Spatiotemporal organization of enteroendocrine peptide expression in Drosophila.

The digestion of food and absorption of nutrients occurs in the gut. The nutritional value of food and its nutrients is detected by enteroendocrine cells, and peptide hormones produced by the enteroendocrine cells are thought to be involved in metabolic homeostasis, but the specific mechanisms are still elusive. The enteroendocrine cells are scattered over the entire gastrointestinal tract and can be classified according to the hormones they produce. We followed the changes in combinatorial expression of regulatory peptides in the enteroendocrine cells during metamorphosis from the larva to the adult fruit fly, and re-confirmed the diverse composition of enteroendocrine cell populations. Drosophila enteroendocrine cells appear to differentially regulate peptide expression spatially and temporally depending on midgut region and developmental stage. In the late pupa, Notch activity is known to determine which peptides are expressed in mature enteroendocrine cells of the posterior midgut, and we found that the loss of Notch activity in the anterior midgut results in classes of enteroendocrine cells distinct from the posterior midgut. These results suggest that enteroendocrine cells that populate the fly midgut can differentiate into distinct subtypes that express different combinations of peptides, which likely leads to functional variety depending on specific needs.

Identification and characterization of GAL4 drivers that mark distinct cell types and regions in the Drosophila adult gut.

The gastrointestinal tract in the adult Drosophila serves as a model system for exploring the mechanisms underlying digestion, absorption and excretion, stem cell plasticity, and inter-organ communication, particularly through the gut-brain axis. It is also useful for studying the cellular and adaptive responses to dietary changes, alterations in microbiota and immunity, and systematic and endocrine signals. Despite the various cell types and distinct regions in the gastrointestinal tract, few tools are available to target and manipulate the activity of each cell type and region, and their gene expression. Here, we report 353 GAL4 lines and several split-GAL4 lines that are expressed in enteric neurons (ENs), progenitors (ISCs and EBs), enterocytes (ECs), enteroendocrine cells (EEs), or/and other cell types that are yet to be identified in distinct regions of the gut. We had initially collected approximately 600 GAL4 lines that may be expressed in the gut based on RNA sequencing data, and then crossed them to UAS-GFP to perform immunohistochemistry to identify those that are expressed selectively in the gut. The cell types and regional expression patterns that are associated with the entire set of GAL4 drivers and split-GAL4 combinations are annotated online at http://kdrc.kr/index.php (K-Gut Project). This GAL4 resource can be used to target specific populations of distinct cell types in the fly gut, and therefore, should permit a more precise investigation of gut cells that regulate important biological processes.

Prognostic factors for the survival of primary molars following pulpotomy with mineral trioxide aggregate: a retrospective cohort study.

OBJECTIVES: The aim of the present study was to evaluate potential factors influencing the success rates of mineral trioxide aggregate (MTA) pulpotomy performed in primary molars. MATERIALS AND METHODS: A total of 347 teeth treated between March 2012 and December 2016 in 258 patients, with a mean age of 5.3 ± 1.7 years, were included in the analysis. Kaplan-Meier analyses were used to analyze were used time to failure. Multivariate Cox regression analysis with shared frailty was used to evaluate the clinical factors associated with failures. RESULTS: The mean (standard deviation) follow-up period was 35.8 (19.6) months. Within 84 months, the survival rate was 87.1%. In multivariate Cox regression, treatment performed in lower primary molars had a lower survival rate than upper primary molars (hazard ratio [HR] = 3.38, P = 0.012). Caries extension below the cemento-enamel junction had more risk of failure (HR = 10.9, P < 0.001). Final restoration using resin-modified glass ionomer or amalgam (direct filling) had a lower survival rate than stainless steel crown (HR = 5.62, P = 0.002). CONCLUSIONS: Clinical variables such as arch type, degree of caries extension, and type of final restoration may affect the survival of primary molars following MTA pulpotomy. CLINICAL RELEVANCE: The results of this study indicate that specific clinical variables can be used to predict the prognosis of MTA pulpotomy in primary teeth, and estimate the risk of treatment failure. Assessments of these variables should be performed in the context of evidence-based clinical decision making.

Deferoxamine Reduces Inflammation and Osteoclastogenesis in Avulsed Teeth.

Replacement and inflammatory resorption are serious complications associated with the delayed replantation of avulsed teeth. In this study, we aimed to assess whether deferoxamine (DFO) can suppress inflammation and osteoclastogenesis in vitro and attenuate inflammation and bone resorption in a replanted rat tooth model. Cell viability and inflammation were evaluated in RAW264.7 cells. Osteoclastogenesis was confirmed by tartrate-resistant acid phosphatase staining, reactive oxygen species (ROS) measurement, and quantitative reverse transcriptase-polymerase chain reaction in teeth exposed to different concentrations of DFO. In vivo, molars of 31 six-week-old male Sprague-Dawley rats were extracted and stored in saline (n = 10) or DFO solution (n = 21) before replantation. Micro-computed tomography (micro-CT) imaging and histological analysis were performed to evaluate inflammation and root and alveolar bone resorption. DFO downregulated the genes related to inflammation and osteoclastogenesis. DFO also reduced ROS production and regulated specific pathways. Furthermore, the results of the micro-CT and histological analyses provided evidence of the decrease in inflammation and hard tissue resorption in the DFO group. Overall, these results suggest that DFO reduces inflammation and osteoclastogenesis in a tooth replantation model, and thus, it has to be further investigated as a root surface treatment option for an avulsed tooth.

Intranuclear Delivery of Nuclear Factor-Kappa B p65 in a Rat Model of Tooth Replantation.

After avulsion and replantation, teeth are at risk of bone and root resorption. The present study aimed to demonstrate that the intra-nuclear transducible form of transcription modulation domain of p65 (nt-p65-TMD) can suppress osteoclast differentiation in vitro, and reduce bone resorption in a rat model of tooth replantation. Cell viability and nitric oxide release were evaluated in RAW264.7 cells using CCK-8 assay and Griess reaction kit. Osteoclast differentiation was evaluated using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and tartrate-resistant acid phosphatase (TRAP) staining. Thirty-two maxillary rat molars were extracted and stored in saline (n = 10) or 10 µM nt-p65-TMD solution (n = 22) before replantation. After 4 weeks, specimens were scored according to the inflammatory pattern using micro-computed tomography (CT) imaging and histological analyses. nt-p65-TMD treatment resulted in significant reduction of nitric oxide release and osteoclast differentiation as studied using PCR and TRAP staining. Further, micro-CT analysis revealed a significant decrease in bone resorption in the nt-p65-TMD treatment group (p < 0.05). Histological analysis of nt-p65-TMD treatment group showed that not only bone and root resorption, but also inflammation of the periodontal ligament and epithelial insertion was significantly reduced. These findings suggest that nt-p65-TMD has the unique capabilities of regulating bone remodeling after tooth replantation.

Effect of Heat and Sonic Vibration on Penetration of a Flowable Resin Composite Used as a Pit and Fissure Sealant.

OBJECTIVE: To evaluate penetration of a flowable resin composite into fissures using three different application methods: (1) conventional, (2) heat, and (3) sonic vibration. STUDY DESIGN: Forty-five sound maxillary third molars were divided randomly into three groups (n=15 per group). The occlusal surfaces of the teeth were etched and flowable resin composites were applied into the fissure using the assigned application method. The crowns were sectioned and examined with an optical microscope to assess penetration. In addition, three-point flexural strength was analyzed. RESULTS: The sonic vibration group exhibited significantly greater penetration into the fissure compared with the other test groups (p<0.001). The heat group exhibited greater penetration into the fissure compared with the conventional group (p=0.003). However, three-point flexural strength was similar among all groups (p>0.05). CONCLUSIONS: Sonic vibration and heat increased penetration into fissures. Notably, sonic vibration exhibited the greatest penetration. We found that the application method did not influence the three-point flexural strength.

Changing the Angulation of the Tooth Germ in the Bony Crypt: A Case Report.

An ankylosed primary molar may cause rotation or ectopic impaction of succedaneous premolar. When conventional treatment modalities such as observation, surgical exposure with or without orthodontic traction, and autotransplantation are not possible, the simple surgical relocation method could be an alternative treatment option for a lingually rotated premolar during the tooth germ stage before opting to extraction. In the case reported herein, the lingually rotated permanent mandibular second premolar tooth germ was surgically relocated within its bony crypt. Continued root development and spontaneous eruption were observed without complications during the 3.5-year follow-up period.

Clinical Assessment of a New Caries Activity Test Using Dental Plaque Acidogenicity in Children under Three Years of Age.

OBJECTIVES: The aim of this study was to assess the validity of a new caries activity test that uses dental plaque acidogenicity in children with deciduous dentition. STUDY DESIGN: Ninety-two children under the age of three years old underwent clinical examination using the dft index and examinations with two caries activity tests. Plaque samples for the new Cariview(®) test and the saliva sample for the conventional Dentocult SM(®) test were collected, incubated, and scored according to each manufacturers' instruction. The data were analysed using ANOVA and Spearman correlation analyses to evaluate the relationships between the test results and the caries experience. RESULTS: The mean dft index of all of the subjects was 4.73, and 17.4% of the subjects were caries-free. The levels of caries risk based on the new Cariview test score significantly increased with the caries experience (p < 0.01). The test results revealed a stronger correlation with caries indices (dft and dt index) than the conventional SM colony counting method (r = 0.43, r = 0.39, p < 0.01). CONCLUSIONS: The new caries activity test to analyse the acidogenic potential of whole microorganisms from dental plaques can be used to evaluate caries risk in children with deciduous teeth.

Molecular and cellular organization of odorant binding protein genes in Drosophila.

Chemosensation is important for the survival and reproduction of animals. The odorant binding proteins (OBPs) are thought to be involved in chemosensation together with chemosensory receptors. While OBPs were initially considered to deliver hydrophobic odorants to olfactory receptors in the aqueous lymph solution, recent studies suggest more complex roles in various organs. Here, we use GAL4 transgenes to systematically analyze the expression patterns of all 52 members of the Obp gene family and 3 related chemosensory protein genes in adult Drosophila, focusing on chemosensory organs such as the antenna, maxillary palp, pharynx, and labellum, and other organs such as the brain, ventral nerve cord, leg, wing, and intestine. The OBPs were observed to express in diverse organs and in multiple cell types, suggesting that these proteins can indeed carry out diverse functional roles. Also, we constructed 10 labellar-expressing Obp mutants, and obtained behavioral evidence that these OBPs may be involved in bitter sensing. The resources we constructed should be useful for future Drosophila OBP gene family research.

Molecular and cellular basis of sodium sensing in Drosophila labellum.

Appropriate ingestion of salt is essential for physiological processes such as ionic homeostasis and neuronal activity. Generally, low concentrations of salt elicit attraction, while high concentrations elicit aversive responses. Here, we observed that sugar neurons in the L sensilla of the Drosophila labellum cf. responses to NaCl, while sugar neurons in the S-c sensilla do not respond to NaCl, suggesting that gustatory receptor neurons involved in NaCl sensing may employ diverse molecular mechanisms. Through an RNAi screen of the entire Ir and ppk gene families and molecular genetic approaches, we identified IR76b, IR25a, and IR56b as necessary components for NaCl sensing in the Drosophila labellum. Co-expression of these three IRs in heterologous systems such as S2 cells or Xenopus oocytes resulted in a current in response to sodium stimulation, suggesting formation of a sodium-sensing complex. Our results should provide insights for research on the diverse combinations constituting salt receptor complexes.

TRPM7 Is Essential for RANKL-Induced Osteoclastogenesis.

The transient receptor potential melastatin type 7 (TRPM7) channel is a widely expressed non-selective cation channel with fusion to the C-terminal alpha kinase domain and regarded as a key regulator of whole body Mg(2+) homeostasis in mammals. However, the roles of TRPM7 during osteoclastogenesis in RAW264.7 cells and bone marrow-derived monocyte/macrophage precursor cells (BMMs) are not clear. In the present study, we investigate the roles of TRPM7 in osteoclastogenesis using methods of small interfering RNA (siRNA), RT-PCR, patch-clamp, and calcium imaging. RANKL (receptor activator of NF-κB ligand) stimulation did not affect the TRPM7 expression and TRPM7-mediated current was activated in HEK293, RAW264.7, and BMM cells by the regulation of Mg(2+). Knock-down of TRPM7 by siTRPM7 reduced intracellular Ca(2+) concentration ([Ca(2+)](i)) increases by 0 mM [Mg(2+)](e) in HEK293 cells and inhibited the generation of RANKL-induced Ca(2+) oscillations in RAW264.7 cells. Finally, knock-down of TRPM7 suppressed RANKL-mediated osteoclastogenesis such as activation and translocation of NFATc1, formation of multinucleated cells, and the bone resorptive activity, sequentially. These results suggest that TRPM7 plays an essential role in the RANKL-induced [Ca(2+)](i) oscillations that triggers the late stages of osteoclastogenesis.

CTTN Overexpression Confers Cancer Stem Cell-like Properties and Trastuzumab Resistance via DKK-1/WNT Signaling in HER2 Positive Breast Cancer.

BACKGROUND: Despite the therapeutic success of trastuzumab, HER2 positive (HER2+) breast cancer patients continue to face significant difficulties due to innate or acquired drug resistance. In this study we explored the potential role of CTTN in inducing trastuzumab resistance of HER2+ breast cancers. METHODS: Genetic changes of CTTN and survival of HER2+ breast cancer patients were analyzed in multiple breast cancer patient cohorts (METABRIC, TCGA, Kaplan-Meier (KM) plotter, and Hanyang University cohort). The effect of CTTN on cancer stem cell activity was assessed using the tumorsphere formation, ALDEFLUOR assay, and by in vivo xenograft experiments. CTTN-induced trastuzumab resistance was assessed by the sulforhodamine B (SRB) assay, colony formation assays, and in vivo xenograft model. RNA-seq analysis was used to clarify the mechanism of trastuzumab resistance conferred by CTTN. RESULTS: Survival analysis indicated that CTTN overexpression is related to a poor prognosis in HER2+ breast cancers (OS, p = 0.05 in the Hanyang University cohort; OS, p = 0.0014 in KM plotter; OS, p = 0.008 and DFS, p = 0.010 in METABRIC). CTTN overexpression-induced cancer stem cell-like characteristics in experiments of tumorsphere formation, ALDEFLUOR assays, and in vivo limiting dilution assays. CTTN overexpression resulted in trastuzumab resistance in SRB, colony formation assays, and in vivo xenograft models. Mechanistically, the mRNA and protein levels of DKK-1, a Wnt antagonist, were downregulated by CTTN. Treatment of the ß-catenin/TCF inhibitor reversed CTTN-induced cancer stem cell-like properties in vitro. Combination treatment with trastuzumab and ß-catenin/TCF inhibitor overcame trastuzumab resistance conferred by CTTN overexpression in in vitro colony formation assays. CONCLUSIONS: CTTN activates DKK-1/Wnt/ß-catenin signaling to induce trastuzumab resistance. We propose that CTTN is a novel biomarker indicating a poor prognosis and a possible therapeutic target for overcoming trastuzumab resistance.

Nanoporous MoS2 Field-Effect Transistor Based Artificial Olfaction: Achieving Enhanced Volatile Organic Compound Detection Inspired by the Drosophila Olfactory System.

Olfaction, a primal and effective sense, profoundly impacts our emotions and instincts. This sensory system plays a crucial role in detecting volatile organic compounds (VOCs) and realizing the chemical environment. Animals possess superior olfactory systems compared to humans. Thus, taking inspiration from nature, artificial olfaction aims to achieve a similar level of excellence in VOC detection. In this study, we present the development of an artificial olfaction sensor utilizing a nanostructured bio-field-effect transistor (bio-FET) based on transition metal dichalcogenides and the Drosophila odor-binding protein LUSH. To create an effective sensing platform, we prepared a hexagonal nanoporous structure of molybdenum disulfide (MoS2) using block copolymer lithography and selective etching techniques. This structure provides plenty of active sites for the integration of the LUSH protein, enabling enhanced binding with ethanol (EtOH) for detection purposes. The coupling of the biomolecule with EtOH influences the bio-FETs potential, which generates indicative electrical signals. By mimicking the sniffing techniques observed in Drosophila, these bio-FETs exhibit an impressive limit of detection of 10-6% for EtOH, with high selectivity, sensitivity, and detection ability even in realistic environments. This bioelectric sensor demonstrates substantial potential in the field of artificial olfaction, offering advancements in VOC detection.

Biocompatibility and mineralization potential of new calcium silicate cements.

Background/purpose: As calcium silicate cements (CSCs) have been successfully used in various types of vital pulp therapy, many new CSC products have been developed. The aim of this study was to evaluate the biocompatibilities and mineralization potential of new CSC. The experimental materials were NeoMTA Plus and EndoSequence Root Repair Material-Fast Set Putty (ERRM-FS) which were compared to ProRoot MTA. Materials and methods: In vitro, the effects of the new CSC on stem cells were evaluated. Each CSC was prepared for cell viability testing, alkaline phosphatase (ALP) assay, and calcium ion release assay. In vivo, the exposed pulp model was used for the partial pulpotomy procedure. Thirty-six teeth were treated with three materials: ProRoot MTA, NeoMTA Plus, or ERRM-FS. After four weeks, the teeth were extracted and processed for histologic analysis. Dentin bridge formation, pulp inflammation, and odontoblastic cell layer were evaluated and the area of newly formed calcific barrier of each group was measured. Results: Three CSCs demonstrated similar cell viability on stem cells and the levels of ALP and calcium release were not significantly different between tested materials. ProRoot MTA and ERRM-FS showed better tissue healing process than NeoMTA Plus after partial pulpotomy, in terms of quality of calcific barrier and pulp inflammation. The outcomes from measuring newly formed calcific area demonstrated no significant differences between the materials. Conclusion: NeoMTA Plus and ERRM-FS displayed similar biocompatibilities and mineralization potential compared to ProRoot MTA. Therefore, these new CSCs can be used as desirable alternatives to ProRoot MTA.

General gene expression patterns and stemness of the gingiva and dental pulp.

BACKGROUND/PURPOSE: Due to the unique properties of healing processes and cellular differentiation, the gingiva and dental pulp have attracted attention as a potential source of mesenchymal stem cells (MSCs). The purpose of this study was to obtain molecular-level information on these tissues in terms of their function and differentiation processes and investigate stemness. MATERIALS AND METHODS: Healthy gingival tissues were collected from patients (n = 9; aged 7-12 years) who underwent simple surgical procedures, and normal dental pulp tissues were obtained from patients (n = 25; aged 11-25 years) undergoing tooth extraction for orthodontic reasons. Complementary DNA microarray, qRT-qPCR, and immunohistochemical staining were performed to assess general and MSC gene expression patterns. RESULTS: In the gingival tissue, genes related to keratinization, the formation of epithelial cells and ectoderm, and immune and/or inflammatory responses were highly expressed. Meanwhile, in the dental pulp tissue, genes related to ion transport, neuronal development and axon guidance, bone and enamel mineralization, extracellular matrix organization, and angiogenesis were highly expressed. When focusing on the expression of MSC genes, induced pluripotent stem (iPS) cell genes, such as Sox2, c-Myc, and KLF4, were expressed at higher levels in the gingival tissue, whereas dental stem cell genes, such as NT5E and VCAM1, were expressed in dental pulp tissue. CONCLUSION: We found different general and MSC gene expression patterns between the gingival and dental pulp tissue. These results have implications for future regenerative medicine, considering the application of gingival tissue as a potential source of iPS cells.

The Piston Elastic: A Novel Device for Treating Entrapped Ectopic Permanent Molars.

Ectopic eruption of the permanent molar may absorb the distal root of the primary second molar and may result in a decreased arch length or delayed eruption of the permanent tooth, requiring timely treatment. Therefore, we devised an effective and convenient method to unlock the entrapped tooth using a novel device called a "piston-elastic". This case report aims to explain the design and clinical application of this piston-elastic and to describe successful cases. Three patients (aged 6, 13, and 16 years) with ectopically erupted maxillary and mandibular molars, respectively, were treated with a piston-elastic. It was bound to the locked molar to improve the eruption path. After a certain time period, the repulsive force pushed the surface of the adjacent tooth, improving the eruption path of the entrapped tooth. The piston-elastic is a novel device that simply and effectively changes the direction of eruption of ectopically entrapped molars. As it can be manufactured and attached to the chair side, impression acquisition on a model cast and laboratory procedures are unnecessary. Compared to existing methods, the piston-elastic can be easily produced and delivered, causes little irritation, and is inexpensive.

Modifications of Rect-Spring to Enhance the Engagement of Ectopically Entrapped Molars with 2 Case Reports.

The Rect-spring appliance, used for the management of ectopically erupting molars, shows weak retention on mesially tilted molars. We present three modifications of the appliance for better engagement and their advantages. We describe cases of two 7-year-old patients with ectopically erupting maxillary first molars with a 2.2 mm and 2.5 mm depth of entrapment, respectively. The modified Rect-spring (mRS) was inserted between the ectopically erupting first molar and adjacent primary second molar, and exerted a distalization force with an interproximal wedging effect at the same time. After 3 months, the ectopically erupting first molars were successfully brought into proper occlusion. No discomfort was reported. The mRS is suitable for various locking cases except for severely tilted molars without requiring any laboratory procedures. We suggest it as the first choice for unlocking the first molars.

In Vivo Evaluation of Decellularized Human Tooth Scaffold for Dental Tissue Regeneration.

Conventional root canal treatment may result in loss of tooth vitality, which can lead to unfavorable treatment outcomes. Notably, a ceased tooth development of immature permanent teeth with open apices, regeneration of periodontal ligaments (PDL), and pulp is highly expected healing process. For regeneration, the scaffold is one of the critical components that carry biological benefits. Therefore, this study evaluated a decellularized human tooth as a scaffold for the PDL and pulp tissue regeneration. A tooth scaffold was fabricated using an effective decellularization method as reported in previous studies. PDL stem cells (PDLSCs) and dental pulp stem cells (DPSCs) obtained from human permanent teeth were inoculated onto decellularized scaffolds, then cultured to transplant into immunosuppressed mouse. After 9 weeks, PDLSCs and DPSCs that were inoculated onto decellularized tooth scaffolds and cultured in an in vivo demonstrated successful differentiation. In PDLSCs, a regeneration of the cementum/PDL complex could be expected. In DPSCs, the expression of genes related to revascularization and the hard tissue regeneration showed the possibility of pulp regeneration. This study suggested that the potential possible application of decellularized human tooth could be a scaffold in regeneration PDL and pulp tissue along with PDLSCs and DPSCs, respectively, as a novel treatment method.

NSD3-Induced Methylation of H3K36 Activates NOTCH Signaling to Drive Breast Tumor Initiation and Metastatic Progression.

Histone methyltransferase NSD3 is frequently dysregulated in human cancers, yet the epigenetic role of NSD3 during cancer development remains elusive. Here we report that NSD3-induced methylation of H3K36 is crucial for breast tumor initiation and metastasis. In patients with breast cancer, elevated expression of NSD3 was associated with recurrence, distant metastasis, and poor survival. In vivo, NSD3 promoted malignant transformation of mammary epithelial cells, a function comparable to that of HRAS. Furthermore, NSD3 expanded breast cancer-initiating cells and promoted epithelial-mesenchymal transition to trigger tumor invasion and metastasis. Mechanistically, the long isoform (full-length transcript) of NSD3, but not its shorter isoform lacking a catalytic domain, cooperated with EZH2 and RNA polymerase II to stimulate H3K36me2/3-dependent transactivation of genes associated with NOTCH receptor cleavage, leading to nuclear accumulation of NICD and NICD-mediated transcriptional repression of E-cadherin. Furthermore, mice harboring primary and metastatic breast tumors with overexpressed NSD3 showed sensitivity to NOTCH inhibition. Together, our findings uncover the critical epigenetic role of NSD3 in the modulation of NOTCH-dependent breast tumor progression, providing a rationale for targeting the NSD3-NOTCH signaling regulatory axis in aggressive breast cancer. SIGNIFICANCE: This study demonstrates the functional significance of histone methyltransferase NSD3 in epigenetic regulation of breast cancer stemness, EMT, and metastasis, suggesting NSD3 as an actionable therapeutic target in metastatic breast cancer.

Physical properties of resin-reinforced glass ionomer cement modified with micro and nano-hydroxyapatite.

Hydroxyapatite is a biologically compatible material and a major component of dental enamel and bone tissue. Because of its biocompatibility and structural similarity to human teeth and the skeletal system, a number of dental studies have evaluated its application as a bone substitute or dental restorative material. This study was to evaluate the differences in bonding strength and resistance to demineralization between micro-hydroxyapatite and nano-hydroxyapatite added to self-cured resin-reinforced/modified glass ionomer cement. RelyX was used as the base glass ionomer cement material and for the control group. 10% micro-hydroxyapatite added glass ionomer cement was named experimental group 1, and 10% nano-hydroxyapatite added glass ionomer cement was named experimental group 2. Physical tests for ISO9917-1:2007 in each group was acceptable, except the setting time of nano-hydroxyapatite added glass ionomer cement, which exceeded maximum setting time. Bonding strength was greatest in nano-hydroxyapatite glass ionomer cement, and cohesive failure was common in all specimens. When fractured surface was observed under SEM, spherical particles were observed in experimental groups containing hydroxyapatite particles, and they were more prevalent in nano-HA added glass ionomer cement group than in micro-hydroxyapatite added group. Both experimental groups exhibited greater resistance to demineralization compared to the control group, and there was no significant difference between the experimental groups. Under SEM, nano-hydroxyapatite added glass ionomer cement exhibited increased resistance to demineralization compared to micro-hydroxyapatite added glass ionomer cement.