytokines secretion and oxidative stress status were measured by use of specific commercial test kits. The hPDLSCs viability was analyzed by Cell Counting Kit-8 assay. The cell apoptosis and cell cycle were assayed with flow cytometry. Expressions levels of proteins involved in apoptosis, osteogenic differentiation, and TLR4/NF-kappaB pathway were evaluated by Western blotting. Alkaline phosphatase (ALP) activity was detected by ALP assay kit and ALP staining. The mineralized nodules formation of hPDLSCs was checked by Alizarin Red S staining. RESULTS Our results showed that LPS induced increased levels of inflammatory cytokines and oxidative stress and mediated the phosphorylation and nuclear translocation of NF­kappaB p65 in hPDLSCs. SAC reversed the abnormal secretion of inflammatory cytokines and inhibited the TLR4/NF­kappaB activation induced by LPS. SAC also upregulated cell viability, ALP activity, and the ability of osteogenic differentiation. The anti-inflammation and TLR4/NF­kappaB inhibition effects of SAC were reversed by TLR4 overexpression. CONCLUSIONS Taken together, our results revealed that SAC effectively attenuates LPS-induced inflammation and apoptosis via the TLR4/NF-kappaB pathway and that SAC is effective in treating periodontitis.

The effects of NELL on corticotomy-assisted tooth movement and osteogenesis in a rat model.

INTRODUCTION: Exogenous Nel-like molecule type 1 (NELL1) represents a potentially attractive clinical treatment option in the orthodontic and other settings because of its osteoinductive and vasculogenic properties. AIMS: To explore effects of NELL1on corticotomy-assisted tooth movement and osteogenesis in a rat model. METHODS: Thirty Sprague-Dawley rats were divided into 6 groups: Control, Sham, Tooth movement only, Vehicle, NELL1-LD (low-dose NELL), NELL1-HD (high-dose NELL). Human recombinant NELL1 protein was applied locally (Groups NELL1-LD and NELL1-HD) into buccal mucosa region of left first upper molar. Then the distance and velocity of tooth movement was measured, animals at 6 weeks after surgery were sacrificed, and was followed by computed tomography and histochemical staining. RESULTS: Both NELL1 groups had higher bone mineral density, greater tooth movement distance and velocity in comparison to the Vehicle group. Proximally and distally, periodontal ligament width was significantly increased in the NELL1-LD and NELL1-HD groups. Decortication enhances remodeling, however, rapid bone formation by high-dose NELL1 may affect bone absorption. CONCLUSION: Appropriate dose of NELL1 can be administrated to reduce the total time for tooth movement, and may shorten the treatment time in select populations.

[Decreased phosphorylation of mitogen activated protein kinase and protein kinase B contribute to the inhibition of osteogenic differentiation mediated by activation of Toll like receptor in human periodontal ligament stem cells].

OBJECTIVE: To investigate the effects of Toll like receptors on the osteogenesis of human pe-riodontal ligament stem cells (hPDLSCs) and probable molecular mechanism. METHODS: Real-time PCR and flow cytometry were applied to test the expression of TLRs in hPDLSCs and the positive cell percentage of TLR. hPDLSCs were cultured in osteogenic medium for 7 to 14 days with different TLR agonists at various concentrations . The effect of different TLR on osteogenic differentiation of hPDLSCs was evaluated by alizarin red S staining, alkaline phosphatase (ALP) staining and ALP activity assay. Western blotting was used to analyze the phosphorylation levels of extracellular regulated protein kinases (ERK), c-Jun N-terminal protein kinase (JNK), P38, AKT and expression of Runx2 an osteogenic related gene after treatment with TLR agonists, compared with the effect of inhibitors of mitogen activated protein kinase (MAPK) or protein kinase B (PKB or AKT) on Runx2 expression of hPDLSCs cultured in osteogenic medium. RESULTS: Higher expressions of TLR1,3,4,6 were found in hPDLSCs through real-time PCR. Positive cell percentage of TLR was determined by flow cytometry and described as TLR1: 2.82%±0.68%; TLR2: 1.26%±0.09%; TLR3: 13.23%±2.05%; TLR4: 3.64%±0.79%; TLR6: 3.21%±1.64%, whose tendency was comparable to their mRNA expression in hPDLSCs. Most TLR ligands had no effect on the ALP staining, activity and mineralization of hPDLSCs at lower concentration except for 0.1 mg/L PolyI:C could induce the osteogenic ability of hPDLSCs. On the contrary, Higher concentration of TLR ligands (PolyI:C: 10 mg/L, LPS: 10 mg/L , Pam3CSK4: 1 mg/L, FSL-1: 50 µg/L) had obviously inhibitory effect on osteogenic differentiation of hPDLSCs. Activation of TLR using higher concentration of TLR ligands could downregulate the phosphorylation levels of ERK, P38, JNK and AKT, and also reduced the expression of Runx2, compared with the untreated control. The inhibitors of MAPK (U0126, SP600125,SB203580) and inhibitor of AKT (perifosine) could also inhibit Runx2 expression. CONCLUSION: Higher concentration of TLR ligands could inhibit osteogenic differentiation of hPDLSCs. This inhibitory effect seemed to be related to decreased phosphorylation of MAPK and AKT.

Decreased phosphorylation of mitogen activated protein kinase and protein kinase B contribute to the inhibition of osteogenic differentiation mediated by activation of Toll like receptor in human periodontal ligament stem cells / 北京大学学报(医学版)

OBJECTIVE@#To investigate the effects of Toll like receptors on the osteogenesis of human pe-riodontal ligament stem cells (hPDLSCs) and probable molecular mechanism.@*METHODS@#Real-time PCR and flow cytometry were applied to test the expression of TLRs in hPDLSCs and the positive cell percentage of TLR. hPDLSCs were cultured in osteogenic medium for 7 to 14 days with different TLR agonists at various concentrations . The effect of different TLR on osteogenic differentiation of hPDLSCs was evaluated by alizarin red S staining, alkaline phosphatase (ALP) staining and ALP activity assay. Western blotting was used to analyze the phosphorylation levels of extracellular regulated protein kinases (ERK), c-Jun N-terminal protein kinase (JNK), P38, AKT and expression of Runx2 an osteogenic related gene after treatment with TLR agonists, compared with the effect of inhibitors of mitogen activated protein kinase (MAPK) or protein kinase B (PKB or AKT) on Runx2 expression of hPDLSCs cultured in osteogenic medium.@*RESULTS@#Higher expressions of TLR1,3,4,6 were found in hPDLSCs through real-time PCR. Positive cell percentage of TLR was determined by flow cytometry and described as TLR1: 2.82%±0.68%; TLR2: 1.26%±0.09%; TLR3: 13.23%±2.05%; TLR4: 3.64%±0.79%; TLR6: 3.21%±1.64%, whose tendency was comparable to their mRNA expression in hPDLSCs. Most TLR ligands had no effect on the ALP staining, activity and mineralization of hPDLSCs at lower concentration except for 0.1 mg/L PolyI:C could induce the osteogenic ability of hPDLSCs. On the contrary, Higher concentration of TLR ligands (PolyI:C: 10 mg/L, LPS: 10 mg/L , Pam3CSK4: 1 mg/L, FSL-1: 50 μg/L) had obviously inhibitory effect on osteogenic differentiation of hPDLSCs. Activation of TLR using higher concentration of TLR ligands could downregulate the phosphorylation levels of ERK, P38, JNK and AKT, and also reduced the expression of Runx2, compared with the untreated control. The inhibitors of MAPK (U0126, SP600125,SB203580) and inhibitor of AKT (perifosine) could also inhibit Runx2 expression.@*CONCLUSION@#Higher concentration of TLR ligands could inhibit osteogenic differentiation of hPDLSCs. This inhibitory effect seemed to be related to decreased phosphorylation of MAPK and AKT.

Developmental changes in primary cilia in the mouse tooth germ and oral cavity.

The primary cilium, a sensory apparatus, functions as both a chemical and mechanical sensor to receive environmental stimuli. The present study focused on the primary cilia in the epithelialmesenchymal interaction during tooth development. We examined the localization and direction of projection of primary cilia in the tooth germ and oral cavity of mice by immunohistochemical observation. Adenylyl cyclase 3 (ACIII)-immunolabeled cilia were visible in the inner/outer enamel epithelium of molars at the fetal stage and then conspicuously developed in the odontoblast layer postnatally. The primary cilia in ameloblasts and odontoblasts-shown by the double staining of acetylated tubulin and γ-tubulin-were regularly arranged from postnatal Day12, projecting apart from each other. The periodontal ligament possessed ACIII-positive cilia, which gathered on both sides of the dentin/cement and alveolar bone in postnatal days. In the oral cavity, numerous long primary cilia immunoreactive for ACIII were condensed at subepithelial stromal cells in the oral processes in fetuses, while postnatally a small number of short cilia were dispersed throughout the stroma of the oral cavity. These findings suggest that the primary cilia showing stage- and regionspecific morphology are involved in the epithelial-mesenchymal interaction during tooth development via mechano- and/or chemoreception for growth factors.

Review of common conditions associated with periodontal ligament widening

PURPOSE: The aim of this article is to review a group of lesions associated with periodontal ligament (PDL) widening. MATERIALS AND METHODS: An electronic search was performed using specialized databases such as Google Scholar, PubMed, PubMed Central, Science Direct, and Scopus to find relevant studies by using keywords such as “periodontium”, “periodontal ligament”, “periodontal ligament space”, “widened periodontal ligament”, and “periodontal ligament widening”. RESULTS: Out of nearly 200 articles, about 60 were broadly relevant to the topic. Ultimately, 47 articles closely related to the topic of interest were reviewed. When the relevant data were compiled, the following 10 entities were identified: occlusal/orthodontic trauma, periodontal disease/periodontitis, pulpo-periapical lesions, osteosarcoma, chondrosarcoma, non-Hodgkin lymphoma, progressive systemic sclerosis, radiation-induced bone defect, bisphosphonate-related osteonecrosis, and osteomyelitis. CONCLUSION: Although PDL widening may be encountered by many dentists during their routine daily procedures, the clinician should consider some serious related conditions as well.

Factors that affect the bite force measurement

Mastication is the process to help digestion by chewing or grinding food. Masticatory system consists of maxilla, mandible, temporomandibular joints, ligaments, dentitions, and musculatures. Assessing the bite force can be one of the methods to estimate the masticatory system. Bite force is influenced by facial morphology, age, sex, periodontal status, temporomandibular joint disorder and dental condition, and so forth. In general, higher maximum bite force is seen in those who have a square-shaped face and in male rather than female. In addition, bite force tends to be increased by age 20, maintained constantly until age 40 - 50, and then decreased. Periodontal disease is known as a causative factor for decreased bite force while temporomandibular disorder (TMD) remains controversial as to whether it affects the force. The status of teeth is considered as an important factor to determine the maximum bite force.

Mesenchymal stem cells of different origin: Comparative evaluation of proliferative capacity, telomere length and pluripotency marker expression.

AIMS: In vitro expansion changes replication and differentiation capacity of mesenchymal stem cells (MSCs), increasing challenges and risks, while limiting the sufficient number of MSCs required for cytotherapy. Here, we characterized and compared proliferation, differentiation, telomere length and pluripotency marker expression in MSCs of various origins. MAIN METHODS: Immunophenotyping, proliferation and differentiation assays were performed. Pluripotency marker (Nanog, Oct-4, SOX-2, SSEA-4) expression was determined by immunofluorescence. Quantitative PCR was performed for relative telomere length (RTL) analyses, while expression of relevant genes for pluripotency markers, differentiation state (Cbfa1, human placental alkaline phosphatase, peroxisome proliferator activated receptor, Sox9 and Collagen II a1), and telomerase reverse transcriptase (hTERT) was determined by semiquantitative RT-PCR. KEY FINDINGS: Peripheral blood MSCs (PB-MSCs) and umbilical cord MSCs (UC-MSCs) showed the highest, while periodontal ligament MSCs (PDL-MSCs) and adipose tissue MSCs (AT-MSCs) the lowest values of both the replication potential and RTL. Although MSCs from exfoliated deciduous teeth (SHEDs), PDL-MSCs and AT-MSCs showed higher mRNA expression of pluripotency markers, all MSCs expressed pluripotency marker proteins. SHEDs and PDL-MSCs showed prominent capacity for osteogenesis, PB-MSCs and UC-MSCs showed strengthened adipogenic differentiation potential, while AT-MSCs displayed similar differentiation into both lines. SIGNIFICANCE: The MSCs populations derived from different sources, although displaying similar phenotype, exhibited high degree of variability regarding biological properties related to their self-renewal and differentiation capacity. These data indicate that for more accurate use in cell therapy, individualities of MSCs isolated from different tissues should be identified and taken into consideration when planning their use in clinical protocols.

Standardized in vivo model for studying novel regenerative approaches for multitissue bone-ligament interfaces.

The regeneration of the original structure and function of bone-ligament interfaces remains a major challenge in biomedical research. A preclinical model that maintains physiologic mechanical loads and controls for other external factors, such as microbial influence, is of great value for testing novel regenerative materials, provided that studies are performed by highly trained researchers with proper regard for animal welfare. The tooth root fenestration preclinical model is an ideal tool for hard tissue evaluation by micro-computed tomography, histological techniques and RNA analyses. The procedure starts with an extraoral incision lateral to the mandible and reflection of the masseter muscle. Superficial lateral mandibular bone is removed with standardized dimensions to expose the roots of the teeth and to eliminate periodontal ligament and cementum to expose the tooth dentin. The testing material can subsequently be applied to the defect and the flap can be repositioned and secured back in place. At specific time points, samples are collected and processed according to the subsequent analyses to be performed, which can include descriptive histology, histomorphometry, immunostaining, 3D bone imaging, electron microscopy, gene expression analyses and safety assessments.