Micrometer-scale structure in shark vertebral centra.

The vertebral centra of sharks consist of cartilage, and many species' centra contain a bioapatite related to that in bone. Centra microarchitectures at the 0.5-50 µm scale do not appear to have been described previously. This study examines centrum microarchitecture in lamniform and carcharhiniform sharks with synchrotron microComputed Tomography (microCT), scanning electron microscopy and spectroscopy and light microscopy. The analysis centers on the blue shark (carcharhiniform) and shortfin mako (lamniform), species studied with all three modalities. Synchrotron microCT results from seven other species complete the report. The main centrum structures, the corpus calcareum and intermedialia, consist of fine, closely-spaced, mineralized trabeculae whose mean thicknesses and spacings range from 4.5 to 11.2 µm and 4.5 to 15.6 µm, respectively. A significant (p = 0.00001) positive linear relationship between and exists for multiple positions within one mako centrum. Carcharhiniform species' and exhibit an inverse linear relationship (p = 0.005) while in lamniforms these variables tend toward a positive relationship which does not reach statistical significance (p = 0.099). In all species, the trabeculae form an uninterrupted, interconnected network, and the unmineralized volumes are similarly interconnected. Small differences in mineralization level are observed in trabeculae. Centrum growth band pairs are found to consist of locally higher /lower mineral volume fraction. Within the intermedialia, radial canals and radial microrods were characterized, and compacted trabeculae are prominent in the mako intermedialia. The centra's mineralized central zones were non-trabecular and are also described. STATEMENT OF SIGNIFICANCE: This study's novel result is the demonstration that the mineralized cartilage of sharks' vertebral bodies (centra) consists of a fine 3D array of interconnected plates (trabeculae) and an interpenetrating network of unmineralized tissue. This microstructure is radically different from that in tesserae or in teeth, the other main mineralized shark tissues. Using volumetric synchrotron microComputed Tomography, numerical values of mean trabecular thickness and spacing and their relationship were measured for nine species. Scanning electron microscopy added a higher resolution view of the microstructures, and histology provided complementary information on cartilage and cells. The present results suggest centra microstructure helps accommodate the very large in vivo strains and may prevent damage accumulation during millions of cycles of swimming-induced loading.

Enamel hypoplasia in molars of sheep and goats, and its relationship to the pattern of tooth crown growth.

Enamel is the most highly mineralized and durable tissue of the mammalian body. As enamel does not undergo remodeling or repair, disturbances of enamel formation leave a permanent record in the tissue that can be used for life history reconstruction. This study reports light and scanning electron microscope findings on hypoplastic enamel defects, and on the chronology of crown growth in the molars of sheep and goats. A marked reduction of enamel extension rates in cervical compared with more cuspal crown portions of sheep and goat molars was recorded, with formation of the cervical 25% of the crown taking about the same time as that of the upper 75% of the crown. This explains the more frequent occurrence of enamel hypoplasia in cervical compared with upper and middle crown portions. Regarding the identification of hypoplastic enamel defects by external inspection, our results suggest a dependence on the type of defect and the associated presence of smaller or larger amounts of coronal cementum. Defects considered to reflect a slight to moderate impairment of secretory ameloblast function can normally be correctly diagnosed as they are not occluded by thick layers of cementum. In contrast, defects denoting a severe impairment of enamel matrix secretion can typically not be correctly identified because they are occluded by large amounts of cementum, so that neither depth nor extension of the defects can be assessed on external inspection. In these cases, microscopic analysis of tooth sections is required for a correct diagnosis of the hypoplastic enamel defects.

Antlers - Evolution, development, structure, composition, and biomechanics of an outstanding type of bone.

Antlers are bony appendages of deer that undergo periodic regeneration from the top of permanent outgrowths (the pedicles) of the frontal bones. Of the "less familiar" bone types whose study was advocated by John Currey to gain a better understanding of structure-function relationships of mineralized tissues and organs, antlers were of special interest to him. The present review summarizes our current knowledge about the evolution, development, structure, mineralization, and biomechanics of antlers and how their formation is affected by environmental factors like nutrition. Furthermore, the potential role of antlers as a model in bone biology and several fields of biomedicine as well as their use as a monitoring tool in environmental studies are discussed.

Insights from the inside: histological analysis of abnormal enamel microstructure associated with hypoplastic enamel defects in human teeth.

We studied the abnormalities in enamel microstructure associated with enamel hypoplasia in human teeth from the early medieval (5th-7th century AD) cemetery of Barbing, Germany, using light and scanning electron microscopy. The main aim of the study was to test the hypothesis that by analyzing the microstructure of fully formed enamel it is possible to reconstruct the reaction pattern of secretory ameloblasts to stress events leading to enamel hypoplasia. From the histological findings, a sequence of increasing impairment of secretory ameloblast function involving three thresholds was deduced. Surpassing of each of these thresholds is assumed to result in characteristic changes in enamel microstructure attributable to specific functional/morphological alterations of secretory ameloblasts. Based on our results we propose a model identifying the principal factors influencing the reaction of secretory ameloblasts to stress. The present study demonstrates that by including microscopic analysis in the study of enamel hypoplasia, it is possible to obtain a more complete picture of the formation of these developmental defects than is possible by inspection of crown surface features alone, and to draw more substantiated conclusions about the possible nature of developmental defects of enamel.

Deposition of cellular cementum onto hypoplastic enamel of fluorotic teeth in wild boars (Sus scrofa L.).

The nature of deposits present in hypoplastic defects of fluorotic enamel of wild boar teeth was studied by light microscopy and scanning electron microscopy. The fluorotic enamel showed different developmental abnormalities, denoting a severe disturbance of ameloblast function during the secretory stage of amelogenesis. These abnormalities included the occurrence of grossly accentuated incremental lines with associated zones of aprismatic enamel and the presence of different forms of hypoplastic defects. Two types of deposits were present on the hypoplastic enamel: cellular cementum and posteruptively acquired, presumably partially mineralized dental plaque. Coronal cementum is not normally formed in pig teeth. Presence of this tissue in fluorotic teeth of wild boars is seen as indicative of a premature disintegration of the enamel epithelium prior to the completion of amelogenesis. This was supposed to have resulted in a contact of mesenchymal cells of the dental follicle with the surface of the immature enamel and, in consequence, in a differentiation of these cells into cementoblasts. To our knowledge, this is the first study reporting the formation of coronal cementum as part of the spectrum of pathological changes in fluorotic teeth in a species whose tooth crowns are normally free of cementum.

Cyproterone acetate reduced antler growth in surgically castrated fallow deer.

We studied the role of androgens in antler growth. In particular, we investigated whether the onset of antler regrowth is triggered by a short-term pulse of testosterone and if low levels of androgens are required for antler growth. The study was conducted on 12 surgically castrated fallow deer bucks (Dama dama) aged approximately 27 months. Six animals (CA group) were given the antiandrogen, cyproterone acetate (CA, 1000 mg/treatment); the others were given vehicle solution only (control). Before each CA treatment, blood was sampled and analysed for testosterone, androstenedione, IGF-1, cortisol, FSH, and LH. CA treatment and blood sampling were performed 2 days before castration, on the day of castration and afterwards at 2-day intervals until day 22. Subsequently, CA treatment and blood sampling continued at weekly intervals until day 270. All animals cast their antlers, followed by antler regrowth in all control bucks, but in only four of the six CA-treated castrates. Plasma testosterone concentrations were low in all animals (between 0.01 and 0.20 ng/ml), but were significantly (P<0001) greater in the controls. In both groups, a temporary increase in testosterone values was recorded around the time of antler regrowth, the peak being significantly (P<0.01) higher in the controls. Androstenedione showed a similar pattern as testosterone. Plasma IGF-1 concentrations increased sharply during the antler growth spurt and did not differ significantly between the two groups throughout the study period. Cortisol concentrations were greater in controls than in the CA group. However, no link with the antler cycle was apparent. FSH and LH concentrations were higher in the controls for most of the study. Antlers produced by the control bucks were significantly larger than those in the CA group (P<0.03). For antler length, testosterone, androstenedione and IGF-1, areas under the curve (AUC) were calculated over the period of antler growth. For the pooled deer (n=12) significant correlations existed between AUCs of antler length and testosterone, but not for antler length and IGF-1. Also, a trend for a positive correlation between AUCs of antler length and androstenedione was noted. It is concluded that a plasma androgen concentration at least above a minimal threshold level is a necessary prerequisite for normal antler regrowth in fallow deer, and that this androgen effect is not mediated via circulating IGF-1. The biological role of low levels of androgens may be to sensitize antler cells to the stimulating effect of IGF.

A scanning electron microscopic study on the distribution of peritubular dentine in cheek teeth of Cervidae and Suidae (Mammalia, Artiodactyla).

Distribution of peritubular dentine was studied in cheek teeth of fallow deer (Dama dama), roe deer (Capreolus capreolus) and wild boar (Sus scrofa). In the two cervid species, especially intense peritubular dentine formation was found in the outer regions of mid and cuspal coronal dentine. In these areas a marked asymmetry occurred, peritubular dentine being predominantly secreted onto the side of the dentinal tubule walls nearest to the dentinoenamel junction. Intensity and asymmetry of peritubular dentine formation decreased cervically. In root dentine, the walls of the dentinal tubules were covered with only a thin peritubular dentine layer of even thickness. Here, in contrast to peripheral coronal dentine, the volume of intertubular dentine far exceeded that of peritubular dentine. In porcine coronal dentine, PTD asymmetry, being of lesser extent than in cervids, was observed only in peripheral areas of cuspal and flank regions of the cheek teeth. Because peritubular is more highly mineralized than intertubular dentine, the relative volume of dentine made up from the two components has an important influence on dentinal wear resistance. The significance of variations in volume and distribution of peritubular dentine between different dentinal regions for achieving and maintaining a functional occlusal surface is shown for cervid cheek teeth. Our results suggest that dentinal structure (in addition to enamel structure) should be taken more into consideration when discussing occlusal surface morphology in herbi- and omnivores from a functional point of view.

Fluoride-induced alterations of enamel structure: an experimental study in the miniature pig.

We studied the structural changes in the enamel of mandibular third molars of miniature pigs administered a daily oral dose of 2 mg NaF (approximately 0.9 mg of fluoride) per kg body weight (added to the feed) for 1 year. The treatment period covered most of the secretory stage and the entire post-secretory stage of amelogenesis of the M(3). The enamel of the molars from the fluoride-fed pigs appeared opaque and chalky, and the erupted portions were stained brown. The underlying histopathological change was a pronounced subsurface hypomineralization of the enamel beneath a thin surface rim of higher mineral content. This enamel hypomineralization was attributed to a fluoride-induced impairment of the process of enamel maturation. The most conspicuous finding in the fluorotic enamel was the presence of numerous pit-type hypoplastic defects, denoting a marked fluoride-induced disturbance also of the secretory stage of amelogenesis. Microradiography and scanning electron microscopy revealed an enhanced incremental pattern in the outer enamel of the fluorotic molars. Typically, the bottom of larger hypoplastic defects was underlain by a broad, grossly accentuated incremental line. Occurrence of larger hypoplasias was further associated with the presence of aprismatic enamel, the formation of which was attributed to a loss of the prism-forming (distal) portion of the Tomes' processes of secretory ameloblasts. The findings in the miniature pigs closely parallel earlier observations on fluorotic enamel of free-ranging deer and wild boar from fluoride-polluted areas.

Occurrence of a postpermanent mandibular fourth premolar in a moose bull (Alces alces L.).

By a number of diagnostic criteria a supplemental premolar in the right hemimandible of a 6-yr-old moose bull from Bielorussia was identified as a postpermanent fourth premolar. Its development, which must have started at least 3 yr after the permanent fourth premolar had reached its functional position, resulted in rotation of the P4 by 80 degrees, as well as extensive resorption on both root and crown.

Fluoride-induced developmental changes in enamel and dentine of European roe deer (Capreolus capreolus L.) as a result of environmental pollution.

Using macroscopic, microradiographic and scanning electron-microscopic methods, the effects of increased fluoride exposure on enamel and dentine formation were studied in fluorosed mandibular premolars and molars of roe deer from the heavily industrialized Ruhr area, Germany. Macroscopically, fluorosed teeth were characterized by opaque and stained enamel and in more severe cases also by enamel surface lesions, reduction or loss of enamel ridges on their occlusal surfaces and increased wear. Microradiographically, fluorosed enamel exhibited different degrees of subsurface hypomineralization, in part apparently indicating a fluoride effect during enamel maturation. In some specimens, a pronounced but varying enhancement of the pattern of Retzius lines was observed throughout the enamel, denoting strongly intermittent fluoride exposure during enamel matrix secretion. This variation in exposure was also reflected histologically in dentine, by bands of interglobular dentine and marked accentuation of incremental lines. Microradiography of sections through enamel surface hypoplastic lesions showed the enamel forming the bottom and partly also the walls of the lesions to be highly mineralized. Scanning electron microscopy showed that the outer enamel along the more pronounced hypoplastic lesions consisted of stacked, thin layers of 'aprismatic' enamel, indicating that the ameloblasts in these areas had lost the distal (rod-forming) regions of their Tomes' processes. These observations demonstrate that the origin of enamel hypoplasias in deer clearly differs from that in rodents, where fluoride induces the formation of subameloblastic cysts. The differences in the degree of fluorotic alteration between the teeth of a single tooth row could be related to the developmental sequence of the dentition in roe deer. The roe deer is thus considered to be a very sensitive and useful bioindicator of environmental pollution by fluorides.

Reconstruction of a decline of ambient lead levels in the Ruhr area (Germany) by studying lead concentrations in antlers of roe deer (Capreolus capreolus).

We determined lead concentrations in antlers (n = 132) of roe bucks > or = 2 years of age, killed between 1951 and 1999 in the northern Ruhr area, Germany. The lead content of the antlers ranged between 0.6 and 19.0 mg/kg dry weight of bone, and overall markedly declined over the study period. As a consequence, mean lead levels in younger samples were always significantly (P<0.001) lower than those in older ones (for the periods: 1951-1969, 1970-1979, 1980-1989 and 1990-1999). Annual mean antler lead concentrations were positively correlated (r(s) = 0.74, P<0.001) with annual mean lead concentrations in airborne particulate matter of the Rhine-Ruhr area during the period 1974-1999. The findings of the present study underscore that roe deer antlers are well suited for a monitoring of temporal trends in environmental lead levels in a cost-effective way.

Monitoring regional fluoride pollution in the Saxonian Ore mountains (Germany) using the biomarker dental fluorosis in roe deer (Capreolus capreolus L.).

The spatial variation of fluoride contamination in the Saxonion part of the Ore mountains (Federal state of Saxony, Germany) was assessed by analysing the prevalence and severity of dental fluorosis in wild male roe deer aged 2 years or more (n = 794) from this region. The study area is exposed to increased atmospheric deposition of fluorides, originating from emission sources in the North-Bohemian brown coal belt. The severity of dental fluorosis in the permanent premolars and molars of one hemimandible per animal was assessed based on a scoring system and a dental lesion index of fluorosis (DLI) assigned to each specimen. On the basis of the mean DLI calculated for the roe deer originating from a certain municipal district, the respective deer sample was assigned to one of seven fluorosis categories, and a map showing the distribution of these categories was produced. Highest fluorosis categories were recorded for roe deer samples from the central and eastern parts of the study area that directly bordered on, or were located near to, the Czech-German border. A close spatial relationship between the main fluoride emission sources in North-Bohemia and the regions with the highest prevalence and severity of dental fluorosis in roe deer was discernible. The observed regional variation of fluoride pollution is in accordance with the results of other studies directly measuring fluoride deposition into the study area or fluoride levels in vegetation. A main advantage of the method used in this study over other ways of recording fluoride contamination of wildlife habitats is that it uses an established and continuously operated system of specimen collection, thereby enabling efficient monitoring with high spatial and temporal resolution at very low cost. Moreover, a clearly recognisable and relevant biological effect is measured which occurs in a dose-dependent manner over a range of environmentally relevant concentrations.

Mandibular bone fluoride levels and occurrence of fluoride induced dental lesions in populations of wild red deer (Cervus elaphus) from Central Europe.

Mandibular bone fluoride concentrations as well as frequency and intensity of fluoride-induced dental lesions were studied in four red deer populations from Czechia and Germany exposed to different levels of environmental fluoride. The degree of fluorosis in the permanent cheek teeth was recorded by a scoring system and the individual mandibles assigned to one of three categories (unfluorosed, slightly to moderately fluorosed, and markedly to severely fluorosed) on the basis of the tooth scores. An increase in the frequency and severity of dental fluorosis was positively associated with an increase in bone fluoride level, which itself was significantly different between the four samples. In all samples, the rate of bone fluoride accumulation was higher in younger than in older deer. The results of this study show that frequency and intensity of dental fluorosis can be used as valid indicators of increased fluoride exposure of deer populations, and thus as a quick, easy, economic and reliable means of monitoring fluoride pollution in areas inhabited by wild deer.

Treatment of the growing pedicle with retinoic acid increased the size of the first antlers in fallow deer (Dama dama L.).

Unilateral injection of 10 mg of all-trans retinoic acid (RA) into the lateral portion of the growing pedicle of fallow bucks (n = 20) led to a significant (P = 0.033, Wilcoxon matched-pairs test) increase in first antler volume (median, 25.5 ml) as compared to the contralateral (control) side, injected with vehicle only (median, 21.5 ml). It is hypothesized that the RA treatment of the developing pedicle exercised a direct or indirect effect on the periosteal/perichondrial cells covering the growing cranial appendage, resulting in an increased proliferation rate of the cells of the antler perichondrium.

Fluoride concentrations in antler bone of roe deer (Capreolus capreolus) indicate decreasing fluoride pollution in an industrialized area of western Germany.

In order to reconstruct temporal changes in ambient fluoride levels in the industrialized Ruhr area (western Germany), we analyzed the bone fluoride content of 167 antlers of roe deer (Capreolus capreolus) killed between 1951 and 1999 in the northern part of this region. Individual values ranged between 110 and 8,178 mg F-/kg ash, and there was an overall marked decrease over the sampling period. Average bone fluoride concentrations in antlers from the periods 1980 through 1989 (geometric mean [95% confidence interval]: 1,490 [1,193-1,861] mg/kg ash) and 1990 through 1999 (753 [644-882] mg/kg ash) differed significantly (p < 0.001) and were both significantly (p < 0.001) lower than those from the periods 1951 through 1969 (3,720 [3,227-4,288] mg/kg ash) and 1970 through 1979 (2,573 [2,203-3,006] mg/kg ash). The findings are seen as indicative of a progressively reduced atmospheric fluoride deposition into the study area, caused by effective emission-control measures in Germany and neighboring countries. Because antlers are replaced annually, grow during a fixed period of some months, and are regularly collected and kept as trophies, they are well suited as monitoring units for analyzing temporal trends in environmental pollution by fluoride and other bone-seeking pollutants.

Effects of retinoic acid on pedicle and first antler growth in a fallow buck (Dama dama L.).

Unilateral injection of 10 mg all-trans retinoic acid (in 1 ml of castor oil) into the early pedicle anlage of a male fallow deer resulted in accelerated growth of the cranial appendage, and altered pedicle and first antler shape on the treatment side, whereas pedicle and antler growth on the control side, injected with 1 ml of vehicle solution only, was normal. It is concluded that retinoic acid is able to alter pedicle and first antler morphogenesis, presumably by affecting the positional information of the periosteal cells covering the incipient pedicle.

Delayed ectopic antler growth and formation of a double-head antler in the metacarpal region of a fallow buck (Dama dama L.) following transplantation of antlerogenic periosteum.

Autologous transplantation of antlerogenic periosteum to the metacarpal region of a fallow buck resulted in the formation of an ectopic pedicle within a few months after transplantation. In the following years the pedicle increased in size, but no ectopic antler development occurred. However, in the ninth year after transplantation a first antler of about 3 cm length grew from the ectopic pedicle. The hard ectopic first antler was not cast, and renewed antler growth, starting in late spring of the following year, led to the formation of a larger subsequent antler of 7.3 cm length. The latter exhibited a distal flattening (palmation) and a rudimentary basal coronet, thereby displaying morphological features typical of the subsequent antlers of fallow bucks. The results of the experiment indicate (1) that transformation from pedicle to antler growth can occur several years after pedicle initiation, when the pedicle has reached a certain threshold size, (2) that species and age specific features of antler morphology tend to be realized even in ectopic locations, and (3) that primary and subsequent antler growth was apparently triggered by cells of the pedicle periosteum, being derived from the transplanted antlerogenic periosteum. The findings thereby emphasize the importance of local factors in the control of pedicle/first antler transformation and antler morphogenesis.

The macroscopic and microscopic structure of double-head antlers and pedicle bone of cervidae (Mammalia, Artiodactyla).

Ortho- and heterotopically formed double-head antlers were studied in red, fallow and roe deer. The malformation was the result of new antler growth without previous casting of the old antlers. Thus, two antler structures belonging to successive antler generations originated from one pedicle. These two structures were always separated by a horizontal groove. Histologically, signs of osteoclastic resorption were observed in the interior and at the outer circumference of the distal parts of the pedicles of the double heads. The resorptive process had, however, not been of an intensity necessary for subsequent antler casting. We also observed that the double-head's second antler generation had developed as a periosteal exostosis of the distal pedicle bone. Thus, we assume that in normogenesis formation of the bony component of subsequent antlers is also probably dependent on cells derived from pedicle periosteum. Finally, the process of antler regrowth in deer is compared with epimorphic regeneration occurring in other vertebrates.

A quantitative backscattered electron imaging study of hypomineralization and hypoplasia in fluorosed dental enamel of deer.

Mineral content and distribution of fluorosed and unfluorosed (control) dental enamel of roe deer and red deer cheek teeth were analyzed using digital backscattered electron (BSE) imaging of PMMA-embedded specimens. Compared to the controls, the fluorosed enamel exhibited various aberrations resulting from a fluoride-induced disturbance of the processes involved in enamel formation. Thus, the presence of surface hypoplasias and an enhancement of the incremental pattern in the fluorosed enamel are evidence of a fluoride impact on the secretory ameloblasts, whereas a (subsurface) hypomineralization of different depth and extent is indicative of a fluoride effect on the maturation stage of amelogenesis. The marked variation in the severity of enamel hypomineralization seen along the coronocervical axis of a specimen pointed to a fluoride impact of varying intensity during this period of tooth development. Our observations further indicated that, in some locations, ameloblasts severely affected by fluoride during enamel matrix formation were able to recover from this insult and to function quite normally during the maturation stage of amelogenesis. A major advantage of the BSE imaging technique used in the present study over other methods is that it allows for a combination of micromorphological information with quantitative data on the mineralization of the analyzed tissue, which proved to be very useful for the characterization of fluoride-induced changes in dental enamel.

Ultrastructural aspects of cartilage formation, mineralization, and degeneration during primary antler growth in fallow deer (Dama dama).

Due to their rapid growth, regular replacement and easy accessibility, deer antlers are considered a useful model for the study of cartilage and bone differentiation and mineralization in mammals. The present study describes, for the first time, the cellular and extracellular matrix changes associated with cartilage formation, mineralization and degeneration in primary antlers on the ultrastructural level. Growing primary antlers of 3 to 4 cm length were obtained from six fallow bucks, aged about 10 months. It was shown that the chondroblasts were derived from progenitor cells of the antler perichondrium and differentiated into mature chondrocytes that subsequently underwent hypertrophic changes. Concomitant with cell hypertrophy, formation of a lacunar and a perilacunar extracellular matrix was observed, the latter containing numerous collagenous fibers. Mineralization of the extracellular matrix occurred via matrix vesicles and the formation of apatite crystals at distinct sites of the collagenous fibers. The hypertrophic chondrocytes of the mineralized cartilage then degenerated, a process that was also occasionally observed in more distally located cells surrounded by still unmineralized matrix. No morphological indications of a transdifferentiation of hypertrophic chondrocytes into bone forming cells, i.e., co-occurrence of a degenerating chondrocyte and a viable osteogenic cell in intact lacunae, were found. The cellular and extracellular matrix changes seen in primary antlers resemble those described for secondary antlers. Our results further indicate that the hypertrophic chondrocytes of primary antlers eventually undergo apoptosis, thereby providing further evidence that metaplastic conversion of cartilage into bone does not play a role in antler growth.