Rabbit Cortical Bone Research Articles

Rabbit cortical bone tissue increases its elastic stiffness but becomes less viscoelastic with age

Bone is dynamic tissue undergoing changes in its composition, structure and functional properties during growth. It has been proposed that especially changes in the collagen phase of bone are responsible for making the bone more fragile, and potentially less viscoelastic with age. Hence, robust methods to measure viscoelasticitiy are needed. This study aimed to characterize the development of the elastic and viscoelastic mechanical properties of rabbit bone during maturation and growth, as assessed by nanoindentation. The humeri from female New Zealand white rabbits of varying age (newborn, 11 days, 4 weeks, 3 and 6 months old, n = 8 per group) were investigated. Mid-diaphyseal cortical bone samples were cut, dehydrated, embedded and polished. Nanoindentation probing, semi-dynamic testing with a frequency of 20 Hz and creep with a dwell time of 60 s were performed under load control to quantify the elastic and the time-dependent viscoelastic mechanical properties of bone. The elastic moduli were evaluated with all three methods and the viscoelastic parameters were assessed using the phase-shift and the creep time constant. The elastic stiffness of bone increased significantly with each consecutive age group, from 11 days to 6 months of age, based on the reduced modulus from the indentation probing, the storage modulus from the semi-dynamic test, and the first elastic parameter from the creep test. These elastic parameters correlated significantly ( R = 0.88–0.94, p < 0.01). The values of viscoelastic parameters, the phase-shift and time creep constant, decreased significantly with age. The viscous properties determined by the creep and the semi-dynamic testing correlated significantly ( R = 0.90, p < 0.01), however, no correlation was found between the phase-shift and the creep time constant. Additionally, the present results showed specific associations with tissue composition, as measured with Fourier Transform Infrared spectroscopy of the same samples. In summary, the present results reveal significant changes in material properties of rabbit cortical bone with age. The elastic modulus of bone tissue increased by approximately 60%, whereas the viscoelastic parameters decreased by 10% to 25% during the first 6 months of the rabbit's life. Together, this indicates significant structural and functional maturation of the bone matrix during growth of the rabbit.

Cluster analysis of infrared spectra of rabbit cortical bone samples during maturation and growth

Bone consists of an organic and an inorganic matrix. During development, bone undergoes changes in its composition and structure. In this study we apply three different cluster analysis algorithms [K-means (KM), fuzzy C-means (FCM) and hierarchical clustering (HCA)], and discriminant analysis (DA) on infrared spectroscopic data from developing cortical bone with the aim of comparing their ability to correctly classify the samples into different age groups. Cortical bone samples from the mid-diaphysis of the humerus of New Zealand white rabbits from three different maturation stages (newborn (NB), immature (11 days-1 month old), mature (3-6 months old)) were used. Three clusters were obtained by KM, FCM and HCA methods on different spectral regions (amide I, phosphate and carbonate). The newborn samples were well separated (71-100% correct classifications) from the other age groups by all bone components. The mature samples (3-6 months old) were well separated (100%) from those of other age groups by the carbonate spectral region, while by the phosphate and amide I regions some samples were assigned to another group (43-71% correct classifications). The greatest variance in the results for all algorithms was observed in the amide I region. In general, FCM clustering performed better than the other methods, and the overall error was lower. The discriminate analysis results showed that by combining the clustering results from all three spectral regions, the ability to predict the correct age group for all samples increased (from 29-86% to 77-91%). This study is the first to compare several clustering methods on infrared spectra of bone. Fuzzy C-means clustering performed best, and its ability to study the degree of memberships of samples to each cluster might be beneficial in future studies of medical diagnostics.

Histomorphometric Analysis of HA-Coated Implants Interface With Irradiated and Nonirradiated Bone

The purpose of the present study was to investigate whether osseointegration of HA-coated implants can be affected by irradiation. A single dose of 15 Gy was applied to the right tibia of 5 rabbits and the left tibia was not irradiated and used as control. After 3 months, the animals were killed and the quantification of the implant-bone contact surfaces was performed using scanning electron micrographs of the resin sections containing the implants in the irradiated and nonirradiated bones. After the healing period, all implants were stable. The results show a mean percentage of bone-implant contact of 45.52% in the irradiated tissue and 47.56% in the nonirradiated tissue, with no statistic significant difference (P > 0.05). In conclusion, radiotherapy may not interfere with osseointegration process around HA-coated implants in rabbit cortical bone.

The response of bone to nanocrystalline hydroxyapatite-coated Ti13Nb11Zr alloy in an animal model

An in vivo study was carried out on uncoated and hydroxyapatite (HA)-coated nanostructured Ti13Nb11Zr alloy in comparison with high-grade Ti6Al4V, to investigate the effect of the different surfaces on osteointegration rate. A highly effective method to obtain a fast biomimetic deposition of a thin layer of nanocrystalline HA was applied to coat both substrates. Cylindrical pins were implanted in rabbit cortical bone and evaluated at 4 and 12 weeks by histomorphometry and microhardness tests. The results confirmed the ability of the slightly supersaturated Ca/P solution to induce a fast deposition of nanocrystalline HA on Ti alloys' surfaces. HA-coated Ti13Nb11Zr had the highest osteointegration rate at 4 and 12 weeks. Both HA-coated surfaces showed an affinity index significantly higher than those of native surfaces at 4 weeks (Ti13Nb11Zr + HA: 37%; Ti6Al4V + HA: 26%). Microhardness test showed a significantly higher bone mineralization index of HA-coated Ti13Nb11Zr in comparison with that of HA-coated Ti6Al4V surface. The study suggests that the HA coating on both alloys enhances bone response around implants and that there is a synergic effect of Ti–Nb–Zr alloy with the HA coating on bone remodeling and maturation.

Measurement of phosphorus content in normal and osteomalacic rabbit bone by solid‐state 3D radial imaging

In osteomalacia decreased mineralization reduces the stiffness and static strength of bone. We hypothesized that hypomineralization in osteomalacic bone could be quantified by solid-state (31)P magnetic resonance imaging (SS-MRI). Hypomineralization was measured with a 3D radial imaging technique at 162 MHz (9.4T) in rabbit cortical bone of hypophosphatemic (HY) and normophosphatemic (NO) animals. The results were compared with those obtained by quantitative micro-CT (micro-CT) and (31)P solution NMR. 3D images of 277 microm isotropic voxel size were obtained in 1.7 hr with SNR approximately 9. Mineral content was lower in the HY relative to the NO group (SS-MRI: 9.48 +/- 0.4 vs. 11.15 +/- 0.31 phosphorus wet wt %, P < 0.0001; micro-CT: 1114.6 +/- 28.3 vs. 1175.7 +/- 23.5 mg mineral/cm(3); P = 0.003). T(1) was shorter in the HY group (47.2 +/- 3.5 vs. 54.1 +/- 2.7 s, P = 0.004), which suggests that relaxation occurs via a dipole-dipole (DD) mechanism involving exchangeable water protons, which are more prevalent in bone from osteomalacic animals.

Nuclear Magnetic Resonance Studies of Bone Water

Mineralized bone tissue has a significant water component. Bone water is associated with the collagen fibers or mineral fraction or occurring as pore water of the Haversian and lacunocanalicular system. Among the multiple physiologic functions that include signaling and providing to bone its viscoelastic properties, bone water enables the transport of ions and nutrients to and waste products from the cells. In addition, it plays a key role during mineralization whereby collagen-bound water is gradually replaced by calcium apatite-like mineral. In this review it is shown how nuclear magnetic resonance (NMR) allows the study of various physiologically relevant properties of bone water nondestructively. Isotope exchange experiments are described from which the apparent water diffusion coefficient can be calculated. The method is based on monitoring the migration of H2O into the D2O after immersion of the specimen in heavy water. Data obtained from rabbit cortical bone in the normal and mineral-depleted skeleton provide evidence for the hypothesized reciprocal relationship between bone water and mineral. Further, from the diffusion coefficient (Da = (7.8+/-1.5) x 10(-7) cm2/s) measured at 40 degrees C it can be inferred that diffusive transport of small molecules from the bone's microvascular system to the osteocytes occurs within minutes. Finally, whereas isotope exchange is not feasible in vivo, it is shown that bone water can be imaged by proton MRI.

Mechanism of Apatite Formation on a Sodium Silicate Glass in a Simulated Body Fluid

29Si and 31P magic‐angle‐spinning nuclear magnetic resonance (MAS‐NMR) spectra were used to analyze 20Na2O·80SiO2 glass particles before and after soaking in a simulated body fluid (SBF; Kokubo solution, pH 7.4) for various periods (up to 14 d). The structure of the bulk glass and the glass surface, as well as the chemical states of the calcium phosphates adsorbed on the glass surface, were examined. The bulk glass before soaking in the SBF showed two 29Si MAS‐NMR resonances, at −100 and −110 ppm, which were attributable to Si(OSi)3ONa and Si(OSi)4 units, respectively. A new ‐105 ppm peak, which was due to Si(OSi)3OH units, appeared after soaking in the SBF and grew as the soaking period increased. Thus, in the SBF, the glass network was degraded and hydrolyzed; that is, dissolution of the Na+ ions from the glass network and the formation of Si‐O‐ groups occurred. After soaking for 1 d, a 31P MAS‐NMR resonance peak of orthophosphate (PO43‐) ions that were deposited on the glass surface appeared at ∼3.0 ppm and grew as the soaking period increased. The chemical shift shows that the local structural environment of phosphorus atoms and ions was very similar to that of hydroxyapatite or rabbit cortical bone. Inductively coupled plasma atomic emission spectroscopic analysis confirmed the adsorption of calcium phosphates on the glasses and the close relationship between the dissolution of the Na+ ions and the precipitation of the calcium phosphates. It was concluded that the layer of hydrated silica gel, which was composed of Si(OSi)3O‐ units, provided the negatively charged sites that induced or promoted the precipitation of calcium phosphates, which led to apatite nucleation and crystallization.

Bone response to surface modified titanium implants - studies on the tissue response after 1 year to machined and electropolished implants with different oxide thicknesses.

The bone formation around titanium implants with varied surface properties was investigated after 1 year in rabbits. Machined and electropolished samples with and without thick, anodically formed surface oxides were prepared, surface characterized and inserted in the cortical bone of rabbits. Scanning electron microscopy, scanning Auger electron spectroscopy and atomic force microscopy revealed marked differences in oxide thickness, surface topography and roughness, but no significant differences in surface chemical composition between the different groups of implants. Light microscopic morphology and morphometry showed that all implants were in contact with bone and had a large proportion of bone within the threads. There were no significant differences between the differently prepared implant groups. Our study shows that a high degree of bone contact and bone formation is achieved after 1 year with titanium implants which are modified with respect to oxide thickness and surface topography. There is no indication that a reduction of surface roughness, which in the initial phase decreases the rate of bone formation, had any influence on the amount of bone after 1 year in rabbit cortical bone.

Structure of the interface between rabbit cortical bone and implants of gold, zirconium and titanium.

The role of surface properties (chemical and structural) for the interaction between biomaterials and tissue is not yet understood. In the present study, implants made of titanium, zirconium (transition metals with surface oxides) and gold (metallic surface) were inserted into the rabbit tibia. Light microscopic (LM) morphometry showed that after 1 and 6 mo the gold implants had less amount of bone within the threads and a lower degree of bone-implant contact than the titanium and zirconium implants, which did not differ from each other. These quantitative differences were supported by LM and ultrastructural observations of the interface. The ultrastructural observations in addition demonstrated that the layer of non-collagenous amorphous material located between the implant and the calcified bone was appreciably thicker around zirconium than around titanium implants. The factors potentially responsible for the observed morphological differences in the bone around the different material surfaces are discussed.

Bone response to surface-modified titanium implants: studies on the early tissue response to machined and electropolished implants with different oxide thicknesses

The bone formation around titanium implants with varied surface properties is investigated. Machined and electropolished samples with and without thick, anodically formed surface oxides were prepared, surface characterized and inserted in the cortical bone of rabbits (1, 3 and 6 weeks). Scanning electron microscopy, scanning Auger electron spectroscopy and atomic force microscopy revealed marked differences in oxide thickness, surface topography and roughness, but no significant differences in surface chemical composition, between the different groups of implants. Light microscopic morphology and morphometry showed that all implants were in contact with bone and had a large proportion of bone within the threads at 6 weeks. The smooth, electropolished implants, irrespective of anodic oxidation, were surrounded by less bone than the machined implants after 1 week. After 6 weeks the bone volume as well as the bone-implant contact were lower for the merely electropolished implants than for the other three groups. Our study shows that a high degree of bone contact and bone formation are achieved with titanium implants which are modified with respect to oxide thickness and surface topography. However, the result with the smooth (electropolished) implants indicates that a reduction of surface roughness, in the initial phase, decreases the rate of bone formation in rabbit cortical bone.

Bone response to surface modified titanium implants: studies on electropolished implants with different oxide thicknesses and morphology

In a series of experimental studies, bone formation was analysed around systematically modified titanium implants. In the present study, machined, electropolished and anodically oxidized implants were prepared, surface characterized and inserted in the cortical bone of rabbits (7wks and 12wks). SEM, scanning Auger electron spectroscopy and atomic force microscopy revealed no differences in surface composition but marked differences in oxide thickness, surface topography and roughness. Light microscopic morphology and morphometry showed that all implants were in contact with bone, and had a large proportion of bone within the threads. The smooth, electropolished implants were surrounded by less bone than the machined implants with similar oxide thickness, (4–5 nm) and the anodically oxidized implants with thicker oxides (21 nm and 180 nm, respectively) after 7wks. These studies show that a high degree of bone contact and bone formation can be achieved with titanium implants which are modified with respect to oxide thickness and surface topography. However, it appears that a reduction of surface roughness may influence the rate of bone formation in rabbit cortical bone. Biomaterials (1994) 15, (13) 1062–1074

Early tissue response to titanium implants inserted in rabbit cortical bone

The tissue response to screw-shaped implants of commercially pure titanium was studied by light microscopy 3–180 days after insertion in the rabbit tibia. The implant site in the tibial metaphysis consisted mainly of cortical bone. Three days after implantation, osteoblasts, producing osteoid, were observed at the endosteal surface and elongated mesenchymal cells were present in the injury area. Some macrophages but rather few other inflammatory cells were identified. Multinuclear giant cells were in direct contact with the implant and formed an almost continuous layer along the surface from the 7th day. The number of giant cells decreased with time and with increased bone-titanium contact. Bone formation was never seen direct on the implant surface but was first observed at day 7 as a woven trabecular bone formed at the endosteal surface and extending towards the implant and as a solitary formation of woven bone close to the implant. The solitary bone matrix served as a base for surface osteoblasts which produced osteoid in a lamellar arrangement. With time the two types of newly formed bone fused and more bone filled the threads and became remodelled by bone remodelling units. Light microscopic morphometry in ground sections demonstrated that the bone/titanium contact and bone area in the threads increased with time up to 6 months after implantation

Early tissue response to titanium implants inserted in rabbit cortical bone

Early tissue response to titanium implants inserted in rabbit cortical bone

Commercially pure titanium and Ti6AI4V implants with and without nitrogen-ion implantation: surface characterization and quantitative studies in rabbit cortical bone

Nitrogen-ion implantation is a surface modification of interest for biomedical materials. In this study screw-shaped commercially pure titanium and Ti6AI4V alloy implants were nitrogen-ion implanted and analysed by scanning electron microscopy (SEM) and scanning Auger electron spectroscopy (AES). The surface topography was essentially similar for treated and non-treated samples. AES survey spectra showed an incorporation of nitrogen into the surface of the nitrogen-ion treated commercially pure titanium and Ti6AI4V implants, as judged by the ratio between the intensities of the peaks at 390 and 420 eV. AES depth profiles revealed a similar oxide thickness (7.5 nm) for the nitrogen-ion implanted and the non-treated samples. In nitrogen-ion implanted screws nitrogen was detected up to depths of about 150 nm below the surface, with a maximum nitrogen concentration at about 50 nm. Light microscopic examination of the 10 μm-thick ground sections 3 months after the insertion of nitrogen-ion implanted and non-treated commercially pure titanium and Ti6AI4V screws in the proximal tibial methaphysis of rabbits showed that bone filled a large portion of the area within the threads. A fibrous capsule was not observed. Light microscopic morphometry did not reveal any statistically significant differences in bone-metal contact or bone area within the threads of nitrogen-ion treated and non-treated implants. The “mirror-image” analysis showed that for all implants examined significantly more bone was present immediately outside than inside the threads. The results from this study indicate that nitrogen-ion implanted, screw-shaped, commercially pure titanium and Ti6AI4V implants heal as well in cortical bone as non-treated samples.

Bone Morphogenesis of Rabbit Bone Morphogenetic Protein-Bound Hydroxyapatite-Fibrin Composite

Hydroxyapatite (HAP) and fibrin have been implanted in patients and observed to be well tolerated in orthotopic sites. This is a report on a composite of HAP, fibrin, and rabbit bone morphogenetic protein and insoluble noncollagenous protein (BMP-iNCP). Drill holes in the femoral condyles of rabbits were packed with granulated HAP (200 mg), fibrin (0.3 ml), BMP-iNCP (5 mg), or various combinations of the two. The fibrin consisted mainly of sterilized human fibrinogen and thrombin, and BMP-iNCP was prepared from demineralized rabbit cortical bone. New bone formation was observed at one, two, four, and eight weeks after implantation. The BMP-iNCP augmented new bone formation in rabbit femoral condyles. Fibrin made the composite easier to manipulate and did not inhibit osteogenesis at any period. The composites of HAP with BMP-iNCP and of HAP with BMP-iNCP and fibrin produced higher yields of new bone than fibrin alone or HAP alone.

Bone Mass Determination from Microradiographs by Computer-Assisted Videodensitometry

Aluminium was evaluated as a reference substance in the assessment of rabbit cortical bone by microradiography followed by videodensitometry. Ten dense, cortical-bone specimens from the same tibia diaphysis were microradiographed using prefiltered 27 kV roentgen radiation together with aluminium step wedges and bone simulating phantoms for calibration. Optimally exposed and processed plates were analysed by previously described computer-assisted videodensitometry. For comparison, the specimens were analysed by physico-chemical methods. A strict proportionality was found between the 'aluminium equivalent mass' and the ash weight of the specimens. The total random error was low with a coefficient of variation within 1.5 per cent. It was concluded that aluminium is an appropriate reference material in the determination of cortical bone, which it resembles in effective atomic number and thus x-ray attenuation characteristics. The 'aluminium equivalent mass' is suitably established as the standard of expressing the results of bone assessment by microradiography.

Keratan sulfate proteoglycan in rabbit compact bone is bone sialoprotein II.

A keratan sulfate proteoglycan was isolated under denaturing conditions from the mineral compartment of rabbit cortical bone. This small proteoglycan (Kd = 0.39 on Superose 6, Mr approximately 20,000 on sodium dodecyl sulfate gels) contained small keratan sulfate chains that were distinctly bimodal in size. The keratanase and endo-beta-galactosidase digestible glycosaminoglycan chains were O-linked to a core protein of Mr approximately 80,000. This core protein had several properties in common with the bone sialoprotein II molecule of bovine and human bone including: a closely spaced doublet band on sodium dodecyl sulfate electrophoresis gels; a high staining intensity with Stains All that was greatly diminished by neuraminidase; a significant amount of small O-linked oligosaccharides; and an amino-terminal amino acid sequence that was nearly identical to human bone sialoprotein II. (In contrast, bone sialoprotein II in human, bovine, and rat bone does not appear to have any keratan sulfate chains.) Antiserum made against the keratan sulfate proteoglycan reacted with its core protein on electrotransfers from sodium dodecyl sulfate-polyacrylamide gels.

A comparative study of different β-whiUockite ceramics in rabbit cortical bone with regard to their biodegradation behaviour

Different β-whitlockite ceramic cylinders of standard size were implanted in the tibiae of rabbits to study the influence of micropores and chemistry on the biodegradation rate. The materials were evaluated by radiography and light microscopy. Surface chemistry was varied by the addition of impurities, while different applied pressures before sintering and different sintering temperatures gave rise to different micropores. Both factors influenced the biodegradation rate.

The small molecular weight, γ-carboxyglutamic acid-containing protein of rabbit bone tissue

The major γ-carboxyglutamic acid-containing protein of rabbit cortical bone isolated and purified from near-neutral (pH 7.5) EDTA-extracts by DEAE-cellulose and Sephadex G 75 column chromatography had a molecular weight of about 5600 based on integral amino-acid composition; this was confirmed by high-performance liquid chromatography. The purified protein had high glutamic acid and aspartic-acid contents, two to three residues of γ-carboxyglutamic acid per molecule and zero levels of serine, threonine, methionine, histidine and tryptophan. Equilibrium dialysis indicated that the protein has a weak affinity for calcium ions with a formation constant of 1515 M −1 at I 0.15, pH 7.5, 25 °C with a binding capacity of 2 mol calcium per mole protein.

The acidic macromolecules in rabbit cortical bone tissue

Rabbit cortical bone powder was extracted with EDTA solutions at neutral pH and the soluble constituents fractionated by DEAE-cellulose chromatography. The acidic fractions obtained were further investigated by gel chromatography and chemical analysis. Two classes of proteoglycans were present; one containing chondroitin sulphate and the other containing material resembling keratan sulphate mixed with a smaller amount of chondroitin sulphate. Sialoglycoproteins were detected by specific chemical introduction of tritium label into the sialic acid residues of the acidic glycoprotein fraction. Following sodium dodecyl sulphatepolyacrylamide gel electrophoresis and fluorography, the presence of numerous distinct sialoglycoprotein components was demonstrated.