Osteocyte lacuna population densities in sheep, elk and horse calcanei

Osteocytes, the most prevalent cell type in bone, appear to communicate via gap junctions. In limb-bone diaphyses, it has been hypothesized that these cellular networks have the capacity to monitor habitual strains, which can differ significantly between cortical locations of the same bone. Regional differences in microdamage associated with prevalent/predominant strain mode (tension, compression, or shear) and/or magnitude may represent an important "variable" detected by this network. This hypothesis was indirectly addressed by examining bones subjected to habitual bending for correlations of osteocyte lacuna population densities (n/mm(2) bone area, Ot.Lc.N/B.Ar) with locations experiencing high and low strain, and/or prevalent/predominant tension, compression, and shear. We examined dorsal ("compression"), plantar ("tension"), and medial/lateral ("shear" or neutral axis) cortices of mid-diaphyseal sections of calcanei of adult sheep, elk, and horses. Ot.Lc.N/B.Ar data, quantified in backscattered electron images, were also evaluated in a context of various additional structural and material variables (e.g. % ash, cortical thickness, porosity, and secondary osteon population). Results showed significant differences in dorsal versus plantar comparisons with the highest Ot.Lc.N/B.Ar in dorsal cortices of sheep and elk (p < 0.0001); but this was a statistical trend in the equine calcanei (p = 0.14). There were no consistent transcortical (pericortical to endocortical) differences, and Ot.Lc.N/B.Ar in neutral axes was not consistently different from dorsal/plantar cortices. Correlations of Ot.Lc.N/B.Ar with structural and material parameters were also poor and/or inconsistent within or between species. These results provide little or no evidence that the number of osteocyte lacunae has a functional role in mechanotransduction pathways that are typically considered in bone adaptation. Although dorsal/plantar differences may be adaptations for prevalent/predominant strain modes and/or associated microdamage, it is also plausible that they are strongly influenced by differences in the bone formation rates that produced the tissue in these locations.     

Strain-mode-specific mechanical testing and the interpretation of bone adaptation in the deer calcaneus

The artiodactyl (deer and sheep) calcaneus is a model that helps in understanding how many bones achieve anatomical optimization and functional adaptation. We consider how the dorsal and plantar cortices of these bones are optimized in quasi-isolation (the conventional view) versus in the context of load sharing along the calcaneal shaft by “tension members” (the plantar ligament and superficial digital flexor tendon). This load-sharing concept replaces the conventional view, as we have argued in a recent publication that employs an advanced analytical model of habitual loading and fracture risk factors of the deer calcaneus. Like deer and sheep calcanei, many mammalian limb bones also experience prevalent bending, which seems problematic because the bone is weaker and less fatigue-resistant in tension than compression. To understand how bones adapt to bending loads and counteract deleterious consequences of tension, it is important to examine both strain-mode-specific (S-M-S) testing (compression testing of bone habitually loaded in compression; tension testing of bone habitually loaded in tension) and non-S-M-S testing. Mechanical testing was performed on individually machined specimens from the dorsal “compression cortex” and plantar “tension cortex” of adult deer calcanei and were independently tested to failure in one of these two strain modes. We hypothesized that the mechanical properties of each cortex region would be optimized for its habitual strain mode when these regions are considered independently. Consistent with this hypothesis, energy absorption parameters were approximately three times greater in S-M-S compression testing in the dorsal/compression cortex when compared to non-S-M-S tension testing of the dorsal cortex. However, inconsistent with this hypothesis, S-M-S tension testing of the plantar/tension cortex did not show greater energy absorption compared to non-S-M-S compression testing of the plantar cortex. When compared to the dorsal cortex, the plantar cortex only had a higher elastic modulus (in S-M-S testing of both regions). Therefore, the greater strength and capacity for energy absorption of the dorsal cortex might “protect” the weaker plantar cortex during functional loading. However, this conventional interpretation (i.e., considering adaptation of each cortex in isolation) is rejected when critically considering the load-sharing influences of the ligament and tendon that course along the plantar cortex. This important finding/interpretation has general implications for a better understanding of how other similarly loaded bones achieve anatomical optimization and functional adaptation.     

Advancing the deer calcaneus model for bone adaptation studies: ex vivo strains obtained after transecting the tension members suggest an unrecognized important role for shear strains

Sheep and deer calcanei are finding increased use as models for studies of bone adaptation, including advancing understanding of how the strain (deformation) environment influences the ontogenetic emergence of biomechanically relevant structural and material variations in cortical and trabecular bone. These artiodactyl calcanei seem ideal for these analyses because they function like simply loaded short‐cantilevered beams with net compression and tension strains on the dorsal and plantar cortices, respectively. However, this habitual strain distribution requires more rigorous validation because it has been shown by limited in vivo and ex vivo strain measurements obtained during controlled ambulation (typically walking and trotting). The conception that these calcanei are relatively simply and habitually loaded ‘tension/compression bones’ could be invalid if infrequent, though biologically relevant, loads substantially change the location of the neutral axis (NA) that separates ‘compression’ and ‘tension’ regions. The effect on calcaneus strains of the tension members (plantar ligament and flexor tendon) is also not well understood and measuring strains after transecting them could reveal that they significantly modulate the strain distribution. We tested the hypothesis that the NA location previously described during simulated on‐axis loads of deer calcanei would exhibit limited variations even when load perturbations are unusual (e.g. off‐axis loads) or extreme (e.g. after transection of the tension members). We also examined regional differences in the predominance of the three strain modes (tension, compression, and shear) in these various load conditions in dorsal, plantar, medial, and lateral cortices. In addition to considering principal strains (tension and compression) and maximum shear strains, we also considered material‐axis (M‐A) shear strains. M‐A shear strains are those that are aligned along the long axis of the bone and are considered to have greater biomechanical relevance than maximum shear strains because failure theories of composite materials and bone are often based on stresses or strains in the principal material directions. We used the same load apparatus from our prior study of mule deer calcanei. Results showed that although the NA rotated up to 8° medially and 15° laterally during these off‐axis loads, it did not shift dramatically until after transection of all tension members. When comparing results based on maximum shear strain data vs. M‐A shear strain data, the dominant strain mode changed only in the plantar cortex – as expected (in accordance with our a priori view) it was tension when M‐A shear strains were considered (shear : tension = 0.2) but changed to dominant shear when maximum shear strain data were considered (shear : tension = 1.3). This difference leads to different conclusions and speculations regarding which specific strain modes and magnitudes most strongly influence the emergence of the marked mineralization and histomorphological differences in the dorsal vs. plantar cortices. Consequently, our prior simplification of the deer calcaneus model as a simply loaded ‘tension/compression bone’ (i.e. plantar/dorsal) might be incorrect. In vivo and in finite element analyses are needed to determine whether describing it as a ‘shear‐tension/compression’ bone is more accurate. Addressing this question will help to advance the artiodactyl calcaneus as an experimental model for bone adaptation studies.     

Analysis of the Effect of Osteon Diameter on the Potential Relationship of Osteocyte Lacuna Density and Osteon Wall Thickness

An important hypothesis is that the degree of infilling of secondary osteons (Haversian systems) is controlled by the inhibitory effect of osteocytes on osteoblasts, which might be mediated by sclerostin (a glycoprotein produced by osteocytes). Consequently, this inhibition could be proportional to cell number: relatively greater repression is exerted by progressively greater osteocyte density (increased osteocytes correlate with thinner osteon walls). This hypothesis has been examined, but only weakly supported, in sheep ulnae. We looked for this inverse relationship between osteon wall thickness (On.W.Th) and osteocyte lacuna density (Ot.Lc.N/B.Ar) in small and large osteons in human ribs, calcanei of sheep, deer, elk, and horses, and radii and third metacarpals of horses. Analyses involved: (1) all osteons, (2) smaller osteons, either ≤150 μm diameter or less than or equal to the mean diameter, and (3) larger osteons (>mean diameter). Significant, but weak, correlations between Ot.Lc.N/B.Ar and On.W.Th/On.Dm (On.Dm = osteon diameter) were found when considering all osteons in limb bones (r values ?0.16 to ?0.40, P < 0.01; resembling previous results in sheep ulnae: r = ?0.39, P < 0.0001). In larger osteons, these relationships were either not significant (five/seven bone types) or very weak (two/seven bone types). In ribs, a negative relationship was only found in smaller osteons (r = ?0.228, P < 0.01); this inverse relationship in smaller osteons did not occur in elk calcanei. These results do not provide clear or consistent support for the hypothesized inverse relationship. However, correlation analyses may fail to detect osteocyte‐based repression of infilling if the signal is spatially nonuniform (e.g., increased near the central canal). Anat Rec,, 2011. © 2011 Wiley‐Liss, Inc.     

Ontogenetic structural and material variations in ovine calcanei: A model for interpreting bone adaptation

Experimental models are needed for resolving relative influences of genetic, epigenetic, and nonheritable functionally induced (extragenetic) factors in the emergence of developmental adaptations in limb bones of larger mammals. We examined regional/ontogenetic morphologic variations in sheep calcanei, which exhibit marked heterogeneity in structural and material organization by skeletal maturity. Cross‐sections and lateral radiographs of an ontogenetic series of domesticated sheep calcanei (fetal to adult) were examined for variations in biomechanically important structural (cortical thickness and trabecular architecture) and material (percent ash and predominant collagen fiber orientation) characteristics. Results showed delayed development of variations in cortical thickness and collagen fiber orientation, which correlate with extragenetic factors, including compression/tension strains of habitual bending in respective dorsal/plantar cortices and load‐related thresholds for modeling/remodeling activities. In contrast, the appearance of trabecular arches in utero suggests strong genetic/epigenetic influences. These stark spatial/temporal variations in sheep calcanei provide a compelling model for investigating causal mechanisms that mediate this construction. In view of these findings, it is also suggested that the conventional distinction between genetic and epigenetic factors in limb bone development be expanded into three categories: genetic, epigenetic, and extragenetic factors. Anat Rec, 2007. © 2007 Wiley‐Liss, Inc.     

Analysis of a tension/compression skeletal system: Possible strain-specific differences in the hierarchical organization of bone

Background: Examination of a simple skeletal cantilevered beam-like bone (artiodactyl calcaneus) suggests that regional differences in strain magnitude and mode (tension vs. compression) reflect regional adaptation in the structural/material organization of bone. The artiodactyl (e.g., sheep and deer) calcaneus has a predominant loading condition typified by the unambiguous presence of prevailing compressive and tensile strains on opposite cortices. Bone habitually loaded in bending may accommodate regional disparities in loading conditions through modifications of various aspects of its organization. These include overall bone build (gross size and shape), cross-sectional shape, cortical thickness, and mineral content. Methods & Results: Cross-sections taken along the calcaneal body exhibited cranial-caudal elongation with the compression (cranial) cortex thicker than the tension cortex (P < 0.01). Mineral content (ash fraction) was significantly greater in the compression cortex (P < 0.01), averaging 6.6% greater than in the tension cortex. Strong positive correlations were found between mineral content and section location in both the tension (r² = 0.955) and compression (r² = 0.812) cortices. These correlations may reflect functional adaptations to the linear increases in stress that are known to occur in the distal-to-proximal direction in simple, unidirectionally loaded cantilevered beams. According to engineering principles, the roughly triangular transverse cross-sectional geometries and thicker compression cortex are features consistent with a short cantilevered structure designed to resist unidirectional bending. Conclusions: Known differences in mechanical properties of bone in tension vs. compression suggest that these regional differences in cortical thickness and mineralization may be related to differences in strain mode. These structural/material dissimilarities, however, may be related to regional variations in strain magnitude, since bending and axially directed stresses in a simple cantilevered structure produce greater strain magnitudes in the compression domain. It is possible that the superimposed habitual strain magnitudes enhance strain-mode-specific adaptive responses. We hypothesize that these structural/material differences reflect the capacity of bone to process local information and produce a regionally heterogeneous organization that is appropriate for prevailing loading conditions. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public dotmain in the United states of America

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Differences in osteonal micromorphology between tensile and compressive cortices of a bending skeletal system: Indications of potential strain-specific differences in bone microstructure

Background: It has been hypothesized that bone has the capacity to accommodate regional differences in tension and compression strain mode and/or magnitude by altering its osteonal microstructure. We examined a simple cantilevered bone to determine whether regional differences in particular strain-related features are reflected in the microstructural organization of compact bone. Methods & Results: The artiodactyl (e.g., sheep and deer) calcaneus has a predominant loading condition which is typified by prevailing compressive and tensile strains on opposite cortices, and variations in strain magnitudes across each of these cortices. Microscopic examination showed osteon density and cortical porosity differences between tension (caudal) and compression (cranial) cortices, averaging 11.4% more osteons in the compression cortex (P < 0.01) and 80.2% greater porosity in the tension cortex (P < 0.01). There is 43.5% more interstitial bone in the compression cortex (P < 0.01). Osteons in the compression cortex also have smaller areas in contrast to the larger area per osteon in the tension cortex. Although no definite transcortical gradient in osteonal density or cortical porosity is found, fractional area of interstitial bone is largest and osteon population density is lowest in the endocortical regions of both tension and compression cortices. The endocortical regions also have greater porosity than their corresponding middle and pericortical regions (P < 0.01). Conclusions: These osteonal microstructure and cortical porosity differences may be adaptations related to regional differences in strain mode and/or strain magnitude. This may be related to the disparity in mechanical properties of compact bone in tension vs. compression. These differences may reflect a capacity of bone to process local and regional strain-related information. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public dotmain in the United states of America

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Do regional modifications in tissue mineral content and microscopic mineralization heterogeneity adapt trabecular bone tracts for habitual bending? Analysis in the context of trabecular architecture of deer calcanei

Calcanei of mature mule deer have the largest mineral content (percent ash) difference between their dorsal 'compression' and plantar 'tension' cortices of any bone that has been studied. The opposing trabecular tracts, which are contiguous with the cortices, might also show important mineral content differences and microscopic mineralization heterogeneity (reflecting increased hemi-osteonal renewal) that optimize mechanical behaviors in tension vs. compression. Support for these hypotheses could reveal a largely unrecognized capacity for phenotypic plasticity - the adaptability of trabecular bone material as a means for differentially enhancing mechanical properties for local strain environments produced by habitual bending. Fifteen skeletally mature and 15 immature deer calcanei were cut transversely into two segments (40% and 50% shaft length), and cores were removed to determine mineral (ash) content from 'tension' and 'compression' trabecular tracts and their adjacent cortices. Seven bones/group were analyzed for differences between tracts in: first, microscopic trabecular bone packets and mineralization heterogeneity (backscattered electron imaging, BSE); and second, trabecular architecture (micro-computed tomography). Among the eight architectural characteristics evaluated [including bone volume fraction (BVF) and structural model index (SMI)]: first, only the 'tension' tract of immature bones showed significantly greater BVF and more negative SMI (i.e. increased honeycomb morphology) than the 'compression' tract of immature bones; and second, the 'compression' tracts of both groups showed significantly greater structural order/alignment than the corresponding 'tension' tracts. Although mineralization heterogeneity differed between the tracts in only the immature group, in both groups the mineral content derived from BSE images was significantly greater (P < 0.01), and bulk mineral (ash) content tended to be greater in the 'compression' tracts (immature 3.6%, P = 0.03; mature 3.1%, P = 0.09). These differences are much less than the approximately 8% greater mineral content of their 'compression' cortices (P < 0.001). Published data, suggesting that these small mineralization differences are not mechanically important in the context of conventional tests, support the probability that architectural modifications primarily adapt the tracts for local demands. However, greater hemi-osteonal packets in the tension trabecular tract of only the mature bones (P = 0.006) might have an important role, and possible synergism with mineralization and/or microarchitecture, in differential toughening at the trabeculum level for tension vs. compression strains.     

Ontogenetic and regional morphologic variations in the turkey ulna diaphysis: implications for functional adaptation of cortical bone

This study examines relationships between bone morphology and mechanically mediated strain/fluid-flow patterns in an avian species. Using mid-diaphyseal transverse sections of domestic turkey ulnae (from 11 subadults and 11 adults), we quantified developmental changes in predominant collagen fiber orientation (CFO), mineral content (%ash), and microstructure in cortical octants or quadrants (i.e., %ash). Geometric parameters were examined using whole mid-diaphyseal cross-sections. The ulna undergoes habitual bending and torsion, and demonstrates nonuniform matrix fluid-flow patterns, and high circumferential strain gradients along the neutral axis (cranial-caudal) region at mid-diaphysis. The current results showed significant porosity differences: 1) greater osteocyte lacuna densities (N.Lac/Ar) (i.e., "non-vascular porosity") in the caudal and cranial cortices in both groups, 2) greater N.Lac/Ar in the pericortex vs. endocortex in mature bones, and 3) greater nonlacunar porosity (i.e., "vascular porosity") in the endocortex vs. pericortex in mature bones. Vascular and nonvascular porosities were not correlated. There were no secondary osteons in subadults. In adults, the highest secondary osteon population densities and lowest %ash occurred in the ventral-caudal, caudal, and cranial cortices, where shear strains, circumferential strain gradients, and fluid displacements are highest. Changes in thickness of the caudal cortex explained the largest proportion of the age-related increase in cranial-caudal breadth; the thickness of other cortices (dorsal, ventral, and cranial) exhibited smaller changes. Only subadult bones exhibited CFO patterns corresponding to habitual tension (ventral) and compression (dorsal). These CFO variations may be adaptations for differential mechanical requirements in "strain-mode-specific" loading. The more uniform oblique-to-transverse CFO patterns in adult bones may represent adaptations for shear strains produced by torsional loading, which is presumably more prevalent in adults. The micro- and ultrastructural heterogeneities may influence strain and fluid-flow dynamics, which are considered proximate signals in bone adaptation.     

Inhalant allergy to wild animals (deer and elk)

We studied 15 highly atopic persons with historic and/or skin test evidence of allergy to deer or elk. All 15 were also reactive to domestic animal danders (dog, cat, or horse). Twelve patients had elevated IgE antibody levels to deer hair/dander and six patients had elevated IgE antibody levels to elk hair/dander. Elevated IgE antibody levels to serum or urine from deer or elk were found in six persons. By using individual patient sera in RAST inhibition experiments we showed that IgE antibodies were directed to allergens common to hair/dander, urine, and serum. One individual reacted to an allergen common to both deer and cat hair/dander. We concluded that atopic individuals sensitive to domestic animals may develop IgE-mediated reactions to deer or elk allergens following recreational or professional hunting contact with these animals.     

Ontogenetic relationships between in vivo strain environment, bone histomorphometry and growth in the goat radius

Vertebrate long bone form, at both the gross and the microstructural level, is the result of many interrelated influences. One factor that is considered to have a significant effect on bone form is the mechanical environment experienced by the bone during growth. The work presented here examines the possible relationships between in vivo bone strains, bone geometry and histomorphology in the radii of three age/size groups of domestic goats. In vivo bone strain data were collected from the radii of galloping goats, and the regional cortical distribution of peak axial strain magnitudes, radial and circumferential strain gradients, and longitudinal strain rates related to regional patterns in cortical growth, porosity, remodelling and collagen fibre orientation. Although porosity and remodelling decreased and increased with age, respectively, these features showed no significant regional differences and did not correspond to regional patterns in the mechanical environment. Thicker regions of the radius's cortex were significantly related to high strain levels and higher rates of periosteal, but not endosteal, growth. However, cortical growth and strain environment were not significantly related. Collagen fibre orientation varied regionally, with a higher percentage of transverse fibres in the caudal region of the radius and primarily longitudinal fibres elsewhere, and, although consistent through growth, also did not generally correspond to regional strain patterns. Although strain magnitudes increased during ontogeny and regional strain patterns were variable over the course of a stride, mean regional strain patterns were generally consistent with growth, suggesting that regional growth patterns and histomorphology, in combination with external loads, may play some role in producing a relatively 'predictable' strain environment within the radius. It is further hypothesized that the absence of correlation between regional histomorphometric patterns and the measured strain environments is the result of the variable mechanical environment. However, the potential effects of other physiological and mechanical factors, such as skeletal metabolism and adjacent muscle insertions, that can influence the gross and microstructural morphology of the radius during ontogeny, cannot be ignored.     

骨微损伤、骨重建与代谢性骨病

骨微损伤能启动骨重建,骨重建障碍而导致微损伤积累可引发骨折。扫描电镜、同步加速器射线μ-CT和高分辨磁共振显像是研究骨微损伤的新方法,骨理化构成和年龄对微损伤发生和发展有重要影响,骨细胞在微损伤修复中起重要作用,骨微损伤研究有利于代谢性骨病防治。     

Histological organization and its relationship to function in the femur of Alligator mississippiensis

Histological analysis of a growth series of alligator femora tests the correlation between strain milieu and microstructure. From mid‐diaphyseal cross‐sections of these femora (n = 7), vascular canal orientation and density as well as collagen fibre organization were recorded. Throughout ontogeny, the proportion of transverse–spiral (TS) collagen in the dorsal cortex is significantly greater than it is in the ventral cortex (P = 0.008). This regional difference in the proportion of TS collagen is correlated with a regional difference in the state of peak principal strain (compressive or tensile). Nevertheless, the predominant orientation of collagen fibres is longitudinal, which is inconsistent with biomechanical hypotheses that involve peak principal or shear strains. Although the density and orientation of vascular canals do not show significant regional differences (P = 0.26 and P = 0.26, respectively), as with collagen orientation, the vascular canal orientation is predominantly longitudinal. The longitudinal organization of both the vascular canals and the collagen fibres is probably a consequence of longitudinal shifting of subperiosteal osteoid during femoral lengthening. When taken together, these data suggest that growth dynamics is the dominant influence on the histological organization of primary bony tissues in alligator femora.     

Characterization of equine E-selectin

Expression of E-selectin on activated endothelium is a critical initial step that leads to extravasation of leucocytes during inflammation, yet E-selectin is largely uncharacterized in several animal species including the horse. We have sequenced and compared E-selectin genes derived from activated cultures of purified equine (horse), cervid (black-tailed deer) and ovine (sheep) pulmonary artery endothelial cells (ECs). Phylogenetic and amino acid sequence comparisons indicate that bovine, cervid and ovine E-selectin are similar, whereas human and equine E-selectin are more closely related to each other than to the ruminant molecules. Human E- and P-selectin-specific monoclonal antibodies that also recognize equine E-selectin were identified and used to characterize its expression. Expression of E-selectin was more readily induced by lipopolysaccharide treatment in equine ECs than in human ECs and supported adhesion and activation of neutrophils, consistent with the extreme sensitivity of horses to endotoxaemia and septic shock.     

Spatial variation in osteon population density at the human femoral midshaft: histomorphometric adaptations to habitual load environment

Intracortical remodeling, and the osteons it produces, is one aspect of the bone microstructure that is influenced by and, in turn, can influence its mechanical properties. Previous research examining the spatial distribution of intracortical remodeling density across the femoral midshaft has been limited to either considering only small regions of the cortex or, when looking at the entirety of the cortex, considering only a single individual. This study examined the spatial distribution of all remodeling events (intact osteons, fragmentary osteons, and resorptive bays) across the entirety of the femoral midshaft in a sample of 30 modern cadaveric donors. The sample consisted of 15 males and 15 females, aged 21–97 years at time of death. Using geographic information systems software, the femoral cortex was subdivided radially into thirds and circumferentially into octants, and the spatial location of all remodeling events was marked. Density maps and calculation of osteon population density in cortical regions of interest revealed that remodeling density is typically highest in the periosteal third of the bone, particularly in the lateral and anterolateral regions of the cortex. Due to modeling drift, this area of the midshaft femur has some of the youngest primary tissue, which consequently reveals that the lateral and anterolateral regions of the femoral midshaft have higher remodeling rates than elsewhere in the cortex. This is likely the result of tension/shear forces and/or greater strain magnitudes acting upon the anterolateral femur, which results in a greater amount of microdamage in need of repair than is seen in the medial and posterior regions of the femoral midshaft, which are more subject to compressive forces and/or lesser strain magnitudes.     

The response of bone, articular cartilage and tendon to exercise in the horse

Horses can gallop within hours of birth, and may begin training for athletic competition while still growing. This review cites studies on the effects of exercise on bone, tendon and articular cartilage, as detected by clinical and research imaging techniques, tissue biochemical analysis and microscopy of various kinds. For bone, alterations in bone mineral content, mineral density and the morphology of the mineralized tissue are the most common end-points. Apparent bone density increases slightly after athletic training in the cortex, but substantially in the major load paths of the epiphyses and cuboidal bones, despite the lower material density of the new bone, which is deposited subperiosteally and on internal surfaces without prior osteoclastic resorption. With training of greater intensity, adaptive change is supervened by patho-anatomical change in the form of microdamage and frank lesions. In tendon, collagen fibril diameter distribution changes significantly during growth, but not after early training. The exact amount and type of protracted training that does cause reduction in mass average diameter (an early sign of progressive microdamage) have not been defined. Training is associated with an increase in the cross-sectional area of some tendons, possibly owing to slightly greater water content of non-collagenous or newly synthesized matrix. Early training may be associated with greater thickness of hyaline but not calcified articular cartilage, at least in some sites. The age at which adaptation of cartilage to biomechanical influences can occur may thus extend beyond very early life. However, cartilage appears to be the most susceptible of the three tissues to pathological alteration. The effect of training exercise on the anatomical or patho-anatomical features of connective tissue structures is affected by the timing, type and amount of natural or imposed exercise during growth and development which precedes the training.     

Cystic echinococcosis in equids in Italy

Between March 2003 and February 2007, the livers and the lungs of 2,231 horses from various Italian regions were examined for cystic echinococcosis presence at the time of slaughter. Hydatid cysts were found in six horses, namely four from Sardinia, one from Sicily, and one from Tuscany. The location, number, morphology, and fertility of the cysts found were determined. DNA was extracted from the germinal layers and protoscoleces of the fertile cysts and polymerase chain reactions (PCR) were performed in order to strain type DNA isolates for reduced nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1), cytochrome c oxidase subunit 1 (CO1) and 12S partial genes. The PCR products were then purified and sequenced in forward and reverse. Hydatid materials obtained from positive animals were identified as Echinococcus granulosus s.s. (old G1, sheep strain) and Echinococcus equinus (old G4, horse strain) for ND1, CO1, and 12S partial genes. This allowed us to record the presence of the E. equinus in Italy for the first time with molecular tools and also to report new data on the epidemiological situation of this parasite in Italy.     

The first detection of nematodes Ashworthius sidemi in elk Alces alces (L.) in Poland and remarks of ashworthiosis foci limitations

Ashworthius sidemi, a nematode belonging to the family of Trichostrongylidae, is a primary parasite of the Asian deer, mainly sika deer (Cervus nippon), with which it was introduced to Ukraine, as well as Slovakia, the Czech Republic and France. Migrating red deer carried this parasite from neighboring countries to Polish territory. Until now, in Poland, this parasite has been recorded in European bison, red deer, roe deer and fallow deer. As a result of post-mortem examinations of 10 elk, 2 of them from the Augustów Forest and Biebrza Marshes, A. sidemi were found in abomasa for the first time in Poland. The intensity of the invasions was 120 and 7 specimens, respectively. This finding of Ashworthius sidemi in elk indicates a further expansion of the focus of ashworthiosis in Bia?owie?a towards the north into the Biebrza Marshes and the Augustowska Forest. The growth of the elk population and their tendency for long distance migrations can contribute to the spread of the parasitosis in much greater distances than deer. On the basis of our own research and data from the literature, the current spread of ashworthiosis in Poland is discussed.     

Pathogenic, immunologic, and molecular differences between two Ehrlichia risticii strains.

Ehrlichia risticii is the causative agent of Potomac horse fever (PHF), an acute infectious disease of horses. In the last few years, there have been several reports of PHF cases occurring even in vaccinated horses. We isolated a new strain of E. risticii (90-12 strain) from a vaccinated horse suffering from clinical PHF. The major pathogenic, immunologic, and molecular differences between the 90-12 strain and the 25-D stain, which was originally isolated during the outbreaks in 1984, were studied. The 90-12 strain was more pathogenic for mice and horses compared with the 25-D strain. In enzyme-linked immunosorbent assay and immunofluorescence assay with mouse and horse antisera of both the strains, two- to fourfold differences were observed between (immunoblot) with mouse and horse antisera and also with the recombinant clone-specific antibodies. Though several antigens were similar in both the strains, there were significant differences between them in the 110-, 85-, 70-, 51-, and 33-kDa antigens. The 85-kDa antigen was present only in the 90-12 strain but cross-reacted with a 50-kDa antigen of the 25-D strain. The 51-kDa antigens of both strains had different migration patterns, Southern blot hybridization of the genome from both the strains with DNA probes made from the 51-, 55-, and clones for both the strains, whereas the probe of the 85-kDa clone showed a completely different pattern. The 16S rRNA gene sequences from the two strains were identical. Neither strain replicated in gamma interferon-treated mouse peritoneal macrophages. In in vitro neutralization assay, sera from the 25-D strain-infected horse neutralized the homologous strain but did not neutralize the 90-12 strain, whereas sera from the 90-12 strain-infected horse neutralized both the strains. In mouse protection experiments, there was complete homologous protection. But in cross-protection, mice immunized with the 25-D strain were only partially protected against challenge with the 90-12 strain, whereas mice immunized with the 90-12 strain were completely protected against the 25-D strain challenge. These results clearly indicate that there are major differences between the 90-12 and 25-D strains which may have implications regarding the vaccine failure for PHF and the development of an efficient vaccine.     

Modeling and remodeling in a developing artiodactyl calcaneus: a model for evaluating Frost's Mechanostat hypothesis and its corollaries

The artiodactyl (mule deer) calcaneus was examined for structural and material features that represent regional differences in cortical bone modeling and remodeling activities. Cortical thickness, resorption and formation surfaces, mineral content (percent ash), and microstructure were quantified between and within skeletally immature and mature bones. These features were examined to see if they are consistent with predictions of Frost's Mechanostat paradigm of mechanically induced bone adaptation in a maturing "tension/compression" bone (Frost, 1990a,b, Anat Rec 226:403-413, 414-422). Consistent with Frost's hypothesis that surface modeling activities differ between the "compression" (cranial) and "tension" (caudal) cortices, the elliptical cross-section of the calcaneal diaphysis becomes more elongated in the direction of bending as a result of preferential (> 95%) increase in thickness of the compression cortex. Regional differences in mineral content and population densities of new remodeling events (NREs = resorption spaces plus newly forming secondary osteons) support Frost's hypothesis that intracortical remodeling activities differ between the opposing cortices: 1.) in immature and mature bones, the compression cortex had attained a level of mineralization averaging 8.9 and 6.8% greater (P < 0.001), respectively, than that of the tension cortex, and 2.) there are on average 350 to 400% greater population densities of NREs in the tension cortices of both age groups (P < 0.0003). No significant differences in cortical thickness, mineral content, porosity, or NREs were found between medial and lateral cortices of the skeletally mature bones, suggesting that no modeling or remodeling differences exist along a theoretical neutral axis. However, in mature bones these cortices differed considerably in secondary osteon cross-sectional area and population density. Consistent with Frost's hypothesis, remodeling in the compression cortex produced bone with microstructural organization that differs from the tension cortex. However, the increased remodeling activity of the tension cortex does not appear to be related to a postulated low-strain environment. Although most findings are consistent with predictions of Frost's Mechanostat paradigm, there are several notable inconsistencies. Additional studies are needed to elucidate the nature of the mechanisms that govern the modeling and remodeling activities that produce and maintain normal bone. It is proposed that the artiodactyl calcaneus will provide a useful experimental model for these studies.