A digital workflow for designing and manufacturing metal frameworks and removable partial dentures: A novel dental technique.

The purpose of this technical report is to demonstrate a fully digital workflow for designing and fabricating metal frameworks and removable partial dentures. After obtaining a digital cast of the dental arch with bilateral distal extension defect, computer-aided design software and 3D printing technology are used for the design and fabrication of the removable partial denture frameworks, denture teeth, and denture bases, instead of the traditional workflow. The assembly of the three components is facilitated through a meticulously structured framework. The technology, which prints metal frameworks, denture bases, and denture teeth through different processes with different materials, achieves full 3D printing technology for making removable partial dentures.

Apoptotic Extracellular Vesicles Induced Endothelial Cell-Mediated Autologous Stem Cell Recruitment Dominates Allogeneic Stem Cell Therapeutic Mechanism for Bone Repair.

Although stem cell therapy is proved to be a promising strategy for bone repair and regeneration, transplanted allogeneic stem cells generally suffer from unfavorable apoptosis instead of differentiation into osteocytes. How the apoptotic stem cells promote bone regeneration still needs to be uncovered. In this work, we found that apoptotic extracellular vesicles released by allogeneic stem cells are critical mediators for promoting bone regeneration. Based on the results of in vivo experiments, a mechanism of apoptotic stem cells determined autologous stem cell recruitment and enhance osteogenesis was proposed. The nanoscaled apoptotic extracellular vesicles released from transplanted stem cells were endocytosed by vascular endothelial cells and preferentially distribute at endoplasmic reticular region. The oxidized phosphatidylcholine enriched in the vesicles activated the endoplasmic reticulum stress and triggered the reflective elevation of adhesion molecules, which induced the recruitment of autologous stem cells located in the blood vessels, transported them into the defect region, and promoted osteogenesis and bone repair. These findings not only reveal the mechanism of stem cell therapy of bone defects but also provide a cue for investigation of the biological process of stem cell therapy for other diseases and develop stem cell therapeutic strategies.

Bone-Targeting Peptide and RNF146 Modified Apoptotic Extracellular Vesicles Alleviate Osteoporosis.

Background: Osteoporosis is a highly prevalent disease that causes fractures and loss of motor function. Current drugs targeted for osteoporosis often have inevitable side effects. Bone marrow mesenchymal stem cell (BMSCs)-derived apoptotic extracellular vesicles (ApoEVs) are nanoscale extracellular vesicles, which has been shown to promote bone regeneration with low immunogenicity and high biological compatibility. However, natural ApoEVs cannot inherently target bones, and are often eliminated by macrophages in the liver and spleen. Thus, our study aimed to reconstruct ApoEVs to enhance their bone-targeting capabilities and bone-promoting function and to provide a new method for osteoporosis treatment. Methods: We conjugated a bone-targeting peptide, (Asp-Ser-Ser)6 ((DSS)6), onto the surface of ApoEVs using standard carbodiimide chemistry with DSPE-PEG-COOH serving as the linker. The bone-targeting ability of (DSS)6-ApoEVs was determined using an in vivo imaging system and confocal laser scanning microscopy (CLSM). We then loaded ubiquitin ligase RING finger protein146 (RNF146) into BMSCs via adenovirus transduction to obtain functional ApoEVs. The bone-promoting abilities of (DSS)6-ApoEVs and (DSS)6-ApoEVsRNF146 were measured in vitro and in vivo. Results: Our study successfully synthesized bone-targeting and gained functional (DSS)6-ApoEVsRNF146 and found that engineered ApoEVs could promote osteogenesis in vitro and exert significant bone-targeting and osteogenesis-promoting effects to alleviate osteoporosis in a mouse model. Conclusion: To promote the bone-targeting ability of natural ApoEVs, we successfully synthesized engineered ApoEVs, (DSS)6-ApoEVsRNF146 and found that they could significantly promote osteogenesis and alleviate osteoporosis compared with natural ApoEVs, which holds great promise for the treatment of osteoporosis.

MSC-derived exosomes protect auditory hair cells from neomycin-induced damage via autophagy regulation.

BACKGROUND: Sensorineural hearing loss (SNHL) poses a major threat to both physical and mental health; however, there is still a lack of effective drugs to treat the disease. Recently, novel biological therapies, such as mesenchymal stem cells (MSCs) and their products, namely, exosomes, are showing promising therapeutic potential due to their low immunogenicity, few ethical concerns, and easy accessibility. Nevertheless, the precise mechanisms underlying the therapeutic effects of MSC-derived exosomes remain unclear. RESULTS: Exosomes derived from MSCs reduced hearing and hair cell loss caused by neomycin-induced damage in models in vivo and in vitro. In addition, MSC-derived exosomes modulated autophagy in hair cells to exert a protective effect. Mechanistically, exogenously administered exosomes were internalized by hair cells and subsequently upregulated endocytic gene expression and endosome formation, ultimately leading to autophagy activation. This increased autophagic activity promoted cell survival, decreased the mitochondrial oxidative stress level and the apoptosis rate in hair cells, and ameliorated neomycin-induced ototoxicity. CONCLUSIONS: In summary, our findings reveal the otoprotective capacity of exogenous exosome-mediated autophagy activation in hair cells in an endocytosis-dependent manner, suggesting possibilities for deafness treatment.

MSC-derived exosomes protect auditory hair cells from neomycin-induced damage via autophagy regulation

BACKGROUND: Sensorineural hearing loss (SNHL) poses a major threat to both physical and mental health; however, there is still a lack of effective drugs to treat the disease. Recently, novel biological therapies, such as mesenchymal stem cells (MSCs) and their products, namely, exosomes, are showing promising therapeutic potential due to their low immunogenicity, few ethical concerns, and easy accessibility. Nevertheless, the precise mechanisms underlying the therapeutic effects of MSC-derived exosomes remain unclear. RESULTS: Exosomes derived from MSCs reduced hearing and hair cell loss caused by neomycin-induced damage in models in vivo and in vitro. In addition, MSC-derived exosomes modulated autophagy in hair cells to exert a protective effect. Mechanistically, exogenously administered exosomes were internalized by hair cells and subsequently upregulated endocytic gene expression and endosome formation, ultimately leading to autophagy activation. This increased autophagic activity promoted cell survival, decreased the mitochondrial oxidative stress level and the apoptosis rate in hair cells, and ameliorated neomycin-induced ototoxicity. CONCLUSIONS: In summary, our findings reveal the otoprotective capacity of exogenous exosome-mediated autophagy activation in hair cells in an endocytosis-dependent manner, suggesting possibilities for deafness treatment.

Developing and validating clinical models to identify candidates for allergic rhinitis pre-exposure prophylaxis.

PURPOSE: Few risk-forecasting models of allergic rhinitis (AR) exist that may aid AR pre-exposure prophylaxis (PrEP) in clinical practice. Therefore, this study aimed to develop and validate an effective clinical model for identifying candidates for AR PrEP using a routine medical questionnaire. METHODS: This study was conducted in 10 Chinese provinces with 13 medical centers (n = 877) between 2019 and 2021. Clinical characteristics and exposure history were collected via face-to-face interviews. Well-trained physicians diagnosed patients with AR based on skin prick test results and clinical performance. The least absolute shrinkage and selection operator model was used to identify potential risk factors for AR, and the logistic regression model was used to construct the risk-forecasting model. Predictive power and model reliability were assessed using area under the receiver operating characteristic curve and calibration curves, respectively. RESULTS: This study diagnosed 625 patients with AR who had positive responses to at least one indoor or outdoor allergen and 460 to at least one outdoor pollen allergen. Two nomograms were established to identify two types of AR with various sensitization patterns. Both models had an area under curve of approximately 0.7 in the development and internal validation datasets. Additionally, our findings found good agreement for the calibration curves of both models. CONCLUSION: Early identification of candidates for AR PrEP using routine medical information may improve the deployment of limited resources and effective health management. Our models showed good performance in predicting AR; therefore, they can serve as potential automatic screening tools to identify AR PrEP candidates.

Patterns of aeroallergen sensitization in asthma patients identified by latent class analysis: A cross-sectional study in China.

BACKGROUND: This cross-sectional study aimed to identify latent sensitization profiles of asthma patients in mainland China, unveiling the association between regional differences and sensitization patterns. METHODS: 1056 asthma participants from 10 medical centers divided into eastern and western cohorts were clustered into four individual sensitization patterns, respectively, by using an unsupervised statistical modeling method, latent class analysis (LCA), based on the levels of 12 aeroallergens specific IgE reactivities. Moreover, differences in clinical characteristics and environmental exposures were compared in different sensitization patterns. RESULTS: Four distinct sensitization patterns in the two cohorts were defined as follows, respectively. Eastern cohort: Class 1: "High weed pollen and house dust mites (HDMs) sensitization" (8.87%), Class 2: "HDMs dominated sensitization" (38.38%), Class 3: "High HDMs and animal dander sensitization" (6.95%), Class 4: "Low/no aeroallergen sensitization" (45.80%). Western cohort: Class 1: "High weed pollen sensitization" (26.14%), Class 2: "High multi-pollen sensitization" (15.02%), Class 3: "HDMs-dominated sensitization" (10.33%), Class 4: "Low/no aeroallergen sensitization" (48.51%). Of note, the significant statistical difference in age, asthma control test score (ACT) and comorbidities were observed within or between different sensitization patterns. Exposure factors in different sensitization patterns were pointed out. CONCLUSIONS: Asthmatic patients with distinct sensitization patterns were clustered and identified through the LCA method, disclosing the relationship between sensitization profiles of multiple aeroallergens and geographical differences, providing novel insights and potential strategies for atopic disease monitoring, management and prevention in clinical practice.

MSCs-derived apoptotic extracellular vesicles promote muscle regeneration by inducing Pannexin 1 channel-dependent creatine release by myoblasts.

Severe muscle injury is hard to heal and always results in a poor prognosis. Recent studies found that extracellular vesicle-based therapy has promising prospects for regeneration medicine, however, whether extracellular vesicles have therapeutic effects on severe muscle injury is still unknown. Herein, we extracted apoptotic extracellular vesicles derived from mesenchymal stem cells (MSCs-ApoEVs) to treat cardiotoxin induced tibialis anterior (TA) injury and found that MSCs-ApoEVs promoted muscles regeneration and increased the proportion of multinucleated cells. Besides that, we also found that apoptosis was synchronized during myoblasts fusion and MSCs-ApoEVs promoted the apoptosis ratio as well as the fusion index of myoblasts. Furthermore, we revealed that MSCs-ApoEVs increased the relative level of creatine during myoblasts fusion, which was released via activated Pannexin 1 channel. Moreover, we also found that activated Pannexin 1 channel was highly expressed on the membrane of myoblasts-derived ApoEVs (Myo-ApoEVs) instead of apoptotic myoblasts, and creatine was the pivotal metabolite involved in myoblasts fusion. Collectively, our findings firstly revealed that MSCs-ApoEVs can promote muscle regeneration and elucidated that the new function of ApoEVs as passing inter-cell messages through releasing metabolites from activated Pannexin 1 channel, which will provide new evidence for extracellular vesicles-based therapy as well as improving the understanding of new functions of extracellular vesicles.

Inflammatory Periodontal Ligament Stem Cells Drive M1 Macrophage Polarization via Exosomal miR-143-3p-Mediated Regulation of PI3K/AKT/NF-κB Signaling.

Macrophage polarization plays an important role in the progression of inflammation. Exosomes derived from stem cells are promising candidates for macrophage immunoregulation. However, how exosomes derived from periodontal ligament stem cells (PDLSCs) in an inflammatory environment influence macrophage polarization has yet to be fully elucidated. In this study, inflammatory PDLSCs were found to downregulate M2 macrophage polarization at the mRNA and protein levels in a Transwell coculture system of PDLSCs and THP-1-derived M0 macrophages. Furthermore, inflammatory PDLSC-derived exosomes shifted macrophages toward the M1 phenotype. The inhibition of inflammatory PDLSC-derived exosomes by GW4869 weakened inflammatory PDLSC-mediated M1 macrophage polarization. A miRNA microarray was used to determine the differential miRNAs shuttled by healthy and inflammatory PDLSC-derived exosomes. Compared with healthy exosomes, miR-143-3p was enriched in inflammatory PDLSC-derived exosomes, which targeted and inhibited the expression of PI3Kγ and promoted M1 macrophage polarization by suppressing PI3K/AKT signaling and activating NF-κB signaling, while an agonist of the PI3K pathway reversed this effect. Moreover, exosome-shuttled miR-143-3p from PDLSCs drove M1 macrophage polarization and aggravated periodontal inflammation in a mouse periodontitis model. In conclusion, these results demonstrate that inflammatory PDLSCs facilitate M1 macrophage polarization through the exosomal miR-143-3p-mediated regulation of PI3K/AKT/NF-κB signaling, providing a potential new target for periodontitis treatment.

Apoptotic vesicles activate autophagy in recipient cells to induce angiogenesis and dental pulp regeneration.

Extracellular vesicles (EVs) derived from living cells play important roles in donor cell-induced recipient tissue regeneration. Although numerous studies have found that cells undergo apoptosis after implantation in an ischemic-hypoxic environment, the roles played by the EVs released by apoptotic cells are largely unknown. In this study, we obtained apoptotic vesicles (apoVs) derived from human deciduous pulp stem cells and explored their effects on the dental pulp regeneration process. Our work showed that apoVs were ingested by endothelial cells (ECs) and elevated the expression of angiogenesis-related genes, leading to pulp revascularization and tissue regeneration. Furthermore, we found that, at the molecular level, apoV-carried mitochondrial Tu translation elongation factor was transported and regulated the angiogenic activation of ECs via the transcription factor EB-autophagy pathway. In a beagle model of dental pulp regeneration in situ, apoVs recruited endogenous ECs and facilitated the formation of dental-pulp-like tissue rich in blood vessels. These findings revealed the significance of apoptosis in tissue regeneration and demonstrated the potential of using apoVs to promote angiogenesis in clinical applications.

Apoptotic extracellular vesicles alleviate Pg-LPS induced inflammatory responses of macrophages via AMPK/SIRT1/NF-κB pathway and inhibit osteoclast formation.

BACKGROUND: Periodontitis is caused by the imbalance of anti-bacteria immune response and excessive inflammation whereas macrophages play an important role in inflammation. Thus, it is critical for finding efficient anti-inflammatory strategies to alleviate periodontal inflammation and prevent bone destruction. Apoptosis of mesenchymal stem cells (MSCs) exerts immune silencing effects, however, using these effects to develop anti-inflammatory strategies remains unknown. In our study, we extracted apoptotic extracellular vesicles (ApoEVs) from bone marrow MSCs (BMMSCs) and found ApoEVs inhibited macrophages polarizing into proinflammatory condition via AMPK/SIRT1/NF-κB pathway. Besides that, we also found ApoEVs inhibited adjacent osteoclast formation by suppressing the secretion of TNF-α of proinflammatory macrophages. METHODS: BMMSCs derived ApoEVs were extracted by gradient centrifugation. Protein expression level and secreted cytokines of ApoEVs treated macrophages were examined by western blot and ELISA, respectively. Besides, the change of NF-κB pathway and related molecules were examined by immunofluorescence and western blot. The osteoclast formation under the different conditioned mediums from macrophages was measured by TRAP staining, MMP-9 expression, and pit assay. RESULTS: ApoEVs were extracted from staurosporine-induced apoptotic BMMSCs and were in sphere shapes whose diameters are between 100 and 1000 nm. ApoEVs could be phagocyted by macrophages and in turn reduce the expression of COX2 in proinflammatory macrophages. Besides that, ApoEVs suppressed the secretions of TNF-α and IL-6 while elevating the secretion of IL-10 in a dose-dependent manner. Further studies revealed that ApoEVs inhibited macrophages polarizing into proinflammatory phenotypes via AMPK/SIRT1/NF-κB pathway. In addition, ApoEVs inhibited osteoclasts differentiation and bone resorption measured by TRAP staining, MMP-9 expression, and pit resorption area by downregulating the secretion of TNF-α of proinflammatory macrophages. CONCLUSIONS: The results suggest that ApoEVs inhibited macrophages to skew into proinflammatory phenotypes via AMPK/SIRT1/NF-κB pathway and suppress adjacent osteoclasts formation by reducing the secretion of TNF-α. Our findings shed a light on the treatment for periodontitis based on EVs therapy.

A novel tunable, highly biocompatible and injectable DNA-chitosan hybrid hydrogel fabricated by electrostatic interaction between chitosan and DNA backbone.

The injectable hydrogel is an ideal reservoir for drug delivery. In this study, a new injectable DNA hydrogel was fabricated. Firstly, the DNA pre-gel was obtained by heat-cool treatments to induce cross-linkage through base-paring. Then, the pre-gel was cross-linked with chitosan (CS) through electrostatic interaction, which was confirmed by ATR-FTIR and XPS analysis. The DNA-CS hybrid gel showed finely tunable various properties such as porosity and viscosity. To simulate the biomedical application, the dexamethasone (Dex) was loaded into the gel and coated onto titanium implant surface to induce macrophages M2 polarization. Due to the excellent biocompatibility and Dex delivery, the decorated implant surface was favorable for RAW264.7 cells growth and showed powerful effects of inducing M2 polarization both in vitro and in vivo. In conclusion, it is the first report of DNA hydrogel synthesis via CS cross-linkage and the injectable DNA-CS hybrid gel was superb for therapeutic delivery.

Apoptotic vesicles restore liver macrophage homeostasis to counteract type 2 diabetes.

Apoptosis is a naturally occurring process generating plenty of apoptotic vesicles (apoVs), but the feature, fate and function of apoVs remain largely unknown. Notably, as an appealing source for cell therapy, mesenchymal stem cells (MSCs) undergo necessary apoptosis and release apoVs during therapeutic application. In this study, we characterized and used MSC-derived apoVs to treat type 2 diabetes (T2D) mice, and we found that apoVs were efferocytosed by macrophages and functionally modulated liver macrophage homeostasis to counteract T2D. We showed that apoVs can induce macrophage reprogramming at the transcription level in an efferocytosis-dependent manner, leading to inhibition of macrophage accumulation and transformation of macrophages towards an anti-inflammation phenotype in T2D liver. At the molecular level, we discovered that calreticulin (CRT) was exposed on the surface of apoVs to act as a critical 'eat-me' signal mediating apoV efferocytosis and macrophage regulatory effects. Importantly, we demonstrated that CRT-mediated efferocytosis of MSC-derived apoVs contributes to T2D therapy with alleviation of T2D phenotypes including glucose intolerance and insulin resistance. These findings uncover that functional efferocytosis of apoVs restores liver macrophage homeostasis and ameliorates T2D.

Anthocyanin attenuates CD40-mediated endothelial cell activation and apoptosis by inhibiting CD40-induced MAPK activation.

CD40-mediated inflammatory signaling is a potent activator of endothelial cells (ECs) and effective in triggering the pathogenesis of atherosclerosis, a chronic inflammatory disease. Anthocyanin is considered to exert potent cardiovascular-protective effect partially through its anti-inflammatory property, however, the precise mechanism is still unknown. Here we chose cultured human umbilical vein endothelial cells (HUVECs) to explore the influence of anthocyanin on CD40-mediated endothelial activation and apoptosis and the underlying mechanism. Stimulation of human primary HUVECs by CD40 with its physiological ligand CD40L not only augmented MMP-1, -9 secretion and promoted MMP-1, -9 activities, but also induced endothelial cell apoptosis and death. Treatment of ECs with anthocyanins cyanidin-3-O-beta-glucoside (Cy-3-g) and peonidin-3-O-beta-glucoside (Pn-3-g) prevents CD40-induced endothelial activation by inhibiting production of proinflammatory cytokines and matrix metalloproteinases (MMPs). In addition, exposure to anthocyanins inhibits CD40-induced endothelial apoptosis. Anthocyanins also decreased activation of JNK and p38 induced by CD40. Collectively, our findings suggested that the inhibition of JNK and p38 activation interrupts CD40 induced endothelial cell activation and apoptosis, which thereby may represent a mechanism that would explain the anti-inflammatory response of anthocyanin and its athero-protective function.

Cyanidin-3-O-beta-glucoside inhibits iNOS and COX-2 expression by inducing liver X receptor alpha activation in THP-1 macrophages.

Anthocyanins belong to a large and widespread group of water-soluble phytochemicals and exhibit potent antioxidative and anti-inflammatory properties; however, the molecular mechanisms of these biochemical actions mediated by anthocyanins remain unclear. In this study, our data show that pretreatment of THP-1 macrophages with Cyanidin-3-O-beta-glucoside (C3G) for 12 h can enhance the expression and transcriptional activities of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) and liver X receptor alpha (LXRalpha). Furthermore, pretreatment of these cells with C3G for 12 h causes dose-dependent inhibition of lipopolysaccharide (LPS)-induced nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at both the mRNA and protein levels together with a decrease in nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production. Consequently, addition of geranylgeranyl pyrophosphate ammonium salt (GGPP), an LXRalpha antagonist, significantly downregulates the inhibitory effect of C3G on LPS-induced iNOS and COX-2 expression in THP-1 macrophages, whereas the PPARgamma antagonist GW9662 has no effect. Further investigation revealed that LXRalpha might interfere with LPS-induced iNOS and COX-2 expression by suppressing the functional activation of nuclear factor-kappaB (NF-kappaB), not - as was previously proposed - by reducing NF-kappaB nuclear translocation. Taken together, these results indicate that LXRalpha activation has an essential role in the anti-inflammatory property of C3G. Moreover, they provide new insight into the molecular basis for the anti-inflammatory property of anthocyanins.