Transmission FT-IR microspectroscopy of mineral phases in calcified tissues

Fourier-transform infrared microspectroscopy (FT-IRM) was used to study bone mineralization processes in an in vivo model and in enamel in osteogenesis imperfecta. Finally, the ability of FT-IRM to map new bone formed in implanted macroporous calcium phosphate biomaterial from sections was reported for the first time. FTIRM allowed the correlation of the microstructure of bone formation in the in vivo model with modifications in carbonate and phosphate environments of the mineral phases during maturation. FT-IRM analysis on enamel sections revealed changes in the mineral environment of carbonate and phosphate ions and probably in the size of enamel crystals. These modifications contributed to the fragility of enamel in osteogenesis imperfecta. The infrared functional group imaging of a part of implanted biomaterial and the bone ingrowth provided the visualization of chemical modifications occurring in biomaterial implants at 20 μm spatial resolution. The use of FT-IRM, in conjunction with appropriate sampling methods and data analysis should provide further insight into the molecular structure of mineral phases of calcified tissues and help to elucidate mineralization processes, skeletal disorders and properties of the biomaterials used as bone substitute.     

Ultrastructural localization and biochemical characterization of vitronectin in developing rat bone

This study has used light and electron microscope immunohistochemical and biochemical methods to localize and characterize vitronectin in early bone formation of developing rat mandible with rabbit antimurine vitronectin IgG. Developing jaws of foetuses were collected at embryonic day 15 (day 15) to day 18 from pregnant Wistar rats. After aldehyde fixation, specimens with and without osmium post-fixation were dehydrated and embedded in paraffin, Spurr's resin or LR gold resin for morphological and immunohistochemical examinations. At the light microscope level, in day 15 samples, positive vitronectin immunostaining was observed in small elongated areas of intercellular matrix and osteoblasts. Concomitant with initiation of matrix mineralization at day 16, vitronectin staining was similarly observed in small elongated areas containing intercellular matrix and osteoblasts but not clearly detected in fully mineralized bone matrix. The same staining profile was observed at days 17 and 18. At the ultrastructural level, immunogold particles were clearly detected over unmineralized matrix and cisterns of the rough-surfaced endoplasmic reticulum and the Golgi apparatus of osteoblasts as well as over demineralized bone matrix at day 16--18. In order to assess the presence of vitronectin in the mineral phase, mineral-binding bone proteins were extracted from fresh day 18 specimens using a three-step technique: 4 m guanidine HCl (G1 extract), aqueous EDTA without guanidine HCl (E extract), followed by guanidine HCl. Subsequent Western blot analysis of sodium dodecyl sulphate (SDS)--polyacrylamide gel electrophoresis revealed that the antibodies produced only a single band at an Mr of approximately 73 000 in both G1 and E extracts, indicating the presence of vitronectin in the mineralized bone matrix. These results indicate that, at the onset of bone formation, osteoblasts synthesize and release vitronectin, which is subsequently incorporated into the bone matrix and becomes a specific component of bone tissues. The observation of vitronectin in these critical stages of bone formation suggests that it may be involved in the regulation of bone formation. © 1998 Chapman & Hall     

Duration of infection of jirds (Meriones unguiculatus) with Trichostrongylus colubriformis

Jirds (Meriones unguiculatus) were infected with 700 infective larvae (L3) of Trichostrongylus colubriformis. The fecal egg counts (epg) were followed regularly until 340 days postinoculation, and worm numbers were determined on necropsy at 315 and 350 days postinoculation. The epg increased to a peak of 2,600 on day 24 postinoculation. Thereafter, the epg showed continuous decrease from day 31 until day 318 postinoculation. The rates of inoculum recovery were 0.29-1.43% on day 315 and 0.142-0.428% on day 350 postinoculation. On day 315 postinoculation, 12 male worms were recovered from 2 of 3 animals necropsied. On day 350 postinoculation, all 6 animals were infected, and 6 female and 4 male worms were recovered. The systemic IgM, IgG, and intestinal mucous IgA responses were significantly higher in infected groups compared with negative control groups necropsied on 315 and 350 days postinoculation (P < 0.001). These results confirm that the T. colubriformis-jird system is a useful model for studies on the various host-parasite relationships of chronic trichostrongylosis.     

Mineral tessellation in bone and the stenciling principle for extracellular matrix mineralization

We review here the Stenciling Principle for extracellular matrix mineralization that describes a double-negative process (inhibition of inhibitors) that promotes mineralization in bone and other mineralized tissues, whereas the default condition of inhibition alone prevents mineralization elsewhere in soft connective tissues. The stenciling principle acts across multiple levels from the macroscale (skeleton/dentition vs soft connective tissues), to the microscale (for example, entheses, and the tooth attachment complex where the soft periodontal ligament is situated between mineralized tooth cementum and mineralized alveolar bone), and to the mesoscale (mineral tessellation). It relates to both small-molecule (e.g. pyrophosphate) and protein (e.g. osteopontin) inhibitors of mineralization, and promoters (enzymes, e.g. TNAP, PHEX) that degrade the inhibitors to permit and regulate mineralization. In this process, an organizational motif for bone mineral arises that we call crossfibrillar mineral tessellation where mineral formations – called tesselles – geometrically approximate prolate ellipsoids and traverse multiple collagen fibrils (laterally). Tesselle growth is directed by the structural anisotropy of collagen, being spatially restrained in the shorter transverse tesselle dimensions (averaging 1.6 × 0.8 × 0.8 μm, aspect ratio 2, length range 1.5–2.5 μm). Temporo-spatially, the tesselles abut in 3D (close ellipsoid packing) to fill the volume of lamellar bone extracellular matrix. Poorly mineralized interfacial gaps between adjacent tesselles remain discernable even in mature lamellar bone. Tessellation of a same, small basic unit to form larger structural assemblies results in numerous 3D interfaces, allows dissipation of critical stresses, and enables fail-safe cyclic deformations. Incomplete tessellation in osteomalacia/odontomalacia may explain why soft osteomalacic bones buckle and deform under loading.     

Magnetic resonance microscopy of collagen mineralization

A model mineralizing system was subjected to magnetic resonance microscopy to investigate how water proton transverse (T₂) relaxation times and magnetization transfer ratios can be applied to monitor collagen mineralization. In our model system, a collagen sponge was mineralized with polymer-stabilized amorphous calcium carbonate. The lower hydration and water proton T₂ values of collagen sponges during the initial mineralization phase were attributed to the replacement of the water within the collagen fibrils by amorphous calcium carbonate. The significant reduction in T₂ values by day 6 (p < 0.001) was attributed to the appearance of mineral crystallites, which were also detected by x-ray diffraction and scanning electron microscopy. In the second phase, between days 6 and 13, magnetic resonance microscopy properties appear to plateau as amorphous calcium carbonate droplets began to coalesce within the intrafibrillar space of collagen. In the third phase, after day 15, the amorphous mineral phase crystallized, resulting in a reduction in the absolute intensity of the collagen diffraction pattern. We speculate that magnetization transfer ratio values for collagen sponges, with similar collagen contents, increased from 0.25 ± 0.02 for control strips to a maximum value of 0.31 ± 0.04 at day 15 (p = 0.03) because mineral crystals greatly reduce the mobility of the collagen fibrils.     

Trabecular bone's mechanical properties are affected by its non-uniform mineral distribution

The bone remodeling process takes place at the surface of trabeculae and results in a non-uniform mineral distribution. This will affect the mechanical properties of cancellous bone, because the properties of bone tissue depend on its mineral content. We investigated how large this effect is by simulating several non-uniform mineral distributions in 3D finite element models of human trabecular bone and calculating the apparent stiffness of these models. In the ‘linear model’ we assumed a linear relation between mineral content and Young's modulus of the tissue. In the ‘exponential model’ we included an empirical exponential relation in the model. When the linear model was used the mineral distribution slightly changed the apparent stiffness, the difference varied between an 8% decrease and a 4% increase compared to the uniform model with the same BMD. The exponential model resulted in up to 20% increased apparent stiffness in the main load-bearing direction. A thin less mineralized surface layer (28 μm) and highly mineralized interstitial bone (mimicking mineralization resulting from anti-resorptive treatment) resulted in the highest stiffness. This could explain large reductions in fracture risk resulting from small increases in BMD. The non-uniform mineral distribution could also explain why bone tissue stiffness determined using nano-indentation is usually higher than finite element (FE)-determined stiffness. We conclude that the non-uniform mineral distribution in trabeculae does affect the mechanical properties of cancellous bone and that the tissue stiffness determined using FE-modeling could be improved by including detailed information about mineral distribution in trabeculae in the models.     

Regulation of expression of collagenase-3 in normal, differentiating rat osteoblasts.

We investigated the regulation of collagenase-3 expression in normal, differentiating rat osteoblasts. Fetal rat calvarial cell cultures showed an increase in alkaline phosphatase activity reaching maximal levels between 7-14 days post-confluence, then declining with the onset of mineralization. Collagenase-3 mRNA was just detectable after proliferation ceased at day 7, increased up to day 21, and declined at later ages. Postconfluent cells maintained in non-mineralizing medium expressed collagenase-3 but did not show the developmental increase exhibited by cells switched to mineralization medium. Cells maintained in non-mineralizing medium continued to proliferate; cells in mineralization medium ceased proliferation. In addition, collagenase-3 mRNA was not detected in subcultured cells allowed to remineralize. These results suggest that enhanced accumulation of collagenase-3 mRNA is triggered by cessation of proliferation or acquisition of a mineralized extracellular matrix and that other factors may also be required. After initiation of basal expression, parathyroid hormone (PTH) caused a dose-dependent increase in collagenase-3 mRNA. Both the cyclic adenosine monophosphate (cAMP) analogue, 8-bromo-cAMP (8-Br-cAMP), and the protein kinase C (PKC) activator, phorbol myristate acetate, increased collagenase-3 expression, while the calcium ionophore, ionomycin, did not, suggesting that PTH was acting through the protein kinase A (PKA) and PKC pathways. Inhibition of protein synthesis with cycloheximide caused an increase in basal collagenase-3 expression but blocked the effect of PTH, suggesting that an inhibitory factor prevents basal expression while an inductive factor is involved with PTH action. In summary, collagenase-3 is expressed in mineralized osteoblasts and cessation of proliferation and initiation of mineralization are triggers for collagenase-3 expression. PTH also stimulates expression of the enzyme through both PKA and PKC pathways in the mineralizing osteoblast.     

Characteristics of mineral particles in the human bone/cartilage interface

Bone and cartilage consist of different organic matrices, which can both be mineralized by the deposition of nano-sized calcium phosphate particles. We have studied these mineral particles in the mineralized cartilage layer between bone and different types of cartilage (bone/articular cartilage, bone/intervertebral disk, and bone/growth cartilage) of individuals aged 54 years, 12 years, and 6 months. Quantitative backscattered electron imaging and scanning small-angle X-ray scattering at a synchrotron radiation source were combined with light microscopy to determine calcium content, mineral particle size and alignment, and collagen orientation, respectively. Mineralized cartilage revealed a higher calcium content than the adjacent bone (p<0.05 for all samples), whereas the highest values were found in growth cartilage. Surprisingly, we found the mineral platelet width similar for bone and mineralized cartilage, with the exception of the growth cartilage sample. The most striking result, however, was the abrupt change of mineral particle orientation at the interface between the two tissues. While the particles were aligned perpendicular to the interface in cartilage, they were oriented parallel to it in bone, reflecting the morphology of the underlying organic matrices. The tight bonding of mineralized cartilage to bone suggests a mechanical role for the interface of the two elastically different tissues, bone and cartilage.     

Examination of bone chemical composition in osteoporosis using fluorescence-assisted synchrotron infrared microspectroscopy.

Although it is clear that osteoporosis is associated with a reduction in bone mass and a fragile skeleton, it is not understood whether the chemical composition of osteoporotic bone is different from normal bone. In this study, cynomolgus monkeys (Macaca fascicularis) were administered fluorochrome labels at one and two years after ovariectomy (Ovx) or Sham ovariectomy (intact), that were taken up into newly remodeled bone. Using fluorescence-assisted synchrotron infrared microspectroscopy, the chemical composition of bone from intact versus Ovx monkeys has been compared. Results from overall composition distributions (labeled + non-labeled bone) reveal similar carbonate/protein and phosphate/protein ratios, but increased acid phosphate content and different collagen structure in the Ovx animals. Analysis of the fluorochrome-labeled bone indicates similar degrees of mineralization in bone remodeled after one year, but decreased mineralization in Ovx bone remodeled two years after surgery. Thus, bone from monkeys with osteoporosis can be characterized as having abnormal collagen structure and reduced rates of mineralization. Coupled with factors such as trabecular architecture and bone shape and size, these ultrastructural factors may play a contributing role in the increased bone fragility in osteoporosis.     

Correlation of the appearance of γ-carboxyglutamic acid with the onset of mineralization in developing endochondral bone

γ-Carboxyglutamic acid (Gla) is a constituent of the non-collagenous bone protein osteocalcin. The appearance of γ-carboxyglutamic acid during $\text{de}\text{novo}$ differentiation and development of endochondral bone has been correlated with the onset of mineralization. Discrete stages of endochondral bone development were studied by subcutaneous implantation of demineralized rat diaphyseal bone matrix. Residual Gla in acid-demineralized bone matrix was lost rapidly on implantation. Gla levels were basal during mesenchymal cell proliferation (day 3) and chondrogenesis (days 5–7). Gla and calcium levels began to increase during cartilage mineralization (day 9) and continuously increased after day 10 concomitant with bone differentiation.     

Rietveld refinement on x-ray diffraction patterns of bioapatite in human fetal bones

Bioapatite, the main constituent of mineralized tissue in mammalian bones, is a calcium-phosphate-based mineral that is similar in structure and composition to hydroxyapatite. In this work, the crystallographic structure of bioapatite in human fetuses was investigated by synchrotron radiation x-ray diffraction (XRD) and microdiffraction ( micro -XRD) techniques. Rietveld refinement analyses of XRD and micro -XRD data allow for quantitative probing of the structural modifications of bioapatite as functions of the mineralization process and gestational age.     

Vitamin K2, a gamma-carboxylating factor of gla-proteins, normalizes the bone crystal nucleation impaired by Mg-insufficiency

It has been reported that the Mg-insufficient bone is fragile upon mechanical loading, despite its high bone mineral density, while vitamin K2 (MK-4: menatetrenone) improved the mechanical strength of Mg-insufficient bone. Therefore, we aimed to elucidate the ultrastructural properties of bone in rats with dietary Mg insufficiency with and without MK-4 supplementation. Morphological examinations including histochemistry, transmission electron microscopy, electron probe microanalysis (EPMA) and X-ray diffraction were conducted on the femora and tibiae of 4-week-old Wistar male rats fed with 1) a normal diet (control group, 0.09% Mg), 2) a Mg-insufficient diet (low Mg group, 0.006% Mg), or 3) a Mg-insufficient diet supplemented with MK-4 (MK-4 group, 0.006% Mg, 0.03% MK-4). MK-4 appeared to inhibit the osteoclastic bone resorption that is stimulated by Mg insufficiency. EPMA analysis, however, revealed an increased concentration of Ca paralleling Mg reduction in the low Mg group. Assessment by X-ray diffraction revealed an abundance of a particular synthetic form of hydroxyapatite in the low Mg group, while control bones featured a variety of mineralized crystals. In addition, Mg-deficient bones featured larger mineral crystals, i.e., crystal overgrowth. This crystalline aberration in Mg-insufficient bones induced collagen fibrils to mineralize easily, even in the absence of mineralized nodules, which therefore led to an early collapse of the fibrils. MK-4 prevented premature collagen mineralization by normalizing the association of collagen fibrils with mineralized nodules. Thus, MK-4 appears to rescue the impaired collagen mineralization caused by Mg insufficiency by promoting a re-association of the process of collagen mineralization with mineralized nodules.     

Constitutive expression of thrombospondin 1 in MC3T3-E1 osteoblastic cells inhibits mineralization

Thrombospondin 1 (TSP1) is a multifunctional extracellular glycoprotein present mainly in the fetal and adult skeleton. Although an inhibitory effect of TSP1 against pathological mineralization in cultured vascular pericytes has been shown, its involvement in physiological mineralization by osteoblasts is still unknown. To determine the role of TSP1 in biomineralization, mouse osteoblastic MC3T3-E1 cells were cultured in the presence of antisense phosphorothioate oligodeoxynucleotides complementary to the TSP1 sequence. The 18- and 24-mer antisense oligonucleotides caused concentration-dependent increases in the number of mineralized nodules, acid-soluble calcium deposition in the cell/matrix layer, and alkaline phosphatase activity within 9 days, without affecting cell proliferation. The corresponding sense or scrambled oligonucleotides did not affect these parameters. In the antisense oligonucleotide-treated MC3T3-E1 cells, thickened extracellular matrix, well-developed cell processes, increased intracellular organelles, and collagen fibril bundles were observed. On the other hand, the addition of TSP1 to the culture decreased the production of a mineralized matrix by MC3T3-E1 cells. Furthermore, MC3T3-E1 clones overexpressing mouse TSP1 were established and assayed for TSP1 protein and their capacity to mineralize. TSP1 dose-dependently inhibited mineralization by these cells both in vitro and in vivo. These results indicate that TSP1 functions as an inhibitory regulator of bone mineralization and matrix production by osteoblasts to sustain bone homeostasis.     

Alterations of the gamma-carboxyglutamic acid and osteocalcin concentrations in vitamin D-deficient chick bone

The content of osteocalcin and protein bound gamma-carboxyglutamic acid (Gla) was studied as a function of bone maturation and mineralization in normal and vitamin D-deficient, rachitic chickens. The Gla/Ca2+ ratio was elevated in rachitic bone, particularly in the most undermineralized regions. For example, there is a 10- to 20-fold elevation in Gla/Ca2+ in the newly synthesized, least mineralized rachitic bone fraction, which progressively decreases to a 1.5-fold elevation in the most highly mineralized areas of rachitic tissue. Osteocalcin, which is the principal Gla-containing protein of mature bone, was quantitated by radioimmunoassay using specific antiserum to the 5670-dalton chicken protein. Surprisingly, the osteocalcin concentration is decreased 50% in vitamin D-deficient bone. From this we infer that accumulated Gla-containing protein in vitamin D-deficient and poorly mineralized bone may possibly represent a precursor of osteocalcin.     

Sulphation of secreted phosphoprotein I (SPPI, osteopontin) is associated with mineralized tissue formation

Secreted phosphoprotein I (SPPI) is a prominent structural protein in mineralized connective tissues. Rat bone cells in culture produce several forms of SPPI that differ in post-translational modifications such as phosphorylation and sulphation. To determine the significance of protein sulphation in bone formation, the synthesis of SPPI was studied in vitro using rat bone marrow cells (RBMC) which form bone-like tissue when grown in the presence of dexamethasone (Dex) and beta-glycerophosphate (beta-GP). In the presence of 10(-7) M Dex SPPI expression was stimulated 4-5-fold. Radiolabelling multilayered RBMCs for 48 h with [35S]-methionine, Na2[35SO4], or Na3[32PO4] revealed that two major phosphorylated forms of SPPI were secreted into the culture medium: a highly phosphorylated form migrating at 44 kDa on 15% SDS-PAGE and a less phosphorylated 55 kDa form. In the mineralized tissue formed in the presence of Dex and beta-GP, both forms of SPPI, in addition to proteoglycans and a 67 kDa protein, incorporated significant amounts of [35SO4]. Sulphation of SPPI was not observed in the absence of mineral formation, indicating that the sulphation of SPPI is closely associated with mineralization and that it can be used as a sensitive and specific marker for the osteoblastic phenotype.     

Notch sensitivity of mammalian mineralized tissues in impact

The toughness of bone is an important feature in preventing it from fracturing. We consider the notch sensitivity in impact, and the associations between brittleness, notch sensitivity and post-yield energy absorption of mammalian mineralized tissues. Specimens of bone-like tissues covering a wide range of mineralization were broken, either notched or un-notched, in impact. The greater the mineral content, the greater was the notch sensitivity. Also, the more brittle tissues dissipated the least post-yield energy and were the most notch sensitive. It is suggested that since antler bone, the least mineralized of all known mammalian mineralized tissues, seems to be notch insensitive in impact, no adaptive purpose would be served by having mineralized tissues of a lower mineralization than antler. This may explain the lower cut-off in mineralization seen in mammals.     

Association of specific proteolytic processing of bone sialoprotein and bone acidic glycoprotein-75 with mineralization within biomineralization foci

Mineral crystal nucleation in UMR 106-01 osteoblastic cultures occurs within 15-25-microm extracellular vesicle-containing biomineralization foci (BMF) structures. We show here that BAG-75 and BSP, biomarkers for these foci, are specifically enriched in laser capture microscope-isolated mineralized BMF as compared with the total cell layer. Unexpectedly, fragments of each protein (45-50 kDa in apparent size) were also enriched within captured BMF. When a series of inhibitors against different protease classes were screened, serine protease inhibitor 4-(2-aminoethyl)benzenesulfonylfluoride HCl (AEBSF) was the only one that completely blocked mineral nucleation within BMF in UMR cultures. AEBSF appeared to act on an osteoblast-derived protease at a late differentiation stage in this culture model just prior to mineral deposition. Similarly, mineralization of bone nodules in primary mouse calvarial osteoblastic cultures was completely blocked by AEBSF. Cleavage of BAG-75 and BSP was also inhibited at the minimum dosage of AEBSF sufficient to completely block mineralization of BMF. Two-dimensional SDS-PAGE comparisons of AEBSF-treated and untreated UMR cultures showed that fragmentation/activation of a limited number of other mineralization-related proteins was also blocked. Taken together, our results indicate for the first time that cleavage of BAG-75 and BSP by an AEBSF-sensitive, osteoblast-derived serine protease is associated with mineral crystal nucleation in BMF and suggest that such proteolytic events are a permissive step for mineralization to proceed.     

Chemical imaging of biological tissue with synchrotron infrared light

Fourier transform infrared micro-spectroscopy (FTIRM) and imaging (FTIRI) have become valuable techniques for examining the chemical makeup of biological materials by probing their vibrational motions on a microscopic scale. Synchrotron infrared (S-IR) light is an ideal source for FTIRM and FTIRI due to the combination of its high brightness (i.e., flux density), also called brilliance, and broadband nature. Through a 10-μm pinhole, the brightness of a synchrotron source is 100-1000 times higher than a conventional thermal (globar) source. Accordingly, the improvement in spatial resolution and in spectral quality to the diffraction limit has led to a plethora of applications that is just being realized. In this review, we describe the development of synchrotron-based FTIRM, illustrate its advantages in many applications to biological systems, and propose some potential future directions for the technique.