Takeyasu Maeda

Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation.

The composite structure of the mammalian skull, which forms predominantly via intramembranous ossification, requires precise pre- and post-natal growth regulation of individual calvarial elements. Disturbances of this process frequently cause severe clinical manifestations in humans. Enhanced DNA binding by a mutant MSX2 homeodomain results in a gain of function and produces craniosynostosis in humans. Here we show that Msx2-deficient mice have defects of skull ossification and persistent calvarial foramen. This phenotype results from defective proliferation of osteoprogenitors at the osteogenic front during calvarial morphogenesis, and closely resembles that associated with human MSX2 haploinsufficiency in parietal foramina (PFM). Msx2−/− mice also have defects in endochondral bone formation. In the axial and appendicular skeleton, post-natal deficits in Pth/Pthrp receptor (Pthr) signalling and in expression of marker genes for bone differentiation indicate that Msx2 is required for both chondrogenesis and osteogenesis. Consistent with phenotypes associated with PFM, Msx2-mutant mice also display defective tooth, hair follicle and mammary gland development, and seizures, the latter accompanied by abnormal development of the cerebellum. Most Msx2-mutant phenotypes, including calvarial defects, are enhanced by genetic combination with Msx1 loss of function, indicating that Msx gene dosage can modify expression of the PFM phenotype. Our results provide a developmental basis for PFM and demonstrate that Msx2 is essential at multiple sites during organogenesis.

Innervation of periodontal ligament and dental pulp in the rat incisor: an immunohistochemical investigation of neurofilament protein and glia-specific S-100 protein

SummaryNervous elements in the periodontal ligament and dental pulp of rat incisors were investigated by means of immunohistochemistry for neurofilament protein (NFP) and glia-specific S-100 protein. The periodontal ligament in the incisors was densely innervated by NFP-immunoreactive nerve fibers; the distribution of the nerve fibers and their terminations differed markedly from those in molars. NFP-positive, thick nerve bundles entered the lingual periodontal ligament through slits located in the mid-region of the alveolar socket, and immediately formed numerous Ruffini-like corpuscles. In the labial periodontal ligament, all of the NFP-immunoreactive nerve fibers terminated in free endings. The restricted location of the stretch receptor, Ruffini-like corpuscle, in the lingual periodontal ligament appears to be an essential element, because this region is regularly extended during mastication. The nervous elements were restricted to the alveolar half of the periodontal ligament in every region; they avoided the dental half of the periodontal ligament, which presumably moves continuously with the tooth. Pulpal nerve fibers in incisors also showed a characteristic distribution different from those in molars; individual nerve fibers with beaded structures ran in the center of the pulp toward the incisai edge, and did not form the subodontoblastic nerve plexus of Raschkow.Immunostaining for S-100 protein revealed a distribution pattern of nervous elements similar to that for NFP, suggesting that the nerves supplying the periodontal ligament and dental pulp were mostly covered by a Schwann sheath.

Distribution of nerve fibers immunoreactive to neurofilament protein in rat molars and periodontium

SummaryThe distribution of nerve fibers in molars, periodontal ligament and gingiva of the rat shows a complex pattern. Decalcified material including the alveolar bone was sectioned in three different planes and stained by means of immunohistochemistry for detection of the neurofilament protein (NFP); the immunoreactive neural elements were clearly visualized in three-dimensional analyses. NFP-positive nerve fibers formed a subodontoblastic plexus in the roof area of the dental pulp; some of them entered the predentin and dentin directly through the dentinal tubules. This penetration was found mainly in the pulp horn, and was limited to a distance of about 100 μm from the pulpo-dentinal junction. In the periodontal ligament, NFP-positive nerve fibers were found densely distributed in the lower half of the alveolar socket. Two types of nerve terminals were recognized in the periodontal ligament: free nerve endings with tree-like ramifications, and expanded nerve terminals showing button- or glove-like shapes. The former tapered among the periodontal fibers, some even reaching the cementoblastic layer. The latter were located, frequently in groups, within the ligament restricted to the lower third of the alveolar socket. A well-developed plexus of NFP-positive nerves was revealed in the lamina propria of the free gingiva, the innervation being denser toward the epithelium of the gingival crevice. The characteristic distribution of NFP-immunoreactive nerve fibers revealed in this study is discussed in relation to region-specific sensations in the teeth and surrounding tissues.

Cooperative Regulation of Chondrocyte Differentiation by CCN2 and CCN3 Shown by a Comprehensive Analysis of the CCN Family Proteins in Cartilage

CCN2 is best known as a promoter of chondrocyte differentiation among the CCN family members, and its null mice display skeletal dysmorphisms. However, little is known concerning roles of the other CCN members in chondrocytes. Using both in vivo and in vitro approaches, we conducted a comparative analysis of CCN2‐null and wildtype mice to study the roles of CCN2 and the other CCN proteins in cartilage development. Immunohistochemistry was used to evaluate the localization of CCN proteins and other chondrocyte‐associated molecules in the two types of mice. Moreover, gene expression levels and the effects of exogenous CCN proteins on chondrocyte proliferation, differentiation, and the expression of chondrocyte‐associated genes in their primary chondrocytes were evaluated. Ccn3 was dramatically upregulated in CCN2‐null cartilage and chondrocytes. This upregulation was associated with diminished cell proliferation and delayed differentiation. Consistent with the in vivo findings, CCN2 deletion entirely retarded chondrocyte terminal differentiation and decreased the expression of several chondrocyte‐associated genes in vitro, whereas Ccn3 expression drastically increased. In contrast, the addition of exogenous CCN2 promoted differentiation strongly and induced the expression of the associated genes, whereas decreasing the Ccn3 expression. These findings collectively indicate that CCN2 induces chondrocyte differentiation by regulating the expression of chondrocyte‐associated genes but that these effects are counteracted by CCN3. The lack of CCN2 caused upregulation of CCN3 in CCN2‐null mice, which resulted in the observed phenotypes, such as the resultant delay of terminal differentiation. The involvement of the PTHrP‐Ihh loop in the regulation of CCN3 expression is also suggested.

The Ruffini Ending as the Primary Mechanoreceptor in the Periodontal Ligament: Its Morphology, Cytochemical Features, Regeneration, and Development

The periodontal ligament receives a rich sensory nerve supply and contains many nociceptors and mechanoreceptors. Although its various kinds of mechanoreceptors have been reported in the past, only recently have studies revealed that the Ruffini endings--categorized as low-threshold, slowly adapting, type II mechanoreceptors--are the primary mechanoreceptors in the periodontal ligament. The periodontal Ruffini endings display dendritic ramifications with expanded terminal buttons and, furthermore, are ultrastructurally characterized by expanded axon terminals filled with many mitochondria and by an association with terminal or lamellar Schwann cells. The axon terminals of the periodontal Ruffini endings have finger-like projections called axonal spines or microspikes, which extend into the surrounding tissue to detect the deformation of collagen fibers. The functional basis of the periodontal Ruffini endings has been analyzed by histochemical techniques. Histochemically, the axon terminals are reactive for cytochrome oxidase activity, and the terminal Schwann cells have both non-specific cholinesterase and acid phosphatase activity. On the other hand, many investigations have suggested that the Ruffini endings have a high potential for neuroplasticity. For example, immunoreactivity for p75-NGFR (low-affinity nerve growth factor receptor) and GAP-43 (growth-associated protein-43), both of which play important roles in nerve regeneration/development processes, have been reported in the periodontal Ruffini endings, even in adult animals (though these proteins are usually repressed or down-regulated in mature neurons). Furthermore, in experimental studies on nerve injury to the inferior alveolar nerve, the degeneration of Ruffini endings takes place immediately after nerve injury, with regeneration beginning from 3 to 5 days later, and the distribution and terminal morphology returning to almost normal at around 14 days. During regeneration, some regenerating Ruffini endings expressed neuropeptide Y, which is rarely observed in normal animals. On the other hand, the periodontal Ruffini endings show stage-specific configurations which are closely related to tooth eruption and the addition of occlusal forces to the tooth during postnatal development, suggesting that mechanical stimuli due to tooth eruption and occlusion are a prerequisite for the differentiation and maturation of the periodontal Ruffini endings. Further investigations are needed to clarify the involvement of growth factors in the molecular mechanisms of the development and regeneration processes of the Ruffini endings.

Innervation of the Incisors and Periodontal Ligament in Several Rodents: an Immunohistochemical Study of Neurofílament Protein and Glia-Specifîc S-100 Protein

The present immunohistochemical study by use of antisera against neurofilament protein (NFP) and S-100 protein dealt with the innervation of the upper incisors and periodontal ligament in five species of rodents including the guinea pig, hamster, Mongolian gerbil (Meriones unguicularis), mouse and squirrel (Tamias sibiricus). The innervation pattern of the periodontal ligament and dental pulp in the incisors of five rodents was fundamentally identical to that in the rat, which we have previously demonstrated by the same method. The NFP-positive Ruffini-like corpuscles were concentrated in the middle region of the lingual periodontal ligament in all the species examined, suggesting that this particular arrangement of Ruffini-like corpuscles, possibly stretch receptors, was essential to the rodent incisor. The labial periodontal ligament, on the other hand, contained less numerous NFP-positive nerves, these terminating among collagen fibers as free endings. The gerbil and squirrel in particular possessed only a few nerve fibers in the labial periodontal ligament. It was thus presumed that the labial periodontal ligament might be less significant as a mechanoreceptive site than the lingual periodontal ligament. The NFP-positive pulpal nerves, beaded or smooth in shape, ran parallel to the tooth axis, but never extended to the odontoblastic layer; no subodontoblastic plexus was found in the incisors of any of the rodents. S-100-immunopositive nervous elements were distributed in the periodontal ligament and dental pulp of all the rodent species examined, showing a distribution pattern similar to the NFP-positive nerves. Only in the squirrel did odontoblasts show an intense S-100 immunoreactivity.

FGF23 is mainly synthesized by osteocytes in the regularly distributed osteocytic lacunar canalicular system established after physiological bone remodeling.

This study aimed to evaluate whether the immunolocalization of fibroblast growth factor (FGF) 23 and dentin matrix protein 1 (DMP1) is associated with the spatial regularity of the osteocyte lacunar canalicular system(s) (OLCS). Femora of 12-weeks-old male ICR mice were fixed with 4% paraformaldehyde, decalcified with a 10% EDTA solution and then embedded in paraffin. We have devised a triple staining procedure that combines silver impregnation, alkaline phosphatase (ALPase) immunohistochemistry and tartrate-resistant acid phosphatase (TRAPase) enzyme histochemistry on a single paraffin section. This procedure permitted the visualization of ALPase-positive plump osteoblasts and several TRAPase-positive osteoclasts on those bone matrices featuring irregularly arranged OLCS, and of ALPase-positive bone lining cells on the bone matrix displaying the well-arranged OLCS. As observations proceeded from the metaphysis toward the diaphysis, the endosteal cortical bone displayed narrower bands of calcein labeling, accompanied by increased regularity of the OLCS. This implies that the speed of bone deposition during bone remodeling would affect the regularity of the OLCS. While DMP1 was evenly localized in all regions of the cortical bones, FGF23 was more abundantly localized in osteocytes of cortical bones with regularly arranged OLCS. In cortical bones, the endosteal area featuring regular OLCS exhibited more intense FGF23 immunoreaction when compared to the periosteal region, which tended to display irregular OLCS. In summary, FGF23 appears to be synthesized principally by osteocytes in the regularly distributed OLCS that have been established after bone remodeling.

Primary leiomyosarcoma of the maxilla with regional lymph node metastasis. Report of a case and review of the literature.

A rare case of oral leiomyosarcoma diagnosed with the aid of immunohistochemical and electron microscopic examinations together with a review of the literature are reported. The patient was a 70-year-old Japanese man. The primary tumor involved the maxillary gingiva and bone and metastasized to the cervical lymph nodes. On histologic examination the tumor showed invasive growth into the maxillary bone. It was composed of interlacing fascicles of spindle-shaped cells with eosinophilic cytoplasm and elongated, blunt-ended nuclei. The tumor formed extensive metastatic foci in the cervical lymph nodes. On immunohistochemical examination most of the tumor cells were positive for desmin, smooth muscle-specific actin, and myosin. The ultrastructural characteristics of the tumor cells were abundant microfilaments, pinocytotic vesicles, and basement membrane formation. The findings were indicative of a tumor demonstrating myogenic differentiation. A review of the literature during the past 50 years disclosed a total of 60 oral leiomyosarcomas, including our case.

RESPONSES OF IMMUNOCOMPETENT CELLS TO CAVITY PREPARATION IN RAT MOLARS : AN IMMUNOHISTOCHEMICAL STUDY USING OX6-MONOCLONAL ANTIBODY

Responses of immunocompetent cells, especially class II major histocompatibility complex (MHC) antigen-expressing cells, were investigated after cavity preparation in the erupted upper first molar teeth of rats, by immunohistochemistry using OX6-monoclonal antibody. In control teeth, OX6-immunopositive cells were predominantly located beneath the odontoblast layer in the dental pulp. Cavity preparation caused an acute edematous reaction between the injured odontoblasts and predentin, and most of OX6-immunopositive cells in the affected site shifted away from the pulp-dentin border. After 12-24 hours, many OX6-immunopositive cells accumulated along the pulp-dentin border and extended their cytoplasmic processes into the exposed dentinal tubules. After 72 hours, newly differentiated odontoblasts replaced the degenerated odontoblasts, and few OX6-immunopositive cells remained along the pulp-dentin border. Our data suggest that some of the class II MHC antigen-expressing cells in the dental pulp participate in the initial defense reaction and presumably serve as a biological sensor for the external stimuli arriving through the exposed dentinal tubules.

Immunohistochemical demonstration of nerves in the predentin and dentin of human third molars with the use of an antiserum against neurofilament protein (NFP)

SummaryImmunohistochemistry by use of an antiserum against neurofilament protein (NFP) was applied for staining nerve fibers in the predentin and dentin of human third molars. By devising methods for fixation, decalcification and immunostaining, nerve fibers were clearly and specifically demonstrated in thick (more than 50 μm) sections of teeth. Numerous NFP-positive fibers were distributed in the predentin throughout the coronal region, while a few positive fibers penetrated only a short distance into the dentin. The NFP-positive nerve fibers in the predentin took transverse and complicated courses across, rather than penetrating longitudinally through, the dentinal tubules. Pain sensation in the teeth might be attributable to these complex nerve fibers showing two or three-dimensional extensions.