Secondary Osteons Research Articles

A Novel Model to Assess the Effects of Osteoporosis Medications on Bone Quality

In order to examine the effects of osteoporosis treatments on bone tissue that forms during drug administration, we have adapted a decades old model in the rat(1, 2). The premise of our work is that bone matrix mineralization within cortical bone is an important but oft‐neglected component of bone remodeling. Aberrant mineralization can reduce bone strength and increase fracture risk independent of changes in bone mass. Sodium fluoride represents an example of an osteoporosis treatment that alters the mineralization process, leading to increased fracture risk in spite of increased bone mass, thereby causing the very thing it was meant to cure. Unfortunately, small animal models to study matrix mineralization during remodeling of cortical bone in the adult skeleton did not exist when this drug was under development; so these unexpected effects were only discovered in patients.We have performed a series of studies to develop and validate a rodent model to study matrix mineralization during remodeling in adult cortical bone. Briefly, remodeling is induced by severely restricting dietary calcium in lactating animals (0.01% as opposed to the normal 0.6%). When the pups are weaned, the dams are immediately returned to the normal Ca diet. We study the bone that forms during the post‐weaning period in the dams. We have performed experiments to assess the most appropriate anatomical site for examining intra‐cortical remodeling and the effects of manipulating dietary Ca on mineral metabolism and mineralization kinetics. We found that the border between the distal femoral diaphysis and metaphysis is the best site for sampling intra‐cortical remodeling because this site had the most induced pores of a size consistent with rat secondary osteons(3). The increase in porosity was resolved 30 days after weaning, suggesting that the induced intra‐cortical resorption spaces had totally infilled. Endocortical remodeling can also be examined at this site. Studies using in vivo fluorochrome labeling, Fourier‐transform infrared microspectroscopy, back‐scatter scanning electron microscopy, microcomputed tomography, histology and serum assays indicate that matrix mineralization occurs normally and largely within a normal endocrine environment despite the manipulation of Ca in the diet. A major advantage of this model is that it provides multiple sites of synchronized matrix mineralization. We are currently using the model to study how common and emerging osteoporosis drugs affect matrix mineralization and mechanical behavior.Support or Funding InformationNIH Grant R21AR065604This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Histomorphological analysis of the variability of the human skeleton: forensic implications.

One of the fundamental questions in forensic medicine and anthropology is whether or not a bone or bone fragment is human. Surprisingly at times for the extreme degradation of the bone (charred, old), DNA cannot be successfully performed and one must turn to other methods. Histological analysis at times can be proposed. However, the variability of a single human skeleton has never been tested. Forty-nine thin sections of long, flat, irregular and short bones were obtained from a well-preserved medieval adult human skeleton. A qualitative histomorphological analysis was performed in order to assess the presence of primary and secondary bone and the presence, absence and orientation of vascular canals. No histological sections exhibited woven or fibro-lamellar bone. Long bones showed a higher variability with an alternation within the same section of areas characterized by tightly packed secondary osteons and areas with scattered secondary osteons immersed in a lamellar matrix. Flat and irregular bones appeared to be characterized by a greater uniformity with scattered osteons in abundant interstitial lamellae. Some cases of "osteon banding" and "drifting osteons" were observed. Although Haversian bone represent the most frequent pattern, a histomorphological variability between different bones of the same individual, in different portions of the same bone, and in different parts of the same section has been observed. Therefore, the present study has highlighted the importance of extending research to whole skeletons without focusing only on single bones, in order to have a better understanding of the histological variability of both human and non-human bone.

Histomorphometry and cortical robusticity of the adult human femur.

Recent quantitative analyses of human bone microanatomy, as well as theoretical models that propose bone microstructure and gross anatomical associations, have started to reveal insights into biological links that may facilitate remodeling processes. However, relationships between bone size and the underlying cortical bone histology remain largely unexplored. The goal of this study is to determine the extent to which static indicators of bone remodeling and vascularity, measured using histomorphometric techniques, relate to femoral midshaft cortical width and robusticity. Using previously published and new quantitative data from 450 adult human male (n=233) and female (n=217) femora, we determine if these aspects of femoral size relate to bone microanatomy. Scaling relationships are explored and interpreted within the context of tissue form and function. Analyses revealed that the area and diameter of Haversian canals and secondary osteons, and densities of secondary osteons and osteocyte lacunae from the sub-periosteal region of the posterior midshaft femur cortex were significantly, but not consistently, associated with femoral size. Cortical width and bone robusticity were correlated with osteocyte lacunae density and scaled with positive allometry. Diameter and area of osteons and Haversian canals decreased as the width of cortex and bone robusticity increased, revealing a negative allometric relationship. These results indicate that microscopic products of cortical bone remodeling and vascularity are linked to femur size. Allometric relationships between more robust human femora with thicker cortical bone and histological products of bone remodeling correspond with principles of bone functional adaptation. Future studies may benefit from exploring scaling relationships between bone histomorphometric data and measurements of bone macrostructure.

Fracture and microstructural study of bovine bone under mixed mode I/II loading

Understanding the fracture behavior and associated crack growth mechanism in bone material is an important issue for biomechanics and biomaterial researches. Fracture of bone often takes place due to complex loading conditions which result in combined tensile-shear (i.e. mixed mode) fracture mechanism. Several parameters such as loading type, applied loading direction relative to the bone axis, loading rate, age and etc., may affect the mixed mode fracture resistance and damage mechanism in such materials. In this research, a number of mixed mode I/II fracture experiments are conducted on bovine femur bone using a sub-sized test configuration called “compact beam bend (CBB)” specimen to investigate the fracture toughness of bone under different mode mixities. The specimen is rectangular beam containing a mid-edge crack that is loaded by a conventional three-point bend fixture. The results showed the dependency of bone fracture toughness on the state of mode mixity. The fracture surfaces of broken CBB specimens under different loading conditions were studied via scanning electron microscopy (SEM) observations. Fracture surface of all investigated cases (i.e. pure mode I, pure mode II and mixed mode I/II) exhibited smooth patterns demonstrating brittle fracture of bovine femur. The higher density of vascular channels and micro-cracks initiated in the weakened area surrounded by secondary osteons were found to be the main cause of the decreased bone resistance against crack growth and brittle fracture.

The effect of T-2 toxin on bone microstructure in rabbits

T-2 toxin is the most toxic of the trichothecene mycotoxins. Its effect on bone microstructure is still unknown. This study focuses on acute effects of the T-2 toxin on compact and trabecular bone tissues structure of rabbits after a single intramuscular administration. Experimental E group (n = 4) consisted of animals which were intramuscularly injected with T-2 toxin at dose 0.08 mg.kg−1 body weight 72 h before slaughter. Group C (n = 4) without T-2 toxin application served as a control. An absence of primary vascular longitudinal bone tissue near endosteal surfaces, its deposition on periosteal surfaces and a lower density of secondary osteons in the middle part of the substantia compacta were observed in both females and males injected with T-2 toxin. On the contrary, morphometrical analysis of the compact bone showed no demonstrable alternations in the sizes of primary osteons’ vascular canals, Haversian canals or secondary osteons between rabbits from E and C groups. Also, no significant effects of the T-2 toxin on trabecular bone morphometry and cortical bone thickness were observed between rabbits of either sex. The single intramuscular application of T-2 toxin at the dose used in our study affects only qualitative histological characteristics of the compact bone in rabbits.

Acrylamide-induced changes in femoral bone microstructure of mice.

Acrylamide (AA) is one of the most common toxins in foods. Its effect on bone microstructure has not been investigated. The aim of our study was to analyze the impact of acute exposure to AA on femoral bone microstructure in mice. Adult animals were treated perorally with 2 doses of AA (E1 group, 1 mg/kg b.w.) in a 24-h period and with 3 doses of AA (E2 group, 1 mg/kg b.w.) in a 48-h period. Mice exposed to AA had smaller sizes of primary osteon's vascular canals. Secondary osteons were significantly smaller in mice from E2 group; however their increased number (from 38 % to 77 %) was identified in both E1 and E2 groups. In these groups, a higher number of resorption lacunae (from 100 % to 122 %) was also found. The values for bone volume, trabecular number were increased and that for trabecular separation was decreased in mice administered AA. Significantly higher value of bone surface was observed in mice from E1 group whereas trabecular thickness was increased in E2 group. The effect of AA on microstructure of compact and trabecular bone tissues is different. In our study, one dose of AA was used and acute effects of AA were investigated. Therefore, further studies are needed to study mechanisms by which AA acts on bone.

Histomorphometric analysis of bone lacunae in human and pig: Can it be useful for species discrimination?

In recent years, several studies have focused on species identification of bone fragments by the assessment of the histomorphological appearance of bone tissue as well as the quantification of histological structures such as Haversian systems. According to literature, the most consistent distinguishing features are Haversian canal and Haversian system areas. Nonetheless, there is a consistent overlap between human and non-human secondary osteon dimensions. One of the structure that has never been analyzed for the purpose of species discrimination is the bone lacuna, a small oblong cavity in which the osteocyte is locked in. The aim of this study is to verify whether there are significant quantitative differences between human and pig lacunae within secondary osteons with similar areas. Study sample comprises the diaphysis of long bones of a medieval human adult and a subadult pig. A total of 68 secondary osteons with similar areas have been selected for each individual. Each osteon was analyzed with a light microscope and the following measurements were taken at 200X magnification using IScapture® software: 1) total number of lacunae within the osteon, 2) minimum and maximum diameter, area and perimeter of nine lacunae divided between inner, intermediate and outer lacunae. T-test was employed to compare the mean values for the two individuals. The mean number of lacunae per osteon does not seem to be significantly different between human (48.9) and pig (46.9). However, human lacunae seem generally bigger when compared with pig lacunae with a mean area respectively of 45.06 (±17.42) μm2 and 39.6 (± 15.52) μm2. Nonetheless, there is a significant overlap between human and pig. Concerning the differences between inner, intermediate and outer lacunae, the ones close to the cement line (outer lacunae) are clearly bigger when compared with lacunae which are close to the Haversian canal (inner lacunae). Statistical analysis showed a significant difference in the maximum diameter, perimeter and area of the lacunae between human and pig. Though results seem promising, this exploratory study need further research on a bigger sample including individuals of different ages and different species in order to verify its potential for species discrimination.

Secondary osteons scale allometrically in mammalian humerus and femur.

Intra-cortical bone remodelling is a cell-driven process that replaces existing bone tissue with new bone tissue in the bone cortex, leaving behind histological features called secondary osteons. While the scaling of bone dimensions on a macroscopic scale is well known, less is known about how the spatial dimensions of secondary osteons vary in relation to the adult body size of the species. We measured the cross-sectional area of individual intact secondary osteons and their central Haversian canals in transverse sections from 40 stylopodal bones of 39 mammalian species (body mass 0.3–21 000 kg). Scaling analysis of our data shows that mean osteonal resorption area (negative allometry, exponent 0.23,R2 0.54,p<0.005) and Haversian canal area (negative allometry, exponent 0.31,R2 0.45,p<0.005) are significantly related to body mass, independent of phylogeny. This study is the most comprehensive of its kind to date, and allows us to describe overall trends in the scaling behaviour of secondary osteon dimensions, supporting the inference that the osteonal resorption area may be limited by the need to avoid fracture in smaller mammalian species, but the need to maintain osteocyte viability in larger mammalian species.

Occurrence of osteon banding in adult human cortical bone.

Differentiating human from nonhuman fragmented bone is often accomplished using histological methods if the observation of gross morphology proves insufficient. Linearly oriented primary and/or secondary osteonal systems, commonly referred to as osteon bands, are described as a strong indicator of nonhuman bone, particularly the occurrence of multiple bands. This phenomenon has been conventionally documented using two-dimensional (2D) histology, but such analyses are destructive and typically limited to a single cross-section. Progressive developments in high-resolution X-ray imaging, however, allow for the nondestructive three-dimensional (3D) visualization of bone microarchitecture. The primary objective of the current research was to visualize and document the occurrence of osteon banding in adult human cortical bone using high-resolution synchrotron radiation-based micro-Computed Tomography (SR micro-CT). Synchrotron radiation-based micro-CT scanning was carried out at the Canadian Light Source (CLS) national synchrotron facility. The presence or absence of osteon banding was visualized in human skeletal elements from three adult males with representative samples from all regions of the skeleton (n = 129). If present, osteon banding was described and quantified. Results indicated that 23 of 129 human cortical bone specimens exhibited osteon banding, representing 18% of the sample. Linear arrangements of primary and/or secondary osteons were observed in the following skeletal elements: temporal, parietal, frontal, occipital, clavicle, mandible, femur, tibia, ulna, second metatarsal, and sacrum. The present work represents the first 3D examination of inter-element variation in osteon banding in adult human cortical bone. Findings indicate that the presence of multiple osteon bands in a single specimen is not diagnostic of nonhuman bone. As such, osteon banding categorically should not be taken as evidence of nonhuman bone in forensic and archaeological contexts.

Intracortical stiffness of mid-diaphysis femur bovine bone: lacunar-canalicular based homogenization numerical solutions and microhardness measurements.

Microscale lacunar-canalicular (L-C) porosity is a major contributor to intracortical bone stiffness variability. In this work, such variability is investigated experimentally using micro hardness indentation tests and numerically using a homogenization scheme. Cross sectional rings of cortical bones are cut from the middle tubular part of bovine femur long bone at mid-diaphysis. A series of light microscopy images are taken along a line emanating from the cross-section center starting from the ring's interior (endosteum) ring surface toward the ring's exterior (periosteum) ring surface. For each image in the line, computer vision analysis of porosity is conducted employing an image segmentation methodology based on pulse coupled neural networks (PCNN) recently developed by the authors. Determined are size and shape of each of the lacunar-canalicular (L-C) cortical micro constituents: lacunae, canaliculi, and Haversian canals. Consequently, it was possible to segment and quantify the geometrical attributes of all individual segmented pores leading to accurate determination of derived geometrical measures such as L-C cortical pores' total porosity (pore volume fraction), (elliptical) aspect ratio, orientation, location, and number of pores in secondary and primary osteons. Porosity was found to be unevenly (but linearly) distributed along the interior and exterior regions of the intracortical bone. The segmented L-C porosity data is passed to a numerical microscale-based homogenization scheme, also recently developed by the authors, that analyses a composite made up of lamella matrix punctuated by multi-inclusions and returns corresponding values for longitudinal and transverse Young's modulus (matrix stiffness) for these micro-sized spatial locations. Hence, intracortical stiffness variability is numerically quantified using a combination of computer vision program and numerical homogenization code. These numerically found stiffness values of the homogenization solution are corroborated experimentally using microhardness indentation measurements taken at the same points that the digital images were taken along a radial distance emanating from the interior (endosteum) surface toward the bone's exterior (periosteum) surface. Good agreement was found between numerically calculated and indentation measured stiffness of Intracortical lamellae. Both indentation measurements and numerical solutions of matrix stiffness showed increasing linear trend of compressive longitudinal modulus (E11) values vs. radial position for both interior and exterior regions. In the interior (exterior) region of cortical bone, stiffness modulus values were found to range from 18.5 to 23.4 GPa (23 to 26.0 GPa) with the aggregate stiffness of the cortical lamella in the exterior region being 12% stiffer than that in the interior region. In order to further validate these findings, experimental and FEM simulation of a mid-diaphysis bone ring under compression is employed. The FEM numerical deflections employed nine concentric regions across the thickness with graded stiffness values based on the digital segmentation and homogenization scheme. Bone ring deflections are found to agree well with measured deformations of the compression bone ring.

Internal Morphology of Osteoderms of Extinct Armadillos and Its Relationship with Environmental Conditions

The most complete and continuous fossil record of armadillos is composed mostly by isolated osteoderms, frequently found in paleontological and archaeological sites that bear continental South American mammals. Their external morphology has been used to define several species. In the last decade, many authors have focused on the internal structure of vertebrate osteoderms using histological and paleohistological studies. These studies allowed identification of useful features in systematic and phylogenetic contexts. In armadillos, osteoderms are constituted by compact bone tissue (primary and secondary osteons, and concentric layers or lamellae) that delimits cavities, which could contain different soft tissues (adipose tissue, hair follicles, bone marrow, and sweat and sebaceous glands). Traditional paleohistological techniques have allowed the recognition of homologous cavities to those found in osteoderms of current species and from comparison deduce which kind of tissue could had occupied them. We have recently utilized 3D reconstructions in osteoderms of extant species of armadillos to analyze the micromorphology, disposition, and the relationship of different cavities and understand them in depth. Here, we present the results of the application of paleohistology and microtomography in osteoderms of representatives of diferent taxa of extinct Dasypodidae (Astegotheriini, Stegotheriini, “Utaetini,” Euphractini, Eutatini), which allowed us to compare homologous structures based on their three-dimensional reconstruction. The results, added to the previous external morphology studies, have allowed us to define morphological patterns (consistent within each linage). The variation of the volume and extension of cavities associated with different tissues could be strongly associated with changes in the climate and environmental conditions of the species distribution areas.

Cortical Histomorphometry of the Human Humerus During Ontogeny.

Modeling and remodeling are two key determinants of human skeletal growth though little is known about the histomorphometry of cortical bone during ontogeny. In this study, we examined the density and geometric properties of primary and secondary osteons (osteon area and diameter, vascular canal area and diameter) in subperiosteal cortical bone from the human humerus (n=84) between birth and age 18years. Sections were removed from the anterior midshaft aspect of humeri from skeletons. Age-at-death was reconstructed using standard osteological techniques. Analyses revealed significant correlation between the histomorphometric variables and age. Higher densities of primary osteons occurred between infancy and 7years of age but were almost completely replaced by secondary osteons after 14years of age. The geometry of primary osteons was less clearly related to age. Secondary osteons were visible after 2years of age and reached their greatest densities in the oldest individuals. Osteon size was positively but weakly influenced by age. Our data imply that modeling and remodeling are age-dependent processes that vary markedly from birth to adulthood in the human humerus.

Bone Matrix Maturation in a Rat Model of Intra-Cortical Bone Remodeling.

Matrix maturation within cortical bone is an important but oft-neglected component of bone remodeling because of the lack of a suitable small animal model. Intra-cortical remodeling can be induced in rodents by feeding virgin or lactating animals a low-calcium diet. The current study aimed to determine which of these two models is most suitable for studying intra-cortical matrix maturation. We compared intra-cortical remodeling in female rats fed a normal calcium diet (virgin/normal Ca), a low-calcium diet (virgin/low Ca), or a low-calcium diet during lactation (lactation/low Ca). The low-calcium diet was administered for 23 days (induction phase) followed by return to normal calcium for 30 days (recovery phase). At the end of induction, the virgin/normal Ca and virgin/low-Ca animals had no difference in cortical porosity, but the lactation/low-Ca animals had elevated cortical porosity at various diaphyseal sites in the femur and tibia. The distal femoral site had the greatest amount of induced porosity in the size range of rat secondary osteons. Neither global mineralization nor tissue age-specific mineral-to-matrix ratio in the bone formed during recovery were affected in the lactation/low-Ca rats. Serum calcium levels did not differ from controls, but phosphate levels were slightly elevated, consistent with the rapid recovery of lost bone mass. We conclude that the lactation/low-Ca model represents a means to increase intra-cortical remodeling in adult rats with no apparent detrimental effect on matrix maturation. This model will provide researchers with a new tool to study matrix maturation throughout the cortex.

Can secondary osteons be used as ontogenetic indicators in sauropods? Extending the histological ontogenetic stages into senescence

AbstractSauropod bone histology has provided a great deal of insight into the life history of these enormous animals. However, because of high growth rates, annual growth rings are not common in sauropod long bones, so directly measuring growth rates and determining sexual maturity require alternative measures. Histological ontogenetic stages (HOS) have been established to describe the changes in bone histology through development for basal Macronaria and Diplodocoidea, and subsequently for Titanosauria. Despite this, the current HOS model is not able to discriminate bone tissues in late ontogeny, when sauropods had reached asymptotic size and continued to live into senescence but their long bones became extensively remodeled by secondary osteons and all primary bone was destroyed. Here we establish remodeling stages (RS) to characterize the Haversian bone development through ontogeny in eight sauropod taxa (Apatosaurinae,Giraffatitan brancai,Camarasaurusspp.,Dicraeosaurusspp.,Ampelosaurus atacis,Phuwiangosaurus sirindhornae,Magyarosaurus dacus, andAlamosaurus sanjuanensis) and find significant correlation of RS with corresponding femur length (CFL) for the studied taxa, with the exception ofDicraeosaurusandMagyarosaurus. Remodeling stages are based on the maximum number of observable generations of crosscutting osteons from the innermost, mid-, and outermost part of the cortex. The correlation with CFL indicates that secondary osteons present an ontogenetic signal that could extend the histological ontogenetic stages. Remodeling stages also provide additional insight into the changes in histology through ontogeny for Sauropoda. This method has the potential to be used in other taxa, such as thyreophorans and many ornithischians, that develop Haversian tissue through development.

Utility of osteon circularity for determining species and interpreting load history in primates and nonprimates.

Histomorphological analyses of bones are used to estimate an individual's chronological age, interpret a bone's load history, and differentiate species. Among various histomorphological characteristics that can influence mechanical properties of cortical bone, secondary osteon (Haversian system) population density and predominant collagen fiber orientation are particularly important. Cross-sectional shape characteristics of secondary osteons (On.Cr = osteon circularity, On.El = osteon ellipticality) are considered helpful in these contexts, but more robust proof is needed. We sought to determine if variations in osteon shape characteristics are sufficient for accurately differentiating species, load-complexity categories, and regional habitual strain-mode distributions (e.g., tension vs. compression regions). Circularly polarized light images were obtained from 100-micron transverse sections from diaphyses of adult deer calcanei; sheep calcanei, radii, and tibiae; equine calcanei, radii, and third metacarpals (MC3s); chimpanzee femora; and human femora and fibulae. Osteon cross-sectional area (On.Ar), On.Cr, and On.El were quantified indiscriminately and in the contexts of load-complexity and regional strain-mode distributions. On.Cr and On.El, when examined independently in terms of all data, or mean (nested) data, for each bone, exceeded 80% accuracy in the inter-species comparisons only with respect to distinguishing humans from nonhumans. Correct classification among the nonhuman species was <70%. When On.Cr and On.El were coupled together and with On.Ar in discriminant function analyses (nested and unnested data) there were high misclassifications in all but human vs. nonhuman comparisons. Frequent misclassifications in nonhuman comparisons might reflect influences of habitual load complexity and/or strain-mode distributions, or other factors not accounted for by these two considerations.

Morphological and Mechanical Biomimetic Bone Structures.

Cortical bone is an example of a mineralized tissue containing a compositional distribution of hard and soft phases in 3-dimensional space for mechanical function. X-ray computed tomography (XCT) is able to describe this compositional and morphological complexity but methods to provide a physical output with comparable mechanical function is lacking. A workflow is presented here to establish a method of using high contrast XCT to establish a virtual model of cortical bone that is manufactured using a multiple material capable 3D printer. Resultant 3D printed structures were produced based on more and less remodelled bone designs exhibiting a range of secondary osteon density. Variation in resultant mechanical properties of the 3D printed composite structures for each bone design was achieved using a combination of material components and reasonable prediction of elastic modulus provided using a Hashin-Shtrikman approach. The ability to 3D print composite structures using high contrast XCT to distinguish between compositional phases in a biological structure promises improved anatomical models as well as next-generation mechano-mimetic implants.

Submicrostructural domains in human secondary osteons

Submicrostructural domains in human secondary osteons

Osteology of a perinatal aristonectine (Plesiosauria; Elasmosauridae)

AbstractPerinatal specimens give valuable information about the first stages of vertebrate ontogeny. Here, the morphology and palaeohistology of an aristonectine perinatal specimen from Seymour Island (Isla Marambio), López de Bertodano Formation are analysed. The palaeohistological analysis shows incomplete endochondral ossification (retention of a calcified cartilaginous core in the medullary region), predominance of primary bone tissue without secondary remodelling, lack of primary or secondary osteons and of growth marks in the cortical bone, and open vascular spaces not surrounded by a thin coat of lamellar bone tissue. General lines of morphological changes were inferred from comparing the fossil with an adult aristonectine specimen indicating i) a tendency of relatively high and broad posterior cervicals to decrease during ontogeny, ii) a decrease of relative size of the dorsolateral process and an increase of the glenoid ramus and iii) the existence of two separate stages in propodial growth divided into an initial elongation followed by a distal expansion. The presence of a perinatal specimen in the James Ross Archipelago indicates that the region was used as a breeding area by the aristonectines during the last part of the Cretaceous.

Histological analysis of femoral bones in rabbits administered by amygdalin

Cyanogenic glycosides are present in several economically important plant foods. Amygdalin, one of the most common cyanoglucoside, can be found abundantly in the seeds of apples, bitter almonds, apricots, peaches, various beans, cereals, cassava and sorghum. Amygdalin has been used for the treatment of cancer, it shows killing effects on cancer cells by release of cyanide. However, its effect on bone structure has not been investigated to date. Therefore, the objective of this study was to determine a possible effect of amygdalin application on femoral bone microstructure in adult rabbits. Four month old rabbits were randomly divided into two groups of three animals each. Rabbits from E group received amygdalin intramuscularly at a dose 0.6 mg.kg-1 body weight (bw) (group E, n = 3) one time per day during 28 days. The second group of rabbits without amygdalin supplementation served as a control (group C, n = 3). After 28 days, histological structure of femoral bones in both groups of rabbits was analysed and compared. Rabbits from E group displayed different microstructure in middle part of the compact bone and near endosteal bone surface. For endosteal border, an absence of the primary vascular longitudinal bone tissue was typical. This part of the bone was formed by irregular Haversian and/or by dense Haversian bone tissues. In the middle part of substantia compacta, primary vascular longitudinal bone tissue was observed. Cortical bone thickness did not change between rabbits from E and C groups. However, rabbits from E group had a significantly lower values of primary osteons' vascular canals and secondary osteons as compared to the C group. On the other hand, all measured parameters of Haversian canals did not differ between rabbits from both groups. Our results demonstrate that intramuscular application of amygdalin at the dose used in our study affects femoral bone microstructure in rabbits.

Quercetin-induced changes in femoral bone microstructure of adult male rabbits

Flavonoids are a group of plant metabolites with antioxidant effects. One of the most abundant flavonoids in the human diet is quercetin. It is found widely in fruits, vegetables and has a lot of beneficial effects on human health. Quercetin has a positive pharmacological effect on bone metabolism and it prevents the organism against bone loss. However, its impact on the size of basic structural units of the compact bone is still unknown. Therefore, the aim of present study was to investigate the impact of the quercetin on femoral bone microstructure in 5-month-old male rabbits. Five rabbits of Californian broiler line were randomly divided into two groups. In the experimental group (E group; n=3), animals were intramuscularly injected with quercetin at dose 1000 μg.kg-1 body weight (bw) for 90 days, 3 times per week. Two rabbits without quercetin administration served as a control group (C group). According to our results, intramuscular application of quercetin had an insignificant effect on cortical bone thickness in male rabbits. In these rabbits, changes in qualitative histological characteristics were present in the middle part of the compacta, where primary vascular longitudinal bone tissue was present and expanded there from the periosteum. Also, a lower number of secondary osteons was found in these animals. From the histomorphometrical point of view, significantly decreased sizes of primary osteons' vascular canals and secondary osteons (p &lt;0.05) were found in rabbits administered by quercetin. Our findings indicate that subchronic administration of quercetin at the dose used in our study had considerable impact on both qualitative and quantitative histological characteristics of the compact bone in adult male rabbits.