Endosteal stem cells at the bone-blood interface: A double-edged sword for rapid bone formation: Bone marrow endosteal stem cells provide a robust source of bone-making osteoblasts both in normal and abnormal bone formation.

Endosteal stem cells are a subclass of bone marrow skeletal stem cell populations that are particularly important for rapid bone formation occurring in growth and regeneration. These stem cells are strategically located near the bone surface in a specialized microenvironment of the endosteal niche. These stem cells are abundant in young stages but eventually depleted and replaced by other stem cell types residing in a non-endosteal perisinusoidal niche. Single-cell molecular profiling and in vivo cell lineage analyses play key roles in discovering endosteal stem cells. Importantly, endosteal stem cells can transform into bone tumor-making cells when deleterious mutations occur in tumor suppressor genes. The emerging hypothesis is that osteoblast-chondrocyte transitional identities confer a special subset of endosteal stromal cells with stem cell-like properties, which may make them susceptible for tumorigenic transformation. Endosteal stem cells are likely to represent an important therapeutic target of bone diseases caused by aberrant bone formation.

Impaired Glycosylation of Gastric Mucins Drives Gastric Tumorigenesis and Serves as a Novel Therapeutic Target.

BACKGROUND & AIMS: Gastric cancer is often accompanied by a loss of mucin 6 (MUC6), but its pathogenic role in gastric carcinogenesis remains unclear. METHODS: Muc6 knockout (Muc6-/-) mice and Muc6-dsRED mice were newly generated. Tff1Cre, Golph3-/-, R26-Golgi-mCherry, Hes1flox/flox, Cosmcflox/flox, and A4gnt-/- mice were also used. Histology, DNA and RNA, proteins, and sugar chains were analyzed by whole-exon DNA sequence, RNA sequence, immunohistochemistry, lectin-binding assays, and liquid chromatography-mass spectrometry analysis. Gastric organoids and cell lines were used for in vitro assays and xenograft experiments. RESULTS: Deletion of Muc6 in mice spontaneously causes pan-gastritis and invasive gastric cancers. Muc6-deficient tumor growth was dependent on mitogen-activated protein kinase activation, mediated by Golgi stress-induced up-regulation of Golgi phosphoprotein 3. Glycomic profiling revealed aberrant expression of mannose-rich N-linked glycans in gastric tumors, detected with banana lectin in association with lack of MUC6 expression. We identified a precursor of clusterin as a binding partner of mannose glycans. Mitogen-activated protein kinase activation, Golgi stress responses, and aberrant mannose expression are found in separate Cosmc- and A4gnt-deficient mouse models that lack normal O-glycosylation. Banana lectin-drug conjugates proved an effective treatment for mannose-rich murine and human gastric cancer. CONCLUSIONS: We propose that Golgi stress responses and aberrant glycans are important drivers of and promising new therapeutic targets for gastric cancer.

Hes1 marks peri-condensation mesenchymal cells that generate both chondrocytes and perichondrial cells in early bone development.

Bone development starts with condensations of undifferentiated mesenchymal cells that set a framework for future bones within the primordium. In the endochondral pathway, mesenchymal cells inside the condensation differentiate into chondrocytes and perichondrial cells in a SOX9-dependent mechanism. However, the identity of mesenchymal cells outside the condensation and how they participate in developing bones remain undefined. Here we show that mesenchymal cells surrounding the condensation contribute to both cartilage and perichondrium, robustly generating chondrocytes, osteoblasts, and marrow stromal cells in developing bones. Single-cell RNA-seq analysis of Prrx1-cre-marked limb bud mesenchymal cells at E11.5 reveals that Notch effector Hes1 is expressed in a mutually exclusive manner with Sox9 that is expressed in pre-cartilaginous condensations. Analysis of a Notch signaling reporter CBF1:H2B-Venus reveals that peri-condensation mesenchymal cells are active for Notch signaling. In vivo lineage-tracing analysis using Hes1-creER identifies that Hes1+ early mesenchymal cells surrounding the SOX9+ condensation at E10.5 contribute to both cartilage and perichondrium at E13.5, subsequently becoming growth plate chondrocytes, osteoblasts of trabecular and cortical bones, and marrow stromal cells in postnatal bones. In contrast, Hes1+ cells in the perichondrium at E12.5 or E14.5 do not generate chondrocytes within cartilage, contributing to osteoblasts and marrow stromal cells only through the perichondrial route. Therefore, Hes1+ peri-condensation mesenchymal cells give rise to cells of the skeletal lineage through cartilage-dependent and independent pathways, supporting the theory that early mesenchymal cells outside the condensation also play important roles in early bone development.

Resting zone of the growth plate houses a unique class of skeletal stem cells.

Skeletal stem cells regulate bone growth and homeostasis by generating diverse cell types, including chondrocytes, osteoblasts and marrow stromal cells. The emerging concept postulates that there exists a distinct type of skeletal stem cell that is closely associated with the growth plate1-4, which is a type of cartilaginous tissue that has critical roles in bone elongation5. The resting zone maintains the growth plate by expressing parathyroid hormone-related protein (PTHrP), which interacts with Indian hedgehog (Ihh) that is released from the hypertrophic zone6-10, and provides a source of other chondrocytes11. However, the identity of skeletal stem cells and how they are maintained in the growth plate are unknown. Here we show, in a mouse model, that skeletal stem cells are formed among PTHrP-positive chondrocytes within the resting zone of the postnatal growth plate. PTHrP-positive chondrocytes expressed a panel of markers for skeletal stem and progenitor cells, and uniquely possessed the properties of skeletal stem cells in cultured conditions. Cell-lineage analysis revealed that PTHrP-positive chondrocytes in the resting zone continued to form columnar chondrocytes in the long term; these chondrocytes underwent hypertrophy, and became osteoblasts and marrow stromal cells beneath the growth plate. Transit-amplifying chondrocytes in the proliferating zone-which was concertedly maintained by a forward signal from undifferentiated cells (PTHrP) and a reverse signal from hypertrophic cells (Ihh)-provided instructive cues to maintain the cell fates of PTHrP-positive chondrocytes in the resting zone. Our findings unravel a type of somatic stem cell that is initially unipotent and acquires multipotency at the post-mitotic stage, underscoring the malleable nature of the skeletal cell lineage. This system provides a model in which functionally dedicated stem cells and their niches are specified postnatally, and maintained throughout tissue growth by a tight feedback regulation system.

A histology guide for performing human cadaveric studies: SQIP 2024 what to look for with light microscopy.

Histological observation under light microscopy has long been used in human cadaveric studies. However, it can distort the interpretations of findings if not used appropriately; there is no guide for its proper use. The aim of this article is to revisit and discuss the correct use of histology in human cadaveric studies, following discussions with experts in multiple fields of medicine, and to create the first guide for such usage. We reached a consensus with the experts, agreeing that when this principle (structure, quantification, interaction, position: SQIP) is applied to histological observations, the findings will be interpreted correctly. Appropriate use of this recommendation can make human cadaveric studies more accurate and informative. This is the first histology guide for human cadaveric studies.

Bone regeneration via skeletal cell lineage plasticity: All hands mobilized for emergencies: Quiescent mature skeletal cells can be activated in response to injury and robustly participate in bone regeneration through cellular plasticity.

An emerging concept is that quiescent mature skeletal cells provide an important cellular source for bone regeneration. It has long been considered that a small number of resident skeletal stem cells are solely responsible for the remarkable regenerative capacity of adult bones. However, recent in vivo lineage-tracing studies suggest that all stages of skeletal lineage cells, including dormant pre-adipocyte-like stromal cells in the marrow, osteoblast precursor cells on the bone surface and other stem and progenitor cells, are concomitantly recruited to the injury site and collectively participate in regeneration of the damaged skeletal structure. Lineage plasticity appears to play an important role in this process, by which mature skeletal cells can transform their identities into skeletal stem cell-like cells in response to injury. These highly malleable, long-living mature skeletal cells, readily available throughout postnatal life, might represent an ideal cellular resource that can be exploited for regenerative medicine.

Single-Cell RNA-Sequencing Reveals the Skeletal Cellular Dynamics in Bone Repair and Osteoporosis.

The bone is an important organ that performs various functions, and the bone marrow inside the skeleton is composed of a complex intermix of hematopoietic, vascular, and skeletal cells. Current single-cell RNA sequencing (scRNA-seq) technology has revealed heterogeneity and sketchy differential hierarchy of skeletal cells. Skeletal stem and progenitor cells (SSPCs) are located upstream of the hierarchy and differentiate into chondrocytes, osteoblasts, osteocytes, and bone marrow adipocytes. In the bone marrow, multiple types of bone marrow stromal cells (BMSCs), which have the potential of SSPCs, are spatiotemporally located in distinct areas, and SSPCs' potential shift of BMSCs may occur with the advancement of age. These BMSCs contribute to bone regeneration and bone diseases, such as osteoporosis. In vivo lineage-tracing technologies show that various types of skeletal lineage cells concomitantly gather and contribute to bone regeneration. In contrast, these cells differentiate into adipocytes with aging, leading to senile osteoporosis. scRNA-seq analysis has revealed that alteration in the cell-type composition is a major cause of tissue aging. In this review, we discuss the cellular dynamics of skeletal cell populations in bone homeostasis, regeneration, and osteoporosis.

Axin2+ Peribiliary Glands in the Periampullary Region Generate Biliary Epithelial Stem Cells That Give Rise to Ampullary Carcinoma.

BACKGROUND & AIMS: Peribiliary glands (PBGs), clusters of epithelial cells residing in the submucosal compartment of extrahepatic bile ducts, have been suggested as biliary epithelial stem/progenitor cell niche; however, evidence to support this claim is limited because of a lack of PBG-specific markers. We therefore sought to identify PBG-specific markers to investigate the potential role of PBGs as stem/progenitor cell niches, as well as an origin of cancer. METHODS: We examined the expression pattern of the Wnt target gene Axin2 in extrahepatic bile ducts. We then applied lineage tracing to investigate whether Axin2-expressing cells from PBGs contribute to biliary regeneration and carcinogenesis using Axin2-CreERT mice. RESULTS: Wnt signaling activation, marked by Axin2, was limited to PBGs located in the periampullary region. Lineage tracing showed that Axin2-expressing periampullary PBG cells are capable of self-renewal and supplying new biliary epithelial cells (BECs) to the luminal surface. Additionally, the expression pattern of Axin2 and the mature ductal cell marker CK19 were mutually exclusive in periampullary region, and fate tracing of CK19+ luminal surface BECs showed gradual replacement by CK19- cells, further supporting the continuous replenishment of new BECs from PBGs to the luminal surface. We also found that Wnt signal enhancer R-spondin3 secreted from Myh11-expressing stromal cells, corresponding to human sphincter of Oddi, maintained the periampullary Wnt signal-activating niche. Notably, introduction of PTEN deletion into Axin2+ PBG cells, but not CK19+ luminal surface BECs, induced ampullary carcinoma whose development was suppressed by Wnt inhibitor. CONCLUSION: A specific cell population receiving Wnt-activating signal in periampullary PBGs functions as biliary epithelial stem/progenitor cells and also the cellular origin of ampullary carcinoma.

Autocrine regulation of mesenchymal progenitor cell fates orchestrates tooth eruption.

Formation of functional skeletal tissues requires highly organized steps of mesenchymal progenitor cell differentiation. The dental follicle (DF) surrounding the developing tooth harbors mesenchymal progenitor cells for various differentiated cells constituting the tooth root-bone interface and coordinates tooth eruption in a manner dependent on signaling by parathyroid hormone-related peptide (PTHrP) and the PTH/PTHrP receptor (PPR). However, the identity of mesenchymal progenitor cells in the DF and how they are regulated by PTHrP-PPR signaling remain unknown. Here, we show that the PTHrP-PPR autocrine signal maintains physiological cell fates of DF mesenchymal progenitor cells to establish the functional periodontal attachment apparatus and orchestrates tooth eruption. A single-cell RNA-seq analysis revealed cellular heterogeneity of PTHrP+ cells, wherein PTHrP+ DF subpopulations abundantly express PPR. Cell lineage analysis using tamoxifen-inducible PTHrP-creER mice revealed that PTHrP+ DF cells differentiate into cementoblasts on the acellular cementum, periodontal ligament cells, and alveolar cryptal bone osteoblasts during tooth root formation. PPR deficiency induced a cell fate shift of PTHrP+ DF mesenchymal progenitor cells to nonphysiological cementoblast-like cells precociously forming the cellular cementum on the root surface associated with up-regulation of Mef2c and matrix proteins, resulting in loss of the proper periodontal attachment apparatus and primary failure of tooth eruption, closely resembling human genetic conditions caused by PPR mutations. These findings reveal a unique mechanism whereby proper cell fates of mesenchymal progenitor cells are tightly maintained by an autocrine system mediated by PTHrP-PPR signaling to achieve functional formation of skeletal tissues.

Synergy of single-cell sequencing analyses and in vivo lineage-tracing approaches: A new opportunity for stem cell biology.

Single-cell sequencing technologies have rapidly progressed in recent years, and been applied to characterize stem cells in a number of organs. Somatic (postnatal) stem cells are generally identified using combinations of cell surface markers and transcription factors. However, it has been challenging to define micro-heterogeneity within "stem cell" populations, each of which stands at a different level of differentiation. As stem cells become defined at a single-cell level, their differentiation path becomes clearly defined. Here, this viewpoint discusses the potential synergy of single-cell sequencing analyses with in vivo lineage-tracing approaches, with an emphasis on practical considerations in stem cell biology.

Clinical Anatomy Research Association in Oral and Maxillofacial Surgery.

ABSTRACT: Sufficient knowledge of anatomy is critical for oral and maxillofacial surgeons to provide the best treatment to their patients. The authors have recently established the "Clinical Anatomy Research Association in Oral and Maxillofacial Surgery." There is no doubt as to the benefits of collaboration between oral and maxillofacial surgeons/radiologists and anatomists. In this article, we share what was accomplished at the first annual online conference and discuss our mission for the future.

Bone regeneration: A message from clinical medicine and basic science.

Population aging is a global phenomenon and with it, the number of bone fractures increases due to higher incidences of osteoporosis. Bone fractures in the elderly increase the risk of bedridden status and mortality. Therefore, the control of osteoporosis and bone fracture is important for healthy life expectancy, and the fundamental understanding of its pathogenesis and its application in treatment is of great social significance. To solve these clinical problems, it is necessary to integrate clinical medicine and basic research. Bone regeneration after a fracture is an essential function of the living body. The prevailing view is that a small number of resident skeletal stem cells are solely responsible for regenerative capacity. Although these cells have long been considered to be in the bone marrow, it has been shown that they are also present in the growth plate and periosteum. More recently, distinct types of cells in the bone marrow, including bone marrow stromal cells, osteoblast progenitor cells, and osteoblasts, have been shown to participate in bone regeneration. Interestingly, the cellular plasticity of differentiated cells, rather than active recruitment of resident stem cell populations, may largely account for regeneration of bone tissues; terminally differentiated cells de-differentiate into a stem cell-like state, and then re-differentiate into regenerating bone. In this review, we discuss the clinical risk and preventive therapy of bone fractures and the current concept of bone regeneration in basic mechanical insights, which may prove useful to both clinicians and researchers.

What are the retromolar and bifid/trifid mandibular canals as seen on cone-beam computed tomography? Revisiting classic gross anatomy of the inferior alveolar nerve and correcting terminology.

PURPOSE: Since cone-beam computed tomography was developed, a number of radiological studies on the bifid mandibular canals (BMCs) and trifid mandibular canals (TMCs) have been reported. However, many of the suggested subtypes of the BMC described in the literature seem to be normal branches of the inferior alveolar nerve. This might be due to a lack of revisiting classic anatomical studies in the field of radiology. Therefore, such studies are revisited here. METHODS: A database search using PubMed and Google Scholar was conducted on BMC and TMC. Eighty-nine articles underwent full-text assessment. The reported three classifications of BMC and the six modified classifications were reviewed and compared to the intramandibular inferior alveolar nerve branches. RESULTS: Some subtypes of BMC and TMC simply represent normal inferior alveolar nerve branches, i.e., retromolar branch, molar branch (alveolar branch/dental branch), large mental branch, or communicating branch. Others such as Naitoh's type III BMC and forward canal might be a true BMC. CONCLUSION: We found that the bifid mandibular canal is an additional intramandibular canal running parallel to the mandibular canal with/without confluence with the main canal through comparison of classifications of BMC/TMC between the radiology and anatomy fields.

GNAO1 organizes the cytoskeletal remodeling and firing of developing neurons.

Developmental and epileptic encephalopathy (DEE) represents a group of neurodevelopmental disorders characterized by infantile-onset intractable seizures and unfavorable prognosis of psychomotor development. To date, hundreds of genes have been linked to the onset of DEE. GNAO1 is a DEE-associated gene encoding the alpha-O1 subunit of guanine nucleotide-binding protein (GαO ). Despite the increasing number of reported children with GNAO1 encephalopathy, the molecular mechanisms underlying their neurodevelopmental phenotypes remain elusive. We herein present that co-immunoprecipitation and mass spectrometry analyses identified another DEE-associated protein, SPTAN1, as an interacting partner of GαO . Silencing of endogenous Gnao1 attenuated the neurite outgrowth and calcium-dependent signaling. Inactivation of GNAO1 in human-induced pluripotent stem cells gave rise to anomalous brain organoids that only weakly expressed SPTAN1 and Ankyrin-G. Furthermore, GNAO1-deficient organoids failed to conduct synchronized firing to adjacent neurons. These data indicate that GαO and other DEE-associated proteins organize the cytoskeletal remodeling and functional polarity of neurons in the developing brain.

Attitude changes toward prenatal testing among women with twin pregnancies after the introduction of noninvasive prenatal testing: A single-center study in Japan.

AIM: This study aimed to evaluate changes in prenatal testing among women with twin pregnancies before and after the introduction of noninvasive prenatal testing (NIPT). To date, no consensus on prenatal testing for twin pregnancies has been reached in Japan. METHODS: Women pregnant with twins who requested prenatal testing at Kyushu Medical Center from 2005 to 2018 were included in this study. Genetic counseling was provided to all participants. Their chosen methods of testing were collected and classified as invasive diagnosis (ID), noninvasive screening (NIS), and no test requested (NR). Parity, chorionicity, and methods of conception were assessed as attributes. The study period was divided into three terms according to testing availability in our center. RESULTS: After NIPT was introduced in our center, the use of ID methods decreased and eventually disappeared while NIS came to the forefront. NR was also the preferred choice of women with twin pregnancies before the introduction of NIPT and decreased but did not disappear after introducing NIPT. Women with twin pregnancies who underwent assisted reproduction initially showed hesitation to undergo testing but showed a strong preference for NIS after the introduction of NIPT. Differences in choice according to parity, chorionicity, and methods of conception were found before the introduction of NIPT but disappeared after introducing NIPT. CONCLUSION: Increasing information about NIPT has apparently influenced the attitudes of women with twin pregnancies to prenatal testing in Japan. In particular, those who conceive through assisted reproductive technologies exhibited a strong preference for NIPT.

Mandibular canal vs. inferior alveolar canal: Evidence-based terminology analysis.

INTRODUCTION: The mandibular canal, as it was formerly named in Terminologia Anatomica (TA), has also been called the inferior alveolar (nerve) canal in many scientific publications. This study was conducted to investigate how these terms have been understood in different regions and different areas of expertise and to discuss the appropriate future application of the term "mandibular canal." METHODS: A literature search was conducted using PubMed, and articles using different terms for this structure were classified into two groups, inferior alveolar canal/inferior alveolar nerve canal (IAC/IANC) and the mandibular canal (MC). The 50 most recent articles in each group were included. Publication year, journal title, country of the first author, and affiliation of all authors were recorded in both groups for all 100 articles. RESULTS: There was a significant difference between the IAC/IANC and MC groups in the numbers of anatomy journals, other journals, and anatomy affiliations. Turkey published most frequently with a total of 15 articles, followed by Iran with 10 articles, and China/India/United States with seven each. When the six countries of the first author that had three or more publications in each group were compared, only Turkey appeared in both groups; otherwise, different countries were in the two groups. CONCLUSIONS: Based on the results of this analysis, and considering that the tentative new term "inferior alveolar foramen" is used in the latest TA, we suggest that the mandibular canal should be renamed the "inferior alveolar canal."

Comprehensive review of lower third molar management: A guide for improved informed consent.

Lower third molar removal is the most commonly performed dental surgical procedure. Nevertheless, it is difficult to ensure that all the informed consent forms given to patients are based on the best evidence as many newer publications could change the conclusions of previous research. Therefore, the goal of this review article is to cover existing meta-analyses, randomized control trials, and related articles in order to collect data for improved and more current informed consent.

Skeletal Stem Cells for Bone Development and Repair: Diversity Matters.

PURPOSE OF REVIEW: Skeletal stem cells (SSCs) are considered to play important roles in bone development and repair. These cells have been historically defined by their in vitro potential for self-renewal and differentiation into "trilineage" cells; however, little is known about their in vivo identity. Here, we discuss recent progress on SSCs and how they potentially contribute to bone development and repair. RECENT FINDINGS: Bone is composed of diverse tissues, which include cartilage and its perichondrium, cortical bone and its periosteum, and bone marrow and its trabecular bone and stromal compartment. We are now at the initial stage of understanding the precise identity of SSCs in each bone tissue. The emerging concept is that functionally dedicated SSCs are encased by their own unique cellular and extracellular matrix microenvironment, and locally support its own compartment. Diverse groups of SSCs are likely to work in concert to achieve development and repair of the highly functional skeletal organ.

A three-dimensional analysis of primary failure of eruption in humans and mice.

OBJECTIVES: Primary failure of eruption (PFE) is a genetic disorder exhibiting the cessation of tooth eruption. Loss-of-function mutations in parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP) receptor (PTH/PTHrP receptor, PPR) were reported as the underlying cause of this disorder in humans. We showed in a PFE mouse model that PTHrP-PPR signaling is responsible for normal dental follicle cell differentiation and tooth eruption. However, the mechanism underlying the eruption defect in PFE remains undefined. In this descriptive study, we aim to chronologically observe tooth eruption and root formation of mouse PFE molars through 3D microCT analyses. SETTING AND SAMPLE POPULATION: Two individuals with PFE were recruited at Showa University. A mouse PFE model was generated by deleting PPR specifically in PTHrP-expressing dental follicle and divided into three groups, PPRfl/fl ;R26RtdTomato/+ (Control), PTHrP-creER;PPRfl/+ ;R26RtdTomato/+ (cHet), and PTHrP-creER;PRRfl/fl ;R26RtdTomato/+ (cKO). MATERIALS AND METHODS: Images from human PFE subjects were acquired by CBCT. All groups of mouse samples were studied at postnatal days 14, 25, 91, and 182 after a tamoxifen pulse at P3, and superimposition of 3D microCT images among three groups was rendered. RESULTS: Mouse and human PFE molars exhibited a similar presentation in the 3D CT analyses. The quantitative analysis in mice demonstrated a statistically significant decrease in the eruption height of cKO first and second molars compared to other groups after postnatal day 25. Additionally, cKO molars demonstrated significantly shortened roots with dilacerations associated with the reduced interradicular bone height. CONCLUSIONS: Mouse PFE molars erupt at a much slower rate compared to normal molars, associated with shortened and dilacerated roots and defective interradicular bones.

Microsurgical Anatomy of the Superior Wall of the Mandibular Canal and Surrounding Structures: Suggestion for New Classifications for Dental Implantology.

Our goal was to clarify the relationship between the superior wall of the mandibular canal and the presence of teeth. We also sought to study the structural changes of the mandibular canal after tooth loss. Twenty sides from 10 dry mandibles derived from six males and four females were used for this study. The age of the specimens at the time of death ranged from 57 to 91 years. The mandibles were cut in the midline resulting in 20 hemi-mandibles. The presence of teeth (from the second premolar to the third molar) was recorded for each hemi-mandible. The mandibular canal in the body of the mandible was divided into four areas, that is, Areas 1-4. The superior wall of the mandibular canal and a cancellous bone pattern above the mandibular canal were observed. Next, the mandibular canal was horizontally cut at its center and the superior wall of the mandibular canal observed inferiorly. A total of 75 areas (20 dentulous areas and 55 edentulous areas) were produced. The distal view was classified into three groups, Type I (trabecular pattern), Type II (osteoporotic pattern), and Type III (dense/irregular pattern). The Type I pattern was found in 60.0% (12/20) of the dentulous areas and 32.7% of the edentulous areas. While the Type II pattern was found in 15.0% (23/55) of the dentulous areas and 41.8% of the edentulous areas. The inferior view was classified into four groups depending on the surface of the superior wall of the mandibular canal, that is, Class I (trabecular pattern), Class II (osteoporotic pattern), Class III (dense/irregular pattern), and Class IV (smooth).The Class I pattern was seen most frequently (55.0%) in dentulous areas and the Class IV pattern (45.5%) most frequently in edentulous areas. Based on these results, we conclude that the superior wall of the mandibular canal could change following tooth loss. Clin. Anat. 33:223-231, 2020. © 2019 Wiley Periodicals, Inc.