Insights into the bisphosphonate holiday: a preliminary FTIRI study.

Bone composition evaluated by FTIRI analysis of iliac crest biopsies from post-menopausal women treated with alendronate for 10 years, continuously or alendronate for 5 years, followed by a 5-year alendronate-holiday, only differed with the discontinued biopsies having increased cortical crystallinity and heterogeneity of acid phosphate substitution and decreased trabecular crystallinity heterogeneity. INTRODUCTION: Bisphosphonates (BP) are the most commonly used and effective drugs to prevent fragility fractures; however, concerns exist that prolonged use may lead to adverse events. Recent recommendations suggest consideration of a BP "holiday" in individuals taking long-term BP therapy not at high risk of fracture. Data supporting or refuting this recommendation based on bone quality are limited. We hypothesized that a "holiday" of 5 years would cause no major bone compositional changes. METHODS: We analyzed the 31 available biopsies from the FLEX-Long-term Extension of FIT (Fracture Intervention Trial) using Fourier transform infrared imaging (FTIRI). Biopsies from two groups of post-menopausal women, a "Continuously treated group" (N = 16) receiving alendronate for ~ 10 years and a "Discontinued group" (N = 15), alendronate treated for 5 years taking no antiresorptive medication during the following 5 years. Iliac crest bone biopsies were provided at 10 years. RESULTS: Key FTIRI parameters, mineral-to-matrix ratio, carbonate-to-phosphate ratio, acid phosphate substitution, and collagen cross-link ratio as well as heterogeneity of these parameters were similar for Continuously treated and Discontinued groups in age-adjusted models. The Discontinued group had 2% greater cortical crystallinity (p = 0.01), 31% greater cortical acid phosphate heterogeneity (p = 0.02), and 24% lower trabecular crystallinity heterogeneity (p = 0.02). CONCLUSIONS: Discontinuation of alendronate for 5 years did not affect key FTIRI parameters, supporting the hypothesis that discontinuation would have little impact on bone composition. Modest differences were observed in three parameters that are not likely to affect bone mechanical properties. These preliminary data suggest that a 5-year BP holiday is not harmful to bone composition.

Chondrogenic ATDC5 cells: an optimised model for rapid and physiological matrix mineralisation.

The development of chondrogenic cell lines has led to major advances in the understanding of how chondrocyte differentiation is regulated, and has uncovered many signalling pathways and gene regulatory mechanisms required to maintain normal function. ATDC5 cells are a well established in vitro model of endochondral ossification; however, current methods are limited for mineralisation studies. In this study we demonstrate that culturing cells in the presence of ascorbic acid and 10 mM ß-glycerophosphate (ßGP) significantly increases the rate of extracellular matrix (ECM) synthesis and reduces the time required for mineral deposition to occur to 15 days of culture. Furthermore, the specific expression patterns of Col2a1 and Col10a1 are indicative of ATDC5 chondrogenic differentiation. Fourier transform-infrared spectroscopy analysis and transmission electron microscopy (TEM) showed that the mineral formed by ATDC5 cultures is similar to physiological hydroxyapatite. Additionally, we demonstrated that in cultures with ßGP, the presence of alkaline phosphatase (ALP) is required for this mineralisation to occur, further indicating that chondrogenic differentiation is required for ECM mineralisation. Together, these results demonstrate that when cultured in the presence of ascorbic acid and 10 mM ßGP, ATDC5 cells undergo chondrogenic differentiation and produce a physiological mineralised ECM from Day 15 of culture onwards. The rapid and novel method for ATDC5 culture described in this study is a major improvement compared with currently published methods and this will prove vital in the pursuit of underpinning the molecular mechanisms responsible for poor linear bone growth observed in a number of chronic diseases such as cystic fibrosis, chronic kidney disease, rheumatological conditions and inflammatory bowel disease.

Comparable outcomes in fracture reduction and bone properties with RANKL inhibition and alendronate treatment in a mouse model of osteogenesis imperfecta.

UNLABELLED: We report a direct comparison of receptor activator of nuclear factor kappa B ligand (RANKL) inhibition (RANK-Fc) with bisphosphonate treatment (alendronate, ALN) from infancy through early adulthood in a mouse model of osteogenesis imperfecta. Both ALN and RANK-Fc decreased fracture incidence to the same degree with increases in metaphyseal bone volume via increased number of thinner trabeculae. INTRODUCTION: The potential therapeutic benefit of RANKL inhibitors in osteogenesis imperfecta (OI) is under investigation. We report a direct comparison of RANKL inhibition (RANK-Fc) with bisphosphonate treatment (ALN) from infancy through early adulthood in a model of OI, the oim/oim mouse. METHODS: Two-week-old oim/oim, oim/+, and wildtype (+/+) mice were treated with RANK-Fc 1.5 mg/kg twice per week, ALN 0.21 mg/kg/week or saline (n = 12-20 per group) for 12 weeks. RESULTS: ALN and RANK-Fc both decreased fracture incidence (9.0 ± 3.0 saline 4.4 ± 2.7 ALN, 4.3 ± 3.0 RANK-Fc fractures per mouse). Serum TRACP-5b activity decreased to 65% after 1 month in all treated mice, but increased sacrifice with RANK-Fc to 130-200% at sacrifice. Metaphyseal density was significantly increased with ALN in +/+ and oim/oim mice (p < 0.05) and tended to increase with RANK-Fc in +/+ mice. No changes in oim/oim femur biomechanical parameters occurred with treatment. Both ALN and RANK-Fc significantly increased trabecular number (3.73 ± 0.77 1/mm for oim/oim saline vs 7.93 ± 0.67 ALN and 7.34 ± 1.38 RANK-Fc) and decreased trabecular thickness (0.045 mm ± 0.003 for oim/oim saline vs 0.034 ± 0.003 ALN and 0.032 ± 0.002 RANK-Fc) and separation in all genotypes (0.28 ± 0.08 mm for oim/oim saline vs 0.12 ± 0.010 ALN and 13 ± 0.03 RANK-Fc)., with significant increase in bone volume fraction (BVF) with ALN, and a trend towards increased BVF in RANK-Fc. CONCLUSION: Treatment of oim/oim mice with either a bisphosphonate or a RANK-Fc causes similar decreases in fracture incidence with increases in metaphyseal bone volume via increased number of thinner trabeculae.

Comparison of mineral quality and quantity in iliac crest biopsies from high- and low-turnover osteoporosis: an FT-IR microspectroscopic investigation.

Fourier-transform infrared microspectroscopy (FTIRM) allows analysis of mineral content, mineral crystal maturity and mineral composition at approximately 10-micron spatial resolution. Previous FTIRM analyses comparing 4-micron thick sections from non-decalcified iliac crest biopsies from women with post-menopausal osteoporosis, as contrasted with iliac crest tissue from individuals without evidence of metabolic bone disease, demonstrated significant differences in average mineral content (decreased in osteoporosis) and mineral crystal size/perfection (increased in osteoporosis). More importantly, these parameters, which vary throughout the tissue in relation to the tissue age in healthy bone, showed no such variation in bone biopsies from patients with osteoporosis. The present study compares the spatial and temporal variation in mineral quantity and properties in trabecular bone in high- and low-turnover osteoporosis. Specifically, six biopsies from women (n=5) and one man with high-turnover osteoporosis (age range 39-77) and four women and two men with low turnover osteoporosis (age range 37-63) were compared to ten "normal" biopsies from three men and seven woman (age range: 27-69). "High turnover" was defined as the presence of increased resorptive surface, higher than normal numbers of osteoclasts and greater than or equal to normal osteoblastic activity. "Low turnover" was defined as lower than normal resorptive surface, decreased osteoclast number and less than normal osteoblastic activity. Comparing variations in FTIR-derived values for each of the parameters measured at the surfaces of the trabecular bone to the maximum value observed in multiple trabeculae from each person, the high-turnover samples showed little change in the mineral: matrix ratio, carbonate: amide I ratio, crystallinity and acid phosphate content. The low-turnover samples also showed little change in these parameters, but in contrast to the high-turnover samples, the low-turnover samples showed a slight increase in these parameters, indicative of retarded, but existent resorption and formation. These data indicate that FTIR microspectroscopy can provide quantitative information on mineral changes in osteoporosis that are consistent with proposed mechanisms of bone loss.

Thermal and chemical modification of titanium-aluminum-vanadium implant materials: effects on surface properties, glycoprotein adsorption, and MG63 cell attachment.

The microstructure, chemical composition and wettability of thermally and chemically modified Ti-6Al-4V alloy disks were characterized and correlated with the degree of radiolabeled fibronectin-alloy surface adsorption and subsequent adhesion of osteoblast-like cells. Heating either in pure oxygen or atmosphere (atm) resulted in an enrichment of Al and V within the surface oxide. Heating (oxygen/atm) and peroxide treatment both followed by butanol treatment resulted in a reduction in content of V, but not in Al. Heating (oxygen/atm) or peroxide treatment resulted in a thicker oxide layer and a more hydrophilic surface when compared with passivated controls. Post-treatment with butanol, however, resulted in less hydrophilic surfaces than heating or peroxide treatment alone. The greatest increases in the adsorption of radiolabeled fibronectin following treatment were observed with peroxide/butanol-treated samples followed by peroxide/butanol and heat/butanol, although binding was only increased by 20-40% compared to untreated controls. These experiments with radiolabeled fibronectin indicate that enhanced adsorption of the glycoprotein was more highly correlated with changes in chemical composition, reflected in a reduction in V content and decrease in the V/Al ratio, than with changes in wettability. Despite promoting only a modest elevation in fibronectin adsorption, the treatment of disks with heat or heat/butanol induced a several-fold increase in the attachment of MG63 cells promoted by a nonadhesive concentration of fibronectin that was used to coat the pretreated disks compared to uncoated disks. Therefore, results obtained with these modifications of surface properties indicate that an increase in the absolute content of Al and/or V (heat), and/or in the Al/V ratio (with little change in hydrophilicity; heat+butanol) is correlated with an increase in the fibronectin-promoted adhesion of an osteoblast-like cell line. It would also appear that the thermal treatment-induced enhancement of cell adhesion in the presence of this integrin-binding protein is due to its increased biological activity, rather than a mass effect alone, that appear to be associated with changes in chemical composition of the metallic surface. Future studies will investigate the influence of the surface chemical composition of various implantable alloys on protein adsorption and receptor-mediated cell adhesion. In addition, by altering the properties of bound osteogenic protein enhancing exposure to cell integrin binding domains, it may be possible to develop implant surfaces which enhance the attachment, adhesion and developmental response of osteoblast precursors leading to accelerated osseointegration.

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

UNLABELLED: 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. INTRODUCTION: 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. MATERIALS AND METHODS: 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. RESULTS AND CONCLUSIONS: 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.

Effect of hormone replacement therapy on bone quality in early postmenopausal women.

HRT is an effective prophylaxis against postmenopausal bone loss. Infrared imaging of paired iliac crest biopsies obtained at baseline and after 2 years of HRT therapy demonstrate an effect on the mineral crystallinity and collagen cross-links that may affect bone quality. Several studies have demonstrated that hormonal replacement therapy (HRT) is an effective prophylaxis against postmenopausal bone loss, although the underlying mechanisms are still debated. Infrared spectroscopy has been used previously for analyzing bone mineral crystallinity and three-dimensional structures of collagen and other proteins. In the present study, the technique of Fourier transform infrared microscopic imaging (FTIRI) was used to investigate the effect of estrogen on bone quality (arbitrarily defined as mineral/matrix ratio, mineral crystallinity/maturity, and relative ratio of collagen cross-links [pyridinoline/ deH-DHLNL]) at the ultrastructural level, in mineralized, thin tissue sections from double (before and after administration of HRT regimen; cyclic estrogen and progestogen [norethisterone acetate]) iliac crest biopsy specimens from 10 healthy, early postmenopausal women who were not on any medication with known influence on calcium metabolism. FTIRI allows the analysis of undemineralized thin tissue sections (each image analyzes a 400 x 400 microm2 area with a spatial resolution of approximately 6.3 mm). For each bone quality variable considered, the after-treatment data exhibited an increase in the mean value, signifying definite changes in bone properties at the molecular level after HRT treatment. Furthermore, these findings are consistent with suppressed osteoclastic activity.

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.

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.

Intermittent and continuous administration of the bisphosphonate ibandronate in ovariohysterectomized beagle dogs: effects on bone morphometry and mineral properties.

Bisphosphonates have emerged as a valuable treatment for postmenopausal osteoporosis. Bisphosphonate treatment is usually accompanied by a 3-6% gain in bone mineral density (BMD) during the first year of treatment and by a decrease in bone turnover. Despite low bone turnover, BMD continues to increase slowly beyond the first year of treatment. There is evidence that bisphosphonates not only increase bone volume but also enhance secondary mineralization. The present study was conducted to address this issue and to compare the effects of continuous and intermittent bisphosphonate therapy on static and dynamic parameters of bone structure, formation, and resorption and on mineral properties of bone. Sixty dogs were ovariohysterectomized (OHX) and 10 animals were sham-operated (Sham). Four months after surgery, OHX dogs were divided in six groups (n = 10 each). They received for 1 year ibandronate daily (5 out of 7 days) at a dose of 0, 0.8, 1.2, 4.1, and 14 microg/kg/day or intermittently (65 microg/kg/day, 2 weeks on, 11 weeks off). Sham dogs received vehicle daily. At month 4, there was a significant decrease in bone volume in OHX animals (p < 0.05). Doses of ibandronate >/= 4.1 microg/kg/day stopped or completely reversed bone loss. Bone turnover (activation frequency) was significantly depressed in OHX dogs given ibandronate at the dose of 14 microg/kg/day. This was accompanied by significantly higher crystal size, a higher mineral-to-matrix ratio, and a more uniformly mineralized bone matrix than in control dogs. This finding lends support to the hypothesis that an increase in secondary mineralization plays a role in gain in BMD associated with bisphosphonate treatment. Moreover, intermittent and continuous therapies had a similar effect on bone volume. However, intermittent therapy was more sparing on bone turnover and bone mineral properties. Intermittent therapy could therefore represent an attractive alternative approach to continuous therapy.

Infrared microspectroscopic imaging of biomineralized tissues using a mercury-cadmium-telluride focal-plane array detector.

A 64 x 64 mercury-cadmium-telluride focal-plane array detector attached to a Fourier transform infrared microscope was used to spectroscopically image 5 microm sections of canine alveolar bone tissue in the fingerprint region of the infrared spectrum. By ratioing the relative intensities of specific bands across the images, it is possible to obtain spatial distributions of the mineral-to-matrix ratio and mineral maturity as a function of distance from an osteon.

Matrix vesicles promote mineralization in a gelatin gel.

Extracellular matrix vesicles (MVs) are associated with initial calcification in a variety of tissues, but the mechanisms by which they promote mineralization are not certain. In this study, MVs isolated from fourth passage rat growth plate chondrocyte cultures were included within a gelatin gel into which calcium and phosphate ions diffused from opposite ends. In this gel, apatite formation occurs by 3.5 days in the absence of mineralization promoters, allowing measurement of the ability of different factors to "nucleate" apatite before this time or to assess the effects of molecules which modulate the rate and extent of mineral deposition. Mineral ion accumulation and crystal type are assayed at 5 days. In this study, MV protein content in the central band of a 10% gelatin gel was varied by including 100 microliters of a Tris-buffered solution containing 0-300 micrograms/ml MV protein. There was a concentration-dependent increase in mineral accretion. Whereas 10 micrograms MV protein in the gel did not significantly promote apatite formation as compared with vesicle-free gels, 20 and 30 micrograms MV protein in the gel did promote apatite deposition. Inclusion of 10 mM beta-glycerophosphate in the gels, along with MVs, did not significantly increase apatite formation despite the demonstrable alkaline phosphatase activity of the MVs. In contrast, MVs at all concentrations significantly increased apatite accumulation when proteoglycan aggregates or ATP, inhibitors of apatite formation and proliferation, were included in the gel. Slight increases in calcium, but not phosphate accumulation, were also noted when an ionophore was included with the MVs to facilitate Ca ion transport into the vesicles. FT-IR analysis of the mineral formed in the vesicle-containing gels revealed the presence of a bone-like apatite. These data suggest that MVs facilitate mineralization by providing enzymes that modify inhibitory factors in the extracellular matrix, as well as by providing a protected environment in which mineral ions can accumulate.

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.

FTIR microspectroscopic analysis of human iliac crest biopsies from untreated osteoporotic bone.

Historically, osteoporosis has been defined as a disease in which there is "too little bone, but what there is, is normal." As a result of research design and sample selection limitations, published data contradict and confirm the historical definition. Because of these limitations, it has been hard to assess the contribution of mineral quality to mechanical properties, and to select therapeutic protocols that optimize bone mineral properties. The coupling of an optical microscope to an infrared spectrometer enables the acquisition of spectral data at known sites in a histologic section of mineralized tissue without loss of topography and/or orientation. The use of second-derivative spectroscopy coupled with curve-fitting techniques allows the qualitative and quantitative assessment of mineral quality (crystallite size and perfection, mineral:matrix ratio) at well-defined morphologic locations. We have previously applied these techniques to the study of normal human osteonal, cortical, and trabecular bone. The results indicated that the newly deposited bone mineral is less "crystalline/mature" than the older one. In the present study, Fourier transform infrared microspectroscopy (FTIRM) was applied to the study of human osteonal and cortical bone from iliac crest biopsies of untreated osteoporotic patients. The hypothesis tested was that osteoporotic bone mineral is monotonically different in its properties expressed as "crystallinity/maturity" than the normal. The results indicate significant differences in the mineral properties as expressed by crystal size and perfection, with the mineral from osteoporotic bone being more crystalline/mature than the normal.

Fourier transform infrared microspectroscopic analysis identifies alterations in mineral properties in bones from mice transgenic for type X collagen.

Type X collagen has been implicated in the morphogenetic events of endochondral ossification (EO), including the calcification of hypertrophic cartilage and trabeculae prior to their replacement by bone and marrow. Recently, transgenic mice, which expressed a truncated collagen X protein, were reported to exhibit morphologic alterations in all tissues arising through EO. Specifically, the growth plates were compressed within the zone of cartilage hypertrophy, and the number and size of calcified trabeculae were reduced. The condition in the mouse is comparable to Schmid metaphyseal chondrodysplasia in humans for which, to date, 20 defined type X collagen mutations have been reported. The transgenic mouse showed no alterations in mineralization by conventional histology, however, it did show a decrease in newly formed bony trabeculae, and a thinning of periosteal bones. Fourier transform infrared (FTIR) spectroscopy has previously been shown to provide quantitative and qualitative information about the relative amount of mineral and carbonate present, mineral composition, and crystal perfection. To determine whether the expression of abnormal collagen X molecules had an effect on mineral properties, the "quality" of mineral crystals was analyzed in thin sections of tibia from day 17 and day 25 genotypically negative (normal) and positive (mutant) mice from several independent transgenic mouse lines showing varying degrees of the mutant phenotype, by means of Fourier transform infrared microscopic analysis (FTIRM). The results indicate definite differences between normal and transgenic mice calcified cartilage mineral, both in the amount present and the "quality" of the crystals. Calcified cartilage mineral from transgenic mice exhibited less crystallinity and higher acidic phosphate content than the corresponding mineral from normal specimens.

Subcutaneous microvascular (capillary) calcification. Another basis for livedo-like skin changes?

A morbidly obese woman with acute renal failure, high serum phosphorus and slightly depressed serum calcium levels, developed areas of induration in the subcutis with associated livedo reticularis. Later, the subcutis became necrotic and the skin ulcerated. The arterioles, and notably widened occluded capillaries, were found to contain calcium and phosphorus as determined by energy dispersive spectrometry. X-ray diffraction of the subcutis in an early stage of this lesion showed that the mineral was most likely a poorly crystalline hydroxyapatite.

Adenosine 5'-triphosphate promotes mineralization in differentiating chick limb-bud mesenchymal cell cultures.

When chick limb-bud mesenchymal cells are plated in micromass culture, they differentiate to form a mineralizable cartilage matrix. Previous studies have demonstrated that, when the total inorganic phosphate concentration of the medium is adjusted to 3-4 mM by adding inorganic phosphate to the basal medium, the mineralized matrix formed resembles that of chick calcified cartilage in ovo. When the high-energy phosphates adenosine 5'-triphosphate (ATP) or creatine phosphate are used as supplements in place of inorganic phosphate, the mineralized matrix as analyzed by electron microscopy and Fourier transform infrared microscopy is also similar to that in ovo. This is in marked contrast to the mineralized matrix formed in the presence of 2.5-5 mM beta-glycerophosphate, where mineral deposition is random and mineral crystal sizes in general are larger. This is also in contrast to the known ability of ATP to inhibit mineral deposition in solution in the absence of cells. In the differentiating mesenchymal cell culture system, ATP does not alter the rate of cell proliferation (DNA content), the rate of matrix synthesis (3H-leucine uptake), the mean crystallite length, or the rate of mineral deposition (45Ca uptake) when contrasted with cultures supplemented with inorganic phosphate. However, ATP does increase the mineral to matrix ratio, especially around the edge of the culture, where a type I collagen matrix is presented. It is suggested that ATP promotes mineral deposition by providing a high-energy phosphate source, which may be used to phosphorylate extracellular matrix proteins and to regulate calcium flux through cell membranes.

Vitamin B6 deficiency experimentally-induced bone and joint disorder: microscopic, radiographic and biochemical evidence.

In the present study the effect of pyridoxine deficiency on the ultrastructure and morphology of bone and its metabolism was examined in the rapidly growing chick. Pyridoxine-deficient animals had tibias of reduced dry weight and cortical thickness. Histomorphometry demonstrated a disproportionately high eroded surface, lower amount of osteoid tissue and reduced mineralized trabecular width. Anterior-posterior radiographs of the tibiotarsometatarsal joint showed reduced secondary ossification centres and coarse trabeculation. Decalcified metaphyseal cartilage showed irregular trabeculas and a markedly reduced amount of Fast-green counterstain matrix suggesting that there is less collagen present and in turn less availability for matrix to be laid down for later calcification. Plasma activity of the bone alkaline phosphatase isoenzyme (EC 3.1.3.1) was decreased. Plasma Ca and PO4 levels did not vary. The present bone study referring to a pseudo-lathyritic state in which collagen maturation is not completely achieved supports the hypothesis that pyridoxine is an essential nutrient for the connective tissue matrix.

Intramedullary deposits of hydroxyapatite crystals in a patient with rheumatoid arthritis.

A 63-year-old women with a 12-year history of rheumatoid arthritis (RA) had calcific deposits in the periarticular soft tissues with subsequent involvement of the medullary canals of adjacent bones. Except for the RA, there was no evidence of other abnormalities of the bones or mineral homeostasis. Chemical analyses of the deposits identified the crystalline material as apatite. Although the deposition of apatite in the soft tissues of RA is known to occur rarely, this is the first known case of intraosseous apatite deposition.

Retinoic acid induces rapid mineralization and expression of mineralization-related genes in chondrocytes.

Numerous studies of experimental hypo- and hypervitaminosis A have long suggested that retinoic acid (RA) is involved in chondrocyte maturation during endochondral ossification and skeletogenesis. However, the specific and direct roles of RA in these complex processes remain unclear. Based on recent studies from our laboratories, we tested the hypothesis that RA induces the expression of genes associated with the terminal mineralization phase of chondrocyte maturation and promotes apatite deposition in the extracellular matrix. Cell populations containing chondrocytes at advanced stages of maturation were isolated from the upper portion of Day 18 chick embryo sterna and grown for 2 weeks in monolayer until confluent. The cells were then treated with low doses (10-100 nM) of RA for up to 6 days in the presence of a phosphate donor (beta-glycerophosphate) but in the absence of ascorbic acid. Within 4 days of treatment, RA dramatically induced expression of the alkaline phosphatase (APase), osteonectin, and osteopontin genes, caused a several-fold increase in APase activity, and provoked massive mineral formation while it left type X collagen gene expression largely unchanged. The mineral had a mean Ca/Pi molar ratio of 1.5; Fourier transform infrared spectra confirmed that it represented hydroxyapatite. Mineralization was completely abolished by treatment with parathyroid hormone; this profound effect confirmed that RA induced cell-mediated mineralization and not nonspecific precipitation. When cultures were treated with both RA and ascorbic acid, there was a slight further increase in APase activity and increased calcium accumulation. The effects of RA were also studied in cultures of immature chondrocytes isolated from the caudal portion of sternum; however, RA only had minimal effects on mineralization and gene expression in these cells. Thus, RA appears to be a rapid, potent, maturation-dependent, ascorbate-independent promoter of terminal maturation and matrix calcification in chondrocytes.