Periodontal Stem Cells Research Articles

Osteogenic differentiation of periodontal membrane stem cells in inflammatory environments.

Periodontitis is a common disease that is difficult to treat, and if not controlled in time, it causes severe conditions, such as alveolar bone resorption and tooth loosening and loss. Periodontal ligament stem cells constitute a promising cell source for regenerative treatment of periodontitis due to their high osteogenic differentiation capacity. PDLSC osteogenesis plays a central role in periodontal regeneration through successive cytokine-mediated signaling pathways and various biochemical and physicochemical factors. However, this process is inhibited in the inflammatory periodontitis environment due to high concentrations of lipopolysaccharide. Here, we review the mechanisms that influence the osteogenic differentiation of periodontal stem cells in this inflammatory microenvironment.

Advances in periodontal stem cells and the regulating niche: From in vitro to in vivo.

Periodontium possesses stem cell populations for its self-maintenance and regeneration, and has been proved to be an optimal stem cell source for tissue engineering. In vitro studies have shown that stem cells can be isolated from periodontal ligament, alveolar bone marrow and gingiva. In recent years, more studies have focused on identification of periodontal stem cells in vivo. Multiple genetic markers, including Gli1, Prx1, Axin2, αSMA, and LepR, were identified with the lineage tracing approaches. Characteristics, functions, and regulatory mechanisms of specific populations expressing one of these markers have been investigated. In vivo studies also revealed that periodontal stem cells can be regulafrted by different niche and mechanisms including intercellular interactions, ECM and multiple secreted factors. In this review, we summarized the current knowledge of in vitro characteristics and in vivo markers of periodontal stem cells, and discussed the specific regulating niche.

Rescuing effects of periostin in advanced glycation end-products (AGEs) caused osteogenic and oxidative damage through AGE receptor mediation and DNA methylation of the CALCA promoter

An in vitro model was established to simulate a diabetes-type environment by treating human periodontal stem cells with advanced glycation end-products (AGEs). Periostin (POSTN) plays a crucial role in maintaining the integrity of periodontal tissues. However, the role of POSTN in human periodontal stem cells stimulated by AGEs remains unknown. Diabetes mellitus is considered a metabolic disease, and DNA methylation of CpG islands is a biomarker of metabolic syndromes. Diabetes has been found to be closely related to the DNA methylation of certain genes. Here, we investigated the protective mechanism and effect of POSTN on osteogenesis and oxidative stress in the AGE environment, and further explored the CpG island methylation of specific genes potentially mediated by POSTN. The optimal concentration of AGEs was screened using CCK8. AGEs were found to contribute to oxidative stress. Conversely, reactive oxygen species production and malondialdehyde and superoxide activity indicated that the AGE + POSTN group decreased oxidative injury. According to an alkaline phosphatase assay, Alizarin Red S staining, and the expression of key genes and proteins involved in osteogenesis, POSTN mitigated the inhibitory effects of AGE on cell proliferation and osteogenic differentiation potential during osteogenic differentiation. In contrast, the growth and osteogenesis of human periodontal stem cells were notably suppressed by POSTN knockdown. Bisulfite sequencing PCR was used to evaluate the DNA methylation status. Moreover, AGE elevated the expression of DNA methyltransferas 1 (DNMT1) and inhibited the activation of CALAL promoter methylation, which was rescued by the addition of POSTN and 5-Azacytidine (5-AZA). In conclusion, POSTN attenuated the AGE-induced inhibition of osteogenesis in periodontal ligament stem cells by reducing AGE receptor levels and DNA methylation of the calcitonin-related polypeptide α (CALCA) promoter. Thus, POSTN is a promising candidate for dental bone regeneration, representing a novel therapeutic agent for diabetic patients. The mechanism underlying these processes may provide new insights into novel therapeutic targets for improving abnormal bone metabolism in patients with diabetes.

Advances in the Regulation of Osteogenic Differentiation of Periodontal Stem Cells by Signalling Pathways

Periodontal ligament stem cells (PDLSCs) are mesenchymal stem cells derived from periodontal tissues with self-renewal and multidirectional differentiation potential, and are an important cell population for periodontal tissue repair. At present, studies have shown that various signaling pathways play a crucial role in the osteogenic differentiation of periodontal stem cells, but the mechanisms involved in the regulation of osteogenic differentiation of periodontal stem cells are still in the early stage of research. Therefore, it is necessary to investigate the interrelationship and regulation of these pathways in the osteogenic differentiation of periodontal stem cells, cell signaling and new bone formation, in order to further reveal their molecular biological mechanisms. In this paper, we review the research on the regulation of osteogenic differentiation of periodontal stem cells by Notch signaling pathway, Wnt signaling pathway, NF-κB (nuclear factor-kappa B) signaling pathway and MARK signaling pathway, and provide a theoretical basis for the clinical treatment of periodontal bone defects by targeting the signaling pathways.

Comparative Analysis of Dental Pulp and Periodontal Stem Cells: Differences in Morphology, Functionality, Osteogenic Differentiation and Proteome.

Dental stem cells are heterogeneous in their properties. Despite their common origin from neural crest stem cells, they have different functional capacities and biological functions due to niche influence. In this study, we assessed the differences between dental pulp stem cells (DPSC) and periodontal ligament stem cells (PDLSC) in their pluripotency and neuroepithelial markers transcription, morphological and functional features, osteoblast/odontoblast differentiation and proteomic profile during osteogenic differentiation. The data were collected in paired observations: two cell cultures, DPSC and PDLSC, were obtained from each donor. Both populations had the mesenchymal stem cells surface marker set exposed on their membranes but differed in Nestin (a marker of neuroectodermal origin) expression, morphology, and proliferation rate. OCT4 mRNA was revealed in DPSC and PDLSC, while OCT4 protein was present in the nuclei of DPSC only. However, transcription of OCT4 mRNA was 1000–10,000-fold lower in dental stem cells than in blastocysts. DPSC proliferated at a slower rate and have a shape closer to polygonal but they responded better to osteogenic stimuli as compared to PDLSC. RUNX2 mRNA was detected by qPCR in both types of dental stem cells but RUNX2 protein was detected by LC-MS/MS shotgun proteomics only in PDLSC suggesting the posttranscriptional regulation. DSPP and DMP1, marker genes of odontoblastic type of osteogenic differentiation, were transcribed in DPSC but not in PDLSC samples. Our results prove that DPSC and PDLSC are different in their biology and therapeutic potential: DPSC are a good candidate for osteogenic or odontogenic bone-replacement cell-seeded medicines, while fast proliferating PDLSC are a prospective candidate for other cell products.

Prospects for the use of fibrin scaffolds populated with pulp and periodontal stem cells: an experimental study

Relevance. Creating three-dimensional scaffolds from biodegradable materials and seeding them with stem cells derived from the oral tissues is a promising tool for guided tissue regeneration. Pulp and periodontal stem cells have a high potential for osteogenic differentiation, which biologically determines their use in surgical bone reconstruction. The experiment shows the result of using fibrin glue seeded with pulp and periodontal stem cells on the mandible of laboratory mice. The article presents the results of computed tomography and histological examination. The data provide evidence of the influence of seeded scaffolds on bone remodeling in the area of the defect.Materials and methods. The Local Ethics Committee of the North-Western State Medical University named after I.I. Mechnikov gave permission for the practical part of the research work. The study included 29 white laboratory mice. Molars were extracted and a bone defect was formed. Pulp and periodontal stem cells were obtained and cell-seeded scaffolds were made, then they were introduced into the defect area. The animals were euthanized, maxillofacial CT scan and histology of the defect area were performed 28 days after the molar extraction.Results. The oral cavity of mice was examined, molars were extracted, and teeth were morphologically examined under anesthesia. Scaffolds were synthesized and bone defects were filled. CT scans and histology results were analyzed. The bone volume increased in the main group compared to the control group.Conclusion. The fibrin glue can be used to obtain a material with mechanical characteristics sufficient for a stable shape scaffold. The study proved that the pulp stem cells enclosed in a fibrin glue-based scaffold can maintain the ability to proliferate and osteogenically differentiate. The scaffold based on fibrin glue, which we used, affected the bone remodeling process in the area of jaw defects.

High-fat diet exacerbates periodontitis: is it because of dysbacteriosis or stem cell dysfunction?

Previous studies have shown that high-fat diet (HFD) may aggravate periodontitis, however the underlining mechanism remains to be further clarified. This study aims to explore whether HFD promotes periodontitis by inducing periodontal microbiota dysbiosis or stem cell dysfunction. A high-fat diet was given to four-week-old male Sprague-Dawley rats for 12 weeks. Periodontitis was induced during the latter 4 weeks. At the end of the 12th week, samples were collected after euthanasia. Maxillae were harvested for histological or microbial analysis. The microbial 16S rRNA gene sequencing was performed with the Illumina MiSeq platform. The data was analyzed through RDP Classifier against the SILVA database. The mandible molars were harvested for isolating periodontal ligament stem cells (PDLSCs). The protein level of p27, p21, and p16, which are negative regulators of the cell cycle, in PDLSCs were detected. Markers of osteogenic differentiation and pro-inflammatory mediators were detected by real-time polymerase chain reaction. Activation of pro-inflammatory signaling pathways was detected by Western blotting. We found that HFD significantly increased ligature-induced alveolar bone loss. HFD resulted in a less diverse periodontal microbiota, with increased proportions of Lactococcus, Bacillus, Alloprevotella, Carnobacterium, and Exiguobacterium and decreased proportion of Nitrospira. HFD increased the protein levels of p27, p16, and p21, and upregulated the expression of osteogenic biomarkers, IL-1β and IL-10 with the ERK1/2 signaling pathway activated in PDLSCs.

Inflammatory periodontal stem cells mediate interleukin-1β secretion of macrophage by regulating macrophage endoplasmic reticulum stress

Objective: To investigate the effect and mechanism of periodontal ligament stem cell (PDLSC) from inflammatory environment on the secretion of interleukin-1β (IL-1β) by macrophages. Methods: PDLSCs were pretreated with lipopolysaccharide (LPS) in order to simulate the inflammatory environment. Human monocyte cell line (THP-1) cells were treated with conditioned media collected from healthy and inflammatory PDLSCs respectively and divided into conditioned medium of health PDLSC (CM-H) group and conditioned medium of LPS-PDLSC (CM-LPS) group. After 24 h of co-culture, the condition media were abandoned and THP-1 cells were then cultured for another 24 h. The expression of IL-1β in THP-1 cells supernatant was detected by enzyme-linked immunosorbent assay (ELISA). Quantitative real time-PCR (qRT-PCR) was used to detect the expression of glucose regulated protein 78 (GRP78), activating transcription factor-6 (ATF6), inositol requiring enzyme 1 (IRE1), protein kinase R-like endoplasmic reticulum kinase (PERK), CCAAT enhancer binding protein homologous protein (CHOP), activating transcription factor-4 (ATF4) and X box binding protein 1 spliced (XBP1s), which were all related with endoplasmic reticulum stress (ERS), in THP-1 cells. The expressions of proteins GRP78 and CHOP were detected by Western blotting. Furthermore, THP-1 cells, which pretreated with ER inhibitor 4-phenylbutyrate (4-PBA) for intervention experiments were grouped by various concentrations of 4-PBA including groups 0 (control group), 1, 10 and 20 mmol/L and treated with condition medium of inflammatory PDLSC. ELISA was used to detect IL-1β expression and qRT-PCR to detect expression of ERS related genes. Results: ELISA results showed that the expression of IL-1β in THP-1 cells of group CM-LPS [(31.35±2.11) ng/L] was significantly higher than group CM-H [(8.19±1.51) ng/L] (t=12.60, P<0.01). qRT-PCR results showed that the relative expressions of GRP78, ATF6, IRE1, PERK, CHOP, ATF4 and XBP1s genes in THP-1 cells of group CM-LPS (1.782±0.070, 1.387±0.204, 1.404±0.119, 1.777±0.187, 1.325±0.156, 1.295±0.066 and 1.137±0.149, respectively) were significantly higher than those in group CM-H (P<0.05). In the 4-PBA intervention experiment, compared with group 0 mmol/L, the expressions of GRP78, IRE-1, ATF-6, PERK and CHOP were significantly lower in group 1, 10 and 20 mmol/L (P<0.05). Moreover, compared with control group [(31.23±1.98) ng/L], the expression of IL-1β in THP-1 cells were significantly lower in group 10 mmol/L [(21.20±0.37) ng/L] and group 20 mmol/L [(23.85±1.80) ng/L] (P<0.05) with ERS inhibited. Conclusions: PDLSC from inflammatory environment could promote IL-1β secretion of macrophages through upregulating macrophages ERS.

PHOTOBIOMODULATION INCREASES THE VIABILITY AND PROLIFERATION OF HUMAN PERIODONTAL LIGAMENT STEM CELLS CULTURED ON THE SURFACE OF POLYLATIC ACID FILMS

<h3>Backgorund</h3> Polylactic acid (PLA) is a biomaterial with diverse biomedical applications and has been a promising scaffold in tissue engineering mainly due to its biocompatibility, easy manipulation and low cost. Photobiomodulation using low-level laser irradiation has been shown to be a powerful tool to promote in vitro biostimulation in several cell types. The aim of this study was to evaluate the effectiveness of photobiomodulation on the viability and proliferation of human periodontal ligament stem cells (hPDLSC) cultured on PLA scaffolds. <h3>Methods</h3> PLA films were produced by solvent casting method and the surface topography was evaluated by scanning electronic microscopy (SEM) and atomic force microscopy (AFM). hPDLSC were isolated, characterized and cultured on the PLA films. Two groups were evaluated: Control - non irradiated; and Laser - irradiated with diode laser (InGaAIP) with wavelength of 660 nm, power of 30 mW, and a single dose of 1 J/cm² with radiation emitted continuously. Cell viability analyzes were performed 24 and 48 hours after irradiation using the the Alamar Blue biochemical assay and Live/Dead assay. Cell cycle events were assessed by flow cytometry and cell-biomaterial morphological interaction was evaluated by SEM. <h3>Results</h3> The films produced showed a flat and regular surface, with the occasional presence of small pores and an average roughness of 59.381 nm. The results of Alamar Blue assay showed a greater cell metabolic activity in irradiated group compared to control at 24 (<i>p</i><0.05) and 48 h (<i>p</i><0.001), which was confirmed in the Live/Dead assay by a higher density of viable cells in the Laser group. In the analysis of the cell cycle, the Laser group showed an increase of cells in the G2/M phase compared to the Control group (<i>p</i> <0.001). SEM images showed a higher cell density in the irradiated group, with maintenance of cell morphology and projections. <h3>Conclusion</h3> Taken together, the findings of this study demonstrated that photobiomodulation has the ability to increase the viability and proliferation of periodontal stem cells cultured on PLA scaffolds, which may contribute to the development of new studies using this protocol in periodontal tissue engineering.

Reactive oxygen species-mediated cytotoxicity of indirect restorative cement on periodontal stem cells

Reactive oxygen species-mediated cytotoxicity of indirect restorative cement on periodontal stem cells

Notch-dependent activation of periodontal cells osteogenic potential

Background and objective . Periodontal stem cells are a promising material for dentistry and maxillofacial surgery. In this work, we sought to find out how Notch signaling pathway affects the osteogenic potential of periodontal ligament stem cells (PDLSC). Design and methods . In order to verify this, we used lentiviral transduction of PDLSC, adding various amounts of the virus bearing the activated intracellular domain of the Notch1 receptor, NICD. Using real-time PCR, we analyzed the dependence of changes in the expression levels of osteo-markers ( RUNX2, COL1A1, OGN, POSTN ) on the activation strength of Notch signaling pathway, based on the expression of the target gene — HEY1 , in the early stages of differentiation, after 48 and 120 hours after induction of osteogenic differentiation. In addition, using the technique of Alizarin red extraction, we were able to quantify the intensity of mineralization of periodontal MSCs at the final stage of osteogenic differentiation. Results . We found that activation of Notch signaling pathway leads to increased expression of osteogenic markers in the early stages of osteogenic differentiation induction, which in turn leads to the development of the final stage of osteogenic differentiation, characterized by the formation of calcification. Moreover, the more intensive the initial level of Notch, the stronger and more effective were the processes of osteogenesis. Conclusion . In this work, we showed that activation of Notch signaling pathway leads to an increase in the osteogenic potential of periodontal PDLSC in a dose-dependent manner and induces in the cells processes associated with the accumulation of calcifications, the intensity of formation of which directly depends on the “strength” of Notch signal. The results obtained in this work convince us that Notch is able to modulate osteogenic differentiation, affecting its effectiveness, apparently due to the strength of the signal that is transmitted to the cells.

VEGF/VEGF-R/RUNX2 Upregulation in Human Periodontal Ligament Stem Cells Seeded on Dual Acid Etched Titanium Disk.

In restorative dentistry, the main implants characteristic is the ability to promote the osseointegration process as the result of interaction between angiogenesis and osteogenesis events. On the other hand, implants cytocompatibility remains a necessary feature for the success of surgery. The purpose of the current study was to investigate the interaction between human periodontal stem cells and two different types of titanium surfaces, to verify their cytocompatibility and cell adhesion ability, and to detect osteogenic and angiogenic markers, trough cell viability assay (MTT), Confocal Laser Scanning Microscopy (CLSM), scanning electron microscopy (SEM), and gene expression (RT-PCR). The titanium surfaces, machined (CTRL) and dual acid etched (TEST), tested in culture with human periodontal ligament stem cells (hPDLSCs), were previously treated in two different ways, in order to evaluate the effects of CTRL and TEST and define the best implant surface. Furthermore, the average surface roughness (Ra) of both titanium surfaces, CTRL and TEST, has been assessed through atomic force microscopy (AFM). The vascular endothelial growth factor (VEGF) and Runt-related transcription factor 2 (RUNX2) expressions have been analyzed by RT-PCR, WB analysis, and confocal laser scanning microscopy. Data evidenced that the different morphology and topography of the TEST disk increased cell growth, cell adhesion, improved osteogenic and angiogenic events, as well osseointegration process. For this reason, the TEST surface was more biocompatible than the CTRL disk surface.

MiR-4262 regulates differentiation and osteogenesis of human periodontal stem cells by targeting suppressor of cytokine signaling 4

MiR-4262 regulates differentiation and osteogenesis of human periodontal stem cells by targeting suppressor of cytokine signaling 4

Rutin promotes osteogenic differentiation of periodontal ligament stem cells under inflammatory microenvironment

To explore the effect of rutin on osteogenic differentiation of periodontal ligament stem cells under inflammatory microenvironment. Periodontal ligament stem cells (PDLSCs) were obtained by limited dilution method in vitro. PDLSCs were identified by flow cytometery. Lipopolysaccharide(LPS) was used to stimulate human periodontal ligament stem cells to establish an inflammation model in vitro. The experiment was divided into 4 groups: in group 1, only α-MEM was used to culture PDLSCs; in group 2, α-MEM medium containing LPS was used to culture PDLSCs, in group 3, rutin was added to α-MEM medium containing LPS to PDLSCs; and in group 4, α-MEM medium containing rutin was used to culture PDLSCs. Cell counting kit-8 was used to detect cell proliferation activity. Alkaline phosphatase(ALP) staining, ALP activity test, alizarin red staining, RT-PCR, and Western blot were used to detect the changes of osteogenic differentiation ability. The data were analyzed by SPSS 17.0 software package. The results of CCK-8 and ALP activity analysis showed that rutin at 10 μmol/L could significantly promote the proliferation and differentiation of periodontal stem cells under inflammatory state. ALP staining and alizarin red staining proved that (10 μmol/L) rutin could improve osteogenic differentiation of periodontal ligament stem cells under inflammatory microenvironment. RT-PCR and Western blot results showed that rutin could enhance the expression of osteogenic genes and proteins such as COL1, ALP, and RUNX2 under inflammatory state. Rutin can promote osteogenic differentiation of periodontal ligament stem cells under inflammatory microenvironment.

Histone Methylation Mechanisms Modulate the Inflammatory Response of Periodontal Ligament Progenitors.

Inflammatory conditions affect periodontal ligament (PDL) homeostasis and diminish its regenerative capacity. The complexity of biological activities during an inflammatory response depends on genetic and epigenetic mechanisms. To characterize the epigenetic changes in response to periodontal pathogens we have focused on histone lysine methylation as a relatively stable chromatin modification involved in the epigenetic activation and repression of transcription and a prime candidate mechanism responsible for the exacerbated and prolonged response of periodontal cells and tissues to dental plaque biofilm. To determine the effect of inflammatory conditions on histone methylation profiles, related gene expression and cellular functions of human periodontal ligament (hPDL) progenitor cells, a hPDL cell culture system was subjected to bacterial cell wall toxin exposure [lipopolysaccharide (LPS)]. Chromatin immunoprecipitation-on-chip analysis revealed that healthy PDL cells featured high enrichment levels for the active H3K4me3 mark at COL1A1, COL3, and RUNX2 gene promoters, whereas there were high occupancy levels for the repressive H3K27me3 marks at DEFA4, CCL5, and IL-1β gene promoters. In response to LPS, H3K27me3 enrichment increased on extracellular matrix and osteogenesis lineage gene promoters, whereas H3K4me3 enrichment increased on the promoters of inflammatory response genes, suggestive of an involvement of epigenetic mechanisms in periodontal lineage differentiation and in the coordination of the periodontal inflammatory response. On a gene expression level, LPS treatment downregulated COL1A1, COL3A1, and RUNX2 expression and upregulated CCL5, DEFA4, and IL-1β gene expression. LPS also greatly affected PDL progenitor function, including a reduction in proliferation and differentiation potential and an increase in cell migration capacity. Confirming the role of epigenetic mechanisms in periodontal inflammatory conditions, our studies highlight the significant role of histone methylation mechanisms and modification enzymes in the inflammatory response to LPS bacterial cell wall toxins and periodontal stem cell function.

Tooth resurrection: Life after death

Tissue engineering is an emerging domain. Very soon it will be possible to produce a complete vital tooth from a single stem cell. Pulp revascularization is based on the ability of residual pulp and apical and periodontal stem cells to differentiate. These cells are highly potent to create a highly vascularized and a vital conjunctive tissue, which can colonize the available pulp space. This technique is specifically indicated for teeth with incomplete root formation as a substitute for the traditional treatment of apexification, which consists of placing calcium hydroxide paste into the root canal for a stipulated period of time in order to trigger the formation of a calcified barrier. Therefore, there has been an indefinite search for alternatives which allow the full development of immature teeth. Revascularization is a recent treatment module for immature necrotic permanent teeth. Keywords: Tissue engineering, Stem cell, Pulp revascularization, Apexification, Immature teeth.

3D Human Periodontal Stem Cells and Endothelial Cells Promote Bone Development in Bovine Pericardium-Based Tissue Biomaterial.

Bone defects repair represents a public and urgent problem in clinical practice, in fact, every year, more than two million patients required new treatments for bone injuries. Today a complete vascularization is strategic in bone formation, representing a new frontier for clinical application. Aim of this research has been developed a three-dimensional (3D) coculture platform using a bovine pericardium collagen membrane (BioR) loaded with human periodontal ligament stem cells (hPDLSCs) and endothelial differentiated cells from hPDLSCs (E-hPDLSCs) able to undergo toward osteoangiogenesis differentiation process. First, we have characterized at confocal laser scanning microscopy (CLSM) level the E-hPDLSCs phenotype profile, through CD31 and CD34 markers expression and the ability to tube vessel formation. Real Time-Polimerase Chain Reaction (RT-PCR) and western blotting analyses revealed the upregulation of Runt-related transcription factor 2 (RUNX2), Collagen 1A1 (COL1A1), Vascular Endothelial Growth Factor-A (VEGF-A) genes and proteins in the living construct composed by hPDLSCs + E-hPDSCs/BioR. Human PDLSCs + E-hPDLSCs/BioR construct showed also an enhacement of de novo synthesis of osteocalcin. Given that, the extracellular-signal-regulated kinase (ERK)/mitogen activated protein kinase (MAPK) transduction signaling was involved in the osteogenesis and angiogenesis process, the ERK1/2 protein level at biochemical level, in our experimental model, has been investigated. Our results evidenced an upregulation of ERK1/2 proteins level born in the living construct. In conclusion, we believe that the use of the hPDLSCs and E-hPDLSCs coculture togheter with BioR as substrate, could represent an efficient model able to activate through ERK1/2 signaling pathway the osteoangiogenesis process, and then representing a new potential engineered platform for surgeons during the repair and the healing of bone defects.

Modulatory effect of curcumin analogs on the activation of metalloproteinases in human periodontal stem cells.

Periodontitis progresses due to increased levels of active metalloproteinases (MMPs) and the imbalance between MMPs and their tissue inhibitors (TIMPs). Natural curcumin limits the lytic activity of MMPs but has low cellular uptake. Use of synthetic curcumin analogs could be a means of overcoming this limitation of treatment efficiency. Human periodontal stem cells were isolated from gingival tissue, gingival ligament fibers, periodontal ligament, and alveolar bone. The effect of five synthetic curcumin analogs was compared with that of natural curcumin by assessing cytotoxicity [by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay], the cellular uptake (by fluorometry), the proteolytic activities of MMP-2 and -9 (by zymography), and the levels of TIMP-1 (by ELISA). Our results indicated increased cytotoxicity of synthetic curcumins for doses between 100 and 250μM. At a concentration of 10μM, cellular uptake of synthetic curcumins varied depending on their chemical structure. The curcumin compounds modulated pro-MMP-2 levels and increased TIMP-1 production. There was no detectable synthesis of pro-MMP-9 and no activation of MMPs 2 and 9. Gingival tissue and gingival ligament fiber stem cells were most responsive to treatment, showing inverse correlations between pro-MMP-2 and TIMP-1 levels. In conclusion, synthetic curcumins influenced the balance between pro-MMP-2 and TIMP-1 in human periodontal stem cells in vitro, and this could open perspectives for their application as adjuvants in periodontal therapy.

Prx1 Expressing Cells Are Required for Periodontal Regeneration of the Mouse Incisor.

Previous studies have shown that post-natal skeletal stem cells expressing Paired-related homeobox 1 (PRX1 or PRRX1) are present in the periosteum of long bones where they contribute to post-natal bone development and regeneration. Our group also identified post-natal PRX1 expressing cells (pnPRX1+ cells) in mouse calvarial synarthroses (sutures) and showed that these cells are required for calvarial bone regeneration. Since calvarial synarthroses are similar to dentoalveolar gomphosis (periodontium) and since there is no information available on the presence or function of pnPRX1+ cells in the periodontium, the present study aimed at identifying and characterizing pnPRX1+ cells within the mouse periodontium and assess their contribution to periodontal development and regeneration. Here we demonstrated that pnPRX1+ cells are present within the periodontal ligament (PDL) of the mouse molars and of the continuously regenerating mouse incisor. By means of diphtheria toxin (DTA)-mediated conditional ablation of pnPRX1+ cells, we show that pnPRX1+ cells contribute to post-natal periodontal development of the molars and the incisor, as ablation of pnPRX1+ cells in 3-days old mice resulted in a significant enlargement of the PDL space after 18 days. The contribution of pnPRX1+ cells to periodontal regeneration was assessed by developing a novel non-critical size periodontal defect model. Outcomes showed that DTA-mediated post-natal ablation of pnPRX1+ cells results in lack of regeneration in periodontal non-critical size defects in the regeneration competent mouse incisors. Importantly, gene expression analysis of these cells shows a profile typical of quiescent cells, while gene expression analysis of human samples of periodontal stem cells (PDLSC) confirmed that Prx1 is highly expressed in human periodontium. In conclusion, pnPRX1+ cells are present within the continuously regenerating PDL of the mouse incisor, and at such location they contribute to post-natal periodontal development and regeneration. Since this study further reports the presence of PRX1 expressing cells within human periodontal ligament, we suggest that studying the mouse periodontal pnPRX1+ cells may provide significant information for the development of novel and more effective periodontal regenerative therapies in humans.

Oxytocin facilitates the proliferation, migration and osteogenic differentiation of human periodontal stem cells in vitro

ObjectiveTo explore the effect of oxytocin (OT) on the proliferation, migration, and osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in vitro. DesignPDLSCs were obtained by limiting dilution method. Immunofluorescence (IF), cell-counting kit-8 (CCK8), cell migration assay, Alizarin Red S staining, cetylpyridinium chloride (CPC) colorimetry, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot analysis were used to examine the effect of OT on oxytocin receptor (OTR) expression, cell proliferation, migration and osteogenic differentiation of PDLSCs. ResultsOur study showed that PDLSCs expressed OTR. One hundred nanomolar OT exhibited the maximal effect on migration, while only 50 nM OT significantly promoted proliferation of PDLSCs, as well as mineralized nodule formation and calcium deposition (p < 0.05). Furthermore, 50 nM OT significantly up-regulated expression of osteogenesis-related genes—alkaline phosphatase (ALP), Collagen I (Col I), runt related transcription factor 2 (Runx 2), osteopontin (OPN) and osteocalcin (OCN)—at specific time points compared with osteogenic inductive medium (p < 0.05). In addition, western blot analysis demonstrated that 50 nM OT enhanced protein levels of ALP, Col I, and Runx 2 at day 7 and day 14 (p < 0.01), as well as activating the phosphorylation of extracellular-signal-regulated kinase (ERK) and protein kinase B (AKT) pathway; notably, 50 nM OT inhibited phosphorylation of the phosphatidylinositol 3-kinase (PI3K) pathway (p < 0.05). ConclusionsOT promoted proliferation, migration, and osteogenic differentiation of PDLSCs in vitro. Furthermore, the effect of OT on osteogenic differentiation was mediated through ERK and AKT pathway. Thus, OT may have potential for use in periodontal regeneration.