Bovine Femoral Head Research Articles

Evaluation of decellularization process for developing osteogenic bovine cancellous bone scaffolds in-vitro.

Current immunological issues in bone grafting regarding the transfer of xenogeneic donor bone cells into the recipient are challenging the industry to produce safer acellular natural matrices for bone regeneration. The aim of this study was to investigate the efficacy of a novel decellularization technique for producing bovine cancellous bone scaffold and compare its physicochemical, mechanical, and biological characteristics with demineralized cancellous bone scaffold in an in-vitro study. Cancellous bone blocks were harvested from a bovine femoral head (18-24 months old) subjected to physical cleansing and chemical defatting, and further processed in two ways. Group I was subjected to demineralization, while Group II underwent decellularization through physical, chemical, and enzymatic treatments. Both were then freeze-dried, and gamma radiated, finally producing a demineralized bovine cancellous bone (DMB) scaffold and decellularized bovine cancellous bone (DCC) scaffold. Both DMB and DCC scaffolds were subjected to histological evaluation, scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDS), fourier-transform infrared spectroscopy (FTIR), quantification of lipid, collagen, and residual nucleic acid content, and mechanical testing. The osteogenic potential was investigated through the recellularization of scaffolds with human osteoblast cell seeding and examined for cell attachment, proliferation, and mineralization by Alizarin staining and gene expression. DCC produced a complete acellular extracellular matrix (ECM) with the absence of nucleic acid content, wider pores with extensive interconnectivity and partially retaining collagen fibrils. DCC demonstrated a higher cell proliferation rate, upregulation of osteogenic differentiation markers, and substantial mineralized nodules production. Our findings suggest that the decellularization technique produced an acellular DCC scaffold with minimal damage to ECM and possesses osteogenic potential through the mechanisms of osteoconduction, osteoinduction, and osteogenesis in-vitro.

Analysis of hydration and subchondral bone density on the viscoelastic properties of bovine articular cartilage

BackgroundArticular cartilage is known to be a viscoelastic material, however little research has explored the impact of cartilage water content and bone density on its viscoelasticity. This study aimed to isolate subchondral bone density and hydration of articular cartilage and analyse their effects on the viscoelastic properties of articular cartilage.MethodsDynamic mechanical analysis was used to test samples at frequencies of 1, 8, 12, 29, 49, 71, and 88 Hz. Synthetic bone material with densities of 663.7 kg/m3 and 156.8 kg/m3 were used to mimic the bone mineral density (BMD). Dehydration occurred in a stepwise manner at relative humidity (RH) levels of 100%, 30%, and 1%. These relative humidity levels led to water contents of approximately 76%, 8.5%, and ≈ 0% by mass, respectively.ResultsSamples from eight bovine femoral heads were tested under a sinusoidal load. Storage stiffness was lower on the lower substrate density. Storage stiffness, though, increased as cartilage samples were dehydrated from a water content of 76% to 8.5%; decreasing again as the water content was further reduced. Loss stiffness was lower on a lower density substrate and decreased as the water content decreased.ConclusionsIn conclusions, a decrease in hydration decreases the loss stiffness, but a non-linear relationship between hydration and storage stiffness may exist. Additionally, higher BMD values led to greater storage and loss stiffnesses.

Indentation method accounting for thickness effect of viscoelastic layer

AbstractIndentation tests have been widely used to evaluate the mechanical properties of industrial and biological materials in many studies. In this study, we obtained an analytical solution for the axisymmetric contact problem for a viscoelastic layer bonded to a rigid substrate and indented by a flat‐ended cylindrical punch using the elastic–viscoelastic correspondence principle. The analytical solution was obtained from a method using an infinite system of simultaneous equations instead of a conventional method using the second kind of Fredholm equation. Relaxation indentation tests were conducted for silicon rubber, polyurethane gel (a soft material used for elastography), and articular cartilage from a bovine femoral head. The articular cartilage was tested as obtained and then tested again after treatment by collagenase. The viscoelastic models in this study were a three‐element model (standard linear solid model) and a five‐element model (generalized Maxwell model), and the viscoelastic parameters for the models were determined by fitting the analytical solution to results for applied load as it varied over time during relaxation indentation tests. The results suggested that the five‐element model was more appropriate than the three‐element model for reproducing the relaxation indentation behavior of silicon rubber, polyurethane gel, and articular cartilage. Furthermore, the viscoelastic parameters for treated articular cartilage clearly decreased compared with those for unaltered articular cartilage. These results suggest that the method for evaluating the time‐dependent mechanical properties of articular cartilage based on analytical solutions of relaxation indentation tests could be a useful tool for evaluating the state of degenerated cartilage.

Applicability of cone beam computed tomography gray values for estimation of primary stability of dental implants

Objective: To evaluate the relationship between bone density measured by computed tomography (CT) and cone beam computed tomography (CBCT) (Sirona’s Galileos scanner) with primary stability of dental implants. Material and methods: 20 fresh bovine femoral heads were prepared by removal of soft tissue, sectioning of the bone, and placement of markers for location and angulation of implants. Bone density of peri-implant areas was determined preoperatively by CT and CBCT scanning of the prepared bone samples represented by Hounsfield units (HUs) and gray values (GVs), respectively. Then, 60 implants in three sizes (diameter = 4 mm, length = 8, 10, and 12 mm) were inserted into the bones and maximum insertion torque (IT) was recorded. Osstell device was also used for determining the implant stability quotient (ISQ) for each implant. Statistical analysis was performed on the data (alpha = 0.05). Results: Mean ± SD values of GV, HU, and ISQ were 1592.75 ± 231.82, 675.26 ± 115.38 and 61.90 ± 10.14, respectively. Moreover, the most frequent IT limit was 30-35 Ncm (41.4%). Significant relationships were observed between HU and IT, GV and IT, HU and ISQ, GV and ISQ, and IT and ISQ in all implant sizes. Moreover, GV and HU also significantly correlated to each other. Conclusion: Bone density values in CBCT and CT scans are positively associated to primary stability of dental implants. Therefore, GVs obtained from Galileos CBCT scanner can be used for preoperative selection of edentulous sites which allow for better implant stability or locations which require further procedures for enhancing the success rate of dental implants. KEYWORDSComputed tomography; Cone beam computed tomography; Dental implant.

Feasibility study for bone health assessment based on photoacoustic imaging method

This study investigated the feasibility of photoacoustic (PA) imaging of bone and characterization of bone features. By conducting the experiments on bovine femoral heads ex vivo, the light and ultrasonic penetration in bones was studied, together with the depth of PA imaging and measurement in bones. Then, the possibility of three-dimensional (3D) PA imaging of bones by raster scanning of the focusing transducer was studied. The micro-computerized tomography images of the bovine ribs with and without ethylenediaminetetraacetic acid (EDTA) treatment indicated that the 3D PA images could present the changes of bone microstructure resulting from the EDTA treatment. By using PA spectral analysis, the bone samples with and without the treatment of EDTA solution can be distinguished, and the microstructures can be characterized. This study was based on the bovine bone whose size is comparable to human bones, suggesting that PA technology can be used as a novel bone diagnostic technique.

Viscoelasticity of articular cartilage: Analysing the effect of induced stress and the restraint of bone in a dynamic environment.

The aim of this study was to determine the effect of the induced stress and restraint provided by the underlying bone on the frequency-dependent storage and loss stiffness (for bone restraint) or modulus (for induced stress) of articular cartilage, which characterise its viscoelasticity. Dynamic mechanical analysis has been used to determine the frequency-dependent viscoelastic properties of bovine femoral and humeral head articular cartilage. A sinusoidal load was applied to the specimens and out-of-phase displacement response was measured to determine the phase angle, the storage and loss stiffness or modulus. As induced stress increased, the storage modulus significantly increased (p < 0.05). The phase angle decreased significantly (p < 0.05) as the induced stress increased; reducing from 13.1° to 3.5°. The median storage stiffness ranged from 548 N/mm to 707 N/mm for cartilage tested on-bone and 544 N/mm to 732 N/mm for cartilage tested off-bone. On-bone articular cartilage loss stiffness was frequency independent (p > 0.05); however, off-bone, articular cartilage loss stiffness demonstrated a logarithmic frequency-dependency (p < 0.05). In conclusion, the frequency-dependent trends of storage and loss moduli of articular cartilage are dependent on the induced stress, while the restraint provided by the underlying bone removes the frequency-dependency of the loss stiffness.

Calculation of cancellous bone elastic properties with the polarization-based FFT iterative scheme.

The Fast Fourier Transform-based method, originally introduced by Moulinec and Suquet in 1994 has gained popularity for computing homogenized properties of composites. In this work, the method is used for the computational homogenization of the elastic properties of cancellous bone. To the authors' knowledge, this is the first study where the Fast Fourier Transform scheme is applied to bone mechanics. The performance of the method is analyzed for artificial and natural bone samples of 2 species: bovine femoral heads and implanted femurs of Hokkaido rats. Model geometries are constructed using data from X-ray tomographies, and the bone tissue elastic properties are measured using microindentation and nanoindentation tests. Computed results are in excellent agreement with those available in the literature. The study shows the suitability of the method to accurately estimate the fully anisotropic elastic response of cancellous bone. Guidelines are provided for the construction of the models and the setting of the algorithm.

Sample thickness dependence of Bayesian and modified least squares Prony’s analysis methods on systematically shortened bovine cancellous bone

The goal of this study was to compare the results from two proposed methods for separating fast and slow waves from mixed-mode signals, one using Bayesian probability theory and one using modified least-squares Prony’s (MLSP) [Wear, J. Acoust. Soc. Am. 133, 2490–2501 (2013)], on measurements of cancellous bone. Ultrasonic through-transmission data were acquired on a bovine femoral head specimen for thicknesses ranging from 15 mm to 6 mm. The thickness was reduced in 1 mm increments and measurements were acquired at each sample thickness [Nagatani et al., Ultrasonics 48, 607–612 (2008)]. A Bayesian parameter estimation analysis was performed on the experimentally acquired signals to isolate the fast and slow waves and to obtain estimates of the fast and slow wave’s ultrasonic properties. Results for the corresponding ultrasonic properties were estimated using the modified least squares Prony’s method plus curve fitting (MLSP + CF) on the same bovine sample by Wear et al. (J. Acoust. Soc. Am. 136, 2015–2024...

Evaluation of Viscoelastic Property of Articular Cartilage Based on Mechanical Model Considering Tissue Microstructure

The mechanical behavior of articular cartilage is strongly dependent on its microstructure characterized by three-dimensional, depth-dependent densities and anisotropic arrangements of cartilage cells and collagen fibres in extracellular matrix. The inclusion of cartilage microstructure to model analysis is essential for the accurate evaluation of the mechanical property. This study proposes a model-based evaluation method, which could predict tissue microstructure (collagen fibres, proteoglycans, and cartilage cells) based on a time history of reaction force obtained from indentation test, thereby estimating the depth-dependent cartilage mechanical property that is difficult to be estimated by experiment in conventional methods. The model was used for evaluating the viscoelasticities of cartilage at medial (load-supporting) and lateral (non load-supporting) regions on bovine femoral head. The present simulation allows fitting the time-history of reaction force measured from indentation test (R2 = 0.993 ± 0.0012 for lateral region and R2 = 0.990 ± 0.0032 for medial region). The differences of the elastic modulus of collagen fibre and the permeability between medial and lateral regions were consistent to those observed from the chemical composition analysis.

Effect of vibration on the shear strength of impacted bone graft in revision hip surgery

We developed a method of applying vibration to the impaction bone grafting process and assessed its effect on the mechanical properties of the impacted graft. Washed morsellised bovine femoral heads were impacted into shear test rings. A range of frequencies of vibration was tested, as measured using an accelerometer housed in a vibration chamber. Each shear test was repeated at four different normal loads to generate stress-strain curves. The Mohr-Coulomb failure envelope from which shear strength and interlocking values are derived was plotted for each test. The experiments were repeated with the addition of blood in order to replicate a saturated environment. Graft impacted with the addition of vibration at all frequencies showed improved shear strength when compared with impaction without vibration, with 60 Hz giving the largest effect. Under saturated conditions the addition of vibration was detrimental to the shear strength of the aggregate. The civil-engineering principles of particulate settlement and interlocking also apply to impaction bone grafting. Although previous studies have shown that vibration may be beneficial in impaction bone grafting on the femoral side, our study suggests that the same is not true in acetabular revision.

Biomechanical Properties of Orthodontic Miniscrews. An In-vitro Study

Comparing five commercially-available miniscrew types for skeletal anchorage with regard to the biomechanical properties influencing their primary stability. Included in this study was a total of 196 self-tapping and self-drilling miniscrews having a diameter of 2 mm (or the largest possible diameter of the manufacturer in question), a thread shaft length of 10 mm, or the longest miniscrew supplied by the manufacturers. The screw types tested were the FAMI 2, Orlus mini-implant, T.I.T.A.N. Pin, tomas-pin and Vector TAS. Insertion and loosening torque measurements, and pullout tests in axial (0 degrees), 20 degrees and 40 degrees directions, as well as test series with and without pilot hole drilling were performed. Bovine femoral heads having the same bone mineral density (BMD) were used as bone-testing material. Higher insertion torques were found for the cylindrical FAMI 2 screw, the conical Orlus mini-implant and the Vector TAS screw (with mean values of 39.2 Ncm, 32.1 Ncm and 49.5 Ncm) than for the cylindrical T.I.T.A.N. pin and tomas-pin. Insertion without predrilling led the insertion torques of all five screws to rise significantly. We noted statistically significant differences among the five screws in the pullout tests. Those highly significant differences at axial (0 degrees) and 20 degrees angles were not apparent at the 40 degrees pullout angle. Compared with the pullout forces (load) in the axial direction, the cylindrical screws' load values decreased markedly according to the angle (by up to -46.6%). The reduction in pullout force in conjunction with an increasing angle was much less pronounced in the conical screws (-0.8% to -29.0%). The tomas-pin demonstrated the highest pullout force and stiffness values throughout the tests. A total of five tomas-pins, two Orlus mini-implants and one FAMI 2 screw fractured during the pullout tests. Results from our insertion torque measurements suggest that a conical screw design will provide greater primary stability than cylindrical screw types. The cylindrical screw design's superiority was evident in the pullout tests. All the miniscrews' primary stability rose after drill-free insertion. The tomas-pin screws, although biomechanically superior to the other screws, were most prone to fracture.

The Biophysical Effects of a Single Impact Load on Human and Bovine Articular Cartilage

Impact injury to a joint is a known risk factor for the subsequent development of secondary osteoarthritis. An in vitro model, employing a drop-tower loading machine, was used to examine the effect of an impact load on isolated articular cartilage explants from human and bovine femoral heads. Two different types of impact experiment were performed. In the first, 4 mm diameter explants were loaded using a plane-ended impactor. In the second, a modified impactor was developed that had a central 4 mm diameter plane-ended indentor which was used to load the centre of 8 mm diameter explants. This enabled the unloaded outer ring of each explant to be compared with the loaded central core. The modulus values measured using the impactor were found to be higher, compared with the indentor in both species. Scanning electron microscopy showed that cartilage surrounding the loaded central region of the 8 mm explants protected the indented tissue, and these explants showed less damage than the 4 mm samples that were fully impacted. In addition, human cartilage was found to be less damaged than bovine, possibly as a consequence of the different structure as well as of a greater thickness. Both the source of the tissue and the nature of the impact affected the type of damage observed.

Development of heating method by microwave for sterilization of bone allografts

The purpose of this study was to develop a disinfection method using a microwave apparatus to treat large bone allografts. Heating of a bone allograft is an effective method for the disinfection of bacteria or inactivation of viruses. However, the size of the bone we can treat is limited, and following the popular method of using a bathtub is a lengthy process. The experimental system described here was designed using a microwave oven, an optical-fiber thermometer, and a power regulator. Large and small specimens, a femoral head, and a metatarsal were harvested from a bovine femur. The influence of size and the electrical or thermal characteristics of the specimens were assessed regarding temperature distribution after microwave irradiation. The effects of humidity or hot-air supply were also assessed. The average temperature of the bovine femoral head became 80°C throughout the 15 min of microwave irradiation, although the temperature in the metatarsal did not attain uniformity. Microwave irradiation with a hot-air supply realized a uniform distribution of temperature at 83.0° ± 0.4°C in the metatarsal within 15 min. Use of microwave irradiation enables quick heating for disinfection of large allograft bones when a hot-air supply was used as well.

Mithramycin downregulates proinflammatory cytokine-induced matrix metalloproteinase gene expression in articular chondrocytes

Interleukin-1 (IL-1), IL-17 and tumor necrosis factor alpha (TNF-α) are the main proinflammatory cytokines implicated in cartilage breakdown by matrix metalloproteinase (MMPs) in arthritic joints. We studied the impact of an anti-neoplastic antibiotic, mithramycin, on the induction of MMPs in chondrocytes. MMP-3 and MMP-13 gene expression induced by IL-1β, TNF-α and IL-17 was downregulated by mithramycin in human chondrosarcoma SW1353 cells and in primary human and bovine femoral head chondrocytes. Constitutive and IL-1-stimulated MMP-13 levels in bovine and human cartilage explants were also suppressed. Mithramycin did not significantly affect the phosphorylation of the mitogen-activated protein kinases, extracellular signal-regulated kinase, p38 and c-Jun N-terminal kinase. Despite effective inhibition of MMP expression by mithramycin and its potential to reduce cartilage degeneration, the agent might work through multiple unidentified mechanisms.

Functional anatomy of articular cartilage under compressive loading Quantitative aspects of global, local and zonal reactions of the collagenous network with respect to the surface integrity

Objective To assess the influence of local compressive loading on the arrangement of the collagenous fibers in intact articular cartilage. To quantitate the zonal deformation of intact cartilage under load. To analyse the influence of removal of the tangential zone on the load-induced changes.Materials and Methods 380 cylinder shaped cartilage-on-bone samples (d=7mm) were harvested from 20 bovine femoral heads. In 120 of them the tangential zone was removed. All samples were loaded for 20min by 0.42MPa or 0.98MPa. After proteoglycan extraction, fixation in 4% formalin, dehydration by increasing concentrations of acetone, critical point drying, freeze-fracturing and gold-coating the samples were analysed by scanning-electron-microscopy.Results Fiber bulging away from the center of load occurred in an area larger than the directly loaded one and its extent increased parallel to loading (P< 0.01). Crimp was seen only under the indenter and spread with increasing load from the intermediate zone into the tangential zone and radial zone. The absolute height of tangential zone and intermediate zone together remained constant under all loading situations at the costs of the radial zone. All changes due to loading were fully reversible. Removal of the tangential zone reduced the area of bulging (P< 0.01) but markedly increased the amount of crimp. Overall radial strain was not altered, but overall superficial tangential strain was increased by up to 20% (P< 0.01) and high peaks in the local distribution of superficial tensile strain developed.Conclusions The collagenous architecture is a dynamic property of the articular cartilage adapting to its respective loading situation. Crimp reflects local compressive strain. Under compressive loading larger portions of cartilage than the directly loaded areas are functionally included in the process of load transmission. During this process the tangential zone and the intermediate zone form a common functional unit providing a high degree of fiber cross-linkage as a possible mechanism to increase zonal compressive stiffness. Removal of the tangential zone seems to impair distribution of a locally applied compressive load sideways and leads to a reduced cartilage volume included in the process of load transmission. An intact tangential zone contributes to prevent peaks of surface tensile strain.

206 マイクロ波照射による皮質骨および海綿骨を含むウシ中足骨の内部温度均一化の検討

Internal temperature equalization of the bovine metatarsal with cortical and cancellous bone during microwave irradiation was investigated. In our previous study, bovine bone more rapidly than methods of conventional thermal process, because the bone was heated from deep part during microwave irradiation. Moreover, humidifying the surface of the bone reduced distribution in the internal bone temperature. Internal temperature equalization in bovine metatarsal bone by microwave irradiation was not successful, but in bovine femoral head (cancellous bone) and diaphysis (cortical bone), a temperature of 80±10℃. So in this study, industrial microwave oven was used. After refining the system using an industrial microwave oven, an internal temperature of the bovine metatarsals (containing both cancellous bone and cortical bone) could be maintained at 82±3℃ and processing time. These results suggest that thermal processing for disinfection using microwave irradiation can heat the bone more quickly by equalizing the internal temperature.

303 骨梁の弾性特性と配向性からみた海綿骨の力学特性

Bone has hierachical and complex structure and multiscale viewpoint is important for bone mechanics because the mechanical property is dependent on the scale factor of bone structure. Macroscopically, some relation is known between the mechanical property and bulk morphology. On the other hand, limited attention has been paid for such structural and mechanical consideration in mesoscopic and microscopic scale. In this article, the macroscopic elastic modulus of cancellous bone is examined by means of the elastic modulus of trabeculae, its orientation and volume fraction. Mean intercept length and fablic ellipsoid analysis and conventional compression test were used for the brick specimen harvested from bovine femoral head, and three point bending test was used for mechanical property of trabeculae specimen obtained from brick specimen. Some dependency of elastic modulus of trabeculae was suggested on the trabeculae orientation, and the complex relation was expected between the elastic moduli of trabeculae and cancellous bone.

Tensile experiments and SEM fractography on bovine subchondral bone

Subchondral bone undecalcified samples, extracted from bovine femoral heads, are subjected to a direct tensile load. The Young's modulus of each sample is determined from repeated tests within the elastic limit. In a last test, the tensile load is increased up to the specimen failure, determining the ultimate tensile strength. The investigation is performed on both dry and wet specimens. The measured Young's modulus for dry samples is 10.3±2.5 GPa, while that of wet samples is 3.5±1.2 GPa. The ultimate tensile strengths are 36±10 and 30±7.5 MPa for dry and wet specimens, respectively. SEM micrographs of failure surfaces show characteristic lamellar bone structures, with lamellae composed of calcified collagen fibers. Rudimentary osteon-like structures are also observed. Failure surfaces of wet samples show a marked fiber pull-out, while delamination predominates in dry samples. The obtained results are interpreted on the basis of the deformation mechanisms typical of fiber-reinforced laminated composite materials.

The distribution of type VI collagen in the developing tissues of the bovine femoral head.

Type VI collagen appears central to the maintenance of tissue integrity. In adult articular cartilage, type VI collagen is preferentially localised in the chondron where it may be involved in cell attachment. In actively remodelling developing cartilage, the distribution is less certain. We have used confocal immunohistochemistry and in situ hybridisation to investigate type VI collagen distribution in third trimester bovine proximal femoral epiphyses. In general, type VI collagen immunofluorescence was concentrated in the chondrocyte pericellular matrix, with staining intensity strongest in regions which persist to maturity and weakest in regions that remodel during development. Type VI collagen was also present in cartilage canals. In the growth plate and around the secondary centre of ossification, the intensity of type VI collagen stain rapidly decreased with chondrocyte maturation and was absent at hypertrophy, except where canal branches penetrated the growth plate and stain was retained around the adjacent chondrocytes. In situ hybridisation confirmed the presence of type VI collagen mRNA in cartilage canal mesenchymal cells but the signal was low in chondrocytes, suggesting minimal levels of synthesis and turnover. The results are consistent with a role for type VI collagen in stabilising the extracellular matrix during development.

Holding Power of Orthopaedic Screws in Bovine Femoral Heads. A Comparison of Three Cannulated Screws

SummaryFemoral capital physeal fractures have been successfully repaired with 7.0 mm stainless steel cannulated screws. The holding power of 7.0 mm stainless steel cannulated screws, 8.0 mm stainless steel cannulated screws and 8.0 mm titanium cannulated screws was compared. The 8.0 mm titanium cannulated screw had the highest holding power, the 7.0 mm stainless steel cannulated screw was intermediate and the 8.0 mm stainless steel cannulated screw had the lowest holding power.When placed in the bovine femoral head, 8.0 mm titanium cannulated screws have holding power greater than 8.0 mm stainless steel cannulated screws. A 7.0 mm stainless steel cannulated screw has intermediate holding power.