Graphene Oxide Quantum Dots-Preactivated Dental Pulp Stem Cells/GelMA Facilitates Mitophagy-Regulated Bone Regeneration.

Background: In bone tissue engineering (BTE), cell-laden scaffolds offer a promising strategy for repairing bone defects, particularly when host cell regeneration is insufficient due to age or disease. Exogenous stem cell-based BTE requires bioactive factors to activate these cells. Graphene oxide quantum dots (GOQDs), zero-dimensional derivatives of graphene oxide, have emerged as potential osteogenic nanomedicines. However, constructing biological scaffolds with GOQDs and elucidating their biological mechanisms remain critical challenges. Methods: We utilized GOQDs with a particle size of 10 nm, characterized by a surface rich in C-O-H and C-O-C functional groups. We developed a gelatin methacryloyl (GelMA) hydrogel incorporated with GOQDs-treated dental pulp stem cells (DPSCs). These constructs were transplanted into rat calvarial bone defects to estimate the effectiveness of GOQDs-induced DPSCs in repairing bone defects while also investigating the molecular mechanism underlying GOQDs-induced osteogenesis in DPSCs. Results: GOQDs at 5 µg/mL significantly enhanced the osteogenic differentiation of DPSCs without toxicity. The GOQDs-induced DPSCs showed active osteogenic potential in three-dimensional cell culture system. In vivo, transplantation of GOQDs-preactivated DPSCs/GelMA composite effectively facilitated calvarial bone regeneration. Mechanistically, GOQDs stimulated mitophagy flux through the phosphatase-and-tensin homolog-induced putative kinase 1 (PINK1)/Parkin E3 ubiquitin ligase (PRKN) pathway. Notably, inhibiting mitophagy with cyclosporin A prevented the osteogenic activity of GOQDs. Conclusion: This research presents a well-designed bionic GOQDs/DPSCs/GelMA composite scaffold and demonstrated its ability to promote bone regeneration by enhancing mitophagy. These findings highlight the significant potential of this composite for application in BTE and underscore the crucial role of mitophagy in promoting the osteogenic differentiation of GOQDs-induced stem cells.

Nano-ZnO-modified hydroxyapatite whiskers with enhanced osteoinductivity for bone defect repair.

Hydroxyapatite (HA) whisker (HAw) represents a distinct form of HA characterized by its high aspect ratio, offering significant potential for enhancing the mechanical properties of bone tissue engineering scaffolds. However, the limited osteoinductivity of HAw hampers its widespread application. In this investigation, we observed HAw-punctured osteoblast membranes and infiltrated the cell body, resulting in mechanical damage to cells that adversely impacted osteoblast proliferation and differentiation. To address this challenge, we developed nano-zinc oxide particle-modified HAw (nano-ZnO/HAw). Acting as a reinforcing and toughening agent, nano-ZnO/HAw augmented the compressive strength and ductility of the matrix materials. At the same time, the surface modification with nano-ZnO particles improved osteoblast differentiation by reducing the mechanical damage from HAw to cells and releasing zinc ion, the two aspects collectively promoted the osteoinductivity of HAw. Encouragingly, the osteoinductive potential of 5% nano-ZnO/HAw and 10% nano-ZnO/HAw was validated in relevant rat models, demonstrating the efficacy of this approach in promoting new bone formation in vivo. Our findings underscore the role of nano-ZnO particle surface modification in enhancing the osteoinductivity of HAw from a physical standpoint, offering valuable insights into the development of bone substitutes with favorable osteoinductive properties while simultaneously bolstering matrix material strength and toughness.

Corrosion and Biological Behaviors of Biomedical Ti-24Nb-4Zr-8Sn Alloy under an Oxidative Stress Microenvironment.

Biomaterials can induce an inflammatory response in surrounding tissues after implantation, generating and releasing reactive oxygen species (ROS), such as hydrogen peroxide (H2O2). The excessive accumulation of ROS may create a microenvironment with high levels of oxidative stress (OS), which subsequently accelerates the degradation of the passive film on the surface of titanium (Ti) alloys and affects their biological activity. The immunomodulatory role of macrophages in biomaterial osteogenesis under OS is unknown. This study aimed to explore the corrosion behavior and bone formation of Ti implants under an OS microenvironment. In this study, the corrosion resistance and osteoinduction capabilities in normal and OS conditions of the Ti-24Nb-4Zr-8Sn (wt %, Ti2448) were assessed. Electrochemical impedance spectroscopy analysis indicated that the Ti2448 alloy exhibited superior corrosion resistance on exposure to excessive ROS compared to the Ti-6Al-4V (TC4) alloy. This can be attributed to the formation of the TiO2 and Nb2O5 passive films, which mitigated the adverse effects of OS. In vitro MC3T3-E1 cell experiments revealed that the Ti2448 alloy exhibited good biocompatibility in the OS microenvironment, whereas the osteogenic differentiation level was comparable to that of the TC4 alloy. The Ti2448 alloy significantly alleviates intercellular ROS levels, inducing a higher proportion of M2 phenotypes (52.7%) under OS. Ti2448 alloy significantly promoted the expression of the anti-inflammatory cytokine, interleukin 10 (IL-10), and osteoblast-related cytokines, bone morphogenetic protein 2 (BMP-2), which relatively increased by 26.9 and 31.4%, respectively, compared to TC4 alloy. The Ti2448 alloy provides a favorable osteoimmune environment and significantly promotes the proliferation and differentiation of osteoblasts in vitro compared to the TC4 alloy. Ultimately, the Ti2448 alloy demonstrated excellent corrosion resistance and immunomodulatory properties in an OS microenvironment, providing valuable insights into potential clinical applications as implants to repair bone tissue defects.

The ATF4-regulated LncRNA MALAT1 promotes odontoblastic differentiation of human dental pulp stem cells via histone demethylase JMJD3: An in vitro study.

AIM: This study aimed to investigate the upstream regulators and specific mechanisms of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in the odontoblastic differentiation of human dental pulp stem cells (hDPSCs). METHODOLOGY: Human dental pulp stem cells were isolated and cultured, followed by conducting loss- or gain-of-function experiments on ATF4 and loss experiments on MALAT1 to elucidate their respective biological functions in odontoblastic differentiation. Chromatin immunoprecipitation assays and RNA immunoprecipitation were performed to uncover the interaction between ATF4-MALAT1 and MALAT1-JMJD3, respectively. The odontoblastic differentiation was estimated by the mRNA and protein of DSPP and DMP1, as well as alkaline phosphatase staining. RESULTS: Expression of MALAT1 was upregulated in the hDPSCs cultured in an odontoblastic medium, and MALAT1 downregulation suppressed the odontoblastic differentiation of the hDPSCs. Subsequent experiments confirmed that ATF4 promoted odontoblastic differentiation and induced MALAT1 expression by binding to the MALAT1 promoter region. Further experiments revealed that nuclear MALAT1 interacted with JMJD3. MALAT1 knockdown decreased the JMJD3 protein level and demethylase activity, and it enhanced H3K27me3 occupancy of the promoter region of DSPP and DMP1, resulting in the inhibition of DSPP and DMP1 transcription. Importantly, JMJD3 overexpression significantly attenuated the inhibition of odontoblastic differentiation induced by MALAT1 knockdown. CONCLUSIONS: ATF4-regulated MALAT1 plays a positive regulatory role in odontoblastic differentiation of hDPSCs through JMJD3-mediated H3K27me3 modifications of the DSPP and DMP1 promoters.

Melatonin attenuates dental pulp stem cells senescence due to vitro expansion via inhibiting MMP3.

OBJECTIVE: We aimed to identify the crucial genes involved in dental pulp stem cell (DPSC) senescence and evaluate the impact of melatonin on DPSC senescence. METHODS: Western blotting, SA-ß-Gal staining and ALP staining were used to evaluate the senescence and differentiation potential of DPSCs. The optimal concentration of melatonin was determined using the CCK-8 assay. Differentially expressed genes (DEGs) involved in DPSC senescence were obtained via bioinformatics analysis, followed by RT-qPCR. Gain- and loss-of-function studies were conducted to explore the role of MMP3 in DPSC in vitro expansion and in response to melatonin. GSEA was employed to analyse MMP3-related pathways in cellular senescence. RESULTS: Treatment with 0.1 µM melatonin attenuated cellular senescence and differentiation potential suppression in DPSCs due to long-term in vitro expansion. MMP3 was a crucial gene in senescence, as confirmed by bioinformatics analysis, RT-qPCR and Western blotting. Furthermore, gain- and loss-of-function studies revealed that MMP3 played a regulatory role in cellular senescence. Rescue assays showed that overexpression of MMP3 reversed the effect of melatonin on senescence. GSEA revealed that the MMP3-dependent anti-senescence effect of melatonin was associated with the IL6-JAK-STAT3, TNF-α-Signalling-VIA-NF-κB, COMPLEMENT, NOTCH Signalling and PI3K-AKT-mTOR pathways. CONCLUSION: Melatonin attenuated DPSC senescence caused by long-term expansion by inhibiting MMP3.

[Regulation mechanism of resveratrol on odontogenic differentiation of human dental pulp stem cells].

PURPOSE: To investigate whether resveratrol promotes odontogenic differentiation of human dental pulp stem cells(DPSCs) by up-regulating the expression of silent information regulator 1 (SIRT1) and activating ß-catenin signaling pathway. METHODS: Different concentrations of resveratrol(0, 10, 15, 20 and 50 µmol/L) were used to treat DPSCs for 7 days and 14 days, and cell proliferative activity was detected by CCK-8. After odontogenic differentiation induced by 15 µmol/L resveratrol for 7 days, alkaline phosphatase(ALP) staining was performed and real-time quantitative reverse transcription PCR(qRT-PCR) was used to detect the mRNA expression of Runt-related transcription factor 2 (Runx2), dentin sialophosphoprotein(DSPP) and dentin matrix protein-1(DMP-1) in DPSCs. Western blot was used to detect the expression of SIRT1 in DPSCs on a specific day (0, 3rd, 5th, 7th and 14th) after differentiation induction. Western blot was also used to detect the expression of SIRT1 and activated ß-catenin during odontogenic differentiation of DPSCs treated by 15 µmol/L resveratrol for 7 days. The experimental data was analyzed with GraphPad Prism 9 software package. RESULTS: 15 µmol/L resveratrol had no significant effect on proliferation of DPSCs on the 7th and 14th day; 15 µmol/L resveratrol promoted odontogenic differentiation of DPSCs and up-regulated mRNA expression of RUNX2, DSPP, and DMP-1 in DPSCs; the expression of SIRT1 was the highest on the 7th day during odontogenic differentiation induction. Resveratrol resulted in the increasing protein expressions of SIRT1 and activated ß-catenin when DPSCs was induced to odontogenic differentiation for 7 days. CONCLUSIONS: Resveratrol promotes odontogenic differentiation of human DPSCs by up-regulating the expression of SIRT1 protein and activating ß-catenin signaling pathway.

[Effects of chitosan oligosaccharide on bone metabolism and IKK/NF-κB pathway in rats with osteoporosis and periodontitis].

PURPOSE: To investigate the effects of chitosan oligosaccharide on bone metabolism and IKK/NF-κB pathway in mice with osteoporosis and periodontitis. METHODS: Thirty rats were randomly divided into 3 groups, with 10 rats in each group. They were divided into control group, ovariectomized periodontitis group and chitosan oligosaccharide treatment group. Except for the control group, the other two groups were ovariectomized and smeared with Porphyromonas gingivalis fluid to establish the model of osteoporosis with periodontitis. Four weeks after ligation, the rats in chitosan oligosaccharide treatment group were gavaged with 200 mg/kg chitosan oligosaccharide, and the other two groups were gavaged with equal volume of normal saline once a day for 90 days. The periodontal tissues of each group were observed before administration, and the bone mineral density of rats was detected by dual energy X-ray animal bone mineral density and body composition analysis system. After 90 days of administration, the bone mineral density was detected again. After administration, blood was collected from tail vein, and the contents of serum alkaline phosphatase (ALP), bone Gla protein (BGP) and tartrate resistant acid phosphatase 5b (TRACP5b) were measured by enzyme-linked immunodeficient assay. The gingival index and periodontal attachment loss of rats in each group were obtained by visual examination and exploratory examination. The maxilla was removed, and the distance from the enamel cementum boundary to the alveolar crest was measured to obtain alveolar bone absorption value. H-E staining was used to observe the pathology of maxilla in each group. RT-PCR and Western blot were used to detect the nuclear factors in periodontal tissue of rats in each group. SPSS 22.0 software package was used for statistical analysis. RESULTS: Before administration, the gums of the control group were pink without bleeding, and the gums of the other two groups were red and swollen with slight bleeding. After administration, compared with the control group, the bone mineral density, serum ALP, BGP of ovariectomized periodontitis group decreased significantly(P＜0.05); while TRACP5b, gingival index, loss of periodontal attachment and alveolar bone resorption, NF-κB and IKK mRNA and protein expression in periodontal tissue increased significantly(P＜0.05). Compared with the ovariectomized periodontitis group, the bone mineral density, serum ALP, BGP were significantly increased(P＜0.05); while TRACP5b, gingival index, periodontal attachment loss and alveolar bone resorption, NF-κB and IKK mRNA and protein expression in periodontal tissue were significantly decreased (P＜0.05). In the ovariectomized periodontitis group, the periodontal tissue combined with epithelium was separated from the tooth surface, the dental pocket was obvious and deep, and the height of alveolar bone decreased. Although dental pocket could be observed in the periodontal tissue of rats treated with chitosan oligosaccharide, it was not obvious, and new bone appeared around the alveolar bone. CONCLUSIONS: Chitosan oligosaccharide can induce biochemical indexes of bone metabolism to become normal, alleviate the symptoms of periodontitis, this may be related to the inhibition of IKK/NF-κB pathway by chitosan oligosaccharide.

[Resveratrol suppressed the expression of MIP-2 induced by lipopolysaccharide in osteoblasts via up-regulating SOCS-3].

PURPOSE: To explore whether resveratrol dependents on the production of suppressor of cytokine signaling suppressor 3 (SOCS-3) in inhibiting mRNA production of macrophage inflammatory protein-2 (MIP-2) in osteoblasts induced by lipopolysaccharides(LPS) extracted from Porphyromonas endodontalis(P.e). METHODS: MC3T3-E1 cells were treated with different concentrations of resveratrol (0, 5, 10 and 20 µmol/L) and 20 µmol/L resveratrol for different time( 0, 10, 30, 60, 120 and 180 min). The expression of SOCS-3 protein was detected by Western blot. MC3T3-E1 cells were transfected with mouse SOCS3 siRNA (si-SOCS-3) and control siRNA(si-control). Reverse transcription real-time PCR(real-time RT-PCR) and Western blot was used to detect the silencing efficiency of SOCS-3. Cells were stimulated by 20 µg/mL P.e-LPS for 24 h after transfection, in the absence or presence of 20 µmol/L resveratrol for 1 h , and the changes of MIP-2 mRNA were determined by real-time RT-PCR. Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS 13.0 software package. RESULTS: Treatment of MC3T3-El cells with different concentrations of resveratrol caused a significant increase in SOCS-3 protein expression in a dose-dependent manner. During the observation time of 180 min, SOCS-3 protein expression was the highest at 20 µmol/L resveratrol-treated osteoblasts for 60 min. The silencing efficiency of SOCS-3 mRNA was 63.7%. Transfection with SOCS-3 siRNA increased MIP-2 mRNA expression in LPS-stimulated MC3T3-E1 cells and negated the inhibitory effects of resveratrol on LPS-induced MIP-2 mRNA expression(P<0.05). CONCLUSIONS: Resveratrol inhibits the expression of MIP-2 mRNA in osteoblasts induced by P.e-LPS by up-regulating the expression of SOCS-3 protein.

Exosome-Based Molecular Transfer Activity of Macrophage-Like Cells Involves Viability of Oral Carcinoma Cells: Size Exclusion Chromatography and Concentration Filter Method.

Extracellular vesicles (EV) heterogeneity is a crucial issue in biology and medicine. In addition, tumor-associated macrophages are key components in cancer microenvironment and immunology. We developed a combination method of size exclusion chromatography and concentration filters (SEC-CF) and aimed to characterize different EV types by their size, cargo types, and functions. A human monocytic leukemia cell line THP-1 was differentiated to CD14-positive macrophage-like cells by stimulation with PMA (phorbol 12-myristate 13-acetate) but not M1 or M2 types. Using the SEC-CF method, the following five EV types were fractionated from the culture supernatant of macrophage-like cells: (i) rare large EVs (500-3000 nm) reminiscent of apoptosomes, (ii) EVs (100-500 nm) reminiscent of microvesicles (or microparticles), (iii) EVs (80-300 nm) containing CD9-positive large exosomes (EXO-L), (iv) EVs (20-200 nm) containing unidentified vesicles/particles, and (v) EVs (10-70 nm) containing CD63/HSP90-positive small exosomes (EXO-S) and particles. For a molecular transfer assay, we developed a THP-1-based stable cell line producinga GFP-fused palmitoylation signal (palmGFP) associated with the membrane. The THP1/palmGFP cells were differentiated into macrophages producing palmGFP-contained EVs. The macrophage/palmGFP-secreted EXO-S and EXO-L efficiently transferred the palmGFP to receiver human oral carcinoma cells (HSC-3/palmTomato), as compared to other EV types. In addition, the macrophage-secreted EXO-S and EXO-L significantly reduced the cell viability (ATP content) in oral carcinoma cells. Taken together, the SEC-CF method is useful for the purification of large and small exosomes with higher molecular transfer activities, enabling efficient molecular delivery to target cells.

The Inhibitory Role of Rab11b in Osteoclastogenesis through Triggering Lysosome-Induced Degradation of c-Fms and RANK Surface Receptors.

Rab11b, abundantly enriched in endocytic recycling compartments, is required for the establishment of the machinery of vesicle trafficking. Yet, no report has so far characterized the biological function of Rab11b in osteoclastogenesis. Using in vitro model of osteoclasts differentiated from murine macrophages like RAW-D cells or bone marrow-derived macrophages, we elucidated that Rab11b served as an inhibitory regulator of osteoclast differentiation sequentially via (i) abolishing surface abundance of RANK and c-Fms receptors; and (ii) attenuating nuclear factor of activated T-cells c1 (NFATc-1) upstream signaling cascades, following RANKL stimulation. Rab11b was localized in early and late endosomes, Golgi complex, and endoplasmic reticulum; moreover, its overexpression enlarged early and late endosomes. Upon inhibition of lysosomal function by a specific blocker, chloroquine (CLQ), we comprehensively clarified a novel function of lysosomes on mediating proteolytic degradation of c-Fms and RANK surface receptors, drastically ameliorated by Rab11b overexpression in RAW-D cell-derived osteoclasts. These findings highlight the key role of Rab11b as an inhibitor of osteoclastogenesis by directing the transport of c-Fms and RANK surface receptors to lysosomes for degradation via the axis of early endosomes-late endosomes-lysosomes, thereby contributing towards the systemic equilibrium of the bone resorption phase.