Mettl3­mediated m6A RNA methylation regulates osteolysis induced by titanium particles.

Peri­prosthetic osteolysis (PPO) induced by wear particles is considered the primary cause of titanium prosthesis failure and revision surgery. The specific molecular mechanisms involve titanium particles inducing multiple intracellular pathways, which impact disease prevention and the targeted therapy of PPO. Notably, N6­methyladenosine (m6A) serves critical roles in epigenetic regulation, particularly in bone metabolism and inflammatory responses. Thus, the present study aimed to determine the role of RNA methylation in titanium particle­induced osteolysis. Results of reverse transcription­quantitative PCR (RT­qPCR), western blotting, ELISA and RNA dot blot assays revealed that titanium particles induced osteogenic inhibition and proinflammatory responses, accompanied by the reduced expression of methyltransferase­like (Mettl) 3, a key component of m6A methyltransferase. Specific lentiviruses vectors were employed for Mettl3 knockdown and overexpression experiments. RT­qPCR, western blotting and ELISA revealed that the knockdown of Mettl3 induced osteogenic inhibition and proinflammatory responses comparable with that induced by titanium particle, while Mettl3 overexpression attenuated titanium particle­induced cellular reactions. Methylated RNA immunoprecipitation­qPCR results revealed that titanium particles mediated the methylation of two inhibitory molecules, namely Smad7 and SMAD specific E3 ubiquitin protein ligase 1, via Mettl3 in bone morphogenetic protein signaling, leading to osteogenic inhibition. Furthermore, titanium particles induced activation of the nucleotide binding oligomerization domain 1 signaling pathway through methylation regulation, and the subsequent activation of the MAPK and NF­κB pathways. Collectively, the results of the present study indicated that titanium particles utilized Mettl3 as an upstream regulatory molecule to induce osteogenic inhibition and inflammatory responses. Thus, the present study may provide novel insights into potential therapeutic targets for aseptic loosening in titanium prostheses.

ATG7-mediated autophagy protects human gingival myofibroblasts from irradiation-induced apoptosis.

BACKGROUND: Apoptosis resistance of myofibroblasts is critical in pathology of irradiation-induced fibrosis and osteoradionecrosis of the jaw (ORNJ). However, molecular mechanism of apoptosis resistance induced by irradiation in oral myofibroblasts remains largely obscure. METHODS: Matched ORNJ fibroblasts and normal fibroblasts pairs from gingival were primarily cultured, and myofibroblast markers of α-SMA and FAP were evaluated by qRT-PCR and western blot. CCK8 assay and flow cytometric analysis were performed to investigate the cell viability and apoptosis under irradiation treatment. Autophagy-related protein LC3 and ATG7, and punctate distribution of LC3 localization were further detected. After inhibition of autophagy with inhibitor CQ and 3-MA, as well as transfected ATG7-siRNA, cell viability and apoptosis of ORNJ and normal fibroblasts were further assessed. RESULTS: Compared with normal fibroblasts, ORNJ fibroblasts exhibited significantly higher α-SMA and FAP expression, increased cell, viability and decreased apoptosis under irradiation treatment. LC3-II and ATG7 were up-regulated in ORNJ fibroblasts with irradiation stimulation. After inhibition of irradiation-induced autophagic flux with lysosome inhibitor CQ, LC3-II protein was accumulated and punctate distribution of LC3 localization was increased in ORNJ fibroblasts. Moreover, autophagy inhibitor CQ and 3-MA enhanced the irradiation-induced apoptosis but inhibited viability of ORNJ fibroblasts. Silencing ATG7 with siRNA could obviously weaken irradiation-induced LC3-II expression, and promoted irradiation-induced apoptosis of ORNJ fibroblasts. After knockdown of ATG7, finally, p-AKT(Ser473) and p-mTOR(Ser2448) levels of ORNJ fibroblasts were significantly increased under irradiation. CONCLUSION: Compared with normal fibroblasts, human gingival myofibroblasts are resistant to irradiation-induced apoptosis via autophagy activation. Silencing ATG7 may evidently inhibit activation of autophagy, and promote apoptosis of gingival myofibroblasts via Akt/mTOR pathway.

Construction of an enzyme-free biosensor utilizing CuO nanoparticles enriched in DNA polymer to catalyze a click chemistry reaction for SERS detection of the p53 gene.

The p53 gene is a known cancer marker. We report a novel protocol for the SERS tandem strategy to detect the p53 gene with high sensitivity. Herein, the click reaction between azide and alkyne was catalyzed by utilizing copper oxide nanoparticles (CuONPs), which were enriched by a T-DNA-triggered hybridization chain reaction (HCR). The T-DNA signal was amplified by establishing the correlation between the T-DNA signal and the concentration of CuONPs in a nonenzymatic isothermal environment. In contrast to other Raman reporters, we used alkynyl compounds as Raman reporters, which showed excellent characteristics in the Raman-silent region (1800-2800 cm-1). Therefore, the highly sensitive and highly selective SERS signals could be obtained in complex biological matrices. Due to utilizing multistep amplification strategies, including the nanoparticle-modified HCR polymer and "click" reaction, the limit of detection (LOD) and the limit of quantification (LOQ) of this sensor could be as low as 0.0174 pM and 0.0583 pM, respectively. The accuracy of the strategy expressed as the RSD was in the range of 3.14%-6.21%. The results indicated that the constructed sensor has excellent performance for the detection of the p53 gene in serum samples in a low concentration range, which suggests that the proposed enzyme-free SERS analytical sensor has good clinical application prospects.

A colorimetric and SERS dual-readout sensor for sensitive detection of tyrosinase activity based on 4-mercaptophenyl boronic acid modified AuNPs.

Tyrosinase (TYR) is as a well-known polyphenol oxidase and important biomarker of melanocytic lesions. Thus, developing powerful methods to determine TYR activity is of great value in the early diagnosis of skin disease. Direct surface-enhanced Raman scattering (SERS) detection of biomolecules is usually affected by non-specific interference and complicate structure of the analytes. It is a challenge to develop Raman-active molecules with specific recognition to analytes in complex media. Here, we report a novel colorimetric and surface-enhanced Raman scattering (SERS) dual-readout assay for the determination of TYR using commercially available and economical 4-mercaptophenyl boronic acid (4-MPBA) as a Raman-active and recognition molecule. 4-MPBA provides a unique interactive boronic acid group to the diol group of TYR substrate and exhibits good SERS signal. Also, the introduction of magnetic beads could promptly improve the anti-interference ability of dual-mode sensor. The TYR-incubated tyramine-modified magnetic beads could obviously change the concentration of 4-MPBA-AuNPs in the presence of O2 and ascorbic acid, where the ultraviolet visible (UV-vis) absorption and SERS intensity were directly related to the concentration of TYR added. The dual-mode sensor had a rapid response to TYR within 1 min under optimized conditions and had high selectivity for TYR with a limit of detection at 0.001 U/mL. In addition, the dual-mode strategy showed promising prospects in the determination of TYR activity in serum samples and could be used to screen TYR inhibitors.

Exosome secreted by human gingival fibroblasts in radiation therapy inhibits osteogenic differentiation of bone mesenchymal stem cells by transferring miR-23a.

Radiation-induced fibrosis is recently established as a main reason for osteoradionecrosis of the jaw (ORNJ), anti-eradiation fibrosis drugs achieve satisfactory therapeutic effects. However, the molecular mechanism remain to be fully elucidated. In this study, we found the inhibitory effect of irradiation activated gingival fibroblasts on osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs). Moreover, irradiation-activated-fibroblasts significantly increased miR­23a expression in hBMSCs. Decreased miR­23a enhanced osteogenic differentiation of BMSCs, and elevated miR­23a inhibited this process via directly targeting CXCL12. Finally, exosome released from irradiation-activated-fibroblasts inhibited osteogenic differentiation of BMSCs, and these exosome mediated delivery of miR-23a and further regulated miR-23a/CXCL12 axis in hBMSCs. Therefore, our findings suggest that by transferring miR-23a, exosome secreted by human gingival fibroblasts in radiation therapy serves a vital role in osteogenic differentiation of hBMSCs, which may provide novel clinical treatments for ORNJ.

Preparation of Aptamer Responsive DNA Functionalized Hydrogels for the Sensitive Detection of α-Fetoprotein Using SERS Method.

Hepatocellular carcinoma (HCC), which is one of the three major cancers, has attracted growing attention due to its high mortality, health care cost, and circumscribed therapeutic methods. Hence, the development of a fast, accurate, and flexible method to detect α-fetoprotein (AFP), the specific marker of HCC, is significant for diagnosis and treatment of cancer. Here, we constructed a novel SERS biosensing platform combining the target-responsive DNA hydrogel for the sensitive detection of AFP. The linker strand in DNA hydrogel is an aptamer that can specifically recognize AFP and accurately control the release of immunoglobulin G (IgG) encapsulated in hydrogel. In the presence of AFP, the hydrogels were disentangled and the IgG was released. Thereafter, the released IgG was captured by SERS probes and biofunctional magnetic beads through formation of sandwich-like structures, resulting in the signal of Raman tags decreasing in the supernatant after magnetic separation. Due to the ultrahigh sensitivity of the SERS biosensor, the proposed method has a wide detection linear range (50 pg/mL to 0.5 µg/mL) and a detection limit down to 50 pg/mL. Moreover, the sequence of the linker strand in the DNA hydrogel can be specifically encoded into a new aptamer that responds to other cancer markers. This convenient and inexpensive detection method provides a new strategy for the detection of tumor markers.

Role of p53 mediated miR-23a/CXCL12 pathway in osteogenic differentiation of bone mesenchymal stem cells on nanostructured titanium surfaces.

Titanium surface modification is widely established and has been proven to improve the osseointegration, but the molecular mechanism remains to be fully elucidated. MicroRNAs serve vital roles in the process of regulating osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). In this study, we report that miR-23a was significantly down-regulated in the osteogenic differentiation process of BMSCs on nanostructured titanium surfaces. Elevated miR-23a inhibited osteogenic differentiation of BMSCs, and decreased miR-23a enhanced this process. In addition, we also observed that CXCL12 was a direct target of miR-23a. Knockdown of CXCL12 inhibited nanotube Ti induced-osteogenic differentiation of BMSCs, similar to the effect of upregulation of miR-23a. Finally, p53 was decreased and it regulated miR-23a/CXCL12 axis during nanotube Ti induced-osteogenic differentiation of BMSCs. Therefore, our findings suggest that by targeting CXCL12, miR-23a serves a vital role in osteogenic differentiation of BMSCs cultured on nanostructured titanium surfaces, which may provide novel clinical treatments for osseointegration.

CXCR4 enhances cisplatin resistance of human tongue squamous cell carcinoma.

BACKGROUND: The chemokine receptor 4 (CXCR4) plays an important role in tumor progression. Overexpressed CXCR4 is associated with a poor prognosis of patient with head and neck squamous cell carcinomas. However, the correlation between CXCR4 and chemotherapy resistance in tongue squamous cell carcinoma (TSCC) remains obscure. METHODS: Stable cisplatin-resistant CAL27 CDDP and SCC25 CDDP cells were established and identified by CCK8 assay, and the CXCR4 expression was detected using qRT-PCR and Western blot. CXCR4-siRNA was transfected into TSCC CDDP cells, whose transfect efficiency was examined. Cisplatin sensitivity was further detected, as well as several proliferation and apoptosis-related proteins. RESULTS: CAL27 CDDP and SCC25 CDDP cells were successfully established, which exhibited significantly higher cell viability and less apoptosis under cisplatin stimulation than that of parental cells. CXCR4 expression was increased in TSCC CDDP cells. After transfection of CXCR4-siRNA, the expression of CXCR4 was reduced by 73% and 78% in CAL27 CDDP and SCC25 CDDP cells, respectively. CCK8 assay and flow cytometry assay revealed that the proliferative capacity under cisplatin stimulation significantly decreased after CXCR4 silencing. Moreover, increased TSCC CDDP cells were arrested in the G0/G1 phase after knockdown of CXCR4. Compared with negative control group, the expression of cyclin D1 and p-AKT decreased, while that of p-caspase-3 and Bax significantly increased. CONCLUSIONS: Silencing CXCR4 may evidently inhibit proliferation, induce apoptosis and enhance cisplatin sensitivity of TSCC CDDP cells by reduced cyclin D1 and p-AKT, and increased p-caspase-3 and Bax.

Cancer-associated fibroblasts confer cisplatin resistance of tongue cancer via autophagy activation.

Cancer-associated fibroblasts (CAFs) play important roles in carcinogenesis and progression of tongue squamous cell carcinoma (TSCC). However, effect of CAFs on chemotherapy resistance of TSCC remains largely obscure. Here, we cultured the matched primary CAFs and normal fibroblasts (NFs) pairs and detected their roles in cisplatin sensitivity of TSCC, as well as autophagy-related protein LC3 and Beclin1 expressions. During exposure to cisplatin, TSCC with CAFs group exhibited significantly increased cell viability and IC50, but reduced apoptosis than that with NFs group. Meanwhile, cisplatin increased the LC3-II and Beclin1 levels of those TSCC co-cultured with CAFs. Activation of cisplatin-induced autophagic flux was inhibited by CQ, which can accumulate LC3-II protein and increase punctate distribution of LC3 localization. Beclin1 siRNA also decreased the cisplatin-induced autophagy. Both CQ and Beclin1 siRNA increased cisplatin-induced apoptosis but inhibited viability of TSCC co-cultured with CAFs. In vivo, combination of cisplatin and CQ significantly inhibited the growth of xenografted tumors than cisplatin alone. Taken together, our findings highlight the important role of CAFs in cisplatin resistance of tongue cancer via autophagy activation, suggesting that inhibition of autophagy could be an optimal strategy for chemoresistance of TSCC.

Effects of hypoxic condition on osteogenic differentiation of human periodontal ligament cells via hypoxia inducible factor-1α / 口腔疾病防治

Objective@#To investigate the effects of hypoxia on osteogenic differentiation of periodontal ligament cells (PDLCs) and the role of hypoxia inducible factor-1α (HIF-1α) in this process. @*Methods @#Human PDLCs were isolated and identified by checking the expression of vimentin and cytokeratin. PDLCs were cultured in normoxia (20% O2) or hypoxia (1% O2) for 12-72 h. Changes of alkaline phosphatase (ALP) activity and mRNA expressions of osteogenic markers ALP, collagen-I (COL1) and runt related transcription factor 2 (RUNX2) were detected. Western blot was used to detect the expression of HIF-1α. After transfected with HIF1α-siRNA, the expressions of HIF-1αand osteogenic differentiation markers were furthered detected. The statistics were analyzed with SPSS13.0. @*Results@#Positive vimentin but negative cytokeratin were observed in primary cultured PDLCs. ALP activity and mRNA expressions of ALP, COL1 and RUNX2 were decreased in PDLCs in hypoxia for 48 h, while HIF-1α expression was increased. After knocking down of HIF-1α with siRNA, HIF-1α was significantly reduced in PDLCs under hypoxia, while ALP activity and mRNA expressions of osteogenic markers were significantly increased. @*Conclusion @#Hypoxia may inhibit osteogenic differentiation of PDLCs via upregulated HIF-1α.

Osseointegration of three-dimensional designed titanium implants manufactured by selective laser melting.

The selective laser melting (SLM) technique is a recent additive manufacturing (AM) technique. Several studies have reported success in the SLM-based production of biocompatible orthopaedic implants and three-dimensional bone defect constructs. In this study, we evaluated the surface properties and biocompatibility of an SLM titanium implant in vitro and compared them with those of a machined (MA) titanium control surface. In addition, we evaluated the osseointegration capability of the SLM implants in vivo and compared it with those of MA and Nobel-speedy (Nobel-S) implants. SLM microtopographical surface analysis revealed porous and high roughness with varied geometry compared with a smooth surface in MA Ti samples but with similar favourable wettability. Osteoblast proliferation and alkaline phosphatase activity were significantly enhanced on the SLM surface. Histological analysis of the bone-implant contact ratio revealed no significant difference among SLM, MA, and Nobel-S implants. Micro-CT assessment indicated that there was no significant difference in bone volume fraction around the implant among SLM implants and other types of surface modification implants. The removal torque value measurement of SLM implants was significantly lower that of than Nobel-S implants P < 0.001 and higher than that of MA implants. The study demonstrates the capability of SLM implants to integrate with living bone. The SLM technique holds promise as a new dental implant manufacturing technique.

Co­delivery and controlled release of stromal cell­derived factor­1α chemically conjugated on collagen scaffolds enhances bone morphogenetic protein­2­driven osteogenesis in rats.

There has been considerable focus in investigations on the delivery systems and clinical applications of bone morphogenetic protein­2 (BMP­2) for novel bone formation. However, current delivery systems require high levels of BMP­2 to exert a biological function. There are several concerns in using of high levels of BMP­2, including safety and the high cost of treatment. Therefore, the development of strategies to decrease the levels of BMP­2 required in these delivery systems is required. In our previous studies, a controlled­release system was developed, which used Traut's reagent and the cross­linker, 4­(N­maleimi­domethyl) cyclohexane­1­carboxylic acid 3­sulfo­N­hydroxysuccinimide ester sodium salt (Sulfo­SMCC), to chemically conjugate BMP­2 directly on collagen discs. In the current study, retention efficiency and release kinetics of stromal cell­derived factor­1α (SDF­1α) cross­linked on collagen scaffolds were detected. In addition, the osteogenic activity of SDF­1α and suboptimal doses of BMP­2 cross­linked on collagen discs following subcutaneous implantation in rats were evaluated. Independent two­tailed t­tests and one­way analysis of variance were used for analysis. In the present study, the controlled release of SDF­1α chemically conjugated on collagen scaffolds was demonstrated. By optimizing the concentrations of Traut's reagent and the Sulfo­SMCC cross­linker, a significantly higher level of SDF­1α was covalently retained on the collagen scaffold, compared with that retained using a physical adsorption method. Mesenchymal stem cell homing indicated that the biological function of the SDF­1α cross­linked on the collagen scaffolds remained intact. In rats, co­treatment with SDF­1α and a suboptimal dose of BMP­2 cross­linked on collagen scaffolds using this chemically conjugated method induced higher levels of ectopic bone formation, compared with the physical adsorption method. No ectopic bone formation was observed following treatment with a suboptimal dose of BMP­2 alone. Therefore, the co­delivery of SDF­1α and a suboptimal dose of BMP­2 chemically conjugated on collagen scaffolds for the treatment of bone injuries reduced the level of BMP­2 required, reducing the risks of side effects.

Tumor necrosis factor α induces myofibroblast differentiation in human tongue cancer and promotes invasiveness and angiogenesis via secretion of stromal cell-derived factor-1.

OBJECTIVES: Cancer-associated fibroblasts (CAFs) play an important role in tumor progression and are associated with a poor prognosis. Tumor necrosis factor α (TNFα) has been involved in growth and metastasis associated with epithelial-mesenchymal transition (EMT) in many types of cancers. However, the relationship among the TNFα, activation of CAFs and their tumor-promoting effects on tongue squamous cell carcinoma (TSCC) remain obscure. MATERIALS AND METHODS: A series of matched primary CAFs and normal fibroblasts (NFs) pairs were cultured, and additionally two TSCC cell lines SCC9 and CAL27, were treated with TNFα or CAFs derived stromal cell-derived factor-1 (SDF1) respectively to study invasion and EMT in vitro. In addition, NFs were treated with TNFα to detect the expression of myofibroblast markers, invasion, induced collagen gel contraction and enhanced cancer metastasis. Finally, invasion and angiogenesis of human vein endothelial cells (HUVECs) treated with TNFα and CAFs derived SDF1 was measured. RESULTS: TNFα and CAFs-derived SDF1 induced TSCC cells metastasis and EMT separately. TNFα facilitated the transformation of NFs to CAFs-like fibroblasts, which was accompanied by acquisition of similar myofibroblast markers expression and malignant function to CAFs. Furthermore, TNFα and CAFs derived SDF1 also promoted invasion and angiogenesis of HUVECs. CONCLUSION: These results suggest that TNFα may not only directly but also indirectly enhance cancer metastasis, EMT and angiogenesis formation in tongue cancer through myofibroblast differentiation via SDF1 secretion.