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Expression of syndecan-1, 2, 3, and 4 mRNAs during the late stages of tooth germ formation was investigated by in situ hybridization, using [

Mandibular condylar cartilage is a representative secondary cartilage, differing from primary cartilage in various ways. Syndecan is a cell-surface heparan sulfate proteoglycan and speculated to be involved in chondrogenesis and osteogenesis. This study aimed to investigate the expression patterns of the syndecan family in the developing mouse mandibular condylar cartilage. At embryonic day (E)13.0 and E14.0, syndecan-1 and -2 mRNAs were expressed in the mesenchymal cell condensation of the condylar anlage. When condylar cartilage was formed at E15.0, syndecan-1 mRNA was expressed in the embryonic zone, wherein the mesenchymal cell condensation is located. Syndecan-2 mRNA was mainly expressed in the perichondrium. At E16.0, syndecan-1 was expressed from fibrous to flattened cell zones and syndecans-2 was expressed in the lower hypertrophic cell zone. Syndecan-3 mRNA was expressed in the condylar anlage at E13.0 and E13.5 but was not expressed in the condylar cartilage at E15.0. It was later expressed in the lower hypertrophic cell zone at E16.0. Syndecan-4 mRNA was expressed in the condylar anlage at E14.0 and the condylar cartilage at E15.0 and E16.0. These findings indicated that syndecans-1 and -2 could be involved in the formation from mesenchymal cell condensation to condylar cartilage. The different expression patterns of the syndecan family in the condylar and limb bud cartilage suggest the functional heterogeneity of chondrocytes in the primary and secondary cartilage.

The synchondrosis in the cranial base is an important growth center for the craniofacial region. Abnormalities in the synchondroses affect the development of adjacent regions, including the craniofacial skeleton. Here, we report that the transcription factor TBX1, the candidate gene for DiGeorge syndrome, is expressed in mesoderm-derived chondrocytes and plays an essential and specific role in spheno-occipital synchondrosis development by inhibiting the expression of genes involved in chondrocyte hypertrophy and osteogenesis. In Tbx1-deficient mice, the spheno-occipital synchondrosis was completely mineralized at birth. TBX1 interacts with RUNX2, a master molecule of osteoblastogenesis and a regulator of chondrocyte maturation, and suppresses its transcriptional activity. Indeed, deleting Tbx1 triggers accelerated mineralization due to accelerated chondrocyte differentiation, which is associated with ectopic expression of downstream targets of RUNX2 in the spheno-occipital synchondrosis. These findings reveal that TBX1 acts as a regulator of chondrocyte maturation and osteogenesis during the spheno-occipital synchondrosis development. Thus, the tight regulation of endochondral ossification by TBX1 is crucial for the normal progression of chondrocyte differentiation in the spheno-occipital synchondrosis.

The incisive canal for nerves and vessels is generally thought to run along a suture between the incisive bone (IN) and maxilla. In contrast, there was a report saying the canal passes through the IN or primary palate in human fetuses. Examination of sagittal and frontal sections from 69 fetuses (31 of gestational age [GA] 9-15 weeks and 38 of GA 26-34 weeks) showed that the canal often penetrated the IN at the nasal half of its course and that, in other fetuses, the canal penetrated the IN along its entire course, irrespective of involvement of the nasopalatine duct. Canals developing in and corresponding to parts of the suture resulted in partial enlargement of the thin and tight sutures, which contained loose tissue, vessels, nerves and even a duct. Small processes of the IN were identified as upper irregular parts continuous with inferior main masses of bone in frontal sections but as bone fragments in sagittal sections. In some sections, a thin layer of the maxilla along the canal covered the medial or inferior aspect of the IN. Therefore, the incisive canal with or without duct exhibited a spectrum of variations in topographical relation to the IN-maxillary border. Because the primitive oronasal communication passes through the suture, the nasopalatine duct may have originated from the secondary developed elongation of the nasal epithelium at midterm. A large incisive fossa along the midline on the oral surface of the palate might make a macroscopic finding of variants difficult even in adults.

The human nervus terminalis (terminal nerve) and the nerves to the vomeronasal organ (VNON) are both associated with the olfactory nerves and are of major interest to embryologists. However, there is still limited knowledge on their topographical anatomy in the nasal septum and on the number and distribution of ganglion cells along and near the cribriform plate of the ethmoid bone. We observed serial or semiserial sections of 30 fetuses at 7-18 weeks (crown rump length [CRL], 25-160 mm). Calretinin and S100 protein staining demonstrated not only the terminal nerve along the anterior edge of the perpendicular lamina of the ethmoid, but also the VNON along the posterior edge of the lamina. The terminal nerve was composed of 1-2 nerve bundles that passed through the anterior end of the cribriform plate, whereas the VNON consisted of 2-3 bundles behind the olfactory nerves. The terminal nerve ran along and crossed the posterior side of the nasal branch of the anterior ethmoidal nerve. Multiple clusters of small ganglion cells were found on the lateral surfaces of the ethmoid's crista galli, which are likely the origin of both the terminal nerve and VNON. The ganglions along the crista galli were ball-like and 15-20 µm in diameter and, ranged from 40-153 in unilateral number according to our counting at 21-µm-interval except for one specimen (480 neurons; CRL, 137 mm). An effect of nerve degeneration with increasing age seemed to be masked by a remarkable individual difference.

Chondrogenesis is accompanied by not only cellular renovation, but also metabolic stress. Therefore, macroautophagy/autophagy is postulated to be involved in this process. Previous reports have shown that suppression of autophagy during chondrogenesis causes mild growth retardation. However, the role of autophagy in chondrocyte differentiation still largely remains unclear. Here, we show the important role of autophagy on chondrogenesis. The transition of mesenchymal cells to chondrocytes was severely impaired by ablation of Atg7, a gene essential for autophagy. Mice lacking Atg7 after the transition exhibited phenotypes severer than mutant mice in which Atg7 was removed before the transition. Atg7-deficient chondrocytes accumulated large numbers of glycogen granules, hardly proliferate and died specifically in the proliferative zone without any ER-stress signal. Our results suggest that the suppression of autophagy in prechondrogenic cells drives compensatory mechanism(s) that mitigate defective chondrogenesis, and that autophagy participates in glycogenolysis to supply glucose in avascular growth plates. Abbreviations: DDIT3/CHOP: DNA damage inducible transcript 3; ER: endoplasmic reticulum; NFE2L2/NRF2: nuclear factor, erythroid derived 2, like 2; SQSTM1/p62: sequestosome 1; STBD1: starch-binding domain-containing protein 1

The main purpose of this in situ hybridization study was to investigate MMPs and TIMPs mRNA expression in developing mandibular condylar cartilage and limb bud cartilage. At E14.0, MMP-2, -14, TIMP-1 and -2 mRNAs were expressed in the periosteum of mandibular bone, and in the condylar anlage. At E15.0 MMP-2, -14, TIMP-1 and -2 mRNAs were expressed in the perichondrium of newly formed condylar cartilage and the periosteum of developing bone collar, whereas, expression of MMP-14 and TIMP-1 mRNAs were restricted to the inner layer of the periosteum/perichondrium. This expression patterns continued until E18.0. Further, from E13.0 to 14.0, in the developing tibial cartilage, MMP-2, -14, and TIMP-2 mRNAs were expressed in the periosteum/perichondrium, but weak MMP-14 and no TIMP-1 mRNA expression was recognized in the perichondrium. These results confirmed that the perichondrium of condylar cartilage has characteristics of periosteum, and suggested that MMPs and/or TIMPs are more actively involved in the development of condylar (secondary) cartilage than tibial (primary) cartilage. MMP-9-positive cells were observed in the bone collar of both types of cartilage, and they were consistent with osteoclasts/chondroclasts. MMP-13 mRNA expression was restricted to the chondrocytes of the lower hypertrophic cell zone in tibial cartilage at E14.0, indicating MMP-13 can be used as a marker for lower hypertrophic cell zone. It was also expressed in chondrocytes of newly formed condylar cartilage at E15.0, and continuously expressed in the lower hypertrophic cell zone until E18.0. These results confirmed that progenitor cells of condylar cartilage are rapidly differentiated into hypertrophic chondrocytes, which is a unique structural feature of secondary cartilage different from that of primary cartilage.

In situ hybridization of decorin and biglycan mRNA, principal members of small leucine-rich proteoglycan, was performed using [35S]-labeled RNA probes, in the context of the hypothesis that they show different expression patterns associated with osteoblast differentiation in mice. We adopted two ossifying sites that can clearly follow the developmental process of bone formation: ossifying tympanic ring and developing bone collar of mandibular condylar cartilage. Decorin mRNA was expressed in osteoblasts of developing tympanic ring at E14.0, as well as of developing bone collar at E15.0, but biglycan mRNA was not, indicating decorin mRNA was expressed earlier in newly differentiating osteoblasts than biglycan. With maturation of osteoblasts, biglycan mRNA became expressed and maintained its expression both in the outer region (periosteum) and in the interior region (endosteum) of bone. By contrast, decorin mRNA expression was maintained in the outer region but diminished in the interior region. These results indicate that decorin and biglycan show differential expression patterns in differentiating osteoblasts and play specific roles in bone formation.

Three vascular routes to the inner ear are known: (a) through the internal acoustic meatus with the vestibulocochlear nerve; (b) from the endolymphatic duct aperture; and (c) along the canal of Cotugno (CC) inserted into the vestibular part of the ear from the superior or brain side. The third is believed to contain only veins. Examinations of 33 human embryos and fetuses at 6-40 weeks demonstrated that (a) the CC appeared as a recess of epidural mesenchymal tissues at the superior aspect of the otic capsule cartilage in embryos and it was inserted deeply to issue multiple peripheral divisions inferolaterally and posteriorly at midterm; (b) the CC consistently passed through a ring of the superior or anterior semicircular canal and contained both, the arteries from the vestibulocochlear nerve origin at the midbrain and the vein draining into the sigmoid sinus or petrosal sinuses; and (c) the CC appeared not to contribute to ossification of the otic capsule cartilage but, after endochondral ossification of the internal ear, woven bone development occurred along a smooth interface of the CC with the ossified ear. In contrast, another interface between the developing bone and the residual cartilage of the otic capsule was rough and wavy with many short bony columns, called osseous globules. In addition, the endolymphatic duct accompanied veins but no arteries. Our results show that the CC is a major vascular route to the vestibular part of the otic capsule cartilage, but its role appears to be limited after ossification.

Chondroitin sulfate (CS) proteoglycan is a major component of the extracellular matrix and plays an important part in organogenesis. To elucidate the roles of CS for craniofacial development, we analyzed the craniofacial morphology in CS N-acetylgalactosaminyltransferase-1 (T1) gene knockout (KO) mice. T1KO mice showed the impaired intramembranous ossification in the skull, and the final skull shape of adult mice included a shorter face, higher and broader calvaria. Some of T1KO mice exhibited severe facial developmental defect, such as eye defects and cleft lip and palate, causing embryonic lethality. At the postnatal stages, T1KO mice with severely reduced CS amounts showed malocclusion, general skeletal dysplasia and skin hyperextension, closely resembling Ehlers-Danlos syndrome-like connective tissue disorders. The production of collagen type 1 was significantly downregulated in T1KO mice, and the deposition of CS-binding molecules, Wnt3a, was decreased with CS in extracellular matrices. The collagen fibers were irregular and aggregated, and connective tissues were dysorganized in the skin and calvaria of T1KO mice. These results suggest that CS regulates the shape of the craniofacial skeleton by modulating connective tissue organization and that the remarkable reduction of CS induces hypoplasia of intramembranous ossification and cartilage anomaly, resulting in skeletal dysplasia.

Objectives: Mouth breathing caused by nasal obstruction (owing to abnormal pressure of masticatory muscles) affects craniofacial growth and development. The influence of unilateral nasal obstruction on jaw-closing muscles was investigated in rats to reveal one of the etiologic mechanisms. Materials and Methods: Forty 8-day-old male Wistar rats were used in this study. Experimental rats were subjected to left-sided nasal obstruction by burning the external nostril tissue at the age of 8 days. Pulse oxygen saturation was recorded each week. Morphologic changes were evaluated by staining with hematoxylin and eosin (to assess the cross-sectional area) and by adenosine triphosphatase activity staining (to assess the myosin heavy chain isoform composition). Immunohistochemical and reverse transcription quantitative real-time polymerase chain reaction analyses of tumor necrosis factor-α and glucose transporter 4 were carried out at 5 and 9 weeks of age. Results: The cross-sectional area of the jaw-closing muscles was lower in the experimental group at 9 weeks of age. The percentage of myosin heavy chain-2a in masseter muscles was increased in the experimental group compared with the control group. An increase in the tumor necrosis factor-α messenger RNA and protein levels and a decrease in the glucose transporter 4 messenger RNA and protein levels at 5 and 9 weeks of age in the jaw-closing muscles in the experimental group were noted. Conclusions: Unilateral nasal obstruction could affect the morphology and contractile characteristics of jaw-closing muscles during growth in rats.

To obtain a better understanding of multi-laminar deep fascia covering skeletal muscles, we examined nondecalcified histological sections of the arm and thigh of 20 human fetuses aged 25-33 weeks. Morphologies of the fasciae varied between sites and specimens, but the initial morphology was most likely to be a thin and loose sheet on the external surface of the muscles (fascia-1 or F1). When the F1 became wavy, thick and tight, it was detached from the muscle surface. Beneath the F1, the second lamina of fascia (F2) appeared on the muscle surface and it was also detached. In this manner at 25-33 weeks' gestation, fasciae covering the triceps and vastus lateralis muscles had a three-layered configuration (F1, F2, and F3). Due to significant individual variations, this process was not correlated to the ages and sizes of specimens. Muscle contractions might facilitate the detachment. In these muscles, the intramuscular tendon joined the F2 or F3 and the latter became thick and aponeurotic. Along the finally developed lamina, muscle fibers carried a desmin-positive spot for insertion. Increased laminae were accompanied by a reduced number of CD68-positive macrophages and, nerves were absent, near the developing fascia. In contrast to skin ligaments or superficial fasciae showing de novo development in loose tissue, a deep or muscle-covering fascia seemed to originate from the skeletal muscle itself at the surface, and this process was repeated to produce multi-layered fascia. Depending on sites, collagen fibers were added by the intramuscular tendon. Anat Rec, 301:1235-1243, 2018. © 2018 Wiley Periodicals, Inc.

The detailed distribution of Pacinian corpuscles was evaluated by viewing the transverse sections of all fingers and thumbs, including the interdigital areas, from eight hands of five fetuses of gestational age 28-33 weeks (crown-rump length 230-290 mm). Among the 40 fingers and thumbs, serial sections were prepared for 3D reconstructions of nerve elements in the distal and middle phalangeal segments of three fifth fingers; in these three fingers, the distal segment contained 45-75 Pacinian corpuscles. These Pacinian corpuscles were 0.2-1.0 mm in length and 0.05-0.3 mm in thickness, oriented along the proximodistal axis and arranged along the palmar digital nerve branches. Other than beneath the digital skin, small corpuscles (

Objective To access detailed distribution and age-dependent changes of oral epithelial pearls. Design Investigation and analysis with human fetal serial sections. Setting Institute of Embryology. Methods This study examined serial frontal sections of the upper and lower jaws of 19 human fetuses at 12 to 18 weeks and of the lower jaws of four late-stage fetuses. Results The upper jaw contained more than 20 midline and more than 60 lateral pearls greater than 20 μm in diameter, whereas the lower jaw contained fewer than 30 pearls of the same size. Midline pearls in the upper jaw were often cylindrical or rugby-ball shaped, whereas all pearls in the lower jaw were small and spherical. Epithelial pearls in the upper jaw started developing along the upper midline until 12 weeks; lateral pearls and additional midline pearls (or strictly, paramedian pearls) developed until 15 weeks. In the lower jaw, however, pearl development started at 18 weeks and was almost always from the dental lamina. Some of the fetuses assessed had an open nasopalatine canal without a duct, but there was no fibrous connection between this canal and pearls. Similarly, the lip frenulum or incisive suture was not connected with these pearls. Conclusion The timing and sequence of development suggest that postfusion rupture of the palate by midline pearls was unlikely.

Using longitudinal semiserial sections of 12 lower extremities from 8 human foetuses at 15-18 weeks, we compared foetal morphologies of the knee in specimens with and without fabellae. We also compared the fabella, if present, with the hallucal sesamoid in the same foetus. Cartilaginous fabella, positive for versican and tenascin by immunohistochemistry, was found in 5 of the 8 foetuses. This structure was embedded in a thick and tight lateral fibrous band, providing a common origin of the plantaris muscle and the lateral head of the gastrocnemius muscle. The plantaris was covered by the lateral head of the gastrocnemius, but these 2 muscles were separated by a distinct fascia or space. Notably, the foetal fabella did not attach to the joint capsule. In the 3 specimens without fabellae, the lateral fibrous band was thin, containing a fibrous mass, negative for versican and tenascin, in place of the fabella. The "medial" head of the gastrocnemius faced or covered the plantaris, while the lateral head was continuous with the plantaris. A hallucal cartilaginous sesamoid, positive for versican and tenascin, was present in all 8 specimens. It carried a flat surface facing the joint cavity and was covered by tendons of the short muscles of the foot. Because of the difference in topographical relation of muscles between specimens with or without fabella, rather than mechanical stress to the tendon, fabella development may require a distinct plantaris muscle independent of the gastrocnemius. We discussed about an evolutionary aspect of the fabella and plantaris muscle.

The gene expression and protein synthesis of small leucine-rich proteoglycans (SLRPs), including decorin, biglycan, fibromodulin, and lumican, was analyzed in the context of the hypothesis that they are closely related to tooth formation. In situ hybridization, immunohistochemistry, and organ culture with metabolic labeling of [35S] were carried out in mouse first molar tooth germs of different developmental stages using ICR mice at embryonic day (E) 13.5 to postnatal day (P) 7.0. At the bud and cap stage, decorin mRNA was expressed only in the surrounding mesenchyme, but not within the tooth germ. Biglycan mRNA was then expressed in the condensing mesenchyme and the dental papilla of the tooth germ. At the apposition stage (late bell stage), both decorin and biglycan mRNA were expressed in odontoblasts, resulting in a switch of the pattern of expression within the different stages of odontoblast differentiation. Decorin mRNA was expressed earlier in newly differentiating odontoblasts than biglycan. With odontoblast maturation and dentin formation, decorin mRNA expression was diminished and localized to the newly differentiating odontoblasts at the cervical region. Simultaneously, biglycan mRNA took over and extended its expression throughout the new and mature odontoblasts. Both mRNAs were expressed in the dental pulp underlying the respective odontoblasts. At P7.0, both mRNAs were weakly expressed but maintained their spatial expression patterns. Immunostaining showed that biglycan was localized in the dental papillae and pulp. In addition, all four SLRPs showed clear immunostaining in predentin, although the expressions of fibromodulin and lumican mRNAs were not identified in the tooth germs examined. The organ culture data obtained supported the histological findings that biglycan is more predominant than decorin at the apposition stage. These results were used to identify biglycan as the principal molecule among the SLRPs investigated. Our findings indicate that decorin and biglycan show spatial and temporal differential expressions and play their own tissue-specific roles in tooth development.

The anatomical position of the vermiform appendix varies among adults, and these variations are responsible for differences in the symptoms of appendicitis. However, to date no study has examined how and when these variations occur during fetal development. The present study examined horizontal sections of 27 midterm fetuses (crown rump length [CRL] 38-97 mm, gestational age approximately 8-15 weeks). There were 10 fetuses (CRL 56 mm or more) in which the cecum and appendix were in a posterosuperior site near the right kidney (postmigration phase), and 12 fetuses (CRL 39-72 mm) in which the ileocecal junction and appendix remained on the visceral surface of the liver in the anterior or anterolateral abdominal cavity (migration phase, after physiological umbilical herniation). Analysis of the 12 fetuses in the migration phase indicated that the appendix extended inferiorly in eight fetuses and superiorly in four fetuses. Likewise, a "preileal" appendix (a morphology in which the distal part of the appendix was in front of the terminal ileum) was present in eight of these fetuses. Extension of the appendix superiorly or inferiorly during the migration phase seems unrelated to the topographical relationship of the appendix with the terminal ileum at the postmigration phase in fetuses and in adults. Conversely, it seems likely that a retroileal appendix leads to a coiled appendix behind the ileocecal junction. "Guidance" by the liver surface seemed to be important for posterior migration, which ended with the ascent of the liver. Clin. Anat., 33:667-677, 2020. © 2019 Wiley Periodicals, Inc.

Matrix components of growth plate cartilage and mandibular condylar cartilage were immunohistochemically analyzed in cartilage calcification insufficient (CCI) rats, a model for dwarf rats. Reduction in total tibial length, elongation of growth plate, and appearance of noncartilaginous regions in the growth plate were observed in CCI rats. Immunoreactivity for type I collagen and hyaluronic acid (HA) staining were observed in the noncartilaginous region. However, weak immunoreactivity was observed for aggrecan, collagen types II and X, and decorin in this region. Transmission electron microscopy indicated that the noncartilaginous region showed a loose network of thin collagen fibrils, indicating that HA is predominantly involved in capturing space of the noncartilaginous region in the growth plate. Meanwhile, the mandibular condylar cartilage in CCI rats also showed elongation of the cartilaginous region and had a noncartilaginous region, predominantly comprising thick collagen fibrils. The structural difference between the two types of cartilages in CCI rats may be due to the presence of the fibrous cell zone and the fibrocartilaginous nature of the normal condylar cartilage. Additionally, the reduction in mandibular length was relatively less than the reduction in tibial length. The outline of the condylar process showed only slight abnormality. These results suggest that the condylar cartilage compensated its growth by supplying the characteristic noncartilaginous region effectively and may adapt to severe structural changes observed in CCI rats.

We assessed the flap valve of the foramen ovale (FO valve) by examining 30 hearts from human fetuses of gestational age 30-40 weeks. We dissected the hearts, examined their macroscopic morphology, and then prepared semiserial sagittal sections across the valve. Although the primary septum is expected to extend along the left atrial face, eight hearts had a superior rim of the fossa ovalis on the left atrial face that was too thick and high, so there was no smooth continuation with the valve. Moreover, three of these eight hearts each had a flap valve that was fused with a long and narrow plate arising from the caval orifice. Histological analysis indicated that 21 specimens each had a candidate primary septum that contained myocardium, although the left sinuatrial valve (LSAV) contained fibrous tissue, but little or no myocardium. In each of 17 hearts, a candidate primary septum was attached to the left atrial face of the fossa, and parts of the LSAV extended to and approached the right atrial face. However, seven of these 17 hearts each had a folded small primary septum. Another four of these 17 hearts each had an LSAV that extended widely to the fossa, and a candidate primary septum (which might be a remnant) attached to the left atrial side of the LSAV. These variations suggest that the LSAV makes a major contribution to the FO valve in some fetal hearts. Consequently, the fetal FO valve appears to have heterogeneous morphology and origin.

Background This study aimed to clarify the effects of underlying diseases on clinical outcomes of patients aboard a world cruise ship. Methods This prospective cohort study included patients who sought physician consultations at an onboard clinic on a 105-day world cruise (September–December 201X) on a ship chartered by a Japanese travel agency. Multivariable logistic regression analysis was performed to ascertain whether any concurrent disease, such as hypertension, was associated with additional onboard treatment by the primary physician or serious events, including unexpected final disembarkation, temporary disembarkation for hospitalization ashore, shore-side referral, and onboard clinic admission. Results Of 313 patients, 182 (58%) had at least one underlying disease. Sixty-eight (22%) required additional treatment, and 24 (8%) experienced serious events. After adjusting for age, sex, and underlying diseases, the 60–69- and 70–74-year age groups had a lower risk of serious events than the ≤59-year age group (odds ratio [OR], 95% confidence interval [CI]: 0.24, 0.069–0.81; p = 0.022 and 0.045, 0.0051–0.47; p = 0.0055). Underlying disease was associated with serious events (OR, 95% CI: 3.2, 1.1–9.5; p = 0.036). Conclusions Unexpected events can occur in patients on world cruises regardless of age. Preexisting diseases may confer higher risk of serious events.

Development of a long muscle belly in foetal extremities generally requires a definite bony insertion of the long tendon. However, in adults, the pes anserinus and the semimembranosus tendon (SMT) are inserted into fasciae. Development of fascial insertions in foetuses was investigated by examining serial histological sections obtained from 7 foetuses at 8-9 weeks and 8 foetuses at 14-16 weeks. The presence of matrix substances and macrophages was also examined by immunohistochemistry. At 8 weeks, the tendons of the semitendinosus, gracilis, sartorius and semimembranosus muscles were straight and inserted into the initial shaft-like proximal end of the tibia on the proximal side of the popliteus muscle. At 9 weeks, however, the medially extending popliteus muscle appeared to push the pes anserinus tendons superficially, with a loss of cartilage insertions. The SMT obtained an attachment to the popliteus muscle. At 14-16 weeks, the SMT divided into thick and thin bundles: the former contained abundant macrophages and inserted into the tenascin-positive perichondrium of the enlarged proximal tibia, while the later without macrophages ended at the joint capsule. The pes anserinus tendons, negative for both versican and tenascin-c, took highly tortuous courses toward the fascia cruris. Because the medial extension of the popliteus muscle was associated with the enlargement of the proximal tibia, the topographical relationship of the popliteus muscle with these 4 tendons changed drastically, resulting in a loss of cartilage insertion of the pes anserinus tendons as well as the division and reconstruction of the SMT.

Background and Objective This study aimed to evaluate the effects of high-frequency pulsed (HiFP) low-level laser therapy (LLLT) on early wound healing of tooth extraction sockets in rats. Study Design/Materials and Methods Bilateral maxillary first molars were extracted from 6-week-old Sprague-Dawley rats. Sockets on the right were treated by HiFP low-level diode laser irradiation (904–910 nm); the left sides served as unirradiated controls. LLLT (0.28 W, 30 kHz, 200-ns pulse, 0.6% duty cycle, 61.2 J/cm2 total power density) was employed immediately after extraction and every 24 hours thereafter. The maxillae including the sockets were resected 3 or 7 days after extraction. Soft-tissue healing was evaluated on days 0, 3, and 7. The bone mineral content (BMC), bone volume (BV), and bone mineral density (BMD) of the extraction sockets were evaluated by microcomputed tomography, and histomorphometric analysis was carried out on day 7. Real-time PCR analysis of osteogenic marker expression and immunohistochemical detection of proliferating cell nuclear antigen (PCNA)-positive cells were performed on day 3. Results Compared with control sites, the un-epithelialized areas of the extracted sites were significantly reduced by irradiation (P = 0.04), and the BMC, BV, and BMD of laser-treated sites were significantly increased (P = 0.004, 0.006, and 0.009, respectively). On day 7, the mean height of newly formed immature woven bone was higher in laser-treated sites (P = 0.24). On day 3, laser-treated sites showed significantly higher osteocalcin mRNA expression (P = 0.04) and PCNA-positive cell numbers (P = 0.01). Conclusion HiFP low-level diode laser irradiation enhanced soft- and hard-tissue healing of tooth extraction sockets. Lasers Surg. Med. 48:955–964, 2016. © 2016 Wiley Periodicals, Inc.

Hyaluronan (HA) is a major constituent molecule in most extracellular matrices and is synthesized by Hyaluronan synthase (Has). In the present study, we examined expression patterns of Has1, -2, -3 mRNA in developing mouse molar and incisor tooth germs from embryonic day (E) 11.5 to postnatal day (P) 7, focusing on Hertwig's epithelial root sheath (HERS) and the apical bud in particular. Has1 mRNA expression was not detected in all tooth germs examined. Has2 mRNA was expressed in the surrounding mesenchyme from E12.0 to 18.0 in both molar and incisor tooth germs, but disappeared after birth. Meanwhile, Has3 mRNA was exclusively expressed within the enamel organ, especially in the inner enamel epithelium (IEE), stellate reticulum (SR), and stratum intermedium (SI) until the early bell stage at E16.0. Has3 mRNA disappeared as IEE differentiated into differentiating ameloblasts (dABs), but remained in SI until the root developmental stage of the molar tooth germ at P7. Has3 mRNA was also expressed in HERS until P7. In incisors, Has3 mRNA was expressed in the apical bud, especially in the transit-amplifying (TA) cell region from E16.0 to P7, and in the papillary layer (PL) adjacent to the mature enamel. These gene expression patterns suggested that Has3 is the main control factor for prenatal and postnatal HA synthesis of the tooth germ, and may in part regulate crown and root formation of the tooth germ, maintenance of stem cell niches in the apical bud as well as mineral transport in PL.

Epidural blocks have been used extensively in infants. However, little histological information is available on the immature neural-dural transition. The neural-dural transition was histologically investigated in 12 late-stage (28–30 weeks) fetuses. The dural sheath of the spinal cord was observed to always continue along the nerve roots with varying thicknesses between specimens and segments, while the dorsal root ganglion sheath was usually very thin or unclear. Immature neural-dural transitions were associated with effective anesthesia. The posterior radicular artery was near the dorsal root ganglion and/or embedded in the nerve root, whereas the anterior radicular artery was separated from the nearest nerve root. The anterior radicular artery was not associated with the dural sheath but with thin mesenchymal tissue. The numbers of radicular arteries tended to become smaller in larger specimens. Likewise, larger specimens of the upper thoracic and lower lumbar segments did not show the artery. Therefore, elimination of the radicular arteries to form a single artery of Adamkiewicz was occurring in late-stage fetuses. The epidural space was filled with veins, and the loose tissue space extended ventrolaterally to the subpleural tissue between the ribs. Consequently, epidural blocks in infants require special attention although immature neural-dural transitions seemed to increase the effect.

Development of mouse gonial bone and initial ossification process of malleus were investigated. Before the formation of the gonial bone, the osteogenic area expressing alkaline phosphatase and Runx2 mRNA was widely recognized inferior to Meckel's cartilage. The gonial bone was first formed within the perichondrium at E16.0 via intramembranous ossification, surrounded the lower part of Meckel's cartilage, and then continued to extend anteriorly and medially until postnatal day (P) 3.0. At P0, multinucleated chondroclasts started to resorb the mineralized cartilage matrix with ruffled borders at the initial ossification site of the malleus (most posterior part of Meckel's cartilage). Almost all CD31-positive capillaries did not run through the gonial bone but entered the cartilage through the site where the gonial bone was not attached, indicating the forms of the initial ossification site of the malleus are similar to those at the secondary ossification center rather than the primary ossification center in the long bone. Then, the reducing process of the posterior part of Meckel's cartilage with extending gonial bone was investigated. Numerous tartrate-resistant acid phosphatase-positive mononuclear cells invaded the reducing Meckel's cartilage, and the continuity between the malleus and Meckel's cartilage was completely lost by P3.5. Both the cartilage matrix and the perichondrium were degraded, and they seemed to be incorporated into the periosteum of the gonial bone. The tensor tympani and tensor veli palatini muscles were attached to the ligament extending from the gonial bone. These findings indicated that the gonial bone has multiple functions and plays important roles in cranial formation. Anat Rec, 302:1916-1933, 2019. © 2019 American Association for Anatomy.

To investigate a role of proteoglycan (PG) on developing mouse mandibular condylar cartilage, effects of β-D-xyloside, which can inhibit normal PG synthesis, was confirmed in organ culture. Anlagen of condylar cartilage at E14.0 were taken and cultured for 6 days with or without β-D-xyloside. Adding β-D-xyloside remarkably reduced condylar cartilage formation, and formed cartilage showed more fibrocartilaginous characteristics. These results indicated that normal PGsynthesis is required for forming intact condylar cartilage. Thickened perichondrium, however, was compensatory formed around cartilage matrix, and consequently outline of mandibular condyle was considerably maintained. This phenomenon might be derived from characteristics of condylar cartilage as secondary cartilage, and condylar cartilage might have ability to manage to execute its own morphogenesis utilizing materials available.

Objective: To clarify composite fibers and cells in the synovial tissues of the cricoarytenoid joint (CA joint). Methods: Routine histology and immunohistrochemistry using sagittal or nearly sagittal sections obtained from 18 elderly cadaveric specimens. Results: The CA joint capsule was thin and contained few elastic fibers. A limited supportive ligament, namely, a thickened fascia of the posterior cricoarytenoid muscles, was sometimes evident on the lateral aspect of the CA joint. However, even in the weaker medial aspect of the joint, no marked destruction of the synovial tissues was found. The CA joint always contained synovial folds—a short medial fold and long lateral folds—but these contained no or few macrophages, lymphocytes, and blood capillaries. In 2 exceptional specimens showing inflammatory cell infiltration in the submucosal tissue of the larynx, the macrophage-rich area extended toward the capsule and medial synovial fold. Conclusions: The lateral aspect of the CA joint was likely to be supported mechanically by the muscle-associated tissues. Strong support of the arytenoid by muscles might reduce the degree of CA joint injury with age. However, some patients with hoarseness due to mucosal inflammation of the larynx might have accompanying synovitis and subsequent cartilage injury in the CA joint.

Matrix components of vascular canals (VCs) in human fetal mandibular condylar cartilage (15–16 weeks of gestation) were analyzed by immunohistochemistry. Prevascular canals (PVCs), consisting of spindle-shaped cells without capillary invasion, were observed within the cartilage. Intense immunoreactivity for collagen type I, weak immunoreactivity for aggrecan and tenascin-C, weak hyaluronan (HA) staining, and abundant argyrophilic fibers in PVCs indicated that they contain noncartilaginous fibrous connective tissues that was different from those in the perichondrium/periosteum. These structural and immunohistochemical features of PVCs are different from those of previously reported cartilage canals of the long bone. Capillaries entered the VCs from the periosteum and ascended through VCs. Following capillary invasion, loose connective tissue had formed in the lower part of VCs, and immunoreactivity for collagen types I and III, tenascin-C, and HA staining was evident in the matrix of loose connective tissue. No chondroclasts or osteogenic cells were seen at the front of capillary invasion, although small, mononuclear tartrate-resistant acid phosphatase (TRAP)-positive cells were present. Meanwhile, TRAP-positive, multinucleated chondroclasts and flattened, osteoblast-like cells were observed in the loose connective tissue at the lower part of VCs. These results may indicate slow progress of endochondral ossification in human fetal mandibular condyle. Further, unique matrix components in PVCs/VCs, which were different from those in cartilage canals in long bone, may reflect the difference of speed of endochondral ossification in cartilage canals and human fetal mandibular condyles. Anat Rec, 298:1560–1571, 2015. © 2015 Wiley Periodicals, Inc.

We investigated the immunohistochemical localization of tenascin-C in 8-week-old rat periodontal ligaments. Tenascin-C immunoreactivity was detected in zones along with cementum and alveolar bone, and more intensely on the resorption surface of alveolar bone than on the formation surface. On the resorbing surface, tenascin-C immunoreactivity was detected in Howship's lacunae without osteoclasts, and in the interfibrous space of the periodontal ligaments, indicating that this molecule works as an adhesion molecule between bone and fibers of periodontal ligaments. Upon experimental tooth movement by inserting elastic bands (Waldo method), the physiological resorption surface of alveolar bone under compressive force showed enhanced bone resorption and enhanced tenascin-C immunoreactivity. However, on the physiological bone formation surface under compressive force, bone resorption was seen only occasionally, and no enhanced tenascin-C immunoreactivity was noted. In an experiment involving excessive occlusal loading to rat molars, transient bone resorption occurred within interradicular septa, but no enhanced tenascin-C immunoreactivity was seen in the periodontal ligaments. These results indicate that tenascin-C works effectively on the bone resorbing surface of physiological alveolar bone remodeling sites, rather than on the non-physiological transient bone resorbing surface. Fibronectin immunoreactivity was distributed evenly in the periodontal ligaments under experimental conditions. Co-localization of tenascin-C and fibronectin immunoreactivity was observed in many regions, but mutually exclusive expression patterns were also seen in some regions, indicating that fibronectin might not be directly involved in alveolar bone remodeling, but may play a role via interaction with tenascin-C.

In human fetuses, the palatine process of the maxilla is attached to the inferior aspect of the horizontal plate of the palatine bone (HPPB). The fetal palatomaxillary suture is so long that it extends along the anteroposterior axis rather than along the transverse axis. The double layered bony palate disappears in childhood and the transverse suture is formed. To better understand the development of the double layered bone palate, we examined histological sections obtained from 25 fetuses of gestational age 9-11, 16-18 and 30 weeks. The double layered palate was seen in all of the specimens examined. Inferior angulation of the posterior end of the HPPB was evident at 9-11 weeks, but the initial palatine aponeurosis did not attach to the angulation but to a slightly anterior site. Both the maxilla and the HPPB were tightly attached to the vomer at 16-18 weeks. In both bones, bilateral plates met at the midline. The palatomaxillary suture was filled with short, randomly arranged collagen fibers. The nasal end of the suture was covered by a tight periosteum. Immunohistochemical examination of 3 fetuses at 16-18 weeks showed: 1) no expression of versican, tenascin-c or type II collagen in the suture; 2) few mitotic cells positive for proliferating cell nuclear antigen; 3) no or few CD34-positive developing vessels; and 4) no CD68-positive macrophages. These findings suggested that the fetal palatomaxillary suture was inactive for reconstruction and growth and that soft palate muscles likely did not contribute to the development of the double layered configuration.

A newly formed mandibular condylar cartilage at 9 weeks of gestation (37mm crown rump length) was analyzed immunohistochemically. The cells in newly formed cartilage and condensed mesenchymal cells posterior to the cartilage showed alkaline phosphatase immunoreactivity, confirming the mandibular condylar cartilage in human is also derived from periosteum-like tissue, as demonstrated in rodents. The newly formed condylar cartilage simultaneously expresses immunoreactivity for collagen types I and II, aggrecan, versican, and tenascin-C as well as hyaluronan staining but not for collagen type X. Thus newly-formed condylar cartilage has fibrocartilaginous characteristics, but the characteristic that rapidly differentiated into hypertrophic chondrocytes, which is recognized in rodent secondary cartilage, is not conspicuous in human.

Objectives: Temporomandibular joint (TMJ) hypofunction secondary to feeding a liquid dietin the growing period leads to morphological hypoplasia. However, few studies haveevaluated the results ofmechanical loading onthe hypoplastic TMJ. Thisstudy investigatedwhether TMJ hypofunction in rats causes osteoarthritis (OA)-like changes when exposed tomechanical loading.Design: Male 21-day-old Wistar rats weredivided intofour groups.The first group (C)servedas the control. In the second group (W), mechanical loading was applied to the TMJ bycontinuous steady mouth opening (3 h/day for 5 days) from 63 days of age. A jaw-openingdevice was used to hold the mandible open in the maximal mouth-opening position with acobalt–chromium (Co–Cr) wire (w: 0.9 mm). Groups C and W both received a normal harddiet. The third group (L) and fourth group (LW) were fed a liquid diet and group LW weresubjected to the same loading as group W. We evaluated the TMJ using micro-CT, toluidineblue staining and immunohistochemistry of matrix metalloproteinase (MMP)-13.Results: In group LW in the superior and posterior regions of the condyle, bone volumefraction, trabecular thickness and trabecular number were significantly decreased andtrabecular spacing was significantly increased. The ratio of MMP-13 immunopositive cellswas significantly higher than in the other groups. OA-like changes were also observed,including reduced thickness of the cartilage, irregularities in the chondrocytic layer, andcell-free areas.Conclusions: TMJ hypofunction in rats is likely to lead to OA-like changes when exposed tomechanical loading.# 2014 Elsevier Ltd. All rights reserved.* Corresponding author. Tel.: +81 3 5803 5530.E-mail addresses: yoneman.orts@tmd.ac.jp, yoneorts@tmd.ac.jp (I. Yonemitsu).

Rats with dwarfism accompanied by skeletal abnormalities, such as shortness of the limbs, tail, and body (dwarf rats), emerged in a Jcl-derived Sprague-Dawley rat colony maintained at the Institute for Animal Experimentation, St. Marianna University Graduate School of Medicine. Since the dwarfism was assumed to be due to a genetic mutation based on its frequency, we bred the dwarf rats and investigated their characteristics in order to identify the causative factors of their phenotypes and whether they could be used as a human disease model. One male and female that produced dwarf progeny were selected, and reproduction was initiated by mating the pair. The incidence of dwarfism was 25.8% among the resultant litter, and dwarfism occurred in both genders, suggesting that it was inherited in an autosomal recessive manner. At 12 weeks of age, the body weights of the male and female dwarf rats were 40% and 57% of those of the normal rats, respectively. In soft X-ray radiographic and histological examinations, shortening and hypoplasia of the long bones, such as the tibia and femur, were observed, which were suggestive of endochondral ossification abnormalities. An immunohistochemical examination detected an aggrecan synthesis disorder, which might have led to delayed calcification and increased growth plate thickening in the dwarf rats. We hypothesized that the principal characteristics of the dwarf rats were systemically induced by insufficient cartilage calcification in their long bones; thus, we named them cartilage calcification insufficient (CCI) rats.

Immunohistochemical localization of versican and tenascin-C were performed; the periosteum of ossifying mandible and the perichondrium of Meckel's cartilage, of vertebral cartilage, and of mandibular condylar cartilage were examined in midterm human fetuses. Versican immunoreactivity was restricted and evident only in perichondrium of Meckel's cartilage and vertebral cartilage; conversely, tenascin-C immunoreactivity was only evident in periosteum. Therefore, versican and tenascin-C can be used as molecular markers for human fetal perichondrium and fetal periosteum, respectively. Meckel's cartilage underwent endochondral ossification when it was incorporated into the ossifying mandible at the deciduous lateral incisor region. Versican immunoreactivity in the perichondrium gradually became weak toward the anterior primary bone marrow. Tenascin-C immunoreactivity in the primary bone marrow was also weak, but tenascin-C positive areas did not overlap with versican-positive areas; therefore, degradation of the perichondrium probably progressed slowly. Meanwhile, versican-positive perichondrium and tenascin-C-positive periosteum around the bone collar in vertebral cartilage were clearly discriminated. Therefore, the degradation of Meckel's cartilage perichondrium during endochondral ossification occurred at a different rate than did degradation of vertebral cartilage perichondrium. Additionally, the perichondrium of mandibular condylar cartilage showed tenascin-C immunoreactivity, but not versican immunoreactivity. That perichondrium of mandibular condylar cartilage has immunoreactivity characteristic of other periosteum tissues may indicate that this cartilage is actually distinct from primary cartilage and representative of secondary cartilage.

The greater omentum covers the transverse colon from the anterior side in adults, but people might believe the morphology stable once established during fetal life. Sections from 49 midterm and 17 late-stage human fetuses, of gestational ages (GA) 8–15 and 30–38 weeks, respectively, showed complete fusion between the greater omentum and transverse mesocolon after physiological herniation at GA 8–9 weeks; the transverse colon attaching to the anterior aspect of the gastric antrum and pylorus at GA 10–15 weeks; the colon pushing the pylorus or superior portion of the duodenum upward (at GA 10–15 weeks and 30–38 weeks); and the greater omentum without covering the greater portion of the jejunum and ileum but shifted leftward (at GA 30–38 weeks). These subsequent topographical variations of the transverse colon with the stomach and duodenum included the colon tightly fusing with the stomach by a fibrous tissue and; the greater omentum and/or the mesocolon wedged between the stomach and transverse colon. Therefore, in combination, the colon was partly separated from the greater omentum. Moreover, at GA 30–38 weeks, the duodenum consistently showed a horizontal loop in contrast to the usual C-loop in the frontal plane. Consequently, after a complete fusion occurred once between the greater omentum and transverse mesocolon, the topographical change of the upper abdominal viscera seemed to modify, change or even break the initial fusion of the peritoneum. A logical lamination of the peritoneum seemed not to simply connect with the surgical application.

Carbonic anhydrase type IX (CA9) is known to express in the fetal joint cartilage to maintain pH against hypoxia. Using paraffin-embedded histology of 10 human fetuses at 10-16 weeks of gestation with an aid of immunohistochemistry of the intermediate filaments, matrix components (collagen types I and II, aggrecan, versican, fibronectin, tenascin, and hyaluronan) and CA9, we observed all joints and most of the entheses in the body. At any stages examined, CA9-poisitive cells were seen in the intervertebral disk and all joint cartilages including those of the facet joint of the vertebral column, but the accumulation area was reduced in the larger specimens. Glial fibrillary acidic protein (GFAP), one of the intermediate filaments, expressed in a part of the CA9-positive cartilages. Developing elastic cartilages were positive both of CA9 and GFAP. Notably, parts of the tendon or ligament facing to the joint, such as the joint surface of the annular ligament of the radius, were also positive for CA9. A distribution of each matrix components examined was not same as CA9. The bone-tendon and bone-ligament interface expressed CA9, but the duration at a site was limited to 3-4 weeks because the positive site was changed between stages. Thus, in the fetal entheses, CA9 expression displayed highly stage-dependent and site-dependent manners. CA9 in the fetal entheses seemed to play an additional role, but it was most likely to be useful as an excellent marker of mechanical stress at the start of enthesis development.

Objective To investigate if mandibular condylar cartilage is derived from the periosteum of the ossifying mandible or from a separate, programmed blastema. Materials and methods Fetal mice at E14.0–16.0, fetal rats at E16.0–18.0, and human embryos at 9 and 10wks of gestation were used. The initial formation of rat condylar cartilage was investigated by using serial sections and enzyme-histochemistry to detect alkaline phosphatase activity. Histological observations of serial sections of human fetuses as well as 3D-reconstruction models were also analyzed. The expression of collagen type mRNA in developing rat condylar cartilage was directly compared with that in mice by performing in situ hybridization. Results An anlage of the rat condylar process (condylar anlage) was clearly identified in the posterior position of the ossifying mandible and was continuous with it at E16.0. Newly formed rat condylar cartilage was observed at E16.5 and was continuous with the ossifying mandible. Mesenchymal cells in the condylar anlage at E16.0 showed alkaline phosphatase activity and chondrocytes in the newly formed condylar cartilage also showed enzymatic activity. Thus, rat mandibular condylar cartilage that derives from alkaline phosphatase-positive periosteum-like cells is continuous with the ossifying mandible, as previously demonstrated in mice, but rapid differentiation into hypertrophic chondrocytes in rats is not remarkable compared to that in mice. The condylar anlage and the newly formed cartilage were also continuous with the ossifying mandible in human embryos. Conclusions Mammalian mandibular condylar cartilage derives from the periosteum of the ossifying mandible in mice, rats, and humans.

Hyaluronan (also called hyaluronic acid or HA) plays a key role in the morphogenesis of the brain, but little is known about its expression in the human fetal neocortex. Using immunohistochemical methods, we assayed the expression of HA, glial fibrillary acidic protein, vimentin, nestin, and proliferating cell nuclear antigen in paraffin-embedded histologic sections of 8 mid-term fetuses (estimated gestational age, 12-16 weeks; crown-rump length, 75-120mm). At 12-13 weeks, HA was expressed strongly along the membranes of many cells in the cortical plate and the layer 1 or marginal zone, but showed weak, spotty expression in a fiber-rich layer adjacent to the cortical plate, called the cortical stratified transitional field-1 (STF-1 or a primitive form of the subplate). At 15-16 weeks, HA was expressed in the layer 1 and in the early subplate or presubplate, but less strongly in cells of the possible STF-5 near the subventricular zone. However, the positive observation in STF-5 was probably a result of individual difference in development. The developing cortical plate seemed to produce HA in the presubplate to harbor axonal plexus of various afferent systems, while Cajal-Retzius cells were likely to accumulate HA in the layer 1. The HA-rich zones, those sandwiched the cortical plate, might avoid further migration of cortical cells.

Summary In the human middle ear, the annular ligament of the incudostapedial joint and the insertions of the tensor tympani and stapedius muscles contain abundant elastic fibers; i.e., the elastic-fiber-mediated entheses. Hyaluronan also coexists with the elastic fibers. In the present study using immunohistochemistry, we demonstrated the distribution of elastin not only in the incudostapedial joint but also in the other two joints of the middle ear in adults and fetuses. In adults, the expression of elastin did not extend out of the annular ligament composed of mature elastic fibers but clearly overlapped with it. Electron microscopic observations of the annular ligament demonstrated a few microfibrils along the elastic fibers. Thus, in contrast to the vocal cord, the middle ear entheses seemed not to contain elaunin and oxytalan fibers. In mid-term fetuses (at approximately 15–16 weeks of gestation) before opening of the external acoustic meatus, the incudostapedial joint showed abundant elastic fibers, but the incudomalleolar and stapediovestibular joints did not. At this stage, hyaluronan was not colocalized, but distributed diffusely in loose mesenchymal tissues surrounding the ear ossicles. Therefore, fetal development of elastin and elastic fibers in the middle ear entheses is unlikely to require acoustic oscillation. In late-stage fetuses (25–30 weeks), whose ear ossicles were almost the same size as those in adults, we observed bundling and branching of elastic fibers. However, hyaluronan expression was not as strong as in adults. Colocalization between elastic fibers and hyaluronan appeared to be a result of postnatal maturation of the entheses.

Objective Planar cell polarity (PCP) refers to the cell polarity across the tissue plane and controls various cell behaviors and structures. Although the expression of several PCP signaling components has been detected in tooth germs, knowledge of the gene expression patterns of these PCP components during tooth development remains incomplete. The aim of this study is to characterize the temporal and spatial changes in PCP gene expression during tooth development. Design Expression of Celsr1 and 2 , Fzd3 and 6 , Vangl1 and 2 , and Dvl1-3 genes was analyzed in mouse molar germs from the bud to the bell stage using in situ hybridization. Results At the bud stage, all target genes were expressed in all areas of the tooth bud. In the enamel organ at the cap stage, expression of Fzd3 was suppressed in the enamel knot, whereas Fzd6 was strongly expressed there. Expression of Vangl2 was strongly expressed in the inner dental epithelium from the cap stage onwards. In the inner dental epithelium, strong expression of Fzd3 , Dvl2 and Vangl2 was noted at the early bell stage, and of Celsr1 , Fzd3 , Fzd6 , Vangl2 and Dvl2 at the bell stage. Furthermore, differentiated odontoblasts strongly expressed Celsr1 , Vangl2 , and Dvl2 . Conclusion The gene expression patterns delineated in this study improve our understanding of the role(s) of PCP components during tooth development.

This study aimed to evaluate the effects of high-frequency pulsed (HiFP) low-level laser therapy (LLLT) on early wound healing of tooth extraction sockets in rats.Bilateral maxillary first molars were extracted from 6-week-old Sprague-Dawley rats. Sockets on the right were treated by HiFP low-level diode laser irradiation (904-910 nm); the left sides served as unirradiated controls. LLLT (0.28 W, 30 kHz, 200-ns pulse, 0.6% duty cycle, 61.2 J/cmCompared with control sites, the un-epithelialized areas of the extracted sites were significantly reduced by irradiation (P = 0.04), and the BMC, BV, and BMD of laser-treated sites were significantly increased (P = 0.004, 0.006, and 0.009, respectively). On day 7, the mean height of newly formed immature woven bone was higher in laser-treated sites (P = 0.24). On day 3, laser-treated sites showed significantly higher osteocalcin mRNA expression (P = 0.04) and PCNA-positive cell numbers (P = 0.01).HiFP low-level diode laser irradiation enhanced soft- and hard-tissue healing of tooth extraction sockets. Lasers Surg. Med. 48:955-964, 2016. © 2016 Wiley Periodicals, Inc.

We examined the effect of hyperbaric oxygen (HBO2) treatment on bone wound healing in a rat calvarial defect. Critical-sized defects were created in the calvaria of adult Wistar rats. The animals were divided into four groups--HBO2, normobaric oxygen, hyperbaric air, and no treatment. Treatments were performed five days a week, for two weeks. Micro-computerized tomography and histological analysis were used to evaluate the bone defects. Regenerated bone areas were calculated as the percentage of new bone in the cross-sectional area of defect. The new bone cross-sectional area was significantly greater in the HBO2 group than in the other groups. There were no significant differences in the numbers of nucleated cells in the new bone areas. Although new bone volume per defect volume was significantly greater in the HBO2 group than in the other groups, no significant differences in bone mineral density in the new bone area were observed. These findings indicate the facilitatory role of HBO2 treatment on bone wound healing in the rat calvarial bone defect, and it does not appear to have any negative effects on bone maturity. We propose that HBO2 treatment would be useful in promoting bone regeneration following injury in the orofacial region.

In the developing human musculoskeletal system, cell death with macrophage accumulation occurs in the thigh muscle and interdigital area. To comprehensively clarify the distribution of macrophages, we immunohistochemically examined 16 pairs of upper and lower extremities without the hip joint (left and right sides) obtained from 8 human fetuses at approximately 10-15 weeks of gestation. Rather than in muscles, CD68-positive macrophages were densely distributed in loose connective tissues of the flexor aspects of the extremities, especially in the wrist, hand and foot. In contrast, no or fewer macrophages were evident in the shoulder and the extensor aspects of the extremities. The macrophages were not concentrated at the enthesis of the tendon and ligament, but tended to be arranged along other connective tissue fibers. Deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling revealed apoptosis in the hand lumbricalis muscles, but not in the area of macrophage accumulation. Likewise, podoplanin-positive lymphatic vessels were not localized to areas of macrophage accumulation. Re-organization of the connective tissue along and around the flexor tendons of the hand and foot, such as development of the bursa or tendon sheath at 10-15 weeks, might require the phagocytotic function of macrophages, although details of the mechanism remain unknown.

出生後発育期間または神経除去後期間の味蕾における活性化β-カテニンの発現の発現機構を明らかにすることを目的として、マウス味蕾におけるβ-カテニン、Wnt10b、Wnt5aおよびWntのレセプターであるFrizzled(Fzd)の発現パターンを調べた。これらパターンはin situハイブリダイゼーションおよび免疫組織化学法を用いて測定した。活性化β-カテニンの細胞質内の蓄積および核内への移行をソニックヘッジホッグ(Shh)免疫反応性基底細胞および味蕾内細胞において観察し、WntおよびそのレセプターであるFzd-1および-3の細胞内挙動を追跡した。これらの結果から、発生および再生の初期段階におけるβ-カテニン転写増大が基底細胞および未熟細胞の味蕾細胞への分化を促進すること、およびマウス味蕾細胞におけるWnt10bおよびFzd-1および-3はWnt/β-カテニン経路を構成し、その経路がβ-カテニンを活性化して、発生および再生の初期段階におけるβ-カテニン遺伝子の発現を上方調節する役割を持つことが示唆された。

マウス臼歯発生時におけるインシュリン様成長因子(IGF)とIGF結合蛋白質(IGFBP)の発現の時間的・空間的パターンを調べた。IGFとIGFBP発現はin situハイブリダイゼーションにて決定した。E14マウス胚の帽状期、E15胚の帽状期後期、E17-P0の鐘状期、P0-P13のクラウン形成期、P5-P13のルート形成期につき、IGF-I、IGF-II、IGFBP-2、-3、-4、-5、IGF-I受容体(IGF-IR)の発現部位と時期を網羅した。IGFおよびその作用調節因子は歯の成長分化の局所仲介因子となり、歯胚の上皮と間充織で局所的に発現するIGFBP-2、-3、-4、-5は歯の成長分化に役割を果たす可能性が示唆された。

CTRP3/cartducin, a novel C1q family protein, is expressed in proliferating chondrocytes in the growth plate and has an important role in regulating the growth of both chondrogenic precursors and chondrocytes in vitro. We examined the expression of CTRP3/cartducin mRNA in Meckel's cartilage and in condylar cartilage of the fetal mouse mandible. Based on in situ hybridization studies, CTRP3/cartducin mRNA was not expressed in the anlagen of Meckel's cartilage at embryonic day (E)11.5, but it was strongly expressed in Meckel's cartilage at E14.0, and then reduced in the hypertrophic chondrocytes at E16.0. CTRP3/cartducin mRNA was not expressed in the condylar anlagen at E14.0, but was expressed in the upper part of newly formed condylar cartilage at E15.0. At E16.0, CTRP3/cartducin mRNA was expressed from the polymorphic cell zone to the upper part of the hypertrophic cell zone, but was reduced in the lower part of the hypertrophic cell zone. CTRP3/cartducin-antisense oligodeoxynucleotide (AS-ODN) treatment of Meckel's cartilage and condylar anlagen from E14.0 using an organ culture system indicated that, after 4-day culture, CTRP3/cartducin abrogation induced curvature deformation of Meckel's cartilage with loss of the perichondrium and new cartilage formation. Aggrecan, type I collagen, and tenascin-C were simultaneously immunostained in this newly formed cartilage, indicating possible transformation from the perichondrium into cartilage. Further, addition of recombinant mouse CTRP3/cartducin protein to the organ culture medium with AS-ODN tended to reverse the deformation. These results suggest a novel function for CTRP3/cartducin in maintaining the perichondrium. Moreover, AS-ODN induced a deformation of the shape, loss of the perichondrium/fibrous cell zone, and disorder of the distinct architecture of zones in the mandibular condylar cartilage. Additionally, AS-ODN-treated condylar cartilage showed reduced levels of mRNA expression of aggrecan, collagen types I and X, and reduced BrdU-incorporation. These results suggest that CTRP3/cartducin is not only involved in the proliferation and differentiation of chondrocytes, but also contributes to the regulation of mandibular condylar cartilage.

Objective Versican is a large, aggregating chondroitin sulphate proteoglycan. In dental tissue, versican expression occurs primarily in mesenchymal tissue but rarely in epithelial tissue. We investigated the expression, localisation and synthesis of versican in the enamel organ of the developing tooth germ. Design To elucidate versican localisation in vivo , in situ hybridisation and immunohistochemistry were conducted in foetal ICR mice at E11.5–E18.5. Epithelium and mesenchyme from the lower first molars at E16.0 were enzymatically separated and versican mRNA expression was investigated by semi-quantitative RT-PCR. Organ culture of the separated samples combined with metabolic labelling with [ 35 S], followed by gel filtration, was performed to analyse secreted proteoglycans. Results Versican mRNA was first expressed in the thickened dental epithelium at E12.0 and continued to be expressed in the enamel organ until the bell stage. Versican immunostaining was detected in the stellate reticulum areas from the bud stage to the apposition stage. The enamel organ at E16.0 expressed versican mRNA at a level comparable to that in dental mesenchyme. Furthermore, when compared to dental mesenchyme, about 1/2–3/4 of the [ 35 S]-labelled versican-like large proteoglycan was synthesised and released into tissue explants by the enamel organ. Conclusions The dental epithelium of developing tooth germ is able to synthesise significant amounts of versican.

Epithelial/ectodermal plug formation in the developing nose, ear, and eye regions is followed by canalization/recanalization mediated by cell death. However, the mechanism is not well understood. Recently, substance P (SP)-mediated cell death, rather than cell apoptosis, has been reported in neuronal and non-neuronal cells. Horizontal paraffin sections of 5 human fetuses at 15-16 weeks of gestation were used to examine the entire area of the nose, ear, eye and perineum with immunohistochemistry for SP and its receptor neurokinin-1 (NK-1), and protein gene product (PGP) 9.5 and S100 protein to identify whether the positive cells had neural origins. The deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) method was also conducted to identify apoptosis. Four SP antibodies were commercially obtained and compared the results. In addition, using the same antibodies for SP, those results were compared with fetal mouse heads (E14-17). Substance P immunoreactivity of one of the 4 antibodies (sc9758) was clearly found in the nasal plug, the epithelium of the anterior nasal cavity, the entire excretory tear duct, the marginal palpebral conjunctiva, the auditory meatal plug, the parotid duct, the external urethral orifice and, the preputial lamella along the future prepuce. Immunoreactivity was usually seen in enlarged round cells in humans. In fetal mouse heads, in spite of negative reaction in all these sites, the midline epithelial seam at the palate fusion and the oral epithelium especially at and near the tooth germ specifically reacted with the sc9758. Nevertheless, the other 3 antibodies did not react at any of those sites both in human and mouse fetuses. NK-1 receptor-positive cells were seen in the nose and meatal plugs and preputial lamella, but not in the tear duct. S100 protein, PGP 9.5, and TUNEL method all demonstrated negative reactivity at any sc9758-positive sites. Consequently, the present immunoreactivity of the sc9758 antibody seemed to be false positive, but it was likely to react with a specific substance in the epithelial or ectodermal cell because of the clearly restricted staining. Which substance it crossed remains unknown.

The continuous eruption of mouse incisors throughout life is maintained by adult stem cells in the apical end. In these teeth, the continuous expression of Fgf10 in the mesenchyme plays a role in the maintenance of the epithelial stem cell compartment, referred to as the "apical bud." However, little is known about the epithelial signaling that induces and maintains Fgf10 expression. Focusing on the epithelial-mesenchymal interaction during tooth development, we thoroughly investigated candidates expressed in the apical bud. In situ hybridization and immunostaining showed that Fgf9 mRNA and protein were detected in the basal epithelium, stellate reticulum, and inner enamel epithelium of the apical bud. Recombinant Fgf9 protein stimulated cell proliferation in cultures of apical end mesenchyme. Furthermore, Fgf9- releasing beads inhibited apoptosis in mesenchymal tissue cultures and maintained the expression of Fgf10. On the other hand, Fgf10-releasing beads induced Fgf9 expression in cultures of apical buds. Taken together, these results suggest that the stem cell niche in growing incisors is maintained by an epithelial mesenchymal interaction via Fgf9 and Fgf10 signaling.