Articular cartilage gene expression patterns in the tissue surrounding the impact site following applications of shear and axial loads.

BACKGROUND: Osteoarthritis is a degradative joint disease found in humans and commercial swine which can develop from a number of factors, including prior joint trauma. An impact injury model was developed to deliver in vitro loads to disease-free porcine patellae in a model of OA. METHODS: Axial impactions (2000 N normal) and shear impactions (500 N normal with induced shear forces) were delivered to 48 randomly assigned patellae. The patellae were then cultured for 0, 3, 7, or 14 days following the impact. Specimens in the tissue surrounding the loading site were harvested and expression of 18 OA related genes was studied via quantitative PCR. The selected genes were previously identified from published work and fell into four categories: cartilage matrix, degradative enzymes, inflammatory response, and apoptosis. RESULTS: Type II collagen (Col2a1) showed significantly lower expression in shear vs. axial adjacent tissue at day 0 and 7 (fold changes of 0.40 & 0.19, respectively). In addition, higher expression of degradative enzymes and Fas, an apoptosis gene, was observed in the shear specimens. CONCLUSIONS: The results suggest that a more physiologically valid shear load may induce more damage to surrounding articular cartilage than a normal load alone.

Influence of gait on bone strength in turkeys with leg defects.

Leg problems have become more frequent in fast-growing turkeys. The objective of this study was to evaluate the effects of common leg defects on kinetic parameters of gait and biomechanical properties of bone. Nine hundred, day-old, male, Large White turkeys were raised in 48 floor pens. At 42 d of age, turkeys were divided into four categories of leg condition as determined by visual evaluation: Normal, Crooked toes, Shaky legs, and Valgus. Fifteen toms were selected from each group and trained to walk on a pressure sensitive walkway. Gait kinetic data were collected at 92, 115 and 144 d of age. At 145 d of age, turkeys were sacrificed and bones were collected and frozen until analysis. Morphological measurements of femur, tibia and tarsus-metatarsus were recorded. Bone mineral density (BMD) and content (BMC) were obtained using DEXA. Bone strength of tibias was evaluated using a four-point bending test and femurs with a torsion test. ANOVA was used to detect differences among groups, and Tukey's test used for mean separation. There were no differences in BW among different leg conditions. Gait parameters changed as turkeys aged, and age-group interactions were observed on peak vertical force (PVF), contact time, step length (SL) and bipedal cycle. No differences (P > 0.05) were detected in morphological measurements of femur or tibia. Relative asymmetry of femur length was lower (P < 0.05) in Normal and Valgus turkeys than in toms with Crooked toes. There were no differences (P > 0.05) among groups for femur BMD, BMC or strength. Tibia BMD and the area moment of inertia of turkeys with Crooked toes were lower (P < 0.05) than in toms with Valgus. With all four leg conditions, femur strength was positively correlated with PVF and negatively correlated with SL; BMD and BMC were correlated with tibia strength and gait kinetic parameters. In conclusion, only crooked toes caused consistent differences in gait patterns, bone properties and bone strength, but in general, gait kinetics was correlated with bone biomechanics in turkeys.

Gait parameters in four strains of turkeys and correlations with bone strength.

Locomotion problems in meat poultry have multifactorial etiology. A better understanding of normal gait and its influences on biomechanical aspects of leg bones among turkey genetic lines is important to prevent skeletal disorders and locomotion issues. The objective of this experiment was to determine the possible differences in gait kinetic and kinematic parameters of turkey strains and their effects on bone biomechanical properties. Four genetic lines, named A, B, C, and D, were obtained and raised in 48 floor pens with new pine shavings. Leg health issues were classified at 16 and 33 d of age. Fifteen turkeys from each strain with apparent normal legs and gait at 33 d of age were selected for gait analysis. These 15 turkeys were trained to walk on a pressure sensitive walkway and video was recorded to calculate articulation movements. These data also were analyzed to obtain kinetic and kinematic parameters of the gait cycle collected at 47, 84, 107, and 145 d of age. At 20 wk all turkeys were sacrificed, and legs were collected and frozen for analysis. Weights and morphologic measurements of the femur, tibia, and shank were recorded. Bone mineral density (BMD) and content (BMC) were obtained using DEXA. Femur and tibia strength were evaluated by a 4-point bending test and torsion test, respectively. Gait parameters changed as toms aged and some differences were observed among lines. Genetic lines differed on BMD, but not on BMC. Strain D had a higher BMD and smaller diaphyseal angle than strain C, characteristics that were correlated with stronger bones. Strain D also had the lowest incidence of leg problems while strain C had the highest. Furthermore, the D strain had a smaller vertical motion of the toe than strains C and B at 47 d and strain A at 145 d, indicating that the D strain had a more efficient gait. In summary, genetic strains differ significantly on gait parameters, which in turn impacts bone biomechanics.

Progression of Gene Expression Changes following a Mechanical Injury to Articular Cartilage as a Model of Early Stage Osteoarthritis.

An impact injury model of early stage osteoarthritis (OA) progression was developed using a mechanical insult to an articular cartilage surface to evaluate differential gene expression changes over time and treatment. Porcine patellae with intact cartilage surfaces were randomized to one of three treatments: nonimpacted control, axial impaction (2000 N), or a shear impaction (500 N axial, with tangential displacement to induce shear forces). After impact, the patellae were returned to culture for 0, 3, 7, or 14 days. At the appropriate time point, RNA was extracted from full-thickness cartilage slices at the impact site. Quantitative real-time PCR was used to evaluate differential gene expression for 18 OA related genes from four categories: cartilage matrix, degradative enzymes and inhibitors, inflammatory response and signaling, and cell apoptosis. The shear impacted specimens were compared to the axial impacted specimens and showed that shear specimens more highly expressed type I collagen (Col1a1) at the early time points. In addition, there was generally elevated expression of degradative enzymes, inflammatory response genes, and apoptosis markers at the early time points. These changes suggest that the more physiologically relevant shear loading may initially be more damaging to the cartilage and induces more repair efforts after loading.

In vitro biomechanical comparison of the flexion/extension mobility of the canine lumbosacral junction before and after dorsal laminectomy and partial discectomy.

The purpose of this canine cadaver study was to evaluate the range of flexion and extension of the canine lumbosacral spine before and after dorsal laminectomy and partial discectomy. Using a cantilever biomechanical system, a 3Nm bending moment was applied to flex and extend the lumbosacral segment. Motion in L7 (total range of motion [ROM] and neutral zone motion [NZ]) was recorded via a rotational potentiometer. There was a significant increase in NZ and ROM after the decompressive procedures (NZ before decompression 6.0±1.2°; NZ after decompression 7.6±2.1°; ROM before decompression 32.8±6.4°; ROM after decompression 40.2±5.6°). It is unknown whether dorsal laminectomy and partial discectomy will induce the same increased motion in clinical cases. Dogs with lumbosacral subluxation, active dogs with little radiographic degenerative changes and working dogs could benefit from lumbosacral stabilization. This cadaver study demonstrated that dorsal laminectomy and partial discectomy at the lumbosacral junction does lead to significant spinal instability.

Organic trace minerals and 25-hydroxycholecalciferol affect performance characteristics, leg abnormalities, and biomechanical properties of leg bones of turkeys.

Leg problems and resulting mortality can exceed 1% per week in turkey toms starting at approximately 15 wk of age. Dietary supplementation of organic trace minerals (MIN) and 25-hydroxycholecalciferol (HyD) may improve performance, decrease incidence of leg abnormalities, and increase bone strength. Nicholas 85X700 toms were assigned to 4 treatments consisting of a factorial arrangement of 2 concentrations of MIN (0 and 0.1% of Mintrex P(Se), which adds 40, 40, 20, and 0.3 mg/kg of Zn, Mn, Cu, and Se, respectively) and 2 concentrations of HyD (0 and 92 microg/kg of HyD). Diets were formulated to be equal in nutrient content and fed ad libitum as 8 feed phases. Feed intake and BW were measured at 6, 12, 15, 17, and 20 wk of age. Valgus, varus, and shaky leg defects were determined at 12, 15, 17, and 20 wk of age. Tibia and femur biomechanical properties were evaluated by torsion and bending tests at 17 wk of age. There were no treatment effects on BW. Only MIN significantly improved feed conversion ratio through to 20 wk of age. Cumulative mortality at 3 wk of age was greater among the MIN birds, but it was lower by 20 wk (P = 0.085). The MIN decreased the incidence of varus defects at 17 wk of age; shaky leg at 12, 15, and 17 wk of age; and valgus defects at 15, 17, and 20 wk of age. There were no MIN x HyD interaction effects on individual gait problems. Maximum load and the bending stress required for tibias to break in a 4-point assay were increased with MIN supplementation, especially when HyD was also added. Maximum shear stress at failure of femoral bones in a torsion assay was increased by supplementation with both MIN and HyD together. Dietary supplementation of MIN and HyD may improve biomechanical properties of bones. Dietary MIN supplementation may improve feed conversion of turkeys, likely by decreasing leg problems.

Gene expression profiling of chondrocytes from a porcine impact injury model.

OBJECTIVE: To identify differentially expressed genes between axially impacted and control articular cartilage taken from porcine patellae maintained in organ culture for 14 days. METHODS: Porcine patellae were impacted perpendicular to the articular surface to create an impact injury. Intact patellae (control and impacted) were maintained in culture for 14 days. Total RNA was then extracted from the articular cartilage beneath the impaction and used to prepare two Serial Analysis of Gene Expression (SAGE) libraries. Approximately 42,500 SAGE long tags were sequenced from the libraries. The expression of select genes was confirmed by quantitative real-time PCR analysis. RESULTS: Thirty-nine SAGE tags were significantly differentially expressed in the impacted and control libraries, representing 30 different annotated pig genes. These genes represented gene products associated with matrix molecules, iron and phosphate transport, protein biosynthesis, skeletal development, cell proliferation, lipid metabolism and the inflammatory response. Twenty-three of the 30 genes were down-regulated in the impacted library and five were up-regulated in the impacted library. Quantitative real-time PCR follow-up of four genes supported the results found with SAGE. CONCLUSION: We have identified 30 putative genes differentially expressed in a porcine impact injury model and validated these findings for four of these genes using real-time PCR. Results using this impact injury model have contributed further evidence that damaged chondrocytes may de-differentiate into fibroblast-like cells and proliferate in an attempt to repair themselves. Additional work is underway to study these genes in further detail at earlier time points to provide a more complete story about the fate of chondrocytes in articular cartilage following an injury.

Two photon induced polymerization of organic-inorganic hybrid biomaterials for microstructured medical devices.

Three-dimensional microstructured medical devices, including microneedles and tissue engineering scaffolds, were fabricated by two photon induced polymerization of Ormocer organic-inorganic hybrid materials. Femtosecond laser pulses from a titanium:sapphire laser were used to break chemical bonds on Irgacure 369 photoinitiator within a small focal volume. The radicalized starter molecules reacted with Ormocer US-S4 monomers to create radicalized polymolecules. The desired structures are fabricated by moving the laser focus in three dimensions using a galvano-scanner and a micropositioning system. Ormocer surfaces fabricated using two photon induced polymerization demonstrated acceptable cell viability and cell growth profiles against B35 neuroblast-like cells and HT1080 epithelial-like cells. Lego-like interlocking tissue engineering scaffolds and microneedle arrays with unique geometries were created using two photon induced polymerization. These results suggest that two photon induced polymerization is able to create medical microdevices with a larger range of sizes, shapes, and materials than chemical isotropic etching, injection molding, reactive ion etching, surface micromachining, bulk micromachining, polysilicon micromolding, lithography-electroforming-replication, or other conventional microfabrication techniques.

Mechanical modulation of growth for the correction of vertebral wedge deformities.

This study tested the following hypotheses: (a) a vertebral wedge deformity created by chronic static asymmetrical loading will be corrected by reversal of the load asymmetry; (b) a vertebral wedge deformity created by chronic static asymmetrical loading will remain if the load is simply removed; and (c) vertebral longitudinal growth rates, altered by chronic static loading, will return to normal after removal of the load. An external fixator was used to impose an angular deformity (Cobb angle of 30 degrees) and an axial compression force (60% body weight) on the ninth caudal (apical) vertebra in two groups of 12 5-week-old Sprague-Dawley rats. This asymmetrical loading was applied to all rats for 4 weeks to create an initial wedge deformity in the apical vertebra. The rats from group I (load reversal) then underwent 1 week of distraction loading followed by 4 weeks of asymmetrical compressive loading with the imposed 30 degree Cobb angle reversed. The rats from group II (load removal) had the apparatus removed and were followed for 5 weeks with no external loading. Weekly radiographs were obtained and serial fluorochrome labels were administered to follow vertebral wedging. After the initial 4-week loading period, the combined average wedge deformity that developed in the apical vertebra of the animals in both groups was 10.7 +/- 4.4 degrees. The group that underwent load reversal showed significant correction of the deformity with the wedging of the apical vertebra decreasing to, on average, 0.1 +/- 1.4 degrees during the 4 weeks of load reversal. Wedging of the apical vertebra in the group that underwent load removal significantly decreased to 7.3 +/- 3.9 degrees during the first week after removal of the load, but no significant changes in wedging occurred after that week. This indicated a return to a normal growth pattern following the removal of the asymmetrically applied loading. The longitudinal growth rate of the apical vertebra also returned to normal following removal of the load. Vertebrae maintained under a load of 60% body weight grew at a rate that was 59.4 +/- 17.0% lower than that of the control vertebrae, whereas after vertebrae were unloaded their growth averaged 102.4 +/- 31.8%. These findings show that a vertebral wedge deformity can be corrected by reversing the load used to create it and that vertebral growth is not permanently affected by applied loading.

Compression-induced changes in intervertebral disc properties in a rat tail model.

STUDY DESIGN: An Ilizarov-type apparatus was applied to the tails of rats to assess the influence of immobilization, chronically applied compression, and sham intervention on intervertebral discs of mature rats. OBJECTIVES: To test the hypothesis that chronically applied compressive forces and immobilization cause changes in the biomechanical behavior and biochemical composition of rat tail intervertebral discs. SUMMARY OF BACKGROUND DATA: Mechanical factors are associated with degenerative disc disease and low back pain, yet there have been few controlled studies in which the effects of compressive forces on the structure and function of the disc have been isolated. METHODS: The tails of 16 Sprague-Dawley rats were instrumented with an Ilizarov-type apparatus. Animals were separated into sham, immobilization, and compression groups based on the mechanical conditions imposed. In vivo biomechanical measurements of disc thickness, angular laxity, and axial and angular compliance were made at 14-day intervals during the course of the 56-day experiment, after which discs were harvested for measurement of water, proteoglycan, and collagen contents. RESULTS: Application of pins and rings alone (sham group) resulted in relatively small changes of in vivo biomechanical behavior. Immobilization resulted in decreased disc thickness, axial compliance, and angular laxity. Chronically applied compression had effects similar to those of immobilization alone but induced those changes earlier and in larger magnitudes. Application of external compressive forces also caused an increase in proteoglycan content of the intervertebral discs. CONCLUSIONS: The well-controlled loading environment applied to the discs in this model provides a means of isolating the influence of joint-loading conditions on the response of the intervertebral disc. Results indicate that chronically applied compressive forces, in the absence of any disease process, caused changes in mechanical properties and composition of tail discs. These changes have similarities and differences in comparison with human spinal disc degeneration.

Progression of vertebral wedging in an asymmetrically loaded rat tail model.

STUDY DESIGN: A rat tail model was used to test the hypothesis that angulation and asymmetric axial compressive loading would lead to vertebral wedging because of asymmetric longitudinal growth in the physes. OBJECTIVES: To study the effect of angulation and asymmetric loading on the progression of spinal curvature in a rat tail model. SUMMARY OF BACKGROUND DATA: Large idiopathic scoliotic curves in children with significant growth remaining are the curves most likely to progress. The mechanism of progression of skeletal deformities is thought to be controlled by the Hueter-Volkmann law, whereby additional axial compression decelerates growth, and reduced axial compression accelerates growth. It has been hypothesized that spinal curvature leads to asymmetric loading transversely along the vertebral growth plate, causing progressive vertebral wedging by means of a vicious cycle. METHODS: Two 32-mm diameter external ring fixators were glued to 0.7-mm pins that had been inserted percutaneously through the eighth and 10th caudal vertebra of 10 6-week-old Sprague-Dawley rats. Calibrated springs and 15 degrees wedges, mounted on stainless steel threaded rods passing through holes distributed around the rings, imposed a 30 degrees Cobb angle and axially compressed the instrumented vertebrae. Fluorochrome labels and radiographs were used to document the progression of vertebral wedging. RESULTS: The wedging initially was entirely in the intervertebral discs, but by 6 weeks the wedging of the discs and vertebrae were approximately equal. Fluorochrome labeling confirmed that the vertebral wedging resulted from asymmetric growth in the physes. CONCLUSIONS: This study shows that vertebrae, when asymmetrically loaded, become wedged. This is consistent with the concept of mechanically provoked progression of scoliotic deformities according to the Hueter-Volkmann law.

Intraoperative force-setting did not improve the mechanical properties of an augmented bone-tendon-bone anterior cruciate ligament graft in a goat model.

It has been hypothesized that load affects the mechanical properties of an anterior cruciate ligament graft while it remodels. The goal of this study was to use an existing goat model to evaluate the effect of intraoperative set force on the postoperative mechanical properties of an autograft that had been augmented with a synthetic segment. The following questions were addressed. Do augmented autografts set with a high force intraoperatively have improved structural and material graft properties and lower anterior-posterior knee laxity at 3 months after surgery, compared with autografts set with a low intraoperative force? How do the structural and material properties of these implanted autografts compare with the mechanical properties of an intact anterior cruciate ligament or an unimplanted control autograft? The anterior cruciate ligament was reconstructed in seven goats with use of a composite graft consisting of a bone-patellar tendon-bone autograft and a synthetic augmentation device. A force-setting technique was used intraoperatively to establish the load-sharing between the autograft and augmentation segments such that the autograft carried either a high (16.5 N in four animals) or low (1.5 N in three animals) level of force, while the total force in the composite graft remained constant. Tensile testing was performed at 3 months after surgery to determine the material and structural properties of the autograft, the intact anterior cruciate ligament from the normal contralateral knee, and a control bone-patellar tendon-bone graft of similar size that was harvested from the contralateral knee at the time of necropsy and had never been implanted in the joint. The structural and material properties of the autografts initially set to high or low loads at surgery were not significantly different after 3 months of implantation. The strength and stiffness of the implanted tendons were an average of 24 and 20% of the strength and stiffness of the normal anterior cruciate ligament and 31 and 62% of the control tendons, respectively. Intraoperative set force in an augmented anterior cruciate ligament graft at the levels chosen in this study did not significantly affect weakening of the autograft at 3 months.

Design and biomechanics of a plate for the distal radius.

A dorsal plate for the distal radius was designed to provide rigid fixation and thus allow early motion. It functions as a blade plate, lessening the role of metaphyseal screws, and providing internal neutralization rather than compression. The rigidity and strength of the plate were compared to the existing T-plate in an unstable, extra-articular fracture model in paired, fresh-cadaver, axially loaded radii. The dorsal plate construct was significantly stronger and more rigid than the T-plate construct. The failure mode was similar for both plate types; 8 of 10 constructs failed with plate bending and screw loosening, while the oldest specimen pair showed primary bone failure. Compared to the T-plate, the dorsal plate transmitted a greater single axial load from the articular surface to the shaft.

Elastic modulus of calcified cartilage is an order of magnitude less than that of subchondral bone.

The elastic moduli of calcified cartilage and subchondral bone tissues were measured experimentally with use of a three-point bending test. Specimens were obtained from a bovine patella and the distal end of a bovine femur, from two different animals. Fifteen specimens were tested as "pure" subchondral bone beams, and 15 were tested as composite calcified cartilage/subchondral bone beams. A least-squares optimization scheme was used to obtain modulus values from the composite beams. The elastic modulus for subchondral bone calculated from the "pure" subchondral bone beams was 2.3 +/- 1.5 GPa (3.9 +/- 1.5 GPa for specimens from the femur and 1.6 +/- 0.7 GPa for specimens from the patella). The composite beam optimization resulted in a modulus for subchondral bone of 5.7 +/- 1.9 GPa and a modulus for calcified cartilage of 0.32 +/- 0.25 GPa. The modulus for the calcified cartilage was more than an order of magnitude lower than the modulus of the underlying subchondral bone. This supports the idea that the zone of calcified cartilage forms a transitional zone of intermediate stiffness between the articular cartilage and the subchondral bone.

Experimental method for the measurement of the elastic modulus of trabecular bone tissue.

A procedure has been developed to measure the elastic modulus of small, irregularly shaped specimens without significantly disturbing the specimen's internal or surface structure. This procedure was developed to measure the average elastic modulus of isolated trabeculae from human cancellous bone tissue. The procedure combines direct testing of a cantilever beam-type specimen, along with finite element modeling of the specimen and the testing conditions. Initial estimates for the bone tissue material properties are input into the finite element model; differences between the calculated finite element displacement and the experimentally observed displacement allows the actual material modulus to be determined. Machined aluminum and cortical bone specimens were used to test the accuracy and repeatability of the procedure. Manipulations of the finite element models were performed to examine the effect that mesh construction errors might have on the accuracy of the results. None of the parameters examined resulted in changes in the measured finite element displacements of greater than 8%. In tests on six trabecular bone specimens, an average elastic modulus of 7.8 GPa was calculated. Even taking into account the possible sources of error, this value remains significantly less than the accepted value for cortical bone.