Human Periodontal Ligament Tissue Research Articles

Orthodontically induced changes to the genetic profile in periodontal ligament tissue and cytokine release in gingival crevicular fluid – A pilot investigation

Background/purposeIt has been known that genetic factors influence orthodontic tooth movement, however, scientific research on humans is lacking. Therefore, this study aimed to investigate dynamic changes to the genetic profile in human periodontal ligament (PDL) tissue and cytokine release in gingival crevicular fluid (GCF) during the first 28 days of orthodontic treatment. Materials and methodsFifteen teeth from three patients were recruited. Full-mouth fixed appliances with extraction of four premolars and one maxillary third molar was planned for orthodontic treatment. GCF collection and tooth extraction were performed following force application for 0, 1, 3, 7, and 28 days. GCF was analyzed using multiplex immunoassay for 27 cytokines. PDL tissue was collected after extraction and submitted for RNA exome-sequencing using Illumina sequencing platform. Further analysis of differentially expressed genes (DEGs), gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and heatmaps were conducted. ResultsGCF cytokine levels varied among three patients; some patients exhibited a peak cytokine level on Day 0 whereas others did so on Days 1–3. In RNA exome sequencing data, GO and KEGG analyses showed that genes associated with sensory receptors were upregulated on Day 1, genes involved in bone remodeling were upregulated on Days 3 and 28, and genes related to osteoclast differentiation were upregulated on Day 7. ConclusionRNA sequencing data demonstrate that the specific types of genes are expressed at different time points, whereas the data on cytokine changes show a large variation in concentration levels and dynamic change patterns among the patients.

Transdifferentiation of periodontal ligament stem cells into acinar cells using an indirect co-culture system.

Serous Acinar Cells (ACs) are mature and functional secretory epithelial cells that develop and complete through other stem cells at the end of the ductal system. So, the regeneration of the salivary gland damaged by radiation does not occur without cell therapy. Todays, an accessible tissue like the Periodontal Ligament (PDL) of the tooth was considered to easily extract the Mesenchymal Stem Cells (MSCs). In-vitro differentiation of stem cells before transplantation to damaged tissue reduces the risk of tumorigenesis. This study was conducted to evaluate the feasibility of differentiation of PDLSCs into salivary acinar cells by a co-culture system. PDLSCs were isolated from adult human PDL tissue and co-cultured with rat parotid ACs using an indirect co-culture system. The transdifferentiation of PDLSCs was evaluated by PCR of Aquaporin 5 (AQP5) and Carbonic anhydrase 6 (CA6) genes, then quantitative real-time PCR was used to measure the gene expression levels. The data were analyzed by ANOVA. Specific bond with the correct size on 6% acrylamide gel and TBE5X buffer showed the expression of AQP5 and CA6 in PDLSCs co-cultured with acinar cells. RT-PCR revealed co-cultured PDLSCs with or without KGF (Keratinocyte Growth Factor) showed significantly increased expression of AQP5 genes in compared to the initial PDLSCs. Expression of AQP5 and CA6, indicating successful transdifferentiation of PDLSCs into ACs, in co-culture system for 3weeks.

ANGPTL4 regulates the osteogenic differentiation of periodontal ligament stem cells

The molecular mechanism of mechanical force regulating the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) has not been clearly elucidated. In this study, two mRNA-seqs, GSE106887 and GSE109167, which contained several samples of PDLSCs under mechanical force, were downloaded from Gene Expression Omnibus. Differential expression analysis was firstly taken between GSE106887 and GSE109167, then the common 84 up-regulated genes and 26 down-regulated genes were selected. Function enrichment analysis was used to identify the key genes and pathways in PDLSCs subjected to the tension and compression force. PDLSCs were isolated from human periodontal ligament tissues. The effects of ANGPTL4 knockdown with shRNA on the osteogenic differentiation of PDLSCs were studied in vitro. Then, the orthodontic tooth movement (OTM) rat model was used to study the expression of HIF-1α and ANGPTL4 in alveolar bone remodeling in vivo. ANGPTL4 and the HIF-1 pathway were identified in PDLSCs subjected to the tension and compression force. alizarin red staining, alcian blue staining, and oil red O staining verified that PDLSCs had the ability of osteogenic, chondrogenic, and adipogenic differentiation, respectively. Verification experiment revealed that the expression of ANGPTL4 in PDLSCs significantly increased when cultured under osteogenic medium in vitro. While ANGPTL4 was knocked down by shRNA, the levels of ALPL, RUNX2, and OCN decreased significantly, as well as the protein levels of COL1A1, ALPL, RUNX2, and OCN. During the OTM, the expression of HIF-1α and ANGPTL4 in periodontal ligament cells increased on the tension and compression sides. We concluded the positive relationship between ANGPTL4 and osteogenic differentiation of PDLSCs.

Identification of Dental Stem Cells Similar to Skeletal Stem Cells

Stem and progenitor cells play important roles in the development and maintenance of teeth and bone. Surface markers expressed in bone marrow–derived mesenchymal stem cells are also expressed in dental tissue–derived stem cells. Mouse skeletal stem cells (mSSCs, CD45−Ter119−Tie2−CD51+Thy−6C3−CD105−CD200+) and human skeletal stem cells (hSSCs, CD45−CD235a−TIE2−CD31−CD146−PDPN+CD73+CD164+) have been identified in bone and shown to play important roles in skeletal development and regeneration. However, it is unclear whether dental tissues also harbor mSSC or hSSC populations. Here, we employed rainbow tracers and found that clonal expansion occurred in mouse dental tissues similar to that in bone. We sorted the mSSC population from mouse periodontal ligament (mPDL) tissue and mouse dental pulp (mDP) tissue in the lower incisors by fluorescence-activated cell sorting (FACS). In addition, we demonstrated that mPDL-derived skeletal stem cells (mPDL-SSCs) and mDP-derived skeletal stem cells (mDP-SSCs) have similar clonogenic capacity, as well as cementogenic and odontogenic potential, but not adipogenic potential, similar to the characteristics of mSSCs. Moreover, we found that the dental tissue–derived mSSC population plays an important role in repairing clipped incisors. Importantly, we sorted the hSSC population from human periodontal ligament (hPDL) and human dental pulp (hDP) tissue in molars and identified its stem cell characteristics. Finally, hPDL-like and hDP-like structures were generated after transplanting hPDL-SSCs and hDP-SSCs beneath the renal capsules. In conclusion, we demonstrated that mouse and human PDL and DP tissues harbor dental stem cells similar to mSSCs and hSSCs, respectively, providing a precise stem cell population for the exploration of dental diseases.

The profiling and analysis of gene expression in human periodontal ligament tissue and fibroblasts.

ObjectivesThe periodontal ligament (PDL) is an important component of periodontium to support dental structure in the alveolar socket. Regeneration of PDL tissue is an effective treatment option for periodontal disease and the profiling of genes involved in this process will be informative. Therefore, our study aims to accurately delineate the profiling of gene expression for PDL tissue regeneration.Materials and MethodsWe isolated PDL tissues and PDL fibroblasts (PDLFs) from premolar teeth, which were extracted from healthy periodontal status patients undergoing orthodontic treatment. Messenger RNA (mRNA) expression in PDL tissue and PDLFs were analyzed using Cap analysis gene expression, which is a second‐generation sequencing technique to create profiling. We also determined the protein expression using Western blot.ResultsCollagens (type I, III, and VI), noncollagenous proteins (periostin and osteonectin), and proteoglycans (asporin, lumican, decorin, and osteomodulin) were highly expressed in PDL tissue. Integrin, β1 was also expressed in PDL tissue. On comparison of gene expression between PDL tissue and PDLFs, four PDL marker genes, osteopontin, asporin, periostin, and osteonectin, were decreased in PDLFs. The genes for gene regulation were also highly expressed.ConclusionsOur study demonstrated the overall profiling of mRNA expression in PDL tissue and analyzed the important genes which may be useful for providing specific information for the reconstruction of PDL. We also identified the difference in gene expression between PDL tissue and PDLFs which might provide insights towards PDL regeneration.

Matrix metalloproteinases and Th17 cytokines in the gingival crevicular fluid during orthodontic tooth movement.

Matrix metalloproteinases (MMPs) contribute to remodeling in orthodontic tooth movement (OTM). Moreover, IL-17 can promote the production of MMPs. This study aimed to investigate the regulation of Th17 on MMPs expression during OTM. Eighteen children undergoing orthodontic treatment were recruited. The gingival crevicular fluid (GCF) was collected at different time points: the day of application (T0), one hour (T1), 24 hours (T2), one week (T3), 4 weeks (T4) and 12 weeks (T5) after the application of orthodontic force. Th17 cell-related cytokines and MMPs expression were measured in GCF by Multiplex Luminex analyser. Human periodontal ligament (hPDL) tissues were stimulated by IL-17. The levels of IL-17 and MMPs (MMP-1, MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13) of the study teeth at T2-T4 were significantly up-regulated compared with that of T0 and T1 and decreased to baseline level at T5. We found that the expression of IL-17 was correlated with MMPs. After rhIL-17 treatment, the expression of MMP-1, MMP-2, and MMP-9 were up-regulated significantly. The IL-17 expression was positively correlated with MMPs. IL-17 promotes the expression of MMP-1, MMP-2, and MMP-9 by hPDL cells, suggesting that IL-17 plays a crucial role in the remodeling during OTM.

Irradiation with red light-emitting diode enhances proliferation and osteogenic differentiation of periodontal ligament stem cells.

This study aimed to evaluate the effects of low-energy red light-emitting diode (LED) irradiation on the proliferation and osteogenic differentiation of periodontal ligament stem cells (PDLSCs). PDLSCs were derived from human periodontal ligament tissues of premolars and were irradiated with 0 (control group), 1, 3, or 5 J/cm2 red LED in osteogenic induction medium. Cell proliferation was analyzed using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Osteogenic differentiation activity was evaluated by monitoring alkaline phosphatase (ALP) activity, alizarin red staining, and real-time polymerase chain reaction (RT-PCR) results. Osteoblast-associated proteins (Runx2, OCN, OPN, and BSP) were detected using western blotting. The results of the MTT assay indicated that PDLSCs in the irradiation groups exhibited a higher proliferation rate than those in the control group (P < 0.05). ALP results showed that after 7 days of illumination, only 5 J/cm2 promoted the expression of ALP of PDLSCs. However, after 14 days of illumination, the irradiation treatments did not increase ALP activity. The results of alizarin red staining showed that red LED promoted osteogenic differentiation of the PDLSCs. The real-time polymerase chain reaction (RT-PCR) results demonstrated that red LED upregulated the expression levels of osteogenic genes. Expression of the proteins BSP, OPN, OCN, and Runx2 in the irradiation groups was higher than that in the control group. Our results confirmed that low-energy red LED at 1, 3, and 5 J/cm2 promotes proliferation and osteogenic differentiation of PDLSCs.

Analysis of lncRNAs-miRNAs-mRNAs networks in periodontal ligament stem cells under mechanical force.

Our study aims to analyze the expression profiles of long non-coding RNAs (lncRNAs) and investigate the potential regulatory networks among lncRNAs, microRNAs (miRNAs), and mRNAs in periodontal ligament stem cells (PDLSCs) under mechanical force (MF). PDLSCs were isolated from human periodontal ligament tissues and identified by flow cytometry analysis. Multidirectional differentiation potential of PDLSCs was obtained by osteogenic and adipogenic induction. High-throughput RNA sequencing was used to identify the expression patterns of lncRNAs and mRNAs in PDLSCs under MF. MF-responsive miRNAs were obtained from the previous microarray data. LncRNAs-miRNAs-mRNAs networks were constructed by Cytoscape. PDLSCs cultured from the periodontal ligament tissues were positive for STRO-1, CD146 and negative for CD45, CD34. Alizarin red staining and Oil Red O staining showed that PDLSCs had the ability of osteogenic and adipogenic differentiation. Then, a total of 1,339 and 1,426 differentially expressed lncRNAs and mRNAs were identified, respectively, in PDLSCs under MF. Based on the previous miRNA microarray analysis, the potential interaction networks of lncRNAs-miRNAs-mRNAs were constructed. It was found that lncRNAs and mRNAs could competitively interact with the same miRNA. LncRNAs-miRNAs-mRNAs networks were involved in PDLSCs under MF, which might provide a novel mechanism in the regulation of clinical orthodontic tooth movement process.

Overview of noncoding RNAs involved in the osteogenic differentiation of periodontal ligament stem cells.

Periodontal diseases are infectious diseases that are characterized by progressive damage to dental support tissue. The major goal of periodontal therapy is to regenerate the periodontium destroyed by periodontal diseases. Human periodontal ligament (PDL) tissue possesses periodontal regenerative properties, and periodontal ligament stem cells (PDLSCs) with the capacity for osteogenic differentiation show strong potential in clinical application for periodontium repair and regeneration. Noncoding RNAs (ncRNAs), which include a substantial portion of poly-A tail mature RNAs, are considered "transcriptional noise." Recent studies show that ncRNAs play a major role in PDLSC differentiation; therefore, exploring how ncRNAs participate in the osteogenic differentiation of PDLSCs may help to elucidate the underlying mechanism of the osteogenic differentiation of PDLSCs and further shed light on the potential of stem cell transplantation for periodontium regeneration. In this review paper, we discuss the history of PDLSC research and highlight the regulatory mechanism of ncRNAs in the osteogenic differentiation of PDLSCs.

Comparative study with the effect of stromal cell derived factor-1 on osteogenic differentiation of human healthy and inflammatory periodontal ligament stem cells

This study aims to compare the osteogenic differentiation capability of stem cells derived from human inflammatory periodontal ligament tissues (iPDLSCs) with those of stem cells derived from healthy periodontal ligament tissues (hPDLSCs). Both types of tissues were induced by stromal cell derived factor (SDF-1) in vitro. iPDLSCs and hPDLSCs were primarily cultured by tissue digestion method and purified by limited dilution cloning. The cells were passaged and identified by stem cell surface marker expression through flow cytometry. Then, we used thiazolyl blue tetrazolium bromide to detect and compare the proliferation capabilities of the iPDLSCs and hPDLSCs. Express of bone volumes were detected by alizarin red staining after SDF-1 was added to the cells. Using alkaline phosphatase, we evaluated the osteogenic differentiation capability of the cells induced by SDF-1. The expression levels of the osteogenesis-related genes of the cells induced by SDF-1 were determined by reverse transcription-polymerase chain reaction. After purification, both iPDLSCs and hPDLSCs expressed stem cell markers. hPDLCSs had a higher proliferation capability than iPDLSCs. Osteogenesis-related genes had higher expression levels in the cells induced by SDF-1 than in those without induction (P<0.05). SDF-1 at 50 and 200 ng·mL⁻¹ concentration greatly affected the differen-tiation capabilities of iPDLSCs and hPDLSCs respectively. iPDLSCs and hPDLSCs had osteogenic differentia-tion capability. The level of osteogenic differentiation in normal and inflamed periodontal ligament stem cells increases after SDF-1 induction.

Accelerated construction of an in vitro model of human periodontal ligament tissue: vacuum plasma combined with fibronectin coating and a polydimethylsiloxane matrix.

Tying shape memory wires to crowded teeth causes the wires to deform according to the dental arch. This deformation results in a resilient force that is delivered to the tooth. The appropriate amount of force can activate the osteogenetic and osteoclastic ability of the periodontal ligament (PDL) and the tooth can be moved. This is the biological basis of orthodontic treatment. To achieve further insight into the mechanisms underlying orthodontic treatment, we examined whether accelerated construction of an in vitro human PDL fibroblast (HPdLF) stretching model can be achieved by combining fibronectin coating and vacuum plasma treatment with polydimethylsiloxane (PDMS) cell-culture chambers. Each chamber was randomly assigned to a no-surface modification (NN), fibronectin coating (FN), vacuum plasma treatment (PN), or vacuum plasma treatment followed by a fibronectin coating (PF) treatment protocol. The physical and chemical features and ability to promote cellular proliferation of the PDMS chamber surfaces were evaluated. Cellular adhesion of four materials were evaluated and two best-proliferated groups were considered as better model-constructing surfaces and used in subsequent experiments and used in subsequent experiments. HPdLFs were cultured on these two kinds of chambers without stretching for 3 days, then with stretching for 7 days. Time-course gene expression cellular morphology were evaluated. Chambers in the PN group had high wettability and surface component changes. The FN and PF chambers had high cellular proliferation ability. They were selected into subsequent experiments. After 3 days of culturing HPdLFs on the PF and PN chambers, the cells in the PF chambers had significantly higher levels of runt-related transcription factor 2 (Runx-2) and osteocalcin (OCN) gene expression compared with the cells in the PN chambers. After cyclic stretch application to the cells in the PN and PF chambers, expression of the type-3 collagen (COL-3) gene in PF group continued to increase for 7 days and was significantly higher than that in the PN group from day 5 onwards. The HPdLFs in the PF group showed parallel alignment from days 3 to 7 after imposition of cyclic stretch, while those in the PN group aligned in parallel from day 5 on. Our results suggested that applying a fibronectin coating to a PDMS chamber after plasma treatment can accelerate establishment of an in vitro PDL stretching model.

Evaluation and comparison of Vitamin D receptors in periodontal ligament tissue of Vitamin D-deficient chronic periodontitis patients before and after supplementation of Vitamin D3.

Background:Vitamin D, an important hormone required by the body, exerts its biological effects through Vitamin D receptors (VDRs) present on target cells. Vitamin D is ineffective in tissues which lack VDR. Various tissues show the presence of VDRs. However, evidence for the presence of VDRs in human periodontal ligament tissue in fully erupted teeth in adults is lacking. The present study intends to evaluate the presence of VDRs in periodontal ligament (PDL) tissue and assess their response to serum Vitamin D3 levels in chronic periodontic patients.Materials and Methods:A total of 19 chronic periodontitis patients were enrolled in the study and tested for serum 25(OH)D3 levels. Deficient patients were supplemented with Vitamin D3. PDL tissue of these patients was isolated after tooth extraction before and after supplementation of Vitamin D3 and analyzed for the presence of VDR in PDL tissue by using enzyme-linked immunosorbent assay.Results:All the chronic periodontitis patients were found to be deficient in Vitamin D3. The mean serum 25(OH)D3 level before supplementation was 13.96 ng/mL which significantly increased to 35.12 ng/mL after supplementation of Vitamin D3 for 6 weeks. VDR analysis determined mean VDR conc. in PDL tissue to be -1.443 ng/mL, which increased to 2.38 ng/mL after supplementation. A concentration dependent correlation was seen between serum 25(OH)D3 levels and VDR conc. in PDL tissue after supplementation.Conclusions:The study determined Vitamin D Receptors (VDR) in PDL tissue after supplementation of Vitamin D. Thus in addition to the standard treatment modalities, Vitamin D3 supplementation would be an important factor for generation of adequate immune response.

Activation of autophagy in periodontal ligament mesenchymal stem cells promotes angiogenesis in periodontitis.

Angiogenesis alteration in tooth support tissue plays an essential role in periodontitis. Mesenchymal stem cells (MSCs) can affect vessel formation by endothelial cells (ECs) through paracrine function. Autophagy is reported to be closely related to cell secretion. Here we investigated the angiogenesis-promoting ability of MSCs that reside in the periodontal ligament (known as periodontal ligament stem cells, PDLSCs) under inflammatory conditions in order to explore the mechanism of angiogenesis alteration in periodontitis. PDLSCs were isolated from healthy and inflamed human periodontal ligament tissues (HPDLSCs and PPDLSCs, respectively). HPDLSCs were subjected to an inflammatory environment (IPDLSCs) in vitro using inflammatory cytokines. Angiogenesis-promoting cytokine expression and autophagy were evaluated in PDLSCs by quantitative reverse transcription-polymerase chain reaction and western-blot analysis before co-culturing them with ECs. The angiogenesis ability of ECs in the co-culture system was examined by a matrigel tube formation test. Rapamycin and pcDNA for Beclin-1 (cDNA-Beclin-1) were used to promote autophagy in PDLSCs and siRNA Beclin-1 (siBeclin-1) was used to repress it. The inflammatory environment increased autophagy and the expression of basic fibroblast growth factor (bFGF) and angiogenin (Ang) in PDLSCs. More tube formation was observed in ECs from the co-culture system which was pretreated with tumor necrosis factor (TNF)-α and interleukin (IL)-1β. PDLSCs treated with rapamycin or transfected with cDNA-Beclin-1 showed higher expression levels of bFGF and Ang that promoted tube formation by the co-cultured ECs. PDLSCs transfected with siBeclin-1 resulted in the opposite results. Autophagy modulates angiogenesis-promoting ability of PDLSCs, which could be increased by an inflammatory environment.

Stemness Characteristics of Periodontal Ligament Stem Cells from Donors and Multiple Sclerosis Patients: A Comparative Study.

Multiple sclerosis (MS) is the most prevalent and progressive autoimmune disease that affects the central nervous system, and currently, no drug is available for the treatment. Stem cell therapy has received substantial attention in MS treatment. Recently, we demonstrated the immunosuppressive effects of mesenchymal stem cells derived from neural crest-originated human periodontal ligament tissue (hPDLSCs) in an in vivo model of MS. In the present study, we comparatively investigated the stemness properties of hPDLSCs derived from healthy donors and relapsing-remitting MS patients. Stem cell marker expression, cell proliferation, and differentiation capacity were studied. We found that both donor- and MS patient-derived hPDLSCs at early passage 2 showed similar expression of surface antigen markers and cell proliferation rate. Significant level of osteogenic, adipogenic, chondrogenic, and neurogenic differentiation capacities was observed in both donor- and MS patient-derived hPDLSCs. Interestingly, these cells maintained the stemness properties even at late passage 15. Senescence markers p16 and p21 expression was considerably enhanced in passage 15. Our results propose that hPDLSCs may serve as simple and potential autologous stem cell niche, which may help in personalized stem cell therapy for MS patients.

Mineral trioxide aggregate enhances the osteogenic capacity of periodontal ligament stem cells via NF-κB and MAPK signaling pathways.

Mineral trioxide aggregate (MTA), as a bioactive material, has a widespread application in clinical practice. To date, the effects of MTA on the proliferation and differentiation of human periodontal ligament stem cells (hPDLSCs) remain unclear. hPDLSCs were isolated from human periodontal ligament tissues and cultured with MTA conditioned media. Cell counting kit-8 (CCK-8) assay was performed to assess the proliferation capacity of MTA-treated hPDLSCs. Immunofluorescence assay, alkaline phosphatase (ALP) activity, alizarin red staining, real-time RT-PCR, and western blot analyses were used to investigate the odonto/osteogenic capacity of hPDLSCs as well as the involvement of NF-κB and MAPK pathways. ALP activity assay revealed that 2 mg/ml was the optimal concentration for the induction of hPDLSCs by MTA. The protein expression of DSP, RUNX2, OCN, OSX, OPN, DMP1, ALP, and COL-I in MTA-treated hPDLSCs was significantly higher than those in control group (p < 0.01). When hPDLSCs were treated with the inhibitors of NF-κB and MAPK pathways (U0126, SP600125, SB203580, and BMS345541), the effects of MTA on the differentiation of hPDLSCs were suppressed. Mechanistically, P65 was detected to transfer from cytoplasm to nuclei, as indicated by western blot and immunofluorescence assay. Moreover, MAPK-related proteins and its downstream transcription factors were also upregulated in MTA-treated hPDLSCs. Together, mineral trioxide aggregate can promote the odonto/osteogenic capacity of hPDLSCs via activating the NF-κB and MAPK pathways.

Establishment and characterization of novel epithelial-like cell lines derived from human periodontal ligament tissue in vitro

In this study, novel human-derived epithelial-like cells (hEPLCs) lines were established from periodontal ligament (PDL) tissues, which were composed of a variety of cell types and exhibited complex cellular activities. To elucidate the putative features distinguishing these from epithelial rest of Malassez (ERM), we characterized hEPLCs based on cell lineage markers and tight junction protein expression. The aim of this study was, therefore, to establish and characterize hEPLCs lines from PDL tissues. The hEPLCs were isolated from PDL of third molar teeth. Cellular morphology and cell organelles were observed thoroughly. The characteristics of epithelial–endothelial-mesenchymal-like cells were compared in several markers by gene expression and immunofluorescence, to ERM and human umbilical-vein endothelial cells (HUVECs). The resistance between cellular junctions was assessed by transepithelial electron resistance, and inflammatory cytokines were detected by ELISA after infecting hEPLCs with periodontopathic bacteria. The hEPLCs developed into small epithelial-like cells in pavement appearance similar to ERM. However, gene expression patterns and immunofluorescence results were different from ERM and HUVECs, especially in tight junction markers (Claudin, ZO-1, and Occludins), and endothelial markers (vWF, CD34). The transepithelial electron resistance indicated higher resistance in hEPLCs, as compared to ERM. Periodontopathic bacteria were phagocytosed with upregulation of inflammatory cytokine secretion within 24 h. In conclusion, hEPLCs that were derived using the single cell isolation method formed tight multilayers colonies, as well as strongly expressed tight junction markers in gene expression and immunofluorescence. Novel hEPLCs lines exhibited differently from ERM, which might provide some specific functions such as metabolic exchange and defense mechanism against bacterial invasion in periodontal tissue.

Conditioned medium of periodontal ligament mesenchymal stem cells exert anti-inflammatory effects in lipopolysaccharide-activated mouse motoneurons

Conditioned medium derived from mesenchymal stem cells (MSCs) shows immunomodulatory and neuroprotective effects in preclinical models. Given the difficulty to harvest MSCs from bone marrow and adipose tissues, research has been focused to find alternative resources for MSCs, such as oral-derived tissues. Recently, we have demonstrated the protective effects of MSCs obtained from healthy human periodontal ligament tissue (hPDLSCs) in murine experimental autoimmune encephalomyelitis model. In the present in vitro study, we have investigated the immunomodulatory and neuroprotective effects of conditioned medium obtained from hPDLSCs of Relapsing Remitting- Multiple sclerosis (RR-MS) patients on NSC34 mouse motoneurons stimulated with lipopolysaccharide (LPS). Immunocytochemistry and western blotting were performed. Increased level of TLR4 and NFκB, and reduced level of IκB-α were observed in LPS-stimulated motoneurons, which were modulated by pre-conditioning with hPDLSC-conditioned medium. Inflammatory cytokines (TNF-α, IL-10), neuroprotective markers (Nestin, NFL 70, NGF, GAP43), and apoptotic markers (Bax, Bcl-2, p21) were modulated. Moreover, extracellular vesicles of hPDLSC-conditioned medium showed the presence of anti-inflammatory cytokines IL-10 and TGF-β. Our results demonstrate the immunosuppressive properties of hPDLSC-conditioned medium of RR-MS patients in motoneurons subjected to inflammation. Our findings warrant further preclinical and clinical studies to elucidate the autologous therapeutic efficacy of hPDLSC-conditioned medium in neurodegenerative diseases.

Cell Responses to Conditioned Media Produced by Patient-Matched Stem Cells Derived From Healthy and Inflamed Periodontal Ligament Tissues.

Periodontal ligament stem cells (PDLSCs) derived from clinically compromised teeth with periodontitis are considered a readily accessible cell source, but their impaired stem cell functionalities, as observed in various in vitro and in vivo models, necessitate further investigation of these inflamed cells before their translation into therapeutic applications. In this study, the effects of conditioned media (CM) produced by stem cells derived from human healthy periodontal ligament tissues (H-PDLSCs) or inflamed periodontal ligament tissues (I-PDLSCs), referred to as H-CM and I-CM, respectively, on the biologic properties of H-PDLSCs and I-PDLSCs from the same donor are compared to explore the extent to which inflamed cells can be rescued by their extrinsic environment (i.e., by H-CM). H-CM and I-CM were prepared from in vitro cell cultures, and the cellular responses of H-PDLSCs and I-PDLSCs to patient-matched H-CM and I-CM were investigated in terms of colony-forming ability, cell proliferation, and adipogenic/osteogenic differentiation. In H-CM and I-CM, H-PDLSCs and I-PDLSCs exhibited similar adipogenic potential. However, when incubated in I-CM, both cell types demonstrated an increased capacity to proliferate but a decreased capacity to differentiate into osteoblasts. Significantly, the impaired osteogenic differentiation of I-PDLSCs was partially rescued by incubation in H-CM under osteo-inducing conditions. The CM of patient-matched H-PDLSCs and I-PDLSCs differed, and the impaired osteogenic differentiation of inflamed stem cells had the potential to be rescued, at least partially, for therapeutic use via changing the cell culture microenvironment in vitro.

The expression of serine protease HtrA1 in human periodontal ligament tissue and the effect of HtrA1 on the proliferation of human periodontal ligament cells

To observe the expression of serine protease HtrA1 in human periodontal ligament tissue and to explore the effect of HtrA1 on the proliferation of human periodontal ligament cells (hPDLC). Six human premolars and three human third molars(patient's ages ranging from 12 to 25, with intact root, without caries and/or periodontitis) were obtained in the Department of Maxillofacial Surgery of Wuhan University Hospital of Stomatology. Reverse transcription-PCR(RT-PCR) and immunohistochemistry analysis were applied to investigate the expression of HtrA1. Primary hPDLC were obtained by tissue-culture method in vitro. The proliferation of hPDLC was determined by methyl thiazolytetrazolium(MTT). Lentivirus-mediated over-expression and reduction of HtrA1 level was performed. An empty vector was used as negative control. On days 1, 3, 5, 7 and 9, the growth of hPDLC was characterized using cell counting kit-8(CCK-8) assay. RT-PCR data indicated that HtrA1 mRNA was expressed in human periodontal ligament tissue. Immunohistochemistry analysis showed HtrA1 was expressed in human periodontal ligament, mainly in the cytoplasm of hPDLC and the extracellular matrix. The MTT result suggested that the growth curve was consistent with the growth characteristics of hPDLC. The stable over-expression and knockdown cell lines was successfully established by lentivirus with more than 90% transfection efficiency. CCK-8 assay showed that HtrA1 over-expression inhibited the proliferation of hPDLC(0.897±0.060, 0.890±0.083, 1.631±0.038, 1.111±0.041, 1.110±0.189), while cell proliferation increased after down-regulation of HtrA1(0.329±0.021, 0.529±0.044, 0.973±0.056, 1.626±0.102, 2.344±0.198)(P<0.05). HtrA1 is expressed in human periodontal ligament tissue at both mRNA and protein levels, and may play an important role in regulating the proliferation of hPDLC.

Cell survival and gene expression under compressive stress in a three-dimensional in vitro human periodontal ligament-like tissue model.

This study investigated cell survival and gene expression under various compressive stress conditions mimicking orthodontic force by using a newly developed in vitro model of human periodontal ligament-like tissue (HPdLLT). The HPdLLT was developed by three-dimensional culturing of human periodontal ligament fibroblasts in a porous poly-L-lactide matrix with threefold increased culture media permeability due to hydrophilic modification. In vitro HPdLLTs in experimental groups were subjected to 5, 15, 25 and 35g/cm(2) compressive stress for 1, 3, 7 or 14days; controls were cultured over the same periods without compressive stress. Cell morphology and cell apoptosis in the experimental and control groups were investigated using scanning electron microscopy and caspase-3/7 detection. Real-time polymerase chain reaction was performed for seven osteogenic and osteoclastic genes. Similar extracellular matrix and spindle-shaped cells were observed inside or on the surface of in vitro HPdLLTs, with no relation to compressive stress duration or intensity. Similar caspase-3/7 activity indicating comparable apoptosis levels was observed in all samples. Receptor activator of nuclear factor kappa-B ligand and bone morphogenetic protein 2 genes showed characteristic "double-peak" expression at 15 and 35g/cm(2) on day 14, and alkaline phosphatase and periodontal ligament-associated protein 1 expression peaked at 5g/cm(2) on day 14; other genes also showed time-dependent and load-dependent expression patterns. The in vitro HPdLLT model system effectively mimicked the reaction and gene expression of the human periodontal ligament in response to orthodontic force. This work provides new information on the effects of compressive stress on human periodontal ligament tissue.