Osteogenesis of the mandibular arch in Ts16 mice.

Males and females homozygous for the Robertsonian translocation specific for chromosomes 16 and 17 Rb(16.17)Bnr and male and females homozygous for the Robertsonian translocation for chromosomes 6 and 16 Rb(6.16)24Lub were bred to produce double heterozygotes [Rb(16.17)Bnr/Rb(6.16)24Lub]. Experimental data were based on 156 features: 70 euploid (control), 86 trisomic. Affected fetuses were identified by decreased size, shortened faces (flattened snouts), oedema, petechiae, open eyelids, and dysplastic ears. Identification of trisomics was substantiated by karyotyping the metaphasic spreads. Five gestational days were studied (14-18). Fetal age was assumed to be accurate as fertilization occurred within half an hour of copulation. Euploid specimens followed normal developmental paths of chondrogenesis, osteogenesis and related tissues. In trisomics, developmental faults increased unequally between gestational days 15 and 17: some tissues were mildly and others acutely affected. Among the trisomic disorders were diminished growth, lagging mitotic activity, and retarded and poorly ordered tissue development, especially of bone. All of these contributed to hypoplasia, hypocellularity, reduced vascular supply and enlarged intercellular spaces. Intensities of the mandibular abnormalities varied among litters and littermates. The severity of the developmental disorders of an individual Ts16 specimen differed among the tissue components studied. Of the trisomic mandibular tissues, bone was most frequently involved and Meckel's cartilage least.

Molar odontogenesis in the trisomic 16 mouse.

Stocks used were male and female monozygotes for Robertsonian translocation specific for chromosomes 16 and 17 Rb(16.17)7Bnr and males and females homozygous for Robertsonian translocation for chromosomes 6 and 16 Rb(6.16)24Lub to produce double heterozygotes characterized as Rb(16.17)Bnr/Rb(6.16)24Lub. This study was based on 156 fetuses, of which 70 were normal (euploid/controls) and 86 were affected trisomics identified grossly by decreased size, shortened faces (flattened snouts), oedema, petechiae, open eyelids and dysplastic ears. Confirmation of trisomics included karyotyping metaphasic spreads. Throughout the five gestational days studied (14-18), trisomic fetuses exhibited developmental delays of up to 24 h. In general, tooth organs were smaller, hypocellular, hypoplastic and had a decreased blood supply. These differences were progressive and more pronounced in the later periods of odontogenesis, especially in the morpho- and histodifferentiation stages.

Synthetic activities of mass cultures and clones of human gingival fibroblasts.

The percentage of synthesis dedicated to collagen is elevated in low-density cultures of human gingival fibroblasts, as is per-cell total protein synthetic activity and glycosaminoglycan accumulation. These observations can be explained, in part, by a decrease in membrane transport of precursor substance in high-density cultures. Synthetic activity by human fibroblasts can be reliably assayed in vitro using as few as 500 cells sparsely seeded. Such low-cell number assay is essential for study of single-cell clones, where replicative life span is limited.

Alcohol induced cardiac malformations in the rat.

A pattern of malformations described as the "fetal alcohol syndrome" (FAS), observed in the progeny of chronic alcoholic women, has been recently reported (Jones et al. 1973). Laboratory investigations in this area have focused primarily on the development of animal models which mimic the syndrome (Randall 1977; Chernoff 1977). In the present investigation, a single, intraperitoneal injection of an alcohol solution (0.03 ml/gm body weight of a 25% solution of 95% ethanol) was administered on day 10 of gestation to pregnant Sprague-Dawley rats. Fetuses were recovered on day 18 of gestation and examined for external malformations. Subsequent to standard fixation procedures utilizing Bouin's fixative, the tissues were serially sectioned at 8 micron and stained with hematoxylin and eosin. External malformations were infrequent in the experimental fetuses consisting of adactyly , wrinkled skin, and gastroschisis. Histological examination, however, revealed severe umbilical hernias and interatrial-septal defect in the experimental fetuses. The interatrial-septal defect was of the ostium secundum type and occurred in 41% of the offspring of the treated animals. It was not present in the control fetuses. This type of defect is recognized by the lack of a complete septum primum in the septal wall, permitting complete communication of the two atria at the level of the foramen ovale. The septum secundum usually remained intact.

Growth pattern and citrate production in organ cultures of adult rat ventral prostate.

Prostate rudiments from Wistar rats were cultured in MEM supplemented with 15% FBS and insulin (2 microgram/ml). In situ prostate acinar epithelium consists of tall columnar and low basal cells. Ultrastructure of the columnar cells is that of typical secretory cells; however, the basal cells are poor in cell organelle. During the first week of culture many secretory cells degenerated and were replaced by cuboidal or low columnar cells. In the presence of testosterone (2 microgram/ml) the secretory cells remained viable for 8-10 days before undergoing necrosis. At the termination of the experiment the acinar epithelium consisted of low cuboidal cells. The cultures showed a pattern of citrate production which reached a maximum at 5 days and remained-relatively constant thereafter. Ultrastructure of 1-week cultures exhibited Golgi complexes with dilated cisternae. Although a paucity of cell organelle was found in 7-day cultures, older cultures (19 days) exhibited morphologic characteristics of normal secretory cells.

Thiamine pyrophosphatase activity of the prenatal mouse molar. A histochemical investigation.

Thiamine pyrophosphatase (TPPase), an enzyme associated with the Golgi apparatus, has been implicated in the regulation of cellular oxidation as well as of transport across cell membranes. This enzyme has been localized in odontogenic tissues of the postnatal mouse and it was the intent of the present study to localize TPPase during prenatal odontogenesis. Mouse fetuses (CDI, Charles River) 14 through 19 days postconception were decapitated, the heads were frozen and mounted on the chuck of a cryostat. Frontal sections, 14 micrometers thick, were air-dried and incubated for TPPase activity. Subsequent to incubation the activity was visualized by immersion in 1% ammonium sulfide. The degree of enzyme activity varied not only with the chronological age of the fetus but also as a function of the tissue's metabolic state. Regions, such as the dental lamina, evidenced decreased TPPase activity with increasing age, while tissue layers such as the IEE displayed greater enzyme activity with increasing age.

The effects of nicotine on mouse first molar tooth germs in organ culture.

Mandibular first molars, extirpated from 18-day mouse fetuses were cultured in BGJb medium (Fitton-Jackson modification) in a Trowell type culture system. Two-day cultures were treated with 78, 117, 156, and 312 microgram/ml nicotine sulfate. Untreated control tooth germs demonstrated normal growth in vitro. The peripheral cell layer of mesenchymal papilla and the inner enamel epithelium differentiated into odontoblasts and ameloblasts respectively with subsequent elaboration of extracellular matrix. Tooth germs treated with 78 microgram/ml nicotine were only slightly affected where as higher doses of the drug produced extensive cell damage. Dental papilla appeared more sensitive than the enamel organ. Large necrotic foci were present in the pulp mesenchyme involving in some cases the odontogenic layer and severely limiting production of predentin. The basement membrane in these areas was partially disrupted and stained poorly for PAS. The inner enamel epithelium was not affected by 78 microgram/ml dose of nicotine, however, higher doses produced extensive cell necrosis in this layer. Although enamel matrix was abundantly present in untreated controls, the tooth germs exposed to 117, 156, and 312 microgram/ml nicotine sulfate failed to elaborate this extracellular matrix. Tooth germs exposed to nicotine for 24 hours followed by culturing in control medium demonstrated complete recovery. Treated cultures maintained in vitro for 7-9 days in control media had replaced the necrotic cells in the ameloblastic and odontoblastic layers and demonstrated abundant dentin and enamel matrices.

SDH activity in the developing incisors or irradiated mouse fetuses.

Pregnant CD1 Swiss albino mice were irradiated with 400 rads of whole body X-irradiation on the twelfth gestational day. The animals were then sacrificed beginning on day 14 through 20 of gestation by chloroform inhalation. The fetuses were extirpated via laparotomy and decapitated. The severed heads were rapidly frozen and sectioned in a cryostat. The sections were affixed to glass slides and incubated for succinic dehydrogenase activity according to the method of Nachlas et al. (1957) and counterstained in Safranin 0, routinely dehydrated and mounted. Data from observations indicated that siccinic dehydrogenase activity appeared normal in the tissue layers of the developing tooth germ when compared to control animals. When the experimental procedure had invoked damage to the developing tooth, succinic dehydrogenase activity was lessened relative to the degree of damage. Presumably the X-irradiation had affected the cellular maturation process thereby reducing the functional competency of the cells as illustrated by the reduced enzyme activity.

Sensitivy of mouse molar tooth germs to x-ray irradiation in vitro.

Molar tooth germs, extirpated from 18-day mouse fetuses were cultured on Millipore filter strips in Falcon organ culture dishes. The tooth germs were exposed to 250 kVcp X-rays at 106 R/min. for a total exposure of 1 600 R. Tissues were harvested on a daily basis for a total period of 12 days and were examined microscopically, utilizing H and E stain. Severe disorganization of the tooth germs was evident within 24 hours of irradiation. The basement membrane became hyalinized; pyknotic nuclei and lysed cells were observed throughout the dental papilla, but mostly in the regions of the presumptive cusps. Although a thin layer of predentin was elaborated by the odontoblasts, the matrix failed to calcify and enamel matrix was not produced. Cultures older than 10 days demonstrated extensive cell death. The entire pulp was reduced to a mass of necrotic cells and the ameloblastic layer consisted of an epithelial remnant covering the cuspal tips.

Development of the squamosomandibular articulation in the Mongolian gerbil (Meriones unguiculatus). II. Succinate dehydrogenase activity.

Succinate dehydrogenase activity has been studied, according to the method of Nachlas et al. (1957), in the developing tissues forming the squamosomandibular articulation in the Mongolian gerbil from its inception through the sixty postnatal day. Increased activity was observed in the chondroblasts, osteoblasts and mesenchymal tissues of the developing articulation. The chondroclasts of the developing mandibular condyle displayed intense reaction as did the osteoclasts of the developing bony articulation. Succinate dehydrogenase activity appeared to be related to the functional maturity of the cellular elements of the developing joint.

Ultrastructural localization of adenosine triphosphatase in the stellate reticulum, stratum intermedium and ameloblasts of the mouse molar.

ATPase activity in the developing first mandibular molar of the mouse was demonstrated at the electron microscopic level with the method of Wachstein & Meisel (1957). It was localized along the cell surfaces of the ameloblast and stratum intermedium interface, the stratum intermedium and the stellate reticulum. The ATPase final reaction product was also present at the cell membranes of the proximal region of adjacent ameloblasts and extended to the level of the nuclei. The demonstration of ATPase mainly on the plasma membranes was similar to the observations by other investigators of various non-odontogenic cell types involved in the exchange of materials across plasma membranes.

A fine-structural analysis of mouse molar odontoblast maturation.

The first mandibular molars of the Swiss albino mice, 1 through 4 days of age, were fixed in glutaraldehyde or Karnovsky's fixative. The tissues were postfixed in OSO4, dehydrated and embedded in Epon. The prepolarizing, polarizing and secretory odontoblasts were described. The prepolarizing cells, located in the vicinity of the cervical loop, were mesenchymal-like in morphology. The cells of the polarizing stage possessed organelles indicative of protein synthesis. The nucleus was located proximally. Aperiodic fibers were evident in the wide basement membrane. The secretory odontoblasts were long, slender, polarized cells closely adjoining one another. Each odontoblast possessed six morphologically discernible regions: (1) an infranuclear region, limited in size and containing few cellular organelles; (2) a nuclear region, housing the oval nucleus and a few associated lamellae of rough endoplasmic reticulum as well as a limited number of mitochondria; (3) a supranuclear rough endoplasmic reticulum region, possessing an abundance of these organelles as well as some mitochondria and secretory vesicles; (4) a Golgi region, occupying the middle third of the cell, housing the elements of an extensive Golgi apparatus which was surrounded by peripherally located profiles of rough endoplasmic reticulum; additionally, this region contained smooth endoplasmic reticulum, mitochondria, numerous secretory granules and vesicles and occasional intracellular collagen fibers; (5) an apical rough endoplasmic reticulum region, containing a rough endoplasmic reticulum component that was less extensive than its supranuclear counterpart; in addition, this region was the one richest in mitochondria and contained a plethora of secretory vesicles and granules; (6) the odontoblastic process, a region mostly void of organelles, containing various secretory products, some of which appeared to be in the process of being released extracellularly into the surrounding dentin matrix.

Electron microscopic localization of 5'-nucleotidase in the stratum intermedium and ameloblasts.

5'-nucleotidase was demonstrated at the fine structural level in the stratum intermedium and ameloblasts of the first mandibular molars of CD-1 mice. The enzyme was localized with the Wachstein & Meisel (1957) method along the plasma membranes of the cells of the stratum intermedium and ameloblasts. While 5'- nucleotidase was present throughout the stratum intermedium, only the proximal region of the plasma membranes of ameloblasts was demonstrably active for this enzyme. 5'-Nucleotidase has been implicated in transport of metabolites across cell membranes, and its localization in the present study supports this implication as well as the transport functions of the stratum intermedium and the stratum intermedium--ameloblastic interface.

Palatal shelf epithelium: a morphologic and histochemical study in X-irradiated and normal mice.

The palatal shelf epithelium of normal and irradiated mice was examined morphologically and histochemically, utilizing the periodic acid-Schiff (PAS) technique for the demonstration of the basement membrane and the Nitro BT method for succinate dehydrogenase activity in order to demonstrate the metabolic competence of its cells. The 'programmed cell death theory' was not supported by the present investigation, since the cells of the medial ridge epithelium retained their structural and metabolic integrity even subsequent to the formation of cell nests. Additionally, the medial ridge epithelium of mice with radiation-induced cleft palates demonstrated normal structural and metabolic integrity long past the prospective time of fusion.

Succinic dehydrogenase activity during palate formation in the Mongolian gerbil.

The palatal shelf epithelium of the Mongolian gerbil was examined for succinic dehydrogenase activity prior to, during and the after palatal fusion (days 18-20 post coitus). Enzyme activity was present during all stages examined, and was noted even in the epithelial pearls of fused palates. The presence of SDH activity in these epithelial pearls lends support to the theory 'epithelial stretching', and questions the theory of 'programmed cell death' in relation to the loss of the epithelium along the plane of fusion.

Histochemical evaluation of thiamine pyrophosphatase activity during first molar odontogenesis of the neonatal hairless mouse.

Localization of thiamine pyrophosphatase activity has been evaluated in the developing first molar of the neonatal hairless mouse. Postnatal animals from parturition to five days of age were decapitated and the severed heads frozen and sectioned in a frontal plane on a cryostat. 14 micron thick sections were fixed and subsequently incubated for thiamine pyrophosphatase activity according to the method of GOLDFISCHER et al. (1971). The tissue was visualized, dehydrated, cleared and mounted. Light microscopy was utilized in evaluating thiamine pyrophosphatase activity. Thiamine pyrophosphatase activity in the first molar of the hairless mouse is presented in tabular form and compared to similar data for the Swiss albino mouse. Enzyme activity increased as the metabolic activities of various cell layers increased. Thus, thiamine pyrophosphatase activity appeared to be related to the degree of differentiation and functional completency of the odontogenic tissues in the hairless mouse.

Molar odontogenesis in the hairless mouse.

Molar odontogenesis was studied in the hairless mouse from the initiation of the dental lamina through apposition. The dental lamina stage of the first molar was recognized on the 13th day, the bud on day 14th, cap on the 16th, bell on the 18th and apposition on the 20th day after conception. The morphology of the various stages and their temporal sequence were compared with those of other rodents.

Effects of ionizing radiation on incisor development of the prenatal mouse.

The effects of 100 rad of X-irradiation of incisor development in CD1 mice were studied. 24 pregnant mice were irradiated on the 12th day post coitum and sacrificed from the 14th through the 20th gestational days. The 191 irradiated fetuses were smaller than those not irradiated, their crania and necks were malformed and their lower extremities were poorly developed. The developing incisors of the irradiated animals were retarded, the pulpal vessels were enlarged and the vessels walls did not maintain their structural integrity. The cells of the future pulp were necrotic, and the basement membranes appeared hyalinized. Ameloblasts and odontoblasts were abnormal in morphology and the formation of dental hard tissue was inhibited in places. Complete absence of incisor tooth germ was noted in three of the fetuses.