Two different correlations between nanoindentation modulus and mineral content in the bone-cartilage interface.

The biomechanical properties of the zone of calcified cartilage (ZCC) in articulating joints are of clinical relevance due to the role ZCC plays in load transfer from cartilage to bone. To determine the micron-level mechanical properties and their correlation to mineral concentration in the ZCC, we combined nanoindentation (for micrometer level stiffness E(r) and hardness H) and quantitative back-scattered electron imaging or qBEI (for micrometer level mean calcium concentration Ca(Mean)) to study the ZCC-subchondral bone junction in 3 embedded human patellae. Nanoindentation line scans were correlated to qBEI analysis in the ZCC. The correlation between local stiffness and local mineral content was different in calcified cartilage compared to bone. The stiffness and hardness of calcified cartilage was typically lower than subchondral bone for the same mineral content. ZCC showed a wider range of variation in calcium content (1-28 wt %) compared to subchondral bone (16-26 wt %). 2D material property maps of the ZCC were generated from the mechanical-mineral correlation, showing that bands of high and low stiffness were found between the bone and tidemark, and between the ZCC and the unmineralized cartilage.

Normal mineralization and nanostructure of sclerotic bone in mice overexpressing Fra-1.

Increased bone mass due to elevated number of active osteoblasts has been reported for transgenic mice overexpressing the transcription factor Fra-1. To explore the potential of the anabolic action of Fra-1 in treatment of osteoporosis, we examined the integrity of bone matrix generated in Fra-1 transgenic mice. Femora from Fra-1 transgenic (Fra-1 tg) and wild-type littermates were analyzed for bone mineralization density distribution (BMDD) and nanostructure using quantitative backscattered electron imaging (qBEI) and scanning small angle X-ray scattering (scanning-SAXS), respectively. For comparison, we studied mice lacking c-Fos (Fos-/-), which develop osteopetrosis because of the absence of osteoclasts. Morphometrical analysis of metaphyseal spongiosa revealed an up to 5-fold increase in bone volume for Fra-1 transgenic compared to wild type. BMDD indicated a transient lower mineralization of bone for Fra-1 transgenic at 5 and 8 weeks, which became comparable to that of wild-type mice by 8 months. The homogeneity of mineralization was not altered in the Fra-1 transgenic mice at any ages examined. However, it was strikingly reduced in Fos-/- due to an abundance of hypermineralized cartilage. The bone nanostructure did not show abnormalities in Fra-1 transgenic or Fos-/-. These results provide a rationale for the development of therapeutic applications involving Fra-1-induced bone formation.

Targeted overexpression of vitamin D receptor in osteoblasts increases calcium concentration without affecting structural properties of bone mineral crystals.

Increased cross-sectional area and strength of long bones has been observed in transgenic mice with 2-fold (OSV9) and 3-fold (OSV3) elevation of osteoblast vitamin D receptor (VDR) levels. In the present study, mineralization density distributions, including typical calcium content (Ca(Peak)) and homogeneity of mineralization (Ca(Width)) of femoral bone and growth plate cartilage, were determined by quantitative backscattered electron imaging (qBEI). Fourier-transform infrared (FTIR) microspectroscopy was used to examine mineral content, collagen and crystal maturation, and scanning small angle X-ray scattering (scanning-SAXS) for studying mineral particle thickness and alignment. In addition, X-ray diffraction (XRD) of distal tibiae revealed mineral particle c-axis size. In trabecular bone, the increase in Ca(Peak) was significant for both OSV9 (+ 3.14%, P = 0.03) and OSV3 (+ 3.43%, P = 0.02) versus controls with 23.61 +/- 0.45 S.D. wt% Ca baseline values. In cortical bone, Ca(Peak) was enhanced for the OSV3 mice (+ 1.84%, P = 0.02) versus controls with 26.61 +/- 0.28 S.D. wt% Ca, and OSV9 having intermediate values. Additionally, there was significantly increased homogeneity of mineralization as denoted by a reduction of Ca(Width) (-8.4%, P = 0.01) in primary spongiosa. FTIR microspectroscopy, with the exception of an increased collagen maturity in OSV3 trabecular bone (+ 9.9%, P = 0.02), XRD, and scanning-SAXS indicated no alterations in the nanostructure of transgenic bone. These findings indicate that elevation of osteoblastic vitamin D response led to formation of normal bone with higher calcium content. These material properties, together with indications of decreased bone resorption in secondary spongiosa and increased cortical periosteal bone formation, appear to contribute to the improved mechanical properties of their long bones and suggest an important physiological role of the vitamin D-endocrine system in normal bone mineralization.

Orientation of mineral crystallites and mineral density during skeletal development in mice deficient in tissue nonspecific alkaline phosphatase.

Tissue nonspecific alkaline phosphatase (TNALP) is thought to play an important role in mineralization processes, although its exact working mechanism is not known. In the present investigation we have studied mineral crystal characteristics in the developing skeleton of TNALP-deficient mice. Null mutants (n = 7) and their wild-type littermates (n = 7) were bred and killed between 8 and 22 days after birth. Skeletal tissues were processed to assess mineral characteristics (small angle X-ray scattering, quantitative backscattered electron imaging), and to analyze bone by light microscopy and immunolabeling. The results showed a reduced longitudinal growth and a strongly delayed epiphyseal ossification in the null mutants. This was accompanied by disturbances in mineralization pattern, in that crystallites were not orderly aligned with respect to the longitudinal axis of the cortical bone. Among the null mutants, a great variability in the mineralization parameters was noticed. Also, immunolabeling of osteopontin (OPN) revealed an abnormal distribution pattern of the protein within the bone matrix. Whereas in the wild-type animals OPN was predominantly observed in cement and reversal lines, in the null mutants, OPN was also randomly dispersed throughout the nonmineralized matrix, with focal densities. In contrast, the distribution pattern of osteocalcin (OC) was comparable in both types of animals. It is concluded that ablation of TNALP results not only in hypomineralization of the skeleton, but also in a severe disorder of the mineral crystal alignment pattern in the corticalis of growing long bone in association with a disordered matrix architecture, presumably as a result of impaired bone remodeling and maturation.

Viscoelastic properties of collagen: synchrotron radiation investigations and structural model.

Collagen type I is the most abundant structural protein in tendon, skin and bone, and largely determines the mechanical behaviour of these connective tissues. To obtain a better understanding of the relationship between structure and mechanical properties, tensile tests and synchrotron X-ray scattering have been carried out simultaneously, correlating the mechanical behaviour with changes in the microstructure. Because intermolecular cross-links are thought to have a great influence on the mechanical behaviour of collagen, we also carried out experiments using cross-link-deficient tail-tendon collagen from rats fed with beta-APN, in addition to normal controls. The load-elongation curve of tendon collagen has a characteristic shape with, initially, an increasing slope, corresponding to an increasing stiffness, followed by yielding and then fracture. Cross-link-deficient collagen produces a quite different curve with a marked plateau appearing in some cases, where the length of the tendon increases at constant stress. With the use of in situ X-ray diffraction, it was possible to measure simultaneously the elongation of the collagen fibrils inside the tendon and of the tendon as a whole. The overall strain of the tendon was always larger than the strain in the individual fibrils, which demonstrates that some deformation is taking place in the matrix between fibrils. Moreover, the ratio of fibril strain to tendon strain was dependent on the applied strain rate. When the speed of deformation was increased, this ratio increased in normal collagen but generally decreased in cross-link-deficient collagen, correlating to the appearance of a plateau in the force-elongation curve indicating creep. We proposed a simple structural model, which describes the tendon at a hierarchical level, where fibrils and interfibrillar matrix act as coupled viscoelastic systems. All qualitative features of the strain-rate dependence of both normal and cross-link-deficient collagen can be reproduced within this model. This complements earlier models that considered the next smallest level of hierarchy, describing the deformation of collagen fibrils in terms of changes in their molecular packing.

Graded microstructure and mechanical properties of human crown dentin.

The mineralized tissue of intertubular dentin is a collagen-mineral composite with considerable local variations of mechanical properties. Area scans of human coronal dentin were made by complementary methods to investigate correlations between local mechanical properties and the density, size, and crystallinity of the mineral particles. Scanning images from the same specimen were collected with Fourier-transform infrared microspectroscopy in reflectance mode (FTIR-RM), small angle X-ray scattering (SAXS), quantitative backscattered electron imaging (qBEI), and Nanoindentation in an atomic force microscope. The mineral content of dentin was found to decrease and the thickness of mineral crystals to increase towards the dentin-enamel junction (DEJ). Hardness and elastic modulus both decreased towards the DEJ. In a correlation analysis, the mineral content and, even more, the thickness of mineral crystals were found as the best predictors of hardness. The dentin layer close to the DEJ corresponds to a local minimum in hardness and elastic modulus, a configuration known to be an effective obstacle for crack propagation. Hence, the observed variations of mechanical and structural properties in an area between 0 and 1.5 mm below the DEJ define crown dentin as a gradient material optimized for its mechanical function.

Structural development of the mineralized tissue in the human L4 vertebral body.

Knowledge of the structural development of the human vertebrae from non-weight-bearing before birth to weight-bearing after birth is still poor. We studied the mineralized tissue of the developing lumbar L4 vertebral body at ages 15 weeks postconception to 97 years from the tissue level (trabecular architecture) to the material level (micro- and nanostructure). Trabecular architecture was investigated by 2D histomorphometry and the material level was examined by quantitative backscattered electron imaging (for typical calcium content, CaMaxFreq) and scanning small-angle X-ray scattering (for mean mineral particle thickness). During early development, the trabecular orientation changed from a radial to a vertical/horizontal pattern. For bone area per tissue area and trabecular width in postnatal cancellous bone, the maximum was reached at adolescence (20 years), while for trabecular number the maximum was reached at childhood (approximately 1 year). CaMaxFreq was lower in early bone (approximately 21 wt%) than in mineralized cartilage (approximately 29 wt%) and adolescent bone (approximately 23 wt%). In conclusion, the changes at the tissue level were observed to continue throughout life while the development of bone at the material level (CaMaxFreq, mineral particle thickness and orientation) is essentially complete after the first years of life. CaMaxFreq and mean particle thickness increase rapidly during the first years and reach saturation. Remarkably, when these parameters are plotted versus logarithm of age, they appear linear.

Evidence of a novel staphylococcal mec-encoded element (mecR) controlling expression of penicillin-binding protein 2'.

A region was identified on the methicillin resistance determinant (mec) isolated from Staphylococcus epidermidis and cloned into Staphylococcus carnosus which was responsible for a novel downregulation of the expression of methicillin resistance. The presence of this region reduced the overall expression of methicillin resistance and the synthesis of the mec-encoded penicillin-binding protein 2' (PBP 2') in S. carnosus. This region was located by Bal31 deletion mutagenesis upstream of the structural gene for PBP 2'. Deletions within this region resulted in higher levels of expression of methicillin resistance and increased levels of PBP 2' synthesis. We tentatively called this region mecR. Analysis of selected Mcr strains of Staphylococcus aureus and S. epidermidis by Southern hybridization suggested that the natural occurrence of two types of mec resistance determinants differ by the presence or absence of mecR-specific sequences.

Cloning and expression of methicillin resistance from Staphylococcus epidermidis in Staphylococcus carnosus.

A 6.2-kilobase chromosomal DNA fragment from a methicillin-resistant Staphylococcus epidermidis strain was cloned into Staphylococcus carnosus by using staphylococcal plasmid pCA44 as the vector. The recombinant plasmid obtained, pBBB21, conferred methicillin resistance on its host and was responsible for the synthesis of a low-affinity penicillin-binding protein (PBP), PBP 2'. PBP 2' determined by the S. epidermidis DNA and expressed as a membrane-bound PBP in S. carnosus reacted with monoclonal antibodies directed against PBP 2' of Staphylococcus aureus origin, and the cloned S. epidermidis DNA hybridized to the methicillin (mec)-specific DNA from S. aureus. These findings point to a common origin of the methicillin resistance determinant in staphylococci.