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Collagen Type I of Rat Cortical and Trabecular Bone Differs in the Extent of Posttranslational Modifications
Abstract
This study sought to evaluate whether the architecture of the matrix of cortical and trabecular bone is exactly the same.
For this purpose we analyzed the extent of some posttranslational modifications of type I collagen, which is the major component
of bone matrix. Ten female and 10 male 100-day-old rats were sacrificed and the content of hydroxylysine, glycosylated hydroxylysine,
and pyridinium cross-links of collagen from cortical and trabecular bone was determined. The amount of each compound was expressed
as a molar ratio with hydroxyproline. The collagen posttranslational modification pattern appears to be the same in both sexes
but with a higher extent of differences in females compared with males. Comparing cortical and trabecular bone, the former
contains a higher amount of hydroxylysine residues whereas in the latter, glycosylation of hydroxylysine is higher and pyridinium
cross-link concentration is lower. Moreover, an inverse linear relationship between glycosylated hydroxylysine and pyridinium
crosslinks concentration was established, both for female (r=−0.455,P=0.04) and male rats (r=−0.426;P=0.06). This paper discusses what these findings may mean in functional terms.
... El proceso de desmineralización fue seguido por un espectrofotómetro, marca Nicolet E. S. P. 560 magna IR laser, se observaron las vibraciones correspondientes al grupo fosfato (PO 4 -3 ) presentes en la fase mineral, se prepararon pastillas de KBr, con 200 mg de la sal y 0.75 mg de la muestra a una presión de 8000 psi, aplicando vacío durante 1 minuto en una prensa hidráulica. ...
... En el diagrama de barras (Figura 3) se observa como cada 12 horas el contenido de mineral disminuye gradualmente, mostrando a las 72 horas una pérdida de 52% de la masa inicial, esta pérdida de masa se atribuye completamente a la hidroxiapatita removida a lo largo del proceso de descalcificación. [4] Fig. 3 Masa retenida (%) vs el tiempo de desmineralización En la Figura 4 se evidencia como disminuye la intensidad de los picos correspondientes a las vibraciones por estiramiento (1035 cm -1 ) y por deformación (603 cm -1 ,564 cm -1 ) del grupo fosfato (PO 4 -3 ), pertenecientes a la hidroxiapatita, lo que indica que el proceso de desmineralización se produ- En la Figura 5, se ve claramente que a medida que se elimina el mineral del hueso, la endoterma que corresponde al pico de desnaturalización del colágeno en 166ºC disminuye hasta 113ºC a las 72 horas de tratamiento, lo que indica que la triple hélice de colágeno va adquiriendo mayor movilidad hasta el punto de romper los enlaces débiles (puentes de hidrogeno) entre las cadenas que conforman la misma, pero los enlaces covalentes de entrecruzamiento intra e intermolecular permanecen intactos. Además, comienza a aparecer una pequeña endoterma relacionada con la degradación del colágeno a 250ºC que disminuye a 230ºC a las 72 horas. ...
... En el diagrama de barras (Figura 3) se observa como cada 12 horas el contenido de mineral disminuye gradualmente, mostrando a las 72 horas una pérdida de 52% de la masa inicial, esta pérdida de masa se atribuye completamente a la hidroxiapatita removida a lo largo del proceso de descalcificación. [4] Fig. 3 Masa retenida (%) vs el tiempo de desmineralización En la Figura 4 se evidencia como disminuye la intensidad de los picos correspondientes a las vibraciones por estiramiento (1035 cm -1 ) y por deformación (603 cm -1 ,564 cm -1 ) del grupo fosfato (PO 4 -3 ), pertenecientes a la hidroxiapatita, lo que indica que el proceso de desmineralización se produ- En la Figura 5, se ve claramente que a medida que se elimina el mineral del hueso, la endoterma que corresponde al pico de desnaturalización del colágeno en 166ºC disminuye hasta 113ºC a las 72 horas de tratamiento, lo que indica que la triple hélice de colágeno va adquiriendo mayor movilidad hasta el punto de romper los enlaces débiles (puentes de hidrogeno) entre las cadenas que conforman la misma, pero los enlaces covalentes de entrecruzamiento intra e intermolecular permanecen intactos. Además, comienza a aparecer una pequeña endoterma relacionada con la degradación del colágeno a 250ºC que disminuye a 230ºC a las 72 horas. ...
... Se sabe además que alteraciones en la disposición espacial (dada por el grado de glicosilación) puede alterar las propiedades de resistencia de las fibrillas de colágeno así como la deposición del mineral del tejido. En alteraciones como la osteoporosis postmenopausal, donde la deprivación de estrógenos causa un incremento en la tasa de degradación ósea versus la formación, ha sido reportado que el grado de glicosilación de la matriz colagénica del hueso trabecular se ve incrementado en comparación con individuos noosteoporóticos [1][2][3]; Sin embargo hasta la fecha no se han realizado estudios in vitro que permitan verificar si efectivamente el grado de glicosilación de la matriz colagénica del hueso puede ser reconocida por las células óseas, respondiendo de forma diferente como consecuencia a estas variaciones. ...
... Fueron sacrificadas a los 45 días luego de la operación tiempo en el cual desarrollaron la condición de osteoporosis. Los huesos largos fueron extraídos, lavados y desmineralizados, empleando el tratamiento descrito por Noris-Suárez et al [2]. Una vez descalcificadas las muestras óseas, se separó el colágeno de la región ósea cortical y el de la región ósea trabecular. ...
... La determinación cuantitativa permitió evidenciar que mientras el tratamiento con DXM parece inducir una limitada mineralización del colágeno óseo cortical, sin diferencias evidentes entre el colágeno de animales control y OVX, en el caso de los cultivos sin DXM si se encuentra una diferencia en la cantidad de mineral depositado, siendo hasta 4 veces mayor el grado de mineralización de la matriz proveniente de animales control en comparación con el mineral depositado en la matriz de animales OVX. Trabajos previos demostraron que el grado de glicosilación del colágeno óseo se incrementa en animales ovariectomizados, especialmente en el tejido óseo trabecular, donde ocurre la pérdida de tejido óseo en forma más pronunciada [1][2][3]. Siendo esta proteína la más abundante en el tejido óseo y debido al papel tan importante que ella ejerce el mantenimiento de este tejido, resulta de gran importancia entender como el grado de glicosilación de la matriz colagénica puede regular las funciones celulares de las células que componen el hueso. ...
In the bone tissue, collagen is the most abundant protein, conforming 90% of the organic matrix of this mineralized structure. Different works indicate that in postmenopausal osteoporosis, the early degradation of the bone tissue seems to be due to the presence of bone collagen in a condition of overglycosylation, especially in the trabecular bone. To understand the role of the glycosylation of the collagen molecule and how it can regulate functions of the bone cells, such as cellular adhesion and biomineralization, is the main objective of the present work. 8 rats were ovariectomized and another 8 were sham-operated (control group). After 45 days the rats were sacrificed, femurs were extracted and the bones were decalcified in order to obtain the collagen matrix mineral free. Primary cultures of bone cells were obtained from calvaria of neonatal rats. The adhesion and differentiation (alkaline phosphatase activity and biomineralization) of these cells were evaluated on the collagen bone matrix obtained of the ovariectomized and control rats. The results of this work show a greater adhesion of the osteoblastic cells on the bone collagen for the control group in comparison with the adhesion found for the ovariectomized group, in the first two hours. Additionally, cell FA activities and mineralization on material differ between control and ovariectomized animals bone collagen samples. In conclusion, in the present work we present evidence that suggest that bone cells can recognize differences in the degree of glycosylation of the collagen matrix in ovariectomized animals in comparison with the control group, and the cellular response is different as a consequence of these variations of the extracellular material.
... Se sabe además que alteraciones en la disposición espacial (dada por el grado de glicosilación) puede alterar las propiedades de resistencia de las fibrillas de colágeno así como la deposición del mineral del tejido. En alteraciones como la osteoporosis postmenopausal, donde la deprivación de estrógenos causa un incremento en la tasa de degradación ósea versus la formación, ha sido reportado que el grado de glicosilación de la matriz colagénica del hueso trabecular se ve incrementado en comparación con individuos noosteoporóticos [1][2][3]; Sin embargo hasta la fecha no se han realizado estudios in vitro que permitan verificar si efectivamente el grado de glicosilación de la matriz colagénica del hueso puede ser reconocida por las células óseas, respondiendo de forma diferente como consecuencia a estas variaciones. ...
... Fueron sacrificadas a los 45 días luego de la operación tiempo en el cual desarrollaron la condición de osteoporosis. Los huesos largos fueron extraídos, lavados y desmineralizados, empleando el tratamiento descrito por Noris-Suárez et al [2]. Una vez descalcificadas las muestras óseas, se separó el colágeno de la región ósea cortical y el de la región ósea trabecular. ...
... La determinación cuantitativa permitió evidenciar que mientras el tratamiento con DXM parece inducir una limitada mineralización del colágeno óseo cortical, sin diferencias evidentes entre el colágeno de animales control y OVX, en el caso de los cultivos sin DXM si se encuentra una diferencia en la cantidad de mineral depositado, siendo hasta 4 veces mayor el grado de mineralización de la matriz proveniente de animales control en comparación con el mineral depositado en la matriz de animales OVX. Trabajos previos demostraron que el grado de glicosilación del colágeno óseo se incrementa en animales ovariectomizados, especialmente en el tejido óseo trabecular, donde ocurre la pérdida de tejido óseo en forma más pronunciada [1][2][3]. Siendo esta proteína la más abundante en el tejido óseo y debido al papel tan importante que ella ejerce el mantenimiento de este tejido, resulta de gran importancia entender como el grado de glicosilación de la matriz colagénica puede regular las funciones celulares de las células que componen el hueso. ...
El grado de glicosilación del colágeno óseo regula la adhesión y la capacidad de biomineralizar de las células óseas Abstract— In the bone tissue, collagen is the most abundant protein, conforming 90% of the organic matrix of this mineral-ized structure. Different works indicate that in postmeno-pausal osteoporosis, the early degradation of the bone tissue seems to be due to the presence of bone collagen in a condition of overglycosylation, especially in the trabecular bone. To understand the role of the glycosylation of the collagen mole-cule and how it can regulate functions of the bone cells, such as cellular adhesion and biomineralization, is the main objective of the present work. 8 rats were ovariectomized and another 8 were sham-operated (control group). After 45 days the rats were sacrificed, femurs were extracted and the bones were decalcified in order to obtain the collagen matrix mineral free. Primary cultures of bone cells were obtained from calvaria of neonatal rats. The adhesion and differentiation (alkaline phos-phatase activity and biomineralization) of these cells were evaluated on the collagen bone matrix obtained of the ovariec-tomized and control rats. The results of this work show a greater adhesion of the osteoblastic cells on the bone collagen for the control group in comparison with the adhesion found for the ovariectomized group, in the first two hours. Addition-ally, cell FA activities and mineralization on material differ between control and ovariectomized animals bone collagen samples. In conclusion, in the present work we present evi-dence that suggest that bone cells can recognize differences in the degree of glycosylation of the collagen matrix in ovariec-tomized animals in comparison with the control group, and the cellular response is different as a consequence of these varia-tions of the extracellular material. Palabras claves— Glicosilación del colágeno, matriz ósea, osteoporosis postmenopausal, osteoblastos.
... This energy dissipation property of type I collagen in bone is largely due to its structure, in which fibrils made up of bundles of triple helical tropocollagen molecules containing a glycine-X-Y motif are organised into macroscale fibres (Currey, 1988;Donnelly et al., 2010;Ramshaw et al., 1998;Shoulders and Raines, 2009). A number of studies have shown that direct alterations to collagen properties implicate the overall mechanical properties of bone and often result in changes to fracture risk (Diebold et al., 1991;Kafantari et al., 2000;Sroga and Vashishth, 2012;Suarez et al., 1996). ...
Collagen assembly during development is essential for successful matrix mineralisation, which determines bone quality and mechanocompetence. However, the biochemical and structural perturbations that drive pathological skeletal collagen configuration remain unclear. Deletion of vascular endothelial growth factor (VEGF) in bone forming osteoblasts (OBs) induces sex-specific alterations in extracellular matrix (ECM) conformation and mineralisation coupled to vascular changes, which are augmented in males. Whether this phenotypic dimorphism arises as a result of the divergent control of ECM composition and its subsequent arrangement is unknown and is the focus of this study. Herein, we have used a murine osteocalcin-specific Vegf knockout (OcnVEGFKO) and performed ex vivo multiscale analysis at the tibiofibular junction of both sexes. Furthermore, we also deleted Vegf in vitro in OBs extracted from male and female mice in an attempt to link sex-specific matrix signatures to deviations in gene expression. Label-free and non-destructive polarisation-resolved second harmonic generation microscopy (p-SHG) revealed a reduction in collagen fibre number in males following the loss of VEGF, complemented by observable defects in matrix organisation by backscattered electron scanning electron microscopy. This was accompanied only in males by localised divergence in collagen orientation, determined by p-SHG anisotropy measurements, as a result of OcnVEGFKO. Raman spectroscopy confirmed the effect on collagen was linked to molecular dimorphic VEGF effects on collagen-specific proline and hydroxyproline, and collagen intra-stand stability, in addition to matrix carbonation and mineralisation. V egf deletion in male and female murine OB cultures in vitro further highlighted divergence in genes regulating local ECM structure including Adamts2 , Spp1 , Mmp9 and Lama1 . The current results demonstrate the utility of macromolecular imaging and spectroscopic modalities for the detection of collagen arrangement and ECM composition in pathological bone. Linking the sex-specific genetic regulators to matrix signatures could be important for treatment of dimorphic bone disorders which clinically manifest in both pathological nano and macro-level disorganisation.
... Khun et al. [11] described the differences in the mineral content and crystal maturation process in young and old animals, which mirror those seen in cortical and trabecular bone, respectively. For this reason, the differences between the mineral crystals may be attributed to their age, as well as to contrasting extents of post-translational modifications in the collagen structure [12,13,19,81]. The higher protein-to-mineral ratio and collagen content in protein seen in cortical bone seems to be similarly linked to its mechanical properties. ...
The aim of this study was to explore the hierarchical arrangement of structural properties in cortical and trabecular bone and to determine a mathematical model that accurately predicts the tissue's mechanical properties as a function of these indices. By using a variety of analytical techniques, we were able to characterize the structural and compositional properties of cortical and trabecular bones, as well as to determine the suitable mathematical model to predict the tissue's mechanical properties using a continuum micromechanics approach. Our hierarchical analysis demonstrated that the differences between cortical and trabecular bone reside mainly at the micro- and ultrastructural levels. By gaining a better appreciation of the similarities and differences between the two bone types, we would be able to provide a better assessment and understanding of their individual roles, as well as their contribution to bone health overall.
© 2015 The Author(s) Published by the Royal Society. All rights reserved.
... It shows a weak immunohistochemical staining in bone matrix [44], being preferably at the pericellular area but not in Sharpey's fibers [45]. The collagen content is present at similar levels between cortical and trabecular bones and between male and female [46]. Therefore, it is thought that the mandibular bone matrix including the trabecular bone is rich in collagen. ...
Cadmium (Cd) is a toxic metal that damages bone tissue by affecting its mineral and organic components. The organic matrix is mainly (90%) composed of collagen, which determines the biomechanical strength of bone. The aim of this study was to evaluate the effect of zinc (Zn) supplementation (30 or 60 mg l–1) under moderate and relatively high exposure to Cd (5 and 50 mg l–1) on collagen in the rat tibia proximal epiphysis and diaphysis (regions abundant in trabecular and cortical bone, respectively). Significant decrease in collagen type I biosynthesis was found in both regions of the tibia in Cd-treated rats, whereas the supplementation with Zn provided significant protection against this effect. Western blot confirmed the presence of the major type I collagen in the tibia epiphysis and diaphysis, but collagen type II was revealed only in the epiphysis. Acetic acid- and pepsin-soluble collagen concentration in the tibia epiphysis and diaphysis was significantly increased due to the exposure to Cd, whereas the supplementation with Zn protected, partially or totally, from these effects, depending on the used concentration. The supplementation with Zn also provided protection from unfavorable Cd impact on the maturation of the bone collagen, as the ratio of cross-links to monomers was higher compared to the Cd-treated group. This report confirms our previous findings on the preventive action of Zn against harmful effects of Cd on bone, but additionally, and to the best of our knowledge for the first time, explains the possible mechanism of the beneficial influence of this bioelement.
In implantology, several techniques such as light and polarizing microscopy and histomorphometry are available for evaluation of the bone reactions. Descriptive histology and histomorphometry are the two main types of histological methods. At the microscopic level, bone consists of two forms: woven (an immature disorganized form of bone) and lamellar. During replacement of biomaterial, the remodeling of woven bone is first. Lamellar bone actively replaces woven bone over time and is found in a several structural and functional units with greater organization. Bone remodeling is characterized by the spatial and temporal coupling of bone formation by osteoblasts and bone resorption by osteoclasts. Histomorphometry can be very valuable in measuring the changes in the tissues that surround an implant. The parameters used in the evaluation procedure must be as simple as possible. However, analysis requires considerable expertise and remains time-consuming, despite the reduction in variables. To overcome the limitations presented by the 2D histological section reconstruction from serial sections is an option.
Bone and cartilage are highly specialized connective tissues that are engineered by nature to perform a variety of specialized tasks. As a result, these tissues have unique cellular constituents and ultrastructural organization that help optimize the biochemical demands and biomechanical loads in vivo. Our understanding of these connective tissues has increased over the past few centuries, and progress continues as techniques are defined and improved or new ones are developed. The understanding of the ultrastructural and mechanical properties of bone and cartilage is a dynamic area that is constantly evolving with new understanding. This chapter departs from traditional overviews of bone and cartilage, and presents an examination of cellular constituents and ultrastructural organization of these tissues in the context of recent experimental and theoretical studies. It is the hope of the authors to bring to light many new and exciting findings.
Fibrillar type I collagen is the major organic component in bone, providing a stable template for mineralization. During collagen biosynthesis, specific hydroxylysine residues become glycosylated in the form of galactosyl- and glucosylgalactosylhydroxylysine. Furthermore, key glycosylated hydroxylysine residues, alpha 1/2-87, are involved in covalent intermolecular cross-linking. Although cross-linking is crucial for the stability and mineralization of collagen, the biological function of glycosylation in cross-linking is not well understood. In this study, we quantitatively characterized glycosylation of non-cross-linked and cross-linked peptides by biochemical and nanoscale liquid chromatography-high resolution tandem mass spectrometric analyses. The results showed that glycosylation of non-crosslinked hydroxylysine is different from that involved in cross-linking. Among the cross-linked species involving alpha 1/2-87, divalent cross-links were glycosylated with both mono- and disaccharides, whereas the mature, trivalent cross-links were primarily monoglycosylated. Markedly diminished diglycosylation in trivalent cross-links at this locus was also confirmed in type II collagen. The data, together with our recent report (Sricholpech, M., Perdivara, I., Yokoyama, M., Nagaoka, H., Terajima, M., Tomer, K. B., and Yamauchi, M. (2012) Lysyl hydroxylase 3-mediated glucosylation in type I collagen: molecular loci and biological significance. J. Biol. Chem. 287, 22998-23009), indicate that the extent and pattern of glycosylation may regulate cross-link maturation in fibrillar collagen.
More than 70 mutations in the two structural genes for type I procollagen (COL1A1 and COL1A2) have been found in probands with osteogenesis imperfecta, a heritable disease of children characterized by fragility of bone and other tissues rich in type I collagen. The mutations include deletions, insertions, RNA splicing mutations, and single-base substitutions that convert a codon for glycine to a codon for an amino acid with a bulkier side chain. With a few exceptions, the most severe phenotypes of the disease are explained largely by synthesis of structurally defective pro alpha chains of type I procollagen that either interfere with the folding of the triple helix or with self-assembly of collagen into fibrils. The results emphasize the extent to which the zipperlike folding of the collagen triple helix and the self-assembly of collagen fibrils depend on the principle of nucleated growth whereby a few subunits form a nucleus and the nucleus is then propagated to generate a large structure with a precisely defined architecture. The principle of nucleated growth is a highly efficient mechanism for the assembly of large structures, but biological systems that depend extensively on nucleated growth are highly vulnerable to mutations that cause synthesis of structurally abnormal but partially functional subunits. Recently, several mutations in three other collagen genes (COL2A1, COL3A1, and COL4A5) have been found in probands with genetic diseases involving tissues rich in these collagens. Most of the probands have rare genetic diseases but a few appear to have phenotypes that are difficult to distinguish from more common disorders such as osteoarthritis, osteoporosis, and aortic aneurysms. Therefore, the results suggest that mutations in procollagen genes may cause a wide spectrum of both rare and common human diseases.
This chapter discusses the formation and stability of dansyl chloride procedure. Dansyl chloride is used for providing fluorescent “handles” for the study of proteins. The reaction of the dye with α-chymotrypsin led them to propose its use as a means of studying the α-amino and other reactive groups of proteins and peptides. It is useful in determining the amino acid sequences of small amounts of peptides. Dansyl chloride is a typical aromatic sulfonyl chloride, and reacts with a wide variety of bases, forming derivatives of differing stabilities. Some recommended procedure for peptides discussed are (1) reagents, buffers, and marker mixtures, (2) labeling of peptide, (3) hydrolysis, (4) visualization of compounds on paper, and (5) interpretation of results. The increased requirement for dansyl chloride brings with it the necessity for removing the large amounts of DNS-OH formed by its hydrolysis. The removal procedures used are labeling, removal of salt, urea, DNS-OH, hydrolysis, and identification of end groups. Some of the advantages and limitations of the method are mentioned. The main advantage is the ease and rapidity with which end groups may be determined on very small amounts of peptides.
IntroductionGenetic Contribution to OsteoporosisCandidate Gene ApproachesGenome-Wide ApproachesStatistical IssuesPharmacogenomicsClinical Use of Genetic InformationThe Way Forward
Ovariectomy results in cancellous osteopenia in rat long bones, a condition that is prevented by treatment with estrogens. The purpose of these studies was to clarify the effects of estrogen on cancellous bone turnover using dynamic bone histomorphometry. Treatment of ovariectomized rats with diethylstilbestrol (DES) reduced the mineral apposition rate, double-label perimeter, osteoblast number, and osteoclast number, suggesting that the hormone had inhibitory effects on bone formation as well as bone resorption. However, we could not estimate the bone formation rate because of rapid resorption of tetracycline-labeled bone in the ovariectomized rat. The magnitude of loss was documented by a time course study: 58% of the tetracycline initially incorporated into the secondary spongiosa of the tibial metaphysis was resorbed after 11 days and 89% was resorbed after 22 days. Similarly, cancellous bone area was decreased by 67% after 11 days and by 88% after 22 days. Administration of either DES or tamoxifen (TAM) dramatically reduced resorption of tetracycline as well as the decrease in cancellous bone area. These results demonstrate that (1) estrogen prevents osteopenia in ovariectomized (OVX) rats, in part by inhibiting bone turnover, (2) TAM is an estrogen agonist on bone resorption, and (3) resorption of tetracycline-labeled bone leads to serious underestimation of the bone formation rate in OVX rats.
The idea that rats cannot model human osteopenias errs. The same mechanisms control gains in bone mass (longitudinal bone growth and modeling drifts) and losses (BMU-based remodeling), in young and aged rats and humans. Furthermore, they respond similarly in rats and man to mechanical influences, hormones, drugs and other agents.
An animal model of postmenopausal bone loss can be defined as a living animal in which spontaneous or induced bone loss due to ovarian hormone deficiency can be studied, and in which the characteristics of the bone loss and its sequalae resemble those found in postmenopausal women in one or more respects. Although in comparison to humans, the skeletal mass of rats remains stable for a protracted period during their lifespan, rats can be ovariectomized to make them sex-hormone deficient, and to stimulate the accelerated loss of bone that occurs in women following menopause. Ovariectomy induced bone loss in the rat and postmenopausal bone loss share many similar characteristics. These include: increased rate of bone turnover with resorption exceeding formation; and initial rapid phase of bone loss followed by a much slower phase; greater loss of cancellous than cortical bone; decreased intestinal absorption of calcium; some protection against bone loss by obesity; and similar skeletal response to therapy with estrogen, tamoxifen, bisphosphonates, parathyroid hormone, calcitonin and exercise. These wide-ranging similarities are strong evidence that the ovariectomized rat bone loss model is suitable for studying problems that are relevant to postmenopausal bone loss.
Groups of 19-day-old rats were ovariectomized or were given sham operations. Measurements in urine of the pyridinium cross-links of collagen, pyridinoline and deoxypyridinoline, 7 weeks after surgery showed significantly higher amounts of cross-links relative to creatinine in the ovariectomized groups compared with the controls. Analyses before and after acid hydrolysis of the urine revealed that the increased excretion was only as free cross-link with no change in the concentrations of the bound forms. The loss of trabecular bone in the ovariectomized group was confirmed by immunocytochemical staining with antibodies to type I collagen. There were no differences between the ovariectomized and control groups in the concentrations of cross-links in the tibial bone or the articular cartilage. Measurements of free pyridinoline and deoxypyridinoline in urine therefore appear to provide a good index of the increased bone resorption induced by estrogen deficiency.