Differences in femoral compact bone tissue microscopic structure between adult cows (Bos taurus) and pigs (Sus scrofa domestica)

The purpose of this investigation was to study in detail compact bone tissue microscopic structure of adult cows and pigs with an emphasis to find an adequate key for the species identification. Altogether 18 femurs were analysed. Each of the bones was sectioned at the smallest breadth of their diaphysis. Specimens were prepared using standard histological equipment, producing thin sections approximately 80-100 mum thick. The qualitative differences between investigated species were examined in anterior, posterior, medial and lateral views of thin sections. The quantitative ones were counted using the specific computer software Scion Image. We measured the area, perimeter, minimal and maximal diameter of the Haversian canals, the Haversian systems and the vascular canals of primary osteons. After that a discriminant function analysis was used for the species identification. According to our results the basic structural pattern of femur diaphysis was primary vascular plexiform in both species. However, non-vascular bone tissue was identified in cows and resorption lacunae were found between the secondary osteons in pigs. The measured variables of the Haversian canals, the Haversian systems and the primary osteon's vascular canals were higher in most cases in cows. Classification functions for investigated species give a correct classification of 64.69% of cases. This percentage value can be increased by integrating conclusions from the qualitative analysis.     

Sex-related variation in compact bone microstructure of the femoral diaphysis in juvenile rabbits

Background

While gross morphological changes in the skeleton between males and females are well know, differences between sexes in the histomorphology are less known. It is important to have knowledge on the bone structure of rabbits, as this is a widely used species in biomedical research. A study was performed to evaluate the association between sex and the compact bone morphology of the femoral diaphysis in juvenile rabbits.

Methods

Seventeen clinically healthy 2–3 month-old rabbits (9 females, 8 males) were included in the study. The rabbits were euthanized and the right femur was sampled for analysis. 70–80 microns thick bone sections of the femoral diaphysis were prepared using standard histological equipment. The qualitative histological characteristics were determined according to internationally accepted classification systems while the quantitative parameters were assessed using the software Scion Image. Areas, perimeters, minimum and maximum diameters of primary osteons'' vascular canals, Haversian canals and secondary osteons were measured. Additionally, blood plasma concentrations of progesterone, corticosterone, IGF-I, testosterone and estradiol were analyzed.

Results

Qualitative histological characteristics were similar for both sexes. However, variations of certain quantitative histological characteristics were identified. Measured parameters of the primary osteons'' vascular canals were higher in males than for females. On the other hand, females had significant higher values of secondary osteons parameters. Differences in Haversian canals parameters were only significant for minimum diameter.

Conclusion

The study demonstrated that quantitative histological characteristics of compact bone tissue of the femoral diaphysis in juvenile rabbits were sex dependent. The variations may be associated with different growth and modeling of the femur through influence by sex-specific steroids, mechanical loads, genetic factors and a multitude of other sources. The results can be applied in experimental studies focusing on comparison of the skeletal biology of the sexes.     

Morphology,Morphometry and Spatial Distribution of Secondary Osteons in Equine Femur

A high number of differences exist in bone histological features depending on the species, breed, age and bone. Moreover, osteon distribution may vary in the different sides of a bone as a consequence of different biomechanical strains. The aim of this work was to study the distribution and morphology of osteons in different sides of the equine femoral diaphysis with the attempt to correlate them to the main strains operating on them. The following parameters of secondary osteons and Haversian canals were measured in the transverse sections of diaphyses: perimeter, area, minimum and maximum diameter, eccentricity and osteon population density. A typical Haversian tissue was observed with elliptic secondary osteons consisting in about 10 well‐defined lamellae surrounding a circular Haversian canal. Quantitative analysis displays a different population density of secondary osteons depending on the side. The caudal and medial sides, where compression strains are higher, have more secondary osteons in comparison with the cranial and lateral sides, where tension strains are prevalent. These data suggest that secondary osteon population density may depend on the predominant strains. Even the elliptical shape of secondary osteons may be related to biomechanical strains, as their major axes are oriented cranio‐caudally parallel to prevalent strains.     

Structural changes in femoral bone tissue of rats after subchronic peroral exposure to selenium

Background

The role of selenium (Se) on bone microarchitecture is still poorly understood. The present study aims to investigate the macroscopic and microscopic structures of femoral bone tissue in adult male rats after subchronic peroral administration of Se.

Methods

Twenty one-month-old male Wistar rats were randomly divided into two experimental groups. In the first group (Se group) young males were exposed to 5 mg Na₂SeO₃/L in drinking water, for 90 days. Ten one-month-old males without Se administration served as a control group. At the end of the experiment, macroscopic and microscopic structures of the femurs were analysed using analytical scales, sliding instrument, and polarized light microscopy.

Results

The body weight, femoral length and cortical bone thickness were significantly decreased in Se group rats. These rats also displayed different microstructure in the middle part of the femur, both in medial and lateral views, where vascular canals expanded into the central area of the bone while, in control rats, these canals occurred only near the endosteal surfaces. Additionally, a smaller number of primary and secondary osteons was identified in Se group rats. Histomorphometric analyses revealed significant increases for area, perimeter, maximum and minimum diameters of primary osteons’ vascular canals but significant reductions for all measured variables of Haversian canals and secondary osteons.

Conclusions

Se negatively affected the macroscopic and microscopic structures of femoral bone tissue in adult male rats. The results contribute to the knowledge on damaging impact of Se on bone.     

Effects of a single intraperitoneal administration of cadmium on femoral bone structure in male rats

Background

Exposure to cadmium (Cd) is considered a risk factor for various bone diseases in humans and experimental animals. This study investigated the acute effects of Cd on femoral bone structure of adult male rats after a single intraperitoneal administration.

Methods

Ten 4-month-old male Wistar rats were injected intraperitoneally with a single dose of 2 mg CdCl₂/kg body weight and killed 36 h after the Cd had been injected. Ten 4-month-old males served as a control group. Differences in body weight, femoral weight, femoral length and histological structure of the femur were evaluated between the two groups of rats. The unpaired Student''s t-test was used for establishment of statistical significance.

Results

A single intraperitoneal administration of Cd had no significant effect on the body weight, femoral weight or femoral length. On the other hand, histological changes were significant. Rats exposed to Cd had significantly higher values of area, perimeter, maximum and minimum diameters of the primary osteons'' vascular canals and Haversian canals. In contrast, a significant decrease in all variables of the secondary osteons was observed in these rats.

Conclusions

The results indicate that, as expected, a single intraperitoneal administration of 2 mg CdCl₂/kg body weight had no impact on macroscopic structure of rat''s femora; however, it affected the size of vascular canals of primary osteons, Haversian canals, and secondary osteons.     

Long-term peroral administration of bitter apricot seeds influences cortical bone microstructure of rabbits

Apricot seeds due to the presence of cyanogenic glycoside amygdalin belong to the popular “alternative cancer cures", although anticancer effect of amygdalin remains controversial. This in vivo study points to the effect of long-term peroral administration of bitter apricot seeds on bone microstructure of rabbits since chronic amygdalin toxicity in relation to bone parameters has not been investigated yet. Rabbits (n = 16) were randomly divided into four experimental groups of 4 animals each. Three experimental groups S1, S2 and S3 received commercial feed for rabbits mixed with crushed bitter apricot seeds at doses 60, 300 and 420 mg/kg bw during five months, respectively. The control (C) group received no apricot seeds. The long-term consumption of apricot seeds had no impact on total body weight, femoral weight and femoral length of rabbits. Also, microcomputed tomography (3D analysis) of cortical and trabecular bone tissues did not reveal any significant impact of amygdalin toxicity on relative bone volume, BMD, cortical bone thickness, bone surface, trabecular number, thickness, and their separation. On the other hand, histological (2D) analysis demonstrated evident changes in cortical bone microstructure consistent with a decreased density of secondary osteons in the middle part of substantia compacta due to a replacement of Haversian bone tissue by plexiform bone tissue, vasoconstriction in the primary osteons' vascular canals, Haversian canals, and decreased sizes of secondary osteons in rabbits from S1, S2 and S3 groups. These negative changes are associated with different vascularization and biomechanical properties of cortical bones.     

Comparative bone histology of adult horses (Equus caballus) and cows (Bos taurus)

Bone microstructure of domestic herbivores is still not completely understood. Indeed, works focused on the bone histology of numerous Mammalian species frequently led to misunderstandings because of the high number of variations such as the kind of bone, section orientation, species, breed and age. Moreover, attempts to identify the species in archaeozoological studies by a mere qualitative approach have not been encouraging and in recent years quantitative methods, based on image processing and statistical analysis, have appeared. The present study was undertaken to determine whether morphometrical and morphological differences exist in the compact bone structure of the femur and humerus between horses and cows. Measurements such as area, perimeter, minimum and maximum diameter of osteons and Haversian canals as well as the osteonal density were carried out on cross sections of eight humeri and eight femurs of the two herbivores investigated. In agreement with other authors, the qualitative investigation confirmed that the compact bone of horses and cows can be classified as dense Haversian tissue. Osteons of the horse were more numerous and composed of a higher number of well-defined lamellae when compared with the cow. Diameter, perimeter and area of osteons and Haversian canals were always higher in horses than in cows and this pattern could be related to the different locomotor behaviour of these animals.     

Changes in compact bone microstructure of rats subchronically exposed to cadmium

Background

Chronic exposure to cadmium (Cd), even at low concentrations, has an adverse impact on the skeletal system. Histologically, primary and secondary osteons as basic structural elements of compact bone can also be affected by several toxicants leading to changes in bone vascularization and mechanical properties of the bone. The current study was designed to investigate the effect of subchronic peroral exposure to Cd on femoral bone structure including histomorphometry of the osteons in adult male rats.In our study, 20 one-month-old male Wistar rats were randomly divided into two experimental groups. In the first group, young males received a drinking water containing 30 mg of CdCl₂/L, for 90 days. Ten one-month-old males without Cd intoxication served as a control group. After 90 days of daily peroral exposure, body weight, femoral weight, femoral length, cortical bone thickness and histological structure of the femora were analysed.

Results

We found that subchronic peroral application of Cd had no significant effect on body weight, femoral length and cortical bone thickness in adult rats. On the other hand, femoral weight was significantly increased (P < 0.05) in Cd-intoxicated rats. These rats also displayed different microstructure in the middle part of the compact bone where vascular canals expanded into central area of substantia compacta and supplied primary and secondary osteons. Additionally, a few resorption lacunae which are connected with an early stage of osteoporosis were identified in these individuals. Histomorphometrical evaluations showed that all variables (area, perimeter, maximum and minimum diameter) of the primary osteons’ vascular canals, Haversian canals and secondary osteons were significantly decreased (P < 0.05) in the Cd group rats. This fact points to alterations in bone vascularization.

Conclusions

Subchronic peroral exposure to Cd significantly influences femoral weight and histological structure of compact bone in adult male rats. It induces an early stage of osteoporosis and causes reduced bone vascularization. Histomorphometrical changes of primary and secondary osteons allow for the conclusion that the bone mechanical properties could be weakened in the Cd group rats. The current study significantly expands the knowledge on damaging action of Cd on the bone.     

Simultaneous subchronic exposure to selenium and diazinon as possible risk factor for osteoporosis in adult male rats

Background

Osteoporosis and its main health outcome, fragility fractures, are large and escalating health problems. Skeletal damage may be the critical result of low-level prolonged exposure to several xenobiotics in the general population, but the mechanisms of their adverse effects are not clearly understood. The current study was aimed to investigate the possible ability of simultaneous subchronic peroral administration of selenium (Se) and diazinon (DZN) to induce changes in bone of adult male rats.In our study, twenty 1-month-old male Wistar rats were randomly divided into two experimental groups. In the first group, young males were exposed to 5 mg Na₂SeO₃/L and 40 mg of DZN/L in drinking water, for 90 days. Ten 1-month-old males without Se and DZN intoxication served as a control group. At the end of the experiment, macroscopic and microscopic structures of the femurs were analysed using analytical scales, sliding instrument, and polarized light microscopy.

Results

The body weight, femoral length and cortical bone thickness were significantly decreased in rats simultaneously exposed to Se and DZN (P < 0.05). These rats also displayed different microstructure in the middle part of the compact bone where vascular canals expanded into central area of substantia compacta. The canals occurred only near endosteal surfaces in rats from the control group. Additionally, a smaller number of primary and secondary osteons, as well as a few resorption lacunae were observed near endosteal surfaces in rats simultaneously administered to Se and DZN. The resorption lacunae as typical structures of bone resorption manifestation are connected with an early stage of osteoporosis. Histomorphometric analysis revealed that area, perimeter, maximum and minimum diameters of primary osteons’ vascular canals were significantly increased (P < 0.05) in the Se-DZN-exposed rats. On the other hand, all measured variables of Haversian canals and secondary osteons were considerable reduced (P < 0.05) in these rats.

Conclusions

Simultaneous subchronic peroral exposure to Se and DZN induces changes in macroscopic and microscopic structures of the femurs in adult male rats, and also it can be considered as possible risk factor for osteoporosis. The current study contributes to the knowledge on damaging impact of several xenobiotics on the bone.     

Efficacy of nano-hydroxyapatite prepared by an aqueous solution combustion technique in healing bone defects of goat

The present study was undertaken to evaluate porous hydroxyapatite (HAp), the powder of which was prepared by a novel aqueous solution combustion technique, as a bone substitute in healing bone defects in vivo, as assessed by radiologic and histopathologic methods, oxytetracycline labeling, and angiogenic features in Bengal goat. Bone defects were created in the diaphysis of the radius and either not filled (group I) or filled with a HAp strut (group II). The radiologic study in group II showed the presence of unabsorbed implants which acted as a scaffold for new bone growth across the defect, and the quality of healing of the bone defect was almost indistinguishable from the control group, in which the defect was more or less similar, although the newly formed bony tissue was more organized when HAp was used. Histologic methods showed complete normal ossification with development of Haversian canals and well-defined osteoblasts at the periphery in group II, whereas the control group had moderate fibro-collagenization and an adequate amount of marrow material, fat cells, and blood vessels. An oxytetracycline labeling study showed moderate activity of new bone formation with crossing-over of new bone trabeculae along with the presence of resorption cavities in group II, whereas in the control group, the process of new bone formation was active from both ends and the defect site appeared as a homogenous non-fluoroscent area. Angiograms of the animals in the control group showed uniform angiogenesis in the defect site with establishment of trans-transplant angiogenesis, whereas in group II there was complete trans-transplant shunting of blood vessel communication. Porous HAp ceramic prepared by an aqueous combustion technique promoted bone formation over the defect, confirming their biologic osteoconductive property.     

Musculoskeletal responses of 2-year-old Thoroughbred horses to early training. 6. Bone parameters in the third metacarpal and third metatarsal bones

AIM: To determine the effect of a known training regimen on the size and mineral content of the third metacarpal (Mc3) and third metatarsal (Mt3) bones of 2-year-old Thoroughbred horses trained on racetracks. METHODS: Mc3 and Mt3 of seven horses trained on grass and sand tracks were scanned at several sites using conventional quantitative and peripheral computed tomography (CT). Bone dimensions and density in the diaphysis and epiphysis were compared with those from seven untrained horses. Calcein label was injected in two clusters, during Weeks 9 and 12. The extent and rate of diaphyseal modelling was determined by confocal fluorescent microscopic examination of thin plane parallel sections of the mid-metacarpal region. RESULTS: Volumetric bone mineral density (BMDv) of the epiphysis was markedly higher and of the diaphysis was slightly higher in trained compared with untrained horses, but greater bone size in the trained horses had the greatest effect on an index of bone strength. Active osteons, defined as Haversian systems containing calcein label, were fewer, of smaller diameter at the time of calcein injection, and had a greater bone apposition rate in trained than in untrained horses. CONCLUSIONS: Conventional training of 2-year-old Thoroughbred racehorses over a 13-week period had a significant effect on bone size, density and strength index when compared to untrained horses. CLINICAL RELEVANCE: Bone responded rapidly to early training. The data provide reference values and sites for use in longitudinal studies of commercial training regimens.     

Musculoskeletal responses of 2-year-old Thoroughbred horses to early training. 6. Bone parameters in the third metacarpal and third metatarsal bones

AIM: To determine the effect of a known training regimen on the size and mineral content of the third metacarpal (Mc3) and third metatarsal (Mt3) bones of 2-year-old Thoroughbred horses trained on racetracks.

METHODS: Mc3 and Mt3 of seven horses trained on grass and sand tracks were scanned at several sites using conventional quantitative and peripheral computed tomography (CT). Bone dimensions and density in the diaphysis and epiphysis were compared with those from seven untrained horses. Calcein label was injected in two clusters, during Weeks 9 and 12. The extent and rate of diaphyseal modelling was determined by confocal fl uorescent microscopic examination of thin plane parallel sections of the mid-metacarpal region.

RESULTS: Volumetric bone mineral density (BMDv) of the epiphysis was markedly higher and of the diaphysis was slightly higher in trained compared with untrained horses, but greater bone size in the trained horses had the greatest effect on an index of bone strength. Active osteons, defined as Haversian systems containing calcein label, were fewer, of smaller diameter at the time of calcein injection, and had a greater bone apposition rate in trained than in untrained horses.

CONCLUSIONS: Conventional training of 2-year-old Thoroughbred racehorses over a 13-week period had a significant effect on bone size, density and strength index when compared to untrained horses.

CLINICAL RELEVANCE: Bone responded rapidly to early training. The data provide reference values and sites for use in longitudinal studies of commercial training regimens.     

Correlation of racing performance with radiographic changes in the proximal sesamoid bones of 487 Thoroughbred yearlings

REASONS FOR PERFORMING STUDY: Proximal sesamoiditis has been a common and confusing radiographic diagnosis in Thoroughbred yearling surveys. This study categorised the radiographic appearance of yearling proximal sesamoid bones and determined their effect on the number of races started and earning potential for racing at ages 2 and 3 years. HYPOTHESIS: Yearlings with moderate to severe proximal sesamoiditis have decreased performance at age 2 and 3 years. METHODS: Survey radiographs of 487 Thoroughbred yearlings were reviewed. Proximal sesamoid bones were examined and findings placed into 7 categories, 5 of which measured size, shape, and number of vascular canals, and 2 examined the radiographic shape of the sesamoid abaxial surface. Records of horses age 2 and 3 years were reviewed to obtain the number of races completed and money won in those races. RESULTS: Vascular canals with parallel sides < 2 mm in width in any number were normal. Yearlings with 1 or 2 abnormally conformed vascular canals (nonparallel sides and > 2 mm in width) had a decrease in number of race starts at age 2 years when compared to normal horses. This same change in the fore- or hindlimbs was associated with decreases in starts and earnings at age 2 years. If uniformly present in both fore- and hindlimbs there was no difference from normal horses. Yearlings with > 2 irregular vascular canals had a decrease in number of race starts and earnings at age 2 and 3 years. Changes in proximal sesamoid bone contour did not affect performance. CONCLUSIONS: Horses with enlarged vascular canals, within their sesamoids as yearlings, start fewer races and earn less prize money than horses with normal vascular canals. POTENTIAL RELEVANCE: One or two enlarged canals affected horses as 2-year-olds. Three or more enlarged vascular canals affected horses at ages 2 and 3 years, which should be taken into account when examining radiographs for potential purchase of young horses.     

Vascular pattern of the developing knee joint in the domestic fowl

Laying strain fowls were reared from hatching till 20 weeks old. Birds were killed throughout the growth period and specimens from the distal femur and proximal tibiotarsus prepared for study. The origin and nature of cartilage canals were established in the two bone extremities. The canals follow a pattern of development which was similar in fowls of the same age. Similarities were noted with the vascular patterns reported in other species. The epiphyseal ossification centre of the proximal tibiotarsus formed in an area extensively perfused by cartilage canals. Anastomoses occurred between epiphyseal vascular canals in association with the formation and growth of the epiphyseal ossification centre in the proximal tibiotarsus. Anastomoses between epiphyseal vascular canals did not occur at any other site. The development of the epiphyseal ossification centre in the proximal tibiotarsus suggests that this is a traction epiphysis.     

Pattern of vascular canals in the bone extremities of the pelvic appendicular skeleton in broiler type fowl

Broiler fowls were reared from hatching till 20 weeks old. Birds were killed throughout the growth period and the bone extremities of the pelvic appendicular skeleton prepared for study. The patterns of vascular canals in these broiler fowls were very similar to earlier reports in laying strain fowls. There was a great range of and variation in the vascular morphology of broiler fowl. The physeal vasculature of the rapidly growing broilers was frequently disrupted, and such areas were often associated with dyschondroplastic lesions. The rapidly growing bone extremities in broiler fowls were susceptible to occlusion of epiphyseal vascular canals. lesions were frequently seen at similar sites in different specimens. Links are suggested between the growth rate, weight and behavioural characteristics of broiler fowls and the development of lesions.     

In vitro comparison of metaphyseal and diaphyseal placement of centrally threaded, positive-profile transfixation pins in the equine third metacarpal bone

OBJECTIVE: To evaluate in vitro holding power and associated microstructural and thermal damage from placement of positive-profile transfixation pins in the diaphysis and metaphysis of the equine third metacarpal bone. SAMPLE POPULATION: Third metacarpal bones from 30 pairs of adult equine cadavers. PROCEDURE: Centrally threaded positive-profile transfixation pins were placed in the diaphysis of 1 metacarpal bone and the metaphysis of the opposite metacarpal bone of 15 pairs of bones. Tensile force at failure for axial extraction was measured with a materials testing system. An additional 15 pairs of metacarpal bones were tested similarly following cyclic loading. Microstructural damage was evaluated via scanning electron microscopy in another 6 pairs of metacarpal bones, 2 pairs in each of the following 3 groups: metacarpal bones with tapped holes and without transfixation pin placement, metacarpal bones following transfixation pin placement, and metacarpal bones following transfixation pin placement and cyclic loading. Temperature of the hardware was measured with a surface thermocouple in 12 additional metacarpal bones warmed to 38 C. RESULTS: The diaphysis provided significantly greater resistance to axial extraction than the metaphysis. There were no significant temperature differences between diaphyseal and metaphyseal placement. Microstructural damage was limited to occasional microfractures seen only in cortical bone of diaphyseal and metaphyseal locations. Microfractures originated during drilling and tapping but did not worsen following transfixation pin placement or cyclic loading. CONCLUSIONS AND CLINICAL RELEVANCE: Centrally threaded, positive-profile transfixation pins have greater resistance to axial extraction in the diaphysis than in the metaphysis of equine third metacarpal bone in vitro. This information may be used to create more stable external skeletal fixation in horses with fractures.     

Morphometric Analysis of the Canal System of Cortical Bone: An Experimental Study in the Rabbit Femur Carried Out with Standard Histology and Micro-CT

The osteonal pattern of cortical bone is gradually built around the intracortical vessels by the progression of the cutting cones (secondary remodelling); therefore, the central canal size can be used as index of the remodelling activity. An experimental model in the rabbit femur was used to investigate, through central canal morphometry and frequency distribution analysis, the remodelling activity, comparing the middle of the diaphysis (mid-shaft) with the extremity (distal-shaft) and at the same level sectors and layers of the cortex in transversal sections. The study documented a higher density of canals in the mid-shaft than in the distal-shaft and a higher remodelling in the distal-shaft. There were no significant differences between dorsal, ventral, medial and lateral sectors at both mid-shaft and distal-shaft levels, while the number of canals was higher in the sub-periosteal layers than in the sub-endosteal. A lower threshold of 40 μm² was observed in the central canal area. Sealed osteons in the midshaft were 22.43% of the total number of osteons of the central canal area between 40 and 200 μm² and 0.44% of those of the distal-shaft. Micro-CT allowed a 3D reconstruction of the vascular canal system, which confirmed the branched network pattern rather than the trim architecture of the traditional representation. Some aspects like the lower threshold of the central canal size and the sealed osteons documented the plasticity of the system and its capacity for adaptation to changes in the haemodynamic conditions.     

Congenital chondrodysplastic dwarfism with dyshematopoiesis in a holstein calf

A holstein calf with congenital chondrodysplastic dwarfism was histopathologically examined. The head of the calf was relatively flat giving a dog-like appearance with its short nose and sloping forehead. Limb bones were dumbbell-like with short diaphysis and hypertrophied metaphyses. Bone marrow was pale, whitish and fatty. In the metaphyseal plates most of chondrocytes were pyknotic with swollen and ghost-like cytoplasm, and were irregularly arranged. Column of calcified cartilage were poorly formed losing comb-like structure. Bone marrow was ischemic with poor hematopoiesis and was moderately replaced by adipose tissue. In articular cartilage, most of chondrocytes were degenerated with ghost-like cytoplasm. Many cartilage canals and occasional bone marrow-like structure were formed. The characteristics lesions of the calf were chondrodysplasia and dyshematopoiesis.     

Development of Galanin-Containing Nerve Fibres in Rat Tibia

Galanin exerts tonic inhibition of nociceptive input to the central nervous system. Recently, this peptide was demonstrated in several neuronal and non-neuronal structures in bones and joints. In this study, the time of appearance and topographic localization of galanin-containing nerve fibres in bone were studied in rats from gestational day 16 (GD16) to postnatal day 21 (PD21). The tibia was chosen as a model of developing long bone and indirect immunofluorescence combined with confocal laser scanning microscopy was used to identify galanin-immunoreactive (GAL-IR) nerve fibres. The earliest, sparse GAL-IR fibres were observed on GD21 in the perichondrium of both epiphyses and in the periosteum of the diaphysis. From PD1 onwards, GAL-IR fibres were also seen in the bone marrow cavity and in the region of the inter-condylar eminence of the knee joint. Intramedullary GAL-IR fibres in proximal and distal metaphyses appeared around PD1. Some of them accompanied blood vessels, although free fibres were also seen. GAL-IR fibres located in the cartilage canals of both epiphyses were observed from PD7, in the secondary ossification centres from PD10 and in the bone marrow of both epiphyses from PD14. The time course and localization of galanin-containing nerve fibres resemble the development of substance P- and CGRP-expressing nerve fibres, thus suggesting their sensory origin.