Comparative SEM and TEM examination of the ultrastructure of the resin-dentin interdiffusion zone.

The resin-dentin interdiffusion zone produced by a dentin-adhesive system that removes the smear layer and concurrently decalcifies superficial dentin was morphologically examined by both scanning and transmission electron microscopy. Cross-sectioned resin-bonded dentin discs were etched with an argon-ion beam to make the resin-dentin interface observable by SEM. For the TEM examination, the sections were partly decalcified by an aqueous EDTA solution to facilitate ultramicrotomy and to disclose the ultrastructure of the interdiffusion zone. Both SEM and TEM confirmed the presence of the resin-dentin interdiffusion zone as the junction between the deep unaltered dentin structure and the restorative resin. Within the interdiffusion zone, three sublayers with characteristic ultrastructure and staining were identified by TEM. An upper diffuse black layer contained few structural features. Underneath, partially-altered collagen fibrils were closely packed, mostly running parallel with the interface and perpendicular to the dentinal tubules. Their outline was electron-dense, forming tunnel-like structures. At the base of the upper layer, several stained projections were found to bulge out into the underlying collagen network and appeared to be confined by obstructive, parallel-running collagen fibrils. Finally, the third dense layer, containing hydroxyapatite crystals, demarcated the superficially demineralized dentin layer from the deeper unaltered dentin. Resin diffusion into the decalcified dentin surface layer was evident, but diminished with depth, presumably reducing deeper resin impregnation into the interfibrillar spaces. The citric acid dentin-pretreatment probably caused denaturation of the superficial collagen fibrils. Its decalcifying effect gradually weakened with depth, leaving behind hydroxyapatite crystals at the base of the interdiffusion zone.(ABSTRACT TRUNCATED AT 250 WORDS)

The mandibular body of the human fetus. Histologic analysis of the basilar part.

Histologic and microradiographic analyses of 47 human mandibles from 42 fetuses and 5 neonates have been carried out to study the development of the mandibular body. This not only entails woven bone formation along Meckel's cartilage but also endochondral ossification in the condylar cartilage, the pillars of which are included in the posterior part of the mandibular body. Progressively, woven bone is replaced by lamellar bone, and typical Haversian systems are already present at the fifth month of fetal life. This early occurrence of bone remodelling is related to the fact that the mandible is subjected to intense activity from sucking and swallowing.

A few observations about Meckel's cartilage in the human.

In the human embryo, fusion of Meckel's cartilages takes place during mandibular development. It seems to be independent of the endochondral ossification with respect to the developmental sequence.

Calcified tissues involved in the ontogenesis of the human cranial vault.

The cranial vault of fifteen human subjects varying in age from 20th week of gestational life to 9th month post-matum were submitted to microradiographic and histological analysis. Different phenomena such as cortical drift, bone cavitation and progressive substitution of different calcified tissues by lamellar bone are illustrated. Moreover, this study reveals in several areas the presence of chondroid tissue; it constitutes the edges of the sutures and is responsible for their growth till the post-natal period. Therefore, it can be supported that the role of chondroid tissue is essential for the harmonious development of the cranial vault.

Effects of retinoic acid on rat bone.

The effects of retinoic acid on rat bones were investigated. Fifteen adult male Sprague-Dawley rats were given various oral doses of retinoic acid (0-20 mg/kg body weight/day, 5 days/wk) and were killed after various treatment periods (1-17 wk). Histological and microradiographic examination of the bones showed the presence of lesions, the development of which was dependent on the dose and the duration of treatment. These lesions were characterized by a dissolution of bone matrix, leaving only fibrils, by osteocytic osteolysis and by an increase in osteoclastic resorption.

Histological observations of palatal malformations in rat embryos induced by retinoic acid treatment.

Malformations of the palate were induced in white rat embryos following maternal exposure to retinoic acid (tretinoin). Five experimental groups and the controls were treated by the following protocol: Group 1: pregnant rats received 100 mg retinoic acid (RA)/kg b.w. suspended in corn oil on gestational day (GD) 11.5; Group 2: 20 mg RA/kg b.w. from GD 8-12; Group 3: 20 mg RA/kg b.w. from GD 7.5-11.5; Group 4: 100 mg RA/kg b.w. on GD 10-11; Group 5: 100 mg RA/kg b.w. on GD 10 and 12; Group 6 received corn oil vehicle from GD 7-14.5; and Group 6: served as non-injected controls. In all retinoic acid treated groups, varying degrees of clefts with occasional attempts of fusion were noted. The severity and frequency of the malformations were dependent on dosage or gestational day of drug treatment. Our results indicate that RA, even at the lowest dose tested (20 mg/kg b.w.) severely affects the various tissues constituting the embryonic palatal shelves by altering cell interaction and possibly programmed cell death. These events would then result in lack of or inadequate differentiation with subsequent formation of aberrant craniofacial architecture.

Craniofacial abnormalities induced by retinoic acid: a preliminary histological and scanning electron microscopic (SEM) study.

Exogenous retinoic acid has been found to be teratogenic in animals and man. Craniofacial defects induced by retinoic acid have stimulated considerable research interest. The present report deals with scanning electron microscopical observations of the craniofacial region concurrent with histological examination of craniofacial dysmorphism induced in rat embryos following maternal treatment treated with varying dosages of all-trans-retinoic acid (tretinoin). Two groups of pregnant rats were treated with rat embryos exposed to retinoic acid suspended in corn oil (100 mg/kg b.w. on gestational day 11.5 and 50 mg/kg b.w. on gestational day 10, 11 and 12 respectively). A third group was treated with corn oil (vehicle) while a fourth group remained untreated. A wide spectrum of congenital abnormalities, including exophthalmos, microphthalmia and anophthalmia, maxillo-mandibular dysostosis, micrognathia of both maxilla and mandible, cleft palate, subdevelopment of ear lobe, preauricular tags and macroglossia, were observed in the offspring of retinoic acid treated animals. The abnormalities were both time and dosage dependent, and characteristic of Treacher Collins syndrome when retinoic-acid was administered on gestational day 11.5. In contrast, when retinoic acid was administered were on gestational days 10-12, the defects were similar to those seen in the first and second pharyngeal arch syndrome, as well as in the oculo-auriculo-vertebral spectrum. Whereas our data support the hypothesis that all-trans retinoic-acid disturbs growth and differentiation of several embryonic cell types essential for normal craniofacial development, its mechanism of action remains unclear.

Skeletal growth and chondroid tissue.

Membranous and endochondral ossification processes are insufficient to describe all the aspects observed in the growing skeleton. The presence of chondroid tissue that we have identified by means of all modern histological techniques, including those able to detect the different types of collagen, has also to be explained. Present in the mandibular symphysis of either the human or cat fetuses, chondroid tissue has also been observed in the other parts of the mandible, in the sutural areas of the skull and in all the bones of both axial and appendicular skeleton. The differentiation of the mesenchyme into chondroid tissue could probably be related with mechanical forces exerted simultaneously in opposite directions or with a transient ischemia.

Anterior neural tube malformations induced after all-trans retinoic acid administration in white rat embryos. I. Macroscopical observations.

For this study all-trans-retinoic acid was administered in pregnant white rats in their "prima gravida" pregnancy. Rats were divided in five groups. The first three groups were treated with 20 mg R.A./kg b.w. at several gestational days. The fourth group was treated with corn oil, while the fifth group remained untreated. All the animals were sacrificed during the first hours of the 21st gestational day. In the first group, three embryos, five absorptions and six compact embryonic masses were counted in litters. All the embryos presented exencephaly, combined with external anopthalmia. They also presented severe craniofacial malformations. In the second group, nine embryos and five compact embryonic masses were counted in litters. Three of the embryos presented exencephaly combined with external anopthalmia, while the six remaining presented complex craniofacial anomalies. In the third group, exencephaly was present in two embryos combined with anopthalmia, seven embryos had complex anomalies and four compact embryonic masses were counted in litters. Our results indicate the teratogenic involvement of all-trans-retinoic acid in anterior neural tube differentiation.

All trans retinoic acid interfering with palatal development. Scanning electron microscopical and light microscopical observations on embryonic rat palate.

Diverse studies on retinoic acid teratogenesis, during the recent years, indicate that the drug's analogues target on diverse cell population during differentiation in mammals. During an extended teratological protocol concerning retinoic acid influence in diverse embryonic tissue differentiation in experimental animals we studied all-trans-retinoic acid's influence on palatal development in the white rat embryo. For this purpose, six groups of white rat embryos were studied: Group 1 was treated with 100 mg/kilogram of body weight (k.b.w.) on gestational days (g.d.) 10th and 11th, Group 2 was treated with 100 mg all-trans-retinoic acid/k.b.w. on g.d. 11.5, Group 3 was treated with 50 mg all-trans retinoic acid/k.b.w. on g.d. 10th, 11th and 12th, Group 4 was treated with 50 mg all-trans-retinoic acid/k.b.w. on g.d. 11th and 12th, Group 5 was treated with 20 mg all-trans-retinoic acid/k.b.w. on g.d. 7.5, 8.5, 9.5, 10.5 and 11.5, Group 6 remained untreated. Embryonic heads aged 20 days were observed by light microscopy and scanning electron microscopy. In all treated groups clefts and malformations concerning the differentiation of palatal cell populations were observed. All our findings were compared with normal palatal morphology of untreated "control" embryos. Among the malformations, median clefts were observed, extended along only a part of the primary and all the secondary palate for group 2, the primary and secondary palate for groups 1, 3 and 5 while on group 4, an irregularity of the median palatal raphe and rugae were combined with a median incomplete cleft extended between the primary and secondary palate. Our results are discussed in relation with the international literature results.

Identification of collagen type I and type II in chondroid tissue.

This paper deals with investigations concerning the matrix of chondroid tissue. Among the 73 human fetus or child mandibles and the 42 cat mandibles we have studied histologically and microradiographically, 8 human and 3 cat mandibles were used to determine the collagen composition in chondroid tissue matrix, and 10 cat mandibles were analyzed in order to have an ultrastructural approach to chondroid tissue. Both in human and cat mandibles, types I and II collagen were identified by indirect immunofluorescence and immunoperoxidase techniques. Electron microscopic analysis shows large collagen fibrils which correspond to type I collagen, and smaller collagen fibrils, principally located at the periphery of the chondroid cells. From our investigations, chondroid tissue should be considered as being different from both bone and cartilage, although it is not a transitional tissue, since no transformation of chondroid tissue into bone is observed; it should be classified as an intermediate tissue between cartilage and bone because of its morphological characteristics.

[Symphysis menti in the human fetus]. / Die Symphysis menti beim menschlichen Feten.

The mandibular symphysis of the human fetus is radiolucent (Fig. 1). The ventral extremity of the hemimandible consists of secondary cartilage (Figs. 2A, 2B, 5, 7), chondroid tissue (Fig. 4) and woven bone (Figs. 2A, 2B). The examination of the decalcified mandible allows not only the identification of secondary cartilage but also the observance of the soft tissue of the mandibular symphysis and particularly the chondriola symphysea (Figs. 9, 10). Only the preparation of undecalcified sections permits the study of chondroid tissue (Figs. 4, 6, 8) because of the particular pattern of calcification present in this tissue.

[The mandibular symphysis of the newborn. Histologic and microradiographic study]. / La symphyse mandibularire du nouveau-né. Etude histologique et microradiographique.

In this histologic and microradiographic study of the mandibular symphysis of eleven newborns, we describe each of the tissues which constitute the mandibular symphysis. To make our observations, we have investigated consecutive sections (fig. 1) of the region. At birth, the mandibular symphysis is forged by several small nodules of calcified tissue, the ossicula mentalia, and by soft tissue invisible in the microradiographs. Therefore, it is necessary to examine first the same field at an identical magnification after methylene blue staining, and then the decalcified slides after coloration according to Masson's trichromic method. Between the two hemimandibles we have observed fibrous tissue and chondriola symphysea (fig. 4 and 5). Figures 2A, 2B and 3 shows secondary cartilage bordering the mandibular symphysis and later replaced with bone by endochondral ossification. In the ossicular mentalia (fig. 6 and 7), chondroid tissue (fig. 7 and 8) is interspersed with secondary cartilage and woven bone, as it is in the extremities of the hemimandibles.

[Influence of masticatory muscle insertions on the mandibular structure of the newborn infant]. / Influence des insertions des muscles masticateurs sur la structure mandibulaire du nouveau-né.

The influence of the muscular insertions on the mandibular structure in new born infants has been studied with the aid of microradiographical analysis applied to 80 microns thick undecalcified sections and in paraffin sections after decalcification. The left side of the mandibles was cut into sections parallel to the vestibular surface of the bone (fig. 1) and the right side, in frontal sections (fig. 2). The tendinous fibres of the temporal muscle are inserted into the apex of the coronoïd process, which is, at this age, constituted by chondroïd tissue (fig. 3). The continued differentiation of this tissue within the coronoïd process, permits the migration of the temporal muscle insertion. Microradiographical analysis has been used to show the changes of the calcified tissue aspects in the masseter and medial pterygoïd muscle insertions (fig. 4 and 5). The insertion of the lateral pterygoïd muscle doesn't transform the bone tissue morphology (fig. 6-A and 6-B).

[Role of the condylar cartilage in mandibular growth (author's transl)]. / Rôle du cartilage condylien dans la croissance mandibulaire.

Our purpose is to bring a contribution to a better understanding of the role played by the condylar cartilage in children's mandibular growth. In the newborn, microradiographical analysis of frontal (fig. 1, 2 and 3 A) or sagittal sections (fig. 4 A) has been used to locate trabecular bone of endochondrial origin. This trabecular bone contains islets of calcified cartilage easy to identify in the microradiograph (fig. 3 B) or in the section itself after methylen blue staining (fig. 4 B) and resulting from the activity of the condylar cartilage (fig. 4 C). At four month of age, the condylar cartilage is still in activity (fig. 5 A and 5 B). From both morphological and physiological points of vue, this work leads to suggest that the condylar cartilage must be compared to a growing epiphyseal nucleus or to a so-called infertile extremity of a small long bone rather than with a true growing cartilage.

Electron microscopic study of chondroid tissue in the cat mandible.

This paper deals with electron microscopic appearance of chondroid tissue. Samples from eight cat mandibles were studied without decalcification. The ultrastructural characteristics of the chondroid tissue cells are common with young osteocytes. The interterritorial matrix of chondroid tissue is mineralized, being constituted of large collagen fibrils and calcospherites. The compositions of these parts of the chondroid tissue matrix and of bony matrix are similar but they are two different tissues. The pericellular matrix of the chondroid tissue consists of finely branched filaments, thin collagen fibrils, and an abundant ground substance. It resembles a cartilage matrix and contains type II collagen which is not present in bony matrix.

Comparison of the calcium content of different tissues present in the human mandible.

Qualitative and semi-quantitative comparisons of calcium content in the developing human mandible were performed by means of microradiographic and histophotometric analysis. Differences in calcium content between enamel, calcified cartilage, chondroid tissue and dentin are significant at the 1% level. Chondroid tissue and woven bone are almost similarly mineralized tissues.

Age changes in human bone: a microradiographic and histological study of subperiosteal and periosteal calcifications.

Age changes in subperiosteal areas of human bones are described in 56 subjects of both sexes aged 18-96 years, using microradiographic and histological analysis of thick undecalcified sections from the midshaft of the femur, the tibia, the third metacarpal bone and the third rib. Till the age of 39, most areas of the subperiosteal bone consist of circumferential fundamental lamellae. From the age of 48, certain areas of the subperiosteal bone appear hypercalcified, while in others mineral precipitations occur in the inner layer of the periosteum close to the bone cortex, involving the deep periosteal fibers. These periosteal calcifications should partially account for the increase in transverse diameter of bones throughout adult life.

Metopic sutural closure in the human skull.

The present study reveals the presence in the sutural area of secondary cartilage, assuring the passive growth of the bones and undergoing an endochondral ossification, but without playing a direct role in the synostosis. The chondroid tissue is responsible for the growth of each frontal bone towards the other and constitutes the first bridge of union between the two bones. It is the most important finding in this study, which provides a description of the closure of the metopic suture and of the maintenance of an open sutural space by a process of active resorption. This new knowledge will help to understand better the whole process of suture closure and its pathology.

Oculo-auriculo-vertebral spectrum: cranial and vertebral malformations due to focal disturbed chondrogenesis.

Microradiographic and histological analyses point out a focal disturbed chondrogenesis of both the skull base and the axial skeleton in a case of oculo-auriculo-vertebral spectrum. Cartilage showed disturbed endochondral ossification with defects in calcification, deficient resorption, and abnormal crumpled areas of mineralized cartilage.