MicroRNA expression in JAG1/Notch-activated periodontal ligament stem cells.

OBJECTIVES: The study explored the expression profile of miRNAs in Notch-activated periodontal ligament stem cells (PDLSCs) and examined their potential cellular targets. METHODS: PDLSCs were cultured and treated with indirect immobilized Jagged1. The miRNA expression profile was examined using NanoString analysis. Bioinformatic analysis was performed together with enrichment, and miRNA expression was evaluated and validated using a quantitative polymerase chain reaction (qPCR). RESULTS: A total of 26 miRNAs were differentially expressed in Jagged1 treated PDLSCs compared with the controls. Pathway analysis revealed that altered miRNAs were significantly associated with the transforming growth factor ß (TGF-ß) signaling pathway. Target prediction analysis demonstrated that 11,170 genes as predictable targets of these altered miRNAs. Enrichment of predicted target genes revealed that they were related to ErbB, Ras and MAPK signaling pathways and small GTPase transduction. CONCLUSIONS: The research concludes that several miRNAs are differentially expressed in jagged-1 treated PDLSCs. In translational terms the differential functionality of these miRNAs offer promise for the development of targeted regenerative materials that are necessary for managing lost tissue replacement in periodontal diseases.

Effects of mechanical loading on matrix homeostasis and differentiation potential of periodontal ligament cells: A scoping review.

Various mechanical loadings, including mechanical stress, orthodontics forces, and masticatory force, affect the functions of periodontal ligament cells. Regulation of periodontal tissue destruction, formation, and differentiation functions are crucial processes for periodontal regeneration therapy. Numerous studies have reported that different types of mechanical loading play a role in maintaining periodontal tissue matrix homeostasis, and osteogenic differentiation of the periodontal ligament cells. This scoping review aims to evaluate the studies regarding the effects of various mechanical loadings on the secretion of extracellular matrix (ECM) components, regulation of the balance between formation and destruction of periodontal tissue matrix, osteogenic differentiation, and multiple differentiation functions of the periodontal ligament. An electronic search for this review has been conducted on two databases; MEDLINE via PubMed and SCOPUS. Study selection criteria included original research written in English that reported the effects of different mechanical loadings on matrix homeostasis and differentiation potential of periodontal ligament cells. The final 204 articles were mainly included in the present scoping review. Mechanical forces of the appropriate magnitude, duration, and pattern have a positive influence on the secretion of ECM components such as collagen, as well as regulate the secretion of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases. Additionally, these forces regulate a balance between osteoblastic and osteoclast differentiation. Conversely, incorrect mechanical loadings can lead to abnormal formation and destruction of both soft and hard tissue. This review provides additional insight into how mechanical loadings impact ECM homeostasis and multiple differentiation functions of periodontal ligament cells (PDLCs), thus making it valuable for regenerative periodontal treatment. In combination with advancing technologies, the utilization of ECM components, application of different aspects of mechanical force, and differentiation potential of PDLCs could bring potential benefits to future periodontal regeneration therapy.

Intermittent compressive force regulates matrix metalloproteinases and tissue inhibitors of metalloproteinases expression in human periodontal ligament cells.

OBJECTIVE: This study aims to evaluate the effects of intermittent compressive force (ICF) on the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) by human periodontal ligament cells (hPDLCs). DESIGN: hPDLCs were subjected to ICF with a magnitude of 1.5 g/cm2 and loaded for 24 h. mRNA and protein expression of several MMPs and TIMPs were assessed using RT-PCR and ELISA analyses. An inhibitor of TGF-ß (SB431542) was used to assess a possible role of TGF-ß in the expression of MMPs and TIMPs under ICF. RESULTS: mRNA and protein analyses showed that ICF significantly induced expression of TIMP1 and TIMP3, but decreased expression of MMP1. Incubation with the TGF-ß inhibitor and applied to ICF showed a downregulation of TIMP3, but expression of MMP1 was not affected. CONCLUSION: ICF is likely to affect ECM homeostasis by hPDLCs by regulating the expression of MMP1 and TIMPs. Moreover, TGF-ß1 regulated expression of TIMP3. These findings suggest ICF may decrease the degradation of ECM and may thus be essential for maintaining PDL homeostasis.

Notch signaling regulates mineralization via microRNA modulation in dental pulp stem cells.

OBJECTIVES: This study investigated the miRNA expression profile in Notch-activated human dental stem pulp stem cells (DPSCs) and validated the functions of miRNAs in modulating the odonto/osteogenic properties of DPSCs. METHODS: DPSCs were treated with indirect immobilized Jagged1. The miRNA expression profile was examined using NanoString analysis. Bioinformatic analysis was performed, and miRNA expression was validated. Odonto/osteogenic differentiation was examined using alkaline phosphatase staining, Alizarin Red S staining, as well as odonto/osteogenic-related gene and protein expression. RESULTS: Fourteen miRNAs were differentially expressed in Jagged1-treated DPSCs. Pathway analysis revealed that altered miRNAs were associated with TGF-ß, Hippo, ErbB signalling pathways, FoxO and Ras signalling. Target prediction analysis demonstrated that 7604 genes were predicted to be targets for these altered miRNAs. Enrichment analysis revealed relationships to various DNA bindings. Among differentially expressed miRNA, miR-296-3p and miR-450b-5p were upregulated under Jagged1-treated conditions. Overexpression of miR-296-3p and miR-450b-5p enhanced mineralization and upregulation of odonto/osteogenic-related genes, whereas inhibition of these miRNAs revealed opposing results. The miR-296-3p and miR-450b-5p inhibitors attenuated the effects of Jagged1-induced mineralization in DPSCs. CONCLUSIONS: Jagged-1 promotes mineralization in DPSCs that are partially regulated by miRNA. The novel understanding of these miRNAs could lead to innovative controlled mechanisms that can be applied to modulate biology-targeted dental materials.

Extracellular adenosine triphosphate regulates inflammatory responses of periodontal ligament cells.

BACKGROUND: Various stimuli, that is, mechanical stresses or inflammation, induce the release of adenosine triphosphate (ATP) by human periodontal ligament cells (HPDLCs). Extracellular adenosine triphosphate (eATP) affects HPDLCs' functions such as immunosuppressive action and inflammatory responses. Lipopolysaccharide (LPS) is the key factor involved in periodontal inflammation. However, the possible correlation and detailed mechanism of inflammation-mediated eATP by LPS and inflammatory cascade formation in HPDLCs is unclarified. This study aims to examine the role of eATP on the HPDLCs' responses concerning inflammatory actions after LPS treatment. METHODS: HPDLCs were stimulated with Porphyromonas gingivalis LPS and polyinosinic:polycytidylic acid (poly I:C). The amount of ATP release was measured at different time points using a bioluminescence assay. HPDLCs were treated with eATP. The expression of pro-inflammatory and anti-inflammatory genes was determined. Specific P2X purinoreceptor 7 (P2X7) inhibitors (brilliant blue G [BBG] and KN62), a specific P2Y purinoreceptor 1 (P2Y1) inhibitors (MRS2179), calcium chelator (EGTA), protein kinase C (PKC) inhibitors, nuclear factor kappa-light-chain-enhancer of activated B cells (NF𝜅B) activation inhibitors, and cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) inhibitors (H89 dihydrochloride) and activators (forskolin) were used to dissect the mechanism of eATP-induced HPDLCs' inflammatory responses. RESULTS: LPS and poly I:C induced ATP release. A low concentration of eATP (50 µM) increased pro-inflammatory genes (COX2, IL1B, IL6, IL8, IL12, and TNFA), while a high concentration (500 µM) enhanced anti-inflammatory genes (IL4 and IL10). BBG, KN62, and NF𝜅B activation inhibitors impeded eATP-induced pro-inflammatory genes. MRS2179 and H89 markedly suppressed eATP-induced anti-inflammatory genes. Forskolin induced IL4 and IL10. CONCLUSION: HPDLCs respond to LPS by releasing ATP. eATP has dose-dependent dual functions on HPDLCs' inflammatory responses via different pathways. As regulation of inflammation is important in regeneration, eATP may help to limit inflammation and trigger periodontal regeneration.

Roles of extracellular adenosine triphosphate on the functions of periodontal ligament cells.

OBJECTIVE: Adenosine triphosphate (ATP) is an essential nucleotide that is normally present in both intracellular and extracellular compartments. Extracellular ATP (eATP) has a pivotal role in both physiological and pathological processes of periodontal ligament tissues. Here, this review aimed to explore the various functions of eATP that are involved in the control of behaviours and functions of periodontal ligament cells. METHODS: To identify the included publications for review, the articles were searched in PubMed (MEDLINE) and SCOPUS with the keywords of adenosine triphosphate and periodontal ligament cells. Thirteen publications were used as the main publications for discussion in the present review. RESULTS: eATP has been implicated as a potent stimulator for inflammation initiation in periodontal tissues. It also plays a role in proliferation, differentiation, remodelling, and immunosuppressive functions of periodontal ligament cells. Yet, eATP has diverse functions in regulating periodontal tissue homeostasis and regeneration. CONCLUSION: eATP may provide a new prospect for periodontal tissue healing as well as treatment of periodontal disease especially periodontitis. It may be utilized as a useful therapeutic tool for future periodontal regeneration therapy.

Intermittent compressive force regulates dentin matrix protein 1 expression in human periodontal ligament stem cells.

Background/purpose: Mechanical force differentially regulates periodontal ligament functions depending on types, magnitudes, and duration of stimulation. Intermittent compressive force (ICF) promotes an in vitro mineralization in human periodontal ligament cells. The present study investigated the effect of ICF on dentin matrix protein-1 (DMP1) expression in human periodontal ligament stem cells (hPDLSCs). Materials and methods: Cells were treated with ICF in a serum-free culture medium for 24 h The mRNA and protein expression were examined using real-time polymerase chain reaction, immunofluorescence staining and Western blot analysis, respectively. Results: The exposure to ICF in a serum-free condition significantly induced DMP1 expression in both mRNA and protein levels. The effect of ICF-induced DMP1 expression was inhibited by pretreatment with cycloheximide, indicating the requirement of the intermediated molecule(s). Pretreatment with transforming growth factor ß (TGF-ß) receptor inhibitor (SB431542) or neutralized antibody against TGF-ß1 prior to ICF application abolished the effect of ICF-induced DMP1 expression. Further, recombinant TGF-ß1 treatment stimulated DMP1 expression. Conclusion: The present study illustrated that ICF induces DMP1 expression in hPDLSCs via the regulation of TGF-ß signaling pathway.

Application of shear stress for enhanced osteogenic differentiation of mouse induced pluripotent stem cells.

The self-organizing potential of induced pluripotent stem cells (iPSCs) represents a promising tool for bone tissue engineering. Shear stress promotes the osteogenic differentiation of mesenchymal stem cells, leading us to hypothesize that specific shear stress could enhance the osteogenic differentiation of iPSCs. For osteogenesis, embryoid bodies were formed for two days and then maintained in medium supplemented with retinoic acid for three days, followed by adherent culture in osteogenic induction medium for one day. The cells were then subjected to shear loading (0.15, 0.5, or 1.5 Pa) for two days. Among different magnitudes tested, 0.5 Pa induced the highest levels of osteogenic gene expression and greatest mineral deposition, corresponding to upregulated connexin 43 (Cx43) and phosphorylated Erk1/2 expression. Erk1/2 inhibition during shear loading resulted in decreased osteogenic gene expression and the suppression of mineral deposition. These results suggest that shear stress (0.5 Pa) enhances the osteogenic differentiation of iPSCs, partly through Cx43 and Erk1/2 signaling. Our findings shed light on the application of shear-stress technology to improve iPSC-based tissue-engineered bone for regenerative bone therapy.

A γ-Secretase Inhibitor Attenuates Cell Cycle Progression and Invasion in Human Oral Squamous Cell Carcinoma: An In Vitro Study.

Notch signaling is associated with many human malignancies, including oral squamous cell carcinoma (OSCC). However, the exact function of Notch signaling in OSCC remains unclear. Here, we investigated the effect of Notch signaling inhibition using a γ-secretase inhibitor (DAPT) on OSCC behaviours in vitro. Bioinformatic analysis of public-available gene expression profiles revealed the dysregulation of the Notch signaling pathway in OSCC compared with normal tissues, indicating the role of Notch signaling in OSCC regulation. RNA sequencing analysis of DAPT-treated human OSCC cells revealed the dysregulation of genes related to cell cycle-related pathways. Blocking Notch signaling significantly inhibited cell proliferation. DAPT-induced G0/G1 cell cycle arrest induced cell apoptosis. Furthermore, cell migration and invasion were also reduced in DAPT-treated cells. These findings indicate that Notch signaling activation participates in OSCC regulation by promoting cell growth, cell cycle progression, cell migration, and invasion. These mechanisms could facilitate OSCC progression. These results imply the potential use of Notch signaling inhibitors as a candidate adjuvant treatment in OSCC patients.

Non-canonical Wnt signaling participates in Jagged1-induced osteo/odontogenic differentiation in human dental pulp stem cells.

Osteoblast differentiation requires the interaction of various cell signaling pathways to modulate cell responses. Notch and Wnt signaling are among the crucial pathways that control numerous biological processes, including osteo/odontogenic differentiation. The aim of the present study was to examine the involvement of Wnt signaling in the Jagged1-induced osteo/odontogenic differentiation in human dental pulp stem cells (hDPSCs). The Wnt-related gene expression was analyzed from publicly available data of Jagged1-treated human dental pulp cells. The mRNA expression of Wnt ligands (WNT2B, WNT5A, WNT5B, and WNT16) and Wnt inhibitors (DKK1, DKK2, and SOST) were confirmed using real-time polymerase chain reaction. Among the Wnt ligands, WNT2B and WNT5A mRNA levels were upregulated after Jagged1 treatment. In contrast, the Wnt inhibitors DKK1, DKK2, and SOST mRNA levels were downregulated. Recombinant WNT5A, but not WNT2B, significantly promoted in vitro mineral deposition by hDPSCs. Wnt signaling inhibition using IWP-2, but not DKK1, inhibited Jagged1-induced alkaline phosphatase (ALP) activity, mineralization, and osteo/odontogenic marker gene expression in hDPSCs. In conclusion, Jagged1 promoted hDPSC osteo/odontogenic differentiation by modulating the non-canonical Wnt pathway.

Specific microRNAs Regulate Dental Pulp Stem Cell Behavior.

INTRODUCTION: MicroRNAs (miRNAs), small noncoding RNAs, control the translation of messenger RNAs into proteins. miRNAs have a crucial role in regulating the diverse biological processes of many physiological and pathological activities. The aim of this systematic review was to explore various functions of miRNAs in the regulation of dental pulp stem cell (DPSC) behavior. METHODS: The articles were searched in PubMed, SCOPUS, and ISI Web of Science database using designated keywords. Full-length manuscripts published in English in peer-reviewed journals relevant to the role of miRNAs in DPSC functions were included and reviewed by 2 independent researchers. RESULTS: The original search of the database generated 299 studies. A total of 102 duplicate studies were removed. After their exclusion, 48 studies were selected for review. miRNAs have shown to modulate the stemness and differentiation of various mesenchymal stem cells. The miRNAs expression profiles in DPSCs were differed compared with other cell types and have been demonstrated to regulate the levels of proteins crucial for promoting or inhibiting DPSC proliferation as well as differentiation. Further, miRNAs also modulate inflammatory processes in dental pulp. CONCLUSION: miRNAs have various functions on the regulation of DPSCs and understanding these roles of miRNAs is crucial for the development of new therapeutics in regenerative dental medicine. With the advancing technologies, the utilization of miRNA technology could revolutionarily change the future of regenerative endodontics.

Intermittent compressive force induces cell cycling and reduces apoptosis in embryoid bodies of mouse induced pluripotent stem cells.

In vitro manipulation of induced pluripotent stem cells (iPSCs) by environmental factors is of great interest for three-dimensional (3D) tissue/organ induction. The effects of mechanical force depend on many factors, including force and cell type. However, information on such effects in iPSCs is lacking. The aim of this study was to identify a molecular mechanism in iPSCs responding to intermittent compressive force (ICF) by analyzing the global gene expression profile. Embryoid bodies of mouse iPSCs, attached on a tissue culture plate in 3D form, were subjected to ICF in serum-free culture medium for 24 h. Gene ontology analyses for RNA sequencing data demonstrated that genes differentially regulated by ICF were mainly associated with metabolic processes, membrane and protein binding. Topology-based analysis demonstrated that ICF induced genes in cell cycle categories and downregulated genes associated with metabolic processes. The Kyoto Encyclopedia of Genes and Genomes database revealed differentially regulated genes related to the p53 signaling pathway and cell cycle. qPCR analysis demonstrated significant upregulation of Ccnd1, Cdk6 and Ccng1. Flow cytometry showed that ICF induced cell cycle and proliferation, while reducing the number of apoptotic cells. ICF also upregulated transforming growth factor ß1 (Tgfb1) at both mRNA and protein levels, and pretreatment with a TGF-ß inhibitor (SB431542) prior to ICF abolished ICF-induced Ccnd1 and Cdk6 expression. Taken together, these findings show that TGF-ß signaling in iPSCs enhances proliferation and decreases apoptosis in response to ICF, that could give rise to an efficient protocol to manipulate iPSCs for organoid fabrication.

Dorsomorphin attenuates Jagged1-induced mineralization in human dental pulp cells.

AIM: To investigate whether TGF-ß/BMP signalling participates in Jagged1-induced osteogenic differentiation in human dental pulp cells (hDPs). METHODOLOGY: Bioinformatic analysis of publicly available RNA sequencing data of Jagged1-treated hDPs was performed using NetworkAnalyst. The mRNA expression was validated using real-time polymerase chain reaction. hDPs were seeded on Jagged1 immobilized surfaces in the presence or absence of TGF-ß or BMP inhibitor. Osteogenic differentiation was evaluated using alkaline phosphatase staining, osteogenic marker gene expression and mineralization assay. Statistical analyses were performed using a Kruskal-Wallis test, followed by a pairwise comparison for more than three group comparison. Mann-Whitney U-test was employed for two group comparison. The statistical significance was considered at p < .05. RESULTS: Jagged1 treatment in growth medium significantly promoted TGFB1, TGFB2 and TGFB3 whilst significantly inhibited BMP2, BMP4 and BMP6 mRNA expression (p < .05). In osteogenic induction medium, Jagged1 significantly up-regulated TGFB1, TGFB2 and TGFB3 at days 1 and 3 (p < .05). Pre-treatment with TGF-ß1, TGF-ß2 or TGF-ß3 prior to osteogenic induction resulted in the significant increase of osteogenic marker gene expression, collagen type 1 protein expression, alkaline phosphatase enzymatic activity and mineral deposition (p < .05). However, TGF-ß signalling inhibition with SB431542 (4 µmol L-1 ) or SB505124 (47 and 129 nmol L-1 ) failed to attenuate the effect of Jagged1-induced osteogenic differentiation in hDPs. Dorsomorphin (4 and 8 µmol L-1 ) treatment significantly abolished the effect of Jagged1 on mineralization by hDPs (p < .05). CONCLUSION: Notch signalling activation by Jagged1 modulated TGF-ß and BMP ligand expression. Dorsomorphin, but not TGF-ß receptor inhibitor, attenuated Jagged1-induced osteogenic differentiation in hDPs.

Interleukin 15 participates in Jagged1-induced mineralization in human dental pulp cells.

OBJECTIVES: Crosstalk between Notch and other cell signaling molecules has been implicated to regulate the osteogenic differentiation. Understanding the interaction between Notch and IL15 is essential to reveal molecular mechanism. Thus, the objective of the present study was to investigate whether IL15 participates in the Notch signaling-induced mineral deposition in human dental pulp cells (hDPs). METHODS: hDPs were explanted from dental pulp tissues. To activate Notch signaling, the cells were seeded on Jagged1-immobilized surfaces. The mRNA expression was evaluated using real-time polymerase chain reaction. hDPs were treated with 5-50 ng/mL IL15. Cell viability and proliferation were determined using an MTT assay. Mineral deposition was examined using alizarin red s and Von Kossa staining. In some experiments, the cells were pretreated with a JAK inhibitor prior to stimulation. RESULTS: Jagged1 induced IL15 and IL15RA expression in hDPs. IL15 treatment significantly increased mineral deposition at 14 d and upregulated ALP, OCN, OSX, ANKH, and ENPP1 mRNA expression. IL15-induced mineralization was attenuated by JAK inhibitor pretreatment. Further, JAK inhibitor pretreatment inhibited the effect of Jagged1 on hDP mineral deposition. CONCLUSION: IL15 promoted the osteogenic differentiation in hDPs. Moreover, IL15 participated in the Jagged1-induced mineralization in hDPs.

Mechanical loading and the control of stem cell behavior.

OBJECTIVE: Mechanical stimulation regulates many cell responses. The present study describes the effects of different in vitro mechanical stimulation approaches on stem cell behavior. DESIGN: The narrative review approach was performed. The articles published in English language that addressed the effects of mechanical force on stem cells were searched on Pubmed and Scopus database. The effects of extrinsic mechanical force on stem cell response was reviewed and discussed. RESULTS: Cells sense mechanical stimuli by the function of mechanoreceptors and further transduce force stimulation into intracellular signaling. Cell responses to mechanical stimuli depend on several factors including type, magnitude, and duration. Further, similar mechanical stimuli exhibit distinct cell responses based on numerous factors including cell type and differentiation stage. Various mechanical applications modulate stemness maintenance and cell differentiation toward specific lineages. CONCLUSIONS: Mechanical force application modulates stemness maintenance and differentiation. Modification of force regimens could be utilized to precisely control appropriate stem cell behavior toward specific applications.

NOTCH2 participates in Jagged1-induced osteogenic differentiation in human periodontal ligament cells.

Jagged1 activates Notch signaling and subsequently promotes osteogenic differentiation in human periodontal ligament cells (hPDLs). The present study investigated the participation of the Notch receptor, NOTCH2, in the Jagged1-induced osteogenic differentiation in hPDLs. NOTCH2 and NOTCH4 mRNA expression levels increased during hPDL osteogenic differentiation. However, the endogenous NOTCH2 expression levels were markedly higher compared with NOTCH4. NOTCH2 expression knockdown using shRNA in hPDLs did not dramatically alter their proliferation or osteogenic differentiation compared with the shRNA control. After seeding on Jagged1-immobilized surfaces and maintaining the hPDLs in osteogenic medium, HES1 and HEY1 mRNA levels were markedly reduced in the shNOTCH2-transduced cells compared with the shControl group. Further, shNOTCH2-transduced cells exhibited less alkaline phosphatase enzymatic activity and in vitro mineralization than the shControl cells when exposed to Jagged1. MSX2 and COL1A1 mRNA expression after Jagged1 activation were reduced in shNOTCH2-transduced cells. Endogenous Notch signaling inhibition using a γ-secretase inhibitor (DAPT) attenuated mineralization in hPDLs. DAPT treatment significantly promoted TWIST1, but decreased ALP, mRNA expression, compared with the control. In conclusion, Notch signaling is involved in hPDL osteogenic differentiation. Moreover, NOTCH2 participates in the mechanism by which Jagged1 induced osteogenic differentiation in hPDLs.

Transcriptome analysis of basic fibroblast growth factor treated stem cells isolated from human exfoliated deciduous teeth.

BACKGROUND: Basic fibroblast growth factor (bFGF) regulates cell proliferation, migration, and differentiation in various cell types. The aim of the present study was to determine the bFGF target genes in stem cells isolated from human exfoliated deciduous teeth (SHEDs). METHODS: Cells were isolated from pulp tissue obtained from exfoliated deciduous teeth. Mesenchymal stem cell surface markers and the differentiation potential toward adipogenic and neurogenic lineages were characterized. The bFGF-treated SHED transcriptome was examined using a high throughput RNA sequencing technique. The mRNA and protein expression of selected genes were evaluated using real-time polymerase chain reaction and immunofluorescence staining, respectively. Cell cycle analysis was performed by flow cytometry. The colony forming unit number was also examined. RESULTS: The isolated cells expressed CD44, CD90, CD105, but not CD45. The upregulation of adipogenic and neurogenic marker genes was observed after culturing cells in the appropriate induction medium. Transcriptome analysis of the bFGF treated cells revealed that the upregulated genes were in the cell cycle related pathways, while the downregulated genes were in the extracellular matrix related pathways. Correspondingly, bFGF induced MKI67 mRNA expression and Ki67 protein expression. Furthermore, bFGF treatment significantly decreased the G0/G1, but increased the G2/M, population in SHEDs. Colony formation was markedly increased in the bFGF treated group and was attenuated by pretreating the cells with FGFR or PI3K inhibitors. CONCLUSION: bFGF controls cell cycle progression in SHEDs. Thus, it can be used to amplify cell number to obtain the amount of cells required for regenerative treatments.

RNA sequencing data of human periodontal ligament cells treated with continuous and intermittent compressive force.

Mechanical force regulates numerous biological functions. Application of different force types leads to different cell responses. This data article describes RNA sequencing data identifying gene expression of human periodontal ligament cells (hPDLs) treated with the continuous or intermittent compressive force. These data could be further utilized to investigate the controlling pathways that regulate hPDLs' behaviors by the different force types. Raw RNA sequencing data were deposited in the NCBI Sequence Read Archive (SRP136155) and NCBI Gene Expression Omnibus (GSE112122).

Intermittent compressive force promotes osteogenic differentiation in human periodontal ligament cells by regulating the transforming growth factor-ß pathway.

Mechanical force regulates periodontal ligament cell (PDL) behavior. However, different force types lead to distinct PDL responses. Here, we report that pretreatment with an intermittent compressive force (ICF), but not a continuous compressive force (CCF), promoted human PDL (hPDL) osteogenic differentiation as determined by osteogenic marker gene expression and mineral deposition in vitro. ICF-induced osterix (OSX) expression was inhibited by cycloheximide and monensin. Although CCF and ICF significantly increased extracellular adenosine triphosphate (ATP) levels, pretreatment with exogenous ATP did not affect hPDL osteogenic differentiation. Gene-expression profiling of hPDLs subjected to CCF or ICF revealed that extracellular matrix (ECM)-receptor interaction, focal adhesion, and transforming growth factor beta (TGF-ß) signaling pathway genes were commonly upregulated, while calcium signaling pathway genes were downregulated in both CCF- and ICF-treated hPDLs. The TGFB1 mRNA level was significantly increased, while those of TGFB2 and TGFB3 were decreased by ICF treatment. In contrast, CCF did not modify TGFB1 expression. Inhibiting TGF-ß receptor type I or adding a TGF-ß1 neutralizing antibody attenuated the ICF-induced OSX expression. Exogenous TGF-ß1 pretreatment promoted hPDL osteogenic marker gene expression and mineral deposition. Additionally, pretreatment with ICF in the presence of TGF-ß receptor type I inhibitor attenuated the ICF-induced mineralization. In conclusion, this study reveals the effects of ICF on osteogenic differentiation in hPDLs and implicates TGF-ß signaling as one of its regulatory mechanisms.

Gene expression profiling of Jagged1-treated human periodontal ligament cells.

OBJECTIVE: Jagged1 regulates several biological functions in human periodontal ligament cells (hPDLs). The present study aimed to evaluate mRNA expression profiling of Jagged1-treated hPDLs using microarray technique. METHODS: Notch ligands, Jagged1, were indirectly immobilized on tissue culture surface. Subsequently, hPDLs were seeded on Jagged1 immobilized surface and maintained in growth medium for 48 hr. Total RNA was collected and processed. Gene expression profiling was examined using microarray technique. Real-time polymerase chain reaction and immunofluorescence staining were employed to determine mRNA and protein expression levels, respectively. Cell proliferation and colony-forming unit assay were performed. Cell cycle was evaluated using propidium iodide staining and flow cytometry analysis. RESULTS: The isolated cells demonstrated fibroblast-like morphology and exhibited the co-expression of CD44, CD90, and CD105 surface markers. After stimulated with Jagged1, the total of 411 genes was differentially expressed, consisting both coding and non-coding genes. For coding genes, 165 and 160 coding genes were upregulated and downregulated, respectively. Pathway analysis revealed that the upregulated genes were mainly involved in cellular interactions, signal transduction, and collagen formation and degradation while the downregulated genes were in the events and phases in cell cycle. Jagged1 significantly decreased cell proliferation, reduced colony-forming unit ability, and induced G0/G1 cell cycle arrest in hPDLs. CONCLUSION: Jagged1 regulates various biological pathways in hPDLs. This gene expression profiling could help to understand the mechanisms potentially involved in the Notch signaling regulation in periodontal homeostasis.