A BMP-controlled metabolic/epigenetic signaling cascade directs midfacial morphogenesis.

Craniofacial anomalies, especially midline facial defects, are among the most common birth defects in patients and are associated with increased mortality or require lifelong treatment. During mammalian embryogenesis, specific instructions arising at genetic, signaling, and metabolic levels are important for stem cell behaviors and fate determination, but how these functionally relevant mechanisms are coordinated to regulate craniofacial morphogenesis remain unknown. Here, we report that bone morphogenetic protein (BMP) signaling in cranial neural crest cells (CNCCs) is critical for glycolytic lactate production and subsequent epigenetic histone lactylation, thereby dictating craniofacial morphogenesis. Elevated BMP signaling in CNCCs through constitutively activated ACVR1 (ca-ACVR1) suppressed glycolytic activity and blocked lactate production via a p53-dependent process that resulted in severe midline facial defects. By modulating epigenetic remodeling, BMP signaling-dependent lactate generation drove histone lactylation levels to alter essential genes of Pdgfra, thus regulating CNCC behavior in vitro as well as in vivo. These findings define an axis wherein BMP signaling controls a metabolic/epigenetic cascade to direct craniofacial morphogenesis, thus providing a conceptual framework for understanding the interaction between genetic and metabolic cues operative during embryonic development. These findings indicate potential preventive strategies of congenital craniofacial birth defects via modulating metabolic-driven histone lactylation.

Nanoparticulate bioceramic putty suppresses osteoclastogenesis and inflammatory bone loss in mice via inhibition of TRAF6-mediated signalling pathways: A laboratory investigation.

AIM: This study aimed to determine the effects of iRoot BP Plus on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis in vitro and inflammation-mediated bone resorption in vivo and investigated the underlying molecular mechanisms. METHODOLOGY: CCK-8 was performed to test cell viability in RANKL-induced RAW 264.7 cells and BMDMs in response to iRoot BP Plus. The effect of iRoot BP Plus on osteoclastogenesis was determined using TRAP staining and phalloidin staining, respectively. Pit formation assay was conducted to measure osteoclast resorptive capacity. Western blot and qPCR were performed to examine osteoclast-related proteins and gene expression, respectively. Western blot was also used to investigate the signalling pathways involved. For in vivo experiments, an LPS-induced mouse calvarial bone resorption model was established to analyse the effect of iRoot BP Plus on bone resorption (n = 6 per group). At 7 days, mouse calvaria were collected and prepared for histological analysis. RESULTS: We identified that iRoot BP Plus extracts significantly attenuated RANKL-induced osteoclastogenesis, reduced sealing zone formation, restrained osteolytic capacity and decreased osteoclast-specific gene expression (p < .01). Mechanistically, iRoot BP Plus extracts reduced TRAF6 via proteasomal degradation, then suppressed the phosphorylation of mitogen-activated protein kinases (MAPKs), blocked the nuclear translocation of c-Fos and diminished nuclear factor-κB (NF-κB) p65 and NFATc1 accumulation. Consistent with the in vitro results, iRoot BP Plus extracts attenuated osteoclast activity thus protecting against inflammatory bone resorption in vivo (p < .05), which was accompanied by a suppression of TRAF6, c-Fos, NFATc1 and cathepsin K expression. CONCLUSION: These findings provide valuable insights into the signalling mechanisms underlying nanoparticulate bioceramic putty-mediated bone homeostasis.

P7C3-A20 treats traumatic brain injury in rats by inhibiting excessive autophagy and apoptosis.

Traumatic brain injury is a severe health problem leading to autophagy and apoptosis in the brain. 3,6-Dibromo-beta-fluoro-N-(3-methoxyphenyl)-9H-carbazole-9-propanamine (P7C3-A20) can be neuroprotective in various diseases, including ischemic stroke and neurodegenerative diseases. However, whether P7C3-A20 has a therapeutic effect on traumatic brain injury and its possible molecular mechanisms are unclear. Therefore, in the present study, we investigated the therapeutic effects of P7C3-A20 on traumatic brain injury and explored the putative underlying molecular mechanisms. We established a traumatic brain injury rat model using a modified weight drop method. P7C3-A20 or vehicle was injected intraperitoneally after traumatic brain injury. Severe neurological deficits were found in rats after traumatic brain injury, with deterioration in balance, walking function, and learning memory. Furthermore, hematoxylin and eosin staining showed significant neuronal cell damage, while terminal deoxynucleotidyl transferase mediated dUTP nick end labeling staining indicated a high rate of apoptosis. The presence of autolysosomes was observed using transmission electron microscope. P7C3-A20 treatment reversed these pathological features. Western blotting showed that P7C3-A20 treatment reduced microtubule-associated protein 1 light chain 3-II (LC3-II) autophagy protein, apoptosis-related proteins (namely, Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 [BNIP3], and Bcl-2 associated x protein [Bax]), and elevated ubiquitin-binding protein p62 (p62) autophagy protein expression. Thus, P7C3-A20 can treat traumatic brain injury in rats by inhibiting excessive autophagy and apoptosis.

Proteolytic regulation of a galectin-3/Lrp1 axis controls osteoclast-mediated bone resorption.

Bone-resorbing osteoclasts mobilize proteolytic enzymes belonging to the matrix metalloproteinase (MMP) family to directly degrade type I collagen, the dominant extracellular matrix component of skeletal tissues. While searching for additional MMP substrates critical to bone resorption, Mmp9/Mmp14 double-knockout (DKO) osteoclasts-as well as MMP-inhibited human osteoclasts-unexpectedly display major changes in transcriptional programs in tandem with compromised RhoA activation, sealing zone formation and bone resorption. Further study revealed that osteoclast function is dependent on the ability of Mmp9 and Mmp14 to cooperatively proteolyze the ß-galactoside-binding lectin, galectin-3, on the cell surface. Mass spectrometry identified the galectin-3 receptor as low-density lipoprotein-related protein-1 (Lrp1), whose targeting in DKO osteoclasts fully rescues RhoA activation, sealing zone formation and bone resorption. Together, these findings identify a previously unrecognized galectin-3/Lrp1 axis whose proteolytic regulation controls both the transcriptional programs and the intracellular signaling cascades critical to mouse as well as human osteoclast function.

A Zeb1/MtCK1 metabolic axis controls osteoclast activation and skeletal remodeling.

Osteoclasts are bone-resorbing polykaryons responsible for skeletal remodeling during health and disease. Coincident with their differentiation from myeloid precursors, osteoclasts undergo extensive transcriptional and metabolic reprogramming in order to acquire the cellular machinery necessary to demineralize bone and digest its interwoven extracellular matrix. While attempting to identify new regulatory molecules critical to bone resorption, we discovered that murine and human osteoclast differentiation is accompanied by the expression of Zeb1, a zinc-finger transcriptional repressor whose role in normal development is most frequently linked to the control of epithelial-mesenchymal programs. However, following targeting, we find that Zeb1 serves as an unexpected regulator of osteoclast energy metabolism. In vivo, Zeb1-null osteoclasts assume a hyperactivated state, markedly decreasing bone density due to excessive resorptive activity. Mechanistically, Zeb1 acts in a rheostat-like fashion to modulate murine and human osteoclast activity by transcriptionally repressing an ATP-buffering enzyme, mitochondrial creatine kinase 1 (MtCK1), thereby controlling the phosphocreatine energy shuttle and mitochondrial respiration. Together, these studies identify a novel Zeb1/MtCK1 axis that exerts metabolic control over bone resorption in vitro and in vivo.

STAT3/Mitophagy Axis Coordinates Macrophage NLRP3 Inflammasome Activation and Inflammatory Bone Loss.

Signal transducer and activator of transcription 3 (STAT3), a cytokine-responsive transcription factor, is known to play a role in immunity and bone remodeling. However, whether and how STAT3 impacts macrophage NLR family pyrin domain containing 3 (NLRP3) inflammasome activation associated with inflammatory bone loss remains unknown. Here, STAT3 signaling is hyperactivated in macrophages in the context of both non-sterile and sterile inflammatory osteolysis, and this was highly correlated with the cleaved interleukin-1ß (IL-1ß) expression pattern. Strikingly, pharmacological inhibition of STAT3 markedly blocks macrophage NLRP3 inflammasome activation in vitro, thereby relieving inflammatory macrophage-amplified osteoclast formation and bone-resorptive activity. Mechanistically, STAT3 inhibition in macrophages triggers PTEN-induced kinase 1 (PINK1)-dependent mitophagy that eliminates dysfunctional mitochondria, reverses mitochondrial membrane potential collapse, and inhibits mitochondrial reactive oxygen species release, thus inactivating the NLRP3 inflammasome. In vivo, STAT3 inhibition effectively protects mice from both infection-induced periapical lesions and aseptic titanium particle-mediated calvarial bone erosion with potent induction of PINK1 and downregulation of inflammasome activation, macrophage infiltration, and osteoclast formation. This study reveals the regulatory role of the STAT3/mitophagy axis at the osteo-immune interface and highlights a potential therapeutic intervention to prevent inflammatory bone loss. © 2022 American Society for Bone and Mineral Research (ASBMR).

Expression of PINK1 and Parkin in human apical periodontitis.

AIM: PTEN-induced putative kinase 1 (PINK1) and Parkin E3 ubiquitin-protein ligase (Parkin) are critical for immune and inflammatory regulation in health and disease. PINK1 and Parkin have been confirmed to be involved in the progression of apical periodontitis by affecting mitophagy-related osteoblast apoptosis; however, the expression of PINK1 and Parkin in macrophages, one of the most important cells in apical periodontitis, remains unknown. This study aimed to investigate the expression of PINK1 and Parkin in human apical periodontitis lesions, as well as their possible localization in macrophages. METHODOLOGY: Thirty-seven human periapical tissues, including periapical granulomas (PGs, n = 12), radicular cysts (RCs, n = 11) and healthy gingival tissues (n = 14) were examined. The inflammatory infiltrates of lesions were evaluated by haematoxylin staining, and the expression of PINK1 and Parkin was detected by immunohistochemistry. Double immunofluorescence was used to explore the colocalization of microtubule-associated protein 1 light chain 3 (LC3) and TOMM20, as well as the localization of PINK1 and Parkin, in macrophages of human apical periodontitis lesions. The ultrastructural morphology of mitochondria in human apical periodontitis lesions was visualized by transmission electron microscopy (TEM). Data were analysed by one-way anova with Student-Newman-Keul's test and the Mann-Whitney test. p < .05 was considered statistically significant. RESULTS: Immunohistochemistry demonstrated a significantly higher expression of PINK1 and Parkin proteins in human apical periodontitis lesions than in healthy gingival tissues (p < .0001), but no significant difference was demonstrated between PGs and RCs (p > .05). The higher expression of LC3 and the presence of more LC3-TOMM20 double-positive cells were also observed in human apical periodontitis. Double-labelling analysis of PINK1, Parkin and LC3 with CD68 indicated that macrophage mitophagy might be present in the progression of human apical periodontitis. Finally, the results of TEM morphological analysis revealed the appearance of double-membraned mitophagosomes and vacuolated mitochondria in macrophage-like cells of apical periodontitis lesions. CONCLUSIONS: Our findings indicated that PINK1 and Parkin proteins were highly expressed in clinical apical periodontitis lesions.

Immunocolocalization of Interferon Regulatory Factory 5 with Tumor Necrosis Factor Receptor-associated Factor 6 and AKT2 in Human Apical Periodontitis.

INTRODUCTION: Interferon regulatory factor 5 (IRF5) is critical for the regulation of immune and inflammatory responses in health and diseases. However, the presence of IRF5 in human apical periodontitis remains unknown. This study aimed to explore the expression and colocalization of IRF5 with tumor necrosis factor receptor-associated factor 6 (TRAF6) and AKT2 in human apical periodontitis. METHODS: A total of 39 human periapical tissues, including healthy gingival tissues (n = 12), periapical granulomas (PGs, n = 13), and radicular cysts (RCs, n = 14), were used in this study. The inflammatory infiltrates of lesions were evaluated by hematoxylin-eosin staining. The expression of IRF5 was detected by immunohistochemistry. Double immunofluorescence assessment was performed to colocalize IRF5 with CD68, TRAF6, and AKT2, respectively. Data were analyzed using the Kruskal-Wallis test. RESULTS: Immunohistochemistry revealed significantly higher expressions of IRF5 in PGs and RCs than the healthy control group. IRF5-CD68 double-positive cells were more predominant in RCs and PGs than the healthy control group. Significant differences of the IRF5-TRAF6 and IRF5-AKT2 double-positive cells were detected in periapical lesions compared with the healthy control tissues. CONCLUSIONS: IRF5 was highly expressed in macrophages of human periapical tissues and was colocalized with TRAF6 or AKT2 in human periapical tissues. These findings may provide new clues for understanding the pathogenesis of periapical diseases.

Matrix remodeling controls a nuclear lamin A/C-emerin network that directs Wnt-regulated stem cell fate.

Skeletal stem cells (SSCs) reside within a three-dimensional extracellular matrix (ECM) compartment and differentiate into multiple cell lineages, thereby controlling tissue maintenance and regeneration. Within this environment, SSCs can proteolytically remodel the surrounding ECM in response to growth factors that direct lineage commitment via undefined mechanisms. Here, we report that Mmp14-dependent ECM remodeling coordinates canonical Wnt signaling and guides stem cell fate by triggering an integrin-activated reorganization of the SCC cytoskeleton that controls nuclear lamin A/C levels via the linker of nucleoskeleton and cytoskeleton (LINC) complexes. In turn, SSC lamin A/C levels dictate the localization of emerin, an inner nuclear membrane protein whose ability to regulate ß-catenin activity modulates Wnt signaling while directing lineage commitment in vitro and in vivo. These findings define a previously undescribed axis wherein SSCs use Mmp14-dependent ECM remodeling to control cytoskeletal and nucleoskeletal organization, thereby governing Wnt-dependent stem cell fate decisions.

Comparison of Needle, Ultrasonic, and Laser Irrigation for the Removal of Calcium Hydroxide from Mandibular Molar Root Canals.

Objective: This study aimed to compare the efficacy of conventional needle irrigation (CI), ultrasonically activated irrigation (UAI), photon-induced photoacoustic streaming (PIPS), and shock wave-enhanced emission photoacoustic streaming (SWEEPS) in removing calcium hydroxide [Ca(OH)2] from root canals of mandibular molars using microcomputed tomography. Background: Various adjunctive irrigation strategies have been recommended to improve the removal of Ca(OH)2. No reports have evaluated the SWEEPS laser-activated method for the removal of Ca(OH)2 from root canals of mandibular molars. Materials and methods: Forty mandibular molars were instrumented and filled with Ca(OH)2. Four irrigation groups (CI, UAI, PIPS, and SWEEPS) were established. The volume of root canals and Ca(OH)2 and the Ca(OH)2 volume reduction percentage (%Rd) were calculated. Data were analyzed by one-way analysis of variance and Kruskal-Wallis analysis of variance. Results: The residual Ca(OH)2 in the apical third was higher than that in the cervical and middle thirds in all groups (p < 0.05). Comparison of the %Rd of Ca(OH)2 in mesial canals revealed that PIPS and SWEEPS removed more Ca(OH)2 than CI and UAI in the cervical third (p < 0.05). The middle third of the mesial canals and the cervical and middle thirds of the distal canals did not show significant differences among groups (p > 0.05). Significant differences in the %Rd of Ca(OH)2 were noted between CI and other groups in the apical third of mesial and distal canals (p < 0.05). No group demonstrated complete removal of Ca(OH)2. Conclusions: UAI and laser-activated irrigation significantly improved Ca(OH)2 removal in the apical third of mesial and distal canals. No agitation technique could completely remove Ca(OH)2.

Augmented BMP signaling commits cranial neural crest cells to a chondrogenic fate by suppressing autophagic ß-catenin degradation.

Cranial neural crest cells (CNCCs) are a population of multipotent stem cells that give rise to craniofacial bone and cartilage during development. Bone morphogenetic protein (BMP) signaling and autophagy have been individually implicated in stem cell homeostasis. Mutations that cause constitutive activation of the BMP type I receptor ACVR1 cause the congenital disorder fibrodysplasia ossificans progressiva (FOP), which is characterized by ectopic cartilage and bone in connective tissues in the trunk and sometimes includes ectopic craniofacial bones. Here, we showed that enhanced BMP signaling through the constitutively activated ACVR1 (ca-ACVR1) in CNCCs in mice induced ectopic cartilage formation in the craniofacial region through an autophagy-dependent mechanism. Enhanced BMP signaling suppressed autophagy by activating mTORC1, thus blocking the autophagic degradation of ß-catenin, which, in turn, caused CNCCs to adopt a chondrogenic identity. Transient blockade of mTORC1, reactivation of autophagy, or suppression of Wnt-ß-catenin signaling reduced ectopic cartilages in ca-Acvr1 mutants. Our results suggest that BMP signaling and autophagy coordinately regulate ß-catenin activity to direct the fate of CNCCs during craniofacial development. These findings may also explain why some patients with FOP develop ectopic bones through endochondral ossification in craniofacial regions.

Osteoclast-mediated bone resorption is controlled by a compensatory network of secreted and membrane-tethered metalloproteinases.

Osteoclasts actively remodel both the mineral and proteinaceous components of bone during normal growth and development as well as pathologic states ranging from osteoporosis to bone metastasis. The cysteine proteinase cathepsin K confers osteoclasts with potent type I collagenolytic activity; however, cathepsin K-null mice, as well as cathepsin K-mutant humans, continue to remodel bone and degrade collagen by as-yet-undefined effectors. Here, we identify a cathepsin K-independent collagenolytic system in osteoclasts that is composed of a functionally redundant network of the secreted matrix metalloproteinase MMP9 and the membrane-anchored matrix metalloproteinase MMP14. Unexpectedly, whereas deleting either of the proteinases individually leaves bone resorption intact, dual targeting of Mmp9 and Mmp14 inhibited the resorptive activity of mouse osteoclasts in vitro and in vivo and human osteoclasts in vitro. In vivo, Mmp9/Mmp14 conditional double-knockout mice exhibited marked increases in bone density and displayed a highly protected status against either parathyroid hormone- or ovariectomy-induced pathologic bone loss. Together, these studies characterize a collagenolytic system operative in mouse and human osteoclasts and identify the MMP9/MMP14 axis as a potential target for therapeutic interventions for bone-wasting disease states.

Overexpression of Cyclophilin A in Human Periapical Lesions.

INTRODUCTION: Cyclophilin A (CypA) is a cytosolic protein involved in multiple biological functions, such as inflammation, tissue remodeling, tumorigenesis, and vascular diseases. Human periapical lesions are induced by bacterial infections. However, the expression of CypA in human periapical lesions remains unclear. This study aimed to investigate the presence of CypA in human periapical lesions and the possible association of CypA with angiogenesis, inflammatory cell infiltration, and alveolar bone degradation during inflammatory development. METHODS: Fifty-eight human periapical tissues, including periapical granulomas (PGs, n = 28), radicular cysts (RCs, n = 24), and healthy control tissues (control group, n = 6) were collected. Samples were fixed and analyzed. CypA expression was detected and analyzed by immunohistochemistry in different cross sections. Double immunofluorescence was assessed to colocalize CypA with CD34, CypA with matrix metalloproteinase 9 (MMP-9), and CD147 with MMP-9. RESULTS: CypA was significantly overexpressed in the RC and PG groups compared with the control group (P < .05), but the difference between the RC and PG groups was insignificant (P > .05). CypA-positive cells were mainly lymphocytes, endothelial cells, epithelial cells, and plasma cells. The double-labeling analysis of CypA with CD34 suggested that CypA expression was associated with angiogenesis during periapical lesions. MMP-9 colocalized with both CypA and CD147 indicated that CypA may colocalize with CD147 and may be associated with the degradation of soft and hard tissues around human periapical lesions. CONCLUSIONS: CypA may be involved in the development of periapical lesions with an increase in inflammatory cell infiltration, angiogenesis acceleration, and alveolar bone degradation.

Licorice isoliquiritigenin-encapsulated mesoporous silica nanoparticles for osteoclast inhibition and bone loss prevention.

Mesoporous silica nanoparticles (MSNs) are extensively used in bone tissue regeneration and local drug delivery. However, the effects of MSNs alone on osteoclast formation and function, as well as the utilization of MSNs to deliver natural molecules against bone resorption, remain unexplored. Here, we report the development of licorice-derived bioactive flavonoid isoliquiritigenin (ISL)-encapsulated MSNs (MSNs-ISL) as a potent bone-bioresponsive nanoencapsulation system for prevention of osteoclast-mediated bone loss in vitro and in vivo. Methods: We synthesized MSNs-ISL and then investigated the drug loading and release characteristics of the resulting nanoparticles. In vitro experiments on osteoclast differentiation and bone resorption were performed using mouse primary bone marrow-derived macrophages (BMMs). In vivo animal experiments were conducted using a lipopolysaccharide (LPS)-mediated calvarial bone erosion model. Results: The resulting MSNs-ISL were spherical and highly monodispersed; they possessed a large specific surface area and superior biocompatibility, and allowed acid-sensitive sustained drug release. Compared with free ISL and MSNs alone, MSNs-ISL significantly and additively inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast generation, decreased the size and quantity of sealing zones, and reduced the osteolytic capacity of osteoclasts in vitro. MSNs-ISL treatment also downregulated RANKL-stimulated mRNA expression of osteoclast-associated genes and transcription factors. Mechanistically, MSNs-ISL remarkably attenuated the RANKL-initiated expression of tumor necrosis factor receptor-associated factor 6 (TRAF6), phosphorylation of mitogen-activated protein kinases (MAPKs), and phosphorylation and degradation of inhibitor of κBα (IκBα), together with the nuclear translocation of nuclear factor-κB (NF-κB) p65 and the activator protein (AP)-1 component c-Fos. Moreover, MSNs-ISL almost completely restrained the expression of nuclear factor of activated T cells (NFATc1). Consistent with the in vitro results, MSNs-ISL could block osteoclast activity; relieve inflammation-related calvarial bone destruction in vivo; and suppress c-Fos, NFATc1, and cathepsin K expression levels. Conclusion: Licorice ISL-encapsulated MSNs exhibit notable anti-osteoclastogenetic effects and protect against inflammatory bone destruction. Our findings reveal the feasibility of applying MSNs-ISL as an effective natural product-based bone-bioresponsive nanoencapsulation system to prevent osteoclast-mediated bone loss.

Anti-osteoclastogenic effect of epigallocatechin gallate-functionalized gold nanoparticles in vitro and in vivo.

Background: Epigallocatechin gallate (EGCG), the major anti-inflammatory compound in green tea, has been shown to suppress osteoclast (OC) differentiation. However, the low aqueous solubility of EGCG always leads to poor bioavailability, adverse effects, and several drawbacks for clinical applications. Purpose: In this study, we synthesized EGCG-capped gold nanoparticles (EGCG-GNPs) to solve the drawbacks for clinical uses of EGCG in bone destruction disorders by direct reduction of HAuCl4 in EGCG aqueous solution. Methods and Results: The obtained EGCG-GNPs were negatively charged and spherical. Theoretical calculation results suggested that EGCG was released from GNPs in an acidic environment. Cellular uptake study showed an obviously large amount of intracellular EGCG-GNPs without cytotoxicity. EGCG-GNPs exhibited better effects in reducing intracellular reactive oxygen species levels than free EGCG. A more dramatic anti-osteoclastogenic effect induced by EGCG-GNPs than free EGCG was observed in lipopolysaccharide (LPS)-stimulated bone marrow macrophages, including decreased formation of TRAP-positive multinuclear cells and actin rings. Meanwhile, EGCG-GNPs not only suppressed the mRNA expression of genetic markers of OC differentiation but also inhibited MAPK signaling pathways. Furthermore, we confirmed that EGCG-GNPs greatly reversed bone resorption in the LPS-induced calvarial bone erosion model in vivo, which was more effective than applying free EGCG, specifically in inhibiting the number of OCs, improving bone density, and preventing bone loss. Conclusion: EGCG-GNPs showed better anti-osteoclastogenic effect than free EGCG in vitro and in vivo, indicating their potential in anti-bone resorption treatment strategy.

Positive Correlation between Activated CypA/CD147 Signaling and MMP-9 Expression in Mice Inflammatory Periapical Lesion.

AIM: Cyclophilin A (CypA)/CD147 signaling plays critical roles in the regulation of inflammation and bone metabolism. This study aimed to investigate the participation of CypA/CD147 in mice periapical lesions progression and its relationship with bone resorption. METHODOLOGY: Periapical lesions were induced by pulp exposure in the first lower molars of 40 C57BL/6J mice. The mice were sacrificed on days 0, 7, 14, 21, 28, 35, 42, and 49. Mandibles were harvested for X-ray imaging, microcomputed tomography scanning, histologic observation, immunohistochemistry, enzyme histochemistry, and double immunofluorescence analysis. Western blot was employed to further detect the related molecular signaling pathways in LPS-stimulated RAW 264.7 cells treated with CypA inhibitor. RESULTS: The volume and area of the periapical lesions increased from day 0 to day 35 and remained comparably stable until day 49. Immunohistochemistry demonstrated that the CypA expression levels also increased from day 0 to day 35 and decreased until day 49, similar to CD147 expression (R 2 = 0.4423, P < 0.05), osteoclast number (R 2 = 0.5101, P < 0.01), and the expression of osteoclastogenesis-related matrix metalloproteinase 9 (MMP-9) (R 2 = 0.4715, P < 0.05). Serial sections further confirmed the colocalization of CypA and CD147 on osteoclasts with immunohistochemistry. And the distribution of CypA-positive or CD147-positive cells was positively correlated with the dynamics of MMP-9-positive cells by using immunofluorescence analysis. Furthermore, CD147 and MMP-9 expression in RAW 264.7 cells were both downregulated with CypA inhibitor treatment (P < 0.05). CONCLUSIONS: The present study reveals the positive correlation of CypA/CD147 signaling and osteoclast-related MMP-9 expression in mice inflammatory periapical lesions progression. Therefore, intervention of CypA/CD147 signaling could probably provide a potential therapeutic target for attenuating inflammatory bone resorption.

The Expression of Interferon Regulatory Factor 8 in Human Periapical Lesions.

INTRODUCTION: Interferon regulatory factor 8 (IRF8) is a critical transcription factor in innate immune responses that regulates the development and function of myeloid cells. Human periapical lesions are caused by endodontic microbial infections. However, the presence of IRF8 in human periapical lesions remains elusive. This study aims to explore the expression of IRF8 in human periapical lesions and the possible association of IRF8 with macrophages, nuclear factor kappa B (NF-κB) signaling, and the autophagy process. METHODS: Thirty-nine human periapical tissues, including healthy control tissues (n = 15), radicular cysts (RCs, n = 11), and periapical granulomas (PG, n = 13), were examined. Tissues were fixed in paraformaldehyde and analyzed. The inflammatory infiltrates of lesions were evaluated by hematoxylin-eosin, and the expression of IRF8 was analyzed by immunohistochemistry. Double immunofluorescence assessment was performed to colocalize IRF8 with CD68, NF-κB p65, and LC3B. RESULTS: The expression of IRF8 was significantly higher in RCs and PGs than in the healthy control group, but no significant difference was found between RCs and PGs. There were significantly more IRF8-CD68 double-positive cells in RCs and PGs than in the healthy control group, but no significant difference was observed between RCs and PGs. Double-labeling analysis of IRF8 with NF-κB and LC3B indicated that IRF8 expression is associated with NF-κB signaling and the autophagy process during periapical lesions. CONCLUSIONS: IRF8 could be observed and might possibly be involved in macrophages in the development of periapical lesions.

SPHK1-S1PR1-RANKL Axis Regulates the Interactions Between Macrophages and BMSCs in Inflammatory Bone Loss.

Accumulating evidence indicates that the immune and skeletal systems interact with each other through various regulators during the osteoclastogenic process. Among these regulators, the bioactive lipid sphingosine-1-phosphate (S1P), which is synthesized by sphingosine kinase 1/2 (SPHK1/2), has recently been recognized to play a role in immunity and bone remodeling through its receptor sphingosine-1-phosphate receptor 1 (S1PR1). However, little is known regarding the potential role of S1PR1 signaling in inflammatory bone loss. We observed that SPHK1 and S1PR1 were upregulated in human apical periodontitis, accompanied by macrophage infiltration and enhanced expression of receptor activator of NF-κB ligand (RANKL, an indispensable factor in osteoclastogenesis and bone resorption) and increased numbers of S1PR1-RANKL double-positive cells in lesion tissues. Using an in vitro co-culture model of macrophages and bone marrow stromal cells (BMSCs), it was revealed that in the presence of lipopolysaccharide (LPS) stimulation, macrophages could significantly induce SPHK1 activity, which resulted in activated S1PR1 in BMSCs. The activated S1P-S1PR1 signaling was responsible for the increased RANKL production in BMSCs, as S1PR1-blockage abolished this effect. Applying a potent S1P-S1PR1 signaling modulator, Fingolimod (FTY720), in a Wistar rat apical periodontitis model effectively prevented bone lesions in vivo via downregulation of RANKL production, osteoclastogenesis, and bone resorption. Our data unveiled the regulatory role of SPHK1-S1PR1-RANKL axis in inflammatory bone lesions and proposed a potential therapeutic intervention by targeting this cell-signaling pathway to prevent bone loss. © 2018 American Society for Bone and Mineral Research.

Anti-osteoclastogenesis of Mineral Trioxide Aggregate through Inhibition of the Autophagic Pathway.

INTRODUCTION: Mineral trioxide aggregate (MTA) regulates bone remodeling, particularly osteoclast differentiation. However, intracellular mechanisms underlying the anti-osteoclastogenesis of MTA remain unclear. This study aimed to evaluate the potential alterations of autophagic pathway during anti-osteoclastogenic effects by MTA in vitro and investigate their underlying mechanisms. METHODS: Osteoclast precursors were treated with MTA extracts containing the receptor activator of nuclear factor-kappa B ligand (RANKL). Rapamycin was used to activate autophagy. RANKL-induced osteoclast differentiation was stained with tartrate-resistant acid phosphatase. Several specific autophagy features in osteoclast precursors were measured by using immunofluorescence, monodansylcadaverine, and transmission electron microscope. Autophagy-related proteins were investigated via Western blot analysis. The mRNA expression involved in autophagic and osteoclastic activities was detected with quantitative real-time polymerase chain reaction. RESULTS: MTA extracts inhibited osteoclast differentiation via preventing the fusion of osteoclast precursors without cytotoxicity. MTA extracts interrupted RANKL-induced acidic vesicular organelle formation and autophagic vacuole appearance in osteoclast precursors. Moreover, autophagic genes and proteins stimulated with RANKL diminished with MTA extracts. Notably, autophagy activation through rapamycin promoted multinucleated osteoclasts formation and increased osteoclastic genes expression, which almost reversed MTA-mediated anti-osteoclastogenic effects. CONCLUSIONS: MTA inhibited osteoclastogenesis for bone repair through attenuating the autophagic pathway.

From the Cover: Activation of NF-κB-Autophagy Axis by 2-Hydroxyethyl Methacrylate Commits Dental Mesenchymal Cells to Apoptosis.

2-hydroxyethyl methacrylate (HEMA) is the major resin monomer that is released from incomplete polymerized dental restorative and adhesive biomaterials during dental therapy. Autophagy and apoptosis are biologically connected and the relationship between autophagy and apoptosis is complex under various circumstances. This study aimed to determine whether autophagy is activated by HEMA and further explore the function of autophagy during the HEMA-induced apoptosis of dental mesenchymal cells (DMCs). We exposed DMCs to different concentrations of HEMA. Cell viability showed a time- and concentration-dependent decrease when exposed to HEMA. We showed that HEMA exposure increased autophagic vacuoles and the expression of autophagic biomarkers (Beclin1, Atg5 and LC3). Pre-incubated with autophagy inhibitors (3-methyladenine and chloroquine) significantly prevented HEMA-induced apoptosis. Interestingly, HEMA initiated nuclear factor-κB (NF-κB) expression and nuclear translocation, whereas the NF-κB inhibitor (Bay 11-7082) markedly suppressed HEMA-induced autophagic activation and apoptosis. As is consistent with the in vitro results, HEMA treatment resulted in dental pulp tissue toxicity and activation of typical autophagic vacuoles in the tooth slice organ culture model ex vivo. In summary, we demonstrated that NF-κB signaling functioned upstream of HEMA-inducecd autophagy in DMCs and that the activation of NF-κB-autophagy axis was responsible for HEMA-induced apoptosis. Our findings provide novel insights into the mechanisms of resin monomer-mediated dental pulp damage during dental treatment, highlighting the activation of NF-κB-autophagy axis as an important mechanism of HEMA-mediated apoptosis.