MAST4 regulates stem cell maintenance with DLX3 for epithelial development and amelogenesis.

The asymmetric division of stem cells permits the maintenance of the cell population and differentiation for harmonious progress. Developing mouse incisors allows inspection of the role of the stem cell niche to provide specific insights into essential developmental phases. Microtubule-associated serine/threonine kinase family member 4 (Mast4) knockout (KO) mice showed abnormal incisor development with low hardness, as the size of the apical bud was decreased and preameloblasts were shifted to the apical side, resulting in amelogenesis imperfecta. In addition, Mast4 KO incisors showed abnormal enamel maturation, and stem cell maintenance was inhibited as amelogenesis was accelerated with Wnt signal downregulation. Distal-Less Homeobox 3 (DLX3), a critical factor in tooth amelogenesis, is considered to be responsible for the development of amelogenesis imperfecta in humans. MAST4 directly binds to DLX3 and induces phosphorylation at three residues within the nuclear localization site (NLS) that promotes the nuclear translocation of DLX3. MAST4-mediated phosphorylation of DLX3 ultimately controls the transcription of DLX3 target genes, which are carbonic anhydrase and ion transporter genes involved in the pH regulation process during ameloblast maturation. Taken together, our data reveal a novel role for MAST4 as a critical regulator of the entire amelogenesis process through its control of Wnt signaling and DLX3 transcriptional activity.

Cyclic stretch induced epigenetic activation of periodontal ligament cells.

Periodontal ligament (PDL) cells play a crucial role in maintaining periodontal integrity and function by providing cell sources for ligament regeneration. While biophysical stimulation is known to regulate cell behaviors and functions, its impact on epigenetics of PDL cells has not yet been elucidated. Here, we aimed to investigate the cytoskeletal changes, epigenetic modifications, and lineage commitment of PDL cells following the application of stretch stimuli to PDL. PDL cells were subjected to stretching (0.1 Hz, 10 %). Subsequently, changes in focal adhesion, tubulin, and histone modification were observed. The survival ability in inflammatory conditions was also evaluated. Furthermore, using a rat hypo-occlusion model, we verified whether these phenomena are observed in vivo. Stretched PDL cells showed maximal histone 3 acetylation (H3Ace) at 2 h, aligning perpendicularly to the stretch direction. RNA sequencing revealed stretching altered gene sets related to mechanotransduction, histone modification, reactive oxygen species (ROS) metabolism, and differentiation. We further found that anchorage, cell elongation, and actin/microtubule acetylation were highly upregulated with mechanosensitive chromatin remodelers such as H3Ace and histone H3 trimethyl lysine 9 (H3K9me3) adopting euchromatin status. Inhibitor studies showed mechanotransduction-mediated chromatin modification alters PDL cells behaviors. Stretched PDL cells displayed enhanced survival against bacterial toxin (C12-HSL) or ROS (H2O2) attack. Furthermore, cyclic stretch priming enhanced the osteoclast and osteoblast differentiation potential of PDL cells, as evidenced by upregulation of lineage-specific genes. In vivo, PDL cells from normally loaded teeth displayed an elongated morphology and higher levels of H3Ace compared to PDL cells with hypo-occlusion, where mechanical stimulus is removed. Overall, these data strongly link external physical forces to subsequent mechanotransduction and epigenetic changes, impacting gene expression and multiple cellular behaviors, providing important implications in cell biology and tissue regeneration.

Macrocyclic Conformational Switch Coupled with Pyridinium-Induced PET for Fluorescence Detection of Adenosine Triphosphate.

The binding of nucleotides is crucial for signal transduction as it induces conformational protein changes, leading to downstream cellular responses. Synthetic receptors that bind nucleotides and transduce the binding event into global conformational rearrangements are highly challenging to design, especially those that operate in an aqueous solution. Much work is focused on evaluating functionalized dyes to detect nucleotides, whereas coupling of a nucleotide-induced conformational switching to a sensing event has not been reported to date. We disclose synthetic receptors that undergo a global conformational rearrangement upon nucleotide binding. Integrating naphthalimide and the pyridinium ion into the structure enables stabilization of the folded conformation and efficient fluorescence quenching. The binding of a nucleotide rearranges the receptor conformation and alters the strong fluorescence enhancement. The methylpyridinium-containing receptor demonstrated high sensing selectivity for adenosine 5'-triphosphate (ATP) and a record 160-fold fluorescence enhancement. It can detect fluctuations of ATP in HeLa cells and possesses low cytotoxicity. The developed systems present an attractive approach for designing ATP-responsive artificial molecular switches that operate in water and integrate a strong fluorescence response.

Mitigation of BMP-induced inflammation in craniofacial bone regeneration and improvement of bone parameters by dietary hesperidin.

Based on anti-inflammatory and osteogenic properties of hesperidin (HE), we hypothesized its systemic administration could be a cost-effective method of improving BMP-induced bone regeneration. Sprague-Dawley rats were allocated into 4 groups (n = 10/group): a 5-mm critical-sized mandible defect + collagen scaffold or, scaffold + 1 µg of BMP2 with and without dietary HE at 100 mg/kg. HE was administered by oral gavage 4 weeks prior to surgeries until euthanasia at day 7 or 14 post-surgery. The healing tissue within the defect collected at day 7 was subjected to gene expression analysis. Mandibles harvested at day 14 were subjected to microcomputed tomography and histology. HE + BMP2-treated rats had a statistically significant decrease in expression of inflammatory genes compared to BMP2 alone. The high-dose BMP2 alone caused cystic-like regeneration with incomplete defect closure. HE + BMP2 showed virtually complete bone fusion. Collagen fibril birefringence pattern (red color) under polarized light indicated high organization in BMP2-induced newly formed bone (NFB) in HE-supplemented group (p < 0.05). Clear changes in osteocyte lacunae as well as a statistically significant increase in osteoclasts were found around NFB in HE-treated rats. A significant increase in trabecular volume and thickness, and trabecular and cortical density was found in femurs of HE-supplemented rats (p < 0.05). Our findings show, for the first time, that dietary HE has a remarkable modulatory role in the function of locally delivered high-dose BMP2 in bone regeneration possibly via control of inflammation, osteogenesis, changes in osteocyte and osteoclast function and collagen maturation in regenerated and native bone. In conclusion, HE had a significant skeletal bone sparing effect and the ability to provide a more effective BMP-induced craniofacial regeneration.

Surface Engineering of Natural Killer Cells with CD44-targeting Ligands for Augmented Cancer Immunotherapy.

Adoptive immunotherapy utilizing natural killer (NK) cells has demonstrated remarkable efficacy in treating hematologic malignancies. However, its clinical intervention for solid tumors is hindered by the limited expression of tumor-specific antigens. Herein, lipid-PEG conjugated hyaluronic acid (HA) materials (HA-PEG-Lipid) for the simple ex-vivo surface coating of NK cells is developed for 1) lipid-mediated cellular membrane anchoring via hydrophobic interaction and thereby 2) sufficient presentation of the CD44 ligand (i.e., HA) onto NK cells for cancer targeting, without the need for genetic manipulation. Membrane-engineered NK cells can selectively recognize CD44-overexpressing cancer cells through HA-CD44 affinity and subsequently induce in situ activation of NK cells for cancer elimination. Therefore, the surface-engineered NK cells using HA-PEG-Lipid (HANK cells) establish an immune synapse with CD44-overexpressing MIA PaCa-2 pancreatic cancer cells, triggering the "recognition-activation" mechanism, and ultimately eliminating cancer cells. Moreover, in mouse xenograft tumor models, administrated HANK cells demonstrate significant infiltration into solid tumors, resulting in tumor apoptosis/necrosis and effective suppression of tumor progression and metastasis, as compared to NK cells and gemcitabine. Taken together, the HA-PEG-Lipid biomaterials expedite the treatment of solid tumors by facilitating a sequential recognition-activation mechanism of surface-engineered HANK cells, suggesting a promising approach for NK cell-mediated immunotherapy.

Anti-inflammatory activity of calmodulin-lysine N-methyltransferase through suppressing the caspase-11 non-canonical inflammasome.

Calmodulin (CaM)-lysine N-methyltransferase (CAMKMT) is a novel methyltransferase that catalyzes lysine trimethylation in CaM. However, its specific roles in inflammatory responses and diseases remain unclear. In this study, we investigated the effects of CAMKMT on caspase-11 non-canonical inflammasomes. CAMKMT expression levels were significantly decreased during inflammatory responses activated by caspase-11 non-canonical inflammasome in macrophages. Moreover, CaM lysine trimethylation was markedly inhibited, but no change was observed in CaM expression during these inflammatory responses in macrophages. Activation of the CaM downstream effectors, CaM-dependent proteinkinase kinase 2 and CaM-dependent proteinkinase type IV, was also inhibited during inflammatory responses activated by caspase-11 non-canonical inflammasome in macrophages. Notably, forced expression of CAMKMT restrained caspase-11 non-canonical inflammasome activation via inhibiting proteolytic activation of caspase-11 and gasdermin D (GSDMD), which in turn suppressed pyroptosis and the release of interleukin (IL)-1ß and IL-18 in macrophages. Finally, an in vivo study revealed that CAMKMT ameliorated lipopolysaccharide (LPS)-stimulated acute lethal sepsis in mice by increasing the survival rate and reducing the serum levels of IL-1 ß. These findings suggest CAMKMT as a novel methyltransferase that plays an anti-inflammatory role through restraining caspase-11 non-canonical inflammasome in macrophages.

Mitigation of BMP-induced Inflammation in Craniofacial Bone Regeneration and Improvement of Bone Parameters by Dietary Hesperidin.

Based on anti-inflammatory and osteogenic properties of hesperidin (HE), we hypothesized its systemic administration could be a cost-effective method of improving BMP-induced bone regeneration. Sprague-Dawley rats were allocated into 4 groups (n=10/group): a 5-mm critical-sized mandible defect + collagen scaffold or, scaffold + 1 µg of BMP2 with and without dietary HE at 100 mg/kg. HE was administered by oral gavage 4 weeks prior to surgeries until euthanasia at day 7 or 14. The healing tissue within the defect collected at day 7 was subjected to gene expression analysis. Mandibles harvested at day 14 were subjected to microcomputed tomography and histology. HE+BMP2-treated rats had a statistically significant decrease in expression of inflammatory genes compared to BMP2 alone. The high-dose BMP2 caused cystic-like regeneration with incomplete defect closure. HE+BMP2 showed virtually complete bone fusion. Red collagen fibrils were significantly higher in BMP2-induced newly formed bone (NFB) in HE-supplemented group (p<0.05) indicating high organization. Clear changes in osteocyte lacunae as well as a statistically significant increase in osteoclasts were found around NFB in HE rats. A significant increase in trabecular volume and thickness, and trabecular and cortical density was found in femurs of HE-supplemented rats (p<0.05). Our findings show, for the first time, that dietary HE has a remarkable modulatory role in locally delivered high-dose BMP2-induced bone possibly via control of inflammation, osteogenesis, changes in osteocyte and osteoclast function and collagen maturation in regenerated and native bone. In conclusion, HE has a significant skeletal bone sparing effect and the ability to provide a more effective BMP-induced craniofacial regeneration.

Harnessing the dental cells derived from human induced pluripotent stem cells for hard tissue engineering.

INTRODUCTION: Most mineralized tissues in our body are present in bones and teeth. Human induced pluripotent stem cells (hiPSCs) are promising candidates for cell therapy to help regenerate bone defects and teeth loss. The extracellular matrix (ECM) is a non-cellular structure secreted by cells. Studies on the dynamic microenvironment of ECM are necessary for stem cell-based therapies. OBJECTIVES: We aim to optimize an effective protocol for hiPSC differentiation into dental cells without utilizing animal-derived factors or cell feeders that can be applied to humans and to mineralize differentiated dental cells into hard tissues. METHODS: For the differentiation of both dental epithelial cells (DECs) and dental mesenchymal cells (DMCs) from hiPSCs, an embryoid body (EB) was formed from hiPSCs. hiPSC were differentiated into neural crest cells with an induction medium utilized in our previous study, and hiPSC-derived DECs were differentiated with a BMP-modulated customized medium. hiPSC-dental cells were then characterized, analyzed, and validated with transcriptomic analysis, western blotting, and RT-qPCR. To form mineralized tissues, hiPSC-derived DECs were recombined with hiPSC-derived DMCs encapsulated in various biomaterials, including gelatin methacryloyl (GelMA), collagen, and agar matrix. RESULTS: These hiPSC-derived dental cells are highly osteogenic and chondro-osteogenic in photocrosslinkable GelMA hydrogel and collagen type I microenvironments. Furthermore, hiPSC-derived dental cells in agar gel matrix induced the formation of a bioengineered tooth. CONCLUSION: Our study provides an approach for applying hiPSCs for hard tissue regeneration, including tooth and bone. This study has immense potential to provide a novel technology for bioengineering organs for various regenerative therapies.

Enhanced Bone Formation by Rapidly Formed Bony Wall over the Bone Defect Using Dual Growth Factors.

BACKGROUND: In guided bone regeneration (GBR), there are various problems that occur in the bone defect after the wound healing period. This study aimed to investigate the enhancement of the osteogenic ability of the dual scaffold complex and identify the appropriate concentration of growth factors (GF) for new bone formation based on the novel GBR concept that is applying rapid bone forming GFs to the membrane outside of the bone defect. METHODS: Four bone defects with a diameter of 8 mm were formed in the calvaria of New Zealand white rabbits each to perform GBR. Collagen membrane and biphasic calcium phosphate (BCP) were applied to the bone defects with the four different concetration of BMP-2 or FGF-2. After 2, 4, and 8 weeks of healing, histological, histomorphometric, and immunohistochemical analyses were conducted. RESULTS: In the histological analysis, continuous forms of new bones were observed in the upper part of bone defect in the experimental groups, whereas no continuous forms were observed in the control group. In the histomorphometry, The group to which BMP-2 0.5 mg/ml and FGF-2 1.0 mg/ml was applied showed statistically significantly higher new bone formation. Also, the new bone formation according to the healing period was statistically significantly higher at 8 weeks than at 2, 4 weeks. CONCLUSION: The novel GBR method in which BMP-2, newly proposed in this study, is applied to the membrane is effective for bone regeneration. In addition, the dual scaffold complex is quantitatively and qualitatively advantageous for bone regeneration and bone maintenance over time.

Imidazole-carbazole conjugate for two-photon-excited photodynamic therapy and fluorescence bioimaging.

A heavy-atom-free photosensitizer (CI) based on an imidazole-carbazole conjugate exhibited strong fluorescence emission and ROS generation via both type I and II mechanisms. In particular, CI showed efficient photodynamic therapy and fluorescence bioimaging under two-photon (TP) excitation (740 nm) toward HeLa cells with negligible dark toxicity.

MAST4 controls cell cycle in spermatogonial stem cells.

Spermatogonial stem cell (SSC) self-renewal is regulated by reciprocal interactions between Sertoli cells and SSCs in the testis. In a previous study, microtubule-associated serine/threonine kinase 4 (MAST4) has been studied in Sertoli cells as a regulator of SSC self-renewal. The present study focused on the mechanism by which MAST4 in Sertoli cells transmits the signal and regulates SSCs, especially cell cycle regulation. The expression of PLZF, CDK2 and PLZF target genes was examined in WT and Mast4 KO testes by Immunohistochemistry, RT-qPCR and western blot. In addition, IdU and BrdU were injected into WT and Mast4 KO mice and cell cycle of SSCs was analysed. Finally, the testis tissues were cultured in vitro to examine the regulation of cell cycle by MAST4 pathway. Mast4 KO mice showed infertility with Sertoli cell-only syndrome and reduced sperm count. Furthermore, Mast4 deletion led to decreased PLZF expression and cell cycle progression in the testes. MAST4 also induced cyclin-dependent kinase 2 (CDK2) to phosphorylate PLZF and activated PLZF suppressed the transcriptional levels of genes related to cell cycle arrest, leading SSCs to remain stem cell state. MAST4 is essential for maintaining cell cycle in SSCs via the CDK2-PLZF interaction. These results demonstrate the pivotal role of MAST4 regulating cell cycle of SSCs and the significance of spermatogenesis.

Epithelial plasticity enhances regeneration of committed taste receptor cells following nerve injury.

Taste receptor cells are taste bud epithelial cells that are dependent upon the innervating nerve for continuous renewal and are maintained by resident tissue stem/progenitor cells. Transection of the innervating nerve causes degeneration of taste buds and taste receptor cells. However, a subset of the taste receptor cells is maintained without nerve contact after glossopharyngeal nerve transection in the circumvallate papilla in adult mice. Here, we revealed that injury caused by glossopharyngeal nerve transection triggers the remaining differentiated K8-positive taste receptor cells to dedifferentiate and acquire transient progenitor cell-like states during regeneration. Dedifferentiated taste receptor cells proliferate, express progenitor cell markers (K14, Sox2, PCNA) and form organoids in vitro. These data indicate that differentiated taste receptor cells can enter the cell cycle, acquire stemness, and participate in taste bud regeneration. We propose that dedifferentiated taste receptor cells in combination with stem/progenitor cells enhance the regeneration of taste buds following nerve injury.

Advances in small molecule two-photon fluorescent trackers for lipid droplets in live sample imaging.

Two-photon fluorescent trackers for monitoring of lipid droplets (LDs) would be highly effective for illustrating the critical roles of LDs in live cells or tissues. Although a number of one-photon fluorescent trackers for labeling LDs have been developed, their usability remains constrained in live sample imaging due to photo damage, shallow imaging depth, and auto-fluorescence. Recently, some two-photon fluorescent trackers for LDs have been developed to overcome these limitations. In this mini-review article, the advances in two-photon fluorescent trackers for monitoring of LDs are summarized. We summarize the chemical structures, two-photon properties, live sample imaging, and biomedical applications of the most recent representative two-photon fluorescent trackers for LDs. Additionally, the current challenges and future research trends for the two-photon fluorescent trackers of LDs are discussed.

Transcriptomic Comparison Analysis between Ameloblastoma and AM-1 Cell Line.

Cancer initiation and progression are profoundly along with the crosstalk between cancer cells and the surrounding stroma. Accumulating evidence has shown that the therapy targeting the extracellular matrix (ECM) would regress tumor growth and invasion in the most common carcinomas. However, it remains largely unexplored in several rare tumors like odontogenic tumors. Ameloblastoma (AM) is the representative odontogenic epithelial tumor in the jawbone, and it usually infiltrates into adjacent bone marrow and has unlimited growth capacity and a high potential for recurrence. This study aims to investigate the role of collagen-rich ECM during the invasion of AM. Transcriptomic analysis revealed that ECM- and epithelial-to-mesenchymal transition (EMT)-related genes were up-regulated in AM compared to ameloblastoma cell line, AM-1. Tumoroid forming analysis showed that Collagen-rich ECM is indispensable for AM progression, especially for aggressive growth patterns and collective invasion.

Unraveled roles of Cav1.2 in proliferation and stemness of ameloblastoma.

BACKGROUND: Transcriptome analysis has been known as a functional tool for cancer research recently. Mounting evidence indicated that calcium signaling plays several key roles in cancer progression. Despite numerous studies examining calcium signaling in cancer, calcium signaling studies in ameloblastoma are limited. RESULTS: In the present study, comparative transcriptome profiling of two representative odontogenic lesions, ameloblastoma and odontogenic keratocyst, revealed that Cav1.2 (CACNA1C, an L-type voltage-gated calcium channel) is strongly enriched in ameloblastoma. It was confirmed that the Ca2+ influx in ameloblastoma cells is mainly mediated by Cav1.2 through L-type voltage-gated calcium channel agonist and blocking reagent treatment. Overexpression and knockdown of Cav1.2 showed that Cav1.2 is directly involved in the regulation of the nuclear translocation of nuclear factor of activated T cell 1 (NFATc1), which causes cell proliferation. Furthermore, a tumoroid study indicated that Cav1.2-dependent Ca2+ entry is also associated with the maintenance of stemness of ameloblastoma cells via the enhancement of Wnt/ß-catenin signaling activity. CONCLUSION: In conclusion, Cav1.2 regulates the NFATc1 nuclear translocation to enhance ameloblastoma cell proliferation. Furthermore, Cav1.2 dependent Ca2+ influx contributes to the Wnt/ß-catenin activity for the ameloblastoma cell stemness and tumorigenicity. Our fundamental findings could have a major impact in the fields of oral maxillofacial surgery, and genetic manipulation or pharmacological approaches to Cav1.2 can be considered as new therapeutic options.

Mast4 determines the cell fate of MSCs for bone and cartilage development.

Mesenchymal stromal cells (MSCs) differentiation into different lineages is precisely controlled by signaling pathways. Given that protein kinases play a crucial role in signal transduction, here we show that Microtubule Associated Serine/Threonine Kinase Family Member 4 (Mast4) serves as an important mediator of TGF-ß and Wnt signal transduction in regulating chondro-osteogenic differentiation of MSCs. Suppression of Mast4 by TGF-ß1 led to increased Sox9 stability by blocking Mast4-induced Sox9 serine 494 phosphorylation and subsequent proteasomal degradation, ultimately enhancing chondrogenesis of MSCs. On the other hand, Mast4 protein, which stability was enhanced by Wnt-mediated inhibition of GSK-3ß and subsequent Smurf1 recruitment, promoted ß-catenin nuclear localization and Runx2 activity, increasing osteogenesis of MSCs. Consistently, Mast4-/- mice demonstrated excessive cartilage synthesis, while exhibiting osteoporotic phenotype. Interestingly, Mast4 depletion in MSCs facilitated cartilage formation and regeneration in vivo. Altogether, our findings uncover essential roles of Mast4 in determining the fate of MSC development into cartilage or bone.

Inhibition of L-type voltage-gated calcium channel-mediated Ca2+ influx suppresses the collective migration and invasion of ameloblastoma.

OBJECTIVES: Ameloblastoma (AM) has been known as a benign but locally invasive tumour with high recurrence rates. Invasive behaviour of the AM results in destruction of the adjacent jawbone and the non-detectable remnants during surgery, interrupting the complete elimination of cancer cells. METHODS: To explore novel targets for the tumour cell invasion, a transcriptomic analysis between AM and odontogenic keratocyst were performed through next-generation sequencing in detail. RESULTS: Enrichment of CACNA1C gene (encoding Cav1.2) in AM, a subunit of the L-type voltage-gated calcium channel (VGCC) was observed for the first time. The expression and channel activity of Cav1.2 was confirmed by immunostaining and calcium imaging in the patient samples or primary cells. Verapamil, L-type VGCC blocker revealed suppression of the Ca2+ -induced cell aggregation and collective invasion of AM cells in vitro. Furthermore, the effect of verapamil in suppressing AM invasion into the adjacent bone was confirmed through orthotopic xenograft model specifically. CONCLUSION: Taken together, Cav1.2 maybe considered to be a therapeutic candidate to decrease the collective migration and invasion of AM.

Ameloblastoma modifies tumor microenvironment for enhancing invasiveness by altering collagen alignment.

Tumor progression is profoundly affected by crosstalk between cancer cells and their stroma. In the past decades, the development of bioinformatics and the establishment of organoid model systems have allowed extensive investigation of the relationship between tumor cells and the tumor microenvironment (TME). However, the interaction between tumor cells and the extracellular matrix (ECM) in odontogenic epithelial neoplasms and the ECM remodeling mechanism remain unclear. In the present study, transcriptomic comparison and histopathologic analysis revealed that TME-related genes were upregulated in ameloblastoma compared to in odontogenic keratocysts. Tumoroid analysis indicated that type I collagen is required for ameloblastoma progression. Furthermore, ameloblastoma shows the capacity to remodel the ECM independently of cancer-associated fibroblasts. In conclusion, ameloblastoma-mediated ECM remodeling contributes to the formation of an invasive collagen architecture during tumor progression.

Effect of the Number of Micro-Osteoperforations on the Rate of Tooth Movement and Periodontal Response in Mice.

Accelerated tooth movement can be achieved using micro-osteoperforations (MOPs) to stimulate regeneration of the alveolar bone during minimally invasive surgical trauma. However, there is currently no standardized protocol and limited reports regarding the side effects of MOPs based on biological evidence. This study sought to evaluate the biological effects of the number of MOPs on orthodontic tooth movement (OTM) and the potential risk for root resorption. Male CD1 mice were divided into 4 groups based on the number of MOPs, as follows: Sham; 0MOP+OTM; 2MOP+OTM; and 4MOP+OTM groups. Tooth movement distance and the number of osteoclasts were higher whereas bone volume and trabecular number were lower in the 4MOP+OTM group compared to those of the 0MOP+OTM group. Immunofluorescent assay analysis indicated that the 4MOP+OTM group was positively associated with rapid cementum regeneration and periodontal ligament tissue formation. Our findings revealed that the MOP procedure affected tooth movement and did not significantly contribute to root resorption, whereas it may promote constitutive activation of cementogenesis.