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This study aimed to investigate the effects of different magnitudes and durations of static tensile strain on human periodontal ligament cells (hPDLCs), focusing on osteogenesis, mechanosensing and inflammation. Static tensile strain magnitudes of 0%, 3%, 6%, 10%, 15% and 20% were applied to hPDLCs for 1, 2 and 3 days. Cell viability was confirmed via live/dead cell staining. Reference genes were tested by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and assessed. The expressions of TNFRSF11B, ALPL, RUNX2, BGLAP, SP7, FOS, IL6, PTGS2, TNF, IL1B, IL8, IL10 and PGE2 were analyzed by RT-qPCR and/or enzyme-linked immunosorbent assay (ELISA). ALPL and RUNX2 both peaked after 1 day, reaching their maximum at 3%, whereas BGLAP peaked after 3 days with its maximum at 10%. SP7 peaked after 1 day at 6%, 10% and 15%. FOS peaked after 3 days with its maximum at 3%, 6% and 15%. The expressions of IL6 and PTGS2 both peaked after 1 day, with their minimum at 10%. PGE2 peaked after 1 day (maximum at 20%). The ELISA of IL6 peaked after 3 days, with the minimum at 10%. In summary, the lower magnitudes promoted osteogenesis and caused less inflammation, while the higher magnitudes inhibited osteogenesis and enhanced inflammation. Among all magnitudes, 10% generally caused a lower level of inflammation with a higher level of osteogenesis.

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.

This study developed a drug-loadable hydrogel system with high plasticity and favorable biological properties to enhance oral bone tissue regeneration. Hydrogels of different calcium alginate concentrations were prepared. Their swelling ratio, degradation time, and bovine serum albumin (BSA) release rate were measured. Human periodontal ligament cells (hPDLCs) and bone marrow stromal cells (BMSCs) were cultured with both calcium alginate hydrogels and polylactic acid (PLA), and then we examined the proliferation of cells. Inflammatory-related factor gene expressions of hPDLCs and osteogenesis-related gene expressions of BMSCs were observed. Materials were implanted into the subcutaneous tissue of rabbits to determine the biosecurity properties of the materials. The materials were also implanted in mandibular bone defects and then scanned using micro-CT. The calcium alginate hydrogels caused less inflammation than the PLA. The number of mineralized nodules and the expression of osteoblast-related genes were significantly higher in the hydrogel group compared with the control group. When the materials were implanted in subcutaneous tissue, materials showed favorable biocompatibility. The calcium alginate hydrogels had superior osteoinductive bone ability to the PLA. The drug-loadable calcium alginate hydrogel system is a potential bone defect reparation material for clinical dental application.

Multiphasic scaffolds that combine different architectural, physical, and biological properties are the best option for the regeneration of complex tissues such as the periodontium. Current developed scaffolds generally lack architectural accuracy and rely on multistep manufacturing, which is difficult to implement for clinical applications. In this context, direct-writing electrospinning (DWE) represents a promising and rapid technique for developing thin 3D scaffolds with controlled architecture. The current study aimed to elaborate a biphasic scaffold using DWE based on two polycaprolactone solutions with interesting properties for bone and cement regeneration. One of the two scaffold parts contained hydroxyapatite nanoparticles (HAP) and the other contained the cementum protein 1 (CEMP1). After morphological characterizations, the elaborated scaffolds were assessed regarding periodontal ligament (PDL) cells in terms of cell proliferation, colonization, and mineralization ability. The results demonstrated that both HAP- and CEMP1-functionalized scaffolds were colonized by PDL cells and enhanced mineralization ability compared to unfunctionalized scaffolds, as revealed by alizarin red staining and OPN protein fluorescent expression. Taken together, the current data highlighted the potential of functional and organized scaffolds to stimulate bone and cementum regeneration. Moreover, DWE could be used to develop smart scaffolds with the ability to spatially control cellular orientation with suitable cellular activity at the micrometer scale, thereby enhancing periodontal and other complex tissue regeneration.

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.

The combination of oral derived stem cells and 3-D scaffolds is considered advantageous in bone repair. In particular, collagen membranes possess ideal biological properties and can support infiltration and proliferation of osteoblasts, promoting bone regeneration. Our study aimed to develop a new biocompatible osteogenic construct composed of a commercially available collagen membrane (Evolution [Evo]), human periodontal-ligament stem cells (hPDLSCs) enriched with extracellular vesicles (EVs), or polyethylenimine (PEI)-engineered EVs (PEI-EVs). Osteogenic ability and expression of osteogenic genes were evaluated in vitro in hPDLSCs cultured with or without Evo, with Evo and EVs, or PEI-EVs. In addition, the bone-regeneration capacity of Evo, Evo enriched with hPDLSCs, Evo enriched with hPDLSCs and EVs/PEI-EVs was investigated in rats subjected to calvarial defects. Our results showed that Evo enriched with EVs and PEI-EVs showed high biocompatibility and osteogenic properties in vitro and in vivo. In addition, quantitative reverse-transcription polymerase chain reaction demonstrated the upregulation of osteogenic genes, such as , , , , , and , in the presence of PEI-EVs. Upregulation of BMP2/4 was confirmed for Evo enriched with PEI-EVs and hPDLSCs both in vitro by Western blot and in vivo by immunofluorescence. Our results indicated that Evo enriched with hPDLSCs and PEI-EVs is able to promote a bone-regeneration process for the treatment of calvarium and ossification defects caused by accidental or surgery trauma. In particular, PEI-EVs had a significant role in activation of the osteogenic process.

Spheroids reproduce the tissue structure that is found in vivo more accurately than classic two-dimensional (2D) monolayer cultures. We cultured human periodontal ligament stem cells (HPLSCs) as spheroids that were embedded in collagen gel to examine whether their cementogenic differentiation could be enhanced by treatment with recombinant human plasminogen activator inhibitor-1 (rhPAI-1). The upregulated expression of cementum protein 1 (CEMP1) and cementum attachment protein (CAP), established cementoblast markers, was observed in the 2D monolayer HPLSCs that were treated with rhPAI-1 for 3 weeks compared with that in the control and osteogenic-induction medium groups. In the embedded HPLSC spheroids, rhPAI-1 treatment induced interplay between the spheroids and collagenous extracellular matrix (ECM), indicating that disaggregated HPLSCs migrated and spread into the surrounding ECM 72 h after three-dimensional (3D) culture. Western blot and immunocytochemistry analyses showed that the CEMP1 expression levels were significantly upregulated in the rhPAI-1-treated embedded HPLSC spheroids compared with all the 2D monolayer HPLSCs groups and the 3D spheroid groups. Therefore, 3D collagen-embedded spheroid culture in combination with rhPAI-1 treatment may be useful for facilitating cementogenic differentiation of HPLSCs.

To investigate the roles of Notoginsenoside R1 (NG-R1) on the proliferation and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and explore its possible mechanism. hPDLSCs were isolated and, then characterized by flow cytometry. Cell-counting kit-8 (CCK-8) and colony assays were used to validate the effect of different NG-R1 concentrations on hPDLSCs proliferation and the optimal concentration was determined. Quantitative detection of alkaline phosphatase (ALP) activity at optimal concentration and the mineralization of the cells was investigated by Alizarin Red S staining. qRT-PCR and Western blot were utilized to examine the factors expression levels of ALP, Runx Family Transcription Factor 2 (RUNX2), Collagen I (Col-1) and catenin beta 1 (CTNNB1; β-catenin). In addition, the tankyrase inhibitor XAV-939 was used to explore NG-R1's role in canonical Wnt signaling. hPDLSCs were positive for surface antigens CD90 while negative for CD34 and CD45, which indicated that we have successfully isolated the hPDLSCs. Furthermore, a concentration of 20μmol NG-R1 dramatically enhanced hPDLSCs proliferation, ALP activity, and mineral deposition. ALP, RUNX2, COL-1, and β-catenin expression were all rised in comparison to control group. After XAV-939 was added to disrupt the canonical Wnt signaling, the impact of NG-R1 appeared to be reversed. These findings suggest that NG-R1 can stimulate osteogenic differentiation of hPDLSCs, which is probably attributable to canonical Wnt signaling activation.

Periodontitis is an inflammatory lesion in the periodontal tissue. The behavior of human periodontal ligament stem cells (hPDLSCs), which play an important role in periodontal tissue regeneration, is restricted by the influence of inflammatory mediators. Photobiomodulation therapy exerts anti-inflammatory effects. The purpose of this study was to investigate the effects of light-emitting diode (LED) irradiation on the inflammatory responses of hPDLSCs. The light source was a red LED (peak wavelength: 650 nm), and the total absolute irradiance was 400 mW/cm2. The inflammatory response in hPDLSCs is induced by tumor necrosis factor (TNF)-α. Adenosine triphosphate (ATP) levels and pro-inflammatory cytokine (interleukin [IL]-6 and IL-8) production were measured 24 h after LED irradiation, and the effects of potassium cyanide (KCN) were investigated. LED irradiation at 6 J/cm2 significantly increased the ATP levels and reduced TNF-α-induced IL-6 and IL-8 production. Furthermore, the inhibitory effect of LED irradiation on the production of pro-inflammatory cytokines was inhibited by KCN treatment. The results of this study showed that high-intensity red LED irradiation suppressed the TNF-α-stimulated pro-inflammatory cytokine production in hPDLSCs by promoting ATP synthesis. These results suggest that high-intensity red LED is a useful tool for periodontal tissue regeneration in chronically inflamed tissues.