[Bionic design, preparation and clinical translation of oral hard tissue restorative materials].

Oral diseases concern almost every individual and are a serious health risk to the population. The restorative treatment of tooth and jaw defects is an important means to achieve oral function and support the appearance of the contour. Based on the principle of "learning from the nature", Deng Xuliang's group of Peking University School and Hospital of Stomatology has proposed a new concept of "microstructural biomimetic design and tissue adaptation of tooth/jaw materials" to address the worldwide problems of difficulty in treating dentine hypersensitivity, poor prognosis of restoration of tooth defects, and vertical bone augmentation of alveolar bone after tooth loss. The group has broken through the bottleneck of multi-stage biomimetic technology from the design of microscopic features to the enhancement of macroscopic effects, and invented key technologies such as crystalline/amorphous multi-level assembly, ion-transportation blocking, and multi-physical properties of the micro-environment reconstruction, etc. The group also pioneered the cationic-hydrogel desensitizer, digital stump and core integrated restorations, and developed new crown and bridge restorative materials, gradient functionalisation guided tissue regeneration membrane, and electrically responsive alveolar bone augmentation restorative membranes, etc. These products have established new clinical strategies for tooth/jaw defect repair and achieved innovative results. In conclusion, the research results of our group have strongly supported the theoretical improvement of stomatology, developed the technical system of oral hard tissue restoration, innovated the clinical treatment strategy, and led the progress of the stomatology industry.

Mycoplasma bovis MBOV_RS02825 Encodes a Secretory Nuclease Associated with Cytotoxicity.

This study aimed to determine the activity of one Mycoplasma bovis nuclease encoded by MBOV_RS02825 and its association with cytotoxicity. The bioinformatics analysis predicted that it encodes a Ca(2+)-dependent nuclease based on existence of enzymatic sites in a TNASE_3 domain derived from a Staphylococcus aureus thermonuclease (SNc). We cloned and purified the recombinant MbovNase (rMbovNase), and demonstrated its nuclease activity by digesting bovine macrophage linear DNA and RNA, and closed circular plasmid DNA in the presence of 10 mM Ca(2+) at 22-65 °C. In addition, this MbovNase was localized in membrane and rMbovNase able to degrade DNA matrix of neutrophil extracellular traps (NETs). When incubated with macrophages, rMbovNase bound to and invaded the cells localizing to both the cytoplasm and nuclei. These cells experienced apoptosis and the viability was significantly reduced. The apoptosis was confirmed by activated expression of phosphorylated NF-κB p65 and Bax, and inhibition of Iκßα and Bcl-2. In contrast, rMbovNase(Δ181-342) without TNASE_3 domain exhibited deficiency in all the biological functions. Furthermore, rMbovNase was also demonstrated to be secreted. In conclusion, it is a first report that MbovNase is an active nuclease, both secretory and membrane protein with ability to degrade NETs and induce apoptosis.

Mitochondria Transplantation to Bone Marrow Stromal Cells Promotes Angiogenesis During Bone Repair.

Angiogenesis is crucial for successful bone defect repair. Co-transplanting Bone Marrow Stromal Cells (BMSCs) and Endothelial Cells (ECs) has shown promise for vascular augmentation, but it face challenges in hostile tissue microenvironments, including poor cell survival and limited efficacy. In this study, the mitochondria of human BMSCs are isolated and transplanted to BMSCs from the same batch and passage number (BMSCsmito). The transplanted mitochondria significantly boosted the ability of BMSCsmito-ECs to promote angiogenesis, as assessed by in vitro tube formation and spheroid sprouting assays, as well as in vivo transplantation experiments in balb/c mouse and SD rat models. The Dll4-Notch1 signaling pathway is found to play a key role in BMSCsmito-induced endothelial tube formation. Co-transplanting BMSCsmito with ECs in a rat cranial bone defect significantly improves functional vascular network formation, and improve bone repair outcomes. These findings thus highlight that mitochondrial transplantation, by acting through the DLL4-Notch1 signaling pathway, represents a promising therapeutic strategy for enhancing angiogenesis and improving bone repair. Hence, mitochondrial transplantation to BMSCS as a therapeutic approach for promoting angiogenesis offers valuable insights and holds much promise for innovative regenerative medicine therapies.

HtrA3 paves the way for MSC migration and promotes osteogenesis.

Mesenchymal stem cell (MSC) migration determines the healing capacity of bone and is crucial in promoting bone regeneration. Migration of MSCs is highly dependent on degradation of extracellular matrix by proteolytic enzymes. However, the underlying mechanisms of how enzymolysis paves the way for MSCs to migrate from their niche to the defect area is still not fully understood. Here, this study shows that high-temperature requirement A3 (HtrA3) overcomes the physical barrier and provides anchor points through collagen IV degradation, paving the way for MSC migration. HtrA3 is upregulated in MSCs at the leading edge of bone defect during the early stage of healing. HtrA3 degrades the surrounding collagen IV, which increases the collagen network porosity and increases integrin ß1 expression. Subsequently, integrin ß1 enhances the mechanotransduction of MSCs, thus remodeling the cytoskeleton, increasing cellular stiffness and nuclear translocation of YAP, eventually promoting the migration and subsequent osteogenic differentiation of MSCs. Local administration of recombinant HtrA3 in rat cranial bone defects significantly increases new bone formation and further validates the enhancement of MSC migration. This study helps to reveal the novel roles of HtrA3, explore potential targets for regenerative medicine, and offer new insights for the development of bioactive materials.

A three-dimensional actively spreading bone repair material based on cell spheroids can facilitate the preservation of tooth extraction sockets.

Introduction: Achieving a successful reconstruction of alveolar bone morphology still remains a challenge because of the irregularity and complex microenvironment of tooth sockets. Biological materials including hydroxyapatite and collagen, are used for alveolar ridge preservation. However, the healing effect is often unsatisfactory. Methods: Inspired by superwetting biomimetic materials, we constructed a 3D actively-spreading bone repair material. It consisted of photocurable polyether F127 diacrylate hydrogel loaded with mixed spheroids of mesenchymal stem cells (MSCs) and vascular endothelial cells (ECs). Results: Biologically, cells in the spheroids were able to spread and migrate outwards, and possessed both osteogenic and angiogenic potential. Meanwhile, ECs also enhanced osteogenic differentiation of MSCs. Mechanically, the excellent physical properties of F127DA hydrogel ensured that it was able to be injected directly into the tooth socket and stabilized after light curing. In vivo experiments showed that MSC-EC-F127DA system promoted bone repair and preserved the shape of alveolar ridge within a short time duration. Discussion: In conclusion, the novel photocurable injectable MSC-EC-F127DA hydrogel system was able to achieve three-dimensional tissue infiltration, and exhibited much therapeutic potential for complex oral bone defects in the future.

Assessing Biomaterial-Induced Stem Cell Lineage Fate by Machine Learning-Based Artificial Intelligence.

Current functional assessment of biomaterial-induced stem cell lineage fate in vitro mainly relies on biomarker-dependent methods with limited accuracy and efficiency. Here a "Mesenchymal stem cell Differentiation Prediction (MeD-P)" framework for biomaterial-induced cell lineage fate prediction is reported. MeD-P contains a cell-type-specific gene expression profile as a reference by integrating public RNA-seq data related to tri-lineage differentiation (osteogenesis, chondrogenesis, and adipogenesis) of human mesenchymal stem cells (hMSCs) and a predictive model for classifying hMSCs differentiation lineages using the k-nearest neighbors (kNN) strategy. It is shown that MeD-P exhibits an overall accuracy of 90.63% on testing datasets, which is significantly higher than the model constructed based on canonical marker genes (80.21%). Moreover, evaluations of multiple biomaterials show that MeD-P provides accurate prediction of lineage fate on different types of biomaterials as early as the first week of hMSCs culture. In summary, it is demonstrated that MeD-P is an efficient and accurate strategy for stem cell lineage fate prediction and preliminary biomaterial functional evaluation.

The Deubiquitinase OTUD1 Suppresses Secretory Neutrophil Polarization And Ameliorates Immunopathology of Periodontitis.

Tissue-infiltrating neutrophils (TINs) secrete various signaling molecules to establish paracrine communication within the inflammatory milieu. It is imperative to identify molecular mediators that control this secretory phenotype of TINs. The present study uncovers a secretory neutrophil subset that exhibits increased pro-inflammatory cytokine production and enhanced migratory capacity which is highly related with periodontal pathogenesis. Further analysis identifies the OTU domain-containing protein 1 (OTUD1) plays a regulatory role in this secretory neutrophil polarization. In human and mouse periodontitis, the waning of inflammation is correlated with OTUD1 upregulation, whereas severe periodontitis is induced when neutrophil-intrinsic OTUD1 is depleted. Mechanistically, OTUD1 interacts with SEC23B, a component of the coat protein II complex (COPII). By removing the K63-linked polyubiquitin chains on SEC23B Lysine 81, the deubiquitinase OTUD1 negatively regulates the COPII secretory machinery and limits protein ER-to-Golgi trafficking, thus restricting the surface expression of integrin-regulated proteins, CD9 and CD47. Accordingly, blockade of protein transport by Brefeldin A (BFA) curbs recruitment of Otud1-deficient TINs and attenuates inflammation-induced alveolar bone destruction. The results thus identify OTUD1 signaling as a negative feedback loop that limits the polarization of neutrophils with secretory phenotype and highlight the potential application of BFA in the treatment of periodontal inflammation.

Mitochondria transfer enhances proliferation, migration, and osteogenic differentiation of bone marrow mesenchymal stem cell and promotes bone defect healing.

BACKGROUND: Bone marrow-derived mesenchymal stem cell (BMSC) transplantation is considered a promising therapeutic approach for bone defect repair. However, during the transplantation procedure, the functions and viability of BMSCs may be impaired due to extended durations of in vitro culture, aging, and disease conditions of patients. Inspired by spontaneous intercellular mitochondria transfer that naturally occurs within injured tissues to rescue cellular or tissue function, we investigated whether artificial mitochondria transfer into pre-transplant BMSCs in vitro could improve cellular function and enhance their therapeutic effects on bone defect repair in situ. METHODS: Mitochondria were isolated from donor BMSCs and transferred into recipient BMSCs of the same batch and passage. Subsequently, changes in proliferative capacity and cell senescence were evaluated by live cell imaging, Cell Counting Kit-8 assay, cell cycle analysis, Ki67 staining, qPCR and Western blot analysis of c-Myc expression, and ß-galactosidase staining. Migration ability was evaluated by the transwell migration assay, wound scratch healing, and cell motility tests. Alkaline phosphatase (ALP) staining, Alizarin Red staining, and combined with qPCR and Western blot analyses of Runx2 and BMP2 were performed to elucidate the effects of mitochondria transfer on the osteogenic potential of BMSCs in vitro. After that, in vivo experiments were performed by transplanting mitochondria-recipient BMSCs into a rat cranial critical-size bone defect model. Micro CT scanning and histological analysis were conducted at 4 and 8 weeks after transplantation to evaluate osteogenesis in situ. Finally, in order to establish the correlation between cellular behavioral changes and aerobic metabolism, OXPHOS (oxidative phosphorylation) and ATP production were assessed and inhibition of aerobic respiration by oligomycin was performed. RESULTS: Mitochondria-recipient BMSCs exhibited significantly enhanced proliferation and migration, and increased osteogenesis upon osteogenic induction. The in vivo results showed more new bone formation after transplantation of mitochondria-recipient BMSCs in situ. Increased OXPHOS activity and ATP production were observed, which upon inhibition by oligomycin attenuated the enhancement of proliferation, migration, and osteogenic differentiation induced by mitochondria transfer. CONCLUSIONS: Mitochondria transfer is a feasible technique to enhance BMSC function in vitro and promote bone defect repair in situ through the upregulation of aerobic metabolism. The results indicated that mitochondria transfer may be a novel promising technique for optimizing stem cell therapeutic function.

Proteomics identification and characterization of MbovP730 as a potential DIVA antigen of Mycoplasma bovis.

Mycoplasma bovis (M. bovis) is an important pathogen of cattle. An attenuated live vaccine has recently been developed by this laboratory. However, an effective assay for the differentiation of infected from vaccinated animals (DIVA) is still lacking. Therefore, a comparative immunoproteomics study of the membrane and membrane associated proteins (MAPs) of M. bovis HB0801 and its attenuated strain (M. bovis-150) was aimed to identify potential antigens for DIVA assay. Triton-X-114 fractionated liposoluble proteins of both the virulent and attenuated strains were separated with 2-DE and proteins reacting with sera against the virulent M. bovis strain were detected by MS. A total of 19 differently expressed proteins were identified by MS, among them twelve proteins were detected by MALDI-TOF MS and seven antigenic proteins were identified by short-gun LC-MS/MS. Furthermore, these findings were confirmed at mRNA level by qRT-PCR. The results demonstrated that a putative lipoprotein encoded by functionally unknown gene Mbov_0730 (MbovP730) is a sensitive and specific antigen for DIVA assay. MbovP730 is absent in M. bovis-150 confirmed with Western blot assay and also didn't cross-react with other antisera against common pathogens including infectious bovine rhinotracheitis virus and bovine viral diarrhea virus by iELISA. Thereby rMbovP730-based iELISA was established. For clinical samples, this ELISA provided a sensitivity of 95.7% (95% CI: 90.4%, 98.2%) and specificity was 97.8% (95% CI: 88.4%, 99.6%). Antisera from vaccinated calves (n = 44) were found negative with rMbovP730 based iELISA, while positive with assays based on whole cell proteins of M. bovis-150 and M. bovis HB0801, respectively. In conclusion, this study identified the differential antigen MbovP730 between virulent and attenuated strains and established rMbovP730-based iELISA as a new DIVA method.

Hospital-based epidemiological and clinical characterisation of the malignant transformation of oral leukoplakia in a Chinese population.

OBJECTIVE: The aim of this review was to analyse, systematically, hospital-based epidemiological information concerning the malignant transformation rate (MTR) of oral leukoplakia (OL) in a Chinese population, as well as the associated risk factors. METHODS: Four electronic databases were searched for studies dealing with OL and related risk factors, including age, gender, type of lesion, site, and smoking and drinking habits. RESULTS: The MTR of OL in the hospital-based Chinese population ranged from 4% to 13%, based on the studies analysed. Regarding risk factors, we found that female patients had a higher MTR than male patients, and that patients older than 50 years of age also had a higher MTR. Patients who smoked had a lower MTR, while alcohol consumption seemed to have no association with MTR. Malignant transformation occurred most commonly on the tongue. Regarding lesion type, non-homogeneous OL had a higher MTR, with the granular type having the highest MTR. Our results regarding the epidemiology of OL showed a similar trend to those reported in western populations and provided preliminary epidemiological information on the Chinese population. CONCLUSIONS: Our findings show that female gender, age >50 years and non-homogeneous OL are risk factors for malignant transformation. It is important to develop clinical strategies to educate, diagnose and treat patients with OL and to minimise the MTR of OL.

Mycoplasma bovis NADH oxidase functions as both a NADH oxidizing and O2 reducing enzyme and an adhesin.

Mycoplasma bovis causes considerable economic losses in the cattle industry worldwide. In mycoplasmal infections, adhesion to the host cell is of the utmost importance. In this study, the amino acid sequence of NOX was predicted to have enzymatic domains. The nox gene was then cloned and expressed in Escherichia coli. The enzymatic activity of recombinant NOX (rNOX) was confirmed based on its capacity to oxidize NADH to NAD+ and reduce O2 to H2O2. The adherence of rNOX to embryonic bovine lung (EBL) cells was confirmed with confocal laser scanning microscopy, enzyme-linked immunosorbent assay, and flow cytometry. Both preblocking EBL cells with purified rNOX and preneutralizing M. bovis with polyclonal antiserum to rNOX significantly reduced the adherence of M. bovis to EBL cells. Mycoplasma bovis NOX-expressed a truncated NOX protein at a level 10-fold less than that of the wild type. The capacities of M. bovis NOX- for cell adhesion and H2O2 production were also significantly reduced. The rNOX was further used to pan phage displaying lung cDNA library and fibronectin was determined to be potential ligand. In conclusion, M. bovis NOX functions as both an active NADH oxidase and adhesin, and is therefore a potential virulence factor.