Assessment of Multipotent Mesenchymal Stromal Cells in Bone Marrow Aspirate From Human Calcaneus.

Bone marrow aspirates (BMAs), owing to their innate osteogenic potential, are well-documented supplements to osteoconductive and/or osteoinductive materials. The calcaneal body provides foot and ankle surgeons a convenient harvest site with low morbidity and minimal cost. In the present study, we sought to identify and characterize multipotent mesenchymal stromal cells (MSCs) in BMAs harvested from the human calcaneal body. Ten healthy patients aged 18 to 65 years were enrolled in the present study. BMAs were harvested from the patients without any reported postoperative complications related to the harvest. Cells isolated from all the aspirates were adherent to culture plates and expressed positive MSC surface markers (CD105, CD90, and CD73) and a low level of negative MSC markers (CD34 and CD45). The cells maintained the ability to proliferate and differentiate into cells of mesenchymal lineages. The BMAs from the human calcaneal body offer a healthy source of multipotent MSCs.

Assessment of Bone Marrow Aspiration Site Pain in Foot and Ankle Surgery.

UNLABELLED: Bone marrow aspiration (BMA) is a validated technique to harvest progenitor cells. BMA has many uses in foot and ankle surgery; however, donor site morbidity is a concern. The purpose of this study was to compare the Visual Analog Scale (VAS) pain scores after BMA at 3 different sites (iliac crest, distal tibia, and calcaneus) over a 12-week postoperative recovery period. This was an institutional review board-approved prospective study of 40 patients who underwent BMA as an adjunct to their primary foot and ankle procedure. Each patient had BMA harvested from the ipsilateral anterior iliac crest, distal tibia, and lateral calcaneus at the time of surgery. Patient follow-up questionnaire forms were filled out at 2, 4, 8, and 12 weeks, with the primary outcome measure being VAS pain scores. Mean VAS scores averaged over the 12-week follow-up period were significantly higher in the calcaneus (20.8 ± 28.6) compared with the distal tibia (7.7 ± 17.6) and the iliac crest (4.2 ± 12.4; P < .05). No significant difference was found between the distal tibia and the iliac crest sites. At 12 weeks, all sites were about equal and without appreciable pain. Our data suggested that donor site selection for BMA affects postoperative pain levels, with BMA from the calcaneus resulting in significantly higher pain scores when compared with the iliac crest or distal tibia. The VAS pain score for the calcaneus was likely confounded by the high number of hindfoot/ankle surgeries performed in the ipsilateral foot. LEVELS OF EVIDENCE: Therapeutic, Level II: Prospective, comparative trial.

Quantitative assessment of the yield of osteoblastic connective tissue progenitors in bone marrow aspirate from the iliac crest, tibia, and calcaneus.

BACKGROUND: It is well known that bone marrow aspirate from the iliac crest contains osteoblastic connective tissue progenitor cells. Alternative harvest sites in foot and ankle surgery include the distal aspect of the tibia and the calcaneus. To our knowledge, no previous studies have characterized the quality of bone marrow aspirate obtained from these alternative sites and compared the results with those of aspirate from the iliac crest. The goal of this study was to determine which anatomic location yields the highest number of osteoblastic progenitor cells. METHODS: Forty patients were prospectively enrolled in the study, and separate bone marrow aspirate samples were harvested from the ipsilateral anterior iliac crest, distal tibial metaphysis, and calcaneal body. The aspirate was centrifuged to obtain a concentrate of nucleated cells, which were plated and grown in cell culture. Colonies that stained positive for alkaline phosphatase were counted to estimate the number of osteoblastic progenitor cells in the initial sample. The anatomic locations were compared. Clinical parameters (including sex, age, tobacco use, body mass index, and diabetes) were assessed as possible predictors of osteoblastic progenitor cell yield. RESULTS: Osteoblastic progenitor cells were found at each anatomic location. Bone marrow aspirate collected from the iliac crest had a higher mean concentration of osteoblastic progenitor cells compared with the distal aspect of the tibia or the calcaneus (p < 0.0001). There was no significant difference in concentration between the tibia and the calcaneus (p = 0.063). Age, sex, tobacco use, and diabetes were not predictive of osteoblastic progenitor cell yield. CONCLUSIONS: Osteoblastic progenitor cells are available in the iliac crest, proximal aspect of the tibia, and calcaneus. However, the iliac crest provided the highest yield of osteoblastic progenitor cells. CLINICAL RELEVANCE: The study demonstrated that osteogenic progenitor cells are available in bone marrow aspirate harvested from the tibia or calcaneus as well as the iliac crest. All three sites are easily accessed, with a low risk of adverse events. However, larger volumes of aspirate may be needed from the tibia or calcaneus to approach the yield of cells from the iliac crest.

A numerical model for investigating the mechanics of calcaneal fat pad region.

The present paper pertains to the definition of a numerical model of the calcaneal fat pad region, considering a structure composed of adipose and connective tissues organized in fibrous septae and adipose chambers. The mechanical response is strongly influenced by the structural conformation, as the dimension of adipose chambers, the thickness of connective septae walls and the mechanical properties of the different soft tissues. In order to define the constitutive formulation of adipose tissues, experimental data from pig specimens are considered, according to the functional similarity, while the mechanical response of connective tissue septae is assumed with regard to the mechanical behaviour that characterize ligaments. Different numerical models are provided accounting for the variation of chambers dimensions, septae wall thickness and tissues characteristics. The spiral angles of collagen fibres within the septae influence the capability of the structure to withstand the bulging of chambers. The analysis considers different orientation of the fibres. The response of calcaneal fat pad region is evaluated in comparison with experimental data from unconfined compression tests. The present work provides a preliminary approach to enhance the correlation between the structural conformation and tissues mechanical properties towards the biomechanical response of overall heel pad region.

A weighted osteon morphotype score outperforms regional osteon percent prevalence calculations for interpreting cortical bone adaptation.

Using circularly polarized light microscopy,we described a weighted-scoring method for quantifying regional distributions of six secondary osteon morphotypes(Skedros et al.: Bone 44 (2009) 392-403). This osteon morphotype score (MTS) strongly correlated with "tension" and "compression" cortices produced by habitual bending. In the present study, we hypothesized that the osteon MTS is superior to a relatively simpler method based on the percent prevalence (PP) of these osteon morphotypes. This was tested in proximal femoral diaphyses of adult chimpanzees and habitually bent bones: calcanei from sheep, deer, and horses, radii from sheep and horses, and third metacarpals (MC3s) from horses. Sheep tibiae were examined because their comparatively greater torsion/shear would not require regional variations in osteon morphotypes. Predominant collagen fiber orientation (CFO), a predictor of regionally prevalent/predominant strain mode, was quantified as image gray levels (birefringence). Ten PP calculations were conducted. Although PP calculations were similar to the osteon MTS in corroborating CFO differences between "tension" and "compression" cortices of the chimpanzee femora and most of the habitually bent bones, PP calculations failed to show a compression/tension difference in equine MC3s and sheep radii. With the exception of the prevalence of the "distributed" osteon morphotype, correlations of PP calculations with CFO were weak and/or negative. By contrast, the osteon MTS consistently showed positive correlations with predominant CFO. Compared with the osteon MTS and predominant CFO, regional variations in PP of osteon morpho types are not stronger predictors of nonuniform strain distributions produced by bending.

Effects of ovariectomy and resistance training on MMP-2 activity in rat calcaneal tendon.

Tendon remodeling relies on extracellular matrix (ECM) restructuring by the matrix metallopeptidases (MMPs). The aim of this study was to investigate MMP-2 activity in different regions of the calcaneal tendon (CT) after resistance training (RT) in ovariectomized rats. Wistar adult female rats were grouped into sedentary (Sed-Intact), ovariectomized sedentary (Sed-Ovx), acute exercise (AcuteEx-Intact), ovariectomized acute exercise (AcuteEx-Ovx), resistance trained (ChronicEx-Intact), and ovariectomized resistance trained (ChronicEx-Ovx) (n = 10 each group). The RT protocol required the animals to climb a 1.1-m vertical ladder with weights attached to their tail. The sessions were performed once every 3 days with 4-9 climbs and 8-12 dynamic movements per scaling. The acute groups performed one session and the chronic groups underwent 12 weeks of RT. There was an increase in total MMP-2 activity in Sed-Ovx, AcuteEx-Intact, and ChronicEx-Intact compared with that in Sed-Intact in the proximal region of CT. AcuteEx-Ovx exhibited higher total MMP-2 than Sed-Ovx and AcuteEx-Intact in the distal region of CT. Chronic-Ovx presented lower total MMP-2 activity than Sed-Ovx and Chronic-Intact in the distal region of tendon. The active MMP-2 was higher for the AcuteEx-Ovx than Sed-Ovx and AcuteEx-Intact in proximal region of tendon. There was higher active MMP-2 in the distal region of tendon in the Acute-Ovx than in the Sed-Ovx and AcuteEx-Intact. Ovariectomy and resistance exercise modulate MMP-2 activity according to specific tendon region, indicating a differentiated tissue remodeling.

Interpreting cortical bone adaptation and load history by quantifying osteon morphotypes in circularly polarized light images.

Birefringence variations in circularly polarized light (CPL) images of thin plane-parallel sections of cortical bone can be used to quantify regional differences in predominant collagen fiber orientation (CFO). Using CPL images of equine third metacarpals (MC3s), R.B. Martin, V.A. Gibson, S.M. Stover, J.C. Gibeling, and L.V. Griffin. (40) described six secondary osteon variants ('morphotypes') and suggested that differences in their regional prevalence affect fatigue resistance and toughness. They devised a numerical osteon morphotype score (MTS) for quantifying regional differences in osteon morphotypes. We have observed that a modification of this score could significantly improve its use for interpreting load history. We hypothesized that our modified osteon MTS would more accurately reveal differences in osteon MTSs between opposing "tension" and "compression" cortices of diaphyses of habitually bent bones. This was tested using CPL images in transverse sections of calcanei from sheep, deer, and horses, and radii from sheep and horses. Equine MC3s and sheep tibiae were examined as controls because they experience comparatively greater load complexity that, because of increased prevalence of torsion/shear, would not require regional mechanical enhancements provided by different osteon morphotypes. Predominant CFO, which can reliably reflect adaptation for a regionally prevalent strain mode, was quantified as mean gray levels from birefringence of entire images (excluding pore spaces) in anterior, posterior, medial, and lateral cortices. Results showed that, in contrast to the original scoring scheme of Martin et al., the modified scheme revealed significant anterior/posterior differences in osteon MTSs in nearly all "tension/compression" bones (p<0.0001), but not in equine MC3s (p=0.30) and sheep tibiae (p=0.35). Among habitually bent bones, sheep radii were the exception; relatively lower osteon populations and the birefringence of the primary bone contributed to this result. Correlations between osteon MTSs using the scoring scheme of Martin et al. with CFO data from all regions of each bone invariably demonstrated weak-to-moderate negative correlations. This contrasts with typically high positive correlations between modified osteon MTSs and regional CFO. These results show that the modified osteon MTS can be a strong correlate of predominant CFO and of the non-uniform strain distribution produced by habitual bending.

Clinical anatomy of the calcaneal tuberosity.

The aim of the study was a qualitative anatomical analysis of the macroscopic features of the surface of the calcaneal tuberosity, of the architecture of its cancellous bone and histological structure of the whole region. Dry human bones and pathological dissection material 24-36 h post mortem were used in the study. On the tuberosity, the variability of its surface relief and the two borders between the superior, middle and inferior facets were studied. More frequent medial declination of the inferior line, corresponding to the distal circumference of the Achilles tendon attachment, was found. Two systems of expressive condensation of cancellous bone just below the surface of the calcaneal tuberosity were described. In the histological part of the study, the distribution and different thickness of the fibrous cartilage layer covering the attachment region of Achilles tendon, the bottom of retro-calcaneal bursa and the whole surface of the calcaneal tuberosity were described. The functional and clinical relevance of results obtained are evaluated from the point of view of disciplines dealing with the pathology and surgery of the heel region. The relationships of official anatomical terms and a wide spectrum of clinical synonyms designating this region are discussed.

Euler(strut.cavity), a new histomorphometric parameter of connectivity reflects bone strength and speed of sound in trabecular bone from human os calcis.

The amount of bone and the trabecular microarchitecture are two determinants of bone strength which can be quantified by bone histomorphometry. Among the parameters of bone microarchitecture, the Euler number developed in our laboratory (E( strut.cavity )) and trabecular bone pattern factor (TBPf) evaluate the connectivity and complexity independently of the bone quantity, and the speed of sound (SOS) measured by quantitative ultrasound (QUS) corroborates E( strut.cavity ). The aim of the present study was to validate E( strut.cavity ), TBPf, and SOS as parameters of bone microarchitecture and their contribution to bone strength. We examined 20 right os calcis taken after necropsy in 11 males and 9 females, aged 52-95 years. At the same anatomic location, we measured SOS and broadband ultrasound attenuation (BUA) using a Hologic Sahara device and bone mineral density (BMD) using a Hologic QDR 1000W. At this site a transcortical cylinder was cut for both apparent density measurement (Ap.Dens) and biomechanical tests (maximum compressive stress (sigma(max)) and Young's modulus (E)), and histomorphometry was performed with an automatic image analyzer (Visiolab, Explora Nova, France). E and sigma(max) were significantly correlated with the parameters of bone quantity, microarchitecture, and QUS. However, after adjustment for the bone quantity, E correlated only with E( strut.cavity ), TBPf, and SOS, and sigma(max) with BUA. In conclusion, the bone connectivity and complexity evaluated by E( strut.cavity ) and TBPf contribute to bone strength, independently of the bone quantity. The bone mechanical properties may be assessed, in os calcis, in the elastic domain by SOS and in the plastic domain by BUA.

The effect of age on the structure and composition of rat tendon fibrocartilage.

Biochemical and morphological aspects of fibrocartilages of calcaneal and deep digital flexor tendons in rats aged 30, 180 and 730 days were analyzed. In both tendons a stronger staining with Alcian blue, indicating the presence of proteoglycans, was detected in rats of 30 and 180 days. In animals 730 days old, it was restricted to the pericellular area. Ultrastructural analysis showed a more prominent pericellular matrix in calcaneal tendon compared to the deep digital flexor tendon. The biochemical analysis showed higher levels of proteins and glycosaminoglycans in the calcaneal tendon of 30-day-old rats compared to older rats. In the deep digital flexor tendon, no significant differences were observed between ages. The small proteoglycan, fibromodulin, was detected in both tendons of all ages, but in young rats it appeared to be running as a 210 kDa component, probably due to the association with collagen chains or self-association.

Autocalibrating parallel imaging of in vivo trabecular bone microarchitecture at 3 Tesla.

In this work the generalized autocalibrating partially parallel acquisition (GRAPPA) technique was implemented with modified reconstruction and applied to in vivo high-resolution (HR) magnetic resonance imaging (MRI) of the trabecular bone microarchitecture at 3 Tesla (T) with a multiple-acquisition balanced steady-state free precession (b-SSFP) sequence. Trabecular bone is made up of a network of microstructures (80-140 microm), and its structural deterioration is associated with the skeletal metabolic disorder osteoporosis. HR-MRI is a promising noninvasive tool for assessing the trabecular microarchitecture in vivo, but it involves long acquisition times. Using partially parallel imaging (PPI) to accelerate the acquisition may help mitigate this shortcoming and allow more flexibility in protocol design. In this study the effects of GRAPPA-based reconstruction on image characteristics and the measurement of trabecular bone structural parameters were evaluated. Initial studies showed that image quality and depiction of microstructure were preserved in the GRAPPA-based reconstruction, indicating the feasibility of PPI in HR-MRI of trabecular bone. The results also demonstrated the potential of PPI for increasing the signal-to-noise ratio (SNR) efficiency of multiple-acquisition b-SSFP imaging protocols.

Comparison of measurements of phase velocity in human calcaneus to Biot theory.

Biot's theory for elastic propagation in porous media has previously been shown to be useful for modeling the dependence of phase velocity on porosity in bovine cancellous bone in vitro. In the present study, Biot's theory is applied to measurements of porosity-dependent phase velocity in 53 human calcanea in vitro. Porosity was measured using microcomputed tomography for some samples (n = 23) and estimated based on bone mineral densitometry for the remaining samples (n = 30). The phase velocity at 500 kHz was measured in a water tank using a through-transmission technique. Biot's theory performed well for the prediction of the dependence of sound speed on porosity. The trend was quasilinear, but both the theory and experiment show similar slight curvature. The root mean square error (RMSE) of predicted versus measured sound speed was 15.8 m/s.

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.

Accuracy of 3D MR microscopy for trabecular bone assessment: a comparative study on calcaneus samples using 3D synchrotron radiation microtomography.

Magnetic resonance (MR) imaging is attractive for a noninvasive and radiation-free assessment of in vivo trabecular bone architecture. However the quantitative evaluation of architectural parameters could be biased by the limited sensitivity of MR. The aim of this study was to determine the accuracy of trabecular bone architectural parameters obtained from 3D high-resolution MR images, by comparison to reference images obtained by high-resolution X-ray microtomography using synchrotron radiation, from 29 samples of human calcaneus. MR images were obtained with a 66 microm x 66 microm x 66 microm voxel size, using a 8.5 T MR microscope. Microtomography images were acquired with a 10 microm x 10 microm x 10 mum voxel size, from the same samples. 3D architectural parameters characterizing the morphometry, topology, anisotropy, and orientation were computed from both modalities and carefully compared. To avoid errors, an identical region of interest was selected in the two corresponding images, and the same algorithms were run at identical spatial resolution. Our results establish that network connectivity, orientation and anisotropy are reliable from the MR data. The bone volume fraction, and morphometric parameters measured from the MR data, were found to be biased with respect to their values from the microtomography data, although there was a significant correlation between the two modalities.

The dependence of ultrasonic backscatter on trabecular thickness in human calcaneus: theoretical and experimental results.

Trabecular thickness within cancellous bone is an important determinant of osteoporotic fracture risk. Noninvasive assessment of trabecular thickness potentially could yield useful diagnostic information. Faran's theory of elastic scattering from a cylindrical object immersed in a fluid has been used to predict the dependence of ultrasonic backscatter on trabecular thickness. The theory predicts that, in the range of morphological and material properties expected for trabecular bone, the backscatter coefficient at 500 kHz should be approximately proportional to trabecular thickness to the power of 2.9. Experimental measurements of backscatter coefficient were performed on 43 human calcaneus samples in vitro. Mean trabecular thicknesses on the 43 samples were assessed using micro computed tomography (CT). A power law fit to the data showed that the backscatter coefficient empirically varied as trabecular thickness to the 2.8 power. The 95% confidence interval for this exponent was 1.7 to 3.9. The square of the correlation coefficient for the linear regression to the log transformed data was 0.40. This suggests that 40% of variations in backscatter may be attributed to variations in trabecular thickness. These results reinforce previous studies that offered validation for the Faran cylinder model for prediction of scattering properties of cancellous bone, and provide added evidence for the potential diagnostic utility of the backscatter measurement.

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.

Microgravity, calcium and bone metabolism: a new perspective.

NASA: Microgravity-related bone demineralization is investigated. Spaceflight and bedrest studies are discussed which explore changes in bone mineral content, especially calcium. The mechanism of demineralization is examined and the possibility of bone mineral metabolism adapting to the weightlessness environment is considered.^ieng

The participation of cartilage canals in the ossification of the human fetal calcaneum.

The relationship of cartilage canals to the developing ossification centres in the human calcaneum was investigated. The cartilage canals were always present in the calcaneum by 78 mm CR length. The calcaneum has two primary ossification centres. The main centre was identified in the deep part of the calcaneum as a spherical zone of cartilage cells in the proliferative phase between 82 and 120 mm CR length and in the hypertrophic phase between 130-156 mm. Numerous cartilage canals entered the calcaneum from its dorsal and ventral surfaces and, between 106 and 156 mm CR length, they formed vascular arcades around this centre. Between 165 and 175 mm, calcification and marrow space formation were noted involving branches from the adjoining cartilage canals within these areas, which provided vascular osteogenic tissue to the early spaces. The inconstant parachondral centre, when present, may appear first during the fourth month as a periosteal reaction on the inferolateral side in the anterior wall of a deep groove in front of the posterior tuberosity of the calcaneum. By 120 mm CR length, a thin layer of subperiosteal bone was present along with a zone of early hypertrophic cartilage cells deep to it. Calcification and marrow space formation occurred by 165 mm and these marrow spaces were supplied by periosteal buds. Osteoid tissue was formed in them between 186 and 206 mm CR length. The main centre was oval in shape due to its posterolateral extension irrespective of the presence or absence of the parachondral centre.(ABSTRACT TRUNCATED AT 250 WORDS)

The structure of the trabeculae of cancellous bone. 1. The calcaneus.

87 either cylindroid or laminar trabeculae, isolated from the perpendicular or inferior bundles of the human calcaneus, were embedded in methylmatecrylate and serially cut along longitudinal and transversal planes with a rotatory-blade saw. The microscopical study of the sections showed that in 83% of the samples secondary osteons run along the longitudinal axis of the trabeculae and their lumina either form a continuous channel network throughout each trabecula (37% of cases) or are restricted to discrete segments (46% of cases). The trabeculae entirely devoid of osteons (17%) are the thinnest, never exceeding 400 microns in thickness. This value is not even exceeded by the segments devoid of osteons in the trabeculae in which the Haversian canals occur only intermittently; conversely, the segments containing Haversian canals can reach and exceed 600 microns in thickness. The maximum distance of the osteocytic lacunae from filtering surfaces--i.e. outer surface of the trabeculae or inner surface of the Haversian canals--was found to be almost the same in the segments of the trabeculae that enclose or not osteons, even though the average trabecular thickness is greater in the former than in the latter regions. On the basis of these findings the formation of endotrabecular osteons may be viewed as a device that indirectly favours the metabolic exchange of deep-seated osteocytes while increasing the free surface area available for bone tissue reconstruction. It remains doubtful whether the Haversian systems may also contribute to improve the mechanical properties of the trabeculae. The arrangement of the collagen fibrils, which differs between cylindroid and laminar trabeculae, is apparently well suited to ensure the resistance of the trabeculae to mechanical loading through the use of the least amount of building material, in accordance with Wolff's law.