Periodontal Ligament Tissue Research Articles

Cryopreservation of human dental roots using vitrification for autologous human tooth tissue banking

This study aims to preliminarily evaluate the feasibility of autologous transplantation of tooth tissues cryopreserved with vitrification, by investigating the influence of cryopreservation with vitrification on human dental root, regarding the morphology, microhardness, cell apoptosis, proliferation and differentiation. Freshly extracted human permanent premolars were collected with crown removed. Dental roots were cryopreserved using a commercial vitrification medium (Kitazatousa). After six-month storage in liquid nitrogen, cryopreserved roots were thawed, and then evaluated using histological and immunohistochemical methods. Microhardness of dentine was measured with a Vickers indenter. Cells in periodontal ligament and dental pulp tissues were isolated and characterized. The proliferation, immunophenotype, apoptosis and differentiation ability of cells isolated from cryopreserved roots were evaluated. The data was analyzed using one-way analysis of variance (ANOVA) and Student's t-test. The gross and histological morphology of dental roots was not significantly changed after vitrification and thawing. A few tiny cracks were found in 3 of all 10 cryopreserved samples. No obvious changes were found in microstructure of dentine under SEM observation. Dental pulp cells and periodontal ligament cells were successfully isolated from tissues of cryopreserved human dental roots. There were also no significant differences of those periodontal ligament cells in the two groups regarding morphology, immunophenotype, viability, proliferation and apoptosis. The osteogenic and adipogenic differentiation capability of periodontal ligament cells was maintained by cryopreservation with vitrification. In the conditions of this study, cryopreservation with vitrification preserves cell survival, hardness and structural integrity of dental roots. Vitrification can be a potential way to preserve tooth tissue for future auto-transplantation and autologous cell therapy.

Decorin Promotes Osteoblastic Differentiation of Human Periodontal Ligament Stem Cells

The aim of this study is to clarify the biological functions of decorin (DCN) in the healing and regeneration of wounded periodontal tissue. We investigated the expression pattern of DCN during the healing of wounded periodontal tissue in rats by immunohistochemistry and the effects of DCN on the osteoblastic differentiation of human periodontal ligament (PDL) stem cells (HPDLSCs) and preosteoblasts by Alizarin red S staining, quantitative reverse transcription-polymerase chain reactions, and western blotting. The expression of DCN was increased around the wounded PDL tissue on day 5 after surgery compared with the nonwounded PDL tissue, whereas its expression was not changed in the osteoblastic layer around the wounded alveolar bone. Furthermore, DCN promoted the osteoblastic differentiation of HPDLSCs, but it did not affect the osteoblastic differentiation of preosteoblasts. ERK1/2 phosphorylation was upregulated during the DCN-induced osteoblastic differentiation of HPDLSCs. DCN did not affect proliferation, migration, or the PDL-related gene expression of HPDLSCs. In conclusion, this study demonstrates that DCN has a role in the healing of wounded periodontal tissue. Furthermore, DCN secreted from PDL cells may contribute to bone healing by upregulating osteoblastic differentiation through ERK1/2 signaling in HPDLSCs, indicating a therapeutic effect of DCN in periodontal tissue regeneration.

Role of biologic modifiers in periodontal regeneration- A review

Regenerative techniques, which aim to replace damaged periodontal ligament, bone, cementum, and connective tissue, are newer approaches to periodontal care. In periodontology and oral implantology, the development of molecular mediators has accelerated substantially over the last decade. At various cellular levels, different growth agents cause distinct reactions in periodontal tissues. The potency of biologics in regenerating the periodontal tissues is the call attention in this review paper, in which it discusses the structure, mechanism of action, indication and FDA (Food and Drug Administration) approval to use in regenerating periodontal tissues. The working activity of biologic agents together with reasons for utilizing them in regenerating tissues of periodontium lost due to disease are discussed, as well as the expected benefits as compared to traditional approaches.

Intermittent compressive force regulates human periodontal ligament cell behavior via yes-associated protein

Intermittent compressive force influences human periodontal ligament (PDL) cell behavior that facilitates periodontal tissue regeneration. In response to mechanical stimuli, Yes-associated protein (YAP) has been recognized as a mechanosensitive transcriptional activator that regulates cell proliferation and cell fate decisions. This study aimed to investigate whether compressive forces influence cell proliferation and cell fate decisions of human PDL cells via YAP signaling. YAP expression was silenced by shRNA. The effect of YAP on cell proliferation, adipogenesis and osteogenesis of PDL cells under ICF loading were determined. Adipogenic differentiation bias upon ICF loading was confirmed by fourier-transform infrared spectroscopy (FTIR). The results revealed that ICF-induced YAP promotes osteogenesis, but it inhibits adipogenesis in PDL cells. Depletion of YAP results in PDL cells that are irresponsive to ICF and, therefore, the failure of the PDL cells to undergo osteogenic differentiation. This was shown by a significant reduction in calcium deposited in the CF-derived osteoblasts of the YAP-knockdown (YAP-KD) PDL cells. As to control treatment, reduction of YAP promoted adipogenesis, whereas ICF-induced YAP inhibited this mechanism. However, the adipocyte differentiation in YAP-KD cells was not affected upon ICF treatment as the YAP-KD cells still exhibited a better adipogenic differentiation that was unrelated to the ICF. This study demonstrated that, in response to ICF treatment, YAP could be a crucial mechanosensitive transcriptional activator for the regulation of PDL cell behavior through a mechanobiological process. Our results may provide the possibility of facilitating PDL tissue regeneration by manipulation of the Hippo-YAP signaling pathway.

PAX9 Is Involved in Periodontal Ligament Stem Cell-like Differentiation of Human-Induced Pluripotent Stem Cells by Regulating Extracellular Matrix.

Periodontal ligament stem cells (PDLSCs) play central roles in periodontal ligament (PDL) tissue homeostasis, repair, and regeneration. Previously, we established a protocol to differentiate human-induced pluripotent stem cell-derived neural crest-like cells (iNCs) into PDLSC-like cells (iPDLSCs) using human PDL cell-derived extracellular matrix (ECM). However, it remained unclear what factors principally regulate the differentiation of iNCs into iPDLSCs. In this study, we aimed to identify the transcription factor regulating production of human PDL cell-derived ECM, which is responsible for the generation of iPDLSCs. We cultured iNCs on ECMs of two human PDL cell lines (HPDLC-3S and HPDLC-3U) and of human dermal fibroblasts (HDF). iNCs cultured on HPDLC-3U demonstrated higher iPDLSC-associated gene expression and mesenchymal differentiation capacity than cells cultured on HDF or HPDLC-3S. The transcription factor PAX9 was highly expressed in HPDLC-3U compared with HDF and HPDLC-3S. iNCs cultured on siPAX9-transfected HPDLC-3U displayed downregulation of iPDLSC-associated marker expression and adipocytic differentiation capacity relative to controls. Our findings suggest that PAX9 is one of the transcription factors regulating ECM production in human PDL cells, which is responsible for the differentiation of iNCs into iPDLSCs.

Actin alpha 2, smooth muscle, a transforming growth factor-β1-induced factor, regulates collagen production in human periodontal ligament cells via Smad2/3 pathway

Actin alpha 2, smooth muscle (ACTA2) is an actin isoform that forms the cytoskeleton. Actin plays a crucial role in numerous cellular functions. ACTA2 is a marker of functional periodontal ligament (PDL) fibroblasts and is upregulated by transforming growth factor-β1 (TGF-β1); however, the underlying function of ACTA2 in PDL tissue is unknown. We aimed to examine the localization and potential function of ACTA2 in PDL tissues and cells. RNA expression was determined using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and quantitative RT-PCR. Protein expression was determined using immunofluorescence staining and Western blot analysis. Soluble and insoluble collagen production was examined using the Sircol collagen assay and picrosirius red staining, respectively. Small interfering RNA (siRNA) was used for knockdown assay to examine the effect of ACTA2 in human PDL cells. ACTA2 expression was observed in human primary PDL cells and PDL cell line (2-23cells). TGF-β1 upregulated ACTA2, collagen type Ⅰ alpha1 chain (COL1A1), periostin (POSTN), and fibrillin-Ⅰ(FBN1) expression and soluble and insoluble collagen production in 2-23cells. However, ACTA2 depletion by siRNA strongly suppressed PDL-related gene expression and collagen production compared with those of TGF-β1-stimulated control cells. Furthermore, ACTA2 knockdown significantly suppressed the phosphorylation of Smad2 and Smad3. ACTA2 plays a crucial role in PDL-related marker expression and collagen production via Smad2/3 phosphorylation. Our findings might contribute to the development of novel and effective periodontal therapies.

Viability and Adhesion of Periodontal Ligament Fibroblasts on a Hydroxyapatite Scaffold Combined with Collagen, Polylactic Acid-Polyglycolic Acid Copolymer and Platelet-Rich Fibrin: A Preclinical Pilot Study.

Background: Conventional periodontal therapy relies on bone regeneration strategies utilizing scaffolds made of diverse materials, among which collagen, to promote cell adhesion and growth. Objective: To evaluate periodontal ligament fibroblast (HPdLF) cell adhesion and viability for periodontal regeneration purposes on hydroxyapatite scaffolds containing collagen (HAp-egg shell) combined with polylactic acid–polyglycolic acid copolymer (PLGA) and Platelet-Rich Fibrin (PRF). Methods: Four variations of the HAp-egg shell were used to seed HPdLF for 24 h and evaluate cell viability through a live/dead assay: (1) (HAp-egg shell/PLGA), (2) (HAp-egg shell/PLGA + collagen), (3) (HAp-egg shell/PLGA + PRF) and (4) (HAp-egg shell/PLGA + PRF + collagen). Cell adhesion and viability were determined using confocal microscopy and quantified using central tendency and dispersion measurements; significant differences were determined using ANOVA (p < 0.05). Results: Group 1 presented low cell viability and adhesion (3.70–10.17%); groups 2 and 3 presented high cell viability and low cell adhesion (group 2, 59.2–11.1%, group 3, 58–4.6%); group 4 presented the highest cell viability (82.8%) and moderate cell adhesion (45%) (p = 0.474). Conclusions: The effect of collagen on the HAp-egg shell/PLGA scaffold combined with PRF favored HPdLF cell adhesion and viability and could clinically have a positive effect on bone defect resolution and the regeneration of periodontal ligament tissue.

Wnt5a-Ror2 signaling mediates root resorption

This study aimed to investigate the role of wingless-type MMTV integration site family member 5a (Wnt5a)-receptor tyrosine kinase-like orphan receptor 2 (Ror2) signaling in root resorption. The messenger RNA (mRNA) expression of Wnt5a, Ror2, and RANKL in periodontal ligament cells (PDLCs) under compression force (CF) with or without Ror2 small interfering RNA (siRNA) were measured by quantitative reverse transcription-polymerase chain reaction, and these proteins released into culture supernatants were measured using enzyme-linked immunosorbent assay. Then these PDLC-conditioned media under CF with or without Ror2 siRNA were used to culture osteoclast precursors to detect osteoclastogenesis effects via tartrate-resistant acid phosphatase staining. In invivo studies, the odontoclast number and the root resorption volume under excessive CF with or without Ror2 siRNA were investigated by tartrate-resistant acid phosphatase immunohistochemical staining and microcomputed tomography. The protein levels for Wnt5a, Ror2, and receptor activator of nuclear factor-kappa B ligand (RANKL) in the periodontal ligament tissues were also detected using immunohistochemical staining. Finally, the odontoclast number, root resorption volume, and the mRNA and protein expressions were compared between immature and mature teeth. The mRNA production and protein release level of Wnt5a, Ror2, and RANKL increased after CF, whereas they were significantly downregulated with Ror2 siRNA. The osteoclast number increased treating with culture medium from PDLC applying CF, but the increase was inhibited after adding Ror2 siRNA. In the animal model, the odontoclast number and root resorption volume significantly increased in the CF group but decreased in the CF with the Ror2 siRNA group. The protein levels of Wnt5a, Ror2, and RANKL in periodontal ligament were upregulated under excessive CF, and the pathway was inhibited with Ror2 siRNA. In the immature tooth group, the odontoclast number, root resorption volume, and the mRNA and protein expressions of Wnt5a-Ror2 signaling were reduced. Wnt5a-Ror2 signaling in PDLCs enhanced by excessive CF could promote RANKL release and induce precursor differentiation, partly leading to increased odontoclast activity and ultimate root resorption. The less resorption of the immature tooth may be due to odontoclastogenesis inhibition by decreased expression of Wnt5a-Ror2 signaling.

Knowledge, Awareness and Perception of Dental Stem Cell and Their Applications in Regenerative Medicine Among Professional Groups

Introduction:Dental stem cell research has become an important field in regenerative medicine. Nevertheless, the depth of knowledge and awareness of dental stem cells remain vague among the professional groups in Malaysia. The objective of this study is to assess the level of knowledge and awareness of professional groups in Malaysia on dental stem cells and their applications in regenerative medicine.Methods:A descriptive cross-sectional survey was implemented using online and hard-copy questionnaires that was distributed among medical doctors, scientists, and dentists to assess the level of knowledge and awareness (n=157). The questionnaire consists of 3 sections consisting of demographics, knowledge assessment and perception among the professional groups. The data were analysed using SPSS analysis.Results:Results showed that professional groups in Malaysia were knowledgeable and aware of dental stem cells and their application. Majority of the respondents were dentists (41.91%), followed by medical doctors (35.29%) and scientists (22.79%). 50.74% of respondents were unsure about the similar characteristics between dental stem cells (DSCs) and bone marrow mesenchymal stem cells (BMMSCs). Most respondents were aware of the derivation of DSCs from pulp and periodontal ligament (PDL) tissues (77.21%) and that DSCs are considered an adult stem cell source (51.11%). Most respondents were familiar with the characteristics of DSCs. They were aware that DSCs have the unique ability to self-renew and differentiate into trilineage differentiation (69.12%). They agreed that DSCs are an excellent cell resource for therapeutic approaches to neural repair and regeneration (63.24%). 63.24% of the respondents demonstrated a positive outcome for dental stem cell research. 59.56% of them would be willing to donate their extracted teeth for research. About 56.62% of respondents would conserve their teeth in stem cell banks and 52.94% are interested in investing in the stem cell industry.Conclusion:A high level of perception and awareness about dental stem cells and its application was noted among professional groups in Malaysia. They are supportive and willing to invest for dental stem cell research.

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.

Role of PI3K in the bone resorption of apical periodontitis

BackgroundPhosphoinositide 3-kinase (PI3K) is located within cells, and is involved in regulating cell survival, proliferation, apoptosis and angiogenesis. The purpose of this study was to investigate the role of PI3K in the process of bone destruction in apical periodontitis, and provide reference data for the treatment of this disease.MethodsThe relative mRNA expression of PI3K, Acp5 and NFATc1 in the normal human periodontal ligament and in chronic apical periodontitis were analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). A mouse model of apical periodontitis was established by root canal exposure to the oral cavity, and HE staining was used to observe the progress of apical periodontitis. Immunohistochemical staining was used to detect the expression of PI3K and AKT in different stages of apical periodontitis, while enzymatic histochemical staining was used for detection of osteoclasts. An Escherichia coli lipopolysaccharide (LPS)-mediated inflammatory environment was also established at the osteoclast and osteoblast level, and osteoclasts or osteoblasts were treated with the PI3K inhibitor LY294002 to examine the role of PI3K in bone resorption.ResultsThe expression of PI3K, Acp5 and NFATc1 genes in chronic apical periodontitis sample groups was significantly increased relative to healthy periodontal ligament tissue (P < 0.05). Mouse apical periodontitis was successfully established and bone resorption peaked between 2 and 3 weeks (P < 0.05). The expression of PI3K and Akt increased with the progression of inflammation, and reached a peak at 14 days (P < 0.05). The gene and protein expression of PI3K, TRAP and NFATc1 in osteoclasts were significantly increased (P < 0.05) in the E. coli LPS-mediated inflammatory microenvironment compared to the normal control group. Meanwhile in osteoblasts, the gene and protein expression of PI3K, BMP-2 and Runx2 were significantly reduced (P < 0.05) in the inflammatory microenvironment. With the addition of LY294002, expressions of bone resorption-related factors (TRAP, NFATc1) and bone formation-related factors (BMP-2, Runx2) significantly decreased (P < 0.05).ConclusionsUnder the inflammatory environment induced by LPS, PI3K participates in the occurrence and development of chronic apical periodontitis by regulating the proliferation and differentiation of osteoclasts and osteoblasts.

Tooth transplantation and replantation: Biological insights towards therapeutic improvements

Transplantation and replantation of teeth are effective therapeutic approaches for tooth repositioning and avulsion, respectively. Transplantation involves transplanting an extracted tooth from the original site into another site, regenerating tissue including the periodontal ligament (PDL) and alveolar bone, around the transplanted tooth. Replantation places the avulsed tooth back to its original site, regenerating functional periodontal tissue. In clinical settings, transplantation and replantation result in favorable outcomes with regenerated PDL tissue in many cases. However, they often result in poor outcomes with two major complications: tooth ankylosis and root resorption. In tooth ankylosis, the root surface and alveolar bone are fused, reducing the PDL tissue between them. The root is subjected to remodeling processes and is partially replaced by bone. In severe cases, the resorbed root is completely replaced by bone tissue, which is called as "replacement resorption." Resorption is sometimes accompanied by infection-mediated inflammation. The molecular mechanisms of ankylosis and root resorption remain unclear, although some signaling mechanisms have been proposed. In this mini-review, we summarized the biological basis of repair mechanisms of tissues in transplantation and replantation and the pathogenesis of their healing failure. We also discussed possible therapeutic interventions to improve treatment success rates.

Receptor activator of nuclear factor-κB ligand and tumor necrosis factor-α promotes osteoclast differentiation through the exosomes of inflammatory periodontal ligament stem cells.

Pathological bone resorption is common in chronic periodontitis. However, the effect of exosomes (Exo) secreted by periodontal ligament stem cells (PDLSCs) on bone resorption is unclear. This study explored the Exo of inflammatory PDLSCs, their protein components, and their effects on osteoclast differentiation. PDLSCs were isolated from the periodontal ligament tissues of orthodontic patients and those with chronic periodontitis. The surface markers of PDLSCs were detected by flow cytometry. Exo were characterized by Western blot, transmission electron microscope (TEM), bicinchoninic acid assay (BCA), nanosight tracking analysis (NTA). The protein components of Exo were detected by protein profiling. The expression levels of differentially expressed proteins tumor necrosis factor-α (TNF-α), receptor activator of nuclear factor-κB ligand (RANKL), interleukin (IL)-1α, transforming growth factor β (TGF-β), and bone morphogenetic protein 2 (BMP-2) were verified by enzyme linked immunosorbent assay (ELISA). Then, 10, 100, and 1 000 μg·mL-1 of Exo-CP or Exo-WT were added to RAW264.7 medium, and the expression levels of osteoclast-related indicators were detected by real time quantitative polymerase chain reaction (RT-qPCR), Western blot, and tartrate resistant acid phosphatase (TRAP) staining at 5 days. Experimental data were statistically analyzed using SPSS 24.0 software. The differentially expressed proteins enriched in Exo-CP were mainly related to the tumor necrosis factor (TNF) signaling, osteoclast differentiation, and nuclear transcription factor κB (NF-κB) signaling pathways. ELISA experiments confirmed Exo-CP had high expression of TNF-α, RANKL, and IL-1α and low expression of TGF-β1 and BMP-2 (P<0.05). Adding Exo-CP to RAW264.7 significantly increased the expression of mRNA and proteins related to osteoclast differentiation of cells. In a concentration-dependent manner, the effect of Exo-CP on osteoclast differentiation at concentrations of 100 and 1 000 μg·mL-1 was significantly higher than that on the 10 μg·mL-1 concentration group (P<0.05). Pathological bone resorption of chronic periodontitis may be caused by the activation of Exo-CP to promote osteoclast differentiation. The main protein in Exo may be RANKL and TNF-α. This research provides a new perspective on pathological bone resorption in chronic periodontitis.

Challenges and Tissue Engineering Strategies of Periodontal-Guided Tissue Regeneration.

Periodontitis is a chronic infectious oral disease with a high prevalence rate in the world, and is a major cause of tooth loss. Nowadays, people have realized that the local microenvironment that includes proteins, cytokines, and extracellular matrix has a key influence on the functions of host immune cells and periodontal ligament (PDL) stem cells during a chronic infectious disease such as periodontitis. The above pathological process of periodontitis will lead to a defect of periodontal tissues. Through the application of biomaterials, biological agents, and stem cell therapy, guided tissue regeneration (GTR) makes it possible to reconstruct healthy PDL tissue after local inflammation control. To date, substantial advances have been made in periodontal GTR. However, the process of periodontal remodeling experiences complex microenvironment changes, and currently periodontium regeneration still remains to be a challenging feat. In this review, we summarized the main challenges in each stage of periodontal regeneration, and try to put forward appropriate biomaterial treatment mechanisms or potential tissue engineering strategies that provide a theoretical basis for periodontal tissue engineering regeneration research.

A Spectral Principal Component Analysis-Based Framework for Composite Hard/Soft Tissue Fluorescence Image Investigation.

Traditional thin sectioning microscopy of large bone and dental tissue samples using demineralization may disrupt structure morphologies and even damage soft tissues, thus compromising the histopathological investigation. Here, we developed a synergistic and original framework on thick sections based on wide-field multi-fluorescence imaging and spectral Principal Component Analysis (sPCA) as an alternative, fast, versatile, and reliable solution, suitable for highly mineralized tissue structure sustain and visualization. Periodontal 2-mm thick sections were stained with a solution containing five fluorescent dyes chosen for their ability to discriminate close tissues, and acquisitions were performed with a multi-zoom macroscope for blue, green, red, and NIR (near-infrared) emissions. Eigen-images derived from both standard scaler (Std) and Contrast Limited Adaptive Histogram Equalization (Clahe) pre-preprocessing significantly enhanced tissue contrasts, highly suitable for histopathological investigation with an in-depth detail for sub-tissue structure discrimination. Using this method, it is possible to preserve and delineate accurately the different anatomical/morphological features of the periodontium, a complex tooth-supporting multi-tissue. Indeed, we achieve characterization of gingiva, alveolar bone, cementum, and periodontal ligament tissues. The ease and adaptability of this approach make it an effective method for providing high-contrast features that are not usually available in standard staining histology. Beyond periodontal investigations, this first proof of concept of an sPCA solution for optical microscopy of complex structures, especially including mineralized tissues opens new perspectives to deal with other chronic diseases involving complex tissue and organ defects. Overall, such an imaging framework appears to be a novel and convenient strategy for optical microscopy investigation.

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.

IFNγ-primed periodontal ligament cells regulate T-cell responses via IFNγ-inducible mediators and ICAM-1-mediated direct cell contact.

Periodontal ligament (PDL) cells help maintain tissue homeostasis by balancing PDL tissue inflammation and regeneration. However, the mechanisms by which interferon γ (IFNγ) modulate this process are not yet fully understood. The present study aimed to examine the effect of primed and non-primed PDL cells with IFNγ on the viability and differentiation of T lymphocytes and its functional consequences. The results showed that IFNγ-primed PDL cells possessed enhanced immunosuppression by suppressing T-lymphocyte viability and directing T-lymphocyte differentiation towards a higher T helper (Th) Th2/Th1 ratio. Suppression of T-cell viability was mainly mediated by IFNγ-inducible secreted mediators, which was prevented in the presence of direct cell contact, probably by intercellular adhesion molecule-1 (ICAM-1)-induced PI3 K-mediated transforming growth factor β1 expression in PDL cells. By contrast, ICAM-1 activation augmented IFNγ-induced IFNγ and interleukin-6 expression in PDL cells, which in turn modulated T-cell differentiation. The resulting interaction between these two cell types activated macrophage and suppressed osteoclast differentiation. In conclusion, the results have shown, for the first time to our knowledge, that primed and non-primed PDL cells with IFNγ differentially control T-cell responses via IFNγ-inducible mediators and ICAM-1-mediated direct cell contact, suggesting the role of PDL cells in shifting an inflammatory phase towards a regenerative phase.

Comparative gene expression analysis of stemness between periodontal ligament and umbilical cord tissues in humans

Background/purposeDue to their regenerative potential, periodontal ligament (PDL) and umbilical cord (UBC) tissues are an attractive potential mesenchymal stem cells (MSCs) source. This study compared the expression patterns of genes related to stemness between fresh PDL and UBC tissues. Materials and methodsPDL tissues were collected from 38 permanent premolars extracted for orthodontic purposes, and UBC tissues were obtained from three newborns. Each sample was immediately frozen to prevent RNA degradation. cDNA microarray analysis, quantitative real-time polymerase chain reaction (PCR), and immunohistochemical staining were performed. Gene expression patterns associated with dental stemness (DS) and induced pluripotent stemness (iPS) were compared between PDL and UBC tissues. ResultsIn the cDNA microarray analyses, the expressions of most iPS genes were greater in the PDL than in the UBC. Meanwhile, the expressions of most DS genes were greater in the UBC than in the PDL. Quantitative real-time PCR analyses showed that the expression levels of matrix metallopeptidase 13 (MMP13), ADAM metallopeptidase domain 22 (ADAM22), vascular cell adhesion protein 1 (VCAM1), and kruppel-like factor 4 (KLF4) genes were greater in the PDL than in the UBC, while the expressions of melanoma cell adhesion molecule (MCAM) and activated leukocyte cell adhesion molecule (ALCAM) were greater in the UBC than in the PDL. ConclusionThese results suggest that UBC and PDL tissues showed slightly different expression patterns of genes related to stemness, which warrants further investigation to use these tissues for future regeneration and implantation therapies.

Inhibition of TRPA1 Ameliorates Periodontitis by Reducing Periodontal Ligament Cell Oxidative Stress and Apoptosis via PERK/eIF2α/ATF-4/CHOP Signal Pathway.

Objective In periodontitis, excessive oxidative stress combined with subsequent apoptosis and cell death further exacerbated periodontium destruction. TRPA1, an important transient receptor potential (TRP) cation channel, may participate in the process. This study is aimed at exploring the role and the novel therapeutic function of TRPA1 in periodontitis. Methods Periodontal ligament cells or tissues derived from healthy and periodontitis (PDLCs/Ts and P-PDLCs/Ts) were used to analyze the oxidative and apoptotic levels and TRPA1 expression. TRPA1 inhibitor (HC030031) was administrated in inflammation induced by P. gingivalis lipopolysaccharide (P.g.LPS) to investigate the oxidative and apoptotic levels of PDLCs. The morphology of the endoplasmic reticulum (ER) and mitochondria was identified by transmission electron microscope, and the PERK/eIF2α/ATF-4/CHOP signal pathways were detected. Finally, HC030031 was administered to periodontitis mice to evaluate its effect on apoptotic and oxidative levels in the periodontium and the relieving of periodontitis. Results The oxidative, apoptotic levels and TRPA1 expression were higher in P-PDLC/Ts from periodontitis patients and in P.g.LPS-induced inflammatory PDLCs. TRPA1 inhibitor significantly decreased the intracellular calcium, oxidative stress, and apoptosis of inflammatory PDLCs and decreased ER stress by downregulating PERK/eIF2α/ATF-4/CHOP pathways. Meanwhile, the overall calcium ion decrease induced by EGTA also exerted similar antiapoptosis and antioxidative stress functions. In vivo, HC030031 significantly reduced oxidative stress and apoptosis in the gingiva and periodontal ligament, and less periodontium destruction was observed. Conclusion TRPA1 was highly related to periodontitis, and TRPA1 inhibitor significantly reduced oxidative and apoptotic levels in inflammatory PDLCs via inhibiting ER stress by downregulating PERK/eIF2α/ATF-4/CHOP pathways. It also reduced the oxidative stress and apoptosis in periodontitis mice thus ameliorating the development of periodontitis.

Extracellular Matrix-Oriented Proteomic Analysis of Periodontal Ligament Under Mechanical Stress.

The periodontal ligament (PDL) is a specialized connective tissue that provides structural support to the tooth and is crucial for oral functions. The mechanical properties of the PDL are mainly derived from the tissue-specific composition and structural characteristics of the extracellular matrix (ECM). The ECM also plays key roles in determining cell fate in the cellular microenvironment thus crucial in the PDL tissue homeostasis. In the present study, we determined the comprehensive ECM profile of mouse molar PDL using laser microdissection and mass spectrometry-based proteomic analysis with ECM-oriented data curation. Additionally, we evaluated changes in the ECM proteome under mechanical loading using a mouse orthodontic tooth movement (OTM) model and analyzed potential regulatory networks using a bioinformatics approach. Proteomic changes were evaluated in reference to the novel second harmonic generation (SHG)-based fiber characterization. Our ECM-oriented proteomics approach succeeded in illustrating the comprehensive ECM profile of the mouse molar PDL. We revealed the presence of type II collagen in PDL, possibly associated with the load-bearing function upon occlusal force. Mechanical loading induced unique architectural changes in collagen fibers along with dynamic compositional changes in the matrisome profile, particularly involving ECM glycoproteins and matrisome-associated proteins. We identified several unique matrisome proteins which responded to the different modes of mechanical loading in PDL. Notably, the proportion of type VI collagen significantly increased at the mesial side, contributing to collagen fibrogenesis. On the other hand, type XII collagen increased at the PDL-cementum boundary of the distal side. Furthermore, a multifaceted bioinformatics approach illustrated the potential molecular cues, including PDGF signaling, that maintain ECM homeostasis under mechanical loading. Our findings provide fundamental insights into the molecular network underlying ECM homeostasis in PDL, which is vital for clinical diagnosis and development of biomimetic tissue-regeneration strategies.