Primary Human Periodontal Ligament Cells Research Articles

Effect of Fibrillin-2 on Differentiation into Periodontal Ligament Stem Cell-Like Cells Derived from Human-Induced Pluripotent Stem Cells.

Periodontal tissue regeneration is important for preserving teeth. Periodontal ligament stem cells (PDLSCs) are useful in periodontal tissue regeneration; however, tooth extraction is required to obtain these cells. Therefore, we focused on induced pluripotent stem (iPS) cells and established a method to obtain PDLSC-like cells from iPS cells. Specifically, we first differentiated iPS cells into neural crest-like cells (iNCs). Next, we obtained PDLSC-like cells (iPDLSCs) by culturing iNCs on extracellular matrix (ECM) derived from human primary periodontal ligament cells (HPDLCs). This differentiation method suggested that ECM derived from HPDLCs is important for iPDLSC differentiation. Thus, we aimed to identify the PDLSC-inducing factor present in HPDLC-derived ECM in this study. We first performed comprehensive analyses of HPDLC genes and identified fibrillin-2 (FBN2), an ECM-related factor. Furthermore, to clarify the effect of FBN2 on iPDLSC differentiation, we cultured iNCs using ECM derived from HPDLCs with FBN2 knocked down. As a result, expression of PDL-related markers was reduced in iNCs cultured on ECM derived from HPDLCs transfected with FBN2 siRNA (iNC-siFBN2) compared with iPDLSCs. Furthermore, the expression of CD105 (a mesenchymal stem cell marker), proliferation ability, and multipotency of iNC-siFBN2 were lower compared with iPDLSCs. Next, we cultured iNCs on FBN2 recombinant protein; however, expression of PDL-related markers did not increase compared with iPDLSC. The present results suggest the critical involvement of FBN2 in inducing iPDLSCs from iNCs when in fact it does not promote iPDLSC differantiation. Therefore, we need to elucidate the entire HPDLC-ECMs, responsible for iPDLSCs induction.

3D bioprinted small extracellular vesicles from periodontal cells enhance mesenchymal stromal cell function

Recent research indicates that combining 3D bioprinting and small extracellular vesicles (sEVs) offers a promising ‘cell-free’ regenerative medicine approach for various tissue engineering applications. Nonetheless, the majority of existing research has focused on bioprinting of sEVs sourced from cell lines. There remains a notable gap in research regarding the bioprinting of sEVs derived from primary human periodontal cells and their potential impact on ligamentous and osteogenic differentiation. Here, we investigated the effect of 3D bioprinted periodontal cell sEVs constructs on the differentiation potential of human buccal fat pad-derived mesenchymal stromal cells (hBFP-MSCs). Periodontal cell-derived sEVs were enriched by size exclusion chromatography (SEC) with particle-shaped morphology, and characterized by being smaller than 200 nm in size and CD9/CD63/CD81 positive, from primary human periodontal ligament cells (hPDLCs) and human gingival fibroblasts (hGFs). The sEVs were then 3D bioprinted in 10 % gelatin methacryloyl (GelMA) via microextrusion bioprinting. Release of sEVs from bioprinted constructs was determined by DiO-labelling and confocal imaging, and CD9 ELISA. Attachment and ligament/osteogenic/cementogenic differentiation of hBFP-MSCs was assessed on bioprinted GelMA, without and with sEVs (GelMA/hPDLCs-sEVs and GelMA/hGFs-sEVs), scaffolds. hBFP-MSCs seeded on the bioprinted sEVs constructs spread well with significantly enhanced focal adhesion, mechanotransduction associated gene expression, and ligament and osteogenesis/cementogenesis differentiation markers in GelMA/hPDLCs-sEVs, compared to GelMA/hGFs-sEVs and GelMA groups. A 2-week osteogenic and ligamentous differentiation showed enhanced ALP staining, calcium formation and toluidine blue stained cells in hBFP-MSCs on bioprinted GelMA/hPDLCs-sEVs constructs compared to the other two groups. The proof-of-concept data from this study supports the notion that 3D bioprinted GelMA/hPDLCs-sEVs scaffolds promote cell attachment, as well as ligamentous, osteogenic and cementogenic differentiation, of hBFP-MSCs in vitro.

Upregulated ATF1 Promotes Lipopolysaccharide Induced Inflammatory Response and Inhibits Osteogenic Differentiation of Human Periodontal Ligament Cells by Regulating NF-κB Pathway.

Periodontitis is a chronic inflammatory disease resulting from bacterial plaque infection. While the involvement of activating transcription factor 1 (ATF1) has been extensively explored in various human diseases, its specific role in periodontitis remains unclear. This study aims to elucidate the expression and biological function of ATF1 in the context of periodontitis. Primary human periodontal ligament cells (hPDLCs) were procured from clinical samples and subsequently characterized. Following treatment with P. gingivalis lipopolysaccharide (LPS, 10 μg/mL), hPDLCs underwent transfection with either ATF1 vector or siRNA. The expression levels of ATF1 in LPS-treated hPDLCs or transfected cells were evaluated through real-time quantitative polymerase chain reaction (RT-qPCR) and western blot assay. Inflammatory factors, including interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-α), and interleukin-1beta (IL-1β), were quantified using Enzyme-linked Immunosorbent Assay (ELISA). The assessment of osteogenic proteins, such as runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteoprotegerin (OPG), as well as noncanonical nuclear factor-kappaB (NF-κB) pathway-related proteins (p65, p-p65, IkBα, p-IkBα), was conducted using western blot assay. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry assays were employed to detect cell viability. LPS induced an inflammatory response and hindered the osteogenic differentiation of hPDLCs (p < 0.05, p < 0.01). Furthermore, ATF1 silencing enhanced cell proliferation and suppressed apoptosis in LPS-stimulated hPDLCs (p < 0.05, p < 0.01). ATF1 silencing not only restrained the inflammatory response but also promoted the osteogenic differentiation of LPS-stimulated hPDLCs (p < 0.05, p < 0.01). Importantly, ATF1 silencing effectively blocked the LPS-induced activation of the NF-κB signaling pathway (p < 0.05, p < 0.01, p < 0.001). ATF1 emerges as a promising treatment option, inhibiting the osteogenic differentiation of hPDLCs and mitigating the inflammatory response by preventing the phosphorylation of the NF-κB signaling pathway.

LncRNA JHDM1D-AS1 promotes osteogenic differentiation of periodontal ligament cells by targeting miR-532-5p to activate IGF1R signaling.

The aim of this study was to explore the mechanism underlying periodontal ligament cells (PDLCs) osteogenic differentiation. Periodontitis causes damage to tooth-supporting apparatus and eventually leads to tooth loss. PDLCs hold great promise in periodontal regeneration due to their osteogenic features. The expression of osteogenic markers, lncRNA JHDM1D-AS1, miR-532-5p and IGF1R was examined. For osteogenic differentiation, primary human PDLCs (hPDLCs) were cultured in an osteogenic medium, and it was assessed by ALP activity and Alizarin Red staining. The interaction between JHDM1D-AS1, miR-532-5p and IGF1R was analyzed via dual luciferase, RIP and RNA pull-down assays. JHDM1D-AS1 was up-regulated during osteogenic differentiation and its silencing inhibited hPDLC osteogenic differentiation. JHDM1D-AS1 worked as a miR-532-5p sponge in hPDLCs. miR-532-5p directly targeted IGF1R to suppress its expression, and miR-532-5p knockdown facilitated osteogenic differentiation of hPDLCs. Overexpression of IGF1R promoted osteogenic differentiation of hPDLCs via activating Notch/HES1 signaling in hPDLCs. JHDM1D-AS1 promotes osteogenic differentiation of hPDLCs via sponging miR-532-5p to facilitate IGF1R expression and activate Notch/HES1 signaling.

Low-Intensity Pulsed Ultrasound Attenuates Periodontal Ligament Cells Apoptosis by Activating Yes-Associated Protein-Regulated Autophagy

The goal of the work described here was to determine if low-intensity pulsed ultrasound (LIPUS) has an anti-inflammatory effect on lipopolysaccharide (LPS)-induced inflammation in periodontal ligament cells (PDLCs). The mechanism underlying this effect remains to be explored and is likely related to PDLC apoptosis regulated by Yes-associated protein (YAP) and autophagy. To verify this hypothesis, we used a rat model of periodontitis and primary human PDLCs. We examined alveolar bone resorption in rats and apoptosis, autophagy and YAP activity in LPS-treated PDLCs with and without application of LIPUS by cellular immunofluorescence, transmission electron microscopy and Western blotting. Then, siRNA transfection was used to decrease YAP expression to confirm the regulatory role of YAP in the anti-apoptotic effect of LIPUS on PDLCs. We found that LIPUS attenuated alveolar bone resorption in rats and this was accompanied by YAP activation. LIPUS inhibited hPDLC apoptosis by YAP activation, and promoted autophagic degradation to help autophagy completion. These effects were reversed after YAP expression was blocked. LIPUS attenuates PDLC apoptosis by activating Yes-associated protein-regulated autophagy.

Novel Epigenetic Modulation Chitosan-Based Scaffold as a Promising Bone Regenerative Material.

Bone tissue engineering is a complicated field requiring concerted participation of cells, scaffolds, and osteoactive molecules to replace damaged bone. This study synthesized a chitosan-based (CS) scaffold incorporated with trichostatin A (TSA), an epigenetic modifier molecule, to achieve promising bone regeneration potential. The scaffolds with various biphasic calcium phosphate (BCP) proportions: 0%, 10%, 20%, and 40% were fabricated. The addition of BCP improved the scaffolds’ mechanical properties and delayed the degradation rate, whereas 20% BCP scaffold matched the appropriate scaffold requirements. The proper concentration of TSA was also validated. Our developed scaffold released TSA and sustained them for up to three days. The scaffold with 800 nM of TSA showed excellent biocompatibility and induced robust osteoblast-related gene expression in the primary human periodontal ligament cells (hPDLCs). To evaluate in vivo bone regeneration potential, the scaffolds were implanted in the mice calvarial defect model. The excellent bone regeneration ability was further demonstrated in the micro-CT and histology sections compared to both negative control and commercial bone graft product. New bone formed in the CS/BCP/TSA group revealed a trabeculae-liked characteristic of the mature bone as early as six weeks. The CS/BCP/TSA scaffold is an up-and-coming candidate for the bone tissue engineering scaffold.

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.

CircSKIL promotes osteoblastic differentiation of periodontal ligament cells by sponging miR-532-5p to activate Notch signaling.

Periodontal ligament cells (PDLCs) possess the capacity to differentiate into a variety of cell types to benefit periodontal regeneration. In this study, we examined the circSKIL/miR-532-5p/Notch1 axis in controlling the osteoblastic differentiation of PDLCs. Primary human PDLCs (hPDLCs) were isolated and induced to differentiate into osteoblasts. Osteogenic responses were assessed for the expressions of osteoblast-related marker proteins (including alkaline phosphatase (ALP), osteocalcin (OCN), bone morphogenetic protein-2 (BMP2), and runt-related transcription factor 2 (RUNX2) by RT-PCR. The formation of mineralized nodules was examined by Alizarin Red S (ARS) staining and ALP activity. Expressions of circSKIL, miR-532-5p, and Notch1 were measured by RT-PCR and western blotting, and their regulations by combining bioinformatic analysis and luciferase reporter assay. Notch signaling was assessed for the expressions of hairy and enhancer of split-1 (HES1) and Notch intracellular domain (NICD). During osteoblastic differentiation of hPDLCs, circSKIL, and Notch1 were up-regulated, while miR-532-5p down-regulated. By sponging miR-532-5p, circSKIL activated Notch signaling, increasing levels of Notch1, HES1, and NICD. Functionally, knocking down circSKIL or overexpressing miR-532-5p inhibited osteoblastic differentiation of PDLCs, down-regulating ALP, OCN, BMP2, and RUNX2, and reducing ARS staining or ALP activity. The impacts of circSKIL knockdown were rescued by miR-532-5p inhibitor or overexpressing Notch1, while those caused by up-regulating miR-532-5p were reversed by overexpressing Notch1. By targeting miR-532-5p and up-regulating Notch1, circSKIL critically controls osteoblastic differentiation of hPDLCs. Therefore, modulating this axis may maximize the differentiation of PDLCs into osteoblasts and benefit periodontal regeneration.

Potential donor-dependent regulative effects of endogenous sclerostin expression and mineralization potential in primary human PDL cells in vitro

ObjectivesThe glycoprotein sclerostin is mostly expressed in osteocytes and plays a central role in human bone metabolism. However, sclerostin and the corresponding SOST gene have been found in periodontal ligament cells under mineralizing conditions as well. The present study aimed to investigate, whether there was a correlation between endogenous SOST expression, the corresponding gene, and mineralization potential in human periodontal ligament cells and to identify different sclerostin expression and secretion patterns in cells derived from individual donors. Material and methodsPrimary human periodontal ligament cells of three different donors were cultivated under control or mineralizing conditions for 6, 13, 15 and 18 days, respectively. Calcium deposits were stained with alizarin red and quantified afterwards. Quantitative expression analysis of the SOST gene encoding sclerostin was performed using quantitative reverse transcription polymerase chain reaction (RT-PCR). Additionally, intracellular sclerostin expression was analyzed using Western blotting and extracellular sclerostin secretion was quantified using Enzyme-linked Immunosorbent Assay (ELISA). ResultsAlizarin red staining identified calcium deposits in periodontal ligament cells under mineralizing conditions beginning from day 13, relative SOST expression occurred on day 6. Whereas staining continued to increase in donor 1 on day 15, it remained stable in donors 2 and 3. Conversely, baseline SOST expression was significantly lower in donor 1 compared to donors 2 and 3. Western blotting and ELISA revealed increased intra- and extracellular sclerostin expression at day 13 under mineralizing conditions. Donor 3 exhibited the highest overall sclerostin levels. ConclusionsOur data emphasize donor-specific characteristics in differentiation potential and sclerostin expression patterns in primary human periodontal ligament cells. Sclerostin might play a central role in modulating osteogenic differentiation in periodontal ligament cells as part of a negative feedback mechanism in avoiding excessive mineralization.

Regulation of gingival fibroblast phenotype by periodontal ligament cells in vitro.

ObjectivesStem cell transplantation has shown modest effects on periodontal tissue regeneration, and it is still unclear how regenerative effects utilizing this modality are mediated. A greater understanding of the basic interactions between implanted and host cells is needed to improve future strategies. The aims of this study were to investigate the effects of periodontal ligament (PDL) cells on expression of periodontal markers and alkaline phosphatase (ALP) activity of gingival fibroblasts (GF).Materials and MethodsPrimary human PDL cells were co‐cultured with primary GF cultures either by direct co‐culture with subsequent FACS sorting or indirect co‐culture using transwell cultures and PDL cell conditioned medium. Expression of periodontal markers, asporin, nestin, and periostin, was assessed by qPCR and immunofluorescence staining. Alkaline phosphatase (ALP) expression was assessed by qPCR, histochemical staining, and activity assessed by para‐nitrophenol enzymatic assay. Single cultures of PDL cells and GF were used as controls. The role of Wnt signaling on ALP activity was assessed via Dkk1‐mediated inhibition.ResultsPDL cells significantly upregulated expression of PDL markers in GF with both direct and indirect co‐culture methods when compared to controls (6.05 vs. 0.73 and 59.48 vs. 17.55 fold change of asporin expression). PDL/GF cell co‐cultures significantly increased ALP activity in GF when compared with single GF cultures. Similar results were obtained when using conditioned medium isolated from PDL cell cultures. Dkk1 caused dose‐dependent reduction in ALP activity of GF cultured in PDL cell conditioned medium.ConclusionsPDL cells stimulate expression of periodontal markers and osteogenic capacity of gingival fibroblasts via paracrine signaling which can be partially inhibited with addition of the Wnt antagonist, Dkk1.Further studies are required to identify specific secreted factors responsible for this activity.

Differential Response of Oral Mucosal and Gingival Cells to Corynebacterium durum, Streptococcus sanguinis, and Porphyromonas gingivalis Multispecies Biofilms.

Polymicrobial interactions with oral mucosal surfaces determine the health status of the host. While a homeostatic balance provides protection from oral disease, a dysbiotic polymicrobial community promotes tissue destruction and chronic oral diseases. How polymicrobial communities transition from a homeostatic to a dysbiotic state is an understudied process. Thus, we were interested to investigate this ecological transition by focusing on biofilm communities containing high abundance commensal species and low abundance pathobionts to characterize the host-microbiome interactions occurring during oral health. To this end, a multispecies biofilm model was examined using the commensal species Corynebacterium durum and Streptococcus sanguinis and the pathobiont Porphyromonas gingivalis. We compared how both single and multispecies biofilms interact with different oral mucosal and gingival cell types, including the well-studied oral keratinocyte cell lines OKF4/TERT-1and hTERT TIGKs as well as human primary periodontal ligament cells. While single species biofilms of C. durum, S. sanguinis, and P. gingivalis are all characterized by unique cytokine responses for each species, multispecies biofilms elicited a response resembling S. sanguinis single species biofilms. One notable exception is the influence of P. gingivalis upon TNF-α and Gro-α production in hTERT TIGKs cells, which was not affected by the presence of other species. This study is also the first to examine the host response to C. durum. Interestingly, C. durum yielded no notable inflammatory responses from any of the tested host cells, suggesting it functions as a true commensal species. Conversely, S. sanguinis was able to induce expression and secretion of the proinflammatory cytokines IL-6 and IL-8, demonstrating a much greater inflammatory potential, despite being health associated. Our study also demonstrates the variability of host cell responses between different cell lines, highlighting the importance of developing relevant in vitro models to study oral microbiome-host interactions.

Treponema denticola-Induced RASA4 Upregulation Mediates Cytoskeletal Dysfunction and MMP-2 Activity in Periodontal Fibroblasts.

The periodontal complex consists of the periodontal ligament (PDL), alveolar bone, and cementum, which work together to turn mechanical load into biological responses that are responsible for maintaining a homeostatic environment. However oral microbes, under conditions of dysbiosis, may challenge the actin dynamic properties of the PDL in the context of periodontal disease. To study this process, we examined host-microbial interactions in the context of the periodontium via molecular and functional cell assays and showed that human PDL cell interactions with Treponema denticola induce actin depolymerization through a novel actin reorganization signaling mechanism. This actin reorganization mechanism and loss of cell adhesion is a pathological response characterized by an initial upregulation of RASA4 mRNA expression resulting in an increase in matrix metalloproteinase-2 activity. This mechanism is specific to the T. denticola effector protein, dentilisin, thereby uncovering a novel effect for Treponema denticola-mediated RASA4 transcriptional activation and actin depolymerization in primary human PDL cells.

Glycogen synthase kinase-3β (GSK-3β) deficiency inactivates the NLRP3 inflammasome-mediated cell pyroptosis in LPS-treated periodontal ligament cells (PDLCs).

Bacterial infection caused cell pyroptosis and gingival inflammation contributes to periodontitis progression, and lipopolysaccharide (LPS) is the main infectious agent of gram-negative bacteria, which is reported to be closely associated with gingival inflammation and periodontitis. In this study, the primary human periodontal ligament cells (PDLCs) were isolated, cultured, and exposed to LPS treatment, and the results suggested that LPS suppressed cell viability and promoted pro-inflammatory cytokines' (IL-1β, IL-18, IL-6, and TNF-α) generation and secretion in the PDLCs and its supernatants in a time- and concentration-dependent manner. Also, we noticed that LPS upregulated NLRP3, Gasdermin D, and cleaved caspase-1 to trigger pyroptotic cell death in the PDLCs. Further experiments identified that glycogen synthase kinase-3β (GSK-3β) was upregulated by LPS treatment, and inhibition of GSK-3β by its inhibitor (GSKI) or GSK-3β downregulation vectors was effective to restore normal cellular functions in LPS-treated PDLCs. Mechanistically, blockage of GSK-3β restrained NLRP3-meidated cell pyroptosis and inflammation, resulting in the recovery of cell viability and inhibition of cell death in PDLCs treated with LPS, which further ameliorated periodontitis progression. Finally, we collected the serum from periodontitis patients and healthy volunteers, and the clinical data supported that those pro-inflammatory cytokines were also upregulated in patients' serum but not in the healthy participants. Taken together, we concluded that targeting the GSK-3β/NLRP3 pathway mediated cell pyroptosis was effective to attenuate LPS-induced cell death and inflammation in PDLCs, and this study firstly investigated this issue, which broadened our knowledge in this field.

Knockdown of MALAT1 Inhibits the Progression of Chronic Periodontitis via Targeting miR-769-5p/HIF3A Axis.

Purpose Chronic periodontitis (CP) is a long-lasting inflammatory disease that seriously affects oral health. This study is aimed at investigating the regulatory mechanism of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in CP. Methods Primary human periodontal ligament cells (PDLCs) were treated with P. gingivalis lipopolysaccharide (LPS) to establish a CP model. Quantitative real-time PCR (qRT-PCR) was used to measure the expression of MALAT1 and miR-769-5p in gingival tissues of patients with CP and LPS-treated PDLCs. Cell viability was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of inflammatory cytokines. The protein levels of caspase-3, Bax, Bcl-2, and hypoxia-inducible factor (HIF) 3A were determined by western blot assay. Dual-luciferase reporter (DLR) assay was applied to validate the target relationships between miR-769-5p and MALAT1/HIF3A. Results The expression of MALAT1 and HIF3A was enhanced, and the expression of miR-769-5p was reduced in gingival tissues of patients with CP and LPS-treated PDLCs. MALAT1 knockdown promoted cell viability and inhibited inflammation and cell apoptosis in LPS-treated PDLCs. MALAT1 targeted miR-769-5p and negatively regulated miR-769-5p expression. miR-769-5p overexpression promoted cell viability and inhibited inflammation and cell apoptosis in LPS-treated PDLCs. Besides, miR-769-5p targeted HIF3A and negatively modulated HIF3A expression. Both miR-769-5p inhibition and HIF3A overexpression reversed the inhibitory effects of MALAT1 silencing on LPS-induced PDLC injury in vitro. Conclusion MALAT1 knockdown attenuated LPS-induced PDLC injury via regulating the miR-769-5p/HIF3A axis, which may supply a new target for CP treatment.

Methanandamide diminish the Porphyromonas gingivalis lipopolysaccharide induced response in human periodontal ligament cells

BackgroundThe endocannabinoid system is involved in the regulation of periodontal tissue homeostasis. Synthetic cannabinoid methanandamide (Meth-AEA) has improved stability and affinity to cannabinoid receptors compared to its endogenous analog anandamide. In the present study, we investigated the effect of methanandamide on the production of pro-inflammatory mediators in primary human periodontal ligament cells (hPdLCs).MethodshPdLCs were treated with Meth-AEA for 24 h, and the resulting production of interleukin (IL)-6, IL-8, and monocyte chemotactic protein (MCP)-1 was measured in the absence or the presence of Porphyromonas gingivalis lipopolysaccharide (LPS). Additionally, the effect of Meth-AEA on the proliferation/viability of hPdLCs was measured by the MTT method.ResultsMethanandamide at a concentration of 10 μM significantly inhibited P. gingivalis LPS induced production of IL-6, IL-8, and MCP-1. Basal production of IL-6 and IL-8 was slightly enhanced by 10 μM Meth-AEA. No effect of Meth-AEA on the basal production of MCP-1 was observed. Meth-AEA in concentrations up to 10 μM did not affect the proliferation/viability of hPdLCs, but significantly inhibited it at a concentration of 30 μM.ConclusionOur study suggests that the inflammatory response in periodontal ligament cells could be influenced by the activation of the cannabinoid system, which might be potentially involved in the progression of periodontal disease.

A novel serum-free medium for the isolation, expansion and maintenance of stemness and tissue-specific markers of primary human periodontal ligament cells

PurposePeriodontal ligament (PDL) cell cultures are classically maintained in serum-containing media. However, unwanted side-effects of these conditions on cellular and molecular characteristics demand a serum-free alternative. Even though these limitations are well known and efforts for the development of adequate serum-free alternatives have been made, these approaches for replacement remained unsuccessful so far. This study aimed at developing a well-defined, serum-free formulation supporting both isolation from tissue samples and efficient expansion of PDL cells. Here, of particular focus was the perpetuation of tissue-characteristic markers detectable in primary tissues and of stemness features. Basic proceduresPrimary PDL cell cultures from generally healthy human donors (n = 3) were maintained in basal media N2B27 and E6 together with different concentrations of growth and attachment factors. Cell proliferation was recorded via microscopy and WST assay. Gene expression of RUNX2, Periostin, ALP, CD73, CD90, CD105, CD45, SOX10 and SOX2 was compared to primary PDL explants via qRT-PCR. Immunocytochemistry was performed for anti-CD105, SSEA-3, CD271, HNK1. Serum-containing sDMEM medium served as control. Main findingsN2B27 medium substituted with 25 ng/mL EGF, 25 ng/mL IGF1, 0.5 mg/mL Fetuin plus gelatine coating (designated N2B27-PDLsf) emerged as potent serum-free formulation ensuring adequate culture isolation and expansion. Here, PDL primary tissue signature markers RUNX2 and Periostin remained stable in N2B27-PDLsf compared to controls (229.0-fold ±101.0 and 83.2-fold ±9.6 increase). Additionally, stemness markers ALP and CD105 were significantly upregulated on transcriptional, and CD105 and SOX2 on protein level. Principal conclusionsThis investigation identified a novel serum-free medium for the isolation, and expansion of primary human PDL cells with constantly high proliferation rates. Here, purity and stemness properties are maintained. Thus, N2B27-PDLsf represents a valid replacement for serum-containing media in PDL cultures.

Phelligridin D maintains the function of periodontal ligament cells through autophagy in glucose-induced oxidative stress.

PurposeThe objective of this study was to investigate whether phelligridin D could reduce glucose-induced oxidative stress, attenuate the resulting inflammatory response, and restore the function of human periodontal ligament cells (HPDLCs).MethodsPrimary HPDLCs were isolated from healthy human teeth and cultured. To investigate the effect of phelligridin D on glucose-induced oxidative stress, HPDLCs were treated with phelligridin D, various concentrations of glucose, and glucose oxidase. Glucose-induced oxidative stress, inflammatory molecules, osteoblast differentiation, and mineralization of the HPDLCs were measured by hydrogen peroxide (H2O2) generation, cellular viability, alkaline phosphatase (ALP) activity, alizarin red staining, and western blot analyses.ResultsGlucose-induced oxidative stress led to increased production of H2O2, with negative impacts on cellular viability, ALP activity, and calcium deposition in HPDLCs. Furthermore, HPDLCs under glucose-induced oxidative stress showed induction of inflammatory molecules (intercellular adhesion molecule-1, vascular cell adhesion protein-1, tumor necrosis factor-alpha, interleukin-1-beta) and disturbances of osteogenic differentiation (bone morphogenetic protein-2, and -7, runt-related transcription factor-2), cementogenesis (cementum protein-1), and autophagy-related molecules (autophagy related 5, light chain 3 I/II, beclin-1). Phelligridin D restored all these molecules and maintained the function of HPDLCs even under glucose-induced oxidative stress.ConclusionsThis study suggests that phelligridin D reduces the inflammation that results from glucose-induced oxidative stress and restores the function of HPDLCs (e.g., osteoblast differentiation) by upregulating autophagy.

In Vivo Bone Regeneration Induced by a Scaffold of Chitosan/Dicarboxylic Acid Seeded with Human Periodontal Ligament Cells.

Chitosan/dicarboxylic acid (CS/DA) scaffold has been developed as a bone tissue engineering material. This study evaluated a CS/DA scaffold with and without seeded primary human periodontal ligament cells (hPDLCs) in its capacity to regenerate bone in calvarial defects of mice. The osteogenic differentiation of hPDLCs was analyzed by bone nodule formation and gene expression. In vivo bone regeneration was analyzed in mice calvarial defects. Eighteen mice were divided into 3 groups: one group with empty defects, one group with defects with CS/DA scaffold, and a group with defects with CS/DA scaffold and with hPDLCs. After 6 and 12 weeks, new bone formation was assessed using microcomputed tomography (Micro-CT) and histology. CS/DA scaffold significantly promoted in vitro osteoblast-related gene expression (RUNX2, OSX, COL1, ALP, and OPN) by hPDLCs. Micro-CT revealed that CS/DA scaffolds significantly promoted in vivo bone regeneration both after 6 and 12 weeks (p < 0.05). Histological examination confirmed these findings. New bone formation was observed in defects with CS/DA scaffold; being similar with and without hPDLCs. CS/DA scaffolds can be used as a bone regenerative material with good osteoinductive/osteoconductive properties.

Immortalization of human periodontal ligament stem cells by transferring human telomerase reverse transcriptase gene

Background: Human periodontal ligament stem cells (hPDLSCs) were considered to be potential seed cells for periodontal tissue engineering, however, it possesses a relatively short replicative lifespan and undergo senescence after several passages. So, this study aimed to establish a cell line of immortalized hPDLSCs to provide a consistent and homogeneous research cell model. Methods: Primary human periodontal ligament cells (hPDLCs) were cultured and hPDLSCs were isolated using magnetic beads. The hPDLSCs were then immortalized by infection with a lentivirus vector carrying the hTERT gene. hTERT transcription was verified with RT-PCR. Characteristics of the immortalized cells, such as morphology, proliferation were evaluated. The expression of the odontoblastic/osteogenic markers dentin matrix protein 1 ( DMP1 ), dentin sialophosphoprotein ( DSPP ), alkaline phosphatase ( ALP ) and osteomodulin ( OMD ) was analyzed with real time RT-PCR. Expression of p53 protein was verified with western blot to find whether the overexpression of hTERT could change the carcinogenicity of hPDLSCs. Results: The results of the study showed that hPDLSCs were successfully isolated and hTERT was successfully integrated into the genomic DNA of hPDLSCs to form the immortalized human dental periodontal ligament stem cells (hPDLSCs- hTERT ), furthermore, the ectogenic hTERT was successfully transcribed into mRNA. In long-term culture, hPDLSCs- hTERT bypassed senescence and continued to grow for more than 49 generations. Moreover, hPDLSCs- hTERT retained the phenotypic characteristics of the primary hPDLSCs. Furthermore, similar to hPDLSCs, hPDLSCs- hTERT expressed increased levels of ALP, DMP1, DSPP, and OMD in the odontoblastic/osteogenic induction medium (P<0.05). The expression levels of p53, which is an anti-oncogene, showed no significant difference between the hPDLSCs and hPDLSCs- hTERT . Conclusions: We established a cell line of immortalized hPDLSCs, which remained in an undifferentiated state, exhibiting odontoblastic/osteogenic differentiation potential and low tumorigenicity.

LIPUS promotes FOXO1 accumulation by downregulating miR-182 to enhance osteogenic differentiation in hPDLCs

Low intensity pulsed ultrasound (LIPUS) promotes bone fracture healing in clinical therapy. Transcription factor Forkhead box O1 (FOXO1) is crucial for bone differentiation. But whether FOXO1 is involved in LIPUS-promoted bone differentiation is largely unknown. In the current study, treatment of human primary periodontal ligament cells (hPDLCs) with LIPUS promoted total and nucleus FOXO1 protein accumulation. LIPUS-induced activation of FOXO1 further lead to higher alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2) expression and matrix mineralization. LIPUS inhibited miR-182 expression, which functioned as a repressor of FOXO1 through post-transcriptional regulation. Overexpression of miR-182 reversed the LIPUS-enhanced FOXO1 expression and osteogenic differentiation. Moreover, LIPUS enhanced Akt phosphorylation, which functioned in preventing active FOXO1 excessive accumulation via inducing the cytoplasm translocation of nucleus FOXO1. In conclusion, our study revealed that FOXO1, which was a target gene of miR-182, played an essential role in LIPUS-promoted osteogenic differentiation. This new molecular insight throws light upon the application of LIPUS therapy on periodontal bone defect.