Development of cartilage and bone tissues of the anterior part of the mandible in cichlid fish: a light and TEM study.

The present paper presents ultrastructural details of chondrogenesis of Meckel's cartilage and of ossification of its associated peri- and parachondral bones in a teleost fish, the cichlid Hemichromis bimaculatus. We have distinguished four stages during chondrogenesis, each of which is characterized by specific cellular and matrix features: blastema, primordium, differentiated cartilage and cartilage surrounded by perichondral bone. The blastema is characterized by prechondroblasts and the lack of cartilage matrix; the primordium by chondroblasts and the onset of secretion of matrix of fibrillar and granular nature; differentiated cartilage is characterized by chondrocytes and larger amounts of typical hyaline cartilage matrix. Once perichondral bone is laid down, the chondrocytes show degenerative features but not true hypertrophy. Differentiation of the cartilage cells is attended with cytoplasmic changes indicative of an increasing secretory activity. There is a regional calcification of the cartilage matrix by fusion of calcospherites. Chondrogenesis of the symphyseal area is continuous with that of the rami but starts slightly later. Formation of perichondral bone at the cartilage surface is attended with the deposition of a transitional zone apparently containing a mixture of the two matrices. The role of the perichondral cells is discussed and it is proposed that they may contribute to the formation of the two matrices. The transitional zone may then result either from a diffusion process or from the simultaneous deposition of elements of the two matrices. Growth of the cartilage is argued to be largely the result of matrix secretion, except in the symphyseal area where appositional growth probably occurs until the region is completely covered by perichondral bone. This paper provides a basis for further studies on the developmental interactions between cartilage, bone and teeth during mandibular development in cichlids.     

Bone and cartilage resorption in relation to tooth development in the anterior part of the mandible in cichlid fish: a light and TEM study.

This paper presents ultrastructural features of the contact region between particular tooth germs and Meckel's cartilage prior to, during, and after initial resorption of the perichondral bone and of the cartilage in the cichlids Hemichromis bimaculatus and Astatotilapia burtoni. Imminent resorption opposite such teeth is announced by the presence, in this region, of a particular cell type, considered to be a stage in the cytodifferentiation of osteoclasts. Slightly later, an osteoclast with typical ruffled border is seen to open a fenestra in the perichondral bone which surrounds Meckel's cartilage. Although the action of the osteoclast is directed primarily towards the bone, it may also affect, to a much lesser extent, the underlying uncalcified cartilage. Typically, fibroblast-like cells invade the resorption cavity along with the osteoclast; the tooth germ soon follows. Capillaries are seen to invade the cartilage only at a later stage when a large cavity has been established. It is proposed that the fibroblast-like cells may have a dual function: degradation of cartilage and deposition of new bone. Although these processes are normally limited to the area surrounding tooth germs at specific loci, tooth germs in other positions may sometimes be seen invade the cartilage. They do so either passively, because of the existence of such a cavity, or as a result of their own resorption-inducing activity. Whatever the mechanism, attachment bone is being deposited within the erosion cavity and on the surface of the exposed perichondral bone. The stimuli possibly eliciting resorption of Meckel's cartilage are discussed. It is hypothesized that pressure exerted by the growing tooth germ may stimulate the osteoblasts covering the bone surface and, in this way, provoke osteoclastic bone resorption.     

Epiphyseal and physeal cartilage vascularization: A light microscopic and tritiated thymidine autoradiographic study of cartilage canals in newborn and young postnatal rabbit bone

The vascular pattern of newborn and early postnatal epiphyseal and physeal cartilage is integral to long bone development and differs from later postnatal patterns. In the present study, we supplement light microscopic histology with tritiated thymidine autoradiography to help assess the position of cartilage canals and the dynamics of cartilage vascularity in relation to growth. Tritiated thymidine labeling studies to assess cell proliferation activity were done by using 2 μc/g body weight intraperitoneal injections into newborn and 3-, 4-, and 7-day-old New Zealand white rabbits that were killed 1 hr after the injection. Proximal humeral, distal femoral, and third metatarsal epiphyses were assessed by routine histology and serial section autoradiography. Cartilage canals were seen in each epiphysis. Transphyseal vessels were seen in each epiphysis continuous from the epiphysis to the metaphysis or were present within the physis traversing the proliferating and hypertrophic cell zones. Histologic sections showed vessels from the perichondrium continuous with those of the epiphyseal cartilage canals at proximal humeral, distal femoral, and metatarsal epiphyses. Serial sections showed vascular buds and connective tissue cells lying in indentations at the periphery of and present within the epiphyseal cartilage. Autoradiographic studies showed extensive labeling of vessel wall cells and surrounding connective tissue cells of the cartilage canals (a) within the epiphyseal cartilage, (b) traversing the physis, and (c) within the epiphyseal cartilage but continuous with the perichondrial vessels. The labeling was always far more extensive than in the surrounding chondrocytes and was always present throughout the entire extent of the canals. In conclusion, the cell labeling activity strongly supports an active dynamic phenomenon underlying the vascularization of epiphyseal and physeal cartilage. Anat. Rec. 252:140–148, 1998. © 1998 Wiley-Liss, Inc.     

Morphometric study of malformations induced by hypervitaminosis A on rat fetus Meckel cartilage, maxillary bone and mandible

Morphological and histological studies were carried out to determine the effects on fetal development in rats of a single intraperitoneal injection of 150,000 I.U. of vitamin A, given to the mothers on the 10th day of gestation. The Meckel cartilage of malformed animals appeared to be diminished, with larger nuclei, less numerous isogenous nests, smaller cellular and thin perichondrium and more abundant cartilaginous matrix, which was less deeply stained by alcian blue. The maxillary bone and mandible of the treated fetuses showed thin and poorly calcified trabeculae, smaller medullary spaces and a less fibrous, and more cellular periosteum. These data were confirmed with morphometric techniques.     

Sensory supply of the anterior uvea: A light and electron microscope study

Summary We have studied the ultrastructural features of sensory nerve fibers in the ciliary body and the iris and their parent trigeminal ganglion cells, using intra-axonally transported horseradish peroxidase as a tracer. Unmyelinated nerve fibers of ipsilateral trigeminal origin were found in stroma of both the ciliary body and the iris. Most appeared in bundles of nerve fibers; but some were found singly. Two distinct types of varicosities were found, one containing both a few irregularly shaped vesicles and mitochondria, the other containing mitochondria only. The thin profiles of the axons between these varicosities or endings contained neurotubules and filaments. The trigeminal ganglion cells supplying the anterior eye were of a relatively small size (15–50 m in diameter) and confined to the anteromedial part of the ipsilateral ganglion. These cells could not be classified according to the arrangement of their subcellular organelles. The morphological characteristics of the sensory nerve fibers and ganglion cells supplying the anterior uvea are consistent with the view that pain may be the predominant sensation mediated by these neurons.     

Neurohormonal regulation of the development and regenerative processes of bone and cartilage tissues]

Neurohumoral regulation of cartilaginous and osseous tissues was investigated in different stages of ontogenesis. It has been shown that these tissues are targets practically for all hormones produced by the body. The reciprocal reaction of osseous and cartilaginous tissues to different concentrations of hormones was estimated. It has been also demonstrated that the action of hormones in these tissues manifests itself in accordance with the general regularities and that their effects are strictly specific for the test target tissues. An opinion is advanced about the breach of the endocrine situation in the body as a risk factor of the development of dystrophic processes in cartilages.     

A light and electron microscopic study of the region of cartilage resorption in the embryonic chick femur

It has long been known that uncalcified cartilage of embryonic chick long bones is removed to make way for invading marrow. However, no one has clearly established which cells are responsible for this erosion. Using the light and electron microscopes, we have studied the cartilage-marrow interface, which we presume to be the region of resorption. Here, we found two types of mononuclear cells in intimate contact with cartilage matrix. 1. The predominate cell type had a euchromatic nucleus with a nucleolus and a cytoplasm containing extensive profiles of rough endoplasmic reticulum; also, processes extended from these cells into the adjoining cartilage matrix. 2. Macrophages containing many lysosomal vesicles, which often became swollen, were found on or near the surface of cartilage. In addition, a few cells with an intermediate appearance were present. A decrease in the amount of sulfated material in a 25–30 μm zone of cartilage in advance of the interface and an alteration in the orientation, and in some cases the integrity, of collagenous fibers were associated with the presence of the above mentioned cells. These alterations in cartilage were not due to the synthesis of sulfated or of collagenous material. The above evidence, although not conclusive, suggests that these mononuclear cells are responsible for cartilage resorption. In this respect, the removal of avian uncalcified cartilage is similar to the resorption of uncalcified articular cartilage which occurs in rheumatoid arthritis.     

Some unusual features of mycobacteriosis in the cichlid fish Oreochromis mossambicus

Adult, aquarium-reared Oreochromis mossambicus (Tilapia mossambica), which were naturally infected with Mycobacterium marinum, displayed non-healing skin ulcers and other clinical signs considered to be typical of piscine mycobacteriosis. However, in addition, they frequently had melanotic foci in the skin and spleen, due to the presence of pigment cells surrounding the cutaneous and splenic inflammation. Such melanotic foci have never been reported in response to mycobacteriosis. All fish also have variable numbers of melanomacrophages which appeared to replace pancreatic acini. The relationship of the pancreatic melanomacrophages to the pathogenesis of mycobacteriosis is uncertain, but such lesions may have contributed to the chronic cachexia associated with this case.     

The role of predictability in the stress response of a cichlid fish

In recent years there has been an increasing interest in the cognitive abilities of fish with implications for animal welfare and management of rearing operations. Although it is known that psychological factors can modulate the stress response in mammals, this aspect has seldom been investigated within stress in fish. In this study we investigate whether the perception (appraisal) that fish make of significant environmental events modifies their behavioural and physiological response. For this purpose we have used a predictable vs. unpredictable paradigm for positive (feeding) and negative (confinement) events using the cichlid fish Oreochromis mossambicus as a model species. Results show that there is a differential effect of predictability for the feeding and confinement events. In the confinement experiment, predictability involved more attention to the visual cue and lower cortisol. The feeding event triggered higher levels of anticipatory behaviour and a tendency for higher cortisol in the predictable group. Therefore, predictable negative events reduce the cortisol response. Predictable positive events may elicit an anticipatory response, and when there is a significant delay between the visual cue and the actual occurrence of the event, it may also contain elements that can be interpreted as a stress response. These findings demonstrate that fish can appraise relevant aspects of the environment, with welfare implications for housing, husbandry and experimental procedures.     

Development of the rat sublingual gland: A light and electron microscopic immunocytochemical study

Cell differentiation in the rat sublingual gland occurs rapidly and is largely complete by birth. To study differentiation of the serous and mucous cells of the sublingual gland, we used antibodies to the secretory proteins CSP‐1, SMGB, PSP, and SMGD, and sublingual mucin as specific cell markers. Glands from rats at ages 18, 19, and 20 days in utero, and postnatal days 0, 1, 5, 9, 14, 18, 25, 40, and 60 were fixed and prepared for morphological analysis and immunocytochemical labeling. At age 18 days in utero, a few cells in the developing terminal bulbs contained mucous‐like apical granules that labeled with anti‐mucin. Other cells had mixed granules with a peripheral lucent region and a dense core of variable size that occasionally labeled with anti‐SMGD. Additionally, presumptive serous cells with small dense granules that contained CSP‐1 and SMGB were present. At age 19 days in utero, the dense granules of these cells also labeled with anti‐SMGD. By age 20 days in utero, mucous cells were filled with large, pale granules that labeled with anti‐mucin, and serous cells had numerous dense granules containing CSP‐1, SMGB, PSP, and SMGD. Fewer cells with mixed granules were seen, but dense regions present in some mucous granules (MGs) labeled with anti‐SMGD. After birth, fewer MGs had dense regions, and serous cells were organized into well‐formed demilunes. Except for PSP, which was undetectable after the fifth postnatal day, the pattern of immunoreactivity observed in glands of neonatal and adult animals was similar to that seen by age 20 days in utero. These results suggest that mucous and serous cells have separate developmental origins, mucous cells differentiate earlier than serous cells, and cells with mixed granules may become mucous cells. Anat Rec 266:30–42, 2002. © 2002 Wiley‐Liss, Inc.     

Comparative study of polarized light propagation in biologic tissues

We report the depolarization of light scattered by a variety of birefringent and nonbirefringent tissues. We used Stokes polarimetry to investigate how scatterer structures in each tissue contribute to the depolarization of linearly versus circularly polarized light propagating through that tissue. Experiments were performed on porcine blood, fat, tendon, artery, and myocardium. The results indicate that the two incident polarization states are depolarized differently depending on the structure of the sample. As seen in sphere suspensions, for tissues containing dilute Mie scatterers, circularly polarized light is maintained preferentially over linearly polarized light. For more dense tissues, however, the reverse is true. The results illustrate situations where polarized light will provide an improvement over unpolarized light imaging, information that is crucial to optimizing existing polarimetric imaging techniques.     

An in situ hybridization study of the Syndecan family in the developing condylar cartilage of fetal mouse mandible

Mandibular condylar cartilage is a representative secondary cartilage, differing from primary cartilage in various ways. Syndecan is a cell-surface heparan sulfate proteoglycan and speculated to be involved in chondrogenesis and osteogenesis. This study aimed to investigate the expression patterns of the syndecan family in the developing mouse mandibular condylar cartilage. At embryonic day (E)13.0 and E14.0, syndecan-1 and -2 mRNAs were expressed in the mesenchymal cell condensation of the condylar anlage. When condylar cartilage was formed at E15.0, syndecan-1 mRNA was expressed in the embryonic zone, wherein the mesenchymal cell condensation is located. Syndecan-2 mRNA was mainly expressed in the perichondrium. At E16.0, syndecan-1 was expressed from fibrous to flattened cell zones and syndecans-2 was expressed in the lower hypertrophic cell zone. Syndecan-3 mRNA was expressed in the condylar anlage at E13.0 and E13.5 but was not expressed in the condylar cartilage at E15.0. It was later expressed in the lower hypertrophic cell zone at E16.0. Syndecan-4 mRNA was expressed in the condylar anlage at E14.0 and the condylar cartilage at E15.0 and E16.0. These findings indicated that syndecans-1 and -2 could be involved in the formation from mesenchymal cell condensation to condylar cartilage. The different expression patterns of the syndecan family in the condylar and limb bud cartilage suggest the functional heterogeneity of chondrocytes in the primary and secondary cartilage.     

Aggressive behavior and reproductive physiology in females of the social cichlid fish Cichlasoma dimerus

The South American cichlid fish Cichlasoma dimerus is a freshwater species that presents social hierarchies, a highly organized breeding activity, biparental care and a high frequency of spawning. Spawning is followed by a period of parental care (about 20 days in aquaria conditions) during which the cooperative pair takes care of the eggs, both by fanning them and by removing dead ones. The different spawning events in the reproductive period were classified as female reproductive stages which can be subdivided in four phases, according to their offspring degree of development: (1) female with prespawning activity (day 0), (2) female with eggs (day 1 after fertilization), (3) female with hatched larvae (day 3 after fertilization) and (4) female with swimming larvae (FSL, day 8 after fertilization). In Perciform species gonadotropin-releasing hormone type-3 (GnRH3) neurons are associated with the olfactory bulbs acting as a potent neuromodulator of reproductive behaviors in males. The aim of this study is to characterize the GnRH3 neuronal system in females of C. dimerus in relation with aggressive behavior and reproductive physiology during different phases of the reproductive period. Females with prespawning activity were the most aggressive ones showing GnRH-3 neurons with bigger nuclear and somatic area and higher optical density than the others. They also presented the highest levels of plasma androgen and estradiol and maximum gonadosomatic indexes. These results provide information about the regulation and functioning of hypothalamus-pituitary-gonads axis during reproduction in a species with highly organized breeding activity.     

A simple method for the cultivation of anterior pituitary tissues: Electron microscopical evaluation of the cultured tissues

Summary Suitability of an ordinary incubation system for the culture of anterior pituitary tissues of mice was examined by electron microscopy. It was found that this system has many advantages over Trowell's standard technique for tissue culture and is particularly suitable for the short-term culture.In this system the pituitary tissue cultures were maintained well for at least two days in a chemically defined tissue culture medium (TC 199) without any additives. Addition of 20% calf serum to the medium extended the period to up to five days. Any attempts to prolong it further, however, failed because of a massive necrosis and a great loss of the histological integrity.In the cultured tissues there an enhancement of the LTH cells and a suppression of the other granulated types of cells were observed. The follicular cells were found to hypertrophy and to actively participate in phagocytosis of cell debris.Grateful acknowledgement is made for the technical services of Mr. S. Terada, Mr. T. Anzai and Mr. S. Sasaki.     

Relations between Meckel's cartilage and the morphogenesis of the mandible in the human embryo

Summary Based on a series of human embryos classified in stages [28], the authors studied the evolution of Meckel's cartilage in its ventral portion and its relations to the morphogenesis of the mandible. Three stages appeared particularly important: stage 16, appearance of Meckel's cartilage; stage 20, beginning of membranous ossification; and stage 23, end of the embryonic period (8th week). The primitive bony nodule which develops from the embryonic mesenchyme appears as a double bony layer forming a groove containing the neurovascular bundle, into which the dental lamina is also invaginated.

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Relations entre le cartilage de Meckel et la morphogénèse de la mandibule chez l'embryon humain

Résumé A partir d'une série d'embryons humains classés en stades [28], les auteurs étudient l'évolution du cartilage de Meckel dans sa portion ventrale et ses relations avec la morphogénèse de la mandibule. Trois stades apparaissent comme particulièrement importants : Stade 16 : Apparition du cartilage de Meckel ; Stade 20 : Début de l'ossification membraneuse. Stade 23 : Fin de la période embryonnaire (8ème semaine). Le nodule osseux primitif, qui s'est formé aux dépens du mésenchyme embryonnaire, se présente sous l'aspect d'une double lame osseuse formant une gouttière, lit du paquet vasculo-nerveux, dans laquelle vient également s'invaginer la lame dentaire.

     

Functional analysis of CTRP3/cartducin in Meckel's cartilage and developing condylar cartilage in the fetal mouse mandible

CTRP3/cartducin, a novel C1q family protein, is expressed in proliferating chondrocytes in the growth plate and has an important role in regulating the growth of both chondrogenic precursors and chondrocytes in vitro. We examined the expression of CTRP3/cartducin mRNA in Meckel's cartilage and in condylar cartilage of the fetal mouse mandible. Based on in situ hybridization studies, CTRP3/cartducin mRNA was not expressed in the anlagen of Meckel's cartilage at embryonic day (E)11.5, but it was strongly expressed in Meckel's cartilage at E14.0, and then reduced in the hypertrophic chondrocytes at E16.0. CTRP3/cartducin mRNA was not expressed in the condylar anlagen at E14.0, but was expressed in the upper part of newly formed condylar cartilage at E15.0. At E16.0, CTRP3/cartducin mRNA was expressed from the polymorphic cell zone to the upper part of the hypertrophic cell zone, but was reduced in the lower part of the hypertrophic cell zone. CTRP3/cartducin-antisense oligodeoxynucleotide (AS-ODN) treatment of Meckel's cartilage and condylar anlagen from E14.0 using an organ culture system indicated that, after 4-day culture, CTRP3/cartducin abrogation induced curvature deformation of Meckel's cartilage with loss of the perichondrium and new cartilage formation. Aggrecan, type I collagen, and tenascin-C were simultaneously immunostained in this newly formed cartilage, indicating possible transformation from the perichondrium into cartilage. Further, addition of recombinant mouse CTRP3/cartducin protein to the organ culture medium with AS-ODN tended to reverse the deformation. These results suggest a novel function for CTRP3/cartducin in maintaining the perichondrium. Moreover, AS-ODN induced a deformation of the shape, loss of the perichondrium/fibrous cell zone, and disorder of the distinct architecture of zones in the mandibular condylar cartilage. Additionally, AS-ODN-treated condylar cartilage showed reduced levels of mRNA expression of aggrecan, collagen types I and X, and reduced BrdU-incorporation. These results suggest that CTRP3/cartducin is not only involved in the proliferation and differentiation of chondrocytes, but also contributes to the regulation of mandibular condylar cartilage.     

Morphological changes in articular cartilage due to static compression: polarized light microscopy study

We studied the deformation of the extracellular matrices in articular cartilage using a new compression-preservation method in histology. A Hoffman clamp was used to compress the tissue, which remained throughout the paraffin procedure and was removed from the embedded tissue block just before microtoming. Then 14 cartilage-bone blocks from 2 canine humeri were compressed for various strain levels from 5% to 65%. The histological sections were studied using a polarized light microscope, which generated a pair of two-dimensional maps of the fibril orientation (angle) and fibril organization (retardance) for each section. Results were 3-fold. One there was little change in the angle and retardance profiles of the tissue for strain levels 0-15% and a significant change in these profiles for strain levels 15% and above. Two for higher compression, more fibrils became aligned parallel to the articular surface; and three at approximately 30% strain, a second "transitional zone" was formed in the deep part of the tissue. We concluded that this novel compression procedure can be used effectively to study the altered architecture of the collagen matrix in compressed cartilage.     

Photoperiodic modulation of reproductive physiology and behaviour in the cichlid fish Cichlasoma dimerus

Timing of breeding to an optimal season is a requirement for a successful reproductive outcome in seasonally breeding species. Photoperiodic signals are capable of modifying the reproductive behaviour and reproductive systems in several vertebrate species. The cichlid fish Cichlasoma dimerus shows highly organized breeding activities and different social status. The aim of this study is to test whether C. dimerus reproductive behaviour (male aggressive behaviour and female choice) and reproductive physiology (GnRH3 morphometric parameters, pituitary hormones content and organ-somatic indexes) are modulated by photoperiod. Before spawning, dominant pairs were isolated and kept in opposite tanks of 20 l for one week, so they could see each other but not physically interact. Afterwards, a group was exposed for four weeks to a short photoperiod (8 h light:16 h dark) (short photoperiod exposed animals: SP) while another group was exposed to a long photoperiod (14 h light:10 h dark) (long photoperiod exposed animals: LP). Temperature was maintained constant. Behavioural experiments showed that male aggression related to territory selection and its defence is reduced in SP males. Further, SP females were never chosen. At the brain level we demonstrated that GnRH3 neuronal optical density of staining was reduced. Finally, at the pituitary level we showed that SP males showed low levels of β-LH, PRL and GH in the pituitary, and that SP females showed no significant differences in the pituitary content of any hormone. Taken all together these results suggest that in C. dimerus the photoperiod is a relevant environmental cue related to reproductive behaviour and physiology.     

The development of bone mass and bone strength in the mandible of the female rat

The present study provides baseline data for a number of mandibular growth dimensions, specially on bone mass and bone strength, that were collected between the 21st and the 180th days of postnatal life, which are intended as a reference for researchers designing experimental studies, specially on mandibular catch-up growth, and as an aid for clinicians who must evaluate results from published animal studies for validity and potential extrapolation to the human clinical situation. Fifty weanling female Wistar (Hsd:Wi) rats were fed ad libitum a diet previously shown to allow normal, undeformed mandibular growth. Five of them were randomly selected at different times between 21 and 180 d of life. Mandibular growth was estimated directly on the right hemimandible by taking measurements between anatomical points; mandibular bone mass (calcium mass) was estimated from the mg of calcium, determined by atomic absorption spectrophotometry, present in the ashes of the left hemimandible; and mechanical properties of the right hemimandible were determined using three-point bending mechanical test. Dimensions, bone calcium mass and bone strength of the female rat mandible increased linearly from day 21 to approximately day 90. Bone growth, as expected, was more than twice when assessed from bone weight than when derived from mandibular area, length or height when the parameters were expressed as the relative increase from the mean infant condition. The growth rate of the posterior part of the mandible (behind the third molar) was almost five times greater than that of the anterior part. The rates of growth of the studied parameters showed a marked decline after day 90. ANOVA indicated that no statistical differences were found between day 90 and day 120 values. It could be concluded that the female rat mandible attains its adult size, peak bone calcium mass and bone structural mechanical properties at some point between 90 and 120 d of postnatal life. Because of the extremely high positive correlation between mandibular bone calcium mass and both mandibular area and mandibular weight, it was possible to calculate the mandibular peak bone mass from the relations 7.69 mgCa/cm2 and 0.19 mgCa/mg bone.