The role and applications of extracellular vesicles in osteoporosis

Osteoporosis is a widely observed condition characterized by the systemic deterioration of bone mass and microarchitecture,which increases patient susceptibility to fragile fractures.The intricate mechanisms governing bone homeostasis are substantially impacted by extracellular vesicles(EVs),which play crucial roles in both pathological and physiological contexts.EVs derived from various sources exert distinct effects on osteoporosis.Specifically,EVs released by osteoblasts,endothelial cells,myocytes,and mesenchymal stem cells contribute to bone formation due to their unique cargo of proteins,miRNAs,and cytokines.Conversely,EVs secreted by osteoclasts and immune cells promote bone resorption and inhibit bone formation.Furthermore,the use of EVs as therapeutic modalities or biomaterials for diagnosing and managing osteoporosis is promising.Here,we review the current understanding of the impact of EVs on bone homeostasis,including the classification and biogenesis of EVs and the intricate regulatory mechanisms of EVs in osteoporosis.Furthermore,we present an overview of the latest research progress on diagnosing and treating osteoporosis by using EVs.Finally,we discuss the challenges and prospects of translational research on the use of EVs in osteoporosis.

Emerging roles of exosomes in oral diseases progression

Oral diseases, such as periodontitis, salivary gland diseases, and oral cancers, significantly challenge health conditions due to their detrimental effects on patient's digestive functions, pronunciation, and esthetic demands. Delayed diagnosis and non-targeted treatment profoundly influence patients' prognosis and quality of life. The exploration of innovative approaches for early detection and precise treatment represents a promising frontier in oral medicine.

LITTIP/Lgr6/HnRNPK complex regulates cementogenesis via Wnt signaling

Orthodontically induced tooth root resorption(OIRR)is a serious complication during orthodontic treatment.Stimulating cementum repair is the fundamental approach for the treatment of OIRR.Parathyroid hormone(PTH)might be a potential therapeutic agent for OIRR,but its effects still lack direct evidence,and the underlying mechanisms remain unclear.This study aims to explore the potential involvement of long noncoding RNAs(lncRNAs)in mediating the anabolic effects of intermittent PTH and contributing to cementum repair,as identifying lncRNA-disease associations can provide valuable insights for disease diagnosis and treatment.Here,we showed that intermittent PTH regulates cell proliferation and mineralization in immortalized murine cementoblast OCCM-30 via the regulation of the Wnt pathway.In vivo,daily administration of PTH is sufficient to accelerate root regeneration by locally inhibiting Wnt/β-catenin signaling.Through RNA microarray analysis,lncRNA LITTIP(LGR6 intergenic transcript under intermittent PTH)is identified as a key regulator of cementogenesis under intermittent PTH.Chromatin isolation by RNA purification(ChIRP)and RNA immunoprecipitation(RIP)assays revealed that LITTIP binds to mRNA of leucine-rich repeatcontaining G-protein coupled receptor 6(LGR6)and heterogeneous nuclear ribonucleoprotein K(HnRNPK)protein.Further cotransfection experiments confirmed that LITTIP plays a structural role in the formation of the LITTIP/Lgr6/HnRNPK complex.Moreover,LITTIP is able to promote the expression of LGR6 via the RNA-binding protein HnRNPK.Collectively,our results indicate that the intermittent PTH administration accelerates root regeneration via inhibiting Wnt pathway.The lncRNA LITTIP is identified to negatively regulate cementogenesis,which activates Wnt/β-catenin signaling via high expression of LGR6 promoted by HnRNPK.

Spatiotemporal cellular dynamics and molecular regulation of tooth root ontogeny

Tooth root development involves intricate spatiotemporal cellular dynamics and molecular regulation.The initiation of Hertwig’s epithelial root sheath(HERS)induces odontoblast differentiation and the subsequent radicular dentin deposition.Precisely controlled signaling pathways modulate the behaviors of HERS and the fates of dental mesenchymal stem cells(DMSCs).Disruptions in these pathways lead to defects in root development,such as shortened roots and furcation abnormalities.Advances in dental stem cells,biomaterials,and bioprinting show immense promise for bioengineered tooth root regeneration.However,replicating the developmental intricacies of odontogenesis has not been resolved in clinical treatment and remains a major challenge in this field.Ongoing research focusing on the mechanisms of root development,advanced biomaterials,and manufacturing techniques will enable next-generation biological root regeneration that restores the physiological structure and function of the tooth root.This review summarizes recent discoveries in the underlying mechanisms governing root ontogeny and discusses some recent key findings in developing of new biologically based dental therapies.

Annexin A5 derived from matrix vesicles protects against osteoporotic bone loss via mineralization

Matrix vesicles(MVs)have shown strong effects in diseases such as vascular ectopic calcification and pathological calcified osteoarthritis and in wound repair of the skeletal system due to their membranous vesicle characteristics and abundant calcium and phosphorus content.However,the role of MVs in the progression of osteoporosis is poorly understood.Here,we report that annexin A5,an important component of the matrix vesicle membrane,plays a vital role in bone matrix homeostasis in the deterioration of osteoporosis.We first identified annexin A5 from adherent MVs but not dissociative MVs of osteoblasts and found that it could be sharply decreased in the bone matrix during the occurrence of osteoporosis based on ovariectomized mice.We then confirmed its potential in mediating the mineralization of the precursor osteoblast lineage via its initial binding with collagen type I to achieve MV adhesion and the subsequent activation of cellular autophagy.Finally,we proved its protective role in resisting bone loss by applying it to osteoporotic mice.Taken together,these data revealed the importance of annexin A5,originating from adherent MVs of osteoblasts,in bone matrix remodeling of osteoporosis and provided a new strategy for the treatment and intervention of bone loss.

Autophagy in bone homeostasis and the onset of osteoporosis

Autophagy is an evolutionarily conserved intracellular process,in which domestic cellular components are selectively digested for the recycling of nutrients and energy.This process is indispensable for cell homeostasis maintenance and stress responses.Both genetic and functional studies have demonstrated that multiple proteins involved in autophagic activities are critical to the survival,differentiation,and functioning of bone cells,including osteoblasts,osteocytes,and osteoclasts.Dysregulation at the level of autophagic activity consequently disturbs the balance between bone formation and bone resorption and mediates the onset and progression of multiple bone diseases,including osteoporosis.This review aims to introduce the topic of autophagy,summarize the understanding of its relevance in bone physiology,and discuss its role in the onset of osteoporosis and therapeutic potential.

DNA N^6-methyladenine demethylase ALKBH1 enhances osteogenic differentiation of human MSCs

ALKBH1 was recently discovered as a demethylase for DNA N6-methyladenine （N6-mA）, a new epigenetic modification, and interacts with the core transcriptional pluripotency network of embryonic stem cells. However, the role of ALKBH1 and DNA N6-mA in regulating osteogenic differentiation is largely unknown. In this study, we demonstrated that the expression of ALKBH1 in human mesenchymal stem cells （MSCs） was upregulated during osteogenic induction. Knockdown of ALKBH1 increased the genomic DNA N6-mA levels and significantly reduced the expression of osteogenic-related genes, alkaline phosphatase activity, and mineralization. ALKBHl-depleted MSCs also exhibited a restricted capacity for bone formation in vivo. By contrast, the ectopic overexpression of ALKBH1 enhanced osteoblastic differentiation. Mechanically, we found that the depletion of ALKBH1 resulted in the accumulation of N6-mA on the promoter region of ATF4, which subsequently silenced ATF4 transcription. In addition, restoring the expression of ATP by adenovirus-mediated transduction successfully rescued osteogenic differentiation. Taken together, our results demonstrate that ALKBH1 is indispensable for the osteogenic differentiation of MSCs and indicate that DNA N6-mA modifications area new mechanism for the epigenetic regulation of stem cell differentiation.

Parathyroid hormone increases alveolar bone homoeostasis during orthodontic tooth movement in rats with periodontitis via crosstalk between STAT3 and β-catenin

Periodontitis patients are at risk of alveolar bone loss during orthodontic treatment.The aim of this study was to investigate whether intermittent parathyroid hormone(1–34)treatment(iPTH)could reduce alveolar bone loss during orthodontic tooth movement(OTM)in individuals with periodontitis and the underlying mechanism.A rat model of OTM in the context of periodontitis was established and alveolar bone loss was observed.The control,iPTH and iPTH+stattic groups received injections of vehicle,PTH and vehicle,or PTH and the signal transducer and activator of transcription 3(STAT3)inhibitor stattic,respectively.iPTH prevented alveolar bone loss by enhancing osteogenesis and suppressing bone resorption in the alveolar bone during OTM in rats with periodontitis.This effect of iPTH was along with STAT3 activation and reduced by a local injection of stattic.iPTH promoted osteoblastic differentiation and might further regulate the Wnt/β-catenin pathway in a STAT3-dependent manner.The findings of this study suggest that iPTH might reduce alveolar bone loss during OTM in rats with periodontitis through STAT3/β-catenin crosstalk.

Advanced smart biomaterials and constructs for hard tissue engineering and regeneration

Hard tissue repair and regeneration cost hundreds of billions of dollars annually worldwide, and the need has substantially increased as the population has aged. Hard tissues include bone and tooth structures that contain calcium phosphate minerals.Smart biomaterial-based tissue engineering and regenerative medicine methods have the exciting potential to meet this urgent need. Smart biomaterials and constructs refer to biomaterials and constructs that possess instructive/inductive or triggering/stimulating effects on cells and tissues by engineering the material's responsiveness to internal or external stimuli or have intelligently tailored properties and functions that can promote tissue repair and regeneration. The smart material-based approaches include smart scaffolds and stem cell constructs for bone tissue engineering; smart drug delivery systems to enhance bone regeneration; smart dental resins that respond to pH to protect tooth structures; smart pH-sensitive dental materials to selectively inhibit acid-producing bacteria; smart polymers to modulate biofilm species away from a pathogenic composition and shift towards a healthy composition; and smart materials to suppress biofilms and avoid drug resistance. These smart biomaterials can not only deliver and guide stem cells to improve tissue regeneration and deliver drugs and bioactive agents with spatially and temporarily controlled releases but can also modulate/suppress biofilms and combat infections in wound sites. The new generation of smart biomaterials provides exciting potential and is a promising opportunity to substantially enhance hard tissue engineering and regenerative medicine efficacy.

Runxl protects against the pathological progression of osteoarthritis

Runt-related transcription factor-1(Runxl)is required for chondrocyte-to-osteoblast lineage commitment by enhancing both chondrogenesis and osteogenesis during vertebrate development.However,the potential role of Runxl in joint diseases is not well known.In the current study,we aimed to explore the role of Runxl in osteoarthritis induced by anterior cruciate ligament transaction(ACLT)surgery.We showed that chondrocyte-specific Runxl knockout(Runx1f/fCol2a1-Cre)aggravated cartilage destruction by accelerating the loss of proteoglycan and collagen II in early osteoarthritis.Moreover,we observed thinning and ossification of the growth plate,a decrease in chondrocyte proliferative capacity and the loss of bone matrix around the growth plate in late osteoarthritis.We overexpressed Runxl by adeno-associated virus(AAV)in articular cartilage and identified its protective effect by slowing the destruction of osteoarthritis in cartilage in early osteoarthritis and alleviating the pathological progression of growth plate cartilage in late osteoarthritis.ChIP-seq analysis identified new targets that interacted with Runxl in cartilage pathology,and we confirmed the direct interactions of these factors with Runxl by ChIP-qPCR.This study helps us to understand the function of Runxl in osteoarthritis and provides new clues for targeted osteoarthritis therapy.

AFF4 regulates osteogenic differentiation of human dental follicle cells

As a member of the AFF(AF4/FMR2)family,AFF4 is a transcription elongation factor that is a component of the super elongation complex.AFF4 serves as a scaffolding protein that connects transcription factors and promotes gene transcription through elongation and chromatin remodelling.Here,we investigated the effect of AFF4 on human dental follicle cells(DFCs)in osteogenic differentiation.In this study,we found that small interfering RNA-mediated depletion of AFF4 resulted in decreased alkaline phosphatase(ALP)activity and impaired mineralization.In addition,the expression of osteogenic-related genes(DLX5,SP7,RUNX2 and BGLAP)was significantly downregulated.In contrast,lentivirus-mediated overexpression of AFF4 significantly enhanced the osteogenic potential of human DFCs.Mechanistically,we found that both the mRNA and protein levels of ALKBH1,a critical regulator of epigenetics,changed in accordance with AFF4 expression levels.Overexpression of ALKBH1 in AFF4-depleted DFCs partially rescued the impairment of osteogenic differentiation.Our data indicated that AFF4 promoted the osteogenic differentiation of DFCs by upregulating the transcription of ALKBH1.

Compliant substratum modulates vinculin expression in focal adhesion plaques in skeletal cells

The biophysical properties of the extracellular matrix (ECM) dictate tissue-specific cell behaviour.In the skeleton system,bone shows the potential to adapt its architecture and contexture to environmental rigidity via the bone remodelling process,which involves chondrocytes,osteoblasts,osteoclasts,osteocytes and even peripheral bone marrow-derived stem/stromal cells (BMSCs).In the current study,we generated stiff (~1 014 ± 56) kPa,Young’s modulus) and soft (~46 ± 11) kPa silicon-based elastomer polydimethylsiloxane (PDMS) substrates by mixing curing agent into oligomeric base at 1:5 and 1:45 ratios,respectively,and investigated the influence of substrate stiffness on the cell behaviours by characterizing cell spreading area,cell cytoskeleton and cell adhesion capacity.The results showed that the cell spreading areas of chondrocytes,osteoblasts,osteoclasts,osteocytes and BMSCs were all reduced in the soft substrate relative to those in the stiff substrate.F-actin staining confirmed that the cytoskeleton was also changed in the soft group compared to that in the stiff group.Vinculin in focal adhesion plaques was significantly decreased in response to soft substrate compared to stiff substrate.This study establishes the potential correlation between microenvironmental mechanics and the skeletal system,and the results regarding changes in cell spreading area,cytoskeleton and cell adhesion further indicate the important role of biomechanics in the cell-matrix interaction.

circ_0003204 regulates the osteogenic differentiation of human adipose-derived stem cells via miR-370-3p/HDAC4 axis

Human adipose-derived stem cells(hASCs)are a promising cell type for bone tissue regeneration.Circular RNAs(circRNAs)have been shown to play a critical role in regulating various cell differentiation and involve in mesenchymal stem cell osteogenesis.However,how circRNAs regulate hASCs in osteogenesis is still unclear.Herein,we found circ_0003204 was significantly downregulated during osteogenic differentiation of hASCs.Knockdown of circ_0003204 by si RNA or overexpression by lentivirus confirmed circ_0003204 could negatively regulate the osteogenic differentiation of hASCs.We performed dual-luciferase reporting assay and rescue experiments to verify circ_0003204 regulated osteogenic differentiation via sponging miR-370-3p.We predicted and confirmed that miR-370-3p had targets in the 3′-UTR of HDAC4 m RNA.The following rescue experiments indicated that circ_0003204 regulated the osteogenic differentiation of hASCs via miR-370-3p/HDAC4 axis.Subsequent in vivo experiments showed the silencing of circ_0003204 increased the bone formation and promoted the expression of osteogenic-related proteins in a mouse bone defect model,while overexpression of circ_0003204 inhibited bone defect repair.Our findings indicated that circ_0003204 might be a promising target to promote the efficacy of hASCs in repairing bone defects.

Microenvironmental stiffness mediates cytoskeleton re-organization in chondrocytes through laminin-FAK mechanotransduction

Microenvironmental biophysical factors play a fundamental role in controlling cell behaviors including cell morphology,proliferation,adhesion and differentiation,and even determining the cell fate.Cells are able to actively sense the surrounding mechanical microenvironment and change their cellular morphology to adapt to it.Although cell morphological changes have been considered to be the first and most important step in the interaction between cells and their mechanical microenvironment,their regulatory network is not completely clear.In the current study,we generated silicon-based elastomer polydimethylsiloxane(PDMS)substrates with stiff(15:1,PDMS elastomer vs.curing agent)and soft(45:1)stiffnesses,which showed the Young’s moduli of~450 k Pa and 46 kPa,respectively,and elucidated a new path in cytoskeleton re-organization in chondrocytes in response to changed substrate stiffnesses by characterizing the axis shift from the secreted extracellular protein lamininβ1,focal adhesion complex protein FAK to microfilament bundling.We first showed the cellular cytoskeleton changes in chondrocytes by characterizing the cell spreading area and cellular synapses.We then found the changes of secreted extracellular linkage protein,lamininβ1,and focal adhesion complex protein,FAK,in chondrocytes in response to different substrate stiffnesses.These two proteins were shown to be directly interacted by Co-IP and colocalization.We next showed that impact of FAK on the cytoskeleton organization by showing the changes of microfilament bundles and found the potential intermediate regulators.Taking together,this modulation axis of lamininβ1-FAK-microfilament could enlarge our understanding about the interdependence among mechanosensing,mechanotransduction,and cytoskeleton re-organization.

Hydrogel platform with tunable stiffness based on magnetic nanoparticles cross-linked GelMA for cartilage regeneration and its intrinsic biomechanism

Cartilage injury affects numerous individuals,but the efficient repair of damaged cartilage is still a problem in clinic.Hydrogel is a potent scaffold candidate for tissue regeneration,but it remains a big challenge to improve its mechanical property and figure out the interaction of chondrocytes and stiffness.Herein,a novel hybrid hydrogel with tunable stiffness was fabricated based on methacrylated gelatin(GelMA)and iron oxide nanoparticles(Fe_(2)O_(3))through chemical bonding.The stiffness of Fe_(2)O_(3)/GelMA hybrid hydrogel was controlled by adjusting the concentration of magnetic nanoparticles.The hydrogel platform with tunable stiffness modulated its cellular properties including cell morphology,microfilaments and Young’s modulus of chondrocytes.Interestingly,Fe_(2)O_(3)/GelMA hybrid hydrogel promoted oxidative phosphorylation of mitochondria and facilitated catabolism of lipids in chondrocytes.As a result,more ATP and metabolic materials generated for cellular physiological activities and organelle component replacements in hybrid hydrogel group compared to pure GelMA hydrogel.Furthermore,implantation of Fe_(2)O_(3)/GelMA hybrid hydrogel in the cartilage defect rat model verified its remodeling potential.This study provides a deep understanding of the bio-mechanism of Fe_(2)O_(3)/GelMA hybrid hydrogel interaction with chondrocytes and indicates the hydrogel platform for further application in tissue engineering.

Hedgehog signaling in tissue homeostasis, cancers, and targeted therapies

The past decade has seen significant advances in our understanding of Hedgehog(HH)signaling pathway in various biological events.HH signaling pathway exerts its biological effects through a complex signaling cascade involved with primary cilium.HH signaling pathway has important functions in embryonic development and tissue homeostasis.It plays a central role in the regulation of the proliferation and differentiation of adult stem cells.Importantly,it has become increasingly clear that HH signaling pathway is associated with increased cancer prevalence,malignant progression,poor prognosis and even increased mortality.Understanding the integrative nature of HH signaling pathway has opened up the potential for new therapeutic targets for cancer.A variety of drugs have been developed,including small molecule inhibitors,natural compounds,and long non-coding RNA(LncRNA),some of which are approved for clinical use.This review outlines recent discoveries of HH signaling in tissue homeostasis and cancer and discusses how these advances are paving the way for the development of new biologically based therapies for cancer.Furthermore,we address status quo and limitations of targeted therapies of HH signaling pathway.Insights from this review will help readers understand the function of HH signaling in homeostasis and cancer,as well as opportunities and challenges of therapeutic targets for cancer.

METTL5 regulates cranial suture fusion via Wnt signaling

METTL5 is a methyltransferase that mediates eukaryotic 18S ribosomal RNA m^(6)A modification,and its mutations lead to intellectual disability,microcephaly,and facial dysmorphism in patients.However,the role of METTL5 in craniofacial development remains poorly understood.This study demonstrates that Mettl5 knockout mice exhibit poor ossification,widened cranial sutures,and a cleidocranial dysplasia-like phenotype.Deletion of Mettl5 leads to increased proliferation and decreased osteogenic differentiation of suture mesenchymal stem cells.Mechanistically,we find that Wnt signaling is significantly downregulated after Mettl5 knockout.Overall,we reveal an essential role of METTL5 in craniofacial development and osteogenic differentiation of suture mesenchymal stem cells,making METTL5 a potential diagnostic and therapeutic target for craniofacial developmental diseases.

Generation and application of replication-competent Venus-expressing H5N1,H7N9,and H9N2 influenza A viruses

The generation and application of replication-competent influenza A virus(IAV) expressing a reporter gene represent a valuable tool to elucidate the mechanism of viral pathogenesis and establish new countermeasures to combat the threat of influenza. Here, replication-competent IAVs with a neuraminidase(NA) segment harboring a fluorescent reporter protein, Venus, were generated in the background of H5N1, H7N9, and H9N2 influenza viruses, the three subtypes of viruses with imminent pandemic potential. All three reporter viruses maintained virion morphology, replicated with similar or slightly reduced titers relative to their parental viruses, and stably expressed the fluorescent signal for at least two passages in embryonated chicken eggs. As a proof of concept, we demonstrated that these reporter viruses,used in combination with a high-content imaging system, can serve as a convenient and rapid tool for the screening of antivirals and host factors involved in the virus life cycle. Moreover, the reporter viruses demonstrated similar growth properties and tissue tropism as their parental viruses in mice, among which the H7N9 NA-Venus virus could potentially be used in ex vivo studies to better understand H7N9 pathogenesis or to develop novel therapeutics.

Klotho in Osx+-mesenchymal progenitors exerts pro-osteogenic and anti-inflammatory effects during mandibular alveolar bone formation and repair

Maxillofacial bone defects are commonly seen in clinical practice.A clearer understanding of the regulatory network directing maxillofacial bone formation will promote the development of novel therapeutic approaches for bone regeneration.The fibroblast growth factor(FGF)signalling pathway is critical for the development of maxillofacial bone.Klotho,a type I transmembrane protein,is an important components of FGF receptor complexes.Recent studies have reported the presence of Klotho expression in bone.However,the role of Klotho in cranioskeletal development and repair remains unknown.Here,we use a genetic strategy to report that deletion of Klotho in Osx-positive mesenchymal progenitors leads to a significant reduction in osteogenesis under physiological and pathological conditions.Klotho-deficient mensenchymal progenitors also suppress osteoclastogenesis in vitro and in vivo.Under conditions of inflammation and trauma-induced bone loss,we find that Klotho exerts an inhibitory function on inflammation-induced TNFR signaling by attenuating Rankl expression.More importantly,we show for the first time that Klotho is present in human alveolar bone,with a distinct expression pattern under both normal and pathological conditions.In summary,our results identify the mechanism whereby Klotho expressed in Osx+-mensenchymal progenitors controls osteoblast differentiation and osteoclastogenesis during mandibular alveolar bone formation and repair.Klotho-mediated signaling is an important component of alveolar bone remodeling and regeneration.It may also be a target for future therapeutics.

Functional non-homologous end joining patterns triggered by CRISPR/Cas9 in human cells

CRISPR/Cas9-mediated genome engineering technologies are now widely applied in various organisms,including mouse and human cells（Cong et al.,2013;Mali et al.,2013;Yang et al.,2013;Hsu et al.,2014）.The most widely used customized CRISPR/Cas9（Sp Cas9）is derived from Streptococcus pyogenes（Cong et al.,2013）.