Human Iliac Crest Biopsies Research Articles

Large osteocyte lacunae in iliac crest infantile bone are not associated with impaired mineral distribution or signs of osteocytic osteolysis.

The enlargement of osteocyte lacunae via osteocytic osteolysis was previously detected in situations of increased calcium demand (e.g., lactation, vitamin D deficiency). However, it is unclear whether similar processes occur also in the growing infantile skeleton and how this is linked to the mineral distribution within the bone matrix. Human iliac crest biopsies of 30 subjects (0-6months, n=14; 2-8years, n=6 and 18-25years, n=10) were acquired. Bone microarchitecture was assessed by micro-CT, while cellular bone histomorphometry was performed on undecalcified histological sections. Quantitative backscattered electron imaging (qBEI) was conducted to determine the bone mineral density distribution (BMDD) as well as osteocyte lacunar size and density. We additionally evaluated cathepsin K positive osteocytes using immunohistochemistry. Infantile bone was characterized by various signs of ongoing bone development such as higher bone (re)modeling, lower cortical and trabecular thickness compared to young adults. Importantly, a significantly higher osteocyte lacunar density and increased lacunar area were detected. Large osteocyte lacunae were associated with a more heterogeneous bone mineral density distribution of the trabecular bone matrix due to the presence of hypermineralized cartilage remnants, whereas the mean mineralization (i.e., CaMean) was not different in infantile bone. Absence of cathepsin K expression in osteocyte lacunae indicated nonexistent osteocytic osteolysis. Taken together, we demonstrated that the overall mineralization distribution in infantile bone is not altered compared to young adults besides high trabecular mineralization heterogeneity. Our study also provides important reference values for bone microstructure, BMDD and osteocyte characteristics in infants, children and young adults. Infantile bone displays large osteocyte lacunae indicating a developmental phenomenon rather than osteocytic osteolysis. Larger osteocytes may have superior mechanosensory abilities to enable bone adaption during growth.

Ten year alendronate use does not adversely affect bone quality compared to 5 years use: a human iliac crest biopsy study

Ten year alendronate use does not adversely affect bone quality compared to 5 years use: a human iliac crest biopsy study

Correlation between Absence of Bone Remodeling Compartment Canopies, Reversal Phase Arrest, and Deficient Bone Formation in Post-Menopausal Osteoporosis

Bone remodeling compartments (BRCs) were recently recognized to be present in patients with primary hyperparathyroidism and critical for bone reconstruction in multiple myeloma and endogenous Cushing's syndrome. The BRCs are outlined by a cellular canopy separating the bone remodeling events on the bone surface from the marrow cavity. The present study on human iliac crest biopsy specimens reveals that BRC canopies appear frequently absent above both eroded and formative surfaces in post-menopausal osteoporosis patients, and that this absence was associated with bone loss in these patients. The absence of BRC canopies above the eroded surfaces was furthermore associated with the accumulation of arrested reversal surfaces and a reduced extent of formative surfaces, which both reflect an increased incidence of aborted remodeling cycles. Moreover, the absence of BRC canopies above formative surfaces was associated with a shift in the osteoblast morphological characteristics, from cuboidal to flattened. Collectively, this study shows that the BRCs are unique anatomical structures implicated in bone remodeling in a widespread disease, such as post-menopausal osteoporosis. Furthermore, it particularly highlights the role of the BRC canopies to make the reversal phase progressing toward initiation of matrix deposition, thereby preventing bone loss.

Osteoblast Recruitment Routes in Human Cancellous Bone Remodeling

It is commonly proposed that bone forming osteoblasts recruited during bone remodeling originate from bone marrow perivascular cells, bone remodeling compartment canopy cells, or bone lining cells. However, an assessment of osteoblast recruitment during adult human cancellous bone remodeling is lacking. We addressed this question by quantifying cell densities, cell proliferation, osteoblast differentiation markers, and capillaries in human iliac crest biopsy specimens. We found that recruitment occurs on both reversal and bone-forming surfaces, as shown by the cell density and osterix levels on these respective surfaces, and that bone formation occurs only above a given cell density. Canopies appeared an important source of osteoprogenitors, because (i) canopy cells proved to be more proliferative and less differentiated than bone surface cells, as shown by the inverse levels of Ki-67 and procollagen-3 N-terminal peptide versus osterix, and (ii) canopy cell densities, found to decline with age, and canopy-capillary contacts above eroded surfaces correlated positively with osteoblast density on bone-forming surfaces. Furthermore, we showed that bone remodeling compartment canopies arise from a mesenchymal envelope surrounding the red bone marrow, which is lifted and hypertrophied on initiation of bone resorption. This study, together with earlier reports, led to a model in which canopies and nearby capillaries are critical for reaching the osteoblast density required for bone formation.

Patient-specific bone modelling and remodelling simulation of hypoparathyroidism based on human iliac crest biopsies

We previously developed a load-adaptive bone modelling and remodelling simulation model that can predict changes in the bone micro-architecture as a result of changes in mechanical loading or cell activity. In combination with a novel algorithm to estimate loading conditions, this offers the possibility for patient-specific predictions of bone modelling and remodelling. Based on such models, the underlying mechanisms of bone diseases and/or the effects of certain drugs and their influence on the bone micro-architecture can be investigated. In the present study we test the ability of this approach to predict changes in bone micro-architecture during hypoparathyroidism (HypoPT), as an illustrative example. We hypothesize that, apart from reducing bone turnover, HypoPT must also lead to increased osteocyte mechanosensitivity in order to explain the changes in bone mass seen in patients. Healthy human iliac crest biopsies were used as the starting point for the simulations that mimic HypoPT conditions and the resultant micro-architectures were compared to age-matched clinical HypoPT biopsies. Simulation results were in good agreement with the clinical data when osteocyte mechanosensitivity was increased by 40%. In conclusion, the results confirm our hypothesis, and also demonstrate that patient-specific bone modelling and remodelling simulations are feasible.

Lathyrism-induced alterations in collagen cross-links influence the mechanical properties of bone material without affecting the mineral

In the present study a rat animal model of lathyrism was employed to decipher whether anatomically confined alterations in collagen cross-links are sufficient to influence the mechanical properties of whole bone.Animal experiments were performed under an ethics committee approved protocol. Sixty-four female (47day old) rats of equivalent weights were divided into four groups (16 per group): Controls were fed a semi-synthetic diet containing 0.6% calcium and 0.6% phosphorus for 2 or 4weeks and β-APN treated animals were fed additionally with β-aminopropionitrile (0.1% dry weight). At the end of this period the rats in the four groups were sacrificed, and L2–L6 vertebra were collected. Collagen cross-links were determined by both biochemical and spectroscopic (Fourier transform infrared imaging (FTIRI)) analyses. Mineral content and distribution (BMDD) were determined by quantitative backscattered electron imaging (qBEI), and mineral maturity/crystallinity by FTIRI techniques. Micro-CT was used to describe the architectural properties. Mechanical performance of whole bone as well as of bone matrix material was tested by vertebral compression tests and by nano-indentation, respectively.The data of the present study indicate that β-APN treatment changed whole vertebra properties compared to non-treated rats, including collagen cross-links pattern, trabecular bone volume to tissue ratio and trabecular thickness, which were all decreased (p<0.05). Further, compression tests revealed a significant negative impact of β-APN treatment on maximal force to failure and energy to failure, while stiffness was not influenced. Bone mineral density distribution (BMDD) was not altered either. At the material level, β-APN treated rats exhibited increased Pyd/Divalent cross-link ratios in areas confined to a newly formed bone. Moreover, nano-indentation experiments showed that the E-modulus and hardness were reduced only in newly formed bone areas under the influence of β-APN, despite a similar mineral content.In conclusion the results emphasize the pivotal role of collagen cross-links in the determination of bone quality and mechanical integrity. However, in this rat animal model of lathyrism, the coupled alterations of tissue structural properties make it difficult to weigh the contribution of the anatomically confined material changes to the overall mechanical performance of whole bone. Interestingly, the collagen cross-link ratio in bone forming areas had the same profile as seen in actively bone forming trabecular surfaces in human iliac crest biopsies of osteoporotic patients.

Involvement of human endogenous retroviral syncytin-1 in human osteoclast fusion

Generation of osteoclasts through fusion of mono-nucleated precursors is a key event of bone physiology and bone resorption is inefficient without osteoclast fusion. Several factors playing a critical role in the fusion process have already been recognized, but the factors involved in the actual fusion of the lipid bilayers of their cell membranes are still unknown. Syncytin-1 is a protein encoded by a human endogenous retroviral gene which was stably integrated into the human ancestor genome more than 24 million years ago. Upon activation, syncytin-1 is able to destabilize the lipid bilayer of the target cell and to force the merging of plasma membranes. This protein is a key player in the fusion of cytotrophoblasts. In the present study, syncytin-1 as well as its putative receptor ASCT2 was found to be expressed in differentiating osteoclasts in vitro, both on mRNA and protein level. This was documented through Q-PCR, Western blot and immunofluorescence analyses. These in vitro findings were confirmed by immunohistochemical stainings in human iliac crest biopsies. A syncytin-1 inhibitory peptide reduced the number of nuclei per osteoclast by 30%, as well as TRACP activity. From a mechanistic point of view, it is interesting that the distribution of syncytin-1 immunoreactivity on the cell surface parallels that of actin, another important player in cell fusion, and that cell–cell proximity induces particular patterns of distribution of syncytin-1 and actin in the respective cells. These complementary observations support a critical role of syncytin-1 in osteoclast fusion, which is of special interest in view of its well-known ability to force the merging of plasma membranes.

Bone Material Properties in Trabecular Bone From Human Iliac Crest Biopsies After 3- and 5-Year Treatment With Risedronate

Long-term effects of risedronate on bone mineral maturity/crystallinity and collagen cross-link ratio in triple iliac crest biopsies of osteoporotic women were evaluated. In this double-blinded study, 3- and 5-year treatment with risedronate arrested the tissue aging encountered in untreated osteoporosis and in osteoporosis treated with other antiresorptives. This effect may be contributing to risedronate's antifracture efficacy. Risedronate is widely used in the treatment of osteoporosis. It reduces bone turnover, increases BMD, and decreases fracture risk. To date, there are no data available on the long-term effects of risedronate on bone material properties in humans. Osteoporotic women enrolled in the VERT-NA trial received either risedronate (5 mg/day, orally) or placebo for up to 5 years. All subjects received calcium. They also received vitamin D supplementation if deficient at baseline. Triple iliac crest biopsies were collected from a subset of these subjects at baseline, 3 years, and 5 years. Mineral maturity/crystallinity and collagen cross-link ratio was measured in these biopsies using Fourier transform infrared imaging. Patients that received placebo exhibited increased mineral maturity/crystallinity and collagen cross-link ratio after 3 and 5 years compared with baseline values. On the contrary, patients that received risedronate retained baseline values in both bone material indices throughout. A more spatially detailed analysis revealed that this was achieved mainly through beneficial effects on active bone-forming areas. Surprisingly, patients that received risedronate achieved premenopausal values at bone-forming areas in both indices after 5 years of treatment. Long-term treatment with risedronate affects bone material properties (mineral maturity/crystallinity and collagen cross-link ratio) and arrests the tissue aging apparent in untreated osteoporosis. These changes at the material level of the bone matrix may contribute to risedronate's rapid and sustained antifracture efficacy in osteoporotic patients.

Bone Fragility and Collagen Cross-Links

Infrared imaging analysis of iliac crest biopsy specimens from patients with osteoporotic and multiple spontaneous fractures shows significant differences in the spatial variation of the nonreducible:reducible collagen cross-links at bone-forming trabecular surfaces compared with normal bone. Although the role of BMC and bone mineral quality in determining fracture risk has been extensively studied, considerably less attention has been paid to the quality of collagen in fragile bone. In this study, the technique of Fourier transform infrared imaging (FTIRI) was used to determine the ratio of nonreducible:reducible cross-links, in 2- to 4-microm-thick sections, from human iliac crest biopsy specimens (N = 27) at bone-forming trabecular surfaces. The biopsy specimens were obtained from patients that had been diagnosed as high- or low-turnover osteoporosis, as well as premenopausal women <40 years of age, with normal BMD and biochemistry, who suffered multiple spontaneous fractures. The obtained values were compared with previously published analyses of trabecular bone from normal non-osteoporotic subjects (N = 14, 6 males and 8 females; age range, 51-70 years). Collagen cross-links distribution within the first 50 microm at forming trabecular surfaces in patients with fragile bone was markedly different compared with normal bone.

Microwave irradiation of ethanol-fixed bone improves preservation, reduces processing time, and allows both light and electron microscopy on the same sample.

Methylmethacrylate (MMA) embedding is routinely used for histomorphometry of undecalcified bone preserved by prolonged immersion in ethanol, a procedure that yields poor ultrastructural detail. Because microwave irradiation (MWI) facilitates penetration of fixatives, we have investigated whether it can improve preservation by ethanol. Rat tibiae, some labeled with tetracycline, and a human iliac crest biopsy were immersed in 70% ethanol and dehydrated, both under MWI, for a total processing time of approximately 7 hr. Controls were not irradiated, and all specimens were embedded in MMA at 4C. They were then processed for histomorphometry, histochemistry, structural analysis, and immunolabeling. The results showed that histological preservation was improved with MWI. Static bone formation and resorption parameters and rate of mineral apposition were similar to those of conventionally processed specimens. Mineral distribution, as visualized by von Kossa staining and backscattered electron imaging, was not affected. Alkaline phosphatase and tartrate-resistant acid phosphatase activity, as well as immunolocalization of bone sialoprotein and osteopontin, were readily visualized. Ultrastructurally, osteopontin exhibited a typical distribution in mineralization foci, between calcified collagen fibrils, and at cement lines. These data show that MWI improves preservation and permits application of a broad spectrum of analytical methodologies on the same bone sample while considerably reducing processing time.

Distribution of collagen cross-links in normal human trabecular bone.

Infrared imaging analysis of normal human iliac crest biopsy specimens shows a characteristic spatial variation in the nonreducible:reducible collagen cross-links at trabecular surfaces, depending on the surfaces' metabolic status. Bone is a composite material consisting of mineral, collagen, non-collagenous proteins, and lipids. Bone collagen, mainly type I, provides the scaffold on which mineral is deposited and imparts specific mechanical properties, determined in part by the amount of collagen present, its orientation and fibril diameter, and the distribution of its cross-links. In this study, the technique of Fourier transform infrared imaging (FTIRI) was used to determine the ratio of nonreducible:reducible cross-links, in 2- to 4-microm-thick sections from human iliac crest biopsy specimens (N = 14) at trabecular surfaces as a function of surface activity (forming versus resorbing), with an approximately 6.3-mm spatial resolution. The biopsy specimens were obtained from patients devoid of any metabolic bone disease based on histomorphometric and bone densitometric parameters. Distributions of collagen cross-links within the first 50 mm at forming trabecular surfaces demonstrated a progressive increase in the nonreducible:reducible collagen cross-link ratio, unlike in the case of resorbing surfaces, in which the collagen cross-links ratio (as defined for the purposes of the present report) was relatively constant.

Spectroscopic characterization of collagen cross-links in bone.

Collagen is the most abundant protein of the organic matrix in mineralizing tissues. One of its most critical properties is its cross-linking pattern. The intermolecular cross-linking provides the fibrillar matrices with mechanical properties such as tensile strength and viscoelasticity. In this study, Fourier transform infrared (FTIR) spectroscopy and FTIR imaging (FTIRI) analyses were performed in a series of biochemically characterized samples including purified collagen cross-linked peptides, demineralized bovine bone collagen from animals of different ages, collagen from vitamin B6-deficient chick homogenized bone and their age- and sex-matched controls, and histologically stained thin sections from normal human iliac crest biopsy specimens. One region of the FTIR spectrum of particular interest (the amide I spectral region) was resolved into its underlying components. Of these components, the relative percent area ratio of two subbands at approximately 1660 cm(-1) and approximately 1690 cm(-1) was related to collagen cross-links that are abundant in mineralized tissues (i.e., pyridinoline [Pyr] and dehydrodihydroxylysinonorleucine [deH-DHLNL]). This study shows that it is feasible to monitor Pyr and DHLNL collagen cross-links spatial distribution in mineralized tissues. The spectroscopic parameter established in this study may be used in FTIRI analyses, thus enabling the calculation of relative Pyr/DHLNL amounts in thin (approximately 5 microm) calcified tissue sections with a spatial resolution of approximately 7 microm.

IR Microscopic Imaging of Pathological States and Fracture Healing of Bone

The application of IR microscopic imaging to the study of bone disease and fracture healing is demonstrated. Samples of normal and osteoporotic human iliac crest biopsies were prepared and examined at ∼6–10 μm spatial resolution and 8 cm−1 spectral resolution with a 64 × 64 MCT focal plane array detector coupled to a Fourier transform infrared (FT-IR) microscope and a step-scanning interferometer. Two spectral parameters, one that monitors the extent of mineral (hydroxyapatite) formation in the tissue and another that monitors the size/perfection of the crystals, were compared in the samples generated from normal and pathological tissues. The average mineral levels in the osteoporotic sample were reduced by ∼ 40% from the normal. In addition, the crystal size/perfection was substantially enhanced in the disease state. The applicability of IR imaging techniques to the study of therapeutic intervention was also investigated in a study of the effects of estrogen therapy on fracture healing in rat femurs. Femurs were examined by IR microscopic imaging 4 weeks after fracture. IR imaging showed that the mineral level was enhanced in estrogen-treated samples. In addition, the crystals were larger/more perfect in the treated specimens. These data demonstrate the utility of IR spectroscopic imaging for the study of pathological states of hard tissue.

Two-dimensional vibrational correlation spectroscopy of in vitro hydroxyapatite maturation.

Two-dimensional (2-D) Raman and 2-D IR correlation spectroscopy are applied to analyze changes in the nu(4) region of the IR spectrum and in the nu(1) region of the Raman spectrum during the maturation of hydroxyapatite (HA) following the solution-mediated conversion of amorphous calcium phosphate (ACP) to HA. The nu(1) region of the Raman spectrum exhibits a frequency shift and sharpening during the maturation. Comparison of the experimental and simulated 2-D plots for this process suggests that the shift of a single peak, rather than a change in the relative intensity of two overlapped bands, is responsible for the observed spectral changes. The nu(4) mode of the PO(3-)(4) ion (T(2) symmetry in the free species) splits into a triplet with components near 563, 575, and 603 cm(-1) in HA. In addition, broad features appear at 540 and 617 cm(-1). During the latest stages of the maturation, an OH(-) librational mode develops at approximately 632 cm(-1). Changes in the relative intensities of three components of the nu(4) mode are not all correlated with each other. The synchronous 2-D plots reveal that the 563 and 603 cm(-1) pair are positively correlated while the feature at 575 cm(-1) is absent. A 587 cm(-1) mode arising from ACP is negatively correlated with the 563 and 603 cm(-1) pair and is both synchronously (positively) and asynchronously correlated with the 540 cm(-1) feature during the early stages of the maturation but is absent from 2-D plots of the later stages of the maturation. Cross correlations between the nu(4) mode and the nu(1),nu(3) contour generally confirm and extend previous assignments for the latter spectral region. Finally, the suitability of the 2-D approach for analysis of IR spectral images is examined through studies of HA crystallinity in a human iliac crest biopsy sample. Trabecular bone contains a fraction of HA that is more crystalline and mature than could be achieved in vitro during the room temperature ACP --> HA interconversion.

Infrared Spectroscopy, Microscopy, and Microscopic Imaging of Mineralizing Tissues: Spectra-Structure Correlations from Human Iliac Crest Biopsies

Infrared microscopic images of the cortical region of human iliac crest biopsies have been obtained at ∼7 μm spatial resolution and 8 cm-1 spectral resolution with a 64×64 mercury-cadmium-telluride focal plane array detector coupled to a Fourier transform infrared microscope and a step scanning interferometer. Images of several spectral parameters provide information about the spatial distribution of the mineral (apatite) and protein (mostly collagen) components of the tissue. In addition, the image of a parameter known to reflect the crystallinity/perfection of the mineral phase, namely, the intensity ratio of bands at 1030 and 1020 cm-1 within the phosphate ν1,ν3 contour, revealed a progressive increase in the apatite crystal size/perfection from the osteonal center to the periphery. Finally, a detailed comparison of the spatial distribution of the I(1020)/I(1030) ratio for the same osteon obtained by array detection and by conventional point-by-point microspectroscopy revealed statistically identical behavior, thereby providing a validation of infrared imaging for structural analysis of apatite forming tissues. © 1999 Society of Photo-Optical Instrumentation Engineers.

FTIR microspectroscopic analysis of normal human cortical and trabecular bone.

Fourier transform infrared microspectroscopy (FTIRM) has been used to study the changes in mineral and matrix content and composition in replicate biopsies of nonosteoporotic human cortical and trabecular bone. Changes in osteonal bone in these same samples were reported previously. Spectral maps along and across the lamellae were obtained from iliac crest biopsies of two necropsy cases. Mineral:matrix ratios, calculated from the integrated areas of the phosphate nu1, nu3 band at 900-1200 cm-1 and the amide I band at approximately 1585-1725 cm-1, respectively, were relatively constant in both directions of analysis, i.e., along and across the lamellae. Analysis of the components of the nu1, nu3 phosphate band with a combination of second-derivative spectroscopy and curve fitting revealed the presence of 11 major underlying moieties. Of these, the ratio of the relative areas of the two underlying bands at approximately 1020 and approximately 1030 cm-1 has been shown to be a sensitive index of variation in crystal perfection in both human osteonal bone and in synthetic, poorly crystalline apatites. This ratio was calculated in both cortical and trabecular bone from human iliac crest biopsies along and across the lamellae. The ratio decreased, going from the periosteum to the medullary cavity in the cortical bone, and from the periphery towards the center of trabeculae. These observations were consistent within serial sections obtained from the same biopsy, multiple biopsies obtained from the same necropsy specimen, and biopsies obtained from the two different necropsy specimens. The results presented here along with previously reported changes in osteonal bone show a relation between bone age and "crystallinity/maturity" (a parameter dependent on crystallite size, hydroxyapatite-like stoichiometry, abundance of substituting ions such as CO32-; the more crystalline/mature, the more hydroxyapatite-like stoichiometry, the bigger the crystallite size, the less the ion substitution by ions such as CO32-) as deduced by the 1020/1030 cm-1 ratio. Invariably, younger normal bone is less mature/crystalline than older. These results provide a "baseline" for description of mineral properties, to which diseased bones may be compared.

New possibilities for structural analysis of bone biopsies using microcomputer tomography (muCT)

The combined histological and microcomputed analysis of human iliac crest biopsies leads to major advances in our understanding of three-dimensional bone architecture. Microcomputed tomography avoids the time-consuming reconstruction and artifacts of serial sections. Furthermore, its high resolution allows the recording of structural differences as low as 10 microns. Thus, three-dimensional analysis in combination with histological evaluation of cellular dynamics facilitates earlier and easier recording of changes of cancellous bone.

Age-related changes in trabecular width and spacing in human iliac crest biopsies

Age-related changes in trabecular width and spacing have been examined in iliac crest bone obtained from 96 normal subjects. The mean trabecular plate density and separation were calculated from values of trabecular bone volume and mean trabecular plate thickness. There was an age-related decrease in mean trabecular plate thickness with age in both sexes; this was statistically significant in males ( P < 0.02) but not in females. In females, there was a significant decrease in mean trabecular plate density ( P < 0.001) and increase in mean trabecular plate separation ( P < 0.001); similar but non-significant trends were observed in the males. These results demonstrate that trabecular thinning contributes to age-related bone loss in both sexes. Loss of trabeculae also occurs in both sexes, but to a greater extent in females than in males.

A new method for the two-dimensional analysis of bone structure in human iliac crest biopsies.

A new method is described for the analysis of the two-dimensional structural pattern of trabecular bone in human iliac crest biopsies. 8 microns thick undecalcified sections stained with the von Kossa technique were examined at a magnification of X 9. Using an Ibas II image analyser, the ratio of nodes to free ends and the length of different strut types (cortex to free end, node or cortex, free end to free end and node to node, loop or free end) expressed as a percentage of total strut length were assessed. The reproducibility of the method was good for most of the measured indices but inter-observer and inter-section variation were greater. Comparison of biopsy sections obtained from eleven young healthy control subjects and eleven patients with hepatic osteoporosis revealed a significantly higher node to free end ratio, node to loop and node to node strut length and significantly lower cortex to free end and free end to free end strut length in the controls. No significant differences were seen in node to free end, cortex to cortex or cortex to node strut length. This approach to trabecular bone structure analysis should prove useful in determining patterns of bone loss in health and disease and in examining the effects of treatment on bone structure in osteoporosis.

Inter-observer and intra-observer variation in bone histomorphometry.

Inter-observer variation has been examined for a number of histomorphometric indices in 20 normal human iliac crest biopsies. Quantitation was performed using an eye-piece graticule and eye-piece micrometer. The same sections were examined by two observers and the methodology was identical. Intra-observer variation was also assessed. Significant inter-observer differences were found for the measurement of total trabecular bone volume, osteoid volume and surface, double plus single and double tetracycline labeled surfaces, and the mean osteoid seam width. The percentage variance due to inter-observer variation was highest for osteoid surface and volume, total resorption surface, and mean osteoid seam width. Intra-observer variation in both observers was small. We conclude that a large inter-observer variation may occur in the measurement of a number of histomorphometric indices, even when section preparation and methodology are identical. Caution should be used in basing the diagnosis of metabolic bone disease on strictly defined control data from other observers, particularly when this has been obtained from centers where the effects of inter-observer variation may be magnified by differences in methodology.