Three-dimensional observation of the mouse embryo by micro-computed tomography: Meckel's cartilage, otocyst, and/or muscle of tongue.

Three-dimensional observation during embryogenesis is possible with micro-computed tomography, but there are no observations of organ size. In this paper, three examples of three-dimensional observation of organs by micro-CT are tried. At 13.0 days post-coitum, mouse embryos were fixed in 4% paraformaldehyde for 24 h and stained enbloc by osmium tetroxide overnight. The embryos were then embedded in paraffin using standard methods for 24 h. Specimens were analyzed by micro-computed tomography and image processing was performed. The entire Meckel's cartilage and its relation in the mandible, as well as the complex structure of the otocyst, are easily visualized. Although it is difficult to extract detailed structures of the tongue muscles, it is possible to identify the inner and external tongue muscles. Relation among the organs and other are easily visualized. Three-dimensional observation by micro-computed tomography is an important technology for visualization of embryogenesis and could be used in organ culture.

An immunohistochemical study on the localization of type II collagen in the developing mouse mandibular condyle.

The present chronological investigation assessed the distribution of type II collagen expression in the developing mouse mandibular condyle using immunohistochemical staining with respect to the anatomy of the anlage of the mandibular condyle, the histological characteristics of which were disclosed in our previous investigation. We analyzed fetuses, obtained by cross breeding of ICR strain mice, between 14.0 and 19.0 days post-conception (dpc) and pups on 1, 3, and 5 days post-natal (dpn) using immunohistochemical staining with 2 anti-type II collagen antibodies. The expression of type II collagen was first detected at 15.0 dpc in the lower part of the hypertrophic chondrocyte zone; thereafter, this type II collagen-positive layer was expanded and intensified (P1 layer). At 17.0 dpc, we identified a type II collagen-negative layer (N layer) around the P1 layer and we also identified another newly formed type II collagen-positive layer (P, layer) on the outer surface of the N layer. The most typical and conspicuous 3-layered distribution was observed at 1 dpn; thereafter, there was a reduction in the intensity of expression, and with it, the demarcation between the layers was weakened by 5 dpn. The P1 layer was derived from the central region of the core cell aggregate of the anlage of the mandibular condyle and participated in endochondral bone formation. The N layer was derived from the fringe of the core cell aggregate of the anlage, formed the bone collar at the side of the condyle by intramembranous bone formation, and showed a high level of proliferative activity at the vault. The P2 layer was formed from the outgrowth of the N layer, and could be considered as the secondary cartilage. The intensive expression of type II collagen from 17.0 dpc to 3 dpn was detected in the fibrous sheath covering the condylar head, which is derived from the peripheral cell aggregate of the anlage. Since its expression in the fibrous sheath was not detected in the neighboring section in the absence of hyaluronidase digestion, some changes in the extracellular matrix of the fibrous sheath appear to participate in the generation of the lower joint space. The results of the present investigation indicate that further studies are required to fully characterize the development of the mouse mandibular condyle.

Three-dimensional observation of the mouse embryo by micro-computed tomography: composition of the trigeminal ganglion.

The purpose of this study was to demonstrate a micro-computed tomography (CT) method for observations of the mouse embryo. At 13.0 days post-coitum, mouse embryos were fixed in 4% paraformaldehyde for 24 h and stained en bloc by osmium tetroxide overnight. The embryos were then embedded in paraffin using standard methods for 24 h. Specimens were analyzed by micro-CT and image processing was performed. Organs containing nervous and blood systems could be viewed as a result of different osmium-staining densities. The trigeminal ganglion was imaged using three-dimensional techniques. Observation of the embryo was possible by micro-CT with osmium tetroxide staining.

The effect of angiotensin II on the proliferation of human gingival fibroblasts.

Angiotensin not only raises blood pressure and modifies body fluids and electrolytes but also induces differentiation and proliferation of fibroblasts in the circulatory system in order to repair damage. The purpose of the present study was to observe the influence of the addition of angiotensin II (AngII) or nifedipine (NIF) alone or both sequentially on proliferative activity, the intracellular Ca(2+) concentration ([Ca(2+)]i), and the inositol-1,4,5-triphosphate (IP3) level in cultivated human gingival fibroblasts. Addition of 10(-8)-10(-4) M NIF or 10(-5)-10(-4) M AngII alone increased the proliferation of cultivated gingival fibroblasts, and the interaction of NIF and AngII suppressed proliferation. Addition of AngII alone increased [Ca(2+)]i, with a peak 60 s afterward and a return to a level slightly higher than the pretreatment level at 120 s. Addition of both AngII and NIF did not increase [Ca(2+)]i as much as the addition of AngII alone. When Ca(2+) was absent from the extracellular environment, the AngII-induced increase in [Ca(2+)]i was suppressed. AngII increased the concentration of IP(3), with a peak at 120 s after its addition. From these results we concluded that AngII increased the proliferation of gingival fibroblasts by causing an influx of Ca(2+), which increased [Ca(2+)]i.

Embryological study of the development of the rat temporomandibular joint: highlighting the development of the glenoid fossa.

Basic embryological findings on the development of the temporomandibular joint have yet to be elucidated sufficiently. This experiment, was undertaken to find the standard time course of the development of the temporomandibular joint in rat fetuses. Serial frontal and sagittal sections of rat fetal heads (between 13.5 and 20.5 days post-conception [p.c.]) were examined microscopically. The condyle was recognized as a mesenchymal condensation at 14.5 days p.c., while the glenoid fossa was recognized at 15.5 days p.c. The mesenchymal condensation of the condyle had differentiated into chondrocytes by 16.5 days p.c., and endochondral ossification was recognized at 17.5 days p.c. The intramembranous ossification of the glenoid fossa was already recognized by 16.5 days p.c.; this started in the posterior region and progressed anteriorly between the zygomatic arch and the squamous part of the temporal bone. Ossification of the condyle had not been completed by 20.5 days p.c., a mass of hypertrophic chondrocytes remained in the center of the condylar head. The glenoid fossa was almost completely ossified by 19.5 days p.c. A coarse region of cells, reminiscent of apoptosis, was recognized in the region of the prospective superior joint space at 17.5 days p.c., and an actual joint space had formed by 18.5 days p.c. The inferior joint space was recognized at 19.5 days p.c. as a fissural cavity, but it was much narrower than its superior counterpart. The prospective meniscus was distinguished on the condylar surface at 17.5 days p.c. by the difference in the shape of its constituent cells. The results obtained here seem to be useful for further experiments and molecular biological studies.