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.

Spectroscopic markers of bone quality in alendronate-treated postmenopausal women.

UNLABELLED: Comparison of infrared spectroscopic images of sections from biopsies of placebo-treated post-menopausal women and women treated for 3 years with 10 mg/day alendronate demonstrated significant increases in cortical bone mineral content, no alterations in other spectroscopic markers of "bone quality," but a decrease in tissue heterogeneity. METHODS: The material properties of thick sections from iliac crest biopsies of seven alendronate-treated women were compared to those from ten comparably aged post-menopausal women without bone disease, using infrared spectroscopic imaging at approximately 7 microm spatial resolution. Parameters evaluated were mineral/matrix ratio, crystallinity, carbonate/amide I ratio, and collagen maturity. The line widths at half maximum of the pixel histograms for each parameter were used as measures of heterogeneity. RESULTS: The mineral content (mineral/matrix ratio) in the cortical bone of the treated women's biopsies was higher than that in the untreated control women. Crystallinity, carbonate/protein, and collagen maturity indices were not significantly altered; however, the pixel distribution was significantly narrowed for all cortical and trabecular parameters with the exception of collagen maturity in the alendronate treatment group. CONCLUSIONS: The increases in mineral density and decreased fracture risk associated with bisphosphonate treatment may be counterbalanced by a decrease in tissue heterogeneity, which could impair tissue mechanical properties. These consistent data suggest that alendronate treatment, while increasing the bone mass, decreases the tissue heterogeneity.

Importance of phosphorylation for osteopontin regulation of biomineralization.

Previous in vitro and in vivo studies demonstrated that osteopontin (OPN) is an inhibitor of the formation and growth of hydroxyapatite (HA) and other biominerals. The present study tests the hypotheses that the interaction of OPN with HA is determined by the extent of protein phosphorylation and that this interaction regulates the mineralization process. Bone OPN as previously reported inhibited HA formation and HA-seeded growth in a gelatin-gel system. A transglutaminase-linked OPN polymer had similar effects. Recombinant, nonphosphorylated OPN and chemically dephosphorylated OPN, had no effect on HA formation or growth in this system. In contrast, highly phosphorylated milk OPN (mOPN) promoted HA formation. The mOPN stabilized the conversion of amorphous calcium phosphate (a non-crystalline constituent of milk) to HA, whereas bone OPN had a lesser effect on this conversion. Mixtures of OPN and osteocalcin known to form a complex in vitro, unexpectedly promoted HA formation. To test the hypothesis that small alterations in protein conformation caused by phosphorylation account for the differences in the observed ability of OPN to interact with HA, the conformation of bone OPN and mOPN in the presence and absence of crystalline HA was determined by attenuated total reflection (ATR) infrared (IR) spectroscopy. Both proteins exhibited a predominantly random coil structure, which was unaffected by the addition of Ca(2+). Binding to HA did not alter the secondary structure of bone OPN, but induced a small increase of beta-sheet (few percent) in mOPN. These data taken together suggest that the phosphorylation of OPN is an important factor in regulating the OPN-mediated mineralization process.

Osteopontin facilitates bone resorption, decreasing bone mineral crystallinity and content during calcium deficiency.

Osteopontin null-mice were previously shown to have bones containing more mineral and larger mineral crystals. These bones were independently seen to be resistant to ovariectomy-induced remodeling. To separate the physicochemical effects of osteopontin, which is an in vitro inhibitor of mineral crystal formation and growth, from effects of osteopontin on in vivo bone remodeling, this study examined mature (5-month-old) osteopontin-null (Opn-/-) and wildtype (WT) mice given a calcium-deficient diet. Biochemical parameters were measured during 4 weeks of Ca deficiency, followed by 1 week of refeeding adequate Ca. Ca deficiency caused a transiently greater rise in bone resorption in WT than Opn-/- mice (P = 0.01), whereas only the Opn-/- mice tended to increase Ca absorption (P = 0.08), yet both groups showed elevated levels of parathyroid hormone (PTH) (P < 0.001). The rise in markers of bone formation due to Ca deficiency was similar in both groups during Ca deficiency. Fourier transform infrared microspectroscopy assessed mineral properties at 20 microm spatial resolution in different anatomic regions of the bone. The Ca-deficient Opn-/- animals had slightly increased mineral content as compared to the WT, and there was a significant increase in the mineral content of older (endosteal) bone, implying that osteoclast recruitment was impaired. Crystallinity in the Ca-deficient Opn-/- bones was increased relative to the Ca-deficient WT at all sites except adjacent to the periosteum (younger mineral). These data suggest that osteopontin has both a physicochemical effect (inhibiting crystal growth and crystal proliferation) and a role in osteoclast recruitment, and in its absence, extraskeletal organs maintain calcium homeostasis.

Osteopontin deficiency increases mineral content and mineral crystallinity in mouse bone.

Fourier transform infrared microspectroscopy (FTIRM) and infrared imaging (FTIRI) were used to characterize the mineral in bones of two different lines of Opn-deficient (Opn-/-) mice and their background-matched wild-type controls (Opn+/+). Sections of tibia and femur from 12-week-old and 16-week-old mice were evaluated with a spatial resolution between 10 microm (FTIRM) and 7 microm (FTIRI). FTIRI was used to examine 400 microm x 400 microm areas in cortical bone and trabecular bone and FTIRM examined selected 20 microm x 20 microm areas at sites within these anatomically defined areas. Despite the absence of an obvious phenotype in Opn-deficient mice, being undetectable by radiographic and histological methods, FTIRM analyses revealed that the relative amount of mineral in the more mature areas of the bone (central cortical bone) of Opn-knockout mice was significantly increased. Moreover, mineral maturity (mineral crystal size and perfection) throughout all anatomic regions of the Opn-deficient bone was significantly increased. The 2-dimensional, color-coded data (images) produced by FTIRI showed similar increases in mineral maturity in the Opn-/- bone, however, the crystallinity parameters were less sensitive, and significance was not achieved in all areas analyzed. Nonetheless, the findings of increased mineral content and increased crystal size/perfection in both lines of Opn-deficient mice at both ages are consistent with in vitro data indicating that Opn is a potent inhibitor of mineral formation and mineral crystal growth and proliferation, and also support a role for Opn in osteoclast recruitment and function.

Optimal methods for processing mineralized tissues for Fourier transform infrared microspectroscopy.

Fourier transform infrared microspectroscopy (FTIRM) and infrared imaging (FTIRI) are techniques utilized in the analysis of bone mineral and matrix properties in health and disease. Since the spatial arrangement of bone tissue is conserved using FTIRM and FTIRI, quantitative data can be obtained on bone mineral (hydroxyapatite) crystalline size and composition, and on matrix structure and composition at discrete anatomic locations with a spatial resolution from approximately 7 mm (FTIRI) to 10 mm (FTIRM). To section bone for FTIRM and FTIRI, it must be preserved ("fixed") to maintain its properties, and embedded in a hard supportive material. Since most of the embedding media have components that spectrally overlap the components of mineralized tissues, it is critical to define optimal embedding and fixation protocols that have the least effect on mineral and matrix spectra. In the current study, the spectra of mouse calvaria in seven different fixatives and six different commonly used embedding media were assessed by FTIRM and FTIRI. The fixatives evaluated were absolute ethanol, 70% ethanol, glycerol, formaldehyde, EM fixative, and formalin in cacodylate or phosphate-buffered saline. The embedding media tested were Araldite, Epon, JB-4, LR White, PMMA, and Spurr. Comparisons were made to FTIR spectra obtained from unprocessed ground calvaria and to spectra of cryosections of unfixed tissue, fast-frozen in polyvinyl alcohol (5% PVA). Non-aqueous fixatives and embedding in LR White, Spurr, Araldite, and PMMA had the least effect on the spectral parameters measured (mineral to matrix ratio, mineral crystallinity, and collagen maturity) compared with cryo-sectioned calvaria and non-fixed, non-embedded calvaria in KBr pellets.

Establishment of an osteoid preosteocyte-like cell MLO-A5 that spontaneously mineralizes in culture.

The mechanisms controlling the initiation of mineralization of bone matrix are not clear. To examine this process, we established a cell line called MLO-A5 that mineralizes in sheets, not nodules, within 3 days of culture in the presence of beta-glycerophosphate (beta-GP) and ascorbic acid and within 7 days in the absence of beta-GP and ascorbic acid. The mineral formed in both cases was shown to be bonelike apatite by Fourier transformed infrared (FTIR) spectroscopy. Mineral-to-matrix ratios (min/matrix) calculated from the FTIR data, which are related directly to ash weight, were approximately 0.4 in the absence of beta-GP and ascorbic acid and approximately 1.2 in the presence of beta-GP and ascorbic acid. By comparison, these ratios in fetal rat calvarial cells without beta-GP equal 0 and with beta-GP 1.9. This cell line and three others (MLO-A2, -D1, and -D6) were isolated from the long bones of transgenic mice expressing the large T-antigen driven by the osteocalcin promoter, the same mice from which the osteocyte-like cell line MLO-Y4 was isolated.(1) The cell lines were selected based on a dendritic or stellate morphology. MLO-A5 cells express high alkaline phosphatase, collagen type 1, parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) receptor, bone sialoprotein (BSP), and osteocalcin (767 ng/10(6) cells compared with <1-2.2 ng/10(6) cell for primary mouse osteoblasts and five osteoblast cell lines). The single unique feature of the MLO-A5 cells compared with the other three nonmineralizing cell lines is the high expression of messenger RNA (mRNA) for BSP. These cell lines may represent stages of osteocyte differentiation and the MLO-A5 cells represent the postosteoblast, preosteocyte responsible for triggering mineralization of osteoid.

Dentin sialoprotein (DSP) has limited effects on in vitro apatite formation and growth.

Sialoproteins such as bone sialoprotein (BSP) and dentin sialoprotein (DSP) accumulate at the mineralization fronts in bone and dentin, respectively, suggesting they have some function in the mineralization process. BSP, a highly phosphorylated protein rich in polyglutamate repeats, is an effective nucleator of hydroxyapatite (HA) formation in vitro. The present study examines the effect of DSP, a low phosphorylated but related sialoprotein, on the formation and growth of HA. In vitro, in a gelatin gel diffusion system, DSP at low concentrations (<25 microg/ml) slightly increased the yield of HA formed at 3.5 and 5 days, while at higher concentrations (50-100 microg/ml) it slightly inhibited accumulation. Fewer mineral crystals were formed in the presence of high concentrations of DSP but they tended to aggregate (making them appear larger by electron microscopic analysis) than those formed in DSP-free gels. X-ray diffraction line broadening analysis failed to show significant changes in c-axis crystal dimensions with increasing DSP concentration. When HA-seed crystals were coated with DSP before inclusion in the gelatin gel there was a reduction in mineral accumulation relative to HA-seeds which had not been coated with DSP, but the extent of inhibition was significantly less than that seen in this system with other mineralized tissue matrix sialoproteins, such as osteopontin or BSP. The low affinity of DSP for well-characterized seed crystals and the limited effect of this protein on HA formation and growth suggest that the role of DSP in dentin is not primarily that of a mineralization regulator.

Effects of bone CS-proteoglycans, DS-decorin, and DS-biglycan on hydroxyapatite formation in a gelatin gel.

The small leucine-rich bone proteoglycans, biglycan and decorin, can be purified by chromatography on hydroxyapatite columns, demonstrating their potential affinities for bone apatite. To determine their effects on in vitro apatite formation and growth, a mixture of the chondroitin-sulfate (CS) bone proteoglycans, or purified fractions of the dermatan sulfate (DS) containing proteoglycans, DS-decorin and DS-biglycan obtained from skin and articular cartilage, respectively, were analyzed in a gelatin gel diffusion system in which apatite formation occurs in the absence of proteins in a 3.5 day period. Low concentrations of the bone CS-proteoglycan mixture and low DS-biglycan concentrations (5-25 microg/ml) increased apatite formation relative to proteoglycan-free controls at 3.5 days. The CS-proteoglycan mixture was less effective at 50 microg/ml than at 10 microg/ml. DS-biglycan was similarly most effective at 5-25 microg/ml. At 5 days, when apatite growth and proliferation were assessed, 10 and 50 microg/ml of both CS-bone proteoglycan and DS-biglycan increased mineral yields. DS-decorin, in contrast, had no significant effect on mineral accumulation at any of these concentrations. In seeded growth experiments, 1 and 10 microg/ml CS-proteoglycan and 10 and 50 microg/ml DS-biglycan were significant effective inhibitors of mineral accretion, whereas DS-decorin showed no tendency to inhibit seeded growth. Using molar extinction coefficients to determine concentrations, the binding of DS-biglycan and DS-decorin to apatite (specific surface 54 m2/g) was determined using a Langmuir adsorption isotherm model. DS-biglycan had a greater affinity for apatite than DS-decorin (0.285 ml/micromol versus 0.0098 ml/micromol). DS-biglycan binding was more specific with fewer binding sites (3.5 micromol/m2 compared with 18. 2 micromol/m2 for DS-decorin). Data suggest that of the small proteoglycans, biglycan may play a more significant role than decorin in the regulation of mineralization.

Persistence of complexed acidic phospholipids in rapidly mineralizing tissues is due to affinity for mineral and resistance to hydrolytic attack: in vitro data.

Acidic phospholipids, complexed with calcium and inorganic phosphate, are components of extracellular matrix vesicles. Both the complexed acidic phospholipids and matrix vesicles have previously been shown to serve as hydroxyapatite (HA) nucleators in solution and when implanted in a muscle pouch. The present study supplies evidence that complexed acidic phospholipids can persist in mineralizing tissues both because of their affinity for HA and because of their resistance to hydrolysis by phospholipase A2. Calcium-phosphatidylserine-phosphate complex (CPLX-PS) synthesized with 14C-labeled phosphatidylserine (PS) was used to measure CPLX-PS affinity for HA using a Langmuir adsorption isotherm model. The affinity was shown to be higher and more specific than that of PS itself (K = 8.66 ml/mumol; N, the number of binding sites = 20.4 mumol/m2 as compared with previously reported values for PS of K = 3.33 ml/mumol, and N = 4.87 mumol/m2). Incorporated into synthetic liposomes and incubated in a calcium phosphate solution in which mineralization is induced by an ionophore, CPLX-PS showed behavior distinct from free PS. As previously reported, PS in these liposomes totally blocked HA formation. On the other hand, CPLX-PS in similar concentrations had a varied response, having no effect, slightly inhibiting, or actually promoting HA formation. CPLX-PS was also shown to be a poorer substrate for phospholipase A2 than PS, with Km = 4.63 mM for CPLX-PS and Km = 0.27 mM for PS; and Vmax = 0.029 ml/minute for CPLX-PS and Vmax = 0.066 ml/minute for PS. These data explain how complexed acidic phospholipids may persist in the growth plate and facilitate initial mineral deposition.