Comparative histomorphometric analysis of cellular phenotype in canine stifle ligaments and tendon.

OBJECTIVE: To measure the density of cellular phenotypes in canine caudal cruciate ligament (CaCL), cranial cruciate ligament (CrCL), medial collateral ligament (MCL), and long digital extensor tendon (LDET). STUDY DESIGN: Ex-vivo study. METHODS: Ten CaCL, CrCL, MCL, and LDET obtained from 1 stifle of 10 dogs with no gross pathology were analyzed histologically. The density of cells with 3 nuclear phenotypes (fusiform, ovoid, and spheroid) was determined within the core region of each specimen. RESULTS: Cells with fusiform nuclei were most dense in the MCL (median [range], 319 [118-538] cells/mm2 ) and LDET (331 [61-463]), whereas cells with ovoid nuclei were most dense in the CaCL (276 [123-368]) and CrCL (212 [165-420]). The spheroid nuclear phenotype had the lowest density in all structures (31 [5-61] in CaCL, 54 [5-90] in CrCL, 2 [0-14] in MCL, and 5 [0-80] in LDET); however, the CrCL contained a denser population of spheroid cells compared with MCL and LDET (P < .05). Total cell densities did not differ among the 4 structures (P > .05). CONCLUSION: Phenotype density varied within the ligaments and tendon tested here. The cell population of CaCL and CrCL differed from that of dense collagenous tissues such as MCL and LDET. CLINICAL SIGNIFICANCE: The relatively higher density of spheroid phenotype in CrCL may reflect a distinctive native cellular population or a cellular transformation secondary to unique mechanical environment or hypoxia. This intrinsic cellular population may explain altered tissue properties prone to pathological rupture or poor healing potential of the canine CrCL.

Method and Instrumented Fixture for Femoral Fracture Testing in a Sideways Fall-on-the-Hip Position.

Mechanical testing of femora brings valuable insights into understanding the contribution of clinically-measureable variables such as bone mineral density distribution and geometry on the femoral mechanical properties. Currently, there is no standard protocol for mechanical testing of such geometrically complex bones to measure strength, and stiffness. To address this gap we have developed a protocol to test cadaveric femora to fracture and to measure their biomechanical parameters. This protocol describes a set of adaptable fixtures to accommodate the various load magnitudes and directions accounting for possible bone orientations in a fall on the hip configuration, test speed, bone size, and left leg-right leg variations. The femora were prepared for testing by cleaning, cutting, scanning, and potting the distal end and greater trochanter contact surfaces in poly(methyl methacrylate) (PMMA) as presented in a different protocol. The prepared specimens were placed in the testing fixture in a position mimicking a sideways fall on the hip and loaded to fracture. During testing, two load cells measured vertical forces applied to the femoral head and greater trochanter, a six-axis load cell measured forces and moments at the distal femoral shaft, and a displacement sensor measured differential displacement between the femoral head and trochanter contact supports. High speed video cameras were used to synchronously record the sequence of fracture events during testing. The reduction of this data allowed us to characterize the strength, stiffness, and fracture energy for nearly 200 osteoporotic, osteopenic, and normal cadaveric femora for further development of engineering-based diagnostic tools for osteoporosis research.

Proximal Cadaveric Femur Preparation for Fracture Strength Testing and Quantitative CT-based Finite Element Analysis.

Cadaveric fracture testing is routinely used to understand factors that affect proximal femur strength. Because ex vivo biological tissues are prone to lose their mechanical properties over time, specimen preparation for experimental testing must be performed carefully to obtain reliable results that represent in vivo conditions. For that reason, we designed a protocol and a set of fixtures to prepare the femoral specimens such that their mechanical properties experienced minimal changes. The femora were kept in a frozen state except during preparation steps and mechanical testing. The relevant clinical measures of total hip and femoral neck bone mineral density (BMD) were obtained with a clinical dual X-ray absorptiometry (DXA) bone densitometer, and the 3D geometry and distribution of bone mineral were obtained using CT with a calibration phantom for quantitative estimations based on the greyscale values. Any possible bone disease, fracture, or the presence of implants or artifacts affecting the bone structure, was ruled out with X-ray scans. For preparation, all bones were carefully cleaned of excess soft tissue, and were cut and potted at the internal rotation angle of interest. A cutting fixture allowed the distal end of the bone to be cut off leaving the proximal femur at a desired length. To allow positioning of the femoral neck at prescribed angles during later CT scanning and mechanical testing, the proximal femoral shafts were potted in polymethylmethacrylate (PMMA) using a fixture designed specifically for desired orientations. The data collected from our experiments were then used for validation of quantitative computed tomography (QCT)-based finite element analysis (FEA), as described in a different protocol. In this manuscript, we present the protocol for the precise bone preparation for mechanical testing and subsequent QCT/FEA modeling. The current protocol was successfully applied to prepare about 200 cadaveric femora over a 6-year time period.

Mechanical and morphological properties of trabecular bone samples obtained from third metacarpal bones of cadavers of horses with a bone fragility syndrome and horses unaffected by that syndrome.

OBJECTIVE: To determine morphological and mechanical properties of trabecular bone of horses with a bone fragility syndrome (BFS; including silicate-associated osteoporosis). SAMPLE: Cylindrical trabecular bone samples from the distal aspects of cadaveric third metacarpal bones of 39 horses (19 horses with a BFS [BFS bone samples] and 20 horses without a BFS [control bone samples]). PROCEDURES: Bone samples were imaged via micro-CT for determination of bone volume fraction; apparent and mean mineralized bone densities; and trabecular number, thickness, and separation. Bone samples were compressed to failure for determination of apparent elastic modulus and stresses, strains, and strain energy densities for yield, ultimate, and failure loads. Effects of BFS and age of horses on variables were determined. RESULTS: BFS bone samples had 25% lower bone volume fraction, 28% lower apparent density, 18% lower trabecular number and thickness, and 16% greater trabecular separation versus control bone samples. The BFS bone samples had 22% lower apparent modulus and 32% to 33% lower stresses, 10% to 18% lower strains, and 41 % to 52% lower strain energy densities at yield, ultimate, and failure loads, compared with control bone samples. Differences between groups of bone samples were not detected for mean mineral density and trabecular anisotropy. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that horses with a BFS had osteopenia and compromised trabecular bone function, consistent with bone deformation and pathological fractures that develop in affected horses. Effects of this BFS may be systemic, and bones other than those that are clinically affected had changes in morphological and mechanical properties.

Biomechanical evaluation of screw-in femoral implant in cementless total hip system.

OBJECTIVE: To compare (1) proximal femoral axial strains, (2) femoral head deflection, and (3) failure mechanical properties, between Helica head and neck prosthesis implanted femora and normal femora. STUDY DESIGN: In vitro study. SAMPLE POPULATION: Cadaveric canine femora (n = 5 pair). METHODS: Femoral bone strains and head displacement during in vitro simulation of midstance of the gallop were evaluated using cadaveric femurs cyclically loaded in vitro. Strains and displacements were compared within femurs, before and after, prosthesis implantation; and throughout cycling to seek evidence of movement with cyclic loading. Subsequently, implanted femurs and contralateral, intact femurs were loaded to failure to compare failure mechanical properties and modes of failure. RESULTS: Proximal femoral axial strains were significantly different between intact and implanted femora on all 4 cortical surfaces (P < .05). Compressive strains were lower in the implanted femur on all cortical surfaces, except on the caudal surface which was higher. No difference was noted for femoral head angle under an axial load corresponding to gallop (P > .05). Vertical head displacement was ∼0.1 mm greater for implanted femora than intact femora (P < .05). Yield and failure loads and yield energy of implanted femora were 39-54% lower than those for intact femora (P < .05). Mode of failure for both the intact and implanted femora did not appear to be different. CONCLUSION: Helica femoral prosthesis alters strain distribution in the proximal aspect of the femur and exhibits initial micromotion. Failure load in axial compression of the Helica-implanted femur is less than that of the normal femur, but greater than that expected in vivo.

Immunohistochemical and histomorphometric evaluation of vascular distribution in intact canine cranial cruciate ligament.

OBJECTIVES: To (1) describe vascular distribution in the grossly intact canine cranial cruciate ligament (CCL) using immunohistochemical techniques specific to 2 components of blood vessels (factor VIII for endothelial cells, laminin for basement membrane); and (2) compare the vascularity in different areas of interest (craniomedial versus caudolateral bands; core versus epiligamentous regions; and proximal versus middle versus distal portions) in the intact normal canine CCL. STUDY DESIGN: In vitro study. ANIMALS: Large, mature dogs (n=7) of breeds prone to CCL disease that were euthanatized for nonorthopedic conditions. METHODS: Intact CCL were collected from fresh canine cadavers free from stifle pathology. CCL tissue was processed for immunohistochemistry and stained for factor VIII and laminin. Vascular density was determined by histomorphometric analysis. RESULTS: Specific vascular staining was sparsely identified throughout the CCL; however, the proximal portion of the CCL appears to have a greater number of vessels than the middle or distal portion of the ligament. CONCLUSIONS: The CCL is a hypovascular tissue and its vascular distribution is not homogeneous.

Vascular distribution in ruptured canine cranial cruciate ligament.

OBJECTIVE: To (1) determine the microanatomic vascular distribution in ruptured canine cranial cruciate ligaments (CCL) using specific vascular immunohistochemical techniques, and (2) compare vessel density between ruptured and intact canine CCL and between different areas of interest in ruptured CCL using histomorphometric analysis. STUDY DESIGN: In vitro study. ANIMALS: Dogs (n=41) admitted for surgical treatment of ruptured CCL and 19 dogs euthanatized for nonorthopedic conditions. METHODS: Diseased (variable CCL rupture) and intact (normal control) CCL were processed for immunohistochemical staining specific to vessels (factor VIII, laminin). Mean vascular density was assessed and compared in areas of interest (torn end versus remaining core regions of CCL, proximal femoral versus distal tibial core CCL regions). RESULTS: Ruptured CCL was more vascular than intact CCL; however there was no difference in vascular density between the torn end and the remaining core area of the ruptured CCL. Ruptured CCL was vascularized to a greater degree at the proximal portion than the distal portion of the CCL. Partially ruptured CCLs had a higher vessel density than completely ruptured CCLs. CONCLUSIONS: Vascular density is increased in diseased CCL compared with intact CCL. It remains to be determined whether this finding is associated with the cause of CCL rupture or is a result of CCL degeneration and rupture.

Robust QCT/FEA models of proximal femur stiffness and fracture load during a sideways fall on the hip.

Clinical implementation of quantitative computed tomography-based finite element analysis (QCT/FEA) of proximal femur stiffness and strength to assess the likelihood of proximal femur (hip) fractures requires a unified modeling procedure, consistency in predicting bone mechanical properties, and validation with realistic test data that represent typical hip fractures, specifically, a sideways fall on the hip. We, therefore, used two sets (n = 9, each) of cadaveric femora with bone densities varying from normal to osteoporotic to build, refine, and validate a new class of QCT/FEA models for hip fracture under loading conditions that simulate a sideways fall on the hip. Convergence requirements of finite element models of the first set of femora led to the creation of a new meshing strategy and a robust process to model proximal femur geometry and material properties from QCT images. We used a second set of femora to cross-validate the model parameters derived from the first set. Refined models were validated experimentally by fracturing femora using specially designed fixtures, load cells, and high speed video capture. CT image reconstructions of fractured femora were created to classify the fractures. The predicted stiffness (cross-validation R (2) = 0.87), fracture load (cross-validation R (2) = 0.85), and fracture patterns (83% agreement) correlated well with experimental data.

Material properties are related to stress fracture callus and porosity of cortical bone tissue at affected and unaffected sites.

Stress fractures are overuse injuries of bone that affect elite athletes and military recruits. One response of cortical bone to stress fracture is to lay down periosteal callus. The objectives of this study were to determine if material properties are different among bones with different stages of stress fracture callus, at both a callus site and at a distal site. Cortical specimens were mechanically tested to determine their stress-strain response. Material property differences were examined using nonparametric and regression analyses. At the callus site, material properties were low during the earliest stages of callus, higher with increasing callus maturity, but dropped at the late stage of callus. At the distal site, the material properties were low during early stages of callus and approached, or returned to, those of bones without callus during the late stages of callus. The effects of stress fracture and bone callus are not limited to the focal site of stress fracture.

Effect of GDF-7 deficiency on tail tendon phenotype in mice.

The subfamily of growth/differentiation factors (GDFs) known as GDFs 5, 6, and 7 appears to be involved in tendon maintenance and repair, although the precise nature of this role has yet to be elucidated. The aim of the present study was to examine the role of GDF-7 in tendon maintenance by studying tail tendon fascicle gene expression, composition, and material property strain rate dependency in 16-week-old male and female GDF-7 deficient mice. GDF-7 deficiency did not affect the biochemical composition of tail tendon fascicles, nor did it significantly affect the tensile material properties obtained at either slow (5%/s) or fast (50%/s) strain rates. Further, no difference was found between genotypes in the strain rate sensitivity of any tensile material property. Consistent with the compositional analyses, QRT-PCR data did not reveal any differences of twofold or greater in the gene expression levels of collagens I, III, V, nor in the proteoglycans decorin, fibromodulin, lumican, biglycan, versican, or aggrecan. Gdf5 expression was upregulated twofold in GDF-7 deficient tail tendons, and Bmp7 expression was downregulated twofold. No notable differences in expression levels for Bmp1-6 or Gdf6 were detected. GDF-5 protein levels were 50% higher in GDF-7 deficient tail tendon compared to wild type tail tendon. The results of this study support the intriguing possibility that compensation by Gdf-5 may be at least in part responsible for the absence of a strong phenotype in GDF-7 deficient mice.