Bovine Cancellous Bone Research Articles

Outcome of Subtalar Fusion Using Bovine Cancellous Bone Graft: A Retrospective Case Series

Solvent preserved bovine cancellous bone graft (Tutobone ®) has been promoted as an alternative to autologous bone graft. The aim of our study was to compare the outcomes of subtalar fusion in patients in whom Tutobone ® was used with the outcomes in patients in whom it was not used. This was a retrospective comparative study. Tutobone ® was used in 9 patients in the test group. Of these repairs, 6 were isolated subtalar fusions, and 3 were performed as a part of triple arthrodesis. A total of 17 patients were included in the control group; 4 underwent autologous iliac crest grafting and 13 received a local bone graft from excised joint surfaces. At 12 months after surgery, 8 of the 9 in the Tutobone ® group had persistent pain and radiologic signs of nonunion confirmed on computed tomography scan. All 17 in the other group had successful clinical and radiologic fusion at 12 months. We believe this is sufficient evidence to advise against the use of bovine cancellous bone graft material for subtalar fusion surgery.

Compressive behaviour of bovine cancellous bone and bone analogous materials, microCT characterisation and FE analysis

Compressive behaviour of bovine cancellous bone and three open-cell metallic foams (AlSi7Mg (30 ppi and 45 ppi); CuSn12Ni2 (30 ppi)) has been studied using mechanical testing, micro-focus computed tomography and finite element modelling. Whilst the morphological parameters of the foams and the bone appear to be similar, the mechanical properties vary significantly between the foams and the bone. Finite element models were built from the CT images of the samples and multi-linear constitutive relations were used for modelling of the bone and the foams. The global responses of the bone and foam samples were reasonably well captured by the FE models, whilst the percentage of yielded elements as a measure of damage evolution during compression seems to be indicative of the micro-mechanical behaviour of the samples. The damage evolution and distribution patterns across the bone and the foams are broadly similar for the strain range studied, suggesting possible substitution of trabecular bones with appropriate foams for biomechanical studies.

De novo Generation of an Axially Vascularized Processed Bovine Cancellous-Bone Substitute in the Sheep Arteriovenous-Loop Model

Background/Aims: The aim of this study was to generate an axially vascularized bone substitute. The arteriovenous (AV)-loop approach in a large-animal model was applied in order to induce axial vascularization in a clinically approved processed bovine cancellous bone (PBCB) matrix of significant volume with primary mechanical stability and to assess the course of increasing axial vascularization. Methods: PBCB constructs were implanted into 13 merino sheep together with a microsurgically created AV loop in an isolation chamber. The vascularization process was monitored by sequential magnetic resonance imaging (MRI) scans. Explants were subjected to micro-computed tomography (micro-CT) analysis, histomorphometry and immunohistochemistry for CD31 and CD45. Results: Increasing axial vascularization in PBCB constructs was quantified by histomorphometry and visualized by micro-CT scans. Intravital sequential MRI scans demonstrated a significant progressive increase in perfused volume within the matrices. Immunohistochemistry confirmed endothelial lining of newly formed vessels. Conclusion: This study demonstrates successful axial vascularization of a clinically approved, mechanically stable bone substitute with a significant volume by a microsurgical AV loop in a large-animal model. Thus microsurgical transplantation of a tissue-engineered, axially vascularized and mechanically stable bone substitute with clinically relevant dimensions may become clinically feasible in the future.

Minerals Form a Continuum Phase in Mature Cancellous Bone

Bone is a hierarchically structured composite consisting of a protein phase (type I collagen) and a mineral phase (carbonated apatite). The objective of this study was to investigate the hierarchical structure of mineral in mature bone. A method to completely deproteinize bone without altering the original structure is developed, and the completion is confirmed by protein analysis techniques. Stereoscopy and field emission electron microscopy are used to examine the structural features from submillimeter- to micrometer- to nanometer-length scales of bovine femur cancellous bone. Stereoscopic images of fully deproteinized and demineralized bovine femur cancellous bone samples show that fine trabecular architecture is unaltered and the microstructural features are preserved, indicating the structural integrity of mineral and protein constituents. SEM revealed that bone minerals are fused together and form a sheet-like structure in a coherent manner with collagen fibrils. Well-organized pore systems are observed at varying hierarchical levels. Mineral sheets are peeled off and folded after compressive deformation, implying strong connection between individual crystallites. Results were compared with commercially available heat-deproteinized bone (Bio-Oss®), and evidence showed consistency in bone mineral structure. A two-phase interpenetrating composite model of mature bone is proposed and discussed.

Bone Formation and Neovascularization Mediated by Mesenchymal Stem Cells and Endothelial Cells in Critical-Sized Calvarial Defects

Bone represents a highly dynamic tissue whose development is strongly dependent on vasculogenic and angiogenic processes. Neovascularization also plays an important role in fracture healing and in tissue engineering applications aiming at restoring bone function. We have previously shown in a heterotopic subcutaneous implantation model of severe combined immunodeficiency (SCID) mice that implanted human umbilical vein endothelial cells (HUVECs) gave rise to the formation of a complex functional human neovasculature. In this study, we investigated the effect of HUVEC coimplantation on mesenchymal stem cell (MSC)-mediated bone regeneration in an orthotopic calvarial bone defect model in immunocompromised mice. For this purpose, human fibrin/Matrigel-immobilized HUVECs and MSCs were seeded alone or in combination into scaffolds consisting of decalcified processed bovine cancellous bone (Tutobone) and implanted into calvarial critical-sized defects. Our results show that implanted HUVECs formed complex three-dimensional networks of perfused human neovessels that were stabilized by recruiting perivascular cells. Neovessel formation was considerably higher in the coimplantation group, suggesting that implanted MSCs supported HUVEC-triggered neovascularization. In addition, implanted MSCs effectively supported bone formation in calvarial defects. However, the HUVEC-derived neovasculature did not improve MSC-triggered bone regeneration in this orthotopic critical-sized defect model.

Histologic and Histometric Evaluation of Bovine Cancellous Bone and Beta-Tricalcium Phosphate 45 Months After Grafting in Maxillary Sinus

The aim of this report is to determine the long-term histologic and histometric evaluation of beta-tricalcium phosphate (beta-TCP) and bovine cancellous bone grafted into the left maxillary sinus of a patient in order to augment the sinus floor to prepare it for receiving an implant. Beta-TCP and bovine cancellous bone (BCB) were grafted into the left maxillary sinus at the extracted sites of maxillary left first and second molars of a 62-year-old male patient. Using osteotome technique, 45 months later, core biopsies were taken for histologic and histometric comparison of the newly formed bone. Histometric and histologic evaluation of both specimens revealed a normal maxillary bone structure with 23.08% calcified bone and 76.92% marrow and connective tissue for the beta-TCP grafted site, and 24.81% calcified bone with 75.19% marrow and connective tissue for the BCB grafted site. No residual grafting material was seen in either specimen. Both beta-TCP and BCB were completely resorbed and replaced by new bone 45 months after grafting. Histologic results of the newly formed bone were similar for both grafting materials.

Cancellous bone analysis with modified least squares Prony’s method and chirp filter: Phantom experiments and simulation

The presence of two longitudinal waves in porous media is predicted by Biot's theory and has been confirmed experimentally in cancellous bone. When cancellous bone samples are interrogated in through-transmission, these two waves can overlap in time. Previously, the Modified Least-Squares Prony's (MLSP) method was validated for estimation of amplitudes, attenuation coefficients, and phase velocities of fast and slow waves, but tended to overestimate phase velocities by up to about 5%. In the present paper, a pre-processing chirp filter to mitigate the phase velocity bias is derived. The MLSP/chirp filter (MLSPCF) method was tested for decomposition of a 500 kHz-center-frequency signal containing two overlapping components: one passing through a low-density-polyethylene plate (fast wave) and another passing through a cancellous-bone-mimicking phantom material (slow wave). The chirp filter reduced phase velocity bias from 100 m/s (5.1%) to 69 m/s (3.5%) (fast wave) and from 29 m/s (1.9%) to 10 m/s (0.7%) (slow wave). Similar improvements were found for 1) measurements in polycarbonate (fast wave) and a cancellous-bone-mimicking phantom (slow wave), and 2) a simulation based on parameters mimicking bovine cancellous bone. The MLSPCF method did not offer consistent improvement in estimates of attenuation coefficient or amplitude.

Das Einwachsverhalten von trikortikalen Beckenkammspänen und lösungsmittelkonservierter, boviner Spongiosa bei ventraler Spondylodese der Lendenwirbelsäule im Schafsmodell

Spinal injuries are common and a standard procedure for the stabilisation of spinal injuries is ventral spondylodesis with an autograft from the iliac crest. Because of the high incidence of harvesting complications there is a need to search for alternative materials. The aim of our study was to evaluate graft integration in the lumbar spine of bovine cancellous bone compared to autologous iliac crest material. Two groups of eight female adult sheep (median age 3 years, range 2.4-3.8 years) received surgical treatment in the form of anterior monosegmental spondylodesis. The spondylodesis was performed in all animals in the motion segment L3/4 through a lateral approach with the animals lying on their right sides. To produce serial sections, the explanted vertebral segments were implanted in methyl methacrylate. On one side the histological preparation was examined qualitatively and in addition we analysed the quantity of the bone structure with special software. The bone structure in both groups did not differ significantly and demonstrated integration of the grafts in the adjacent vertebral bodies. Fractures and lysis occurred in the region of the intervertebral disc and were more frequent in the group with the bovine graft. The bony integration of the grafts of both groups was not significantly different and showed good results. Almost all of the bovine grafts fractured or presented regions of lysis. In our opinion bovine cancellous bone graft is not a good alternative to autologous iliac crest.

Effects of dehydration-induced structural and material changes on the apparent modulus of cancellous bone

Dehydration is known to cause an increase in the elastic modulus of bone tissue. However, it also causes structural changes (i.e. shrinkage) which can themselves significantly alter the mechanical properties, particularly in cancellous bone. The current study attempts to estimate the contribution of these two competing factors to the net change of dehydration on the apparent modulus of bovine cancellous bone. Cylindrical cores from the lumbar vertebrae were tested in tension, while hydrated and again after dehydration. The bone volume fractions (BV/TV) were measured in both conditions. The results indicate that the average overall increase in the apparent modulus after dehydration is 14 ± 14% (mean ± SD), which represents the net effect of a 27% increase in modulus due to increased tissue modulus offset by a modulus decrease of 13% due to reductions in bone volume fraction. These observations underscore the need to consider both structural and material changes when comparing hydrated and dehydrated mechanical behaviour.

Bone–cement interfacial behaviour under mixed mode loading conditions

Interfacial behaviour of the bone–cement interface has been studied under tensile, shear and mixed mode loading conditions. Bovine cancellous bone was used to bond with acrylic bone cement to form bone–cement interface samples, which were mechanically tested under selected tensile, shear and mixed mode loading conditions. The influence of the loading angle and the extent of the cement penetration on the interfacial behaviour were examined. The failure mechanisms with regard to loading mode were examined using micro-focus computed tomography. The measured tensile and shear responses were utilized in a cohesive zone constitutive model, from which the pre-yield linear and the post-yield exponential strain softening behaviour under mixed mode loading conditions was predicted. The implications of the work on the studies of cemented joint replacements are also discussed.

The effect of structural anisotropy on the fast wave propagation in cancellous bone.

QUS parameters are closely related to the structural properties and elastic properties of bone, which can provide important information related to bone quality and bone strength. One should be very careful, however, that bone contains complicated structure from microscopic to macroscopic levels. For example, the cancellous bone inside the epiphysis is composed of a complicated trabecular network in the bone marrow, showing strongly anisotropic and heterogeneous structure. We have then observed the longitudinal wave propagation in this complicated medium, paying special attention to the two wave phenomenon. The fast wave, which mainly propagates in the trabecular part of the cancellous bone, is more sensitive to the network structure. With the help of x-ray micro-computed tomography, three dimensional anisotropic trabecular structure and fast wave propagation were investigated using bovine cancellous bones. We can find the strong effect of anisotropic trabecular structure on the fast wave velocity. The mean intercept length (the information of average trabecular length) was a good parameter to describe the three dimensional trabecular structure and showed good correlation with the fast wave velocity.

Quantifying migration and polarization of murine mesenchymal stem cells on different bone substitutes by confocal laser scanning microscopy

Cell migration is preceded by cell polarization. The aim of the present study was to evaluate the impact of the geometry of different bone substitutes on cell morphology and chemical responses in vitro. Cell polarization and migration were monitored temporally by using confocal laser scanning microscopy (CLSM) to follow green fluorescent protein (GFP)±mesenchymal stem cells (MSCs) on anorganic cancellous bovine bone (Bio-Oss(®)), β-tricalcium phosphate (β-TCP) (chronOS(®)) and highly porous calcium phosphate ceramics (Friedrich-Baur-Research-Institute for Biomaterials, Germany). Differentiation GFP±MSCs was observed using pro-angiogenic and pro-osteogenic biomarkers. At the third day of culture polarized vs. non-polarized cellular sub-populations were clearly established. Biomaterials that showed more than 40% of polarized cells at the 3rd day of culture, subsequently showed an enhanced cell migration compared to biomaterials, where non-polarized cells predominated (p<0.003). This trend continued untill the 7th day of culture (p<0.003). The expression of vascular endothelial growth factor was enhanced in biomaterials where cell polarization predominated at the 7th day of culture (p=0.001). This model opens an interesting approach to understand osteoconductivity at a cellular level. MSCs are promising in bone tissue engineering considering the strong angiogenic effect before differentiation occurs.

Decomposition of two-component pulses: Simulation and phantom experiment.

Porous media often support propagation of two compressional waves. When cancellous bone samples are interrogated in through-transmission with broadband sources, these two waves often overlap in time. A method for measuring attenuation and velocity of the two component waves was developed. The method (1) assumes that the transfer function is the sum of two exponentially modulated sinusoids and (2) minimizes the sum of the squared error between a model fit and the data. The method was tested for decomposing a 500-kHz-center-frequency signal containing two overlapping components: one passing through a polycarbonate plate (fast wave) and another passing through a cancellous-bone-mimicking phantom (slow wave). The method yielded estimates of attenuation slopes accurate to within 7% (polycarbonate plate) and 2% (cancellous bone phantom). The method yielded estimates of phase velocities accurate to within 1.5% (both media). The method was tested on simulated data generated using attenuation slopes and phase velocities corresponding to bovine cancellous bone. Throughout broad ranges of signal-to-noise ratio and fast-slow-wave-velocity differential, the method yielded estimates of attenuation slope that were accurate to within 10% and estimates of phase velocity that were accurate to within 5% (fast wave) and 2% (slow wave).

Ectopic osteogenesis of hBMP-2 gene-transduced human bone mesenchymal stem cells/BCB

We determined the feasibility of using scaffolds of adenoviral human BMP2 gene (AdBMP2)-modified human bone marrow mesenchymal stem cells (hBMSCs) and antigen-free bovine cancellous bone (BCB) to construct bone tissue. hMSCs were infected with AdBMP-2. Expression of BMP-2 and alkaline phosphatase confirmed successful secretion of active BMP-2. The osteogenic capability of a composite of AdBMP2-modified hMSCs with BCB was evaluated in athymic mice (group A). BCB (group B), hMSCs/BCB (group C), adenoviral β‐galactosidase genes (Adβgal)-transfected hMSCs/BCB (group D) were controls. Formation of bone tissue was assessed by histological methods 4 weeks and 8 weeks after implantation. Implanted cells were identified by human Y-chromosome-specific fluorescence in-situ hybridization (FISH). hMSCs differentiated into osteogenic cells, and bone formation was observed. Obvious bone formation was not noted at any time point in control groups. We hypothesize that the described method is a promising method for bone regeneration.

Decomposition of Two-Component Ultrasound Pulses in Cancellous Bone Using Modified Least Squares Prony Method – Phantom Experiment and Simulation

Porous media such as cancellous bone often support the simultaneous propagation of two compressional waves. When small bone samples are interrogated in through-transmission with broadband sources, these two waves often overlap in time. The modified least-squares Prony's (MLSP) method was tested for decomposing a 500 kHz-center-frequency signal containing two overlapping components: one passing through a polycarbonate plate (to produce the “fast” wave) and another passing through a cancellous-bone-mimicking phantom (to produce the “slow” wave). The MLSP method yielded estimates of attenuation slopes accurate to within 7% (polycarbonate plate) and 2% (cancellous bone phantom). The MLSP method yielded estimates of phase velocities accurate to within 1.5% (both media). The MLSP method was also tested on simulated data generated using attenuation slopes and phase velocities corresponding to bovine cancellous bone. Throughout broad ranges of signal-to-noise ratio (SNR), the MLSP method yielded estimates of attenuation slope that were accurate to within 1.0% and estimates of phase velocity that were accurate to within 4.3% (fast wave) and 1.3% (slow wave). (E-mail: keith.wear@fda.hhs.gov).

Preparation of porous biphasic .BETA.-TCP/HA bioceramics with a natural trabecular structure from calcined cancellous bovine bone

Porous biphasic β-TCP/HA bioceramics were fabricated by sintering of calcined cancellous bovine bone (CBB) treated with different quantities of NH4H2PO4 at various temperatures. The obtained materials had a porous structure (macropores within 200-800 μm and micropores within 1-5 μm) of natural trabecular bones. The specimens were characterized by X-ray diffraction, scanning electron microscopy (SEM), and gravimetric thermal analysis (TG). The results revealed that porous biphasic β-TCP/HA bioceramics were successfully prepared at sintering temperatures within 762°C-829°C. Biological apatite of calcined CBB was found to transform partly into Ca2P2O7 at heating temperatures between 503°C and 762°C. As the ratio NH4H2PO4/calcined-CBB increased, bioceramics with different phase compositions of β-TCP/HA, β-TCP/HA/Ca2P2O7 or β-TCP/Ca2P2O7 were produced after sintering.

Mineral status and mechanical properties of cancellous bone exposed to hydrogen peroxide for various time periods

Processed cancellous bone has been regarded as one alternative for the treatment of bone defects. In order to avoid immunogenic effects and preserve the natural properties of the bone, the optimal processing method should be determined. To observe the influence of hydrogen peroxide on the mineral status and mechanical properties of cancellous bone for various time periods and find the optimal processing time. Cancellous bone granules from bovine femur condyles were treated with 30% hydrogen dioxide for 0, 12, 24, 36, 48, 60 and 72h separately. The microstructure and mineral content of the granules were evaluated by ash analysis, Micro-CT, scanning electron micrograph and energy dispersive X-ray. The biomechanical properties were analyzed by applying cranial-caudal compression in a materials testing machine. With increasing exposure to hydrogen peroxide, the BMD and BMC of granules gradually decreased, and the Ca/P molar ratios clearly increased (P<0.05). Meanwhile, the mineral content of the granules increased from 48.5 ±1.3 to 79.5±2.1%. Substantial decreases in the strength of the granules were observed, and after 48h severe decreases were noted. The decrease in strength was also evident after normalizing the parameters to the cross-sectional area. Granules of bovine cancellous bone matrix should be processed by hydrogen peroxide for 12 to 36h to fulfill the basic requirements of a bone tissue engineering scaffold. These granules could potentially be useful during orthopedic operations.

Post-yield relaxation behavior of bovine cancellous bone

Relaxation studies were conducted on specimens of bovine cancellous bone at post-yield strains. Stress and strain were measured for 1000 s and the relaxation modulus was determined. Fifteen cylindrical, cancellous bone specimens were removed from one bovine femur in the anterior–posterior direction. The relaxation modulus was found to be a function of strain. Therefore cancellous bone is non-linearly viscoelastic/viscoplastic in the plastic region. A power law regression was fit to the relaxation modulus data. The multiplicative constant was found to be statistically related through a power law relationship to both strain ( p<0.0005) and apparent density ( p<0.0005) while the power coefficient was found to be related through a power law relationship, E( t, ε)= A( ε) t − n( ε) , to strain ( p<0.0005), but not apparent density.

In vivo engineering of a human vasculature for bone tissue engineering applications

The neovascularization of three-dimensional voluminous tissues, such as bone, represents an important challenge in tissue engineering applications. The formation of a preformed vascular plexus could maintain cell viability and promote vascularization after transplantation. We have developed a three-dimensional spheroidal coculture system consisting of human primary endothelial cells and human primary osteoblasts (hOBs) to improve angiogenesis in bone tissue engineering applications. In this study, we investigated the survival and vascularization of the engineered implants in vivo. Endothelial cell spheroids were cocultured with hOBs in fibrin and seeded into scaffolds consisting of processed bovine cancellous bone (PBCB). The cell-seeded scaffolds were evaluated for their angiogenic potential in two different in vivo assays: the chick embryo chorioallantoic membrane (CAM) model and the severe combined immunodeficiency disorder (SCID) mouse model. In both assays, the development of a complex three-dimensional network of perfused human neovessels could be detected. After subcutaneous implantation into immunodeficient mice, the newly formed human vasculature was stabilized by the recruitment of murine smooth muscle α-actin-positive mural cells and anastomoses with the mouse vasculature. We conclude that this endothelial cell spheroid system can be used to create a network of functional perfused blood vessels in vivo. The finding that this process takes place with high efficacy in the presence of co-implanted primary osteoblasts and in an osteoconductive environment provided by the PBCB scaffold, suggests that this system may be suitable for improving vascularization in bone tissue engineering.

Observation of Ultrasonic Fields Transmitted through Bovine Cortical or Cancellous Bones by the Schlieren Method

Ultrasonic wave fields at 1.03 and 3.5 MHz were visualized by the schlieren method after they had traveled through bovine cortical or cancellous bones. Schlieren images showed that wavefronts were distorted at both frequencies when they traveled through a cortical bone of a bovine limb. After traveling through cancellous bones, wavefronts at 1.03 MHz were almost undistorted, but those at 3.5 MHz showed distortions depending on the condition of the bone. These results indicate that correction is required with regard to ultrasound-receiving geometry to improve the accuracy of attenuation measurement in ultrasonic bone densitometry.