The H2O2 Self-Sufficient 3D Printed ß-TCP Scaffolds with Synergistic Anti-Tumor Effect and Reinforced Osseointegration.

Tumor recurrence and massive bone defects are two critical challenges for postoperative treatment of oral and maxillofacial tumor, posing serious threats to the health of patients. Herein, in order to eliminate residual tumor cells and promote osteogenesis simultaneously, the hydrogen peroxide (H2O2) self-sufficient TCP-PDA-CaO2-CeO2 (TPCC) scaffolds are designed by preparing CaO2 or/and CeO2 nanoparticles (NPs)/chitosan solution and modifying the NPs into polydopamine (PDA)-modified 3D printed TCP scaffolds by rotary coating method. CaO2 NPs loaded on the scaffolds can release Ca2+ and sufficient H2O2 in the acidic tumor microenvironment (TME). The generated H2O2 can further produce hydroxyl radicals (·OH) under catalysis effect by peroxidase (POD) activity of CeO2 NPs, in which the photothermal effect of the PDA coating enhances its POD catalytic effect. Overall, NPs loaded on the scaffold chemically achieve a cascade reaction of H2O2 self-sufficiency and ·OH production, while functionally achieving synergistic effects on anti-tumor and bone promotion. In vitro and in vivo studies show that the scaffolds exhibit effective osteo-inductivity, induced osteoblast differentiation and promote osseointegration. Therefore, the multifunctional composite scaffolds not only validate the concept of chemo-dynamic therapy (CDT) cascade therapy, but also provide a promising clinical strategy for postoperative treatment of oral and maxillofacial tumor.

Identifying genes for regulating osteogenic differentiation of human periodontal ligament stem cells in inflammatory environments by bioinformatics analysis.

BACKGROUND AND OBJECTIVES: Periodontitis is an immuno-inflammatory disease caused by dental plaque biofilms and inflammations. The regeneration of bone tissue in inflammatory environment is of great significance for the treatment of periodontal disease, but the specific molecular mechanism of bone formation in periodontitis still needs further exploration. The objective of this study was to identify key osteogenesis-related genes (ORGs) in periodontitis. METHODS: We used two datasets from the Gene Expression Omnibus (GEO) database to find differentially expressed mRNAs and miRNAs, further performed Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Then we predicted the downstream genes of the differentially expressed miRNAs (DEMs) by the TargetScan database and established a miRNA-mRNA regulatory network. Finally, the osteogenic mechanism of periodontitis was explored through quantitative real-time PCR (qRT-PCR) by inducing inflammatory environment and osteogenic differentiation of hPDLSCs. RESULTS: Through differential expression analysis and prediction of downstream target genes of DEMs, we created a miRNA-mRNA regulatory network consisting of 29 DEMs and 11 differentially expressed osteogenesis-related genes (DEORGs). In addition, the qRT-PCR results demonstrated that BTBD3, PLAT, AKAP12, SGK1, and GLCE expression levels were significantly upregulated, while those of TIMP3, ZCCHC14, LIN7A, DNAH6, NNT, and ITGA6 were downregulated under the dual effects of inflammatory stimulation and osteogenic induction. CONCLUSION: DEORGs might be important factors in the osteogenic phase of periodontitis, and the miRNA-mRNA network may shed light on the clarification of the role and mechanism of osteogenesis in periodontitis and contribute to the development of novel therapeutic strategies.

Zoledronic acid regulates the synthesis and secretion of IL-1ß through Histone methylation in macrophages.

Long-term administration of nitrogen-containing bisphosphonates increases the risk of detrimental side effects, such as bisphosphonate-related osteonecrosis of the jaw (BRONJ). BRONJ development is associated with inflammation, but its pathophysiology remains unknown. Here, we examined whether histone methylation is responsible for zoledronic acid (Zol)-induced inflammatory responses. We found that Kdm6a and Kdm6b markedly increased interleukin 1ß expression and Gasdermin D cleavage, which are both activated by Caspase 1, in macrophages. Inhibitors of Kdm6a and Kdm6b robustly abolished Zol-enhanced interleukin 1ß synthesis and secretion from macrophages. When Kdm6a and Kdm6b were pharmacologically inhibited in vivo, poor healing of the alveolar socket and inflammatory responses were ameliorated in Zol-treated mice. Taken together, we showed the pathologic role of Kdm6a and Kdm6b in Zol-promoted inflammatory responses and demonstrated that Kdm6a and Kdm6b are potential therapeutic targets for the treatment of BRONJ.

ARHGAP17 inhibits pathological cyclic strain-induced apoptosis in human periodontal ligament fibroblasts via Rac1/Cdc42.

Rho GTPase-activating protein (Rho-GAP) and Rho GDP dissociation inhibitor (Rho- GDI) are two main negative regulators of Rho GTPase. Our previous work has found that Rho-GDI and Rho GTPase are involved in the response of human periodontal ligament (PDL) cells to mechanical stress. However, whether Rho-GAP also has a role in this process remains unknown. Here, we attempted to find the Rho-GAP gene that may be involved in pathological stretch-induced apoptosis of PDL cells. Human PDL fibroblasts were exposed to 20% cyclic strain for 6 hours or 24 hours, after which the expression levels of ARHGAP10, ARHGAP17, ARHGAP21, ARHGAP24 and ARHGAP28 were determined. Results showed that ARHGAP17 expression decreased the most obviously after treatment of stretch. In addition, ARHGAP17 overexpression abolished 20% cyclic strain-induced apoptosis. Therefore, ARHGAP17 has an important role in pathological stretch-induced apoptosis of human PDL fibroblasts. Moreover, we found that ARHGAP17 overexpression also alleviated cyclic strain-induced activation of Rac1/Cdc42, a major downstream target of ARHGAP17. Furthermore, two Rac1 inhibitors, NSC23766 and EHT 1864, both attenuated ARHGAP17 knockdown-mediated apoptosis in human PDL fibroblasts. Collectively, our data demonstrate that ARHGAP17 inhibits pathological cyclic strain-induced apoptosis in human PDL fibroblasts through inactivating Rac1/Cdc42. This study highlights the importance of Rho signalling in the response of human PDL fibroblasts to mechanical stress.

A 20(S)-protopanaxadiol derivative PPD12 reverses ABCB1-mediated multidrug resistance with oral bioavailability and low toxicity.

The ATP-binding cassette subfamily B member 1 (ABCB1) is a transporter that mediates multidrug resistance (MDR) against chemotherapy, which leads to decreased patient survival. To inhibit ABCB1 activity in MDR cancer cells, the authors previously designed and synthesized a derivative from 20(S)-protopanaxadiol (PPD) PPD12 and verified its efficacy in ABCB1-overexpressing cancer cells. In the present study, the reversal effect of PPD12 on MDR was further evaluated and its pharmacokinetics and toxicity in vitro and in vivo were investigated. Incubation with PPD12 may significantly ameliorate the drug resistance of KB/VCR cells in a short time and maintain its reversed MDR ability for increasing time periods. In assays on a series of CYP450 activities, PPD12 demonstrated slight inhibition effects on the majority of enzymes. The bioavailability of PPD12 was nearly 100% by oral administration in a mouse model. Single PPD12 oral gavage at either high doses or subchronic low doses, was well tolerated by the mice. In addition, PPD12 at the therapeutic dosage did not significantly increase the toxicity of the chemotherapeutic agent Adriamycin when mice received a combination of the two compounds. In conclusion, PPD12 represents a novel type of ABCB1 inhibitor that has significant bioactivity in terms of MDR, high oral bioavailability and low toxicity.

[Preliminary study of Simplant software in mandibular molar implantation].

PURPOSE: The purpose of the study was to investigate the clinical effect and implementation method of mandibular molar implantation using a CT machine combined with Simplant software so as to provide a safer and more reliable method for preoperative preparation of mandibular molar implantation. METHODS: CT/cone-beam CT (CBCT) examinations were applied among 10 cases selected into the study. The Simplant software was used to measure the distances between implantation point center and superior wall of inferior alveolar nerve canal (H1), buccolingual diameter (H2), mesiodistal diameter (H3), ginginal thickness(H4) and occlusal distance (H5). Simplant software was also applied to calculate the bone mineral density of implantation areas and trace the inferior alveolar nerve canal. RESULTS: H1 was between 10.7 mm-17.6 mm, H3 was between 5.4mm-8.3mm.The 10 selected cases undertook implantations according to the results of simulation and all of them succeed after a one year's clinical follow-up. CONCLUSIONS: According to its accuracy in measuring data of the implantation area, Simplant software can be routinely used in preoperative preparation of mandibular molar implantation, which will make the process safer and more controllable.

The Rho-mDia1 signaling pathway is required for cyclic strain-induced cytoskeletal rearrangement of human periodontal ligament cells.

Tooth movement is the result of periodontal tissue reconstruction. The biomechanical effects produced by orthopedic forces can affect the cytoskeletal rearrangement of human periodontal ligament cells (hPDLCs). However, the mechanisms responsible for the cytoskeletal rearrangement are not completely understood. To analyze the effect, we investigated the role of the Rho-mDia1 signaling pathway in cyclic strain-induced cytoskeletal rearrangement of hPDLCs in detail. We cultured hPDLCs on collagen I-coated six-well Bioflex plates and then exposed them to cyclic strain with physiological loading (10%) at a frequency of 0.1Hz for 6 or 24h using a Flexercell Tension Plus system. Notably, the cells cultured on the Bioflex plates showed increased expression levels of RhoA-GTP, profilin-1 protein, and the combination of RhoA and mDia1, whereas the expression levels of Rho-GDIa were reduced compared with a static control group. Furthermore, the cytoskeletal rearrangement of cells was enhanced. However, profilin-1 protein expression and cytoskeletal reorganization under cyclic strain can decrease due to the overexpression of Rho-GDIa or mDia1-siRNA transfection, whereas Rho-GDIa siRNA transfection has the opposite effect on hPDLCs. Together, our results demonstrate that the Rho-mDia1 signaling pathway is involved in the cytoskeletal rearrangement of hPDLCs induced by cyclic strain. These observations may enable a more in-depth understanding of orthodontic tooth movement and the reconstruction of PDL and alveolar bone.

Rho is involved in periodontal tissue remodelling with experimental tooth movement in rats.

OBJECTIVE: To characterise in vivo the close relationship between the Rho signalling pathway and periodontal tissue remodelling in experimental tooth movement in rats. MATERIALS AND METHODS: In total, 24 male Sprague Dawley rats were divided randomly into four groups. Closed-coil springs were used to create a 40-g mesial force to move the right upper first molars in anaesthetised rats. The untreated contralateral side served as a control. On days 3, 7, 10, and 14 after force application, paraffin wax-embedded sections of the dissected maxilla were prepared for immunohistochemistry to localise Rho kinase (ROCK), LIM kinase (LIMK), and its downstream effector (cofilin). The immunoreactivity of the molecules investigated in the periodontal ligament area was converted into grey-scale values. RESULTS: The expression of Rho and its signalling were detected mainly in the disto-coronal areas of the root in the periodontal ligament area of the control and loaded teeth. In contrast, ROCK-, LIMK-, and cofilin-positive cells were rare in the mesio-coronal areas. In the experimental group, the expression of ROCK, LIMK, and cofilin on the tension side (disto-coronal areas) increased significantly on days 7, 10, and 14, compared with those of untreated control teeth. CONCLUSIONS: These data suggest that Rho is involved in periodontal tissue remodelling during orthodontic tooth movement, modulating ROCK, LIMK, and cofilin activity.

Cyclic strain-induced cytoskeletal rearrangement of human periodontal ligament cells via the Rho signaling pathway.

BACKGROUND: Although mechanical stimulations are known have a significant impact on cytoskeletal rearrangement, little is known regarding the behavioral alteration of human periodontal ligament cells (hPDLCs) under cyclic strain. The aim of this study was to elucidate the role of the Rho signaling pathway on cyclic strain-induced cytoskeletal rearrangement of hPDLCs. METHODS: Healthy hPDLCs obtained from teeth extracted for orthodontic purposes were subjected to cyclic strain with physiological loading (10%) at a frequency of 0.1 Hz for 6 h or 24 h using a FX-5000T system. Changes in cell morphology were examined by phase-contrast microscopy, while F-actin reorganization was observed by phalloidin staining and confocal microscopy. Protein expression was analyzed through western blot analysis. RESULTS: Significant enhancement of cytoskeletal reorganization was observed following exposure to the cyclic strain. In addition, a significant increase was noted in the expression levels of GTP-Rho, Rho-associated protein kinase (ROCK) and p-cofilin, whereas the expression levels of Rho GDP dissociation inhibitors alpha (Rho-GDIa) were reduced in the hPDLCs, compared with the static control cells. More importantly, the Rock inhibitor Y-27632 suppressed cyclic strain-induced cytoskeletal rearrangement of hPDLCs. Additionally, Y-27632 and overexpression of Rho-GDIa were found to lower p-cofilin protein expressions under cyclic strain, while Rho-GDIa siRNA transfection had the opposite effect on the hPDLCs. CONCLUSION: Cyclic strain promotes cytoskeletal rearrangement of hPDLCs by downregulating the expression levels of Rho-GDIa and upregulating the expression levels of GTP-Rho, Rock and p-cofilin. These observations may provide valuable insight into understanding orthodontic tooth movement as well as alveolar bone remodeling.

Rho plays a key role in TGF-ß1-induced proliferation and cytoskeleton rearrangement of human periodontal ligament cells.

Human periodontal ligament cells (hPDLCs) form specialised connective tissues that influence the lifespan of the tooth. Periodontal disease is a chronic infectious disease of the periodontal supporting tissues caused by a variety of factors, particularly the loss of hPDLCs. Transforming growth factor-ß1 (TGF-ß1) is a multifunctional cytokine known to play an important role in periodontal disease, but little is known about the effects of TGF-ß1 on human PDL cells. To determine how TGF-ß1 mediates the changes in hPDLCs, we characterised the effects of TGF-ß1 treatment on hPDLCs. We then elucidated the signalling pathway that mediates these effects. Serum-starved hPDLCs were incubated with 10ng/mL TGF-ß1, and their proliferation was examined using the Cell Counting Kit-8, while their morphological changes were examined by phase-contrast microscopy. F-actin reorganisation was visualised by phalloidin staining and confocal microscopy. Protein expression was analysed by western blotting. We found that TGF-ß1 treatment induced proliferation and cytoskeletal reorganisation, decreased Rho-GDIa protein expression, activated ROCK protein expression, and increased the phosphorylation of LIM kinase and cofilin. Proliferation and cytoskeletal rearrangement were suppressed by pre-treatment with the ROCK inhibitor Y-27632; additionally, expression of ROCK protein and phosphorylation of LIM kinase and cofilin were decreased by Y-27632, while Rho-GDIa knockdown by targeted siRNA transfection causes opposite effects. Therefore, we propose that TGF-ß1 induces proliferation and cytoskeletal rearrangement in hPDLCs via Rho GTPase-dependent pathways that modulate ROCK, LIM kinase, and cofilin activity.

Pathological cyclic strain-induced apoptosis in human periodontal ligament cells through the RhoGDIα/caspase-3/PARP pathway.

AIM: Human periodontal ligament (PDL) cells incur changes in morphology and express proteins in response to cyclic strain. However, it is not clear whether cyclic strain, especially excessive cyclic strain, induces PDL cell apoptosis and if so, what mechanism(s) are responsible. The aim of the present study was to elucidate the molecular mechanisms by which pathological levels of cyclic strain induce human PDL cell apoptosis. MATERIALS AND METHODS: Human PDL cells were obtained from healthy premolar tissue. After three to five passages in culture, the cells were subjected to 20% cyclic strain at a frequency of 0.1 Hz for 6 or 24 h using an FX-5000T system. Morphological changes of the cells were assessed by inverted phase-contrast microscopy, and apoptosis was detected by fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide staining followed by flow cytometry. Protein expression was evaluated by Western blot analysis. RESULTS: The number of apoptotic human PDL cells increased in a time-dependent manner in response to pathological cyclic strain. The stretched cells were oriented parallel to each another with their long axes perpendicular to the strain force vector. Cleaved caspase-3 and poly-ADP-ribose polymerase (PARP) protein levels increased in response to pathological cyclic strain over time, while Rho GDP dissociation inhibitor alpha (RhoGDIα) decreased. Furthermore, knock-down of RhoGDIα by targeted siRNA transfection increased stretch-induced apoptosis and upregulated cleaved caspase-3 and PARP protein levels. Inhibition of caspase-3 prevented stretch-induced apoptosis, but did not change RhoGDIα protein levels. CONCLUSION: The overall results suggest that pathological-level cyclic strain not only influenced morphology but also induced apoptosis in human PDL cells through the RhoGDIα/caspase-3/PARP pathway. Our findings provide novel insight into the mechanism of apoptosis induced by pathological cyclic strain in human PDL cells.

[Cyclic strain promotes migration of human periodontal ligament cell via extracellular signal-regulated kinase (ERK) signaling pathway].

OBJECTIVE: To study the effect of cyclic strain on migration of human periodontal ligament cell(hPDLC) and underlying mechanism. METHODS: The cultured hPDLC were subjected to 10% or 20%-elongation magnitude cyclic strain at frequency of 0.1 Hz by FX-4000T system for 6 or 24 hours-duration respectively, while the static group serves as control. hPDLC migration was assayed by wound healing method. The expressions of matrix metalloproteinases-9 (MMP-9) and p-ERK1/2 in hPDLC without or with cyclic strain were analyzed by Western blotting. To investigate the effect of ERK signaling pathway and MMP-9 on migration of hPDLC, the cells were incubated with PD98059, a specific extracellular signal-regulated kinase (ERK) kinase inhibitor, or doxycycline, a MMP inhibitor. Then the expressions of p-ERK1/2 and MMP-9 and hPDLC migration were analyzed. RESULTS: In wound healing tests, the migration of hPDLC exposed to 10% or 20%-cyclic strain at 0.1 Hz-frequency for 6 hours was not apparent but became significantly different for 24 hours (P < 0.05) compared to control. Furthermore, the 20%-elongation magnitude of cyclic strain had more remarkable effect on migration of hPDLC than 10%-elongation magnitude at 24 hours-duration (P < 0.05). Cyclic strain obviously increased the expression of MMP-9 in hPDLC (P < 0.05). PD98059 could repress not only the activation of p-ERK1/2 but also the expression of MMP-9 induced by cyclic strain in hPDLC. The migration of hPDLC enhanced by cyclic strain was repressed by DOX or PD98059 in wound healing tests. CONCLUSIONS: Cyclic strain promotes the migration of hPDLC through activating ERK signaling pathway and inducing the expression of MMP-9.