Human Periodontal Ligament Mesenchymal Stem Research Articles

Effects of Photobiomodulation Therapy with Various Laser Wavelengths on Proliferation of Human Periodontal Ligament Mesenchymal Stem Cells.

Several methods have been proposed to enhance the regeneration and healing time in periodontal therapy. Photobiomodulation therapy (PBMT) is a recently suggested novel technique for this purpose. This study aimed to compare the efficacy of PBMT with various laser wavelengths and energy densities on proliferation of human periodontal ligament mesenchymal stem cells (PDLMSCs). The wells containing PDLMSCs were subjected to laser irradiation at 635, 660, 808 and 980 nm wavelengths with 1, 1.5, 2.5 and 4 J cm-2 energy densities. Cell proliferation and viability were evaluated after 1, 3 and 5 days with the methyl thiazolyl tetrazolium (MTT) assay and 4,6-diamidino-2-phenylindole (DAPI) staining. No significant difference was observed among the experimental and the control groups on day 1 (P > 0.05). On day 3, 808 nm laser at 4 J cm-2 energy density and 980 nm laser at all densities had significant differences with control group. On day 5, the control group had significant differences in cell proliferation with 808 nm laser at 2.5 and 4 J cm-2 energy densities, and 980 nm laser at all densities. PBMT with 635, 660, 808 and 980 nm wavelengths increased the proliferation of PDLMSCs but the maximum cell viability was prominent after irradiation by 980 nm laser with energy density of 4 J cm-2 on day 3.

Characterization and Study of Gene Expression Profiles of Human Periodontal Mesenchymal Stem Cells in Spheroid Cultures by Transcriptome Analysis.

A spheroid is known as a three-dimensional culture model, which better simulates the physiological conditions of stem cells. This study is aimed at identifying genes specifically expressed in spheroid-cultured human periodontal ligament mesenchymal stem cells (hPDLMSCs) using RNA-seq analysis to evaluate their functions. Transcriptome analysis was performed using spheroid and monolayer cultures of hPDLMSCs from four patients. Cluster and Gene Ontology analyses revealed that genes involved in cell-cell adhesion as well as the G2/M and G1/S transitions of mitotic cell cycles were strongly expressed in the monolayer culture group. However, genes involved in the negative regulation of cell proliferation, histone deacetylation, and bone morphogenetic protein signaling were strongly expressed in the spheroid culture group. We focused on the transcription factor nuclear receptor subfamily 4 group A member 2 (NR4A2) among the genes that were strongly expressed in the spheroid culture group and analyzed its function. To confirm the results of the transcriptome analysis, we performed real-time polymerase chain reaction and western blotting analyses. Interestingly, we found that the mRNA and protein expressions of NR4A2 were strongly expressed in the spheroid-cultured hPDLMSCs. Under osteogenic differentiation conditions, we used siRNA to knock down NR4A2 in spheroid-cultured hPDLMSCs to verify its role in osteogenesis. We found that NR4A2 knockdown significantly increased the levels of mRNA expression for osteogenesis-related genes alkaline phosphatase (ALP), Osteopontin (OPN), and type 1 collagen (COL1) (Student's paired t-test, p < 0.05). ALP activity was also significantly increased when compared to the negative control group (Student's paired t-test, p < 0.05). Additionally, spheroid-cultured hPDLMSCs transfected with siNR4A2 were cultured for 12 days, resulting in the formation of significantly larger calcified nodules compared to the negative control group (Student's paired t-test, p < 0.05). On the other hand, NR4A2 knockdown in hPDLMSC spheroid did not affect the levels of chondrogenesis and adipogenesis-related genes under chondrogenic and adipogenic conditions. These results suggest that NR4A2 negatively regulates osteogenesis in the spheroid culture of hPDLMSCs.

Effects of different detergent-containing children's toothpastes on the viability, osteogenic and chondrogenic differentiation of human dental periodontal ligament stem cells and gingival stem cells in vitro

BackgroundDetergents are the most commonly used compounds in toothpastes due to their foaming and cleaning peoperties. This study aimed to investigate the effects of children’s toothpastes with different detergent content on the viability, the osteogenic and chondrogenic differentiation potentials of human mesenchymal stem cells. MethodsThe necessary tissues for human periodontal ligament mesenchymal stem cells (hPDLMSCs) and human gingival mesenchymal stem cells (hGMSCs) isolation were obtained during extraction of 10 impacted third molar teeth. The viability of the cells stimulated with different concentratiaons of Colgate, Sensodyne, Splat, Nenedent, Perlodent toothpaste solutions and complete Dulbocco’s modified eagle medium (control group) were evaluated by using the flow cytometer. In addition, the osteogenic and chondrogenic differentiation potential of human gingival and periodontal ligament mesenchymal stem cells exposed to toothpaste solutions were examined morphologically. Datas were analyzed with IBM SPSS V23. One way ANOVA test was used to determine the differences between the groups for multiple comparisons, while the Tukey post-hoc test was used for pair wise comparisons in determining which groups differed. ResultsA higher percentage of cell viability was detected in Control group at 20 %, 50 % and 80 % (p = 0.000) on hGMSCs. After the Control group, the highest cell viability ratios were observed in the detergent-free Splat group (p = 0.000) followed by the Sensodyne experimental group containing CABP (p = 0.000). While the cell viability rates in Nenedent group was found significantly higher than the Perlodent group at other concentrations except for 20 % concentration (p = 0.000). Colgate group had the lowest percentage of cell viability among the experimental groups at all concentrations on hPDMSCs (p = 0.000). The highest live cell ratios was detected in Control group (p = 0.000), followed by Splat and Sensodyne groups (p = 0.000). The cell viability ratios at 50 % concentration were higher in Perlodent group than Nenedent group (p = 0.000). The highest osteogenic and chondrogenic differentiation potential of mesenchymal stem cells stimulated with different toothpaste was determined in Control and Splat group. ConclusionsAs a result of the findings, it was observed that toothpaste containing SLS had a more negative effect on the viability of the cells and the differentiation potentials than the other groups.

Co-cultured spheroids of human periodontal ligament mesenchymal stem cells and vascular endothelial cells enhance periodontal tissue regeneration.

IntroductionHuman periodontal ligament mesenchymal stem cells (hPDLMSCs) have been known that they play important roles in homeostasis and regeneration of periodontal tissues. Additionally, spheroids are superior to monolayer-cultured cells. We investigated the characteristics and potential of periodontal tissue regeneration in co-cultured spheroids of hPDLMSCs and human umbilical vein endothelial cells (HUVECs) in vitro and in vivo.MethodsCo-cultured spheroids were prepared with cell ratios of hPDLMSCs: HUVECs = 1:1, 1:2, and 2:1, using microwell chips. Real-time polymerase chain reaction (PCR) analysis, Enzyme-Linked Immuno Sorbent Assay (ELISA), and nodule formation assay were performed to examine the properties of co-cultured spheroids. Periodontal tissue defects were prepared in the maxillary first molars of rats and subjected to transplantation assay.ResultsThe expression levels of stemness markers, vascular endothelial growth factor (VEGF), osteogenesis-related genes were up-regulated in co-cultured spheroids, compared with monolayer and spheroid-cultured hPDLMSCs. The nodule formation was also increased in co-cultured spheroids, compared with monolayer and spheroid cultures of hPDLMSCs. Treatment with co-cultured spheroids enhanced new cementum formation after 4 or 8 weeks of transplantation, although there was no significant difference in the new bone formation between co-cultured spheroids and hPDLMSC spheroids.ConclusionsWe found that co-cultured spheroids enhance the periodontal tissue regeneration. Co-cultured spheroids of hPDLMSCs and HUVECs may be a useful therapy that can induce periodontal tissue regeneration.

Spheroid culture enhances osteogenic potential of periodontal ligament mesenchymal stem cells.

Human periodontal ligament mesenchymal stem cells (hPDLMSCs) are reported to be responsible for homeostasis and regeneration of periodontal tissue. Although hPDLMSCs are commonly cultured in monolayers, monolayer cultures have been reported as inferior to 3-dimensional cultures such as spheroids, which are spherical clusters of cells formed by self-assembly. The aim of this study was to examine the osteogenic phenotype of spheroids of hPDLMSCs, compared with monolayer cultures of hPDLMSC, in vitro and in vivo. Spheroids were formed using microwell chips that were tagged with polyethylene glycol. Mesenchymal stem cell (MSC) markers in hPDLMSC spheroids were examined by flow cytometer. Real-time polymerase chain reaction analysis was examined to measure the expressions of stemness markers and osteogenesis-related genes in monolayer and spheroid-cultured hPDLMSCs. Immunofluorescence analysis was performed to confirm protein expressions of stemness markers in PDLMSC spheroids. Nodule formation assay, alkaline phosphatase (ALP) activity assay and transplantation assay in a mouse calvarial defect model were performed to confirm the osteogenic potential of hPDLMSC spheroids. To elucidate the mechanism of spheroid culture enhanced osteogenesis in hPDLMSCs with osteoinductive medium (OIM), a small interfering RNA (siRNA) assay targeted with secreted frizzled-related protein 3 (SFRP3) was examined. The levels of SFRP3 expression in monolayer and spheroid-cultured hPDLMSCs with OIM were measured by real-time polymerase chain reaction and western blotting analysis. ALP gene expression and ALP activity were examined in SFRP3-deficient hPDLMSC spheroids. The hPDLMSC spheroids expressed MSC markers, which were similar to hPDLMSCs grown in monolayer cultures. Intriguingly, the protein and mRNA expressions of transcription factors that regulate "stemness" were significantly increased in hPDLMSC spheroids, compared with hPDLMSCs in monolayer cultures. Nodule formation by hPDLMSCs was significantly increased in spheroid cultures grown with OIM, compared with monolayer-cultured hPDLMSCs. ALP activity and expression of osteogenesis-related genes were also significantly enhanced in hPDLMSC spheroids, compared with monolayer cultures. Treatment with hPDLMSC spheroids significantly enhanced new bone formation in a murine calvarial defect model, compared with hPDLMSCs in monolayer culture. Finally, to elucidate mechanisms by which spheroid culture enhances ALP activation in hPDLMSCs grown with OIM, an siRNA assay was used to manipulate expression of SFRP3, a Wnt signaling antagonist. Knockdown of SFRP3 suppressed ALP gene expression in hPDLMSCs grown in OIM; further, it suppressed ALP activity in spheroid culture. These data suggest that the enhancement of osteogenic potential in hPDLMSC spheroids is regulated through SFRP3-mediated ALP activation. Spheroid cultures of hPDLMSCs may be a novel and useful tool in regenerative medicine.

Effect of cDMEM media containing Ectoine on human periodontal ligament mesenchymal stem cell survival and differentiation.

Ectoine is an amino acid that can preserve osmotic balance and has more preservative activity than other osmoregulators. There are no published reports on the osmoregulators' effects on viability or differentiation of dental stem cells. The aim of this study was to investigate the effect of Ectoine as a storage media on the viability and differentiation potential of human periodontal ligament mesenchymal stem cells (hPDLMSCs). hPDLMSCs were obtained from impacted third molar teeth. The cells were isolated, submitted to trilineage differentiation, and characterized by flow cytometer (FC). hPDLMSCs were incubated with different media containing Ectoine, complete DMEM (cDMEM), Ectoine+cDMEM, milk, and tap water for 2, 6, 12, and 24h. The cells were analyzed by FC for viability, early-apoptosis, late apoptosis, and necrosis rates. Osteogenic and fibroblastic differentiation in hPDLMSCs were investigated by Alizarin red stain and vimentin expression. All treated groups showed significant decreases in cell viability after 2h. Significant increases were detected in the number of dead cells between 2 and 12h in all groups except the Ectoine+cDMEM group. The deposition of mineral matrix nodules was significantly higher in cells cultured with Ectoine+cDMEM compared with the other media. Higher vimentin expressions were detected in cells cultured with cDMEM and Ectoine+cDMEM media, respectively. Ectoine added to cDMEM media, promoted cell survival plus osteogenic and fibroblastic differentiation of hPDLMSCs.

Two Transcripts of FBXO5 Promote Migration and Osteogenic Differentiation of Human Periodontal Ligament Mesenchymal Stem Cells.

Objectives Enhanced migration and osteogenic differentiation of mesenchymal stem cells (MSCs) are beneficial for MSC-mediated periodontal tissue regeneration, a promising method for periodontitis treatment. FBXO5, a member of the F-box protein family, is involved in the osteogenic differentiation of MSCs. Here, we investigated the effect of FBXO5 on human periodontal ligament stem cells (hPDLSCs). Materials and Methods hPDLSCs were isolated from periodontal ligament tissue. Lentivirus FBXO5 shRNA was used to silence FBXO5 expression. Two transcripts of FBXO5 were overexpressed and transduced into hPDLSCs via retroviral infection. Migration and osteogenic differentiation of hPDLSCs were evaluated using the scratch migration assay, alkaline phosphatase (ALP) activity, ALP staining, alizarin red staining, western blotting, and real-time polymerase chain reaction. Results The expression of FBXO5 was upregulated after osteogenic induction in hPDLSCs. FBXO5 knockdown attenuated migration, inhibited ALP activity and mineralization, and decreased RUNX2, OSX, and OCN expression, while the overexpression of two transcript isoforms significantly accelerated migration, enhanced ALP activity and mineralization, and increased RUNX2, OSX, and OCN expression in hPDLSCs. Conclusions Both isoforms of FBXO5 promoted the migration and osteogenic differentiation potential of hPDLSCs, which identified a potential target for improving periodontal tissue regeneration.

Periostin promotes migration, proliferation, and differentiation of human periodontal ligament mesenchymal stem cells

ABSTRACTOverview: Periostin (POSTN) is critical to bone and dental tissue morphogenesis, postnatal development, and maintenance; however, its roles in tissue repair and regeneration mediated by human periodontal ligament mesenchymal stem cells (PDLSCs) remain unclear. The present study was designed to evaluate the effects of POSTN on hPDLSCs in vitro. Materials and Methods: hPDLSCs were isolated and characterized by their expression of the cell surface markers CD44, CD90, CD105, CD34, and CD45. Next, 100 ng/mL recombinant human POSTN protein (rhPOSTN) was used to stimulate the hPDLSCs. Lentiviral POSTN shRNA was used to knockdown POSTN. The cell counting kit-8 (CCK8) and scratch assay were used to analyze cell proliferation and migration, respectively. Osteogenic differentiation was investigated using an alkaline phosphatase (ALP) activity assay, alizarin staining, and quantitative calcium analysis and related genes/protein expression assays. Results: Isolated hPDLSCs were positive for CD44, CD90, and CD105 and negative for CD34 and CD45. In addition, 100 ng/mL rhPOSTN significantly accelerated scratch closure, and POSTN-knockdown cells presented slower closure at 24 h and 48 h. Furthermore, the integrin inhibitor Cilengitide depressed the scratch closure that was enhanced by POSTN at 24 h. The CCK8 assay showed that 100 ng/mL rhPOSTN promoted hPDLSC proliferation. Moreover, 100 ng/mL rhPOSTN increased the expression of RUNX2, OSX, OPN, OCN, and VEGF and enhanced ALP activity and mineralization. POSTN silencing decreased the expression of RUNX2, OSX, OPN, OCN, and VEGF and inhibited ALP activity and mineralization. Conclusions: POSTN accelerated the migration, proliferation, and osteogenic differentiation of hPDLSCs.

P164 Evaluation of pro-inflammatory cytokines, and cell growth inhibition in primary human cells culture from oral tissue treated with uretandimethacrylate (UDMA)

Introduction Dental endodontic sealers are in intimate contact with tissues around the root apex for extended periods. New endodontic sealers have been developed in the past decade, but the biological responses to many new products are not well documented.After exposure to endodontic sealer based on Uretandimethacrylate (UDMA) in ex vivo expanded human dental pulp mesenchymal stem cells (DP-MSCs), human periodontal ligament mesenchymal stem cells (PDL-MSCs), human osteoblasts (HOSTs) and human gingival fibroblasts (HGF) were evaluated: morphological features and cells growth. Moreover, the secretion of pro-inflammatory cytokines as: interleukin-1 Beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-12 (IL-12), tumor necrosis factor-Alpha (TNF-α) at 24 h of UDMA treatment was assessed. Methods DP-MSCs and PDL-MSCs were derived from the dental pulps and the periodontal ligament of 10 young donors. HGF was derived from gingival biopsy. HOSTs were derived from maxilla periosteum. After in vitro isolation, DP-MSCs, PDL-MSCs, HGF and HOSTs were treated with UDMA, and after 24, 48 and 72 h and 1 week of incubation, their morphological features were analysed by light and confocal microscopy. To evaluate the, cell growth an MTT assay was performed. The release of IL-1β, IL-6, IL-8, IL-10, IL-12, TNF-alpha was analysed by ELISA test. Differences in the cell growth and in the interleukin secretion were analysed for statistical significance with two-way anova tests and the Holm-Sidak method for multiple comparisons. Results The UDMA treatment induces a cells growth inhibition in all the different analysed cells line. At microscopic level the cells don’t showed morphological changes, in fact the fibroblastoid features with cytoplasmic processes was observable. An up-regulation of IL-6 IL-8 and TNF-α was present in DP-MSCs, PDL-MSCs, HOSTs and HGF at 24 h of treatment. On the contrary IL-12 was over-expressed only in the PDL-MSCs, while the IL-1β in the DP-MSCs. Conclusion This work shows endodontic sealers may alter the cell growth, and in treated samples to induce an up-regulation of soluble mediators of inflammation such as IL-6, IL-8 and TNF-α cytokines. The direct application of UDMA potentially induces an inflammation process that could be the starting point for toxic response and cell damage in DP-MSCs, PDL-MSCs, HGF and HOSTs.

P024 LPS-P. gingivalis stimulate interleukin-6, −8 and CCL-20 release, and NF-kB translocation in human periodontal ligament mesenchymal stem cells

Introduction Periodontal diseases are clinically characterized by inflammation of the periodontal connective tissue that ultimately induces the destruction of periodontal tissue and the loss of alveolar bone. In chronic periodontitis, the anaerobic gram-negative bacterium Porphyromonas gingivalis (P. gingivalis) is implicated. The pathogenicity of P. gingivalis is exerted by a wide variety of factors, among which the lipopolysaccharides (LPSs). In this study, the expression of TLR-4, the cell growth, the cytokines release, and the nuclear factor-KB (NF-kB) transcription factor expression in response to LPS-P.Gingivalis (LPS-G) were examined in Human Periodontal Ligament Mesenchymal Stem Cells (PDL-MSCs). In this study, to understand the role and the response to LPS-G we examined cell growth, the IL-6, IL-8 and CCL-20 release and the NF-kB signaling pathway in periodontal stem cells. Methods PDL-MSCs were derived from the periodontal ligament of 10 young donors. After in vitro isolation, PDL-MSCs were incubated in the absence (controls) or presence of 5 μg/mL of LPS from P. gingivalis and after 12, 24, 48, and 7 h of incubation, their morphological features were analysed by light and confocal microscopy. PDL-MSCs were characterized by flow cytometry using against TRL-4 PE-conjugated monoclonal antibody.To evaluate the cell growth an MTT assay was performed. The release of IL-6, IL-8, and CCL-20 was analysed by ELISA test. Differences in the cell growth and in the interleukin secretion were analysed for statistical significance with two-way anova tests and the Holm-Sidak method for multiple comparisons. Results In basal conditions, human PDL-MSCs express high levels of TLR-4. In inflammatory conditions mimicked by LPS-Gchallenge, the MTT assay carried out at different treatment times evidenced the decrease of the cell growth. Moreover, the recognition of P. gingivalis components by TLR-4 culminated with the activation of secretion of inflammatory mediators such as: IL-6, IL-8 and CCL-20, and with the up-regulation of NF-kB, which was translocated into the nucleus. Our data intended to specify that TLR-4 expressed by PDL-MSCs is functional and plays a key role in inflammation. Conclusion The data obtained evidenced that the periodontal stem cells express a functional TLR-4 receptor, and the binding of LPS receptor activates the NF-kB signaling pathways. In fact, an increased level of expression and a nuclear translocation of the transcription factor is evident in treated cells. Moreover, a substantial increase of pro-inflammation cytokines IL-6 and IL-8 and CCL-20 chemokine production is visible after 24 h of stimulation with 5μg/ml LPS.In synthesis, our cells model seems to be a valuable tool for the evaluation of the response of stem cells to LPS-G, compounds that almost certainly are responsible for influencing the periodontal diseases. The distinct ability of the periodontal ligament cells to secrete IL-6, IL-8 and CCL-20 emphasizes that these cells may contribute to the release of cytokines in LPS-challenged periodontium and offer the opportunity to better comprehend the degenerative processes in periodontal diseases.

Production of polymeric micelles by microfluidic technology for combined drug delivery: Application to osteogenic differentiation of human periodontal ligament mesenchymal stem cells (hPDLSCs)

The current paper reports the production of polymeric micelles (PMs), based on pluronic block-copolymers, as drug carriers, precisely controlling the cellular delivery of drugs with various physico-chemical characteristics. PMs were produced with a microfluidic platform to exploit further control on the size characteristic of the PMs. PMs were designed for the co-delivery of dexamethasone (Dex) and ascorbyl-palmitate (AP) to in vitro cultured human periodontal ligament mesenchymal stem cells (hPDLSCs) for the combined induction of osteogenic differentiation. Mixtures of block-copolymers and drugs in organic, water miscible solvent, were conveniently converted in PMs within microfluidic channel leveraging the fast mixing at the microscale. Our results demonstrated that the drugs can be efficiently co-encapsulated in PMs and that different production parameters can be adjusted in order to modulate the PM characteristics. The comparative analysis of PM produced by microfluidic and conventional procedures confirmed that the use of microfluidics platforms allowed the production of PMs in a robust manner with improved controllability, reproducibility, smaller size and polydispersity. Finally, the analysis of the effect of PMs, containing Dex and AP, on the osteogenic differentiation of hPDLSCs is reported. The data demonstrated the effectiveness and safety of PM treatment on hPDLSC. In conclusion, this report indicates that microfluidic approach represents an innovative and useful method for PM controlled preparation, warrant further evaluation as general methodology for the production of colloidal systems for the simultaneous drug delivery.

Toll-like Receptor 4 Expression, Interleukin-6, -8 and Ccl-20 Release, and NF-KB Translocation in Human Periodontal Ligament Mesenchymal Stem Cells Stimulated with LPS-P. Gingivalis

Periodontal diseases, the major public health problem of the oral cavity, are clinically characterized by inflammation of the periodontal connective tissue that ultimately induces the destruction of periodontal tissue and the loss of alveolar bone. In chronic periodontitis, as well as aggressive periodontitis, the anaerobic gram-negative bacterium Porphyromonas gingivalis (P. gingivalis) is implicated. The pathogenicity of P. gingivalis is exerted by a wide variety of factors, including lipopolysaccharides (LPSs). LPSs activate the innate immune response during Gram-negative bacterial infections through the Toll-like receptor 4 (TLR-4)/myeloid differentiation protein 2 (MD-2) complex. In this study, the expression of TLR-4, the cell growth, the cytokine release, and the nuclear factor-KB (NF-kB) transcription factor expression in response to LPS- P.Gingivalis (LPS-G) were examined in Human Periodontal Ligament Mesenchymal Stem Cells (PDL-MSCs). The results obtained demonstrate that, in basal conditions, human PDL-MSCs express high levels of TLR-4. In inflammatory conditions mimicked by LPS-G challenge, the MTT assay carried out at different treatment times demonstrated the decrease of the cell growth. Moreover, the recognition of P. gingivalis components by TLR-4 culminated with the activation of secretion of inflammatory mediators such as: IL-6, IL-8 and CCL-20, and with the up-regulation of NF-kB, which was translocated into the nucleus. Our data intended to specify that TLR-4 expressed by PDL-MSCs is functional and plays a key role in inflammation.

Nitric oxide production during the osteogenic differentiation of human periodontal ligament mesenchymal stem cells

The critical tissues that require regeneration in the periodontium are of mesenchymal origin; therefore, the ability to identify, characterize and manipulate mesenchymal stem cells within the periodontium is of considerable clinical significance. In particular, recent findings suggest that periodontal ligament cells may possess many osteoblast-like properties. In the present study, periodontal ligament mesenchymal stem cells obtained from healthy volunteers were maintained in culture until confluence and then induced to osteogenic differentiation. Intracellular calcium ([Ca2+]i) concentration and nitric oxide, important signalling molecules in the bone, were measured along with cell differentiation. Alkaline phosphatase activity was assayed and bone nodule-like structures were evaluated by means of morphological and histochemical analysis. Our results showed that the periodontal ligament mesenchymal stem cells underwent an in vitro osteogenic differentiation, resulting in the appearance of active osteoblast-like cells together with the formation of calcified deposits. Differentiating cells were also characterized by an increase of [Ca2+]i and nitric oxide production. In conclusion, our data show a link between nitric oxide and the osteogenic differentiation of human periodontal ligament mesenchymal stem cells, thus suggesting that local reimplantation of expanded cells in conjugation with a nitric oxide donor could represent a promising method for treatment of periodontal defects.