Establishing legal threshold of 18-years based on the assessment of mandibular molars using three different methods - An observational study.

BACKGROUND: The study evaluates the feasibility of employing the radiographic visibility of the root pulp and periodontal ligament in mandibular molars for age estimation, particularly focusing on the 18 years of age threshold. This study additionally investigates the potential of root canal width reduction in mandibular molars, as a reliable method for forensic age estimation in living individuals. MATERIALS AND METHODS: A cross-sectional study was conducted to assess the radiographic visibility of the root pulp (RPV) and the root canal width (RCW) of mandibular first, second, and third molars along with the radiographic visibility of the periodontal ligament (PLV) of mandibular third molars, in a sample of 403 individuals aged 16-25 years (220 males and 183 females). Data regarding age for different stages of RPV and PLV and various types of RCW were recorded and observed for sex-based differences. Results obtained were tabulated and descriptive statistics were applied to summarise the findings. RESULTS: Individuals over 18 years old were classified with higher accuracy using stage 3 of the RPV scoring system in all mandibular molars (first, second, and third) compared to stage 2, which was also effective for the second and third molars. This result held regardless of sex and side examined. Additionally, root canal width (RCW) assessment demonstrated that individuals with RCW types A, B, and C were more likely to be under 18 years old in both sexes. Conversely, individuals with RCW type U on the right side for males and the left side for females exhibited a higher likelihood of being above 18 years old. CONCLUSION: The study suggests that the assessment of mandibular molars could potentially serve as an auxiliary tool in age estimation methods, particularly for approximating individuals around the 18 years of age threshold. Further investigation is warranted to explore the potential application of root canal width measurements in forensic age estimation.

Stress induced on permanent mandible first molar and space maintainer under normal masticatory forces: a finite element study.

Background: The band and loop space maintainer is used to maintain the missing space of deciduous molars which are lost early. When the second deciduous molar is lost prematurely, the stress on the first permanent molar during different degrees of development may vary when it is the abutment. The design and use of the space maintainer may also lead to damage of the loop. The purpose of this article is to use the finite element method to study the stress on the first permanent molar and the loop with or without occlusal contact, with the first permanent molar of four different degrees of development serving as the abutment. We aimed to guide the clinical design and use of the space maintainer. Methods: We developed finite element models of the mandibular first permanent molar and the band and loop space maintainer, and simulated alveolar bone, periodontal ligament (PDL), enamel and dentin. The four developmental stages were 1/2 (I), 2/3 (II), 3/4 (III) and full development (IV). Ansys Workbench was used to analyze the effects of root development and occlusal contact between the loop and the opposite jaw on abutment teeth and the loop. Abutment teeth were statically loaded vertically and obliquely with a force of 70 N. The loop was statically loaded vertically with a force of 14 N. The stress on all structures and the displacement trends of the loop were calculated. Results: The stress on enamel, dentin, PDL and alveolar bone were similar, and the concentration was consistent. But if there was occlusal contact, the loop produced maximum displacement at the near middle edge of contact with the anterior teeth. When the loop was in occlusal contact with the opposing occlusal tooth, the peak value of the equivalent stress on the space maintainer under vertical load was: group I > group IV > group III > group II, and the maximum principal stress peak change was: group I > group III > group II > group IV. The change of the equivalent stress peak value of the loop under oblique load was: group I > group III > group IV > group II, and the maximum principal stress peak change was: group III > group I > group II > group IV. When the loop was not in occlusal contact with the opposing occlusal tooth, the peak value of the equivalent stress on the space maintainer under vertical load was: group IV > group I > group II > group III, and the maximum principal stress peak change was: group IV > group I > group II > group III. The change of the equivalent stress peak value of the space maintainer under oblique load was: group I > group IV > group II > group III, and the maximum principal stress peak change was: group I > group IV > group II > group III. Conclusions: Our results suggested that whenever possible, choosing the teeth with nearly complete root development as the abutment of the space maintainer is advisable. The design and use of the band and loop space maintainer should avoid occlusal contact with the occlusal teeth to prevent deformation of the loop.

LiCl-induced immunomodulatory periodontal regeneration via the activation of the Wnt/ß-catenin signaling pathway.

BACKGROUND: Growing evidence suggests that excessive inflammation hampers the regenerative capacity of periodontal ligament cells (PDLCs) and that activation of the Wnt/ß-catenin pathway is crucial in suppressing immune dysregulation. OBJECTIVE: This study aimed to establish the role of the Wnt/ß-catenin in regulating the immune microenvironment and its subsequent impact on periodontal regeneration. METHODS: Lithium chloride (LiCl, Wnt activator) was administered daily into the standard periodontal defects created in 12-week-old Lewis rats. Harvested at 1-week and 2-week post-surgery, samples were then subjected to histological and immunohistochemical evaluation of macrophage distribution and phenotype (pro-inflammatory M1 and anti-inflammatory M2). A murine macrophage cell line, RAW 264.7, was stimulated with LiCl to activate Wnt/ß-catenin. Following treatment with the conditioned medium derived from the LiCl-activated macrophages, the expression of bone- and cementum-related markers of the PDLCs was determined. The involvement of Wnt/ß-catenin in the immunoregulation and autophagic activity was further investigated with the addition of cardamonin, a commercially available Wnt inhibitor. RESULTS: A significantly increased number of macrophages were detected around the defects during early healing upon receiving the Wnt/ß-catenin signaling cue. The defect sites in week 2 exhibited fewer M1 and more M2 macrophages along with an enhanced regeneration of alveolar bone and cementum in the Wnt/ß-catenin activation group. LiCl-induced immunomodulatory effect was accompanied with the activation Wnt/ß-catenin signaling, which was suppressed in the presence of Wnt inhibitor. Exposure to LiCl could induce autophagy in a dose-dependent manner, thus maintaining macrophages in a regulatory state. The expression level of bone- and cementum-related markers was significantly elevated in PDLCs stimulated with LiCl-activated macrophages. CONCLUSION: The application of Wnt activator LiCl facilitates the recruitment of macrophages to defect sites and regulates their phenotypic switching in favor of periodontal regeneration. Suppression of Wnt/ß-catenin pathway could attenuate the LiCl-induced immunomodulatory effect. Taken together, the Wnt/ß-catenin pathway may be targeted for therapeutic interventions in periodontal diseases.

Axin2-expressing cells in the periodontal ligament are regulated by bone morphogenetic protein signalling and play a pivotal role in periodontium development.

AIM: To date, controversies still exist regarding the exact cellular origin and regulatory mechanisms of periodontium development, which hinders efforts to achieve ideal periodontal tissue regeneration. Axin2-expressing cells in the periodontal ligament (PDL) have been shown to be a novel progenitor cell population that is essential for periodontal homeostasis. In the current study, we aimed to elucidate the regulatory role of bone morphogenetic protein receptor type 1A (BMPR1A)-mediated BMP signalling in Axin2-expressing cells during periodontium development. MATERIALS AND METHODS: Two strains of Axin2 gene reporter mice, Axin2lacZ/+ and Axin2CreERT2/+ ; R26RtdTomato/+ mice, were used. We next generated Axin2CreERT2/+ ; R26RDTA/+ ; R26RtdTomato/+ mice to genetically ablate of Axin2-lineage cells. Axin2CreERT2/+ ; Bmpr1afl/fl ; R26RtdTomato/+ mice were established to conditionally knock out Bmpr1a in Axin2-lineage cells. Multiple approaches, including micro-computed tomography, calcein green, and alizarin red double-labelling, scanning electron microscopy, and histological and immunostaining assays, were used to analyse periodontal phenotypes and molecular mechanisms. RESULTS: X-gal staining revealed that Axin2-expressing cells in the PDL were mainly distributed along the alveolar bone and cementum surface. Cell lineage tracing and cell ablation assays further demonstrated the indispensable role of Axin2-expressing cells in periodontium development. Next, we found that conditional knockout of Bmpr1a in Axin2-lineage cells led to periodontal defects, which were characterized by alveolar bone loss, impaired cementogenesis, and abnormal Sharpey's fibres. CONCLUSIONS: Our findings suggest that Axin2-expressing cells in the PDL are essential for periodontium development, which is regulated by BMP signalling.

Application of cryopreservation to tooth germ transplantation for root development and tooth eruption.

We cryopreserved mouse tooth germs with widely open cervical margins of the enamel organ to overcome difficulties in cryoprotectant permeation and tested their efficacy by transplanting them into recipient mice. The upper right first molar germs of 8-day-old donor mice were extracted and categorized into the following four groups according to cryopreservation time: no cryopreservation, 1 week, 1 month, and 3 months. The donor tooth germs were transplanted into the upper right first molar germ sockets of the 8-day-old recipient mice. The upper left first molars of the recipient mice were used as controls. The outcome of the transplantation was assessed at 1, 2, and 3 weeks after transplantation. Stereomicroscopic evaluation revealed that most of the transplanted teeth erupted by 3 weeks after transplantation. Micro-computed tomography analysis revealed root elongation in the transplanted groups as well as in the controls. There was no significant difference between the cryopreserved and non-cryopreserved transplanted teeth, but the roots of the cryopreserved teeth were significantly shorter than those of the control teeth. Histological examination revealed root and periodontal ligament formations in all the transplanted groups. These results suggest that the transplantation of cryopreserved tooth germs facilitates subsequent root elongation and tooth eruption.

Zbp1-positive cells are osteogenic progenitors in periodontal ligament.

Periodontal ligament (PDL) possesses a stem/progenitor population to maintain the homeostasis of periodontal tissue. However, transcription factors that regulate this population have not yet been identified. Thus, we aimed to identify a molecule related to the osteogenic differentiation of PDL progenitors using a single cell-based strategy in this study. We first devised a new protocol to isolate PDL cells from the surface of adult murine molars and established 35 new single cell-derived clones from the PDL explant. Among these clones, six clones with high (high clones, n = 3) and low (low clones, n = 3) osteogenic potential were selected. Despite a clear difference in the osteogenic potential of these clones, no significant differences in their cell morphology, progenitor cell marker expression, alkaline phosphatase activity, proliferation rate, and differentiation-related gene and protein expression were observed. RNA-seq analysis of these clones revealed that Z-DNA binding protein-1 (Zbp1) was significantly expressed in the high osteogenic clones, indicating that Zbp1 could be a possible marker and regulator of the osteogenic differentiation of PDL progenitor cells. Zbp1-positive cells were distributed sparsely throughout the PDL. In vitro Zbp1 expression in the PDL clones remained at a high level during osteogenic differentiation. The CRISPR/Cas9 mediated Zbp1 knockout in the high clones resulted in a delay in cell differentiation. On the other hand, Zbp1 overexpression in the low clones promoted cell differentiation. These findings suggested that Zbp1 marked the PDL progenitors with high osteogenic potential and promoted their osteogenic differentiation. Clarifying the mechanism of differentiation of PDL cells by Zbp1 and other factors in future studies will facilitate a better understanding of periodontal tissue homeostasis and repair, possibly leading to the development of novel therapeutic measures.

A Novel Volume-Stable Collagen Matrix Induces Changes in the Behavior of Primary Human Oral Fibroblasts, Periodontal Ligament, and Endothelial Cells.

The aim of the present study was to investigate the influence of a novel volume-stable collagen matrix (vCM) on early wound healing events including cellular migration and adhesion, protein adsorption and release, and the dynamics of the hemostatic system. For this purpose, we utilized transwell migration and crystal violet adhesion assays, ELISAs for quantification of adsorbed and released from the matrix growth factors, and qRT-PCR for quantification of gene expression in cells grown on the matrix. Our results demonstrated that primary human oral fibroblasts, periodontal ligament, and endothelial cells exhibited increased migration toward vCM compared to control cells that migrated in the absence of the matrix. Cellular adhesive properties on vCM were significantly increased compared to controls. Growth factors TGF-ß1, PDGF-BB, FGF-2, and GDF-5 were adsorbed on vCM with great efficiency and continuously delivered in the medium after an initial burst release within hours. We observed statistically significant upregulation of genes encoding the antifibrinolytic thrombomodulin, plasminogen activator inhibitor type 1, thrombospondin 1, and thromboplastin, as well as strong downregulation of genes encoding the profibrinolytic tissue plasminogen activator, urokinase-type plasminogen activator, its receptor, and the matrix metalloproteinase 14 in cells grown on vCM. As a general trend, the stimulatory effect of the vCM on the expression of antifibrinolytic genes was synergistically enhanced by TGF-ß1, PDGF-BB, or FGF-2, whereas the strong inhibitory effect of the vCM on the expression of profibrinolytic genes was reversed by PDGF-BB, FGF-2, or GDF-5. Taken together, our data strongly support the effect of the novel vCM on fibrin clot stabilization and coagulation/fibrinolysis equilibrium, thus facilitating progression to the next stages of the soft tissue healing process.

1α,25-dihydroxyvitamin D3 promotes early osteogenic differentiation of PDLSCs and a 12-year follow-up case of early-onset vitamin D deficiency periodontitis.

Periodontitis is a chronic periodontal disease that contributes to tooth loss. In recent years, many animal studies have reported that vitamin D (VitD) deficiency results in chronic periodontitis. However, no studies have reported cases of early-onset periodontitis with VitD deficiency. This study reports a 5-year-old male patient with early-onset periodontitis, VitD deficiency and VitD receptor (VDR) mutation. The patient was treated with VitD and calcium, and received systematic periodontal treatment. During the 12-year treatment, the periodontal conditions of this patient were stable. Our in vitro study found that VitD could promote the expression of alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), bone morphogenetic protein 2 (BMP2), bone gamma-carboxyglutamate protein (BGLAP), and VDR in the early osteogenic differentiation of periodontal ligament stem cells (PDLSCs). Meanwhile, VitD could downregulate mRNA expression levels of Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin-1ß (IL-1ß) and protein levels of IL-6 in the tumor necrosis factor-α (TNF-α) -induced inflammation of PDLSCs. Therefore, sufficient VitD supply can be a potential treatment for VitD deficiency induced early-onset periodontitis.

Molecular Mechanisms of Periodontal Disease.

Periodontal disease, one of the most prevalent human infectious diseases, is characterized by chronic inflammatory tissue destruction of the alveolar bone and the connective tissues supporting the tooth [...].

Vitamin C alleviates the senescence of periodontal ligament stem cells through inhibition of Notch3 during long-term culture.

Periodontal ligament stem cells (PDLSCs), as potential "seed cells" for periodontal tissue repair and regeneration, require to be expanded in vitro for a large scale. Senescence of PDLSCs occurred during long-term culture may compromise the therapeutic effects of PDLSCs. Medium supplements may be useful in antisenescence. However, the effects and mechanisms of vitamin C (Vc) treatment on PDLSCs during long-term culture are still unclear. In this study, we identified that Vc-treated PDLSCs cells maintained a slender morphology, higher growth rate and migration capacity, stemness, and osteogenic differentiation capability during a long-term culture. Moreover, we also identified that Notch3 was significantly upregulated during the cell senescence, and Vc treatment alleviated the senescence of PDLSCs through inhibition of Notch3 during long-term culture. In summary, Vc treatment suppressed PDLSCs senescence by reducing the expression of Notch3 and might be a simple and useful strategy to inhibit cellular senescence during the cell long-term culture.

Tooth-Supporting Hard Tissue Regeneration Using Biopolymeric Material Fabrication Strategies.

The mineralized tissues (alveolar bone and cementum) are the major components of periodontal tissues and play a critical role to anchor periodontal ligament (PDL) to tooth-root surfaces. The integrated multiple tissues could generate biological or physiological responses to transmitted biomechanical forces by mastication or occlusion. However, due to periodontitis or traumatic injuries, affect destruction or progressive damage of periodontal hard tissues including PDL could be affected and consequently lead to tooth loss. Conventional tissue engineering approaches have been developed to regenerate or repair periodontium but, engineered periodontal tissue formation is still challenging because there are still limitations to control spatial compartmentalization for individual tissues and provide optimal 3D constructs for tooth-supporting tissue regeneration and maturation. Here, we present the recently developed strategies to induce osteogenesis and cementogenesis by the fabrication of 3D architectures or the chemical modifications of biopolymeric materials. These techniques in tooth-supporting hard tissue engineering are highly promising to promote the periodontal regeneration and advance the interfacial tissue formation for tissue integrations of PDL fibrous connective tissue bundles (alveolar bone-to-PDL or PDL-to-cementum) for functioning restorations of the periodontal complex.

Biomimetic collagen-sodium alginate-titanium oxide (TiO2) 3D matrix supports differentiated periodontal ligament fibroblasts growth for periodontal tissue regeneration.

Fabrication of biomaterial that mimics a suitable biological microenvironment is still a major challenge in the field of periodontitis treatment. Hence, in this report, we presented for the first time the fabrication of a novel biomaterial 3D matrix using collagen combined with sodium alginate and titanium oxide (TiO2) to recreate the in-vivo microenvironment and to act as a platform for the culture of human periodontal ligament fibroblasts (HPLF) towards osteogenic differentiation. Further, we explored the changes of differentiated and undifferentiated HPLF cells in morphological and cellular level comparing 2D (standard culture plates) and 3D cell culture systems. The physicochemical parameters such as stiffness, water binding capacity, swelling, shrinkage factor, porosity and in-vitro biodegradation show the suitability of this 3D matrix to act as a scaffold for in-vitro periodontal regeneration. The differentiated HPLF cells in the 3D matrix secrete high levels of collagen, osteocalcin, alkaline phosphatase compared to the conventional 2D cell culture. Morphological analysis revealed the structural changes of HPLF cells before and after differentiation in 2D and 3D cell culture. In this study, we find that the level of osteocalcin secretion towards osteogenic differentiation was enhanced in HPLF cells by 3D matrix as compared with 2D cell culture, which demonstrates the osteogenic stimulatory potential of 3D matrix. Overall, the fabricated 3D matrix supports the differentiation of the HPLF cells into osteoblastogenic lineage cells in-vitro and is a promising approach for further investigations in in-vivo treatment of periodontal tissue impairment.

Survival and complication analyses of avulsed and replanted permanent teeth.

This retrospective clinical study investigated the survival probability of avulsed and replanted permanent teeth in relation to functional healing, replacement and inflammatory resorption. The explorative data analysis included data from 36 patients and 49 replanted permanent teeth with a minimum observation time of 60 days; the patients were generally treated according to the current guidelines of the International Association of Dental Traumatology at the university hospital in Munich, Germany, between 2004 and 2017. The mean observation period was 3.5 years. Functional healing was observed in 26.5% (N = 13/49) of the included avulsion cases. In comparison, replacement resorption affected 51.0% (N = 25/49) of the replanted teeth, of which 24.0% (N = 6/25) were lost over the course of years (mean, 6.1 years). In contrast, inflammatory resorption resulted in the early loss of all replanted teeth (mean, 1.7 years) and affected 22.5% (N = 11/49) of all the monitored teeth. Therefore, it can be concluded that tooth avulsion remains a severe dental injury with an unpredictable prognosis. This topic demands further fundamental research aiming to maintain and/or regenerate the periodontal ligament after tooth avulsion, particularly in association with non-physiological tooth rescue.

Carboxy-Terminal Cementum Protein 1-Derived Peptide 4 (cemp1-p4) Promotes Mineralization through wnt/ß-catenin Signaling in Human Oral Mucosa Stem Cells.

Human cementum protein 1 (CEMP1) is known to induce cementoblast and osteoblast differentiation and alkaline phosphatase (ALP) activity in human periodontal ligament-derived cells in vitro and promotes bone regeneration in vivo. CEMP1's secondary structure analysis shows that it has a random-coiled structure and is considered an Intrinsic Disordered Protein (IDP). CEMP1's short peptide sequences mimic the biological capabilities of CEMP1. However, the role and mechanisms of CEMP1's C-terminal-derived synthetic peptide (CEMP1-p4) in the canonical Wnt/ß-catenin signaling pathway are yet to be described. Here we report that CEMP1-p4 promotes proliferation and differentiation of Human Oral Mucosa Stem Cells (HOMSCs) by activating the Wnt/ß-catenin pathway. CEMP1-p4 stimulation upregulated the expression of ß-catenin and glycogen synthase kinase 3 beta (GSK-3B) and activated the transcription factors TCF1/7 and Lymphoid Enhancer binding Factor 1 (LEF1) at the mRNA and protein levels. We found translocation of ß-catenin to the nucleus in CEMP1-p4-treated cultures. The peptide also penetrates the cell membrane and aggregates around the cell nucleus. Analysis of CEMP1-p4 secondary structure revealed that it has a random-coiled structure. Its biological activities included the induction to nucleate hydroxyapatite crystals. In CEMP1-p4-treated HOMSCs, ALP activity and calcium deposits increased. Expression of Osterix (OSX), Runt-related transcription factor 2 (RUNX2), Integrin binding sialoproptein (IBSP) and osteocalcin (OCN) were upregulated. Altogether, these data show that CEMP1-p4 plays a direct role in the differentiation of HOMSCs to a "mineralizing-like" phenotype by activating the ß-catenin signaling cascade.

Periodontal ligament cells regulate osteogenesis via miR-299-5p in mesenchymal stem cells.

BACKGROUND: The periodontal ligament contains periodontal ligament cells, which is a heterogeneous cell population, and includes progenitor cells that can differentiate into osteoblasts/cementoblasts. Mesenchymal stem cells (MSCs) can differentiate into various cells and can be used for periodontal regenerative therapy. Therefore, transplanted MSCs can be affected by humoral factors from periodontal ligament cells via the transcription factors or microRNAs (miRNAs) of MSCs. In addition, periostin (POSTN) is secreted from HPL cells and can regulate periodontal regeneration and homeostasis. To clarify the regulatory mechanism of humoral factors from periodontal ligament cells, we attempted to identify key genes, specifically microRNAs, involved in this process. METHODS: Human MSCs (hMSCs) were indirectly co-cultured with human periodontal ligament cells (HPL cells) and then evaluated for osteogenesis, undifferentiated MSCs markers, and miRNA profiles. Furthermore, hMSCs were indirectly co-cultured with HPL cells in the presence of anti-POSTN monoclonal antibody (anti-POSTN Ab) to block the effect of POSTN from HPL cells, and then evaluated for osteogenesis or undifferentiated MSC markers. Moreover, hMSCs showed alterations in miRNA expression or cultured with HPL were challenged with POSTN during osteogenesis, and cells were evaluated for osteogenesis or undifferentiated MSC markers. RESULTS: hMSCs co-cultured with HPL cells showed suppressed osteogenesis and characteristic expression of SOX11, an undifferentiated MSC marker, as well as miR-299-5p. Overexpression of miR-299-5p regulated osteogenesis and SOX11 expression as observed with indirect co-culture with HPL cells. Furthermore, MSCs co-cultured with HPL cells were recovered from the suppression of osteogenesis and SOX11 mRNA expression by anti-POSTN Ab. However, POSTN induced miR-299-5p and SOX11 expression, and enhanced osteogenesis. CONCLUSION: Humoral factors from HPL cells suppressed osteogenesis in hMSCs. The suppressive effect was mediated by miR-299-5p and SOX11 in hMSCs.

Dental age estimation based on the radiographic visibility of the periodontal ligament in the lower third molars: application of a new stage classification.

The purpose of the present study was to test whether a new stage classification based on radiographic visibility of the periodontal ligament in lower third molars in a Chinese population can be used for the 18- and 21-year thresholds. A total of 1300 orthopantomograms, including equal numbers of northern Chinese males and females evenly distributed between the ages of 15 and 40 years, were analyzed. The stages were defined according to the visibility of periodontal ligament for the outer parts of lower third molar roots because the visibility status of the periodontal ligament between the roots of lower third molars is none valuable in many Chinese individuals. Stage 0 was first achieved at the age of 17.05 years in males and 17.46 years in females. The earliest appearance of stage 1 was 17.47 years in males and 17.86 years in females. Stage 2 was first observed in males at the age of 21.43 years and in females at the age of 21.96 years. The onset of stage 3 was first observed at the age of 25.83 years in males and 23.14 years in females. Compared with the stage classification of Olze et al., which also considers the mesial parts of the roots, the number of assessable cases could be significantly increased. Therefore, our novel approach is effective for age estimation in the Chinese population.

Short Peptides Protect Oral Stem Cells from Ageing.

Primary stem cells, after several cell divisions, enter into a senescence state, that is characterized by alterations to spindle-shape typical morphology. This concern is one of the main problems in the use of human mesenchymal stem cells (hMSCs) in clinical applications which demand cells in large numbers. Short peptides had geroprotective properties and stimulated stem cell differentiation. The aim of the study is to demonstrate the role of AEDG and KED peptides in maintaining oral hMSCs morphology and functions over long-term expansion. 2 types of hMSCs were investigated: human periodontal ligament stem cells (hPLSCs) and human gingival mesenchymal stem cells (hGMSCs). Cells at the 25th passage were divided into 3 groups: 1 - control (without adding peptide), 2 - treated with AEDG peptide, 3 - treated with KED peptide. Cell cultures were analyzed by an immunofluorescence method and RT-PCR on the p16 and p21 senescence markers expression. AEDG peptide decreased p16 and p21 mRNA expression by 1.56-2.44 times in comparison with the control group. KED peptide decreased p16 and p21 mRNA expression by 1.82-3.23 times in comparison with the control group. These results were confirmed by immunofluorescent visualization. AEDG and KED peptides could be used as supplementary substances in a culture medium to delay the expression of senescence markers in long term stem cell cultivation in order to promote the large-scale in vitro expansion necessarily required for stem cell therapy clinical application. The data obtained confirm the geroprotective effect of AEDG and KED peptide, which was shown early in animal and cells models.

Periostin reverses high glucose-inhibited osteogenesis of periodontal ligament stem cells via AKT pathway.

AIMS: Diabetes mellitus leads to impaired osteogenic differentiation and alveolar bone absorption. Periostin (POSTN) is important for bone and tooth maintenance. This study aims to elucidate the expression of POSTN in high glucose and the effects of both high glucose and POSTN on osteogenesis in hPDLSCs, as well as the underlying mechanism. MAIN METHODS: Cells were incubated with glucose under physiological (5.5 mM normal glucose) or diabetic (30 mM high glucose) conditions in the presence or absence of recombinant human POSTN (rPOSTN). Cell migration was assessed by a scratch assay. Reactive oxygen species (ROS) was used to assess HG-induced oxidative damage. Osteogenesis was evaluated by alkaline phosphatase (ALP) activity and ALP staining, Alizarin Red staining (ARS), as well osteogenic related genes and proteins. KEY FINDINGS: POSTN expression was inhibited during a long-term culture with HG. HG diminished the migration and osteogenesis of hPDLSCs as indicated by decreases in ALP activity and ALP staining, ARS and expression of COL I, RUNX2, OSX, OPN and OCN, but an increase in reactive oxygen species overproduction. All of which were reversed by addition of rPOSTN. POSTN knockdown suppressed migration and osteogenesis of hPDLSCs. Moreover, HG inhibited activation of AKT, which was rescued by addition of POSTN. AKT inhibitor significantly reduced POSTN-mediated osteogenic differentiation. SIGNIFICANCE: rPOSTN could be a therapeutic regime for defective periodontal and peri-implant bone regeneration in diabetes mellitus.

Pivotal Role of Tenascin-W (-N) in Postnatal Incisor Growth and Periodontal Ligament Remodeling.

The continuously growing mouse incisor provides a fascinating model for studying stem cell regulation and organ renewal. In the incisor, epithelial and mesenchymal stem cells assure lifelong tooth growth. The epithelial stem cells reside in a niche known as the cervical loop. Mesenchymal stem cells are located in the nearby apical neurovascular bundle and in the neural plexus. So far, little is known about extracellular cues that are controlling incisor stem cell renewal and guidance. The extracellular matrix protein tenascin-W, also known as tenascin-N (TNN), is expressed in the mesenchyme of the pulp and of the periodontal ligament of the incisor, and is closely associated with collagen 3 fibers. Here, we report for the first time the phenotype of tenascin-W/TNN deficient mice, which in a C57BL/6N background exhibit a reduced body weight and lifespan. We found major defects in the alveolar bone and periodontal ligament of the growing rodent incisors, whereas molars were not affected. The alveolar bone around the incisor was replaced by a dense scar-like connective tissue, enriched with newly formed nerve fibers likely leading to periodontal pain, less food intake and reduced body weight. Using soft food to reduce mechanical load on the incisor partially rescued the phenotype. In situ hybridization and Gli1 reporter mouse experiments revealed decreased hedgehog signaling in the incisor mesenchymal stem cell compartment, which coordinates the development of mesenchymal stem cell niche. These results indicate that TNN deficiency in mice affects periodontal remodeling and increases nerve fiber branching. Through periodontal pain the food intake is reduced and the incisor renewal and the neurovascular sonic hedgehog secretion rate are reduced. In conclusion, tenascin-W/TNN seems to have a primary function in rapid periodontal tissue remodeling and a secondary function in mechanosensation.

Combination of SDF-1 and bFGF promotes bone marrow stem cell-mediated periodontal ligament regeneration.

Stromal cell derived factor-1 (SDF-1) and basic fibroblast growth factor (bFGF) were reported to induce the differentiation of bone marrow stem cells (BMSCs) into cells with characteristics of periodontal ligament fibroblasts. Thus SDF-1 and bFGF may play a positive role in BMSCs-mediated periodontal ligament regeneration. Here, the methylthiazolyldiphenyl tetrazolium bromide (MTT) assay was used to investigate the effect of scaffolds, SDF-1 and bFGF on BMSCs proliferation. RT-PCR and Western blot were used to evaluate gene and protein expression. Beagle dogs were used to establish an animal model of tooth reimplantation and to investigate the effects of scaffolds, BMSCs, SDF-1 and bFGF on periodontal ligament regeneration. X-ray images and micro computed tomography (micro CT) were used to assess morphological changes in replanted teeth and surrounding alveolar bone. H&E staining and Masson's staining were also performed. BMSCs from Beagle dogs growth on scaffolds consisted of dense structured collagens. SDF-1 and bFGF effectively promoted the differentiation of BMSCs into fibroblasts, periodontal membrane reconstruction, and cell proliferation in vitro. SDF-1 and bFGF also stimulated the expression of type I collagen (Col I), type III collagen (Col III), CXC family chemokine receptor 4 (CXCR4), and S100 calcium binding protein A4 (S100A4), and decreased the expression of alkaline phosphatase (ALP). In our experimental Beagle dog model of tooth extraction and replantation, application of SDF-1 and bFGF significantly elevated periodontal membrane reconstruction and thus supported the survival of replanted teeth. In conclusion, the findings from the present study demonstrated that SDF-1 and bFGF enhance the process of periodontal ligament reconstruction, and provide a basis and reference for the use of stem cell tissue engineering in promoting periodontal membrane regeneration.