The successful regeneration of complex tooth structures based on tissue-engineering principles was recently reported. The process of this regeneration, however, remains poorly characterized. In this study, we have used histochemistry to examine the regeneration process of tissue engineered teeth in order to determine the cell types that give rise to these engineered tooth structures. Porcine third molar tooth buds were dissociated into single-cell suspensions and seeded onto a biodegradable polyglycolic acid polymer scaffold. Following varying periods of growth in rat hosts, the specimens were evaluated by histology and immunohistochemistry. Aggregates of epithelial cells were first observed 4-6 weeks after implantation. These aggregates assumed three different shapes: a natural tooth germ-like shape, a circular shape, or a bilayer-bundle. Based on the structure of the stellate reticulum in the dental epithelium, the circular and bilayer-bundle aggregates could be clearly classified into two types: one with extensively developed stellate reticulum, and the other with negligible stellate reticulum. The epithelial cells in the circular aggregates differentiated into ameloblasts. The continuous bilayer bundles eventually formed the epithelial sheath, and dentin tissue was evident at the apex of these bundles. Finally, enamel-covered dentin and cementum-covered dentin formed, a process most likely mediated by epithelial-mesenchymal interaction. These results suggest that the development of these engineered teeth closely parallels that of natural odontogenesis derived from the immature epithelial and mesenchymal cells.
To understand the bone resorption process on the basis of the morphology of bone resorption lacunae, the inner surface of parietal bones in juvenile mice was exposed with a treatment of ultrasonic waves or NaOCl treatment and examined by scanning electron microscopy (SEM). The bone resorption lacunae were divided into two types (I and II) according to differences in morphological features of their walls; the wall of type I lacunae was covered with loose collagen fibrils, while that of type II lacunae was smooth with almost no fibrillar structures. Collagen fibrils in type I lacunae treated with ultrasonic waves differed in appearance from those treated with NaOCl; the collagen fibrils were thin and displayed a smooth surface in type I lacunae treated with ultrasonic waves, while they were thick and showed a rough surface in those treated with NaOCl—probably because superficial uncalcified collagen fibrils were digested with the chemical. The results indicated that type I lacunae occupied 77% of all of the bone resorption lacunae treated with ultrasonic waves, but 51% of those treated with NaOCl. This finding led to the idea that type I lacunae can be subdivided into two: lacunae (Ia), covered with partially calcified fibrils as well as superficial uncalcified fibrils; and lacunae (Ib), covered only with uncalcified fibrils. The presence of uncalcified fibrils in the bone resorption lacunae was further confirmed by backscattered electron (BSE) imaging of SEM. Histochemistry for acid phosphatase or immuno-histochemistry for cathepsin B or carbonic anhydrase in combination with SEM revealed that type I lacunae were located under osteoclasts but type II lacunae were not. These findings indicate that type I lacunae are in the process of bone resorption by osteoclasts, while type II lacunae are in the final stage of bone resorption and free from osteoclasts. Bone resorption may thus proceed in the order of Ia, Ib, and II.
The hybridization site of a DNA probe was detected using a scanning electron microscope (SEM), modifying the standard in situ hybridization (ISH) method. The experiments were performed on human metaphases obtained from lymphocyte cultures of human peripheral blood. The libraries and probes used were: 1-chromosome library for the painting of chromosome 1 (wcp 1), an alphoid centromere-specific probe of chromosome 8 (pZ8.4), and the yeast artificial chromosome (YAC) 964-C10 mapped at band p13 on chromosome 12. These probes were labeled by nick translation with biotin and displayed with a gold-conjugated anti biotin goat antibody. The gold signal was amplified by silver enhancement. The chromatides appeared as packages of thin filaments 120 nm high; some of them collapsed, probably due to ISH procedures. All the probes were clearly detected as small gold particles grouped on the surface of the target chromosomes and chromosome sites. Thus, this procedure is useful to clarify the positional relationship between the chromatin filaments and the probe.
To better understand the relationship between innervation in the sphincter of Oddi and pancreatobiliary diseases, nerve cells which possess nitric oxide synthase (NOS) and/or vasoactive intestinal polypeptide (VIP) were studied immunohistochemically in the sphincter of Oddi and duodenum of humans. Specimens from autopsies included 11 cases with pancreatobiliary diseases and 7 cases without such diseases. An elaborate nerve network was revealed with an anti-S-100 antibody in the sphincter of Oddi and duodenum of all specimens. In the sphincter of Oddi of the control group, approximately 47% of the myenteric nerve cells were NOS positive, whereas 54% were VIP positive. Of the NOS positive nerve cells, 21% were also VIP positive. In contrast, 11% of the nerve cells in the sphincter of Oddi of the disease group were NOS positive while 32% were VIP positive. Within the duodenal myenteric plexus of the control group, 35% of all nerve cells were NOS positive while 40% was VIP positive; among them, 23% of the NOS positive cells were VIP positive. Similar results were observed in the duodenum of the disease group. These data indicate that abundant NOS and VIP positive innervation is present in the sphincter of Oddi and duodenum in humans. The lower proportion of NOS positive or VIP positive nerve cells of the disease group may suggest an inadequacy of the sphincter of Oddi to relax.
Human zona pellucida (ZP) is maintained up to the blastocyst stage prior to hatching. In in vitro fertilized (IVF) embryos, it eventually acts as a morphodynamic interface between the cultured embryo and its microenvironment. Ultrastructural data on the ZP of IVF blastocysts are scarce in humans. We employed correlated phase contrast microscopy (PCM) and scanning electron microscopy (SEM) to study retrospectively the ultrastructural morphology of the ZP outer surface of 20 IVF human blastocysts from 16 Japanese patients (28-44 years of age, average 36.7±4.2) with a history of infertility. Blastocysts were derived from conventional in vitro fertilization (cIVF) (n = 10) and from intracytoplasmic sperm injection (ICSI) (n = 10). Both cIVF and ICSI groups included “clear blastocysts” (n = 5) and “dark blastocysts” (n = 5). By PCM, the clear blastocysts exhibited a regular, round-shaped contour and consisted of clear and voluminous cells. By SEM, they displayed a spongy ZP with numerous fenestrations formed by networked filaments. By PCM, dark blastocysts appeared irregularly shaped and often collapsed, and comprised dark cells and debris. By SEM, their ZP were smooth with remnants of compact fenestrations. In conclusion, viable blastocysts presented a normal ZP outer surface ultrastructure, whereas unhealthy blastocysts showed an altered ZP outer surface, comparable to that of immature/atretic oocytes. Such alterations could reflect sub-optimal culture conditions and/or could be related to blastocyst degenerative processes. The blastocyst ZP surface ultrastructure was unaffected by the fertilization technique (cIVF or ICSI). These data suggest that blastocyst survival in vitro is related to ZP ultrastructure maintenance.
Scanning electron microscopy (SEM) was employed to study the effect of calcitonin on the distribution of actin filaments in osteoclasts obtained from rat tibiae. Fluorescent microscopy was also applied to examine calcitonin-induced changes in the distribution of actin filaments, non-muscle myosin, M-Ras, and extracellular signal-regulated kinase (ERK) to clarify the role of ERK in the cytoskeleton of osteoclasts. SEM of control osteoclasts revealed a ring-like structure in the peripheral region. Labeled actin filaments and non-muscle myosin were detected in the peripheral region and exhibited a ring-like pattern. Immunoreactivity indicating M-Ras and ERK was also detected in the vicinity of the actin ring. After calcitonin treatment, many osteoclasts exhibited a retracted appearance and lacked a discernible actin ring. Numerous retraction fibers were found at the edge of calcitonin-treated osteoclasts. Actin filaments and non-muscle myosin were concentrated in the cytoplasm of calcitonin-treated osteoclasts, and exhibited a filamentous pattern. Labeled M-Ras and ERK also accumulated in the central region of these osteoclasts. These findings suggest that actin-myosin interaction plays an essential role in the retraction of osteoclasts induced by calcitonin. ERK may play a role in this interaction by activating myosin light chain kinase, as previously observed in smooth muscle cells.