Lyudmila Spevak

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.

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 noncrystalline 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 Ca2+. Binding to HA did not alter the secondary structure of bone OPN, but induced a small increase of β-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.

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 β‐glycerophosphate (β‐GP) and ascorbic acid and within 7 days in the absence of β‐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 β‐GP and ascorbic acid and approximately 1.2 in the presence of β‐GP and ascorbic acid. By comparison, these ratios in fetal rat calvarial cells without β‐GP equal 0 and with β‐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/106 cells compared with <1–2.2 ng/106 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.

Effects of Bone CS-Proteoglycans, DS-Decorin, and DS-Biglycan on Hydroxyapatite Formation in a Gelatin Gel

Abstract. 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 μg/ml) increased apatite formation relative to proteoglycan-free controls at 3.5 days. The CS-proteoglycan mixture was less effective at 50 μg/ml than at 10 μg/ml. DS-biglycan was similarly most effective at 5–25 μg/ml. At 5 days, when apatite growth and proliferation were assessed, 10 and 50 μg/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 μg/ml CS-proteoglycan and 10 and 50 μg/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/μmol versus 0.0098 ml/μmol). DS-biglycan binding was more specific with fewer binding sites (3.5 μmol/m2 compared with 18.2 μmol/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.

Use of FTIR Spectroscopic Imaging to Identify Parameters Associated With Fragility Fracture

BMD does not entirely explain an individuals risk of fracture. The purpose of this study was to assess whether specific differences in spatially resolved bone composition also contribute to fracture risk. These differences were assessed using Fourier transform infrared spectroscopic imaging (FTIRI) and analyzed through multiple logistic regression. Models were constructed to determine whether FTIRI measured parameters describing mineral content, mineral crystal size and perfection, and collagen maturity were associated with fracture. Cortical and cancellous bone were independently evaluated in iliac crest biopsies from 54 women (32 with fractures, 22 without) who had significantly different spine but not hip BMDs and ranged in age from 30 to 83 yr. The parameters that were significantly associated with fracture in the model were cortical and cancellous collagen maturity (increased with increased fracture risk), cortical mineral/matrix ratio (higher with increased fracture risk), and cancellous crystallinity (increased with increased fracture risk). As expected, because of its correlation with cortical but not cancellous bone density, hip BMD was significantly associated with fracture risk in the cortical but not the cancellous model. This research suggests that additional parameters associated with fracture risk should be targeted for therapies for osteoporosis.

Dentin Sialoprotein (DSP) Has Limited Effects on In Vitro Apatite Formation and Growth

Abstract. 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 μg/ml) slightly increased the yield of HA formed at 3.5 and 5 days, while at higher concentrations (50–100 μg/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.

Bone Morphogenetic Protein‐2 Restores Mineralization in Glucocorticoid‐Inhibited MC3T3‐E1 Osteoblast Cultures

The anti‐glucocorticoid potential of BMP‐2 in osteoblasts was tested in MC3T3‐E1 cells using dexamethasone (1 μM) and rhBMP‐2 (10 or 100 ng/ml). rhBMP‐2 restored mineralization but not condensation or collagen accumulation. These results demonstrate the potential and limitations of BMPs in counteracting glucocorticoids.

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 µm 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.

DSPP effects on in vivo bone mineralization

Dentin sialophosphoprotein has been implicated in the mineralization process based on the defective dentin formation in Dspp null mice (Dspp-/-). Dspp is expressed at low levels in bone and Dspp-/- femurs assessed by quantitative micro-computed tomography (micro-CT) and Fourier transform infrared spectroscopic imaging (FTIRI) exhibit some mineral and matrix property differences from wildtype femurs in both developing and mature mice. Compared to wildtype, Dspp-/- mice initially (5 weeks) and at 7 months had significantly higher trabecular bone volume fractions and lower trabecular separation, while at 9 months, bone volume fraction and trabecular number were lower. Cortical bone mineral density, area, and moments of inertia in Dspp-/- were reduced at 9 months. By FTIRI, Dspp-/- animals initially (5 months) contained more stoichiometric bone apatite with higher crystallinity (crystal size/perfection) and lower carbonate substitution. This difference progressively reversed with age (significantly decreased crystallinity and increased acid phosphate content in Dspp-/- cortical bone by 9 months of age). Mineral density as determined in 3D micro-CT and mineral-to-matrix ratios as determined by 2D FTIRI in individual cortical and trabecular bones were correlated (r(2)=0.6, p<0.04). From the matrix analysis, the collagen maturity of both cortical and trabecular bones was greater in Dspp-/- than controls at 5 weeks; by 9 months this difference in cross-linking pattern did not exist. Variations in mineral and matrix properties observed at different ages are attributable, in part, to the ability of the Dspp gene products to regulate both initial mineralization and remodeling, implying an effect of Dspp on bone turnover.

Fourier Transform Infrared Imaging Microspectroscopy and Tissue-Level Mechanical Testing Reveal Intraspecies Variation in Mouse Bone Mineral and Matrix Composition

Fracture susceptibility is heritable and dependent upon bone morphology and quality. However, studies of bone quality are typically overshadowed by emphasis on bone geometry and bone mineral density. Given that differences in mineral and matrix composition exist in a variety of species, we hypothesized that genetic variation in bone quality and tissue-level mechanical properties would also exist within species. Sixteen-week-old female A/J, C57BL/6J (B6), and C3H/HeJ (C3H) inbred mouse femora were analyzed using Fourier transform infrared imaging and tissue-level mechanical testing for variation in mineral composition, mineral maturity, collagen cross-link ratio, and tissue-level mechanical properties. A/J femora had an increased mineral-to-matrix ratio compared to B6. The C3H mineral-to-matrix ratio was intermediate of A/J and B6. C3H femora had reduced acid phosphate and carbonate levels and an increased collagen cross-link ratio compared to A/J and B6. Modulus values paralleled mineral-to-matrix values, with A/J femora being the most stiff, B6 being the least stiff, and C3H having intermediate stiffness. In addition, work-to-failure varied among the strains, with the highly mineralized and brittle A/J femora performing the least amount of work-to-failure. Inbred mice are therefore able to differentially modulate the composition of their bone mineral and the maturity of their bone matrix in conjunction with tissue-level mechanical properties. These results suggest that specific combinations of bone quality and morphological traits are genetically regulated such that mechanically functional bones can be constructed in different ways.