Microvessel network, CGRP and NPY distribution, and CBCT image assessments of the mandibular canal between dentulous and edentulous cadavers using principal component analysis

doi:10.21203/rs.3.rs-1989083/v1

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Research Article

Microvessel network, CGRP and NPY distribution, and CBCT image assessments of the mandibular canal between dentulous and edentulous cadavers using principal component analysis

https://doi.org/10.21203/rs.3.rs-1989083/v1

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Objectives

This study focused on the detailed distribution of microvessels in the mandibular canal (MC) and the localized expression of neurotransmitters to assess the relationship between microvessel supply and MC structure using cone-beam computed tomography (CBCT) for safe and reliable dental implant treatment.

Materials and methods

In this study, mandibles from 45 sides of 23 human cadavers aged 76–104 years were examined microscopically, immunohistochemically and by CBCT analysis. These data were further evaluated by principal component analysis (PCA).

Results

The MC structure was classified into three types, including complete (57.0%, 228/400), partial (33.8%, 135/400) and unclear (9.2%, 37/400), for dentulous and edentulous subjects. Calcitonin gene-related peptide- and neuropeptide Y-positive reactions were mainly found in the inferior region of the MC. PCA results revealed that developed capillaries were mainly localized in the molar regions.

Conclusions

Our findings indicate that microvessels express neurotransmitters on the vasa nervorum of the inferior alveolar nerve and vein and artery. These irregular large microvessels are mainly located in dentulous subjects, which may provide useful information for dental implant treatment.

Clinical Relevance:

The microvessel supply pattern might signify a risk of pain in the molar and premolar regions in dentulous and edentulous subjects undergoing CBCT analysis of the MC for dental implant surgery based on oral health-related quality of life.

mandibular canal

microvessels

CGRP

NPY

CBCT

Numerous studies of the inferior nerve of the mandibular canal (MC) have reported the size and location of the MC within the mandible [1–5]. The position of the MC is important in dental surgical treatment because the mandibular nerve, inferior alveolar artery, and vein contained in the MC pass through the canal from the middle of the MC and are distributed in the alveolar bone. The distribution and location of these structures in the MC have been considered important for the assessment of dental treatment risk [2, 3], pain [6] and oral health-related quality of life [7–9]. Moreover, a reduced trabecular bone volume is associated with a reduced amount of bone surrounding the alveolar nerve [10]. However, these previous findings about the inferior alveolar nerve, artery and vein were mainly obtained through intramandibular measurements. Recently, we clarified the existence of a lateral branch of the inferior alveolar nerve, artery and vein that emerges from this MC and reported that it exhibits specific directionality [11].

However, blood vessels exiting from the MC towards the alveolar bone could not be clearly confirmed in the bone images obtained by cone beam computed tomography (CBCT). It is important to define these blood vessel exitances through cadaver dissection. The microvessels also form a complex supply system in the ganglion nerve [12, 13]. The complex microvessel supply in the spinal cord is important and useful information for clinical treatment [14]. The MC also differs from the circulatory system, which has blood vessels entering and exiting from the spinal canal [15] that nourish the spinal cord. The MC structure does not allow nerves and arteries to freely enter and exit. However, the nerves, arteries and veins contained in the MC are surrounded by the serosa. Therefore, the complex microvessel supply in the circulatory system of the intermandibular canal components needs to be defined. Moreover, the inferior alveolar nerve exhibits a complex course with twisting, and the mandibular nerve runs through the canal from the mandibular foramen to the mental foramen or the incisive canal, which can also affect the location of the blood vessels that the branches emerge from the MC [16]. However, there are few reports about the vascular network of the MC-related mandibular alveolar nerve, artery, and vein. Nerves, arteries, and veins may enter and exit through the serosa membranes in the middle of the passage from the mandibular foramen to the mental foramen of the MC. Therefore, we need to examine the microvessel supply and structure of the MC in order to avoid bleeding and pain in dental treatment, especially dental implant treatment, which would also contribute to the improvement of oral health-related quality of life. The neurotransmitter, such as calcitonin gene-related peptide (CGRP) and neuropeptide Y (NPY), are related to remodelling of the perivascular nerve [17] and nervous supply [18, 19]. Therefore, in this study, we morphologically clarified the existence of the vascular network existing in the serous membrane surrounding the nerves and arteries/veins in the MC, and we further defined the localization of neuropeptides, such as NPY and CGRP, based on vascular features. These research data are useful for the purpose of elucidating the role of small blood vessels in the passageway and providing useful information for evaluating hypersensitivity reactions and risks in dental treatment and posttreatment care.

Subjects

A total of 45 mandibles from 23 cadavers preserved at Tokyo Medical University were used in this study. The participant ages ranged from 76 to 104 years (mean: 84.3±7.2) in this study. These cadaver samples were injected with 10% formalin with return perfusion via the femoral artery and then stored in the refrigerator (4°C) for one year. The head and neck were removed from human cadavers, dissected and used for CBCT analysis (n=20) and macroscopic observations (n=6), and whole-mount immunohistochemical observations (n=9) and immunohistochemical sections (n=10) were performed in this study.

CBCT scanning

Human head images were acquired using CBCT (AZ 3000CT; Asahi Roentgen Industry, Kyoto, Japan). The images of the MC and surrounding structures of the mandible were also acquired from the samples at premolar and molar regions. The CBCT images were obtained using a tube potential of 85 kV and a tube current of 4 mA, and the scans encompassed cylindrical areas of 79φ×71 mm at high resolution (voxel size=0.155 mm). From the three-dimensional CBCT images, the measurements of the mandibular elements of the MC were collected using NEOPREMIUM (ASAHIRENTOGEN Ind. Co., Ltd., Kyoto, Japan). The median sagittal plane and the Frankfort plane were defined as reference planes. We defined the locations in detail in 5 five regions (1-5). The measurement area was obtained by randomly sampling from regions the four sides (superior, inferior, buccal, and lingual surfaces) of the five measured regions (1)-(5): (1) the region from the mental foramen to the first premolar (SupMe, InfMe, BucMe, and LingMe); (2) the first and second premolar regions (Sup1-2PM, Inf1-2PM, Buc1-2PM, and Ling1-2PM); (3) the first and second molar regions (Sup1-2M, Inf1-2M, Buc1-2M, and Ling1-2M); (4) the third molar region (Sup3rdM, Inf3rdM, Buc3rdM, and Ling3rdM); and (5) the region from the third molar to the mandibular foramen (SupMf, InfMf, BucMf, and LingMf) in the MC (Fig. 1a, d). The mandible lower edge plane was positioned parallel to the floor, the midsagittal plane was positioned perpendicular to the floor, and the position where the mental foramen was clearly noted mesiodistally served as the measurement position. We also classified three types of MC structures according to CBCT image analysis. Three radiology specialists defined the MC structure classification three times.

Measurement points and identification of microvessel patterns on the serous membrane in the MC by macroscopic observation

We observed and measured the four sides of the indicated CBCT regions in the MC (Fig. 1). On these sides of the five regions of the mandible, we measured four surface sides (superior, inferior, buccal, lingual sides) of the mandibular nerve to determine the blood vessel supply ratio. We measured the ratio of micro blood vessels in the MC (% 0.1 mm per square) using image-analysing computer software (WinROOF; MITANI Co. Japan), which employed measurement methods of Mitsuoka et al. with slight modifications (2018). The average positive intensity (OD) was defined based on the percentage of blood vessels exhibiting a weak (1,999≤ OD) colour level (Figs. 1b, c)

Whole-mount immunohistochemical analysis of CGRP and NPY on the serous membrane in the MC

Whole-mount specimens were washed with distilled water for 24 hr, incubated with 3% H₂O₂ for 20 min to eliminate endogenous peroxidase activity and digested with 0.02% proteinase K (Wako, Tokyo, Japan) for 1 hr at 38°C. After overnight fixation in 4% paraformaldehyde, the samples were washed with distilled water for 50 min, and the proteinase K digestion and overnight fixation steps were repeated. The samples were then washed with phosphate-buffered saline (PBS) for 30 min. Then, samples were sequentially incubated in 2.5%, 5% and 10% sucrose in PBS and then subjected to three freeze/thaw cycles. After overnight incubation with 2% Triton X-100 in PBS at 4°C, the samples were washed three times with PBS for 1 hr and then incubated for 1 hr at room temperature with 2% normal goat serum/PBS (pH 7.2) containing 0.05% Tween 20 to prevent nonspecific antibody binding. Samples were then incubated with rabbit polyclonal antibodies against NPY (diluted 1:100; Lab Vision, CA, USA) and CGRP (1:1000; Biogenesis, NH, USA) or normal goat serum as a negative control (n=1). The samples were washed three times with PBS for 1 hr. Following the manufacturer’s instructions, sections were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, USA). The samples were then washed three times with PBS for 1 hr. The staining was visualized using 0.02% H₂O₂ and 0.1% (1 mg/ml) diaminobenzidine tetrahydrochloride in 0.1 M Tris-HCl, pH 7.2. Images were acquired using a stereomicroscope (Leica MZ 16FA; Leica Microsystems, USA) with the Leica Application Suite software (Leica Microsystems).

Sectional immunohistochemical analysis of CGRP

The molar region of the mandible of each cadaver was used for immunohistochemical observation following CBCT scanning. Mandibles containing the MC were fixed with tissue fixative (Genostaff Co., Ltd.). Decalcified and calcified specimens were prepared and used for analyses. The specimens were cut using a diamond cutter (IsoMet^TM Low Speed; Berg Engineering & Sales Company, Inc.) at 200 μm or sectioned at 5 μm using a rotary microtome. Sections for immunohistochemical analyses were incubated overnight at 4°C with rabbit polyclonal antibodies against CGRP (1:1,000; ab47027, Kenbridge). The sections were counterstained with Mayer’s haematoxylin, dehydrated, and mounted with Malinol (Muto Pure Chemicals Co., Ltd.). Other serial sections were subject to haematoxylin and eosin double staining. Images were acquired using a microscope (Leica DM 2500 16; Leica Microsystems) equipped with Leica Application Suite software (Leica Microsystems).

Statistical analysis

Pearson’s correlation coefficient was used to determine the correlations. The level of significance was set at P<0.05. Multivariate modelling in principal component analysis (PCA) was used to estimate the interactions of the CBCT measured data, microvessel pattern and density data differences between left and right (LR), age, sex, right and left sides based on the significant components of PCA that were performed on individuals (Cassar-Malek et al., 2007). We also performed cluster analyses using the average linkage between groups (hierarchical cluster analysis algorithms). All statistical analyses were performed using IBM SPSS Statistics Base version 26 (IBM Corporation, New York, USA).

Coronal CBCT images of the MC

We measured 400 superior, inferior, buccal, and lingual surface regions from the mental foramen region to the lingual foramen region. The MC was classified into three types, including complete (type 1, 57.0%, 228/400), partial (type 2, 33.8%, 135/400) and unclear (type 3, 9.2%, 37/400), in the four surface regions, including superior, inferior, buccal, and lingual (Fig. 2). The four surface regions, including the superior surface region (type 1, 53%, 53/100; type 2, 38%, 38/100; type 3, 9%, 9/100), inferior surface region (type 1, 69%, 69/100; type 2, 23%, 23/100; type 3, 8%, 8/100), buccal surface region (type 1, 44%, 44/100; type 2, 45%, 45/100; type 3, 11%, 11/100), and lingual surface region (type 1, 61%, 3/100; type 2, 30%, 30/100; type 3, 9%, 9/100), were found in the mandible (Fig. 2). Comparing the MC structure types between dentulous and edentulous subjects, significant differences were not found (Fig. 4a). PCA was performed for 24 parameters of the structure ratio in the MC, and the results were plotted in two-dimensional space defined by the two axes of component 1 (x-axis) and component 2 (y-axis). The two principal components significantly explained 32.7% (component 1, 17.8%; component 2, 14.9%) of the information in the data set of the human MC structure. The positive contributions from four elements (Lig1-2PM, 0.854; Buc1-2PM, 0.835; Inf1-2PM, 0.800; Sup1-2PM, 0.678) and a negative contribution from one element (RL, -0.536) explained component 1. Positive contributions from seven elements (InfMf, 0.843; BucmMf, 0.790; LigMf, 0.744; SupMf, 0.638; Inf3rdM, 0.602; sex, 0.515; teeth, 0.509) were noted for component 2 (Fig. 5a).

Microvessel patterns on the serous membrane of the MC in dentulous and edentulous subjects

We measured 667 collagenous bundles in the inferior, superior, lingual, and buccal surface regions in the MC. On the inner side of the MC, small artery branches were located on the inner side of the MC in contrast to some veins noted in a slightly larger cross-sectional image near the small artery (Fig. 3). We classified 5 types of microvessels in the serous membrane of the inferior alveolar nerve, artery, and vein. These 5 types include the following: a network of complex large microvessels (diameter, >0.5 mm) (type a) (4.19%, 28/667), a network of irregular large vessels (diameter, >0.5 mm) with numerous fine vessels (diameter, > 0.5 mm) (type b) (7.35%, 49/667), a network of numerous vessels of intermediate thickness (diameter, 0.5-0.1 mm) (type c) (29.23%, 195/667), a network of irregular intermediate vessels (0.5-0.1 mm) with numerous fine vessels (type d) (29.23%, 195/667), and a structure with scattered fine microvessels (diameter, <0.1 mm) (type e) (30.0%, 200/667) (Fig. 3). Comparing the microvessel patterns between dentulous and edentulous subjects, a significant difference in type b was found in dentulous subjects, in contrast to Type e, which was commonly found in edentulous subjects (Fig. 4b). Among the five regions, the highest number of blood vessel patterns was found in the first and second molar regions (21.9%, 146/667). Among the surface regions, the highest numbers of blood vessel patterns exhibited the following order: the superior surface region (26.5%, 177/667), inferior surface region (25.6%, 171/667), lingual surface (24.7%, 165/667) and buccal surface regions (23.2%, 154/667) of the inferior alveolar nerve. Moreover, compared to the microvessel density for each measurement section, significant differences in microdensity were found in the InfMF, Inf1-2M, SupMe, and LingMe groups in dentulous subjects in contrast to edentulous subjects (Fig. 4c).

PCA of microvessel pattern types

The variables were plotted in a two-dimensional space defined by two axes, including component 1 (x-axis) and component 2 (y-axis) of the ratio of microvessel density of the serous membrane of the inferior alveolar nerve, artery. The two principal components significantly explained 45.1% (component 1, 24.6%; component 2, 20.5%) of the information in the data set of the human microvessels on the serous membrane of the inferior alveolar nerve, artery, and vein. PCA was performed for 24 parameters associated with the microvessel density ratio in the mandible. These parameters were plotted in two-dimensional space defined by two axes of component 1 (x-axis) and component 2 (y-axis). Component 1 was explained by the positive contributions of five elements (Sup1-2PM, 0.689; Inf1-2PM, 0.655; BucMf, 0.625; Sup1-2M, 0.528; teeth, 0.538) and the negative contributions of five elements (Buc3rdM, -0.846; age -0.707SupMe, -0.675; Inf3rdM, -0.530; RL, -0.523). Component 2 was explained by the positive contributions from seven elements (SupMf, 0.842; LingMf, 0.791; sex, 0.768; Inf3rdM, 0.765; BucMe, 0.751; Buc1-2PM, 0.573; BucMf, 0.553) and a negative contribution from one element (Sup1-2M, -0.613) (Fig. 5b).

PCA of microvessel density on four sides (superior, inferior, buccal, and lingual surfaces) in the MC

The variables were plotted in a two-dimensional space defined by two axes: component 1 (x-axis) and component 2 (y-axis) of the microvessel density ratio of the serous membrane of the inferior alveolar nerve, and artery. The two principal components significantly explained 64.9% (component 1, 44.0%; component 2, 20.9%) of the information in the data set of the human microvessels on the serous membrane of the inferior alveolar nerve, artery, and vein. PCA was performed for 24 parameters in the mandible of ratio microvessel density, which were plotted in two-dimensional space defined by the two axes of component 1 (x-axis) and component 2 (y-axis). The positive contributions of fifteen elements (Buc3rdM, 0.934; Lig1-2M, 0.895; Lig1-2PM, 0.888; LIg3rdM, 0.870; BucMf, 0.843; LigMf, 0.840; Buc1-2PM, 0.825; Buc1-2M, 0.824; Inf1-2PM, 0.761; SupMf, 0.717; sex, 0.697; Sup1-2M, 0.665; BucMe, 0.644; LigMe, 0.645; Sup3rdM, 0.562) and a negative contribution from one element (age, -0.851) explained component 1. Positive contributions from five elements (SupMe, 0.852; BucMe, 0.687; LigMe, 0.682; RL, 0.613; Sup1-2PM, 0.561) were found, and a negative contribution from four elements (Inf1-2M, -0.756; InfMf, -0.654; teeth, -0.650; Inf3rdM, -0.614) was found in component 2 (Fig. 5c).

Immunohistochemical staining of CGRP and NPY on the serous membrane of the inferior alveolar artery and vein

Upon macroscopic observation, strong CGRP- and NPY-positive reactions were found on the small vessels on the serous membrane surrounding the inferior alveolar nerve, artery, and vein in the MC. Both CGRP- and NPY-positive reactions were mainly found in the inferior region of the serous bundles containing the inferior alveolar nerve, artery, and vein. Weak staining was noted in other regions (Fig. 6). In the frontal calcified section, CGRP-positive reactions were mainly found in the inner epithelium cell body of branches of the inferior artery and vein (Fig. 6).

The characteristics of the regions noted along the MC of the inferior alveolar nerve, arteries, and veins are important in implant treatment, as noted in previous reports [20-25]. In conventional dentistry, little attention has been paid to the small capillaries and nerves that emerge from the MC. Furthermore, in order to avoid bleeding and pain in dental treatment, especially in dental implant treatment, investigating the supply and structure of microvessels in the MC contributes to the improvement of oral health-related QOL for patients [7-9].

Microvessel distribution in the MC

Hou-Qing Long et al [26] investigated changes in microvessel density using a chronic spinal cord compression rat model, and its dynamics are oriented to reflect spinal cord compression. Numerous capillary nerves are located on the surface, and these nerves also form a plexus on the surface side of the geniculate ganglion in the facial nerve canal of the temporal bone [13]. In our study, many capillaries were noted on the surface of the inferior alveolar nerve, arteries, and veins in the MC, as well as a few microvessels along the long axis in the mandible. Most of the microvessels running along the long axis in the MC were 0.5-0.1 mm in diameter, and many of these microvessels exhibited scattered thin vascular networks on the collagenous bundle in the MC. The mandibular foramen and the molars, the lower part of the molars, and the upper part of the molars had many small vessels with a diameter of 0.5-0.1 mm. This finding suggests that the inferior alveolar nerves, arteries, and veins that enter through the mandibular foramen in the ramus immediately exit many other blood vessels from the MC into the bone. Moreover, our CBCT observations also support our microvessel analysis results, which indicate that the inferior alveolar nerve sends some branches to the molar region in the MC. There is a possible risk for complications in the mandible associated with clinical treatment given that the MC is completely open at this site with a complex large microvessel network (Type a) on the inferior alveolar nerve, artery and vein. Chronic inflammatory demyelinating polyradiculoneuropathy causes nerve enlargement, and other causes for MC enlargement have been reported [27-30]. These results suggest the possibility for intraosseous neuropathy and sensory deficits [31]. Thus, morphological changes, such as enlargement of the MC, are affected by changes in blood vessels and the nerves present inside. In our study, many of the blood vessels (0.1-0.5 mm or more in diameter) were adjacent to or above the serosa of the inferior alveolar nerve, artery, and vein in MC from the mandibular foramen to the molar or premolar region of the mandible. This finding suggests that blood vessels developed and were distributed from the ramus of the mandible to the molars, and careful attention should be given when performing dental clinical treatments at these sites. More Type c-e microvessels were noted in the serous membrane in both dentulous and edentulous subjects. However, when comparing dentulous and edentulous subjects, significantly more irregular large vessels (Type b) are noted in the MC in dentulous subjects. Significantly more scattered fine microvessels (Type e) were found in edentulous subjects. According to our CBCT results, numerous branch canals also appear in the inferior, superior, lingual, or labial side regions of the MC. These findings suggest that blood vessels and extremely small blood vessels may exit the MC. Large blood vessels emerge as side branches, and the risk for clinical complications for dental implants located around these vessels is presumed to be high. Moreover, importantly, this risk is higher in dentulous subjects. Previous reports suggested the presence of many lateral branches in the molars of the mandible [11], and high vascular density is noted from the third to first molar of the mandible. These results suggest the risk of clinical complications at these molars. Furthermore, comparisons between dentulous and edentulous subjects reveal a high vascular density in the superior region of the MC in dentulous subjects, whereas a high vascular density was noted in the upper part of the MC and the lingual side of the mental region of the mandible in edentulous subjects. These findings suggest that the area around the mental region of the mandible is a clinically high-risk area in edentulous subjects. PCA revealed that the capillaries in the molars developed well when the occlusion was maintained, and the proportion of capillaries decreased with ageing. Moreover, in cluster analysis, the density of blood vessels in the inferior region of the molar sand near the mandibular foramen region and the inferior region of the premolars was affected by the occlusal state. On the other hand, the size of the capillaries is similar to that noted in the mandibular foramen, inferior and superior regions of the premolars, and lingual side of the mandibular foramen region, which contain microvessels. According to PCA of the CBCT canal structure of the mandible, the canal structure of the edentulous subject is most affected at the mental foramen region of the mandible. The findings in these regions are also related to ageing and age. These findings also suggest that ageing female edentulous subjects exhibit the greatest risk for dental implant treatment. In contrast, PCA of the microvessel structure and density and teeth element reveal that these factors are important in the first to second molars, third molar, and mental foramen regions of the mandible. The molars present in these regions are potentially at risk for dental treatments based on our results.

CGRP and NPY distribution

Immunohistochemical staining for CGRP and NPY on the serous membrane revealed similar staining in the same regions. In general, blood flow is controlled by the sympathetic nerve and the parasympathetic nerve. CGRP controls vasodilatation [32, 33]. In addition, CGRP controls smooth muscle by reducing blood flow, which restricts the blood supply to the inferior alveolar nerve, artery, and vein. Microfil perfusion followed by micro-CT analysis showed that CGRP overexpression enhanced peri-implant angiogenesis via increased vessel volume and thickness in streptozotocin-induced diabetic rats [34]. The osteoclasts at the epiphyseal trabeculae facing the growth plate are partly regulated by CGRP-positive nerve fibres, and CGRP is a crucial element in bone metabolism during bone growth and development [35]. The effect of CGRP on bone remodelling could partly occur through its action on blood vessels, thereby regulating local blood flow [36]. This mechanism demonstrates that CGRP exhibits the potential to regulate diabetes-induced dysfunction in angiogenesis and osseointegration; moreover, the promoting role of CGRP can be targeted [37]. CGRP plays an important role in bone formation based on vasodynamics. Therefore, our results reveal many structures forming a plexus with developed CGRP-positive capillaries in the molar region, which is an important region in dental treatment. Similar to the evaluation of capillaries in the molar region of the PCA, the presence of the molar branch, which is a branch of the inferior alveolar nerve, arteries, and veins, supports the strong CGRP expression noted at this site. The implantation of a vascular bundle with CGRP and NPY expression induced an significant effect in vivo [38]. In general, NPY has pleiotropic activities ranging from the control of obesity to anxiolysis and cardiovascular function [39]. Bjurholm et al. [40] suggested that NPY exhibits specific roles in the local regulation of bone physiology, such as blood flow, bone formation or resorption for NPY. NPY was almost exclusively expressed close to or within the blood vessel walls, mostly in the vicinity of the epiphyseal plate. Nerve fibres immunoreactive for the sensory neuro-marker CGRP and sympathetic neuro-marker NPY affect the periodontal ligament during tooth movement through the stimulation of bone remodelling [41]. In our results, NPY was observed on the developed microvessels on the serous membrane of the inferior alveolar nerve, artery, and vein of the MC. Importantly, CGRP-positive reactions generally occurred in the same region. Moreover, some small vessels were located at the nonmineralized MC zone and inner border of the MC as demonstrated by haematoxylin and eosin double staining section of frontal section of the mandible. These results suggested that strong CGRP and NPY expression may facilitate maintenance of the path to the molar region and teeth by bone remodelling. These sensory and sympathetic neuro-markers may be regulated by bone remodelling during blood vessel development. Therefore, the examination of microvessel structure, density and related neurotransmitters, such as CGRP and MPY, in our study provided information to support safe dental implant treatment and could contribute to the improvement of oral health-related QOL for patients.

The calcified area in the MC mostly appeared at the premolar and molar regions. In addition, complex fine microvessels formed a network at the vasa nervorum of the serous membrane in the whole MC. CGRP and NPY expression was mainly found at these vasa nervorum sites of the MC, and the expression of these factors affect microcirculation. These results regarding the different distributions of the dental canal and microvessel network structures in dentulous and edentulous subjects may provide useful information for dental implant treatment. Moreover, these morphological evaluations suggest the possibility of providing useful information for dental treatments.

MC, mandibular canal

SupMf, superior surface of the mental foramen region

InfMf, inferior surface of the mental foramen region

BucMf, buccal surface of the mental foramen region

LigMf, lingual surface of the mental foramen region

Sup3rdM, superior surface of the third molar region

Inf3rdM, inferior surface of the third molar region

Buc3rdM, buccal surface of the third molar region

Lig3rdM, lingual surface of the third molar region

Sup1-2M, superior surface of the first and second molar regions

Inf1-2M, inferior surface of the first and second molar regions

Buc1-2M, buccal surface of the first and second molar regions

Lig1-2M, lingual surface of the first and second molar regions

Sup1-2PM, superior surface of the first and second premolar regions

Inf1-2PM, inferior surface of the first and second premolar regions

Buc1-2PM, buccal surface of the first and second premolar regions

Lig1-2PM, lingual surface of the first and second premolar regions

SupMe, superior surface of the mental foramen region

InfMe, inferior surface of the mental foramen region

BucMe, buccal surface of the mental foramen region

LigMe, lingual surface of the mental foramen region

Acknowledgements

We thank members of the Department of Anatomy, Tokyo Medical University and Dr. Rieko Asaumi of the Department of Oral and Maxillofacial Radiology, School of Life Dentistry at Tokyo, Nippon Dental University for their contribution to this research.

Compliance with ethical standards

Conflict of interest the authors declare that they have no conflicts of interest.

Ethics approval

This study was performed in line with the principles of the Declaration of Helsinki (as revised in 2013). The cadavers used in the present study were donated to Tokyo Medical University and Nippon Dental University, Tokyo, Japan, based on the Act on Body Donation for Medical and Dental Education. All the donors willingly signed a form consenting to body donation for education and research, and all the donors could revoke the intended donation at any time without any disadvantages. The collection and use of mucosa data from all cadavers were permitted by the families of the cadavers and then performed according to the Low Concerning Cadaver Dissection and Preservation enacted in Japan in 1949. The study was approved by the Ethics Review Committee of Nippon Dental University (July 21, 2018; no. NDU-T2015-20) and the Institutional Review Board of Tokyo Medical University (February 6, 2019; TMU, no. T2018-0042).

Informed Consent

Formal consent is not required for this type of study.

Author Contributions

Masachika Takiguchi, DDS, PhD: study conception and design; data interpretation; manuscript revision and final approval of the version to be published; agreement to be accountable for all aspects of the work

Iwao Sato, PhD: study conception and design; data acquisition and interpretation; manuscript revision and final approval of the version to be published; agreement to be accountable for all aspects of the work

Yoko Ueda, DDS, PhD: data acquisition and interpretation; manuscript revision and final approval of the version to be published; agreement to be accountable for all aspects of the work

Kawata Shinichi, PhD: data acquisition and interpretation; manuscript revision and final approval of the version to be published; agreement to be accountable for all aspects of the work

Kenta Nagahori, PhD: data acquisition and interpretation; manuscript revision and final approval of the version to be published; agreement to be accountable for all aspects of the work

Takuya Omotehara, PhD: data acquisition and interpretation; manuscript revision and final approval of the version to be published; agreement to be accountable for all aspects of the work

Tomiko Yakura, DDS, PhD: data acquisition and interpretation; manuscript revision of work and final approval of the version to be published; agreement to be accountable for all aspects of the workZhong-Lian Li MD, PhD.: data acquisition and interpretation; revising of work and final approval of the version to be published; agreement to be accountable for all aspects of the work

Masahiro Itoh MD, PhD: study conception; manuscript drafting and revision and final approval of the version to be published; agreement to be accountable for all aspects of the work

ORCID

Masachika Takiguchi: https://orcid.org/0000-0001-6680-2207

Iwao Sato: https://orcid.org/0000-0002-7035-2525

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Microvessel network, CGRP and NPY distribution, and CBCT image assessments of the mandibular canal between dentulous and edentulous cadavers using principal component analysis

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