Effect of Heparan Sulfate on Vasculogenesis and Dentinogenesis of Dental Pulp Stem Cells.

INTRODUCTION: Heparan sulfate (HS) is a major component of dental pulp tissue. We previously reported that inhibiting HS biosynthesis impedes endothelial differentiation of dental pulp stem cells (DPSCs). However, the underlying mechanisms by which exogenous HS induces DPSC differentiation and pulp tissue regeneration remain unknown. This study explores the impact of exogenous HS on vasculogenesis and dentinogenesis of DPSCs both in vitro and in vivo. METHODS: Human-derived DPSCs were cultured in endothelial and odontogenic differentiation media and treated with HS. Endothelial differentiation of DPSCs was investigated by real-time polymerase chain reaction and capillary sprouting assay. Odontogenic differentiation was assessed through real-time polymerase chain reaction and detection of mineralized dentin-like deposition. Additionally, the influence of HS on pulp tissue was assessed with a direct pulp capping model, in which HS was delivered to exposed pulp tissue in rats. Gelatin sponges were loaded with either phosphate-buffered saline or 101-102 µg/mL HS and placed onto the pulp tissue. Following a 28-day period, tissues were investigated by histological analysis and micro-computed tomography imaging. RESULTS: HS treatment markedly increased expression levels of key endothelial and odontogenic genes, enhanced the formation of capillary-like structures, and promoted the deposition of mineralized matrices. Treatment of exposed pulp tissue with HS in the in vivo pulp capping study induced formation of capillaries and reparative dentin. CONCLUSIONS: Exogenous HS effectively promoted vasculogenesis and dentinogenesis of DPSCs in vitro and induced reparative dentin formation in vivo, highlighting its therapeutic potential for pulp capping treatment.

Characteristic craniofacial defects associated with a novel USP9X truncation mutation.

Germline loss-of-function mutations in USP9X have been reported to cause a wide spectrum of congenital anomalies. Here, we report a Japanese girl with a novel heterozygous nonsense mutation in USP9X who exhibited intellectual disability with characteristic craniofacial abnormalities, including hypotelorism, brachycephaly, hypodontia, micrognathia, severe dental crowding, and an isolated submucous cleft palate. Our findings provide further evidence that disruptions in USP9X contribute to a broad range of congenital craniofacial abnormalities.

The significant role of glycosaminoglycans in tooth development.

This review delves into the roles of glycosaminoglycans (GAGs), integral components of proteoglycans, in tooth development. Proteoglycans consist of a core protein linked to GAG chains, comprised of repeating disaccharide units. GAGs are classified into several types, such as hyaluronic acid, heparan sulfate, chondroitin sulfate, dermatan sulfate, and keratan sulfate. Functioning as critical macromolecular components within the dental basement membrane, these GAGs facilitate cell adhesion and aggregation, and play key roles in regulating cell proliferation and differentiation, thereby significantly influencing tooth morphogenesis. Notably, our recent research has identified the hyaluronan-degrading enzyme Transmembrane protein 2 (Tmem2) and we have conducted functional analyses using mouse models. These studies have unveiled the essential role of Tmem2-mediated hyaluronan degradation and its involvement in hyaluronan-mediated cell adhesion during tooth formation. This review provides a comprehensive summary of the current understanding of GAG functions in tooth development, integrating insights from recent research, and discusses future directions in this field.

Three-dimensional changes in the cranial base associated with soft-diet feeding.

BACKGROUND: Masticatory activity affects the morphology of the maxillo-mandibular complex, however, its influence on the cranial base remains to be elucidated. The recent integration of quantitative morphometric analysis with 3D imaging enabled a comprehensive and high-resolution morphological characterization of the craniofacial complex. We aimed to investigate the influence of masticatory activity on the morphology of the growing cranial base by three-dimensional (3D) geometric morphometric approach using micro-CT. METHODS: The micro-CT data was reanalyzed to illustrate the 3D shape of the cranial base, and wireframe models were generated by connecting landmarks on the images. In the original study, mice were fed a soft diet (SD) of powdered pellets or a conventional hard diet (HD) for 6 weeks from 3 to 9 weeks of age, immediately after weaning. A principal component (PC) analysis analyzed shape variations and assessed their significance, while canonical variate (CV) analysis facilitated the comparison and differentiation of groups based on shape, unveiling meaningful shape distinctions. RESULTS: Three PCs were extracted that significantly separated the SD and HD groups among those explaining variations in shape. These PCs were related to the length of the sphenoid bone, the width of the anterior part of the sphenoid bone, and the length of the cranial base. Furthermore, one CV effectively distinguished SD from HD, and CV analysis showed that the sphenoid was shortened in the length and narrowed at the border of the temporal bone in SD mice. CONCLUSIONS: Masticatory loading affects the skeletal development of the cranial base. The morphology of the sphenoid bone was affected in both the sagittal and transverse axes.

Current Status and Molecular Mechanisms of Resistance to Immunotherapy in Oral Malignant Melanoma.

Immune checkpoint inhibitors (ICIs), including anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and anti-programmed death-1 (PD-1) antibodies, have initiated a new era in the treatment of malignant melanoma. ICIs can be used in various settings, including first-line, adjuvant, and neo-adjuvant therapy. In the scope of this review, we examined clinical studies utilizing ICIs in the context of treating oral mucosal melanoma, a rare disease, albeit with an extremely poor prognosis, with a specific focus on unraveling the intricate web of resistance mechanisms. The absence of a comprehensive review focusing on ICIs in oral mucosal melanoma is notable. Therefore, this review seeks to address this deficiency by offering a novel and thorough analysis of the current status, potential resistance mechanisms, and future prospects of applying ICIs specifically to oral malignant melanoma. Clarifying and thoroughly understanding these mechanisms will facilitate the advancement of effective therapeutic approaches and enhance the prospects for patients suffering from oral mucosal melanoma.

On-demand chlorine dioxide solution enhances odontoblast differentiation through desulfation of cell surface heparan sulfate proteoglycan and subsequent activation of canonical Wnt signaling.

Heparan sulfate proteoglycans (HSPGs) surround the surface of odontoblasts, and their modification affects their affinity for Wnt ligands. This study proposes applying Matching Transformation System® (MA-T), a novel chlorinated oxidant, to enhance dentinogenesis. MA-T treatment in odontoblasts decreased sulfation of HSPG and upregulated the expression of dentin sialophosphoprotein (Dspp) and Dentin Matrix Protein 1 (Dmp1) via activation of canonical Wnt signaling in vitro. Ex vivo application of MA-T also enhanced dentin matrix formation in developing tooth explants. Reanalysis of a public single-cell RNA-seq dataset revealed significant Wnt activity in the odontoblast population, with enrichment for Wnt10a and Wnt6. Silencing assays showed that Wnt10a and Wnt6 were redundant in inducing Dspp and Dmp1 mRNA expression. These Wnt ligands' expression was upregulated by MA-T treatment, and TCF/LEF binding sites are present in their promoters. Furthermore, the Wnt inhibitors Notum and Dkk1 were enriched in odontoblasts, and their expression was also upregulated by MA-T treatment, together suggesting autonomous maintenance of Wnt signaling in odontoblasts. This study provides evidence that MA-T activates dentinogenesis by modifying HSPG and through subsequent activation of Wnt signaling.

The roles of JAK2/STAT3 signaling in fusion of the secondary palate.

Cleft palate has a multifactorial etiology. In palatal fusion, the contacting medial edge epithelium (MEE) forms the epithelial seam, which is subsequently removed with the reduction of p63. Failure in this process results in a cleft palate. We herein report the involvement of janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling in palatal fusion and that folic acid rescues the fusing defect by reactivating JAK2/STAT3. In closure of bilateral palatal shelves, STAT3 phosphorylation was activated at the fusing MEE and mesenchyme underlying the MEE. JAK2 inhibition by AG490 inhibited STAT3 phosphorylation and resulted in palatal fusion failure without removal of the epithelial seam, in which p63 and keratin 17 (K17) periderm markers were retained. Folic acid application restored STAT3 phosphorylation in AG490-treated palatal explants and rescued the fusion defect, in which the p63- and K17-positive epithelial seam were removed. The AG490-induced palatal defect was also rescued in p63 haploinsufficient explants. These findings suggest that JAK2/STAT3 signaling is involved in palatal fusion by suppressing p63 expression in MEE and that folate restores the fusion defect by reactivating JAK2/STAT3.

Orthodontic Management of Severe Hypodontia and Impacted Maxillary Second Molars in a Patient with Sotos Syndrome.

Sotos syndrome is a genetic disorder characterized by distinct craniofacial features, overgrowth in childhood, and impaired intellectual development. We herein report the successful orthodontic treatment of a 14-year-old boy with Sotos syndrome caused by a heterozygous mutation in the NSD1 gene. He showed severe hypodontia, impaction of the maxillary second molars and a skeletal Class III jaw-base relationship. Orthodontic management, including space control by protraction of the maxillary first molars and traction of the impacted molars, was performed using fixed appliances and miniscrews. As a result, acceptable occlusion was obtained without any discernible relapse 18 months postretention.

Craniofacial and dental characteristics of three Japanese individuals with genetically diagnosed SATB2-associated syndrome.

Craniofacial defects are one of the most frequent phenotypes in syndromic diseases. More than 30% of syndromic diseases are associated with craniofacial defects, which are important for the precise diagnosis of systemic diseases. Special AT-rich sequence-binding protein 2 (SATB2)-associated syndrome (SAS) is a rare syndromic disease associated with a wide variety of phenotypes, including intellectual disability and craniofacial defects. Among them, dental anomalies are the most frequently observed phenotype and thus becomes an important diagnostic criterion for SAS. In this report, we demonstrate three Japanese cases of genetically diagnosed SAS with detailed craniofacial phenotypes. The cases showed multiple dental problems, which have been previously reported to be linked to SAS, including abnormal crown morphologies and pulp stones. One case showed a characteristic enamel pearl at the root furcation. These phenotypes add new insights for differentiating SAS from other disorders.

Molecular Mechanisms of Inhibitory Effects of Bovine Lactoferrin on Invasion of Oral Squamous Cell Carcinoma.

Lactoferrin (LF), an iron-binding glycoprotein, has been reported to have anticancer properties. However, the molecular mechanisms behind its anticancer effects on oral squamous cell carcinoma (OSCC) have not yet been elucidated. Therefore, we aimed to clarify the effects of LF on invasion of OSCC, and its underlying molecular mechanism. OSCC cell lines, HSC2 and HOC313, were treated with bovine LF (bLF). The effects of bLF on cell invasion were examined by a chamber migration assay, wound healing assay, and Boyden chamber method with a basement-membrane-analogue. Expression levels of MMP-1, MMP-3, and AP-1 were examined using RT-PCR, qRT-PCR, and western blotting. Roles of LRP1, a receptor of bLF, on cell invasion were analyzed using siLRP1 knockdown cells. Furthermore, to clarify the importance of LRP1 in invasion, the effects of bLF on tPA-induced invasion of OSCC cells were examined. The invasion assays showed that bLF suppressed invasion of the OSCC cells. Moreover, bLF down-regulated AP-1, and resulted in reductions of MMP-1 and MMP-3. With SiLRP1 knockdown, OSCC cells failed to induce their invasion, and bLF was not able to exert its effects on invasion. Furthermore, bLF remarkably inhibited tPA-induced cell invasion. These findings suggest the importance of LRP1 in bLF-suppressed invasion of OSCC cells via the reduction of AP-1 and MMP production.

In Vitro Investigation of the Effects of the Hyaluronan-Rich Extracellular Matrix on Neural Crest Cell Migration.

Neural crest cells (NCCs) are highly migratory cells that originate from the dorsal region of the neural tube. The emigration of NCCs from the neural tube is an essential process for NCC production and their subsequent migration toward target sites. The migratory route of NCCs, including the surrounding neural tube tissues, involves hyaluronan (HA)-rich extracellular matrix. To model NCC migration into these HA-rich surrounding tissues from the neural tube, a mixed substrate migration assay consisting of HA (average molecular weight: 1,200-1,400 kDa) and collagen type I (Col1) was established in this study. This migration assay demonstrates that NCC cell line, O9-1, cells are highly migratory on the mixed substrate and that the HA coating is degraded at the site of focal adhesions in the course of migration. This in vitro model can be useful for further exploration of the mechanistic basis involved in NCC migration. This protocol is also applicable for evaluating different substrates as scaffolds to study NCC migration.

Bovine Lactoferrin Suppresses Tumor Angiogenesis through NF-κB Pathway Inhibition by Binding to TRAF6.

Tumor angiogenesis is essential for tumor progression. The inhibition of tumor angiogenesis is a promising therapy for tumors. Bovine lactoferrin (bLF) has been reported as an anti-tumor agent. However, bLF effects on tumor angiogenesis are not well demonstrated. This study evaluated the inhibitory effects of bLF on tumor angiogenesis in vivo and in vitro. Herein, tumor endothelial cells (TECs) and normal endothelial cells (NECs) were used. Proliferation, migration, tube formation assays, RT-PCR, flow cytometry, Western blotting, siRNA experiments and immunoprecipitation were conducted to clarify the mechanisms of bLF-induced effects. CD-31 immunoexpression was examined in tumor tissues of oral squamous cell carcinoma mouse models with or without Liposomal bLF (LbLF)-administration. We confirmed that bLF inhibited proliferation/migration/tube formation and increased apoptosis in TECs but not NECs. TNF receptor-associated factor 6 (TRAF6), p-p65, hypoxia inducible factor-α (HIF-1α) and vascular endothelial growth factor (VEGF) were highly expressed in TECs. In TECs, bLF markedly downregulated VEGF-A, VEGF receptor (VEGFR) and HIF-1α via the inhibition of p-p65 through binding with TRAF6. Since NECs slightly expressed p-p65, bLF-TRAF-6 binding could not induce detectable changes. Moreover, orally administrated LbLF decreased CD31-positive microvascular density only in TECs. Hence, bLF specifically suppressed tumor angiogenesis through p-p65 inhibition by binding to TRAF6 and suppressing HIF-1α activation followed by VEGF/VEGFR down-regulation. Collectively, bLF can be an anti-angiogenic agent for tumors.

The cell surface hyaluronidase TMEM2 plays an essential role in mouse neural crest cell development and survival.

Hyaluronan (HA) is a major extracellular matrix component whose tissue levels are dynamically regulated during embryonic development. Although the synthesis of HA has been shown to exert a substantial influence on embryonic morphogenesis, the functional importance of the catabolic aspect of HA turnover is poorly understood. Here, we demonstrate that the transmembrane hyaluronidase TMEM2 plays an essential role in neural crest development and the morphogenesis of neural crest derivatives, as evidenced by the presence of severe craniofacial abnormalities in Wnt1-Cre-mediated Tmem2 knockout (Tmem2CKO) mice. Neural crest cells (NCCs) are a migratory population of cells that gives rise to diverse cell lineages, including the craniofacial complex, the peripheral nervous system, and part of the heart. Analysis of Tmem2 expression during NCC formation and migration reveals that Tmem2 is expressed at the site of NCC delamination and in emigrating Sox9-positive NCCs. In Tmem2CKO embryos, the number of NCCs emigrating from the neural tube is greatly reduced. Furthermore, linage tracing reveals that the number of NCCs traversing the ventral migration pathway and the number of post-migratory neural crest derivatives are both significantly reduced in a Tmem2CKO background. In vitro studies using Tmem2-depleted mouse O9-1 neural crest cells demonstrate that Tmem2 expression is essential for the ability of these cells to form focal adhesions on and to migrate into HA-containing substrates. Additionally, we show that Tmem2-deficient NCCs exhibit increased apoptotic cell death in NCC-derived tissues, an observation that is corroborated by in vitro experiments using O9-1 cells. Collectively, our data demonstrate that TMEM2-mediated HA degradation plays an essential role in normal neural crest development. This study reveals the hitherto unrecognized functional importance of HA degradation in embryonic development and highlights the pivotal role of Tmem2 in the developmental process.

Development of dentition: From initiation to occlusion and related diseases.

BACKGROUND: The development of dentition begins in the embryonic oral cavity and progresses in the branchial arches and alveolar bone. Continuous cellular and molecular crosstalk occurs during crown formation, after which the tooth germ begins to migrate apically through the alveolar process into the oral cavity. It eventually comes in contact with its antagonist in the contralateral jaw to establish functional occlusion. Any defect in either step can result in delayed tooth development, the spectrum of which varies from a congenitally missing tooth to an impacted tooth (infraocclusion) with an eruption problem, both of which can impair oral function. HIGHLIGHT: Congenitally missing teeth or eruption problems may result from genetic mutations. Several different mutations have been identified, each causing a distinct phenotype. Thus, it is imperative that medical providers understand the fundamentals of these genetic principles that govern such dental diseases. CONCLUSION: In this review, we focus on several diseases, including congenitally missing teeth and tooth eruption problems. We review these diseases with aspect to their association with a particular syndrome, as well as independently in a non-syndromic capacity. We also review previously identified genetic mutations and discuss the possible mechanisms that cause individual phenotypes by analyzing previous investigations. We also discuss future prospects of how genetic diagnosis and precision medicine could impact the clinical environment in the field of dentistry. ETHICAL APPROVAL: Present study has been carried out in accordance with The Code of Ethics of the World Medical Association and approved by Institutional Review Board of Osaka University Graduate School of Dentistry.

Oral administration of bovine lactoferrin suppresses the progression of rheumatoid arthritis in an SKG mouse model.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammatory bone destruction in which tumor necrosis factor alpha (TNF-α) plays a key role. Bovine lactoferrin (bLF) is a multifunctional protein with anti-inflammatory and immunomodulatory properties. This study aimed to clarify the inhibitory effects of bLF on the pathological progression of RA. The mannan-induced arthritis model in SKG mice (genetic RA model) was used. Orally applied liposomal bLF (LbLF) markedly reduced ankle joint swelling and bone destruction. Histologically, pannus formation and osteoclastic bone destruction were prevented in the LbLF-treated animals. Moreover, orally administered LbLF improved the balance between Th17 cells and regulatory T cells isolated from the spleen of mannan-treated SKG mice. In an in vitro study, the anti-inflammatory effects of bLF on TNF-α-induced TNF-α production and downstream signaling pathways were analyzed in human synovial fibroblasts from RA patients (RASFs). bLF suppressed TNF-α production from RASFs by inhibiting the nuclear factor kappa B and mitogen-activated protein kinase pathways. The intracellular accumulation of bLF in RASFs increased in an applied bLF dose-dependent manner. Knockdown of the lipoprotein receptor-related protein-1 (LRP1) siRNA gene reduced bLF expression in RASFs, indicating that exogenously applied bLF was mainly internalized through LRP-1. Immunoprecipitated proteins with anti-TNF receptor-associated factor 2 (TRAF2; an adapter protein/ubiquitin ligase) included bLF, indicating that bLF binds directly to the TRAF2-TRADD-RIP complex. This indicates that LbLF may effectively prevent the pathological progression of RA by suppressing TNF-α production by binding to the TRAF2-TRADD-RIP complex from the RASFs in the pannus. Therefore, supplemental administration of LbLF may have a beneficial effect on preventive/therapeutic reagents for RA.

Ras signaling and RREB1 are required for the dissociation of medial edge epithelial cells in murine palatogenesis.

Cleft palate is one of the major congenital craniofacial birth defects. The etiology underlying the pathogenesis of cleft palate has yet to be fully elucidated. Dissociation of the medial edge epithelium (MEE) at the contacting region of palatal shelves and subsequent migration or apoptosis of MEE cells is required for proper MEE removal. Ras-responsive element-binding protein 1 (RREB1), a RAS transcriptional effector, has recently been shown to play a crucial role in developmental epithelial-mesenchymal transition (EMT), in which loss of epithelial characteristics is an initial step, during mid-gastrulation of embryonic development. Interestingly, the involvement of RREB1 in cleft palate has been indicated in humans. Here, we demonstrated that pan-Ras inhibitor prevents the dissociation of MEE during murine palatal fusion. Rreb1 is expressed in the palatal epithelium during palatal fusion, and knockdown of Rreb1 in palatal organ culture resulted in palatal fusion defects by inhibiting the dissociation of MEE cells. Our present findings provide evidence that RREB1-mediated Ras signaling is required during palatal fusion. Aberrant RREB1-mediated Ras signaling might be involved in the pathogenesis of cleft palate.

Zfhx4 regulates endochondral ossification as the transcriptional platform of Osterix in mice.

Endochondral ossification is regulated by transcription factors that include SRY-box transcription factor 9, runt-related protein 2 (Runx2), and Osterix. However, the sequential and harmonious regulation of the multiple steps of endochondral ossification is unclear. This study identified zinc finger homeodomain 4 (Zfhx4) as a crucial transcriptional partner of Osterix. We found that Zfhx4 was highly expressed in cartilage and that Zfhx4 deficient mice had reduced expression of matrix metallopeptidase 13 and inhibited calcification of cartilage matrices. These phenotypes were very similar to impaired chondrogenesis in Osterix deficient mice. Coimmunoprecipitation and immunofluorescence indicated a physical interaction between Zfhx4 and Osterix. Notably, Zfhx4 and Osterix double mutant mice showed more severe phenotype than Zfhx4 deficient mice. Additionally, Zfhx4 interacted with Runx2 that functions upstream of Osterix. Our findings suggest that Zfhx4 coordinates the transcriptional network of Osterix and, consequently, endochondral ossification.

Synergistic role of retinoic acid signaling and Gata3 during primitive choanae formation.

Developmental defects of primitive choanae, an anatomical path to connect the embryonic nasal and oral cavity, result in disorders called choanal atresia (CA), which are associated with many congenital diseases and require immediate clinical intervention after birth. Previous studies revealed that reduced retinoid signaling underlies the etiology of CA. In the present study, by using multiple mouse models which conditionally deleted Rdh10 and Gata3 during embryogenesis, we showed that Gata3 expression is regulated by retinoid signaling during embryonic craniofacial development and plays crucial roles for development of the primitive choanae. Interestingly, Gata3 loss of function is known to cause hypoparathyroidism, sensorineural deafness and renal disease (HDR) syndrome, which exhibits CA as one of the phenotypes in humans. Our model partially phenocopies HDR syndrome with CA, and is thus a useful tool for investigating the molecular and cellular mechanisms of HDR syndrome. We further uncovered critical synergy of Gata3 and retinoid signaling during embryonic development, which will shed light on novel molecular and cellular etiology of congenital defects in primitive choanae formation.

Progression of melanoma is suppressed by targeting all transforming growth factor­ß isoforms with an Fc chimeric receptor.

Melanoma is an aggressive type of cancer originating from the skin that arises from neoplastic changes in melanocytes. Transforming growth factor­ß (TGF­ß) is a pleiotropic cytokine and is known to contribute to melanoma progression by inducing the epithelial­mesenchymal transition (EMT) program and creating an environment that favors tumor progression. There are three TGF­ß isoforms, TGF­ß1, TGF­ß2 and TGF­ß3, all of which engage in pro­tumorigenic activities by activating SMAD signaling pathways. All TGF­ß isoforms activate signaling pathways by binding to their TGF­ß type I (TßRI) and type II (TßRII) receptors. Thus, effective targeting of all TGF­ß isoforms is of great importance. In the present study, chimeric proteins comprising the extracellular domains of TßRI and/or TßRII fused with the Fc portion of human immunoglobulin (IgG) were validated in the melanoma context. The Fc chimeric receptor comprising both TßRI and TßRII (TßRI­TßRII­Fc) effectively trapped all TGF­ß isoforms. Conversely, TßRII­Fc chimeric receptor, that comprises TßRII only, was able to interact with TGF­ß1 and TGF­ß3 isoforms, but not with TGF­ß2, which is a poor prognostic factor for melanoma patients. Accordingly, it was revealed that TßRI­TßRII­Fc chimeric receptor suppressed the EMT program in melanoma cells in vitro induced by any of the three TGF­ß isoforms, as revealed by decreased expression of mesenchymal markers. Conversely, TßRII­Fc chimeric receptor inhibited the EMT program induced by TGF­ß1 and TGF­ß3. In addition, it was established that tumor growth in subcutaneous mouse melanoma was inhibited by TßRI­TßRII­Fc chimeric receptor indicating that Fc chimeric receptor could be applied to modify the tumor microenvironment (TME) of melanoma. Therefore, designing of Fc chimeric receptors targeting TGF­ß signals that affect various components of the TME may result in the development of effective anti­melanoma agents.

Senescence-accelerated mouse prone 8 (SAMP8) mice exhibit reduced entoconid in the lower second molar.

OBJECTIVE: The position and size of the major cusps in mammalian molars are arranged in a characteristic pattern that depends on taxonomy. In humans, the cusp which locates distally within each molar is smaller than the mesially located cusp, which is referred to as "distal reduction". Although this concept has been well-recognized, it is still unclear how this reduction occurs. Current study examined whether senescence-accelerating mouse prone 8 (SAMP8) mice could be a possible animal model for studying how the mammalian molar cusp size is determined. DESIGN: SAMP8 mice were compared with parental control (SAMR1) mice. Microcomputed tomography images of young and aged mice were captured to observe molar cusp morphologies. Cusp height from cement-enamel junction and mesio-distal length of molars were measured. The statistical comparison of the measurements was performed by Mann-Whitney U test. RESULTS: SAMP8 mice showed reduced development of the disto-lingual cusp (entoconid) of lower second molar when compared with SAMR1 mice. The enamel thickness and structure was disturbed at entoconid, and aged SAMP8 mice displayed severe wear of the entoconid in lower second molar. These phenotypes were observed on both sides of the lower second molar. CONCLUSIONS: In addition to the general senescence phenotype observed in SAMP8 mice, this strain may genetically possess molar cusp phenotypes which is determined prenatally. Further, SAMP8 mice would be a potential model strain to study the genetic causes of the distal reduction of molar cusp size.