Adult stem cells have the capacity to self-renew and differentiate along multiple lineages in addition to contributing to ongoing tissue maintenance and regeneration after injury. They reside in specific locations called stem cell niches. In biology of the tooth, the discovery of dental epithelial stem cells in continuously growing teeth has been a recent breakthrough. The niche for the adult stem cells of these teeth is formed at the region of the apical end in tooth development. The region possesces a commonly specialized histological structure for the maintenance of adult stem cells and the production of various progenitor cells producing dental tissues. The molecular signals regulating the maintenance and cell fate decision of adult stem cells, such as Notch1, Lunatic fringe, fibroblast growth factor (FGF)-10, are expressed in the epithelial structure and the surrounding mesenchyme. Based on histological and molecular biological studies, we propose a new concept that the eternal tooth buds producing various dental progeny are formed at the apical end in the development of continuously growing teeth, and coin a new term of “apical bud” for indicating this specialized epithelial structure. Furthermore, the relationship between signaling centers and the expression of FGF-10 mRNA as the determinant of morphogenesis is discussed with an emphasis on tooth and limb development, taking note that the expression pattern of FGF-10 is an important key for understanding the mechanisms for the diversity of cusp patterns and between continuous and limited growth.

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The repair process of a full-thickness osteochondral defect was observed in a rat model using Hoechst 33342 as a cell tracer. The osteochondral defect was created at the medial femoral condyle of the right knee joints of twelve 11-week-old male rats. Three weeks after the surgery, Hoechst 33342 was injected into the same knee joints. Calcein, a marker of the mineralization front, was then injected subcutaneousely twice at seven days and one day before harvesting of the tissue. At six, ten, and fourteen weeks and one year after the surgery, femoral condyles were obtained from the operated knee joints, fixed by alcohol, and embedded in polymethylmethacrylate. The sections were examined by fluorescent and then light microscopy. In the lateral femoral condyle cartilage, Hoechst 33342 labeling of chondrocyte nuclei was observed in all layers of the intact cartilage, and the dye never infiltrated beneath the subchondral bone plate. At 6 weeks after the surgery, Hoechst 33342-positive cells were observed not only in the regenerated fibrous cartilage, but also in the newly formed mineralized tissue in the medial femoral condyle. Interestingly, Hoechst 33342 labeling remained undiminished even one year after the intra-articular injection. The findings of the present study suggest that intra-articular injection of Hoechst 33342 is a useful tracer for long-term investigations of chondrocyte differentiation in vivo.

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Chick serum contains a factor that stimulates adipogenesis in Meckel’s chondrocytes in vitro. The present study examined whether chick serum has a capacity for adipogenic induction in vivo, by transplanting serum clots (created by drying chick serum for up to 4 weeks) into mouse spleens. Specimens were harvested for histological analyses, which included light and electron microscopy and immunohistochemistry. The transplanted serum clots induced the appearance of lipid droplet-containing cells in splenic cords and sinus. Almost all the lipid droplet-containing cells were positive for sudan staining and consisted of multilocular lipid vacuoles. Immunostaining showed that the adipocytes induced by transplantation of the serum clots initially appeared as peroxisome proliferator-activated receptor-γ(PPARγ)-positive cells and developed into leptin and α-glycerophosphate dehydrogenase (GPDH)-producing cells, in addition to type III collagen synthesis. Furthermore, double immunofluorescence staining revealed that the immunoreactivity for GPDH was detected not only in stromal cells but also in macrophages. It was thus confirmed that stromal cells and macrophages in the spleen contain lipid droplets as seen in intact white adipose cells. The present results suggest that chick serum contains factors for adipocyte induction not only in vitro but also in vivo, and that the adipogenic potential does not depend on the supplements used during the cell culture.

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Collagen and elastin fibers are the major components of the lung connective tissue, but their spatial organization has not been well documented. We have demonstrated the three-dimensional architecture of collagen and elastin fiber networks in the human and rat lung using scanning electron microscopy. These networks in their original forms were extracted by an alkali-water maceration technique and a formic acid treatment, respectively. The collagen fibers formed a continuum extending throughout the lung and pleura. They were condensed in the alveolar mouth and subdivided into smaller fibers in the alveolar septa, thus forming basket-like networks. Sizes of the alveolar pores in the collagen fiber network of the alveolar septa became larger with age. In the collapsed lung, collagen fibers in the alveolar mouths and septa took on wavelike configurations, while in the inflated lung they became straight. The elastin fibers also formed a continuum, rich in the alveolar mouths and poor in the alveolar septa, were quite straight without any wavelike configuration. Transmission electron microscopy showed that collagen and elastin fibers were intermingled, suggesting that both fiber systems may act as parallel mechanical elements to stress or strain applied. Our results suggest that at low levels of strain the wavy collagen fibers are easily extended to allow alveolar mouths and alveoli to expand, with most of the stress being borne by adjacent elastin fibers, while at higher levels collagen fibers become straight and limit any further distension of alveolar ducts and alveoli. The elastin fiber continuum appears to permit the lung to effectively recoil or retract. The present study has also shown that alveolar pores enlarge with age, suggesting that collagen remodeling may be related to the pathogenesis of emphysema.

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Intrinsic nerve plexuses of the rat trachea and extrapulmonary bronchi were examined by immunohistochemistry. Three nerve plexuses—peritracheal and peri-bronchial, intramuscular, and submucosal—were found in the wall of the trachea and bronchi. Nerve cell bodies were located in the peritracheal and peribronchial nerve plexuses. They occurred singly or formed ganglia in the plexus, and regional differences in cell numbers were found in the cervical and thoracic portions of the trachea and in the extrapulmonary bronchia. In total, 83.5±28.3 ganglia (mean±SD, 57-131, n=5) and 749.8 ±221.1 nerve cell bodies (540-1,080, n=5) were found in the nerve plexus. The mean densities of ganglia were 0.31, 0.97 and 1.15/mm², and the mean densities of the nerve cell bodies were 1.82, 9.26 and 11.54/mm² in the cervical region, thoracic region of trachea, and extrapulmonary bronchi, respectively. Almost all nerve cell bodies in ganglia were positive for choline acetyltransferase and neuropeptide Y (NPY), and a few cells were positive for vasoactive intestinal peptide (VIP). In addition, in cholinergic nerves, a few nerve fibers in the smooth muscles were positive for substance P (SP), calcitonin gene-related peptide (CGRP), and VIP, and a moderate number of fibers were positive for NPY. Tyrosine hydroxylase-immunoreactive nerve fibers were observed around blood vessels and within nerve bundles in the tunica adventitia. In the epithelium, nerve fibers were positive for SP and CGRP. Our results indicate that postganglionic neurons form three layers of cholinergic plexuses in the rat trachea and extrapulmonary bronchi, and that all of these possess intrinsic and extrinsic peptidergic innervation.

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Secretogranin III (SgIII), a member of the granin protein family, is expressed specifically in neuronal and endocrine cells. To examine the precise localization of SgIII in the endocrine pancreas, pancreatic tissues of rats were analyzed immunocytochemically with a polyclonal antiserum raised against rat SgIII. By light microscopy of semithin sections, the immunoreactivity for SgIII was readily detected in pancreatic A- and B-cells, faintly so in D- cells, and not at all in the exocrine pancreas. By immunoelectron microscopy, immunogold particles indicative of SgIII were observed in the peripheral regions of secretory granules, and universally in the pancreatic endocrine cells. Morphometrical analyses indicated that SgIII is most preferentially localized in the periphery of the secretory granule among granins. These findings suggest that SgIII is closely associated with the secretory granule membrane, serving to anchor the aggregates of other soluble constituents to the membrane.

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Tooth induction by xenogenic graft of reconstructed human tooth germ components has never been attempted. Here we report our first attempt at a transplantation of human tooth germ components, heterologously recombined with mouse dental epithelia, into immunocompromised animals.
Human third molar tooth germs enucleated from young patients as prophylactic treatment for orthodontic reasons were collected. The whole or minced human dental papilla was reconstructed with human- or mouse molar enamel epithelium, and transplanted in the dorsal aspect of C.B-17/Icr-scid Jcl mice. The transplant of human dental papilla reconstructed with human enamel epithelium formed thin dentin and immature enamel layers by 3 to 4 weeks, but remained extremely small in quantity due to a shortage of epithelial components in the graft. The addition of E16 mouse molar enamel organs (n=10-12) to each graft augmented the formation of tooth germ-like structures, but the differentiation of mouse molar ameloblasts was suppressed. However, once a solid layer of mineralized dentin was established, mouse ameloblasts accelerated their differentiation, and completed the enamel matrix formation and maturation within the following 4 weeks, whereas human ameloblasts, which had interacted with human dental papilla, remained in the stage of matrix formation during the same period.
These data imply that, in reconstructed transplants, the differentiation of mouse dental epithelia is restrained by putative suppressive factors derived from human dental papilla until they are separated by mineralized dentin layers that serve as a diffusion barrier. The mouse enamel organ nevertheless retains its own phenotypic characteristics and intrinsic timing of cell differentiation and function.

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The rat insulinoma cell line RINm5F, an insulin secreting pancreatic beta cell line, has been used as an attractive model for basic studies of the mechanisms of insulin secretion and, more recently, as a model for the development of alternative methods for the treatment of diabetes. To elucidate the cytological properties and expression patterns of hormones of the gastro-entero-pancreatic system, suspensions of RINm5F cells were investigated by various methods including immunocytochemistry on serial semithin sections, quantitative immunocytochemistry, routine electron microscopy, immuno-electron microscopy, in situ hybridization, and TUNEL technique. At the ultrastructural level, several phenotypes of RINm5F cells were characterized by differences in the number, shape, size, and density of their secretory granules. The most common type contained a mixture of round granules varying in size and electron density. A second type predominantly contained relatively large, moderately dense granules. Moreover, a minority of cells was characterized by the occurrence of polymorphous electron dense granules or the complete absence of any secretory granules. The immunohistochemical data showed that, among the established islet hormones, insulin was present in more than 50% of cells, whereas glucagon and somatostatin occurred only sporadically. Though cells positive for pancreatic polypeptide (PP) were not found, PP-related peptides (NPY and PYY) however could be detected in a minority of cells. The great majority of RINm5F cells were immunoreactive for chromogranin B (CgB), followed by insulin, chromogranin A (CgA), and serotonin (5-HT). In addition to intercellular differences in the density of immunostaining, numerous colocalizations of immunoreactivities were found, suggesting that RINm5F cells represent a mixture of subtypes concerning the individual pattern of hormone expression. The present results reveal a wide range of heterogeneity with respect to the morphology and especially the hormone content between individual RINm5F cells.

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In the vascular wall, adenosine-5’-triphosphate (ATP) released along with noradrenaline from sympathetic nerve terminals is considered to play an important role in controlling intracellular calcium ion ([Ca²⁺] _i) levels in arteries. The present study examined how vascular smooth muscle cells in coronary arterioles respond to ATP in relation to [Ca²⁺] _i dynamics. For this purpose, the dynamics of [Ca²⁺] _i in the coronary arterioles of golden hamsters was examined by real-time laser scanning confocal microscopy. This technique enabled the visualization of [Ca²⁺] _i changes in response to ATP in the intact coronary arterioles, the ultrastructure of which was well preserved. It was shown that an increase in [Ca²⁺] _i in the arteriole smooth muscle cells was elicited by ATP. While P1 purinoceptor agonists have no effect on this process, P2 purinoceptor agonists were found to induce a [Ca²⁺] _i increase in the smooth muscle cells. Suramin (an antagonist of P2X and P2Y receptors) completely inhibited ATP-induced [Ca²⁺] _i dynamics, but reactive blue 2 (a P2Y receptor antagonist) did not. Uridine-5’-triphosphate (a P2Y receptor agonist) had no effect on [Ca²⁺] _i, but α,β- methylene ATP (a P2X receptor agonist) caused a strong increase in [Ca²⁺] _i. We conclude that smooth muscle cells of the hamster coronary arterioles possess P2X, but not P1 or P2Y purinoceptors. The smooth muscle cells probably respond to extracellular ATP via P2X purinoceptors, resulting in the contraction of the coronary arterioles.

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The present study was performed to investigate morphologically the process of neutrophil extravasation induced by the synthetic bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) in venules of the rat mesentery by the combined use of intravital microscopy and transmission electron microscopy (TEM). Special attention was given to the interaction of the neutrophils with the endothelial cells and endothelial basal lamina. By intravital microscopy, the rolling and adhesion of leukocytes were observed within 3 min in preparations activated by fMLP. Neutrophils remained in the vascular wall for more than 30 min even after penetration of the endothelium. In this period, neutrophils migrating between endothelial cells and their basal lamina were often observed by TEM. After 40 min application of fMLP, some of the migrating neutrophils parted from the vessel wall into the surrounding connective tissues. There were neutrophils which passed through the small pore of the basal lamina with a cytoplasmic constriction. These findings indicate that the endothelial basal lamina acts as a physical barrier against neutrophil extravasation, thus resulting in the transient retainment of neutrophils beneath the endothelial cells of the venular wall.

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