A clinically realistic large animal model of intra-articular fracture that progresses to post-traumatic osteoarthritis.

OBJECTIVE: Translation of promising treatments for post-traumatic osteoarthritis (PTOA) to patients with intra-articular fracture (IAF) has been limited by the lack of a realistic large animal model. To address this issue we developed a large animal model of IAF in the distal tibia of Yucatan minipigs and documented the natural progression of this injury. DESIGN: Twenty-two fractures were treated using open reduction and internal fixation with either an anatomic reduction or an intentional 2-mm step-off. Pre-operatively, and 3 days, 1, 2, 4, 8, and 12 weeks post-operatively, animals were sedated for synovial fluid draws and radiographs. Limb loading was monitored at the same time points using a Tekscan Walkway. Animals were sacrificed at 12 weeks and the limbs were harvested for histological evaluation. RESULTS: All animals achieved bony union by 12 weeks, facilitating nearly complete recovery of the initial 60% decrease in limb loading. TNFα, IL1ß, IL6, and IL8 concentrations in the fractured limbs were elevated (P < 0.05) at specific times during the 2 weeks after fracture. Histological cartilage degeneration was more severe in the step-off group (0.0001 < P < 0.27 compared to normal) than in the anatomic reconstruction group (0.27 < P < 0.99 compared to normal). CONCLUSIONS: This model replicated key features of a human IAF, including surgical stabilization, inflammatory responses, and progression to osteoarthritic cartilage degeneration, thereby providing a potentially useful model for translating promising treatment options to clinical practice.

An instrumented pendulum system for measuring energy absorption during fracture insult to large animal joints in vivo.

For systematic laboratory studies of bone fractures in general and intra-articular fractures in particular, it is often necessary to control for injury severity. Quantitatively, a parameter of primary interest in that regard is the energy absorbed during the injury event. For this purpose, a novel technique has been developed to measure energy absorption in experimental impaction. The specific application is for fracture insult to porcine hock (tibiotalar) joints in vivo, for which illustrative intra-operative data are reported. The instrumentation allowed for the measurement of the delivered kinetic energy and of the energy passed through the specimen during impaction. The energy absorbed by the specimen was calculated as the difference between those two values. A foam specimen validation study was first performed to compare the energy absorption measurements from the pendulum instrumentation versus the work of indentation performed by an MTS machine. Following validation, the pendulum apparatus was used to measure the energy absorbed during intra-articular fractures created in 14 minipig hock joints in vivo. The foam validation study showed close correspondence between the pendulum-measured energy absorption and MTS-performed work of indentation. In the survival animal series, the energy delivered ranged from 31.5 to 48.3 Js (41.3±4.0, mean±s.d.) and the proportion of energy absorbed to energy delivered ranged from 44.2% to 64.7% (53.6%±4.5%). The foam validation results support the reliability of the energy absorption measure provided by the instrumented pendulum system. Given that a very substantial proportion of delivered energy passed--unabsorbed--through the specimens, the energy absorption measure provided by this novel technique arguably provides better characterization of injury severity than is provided simply by energy delivery.

A novel impaction technique to create experimental articular fractures in large animal joints.

OBJECTIVE: A novel impaction fracture insult technique, developed for modeling post-traumatic osteoarthritis in porcine hocks in vivo, was tested to determine the extent to which it could replicate the cell-level cartilage pathology in human clinical intra-articular fractures. DESIGN: Eight fresh porcine hocks (whole-joint specimens with fully viable chondrocytes) were subjected to fracture insult. From the fractured distal tibial surfaces, osteoarticular fragments were immediately sampled and cultured in vitro for 48 h. These samples were analyzed for the distribution and progression of chondrocyte death, using the Live/Dead assay. Five control joints, in which "fractures" were simulated by means of surgical osteotomy, were also similarly analyzed. RESULTS: In the impaction-fractured joints, chondrocyte death was concentrated in regions adjacent to fracture lines (near-fracture regions), as evidenced by fractional cell death significantly higher (P < 0.0001) than in central non-fracture (control) regions. Although nominally similar spatial distribution patterns were identified in the osteotomized joints, fractional cell death in the near-osteotomy regions was nine-fold lower (P < 0.0001) than in the near-fracture regions. Cell death in the near-fracture regions increased monotonically during 48 h after impaction, dominantly within 1 mm from the fracture lines. CONCLUSION: The impaction-fractured joints exhibited chondrocyte death characteristics reasonably consistent with those in human intra-articular fractures, but were strikingly different from those in "fractures" simulated by surgical osteotomy. These observations support promise of this new impaction fracture technique as a mechanical insult modality to replicate the pathophysiology of human intra-articular fractures in large animal joints in vivo.

The effect of incongruity and instability on contact stress directional gradients in human cadaveric ankles.

OBJECTIVE: Measure incongruity and instability-associated changes in transient contact stress directional gradients in a human cadaveric ankle model. METHODS: Seven cadaveric ankles were subjected to quasi-physiologic forces and motion under intact conditions and with a stepoff incongruity of the anterior one-third of the distal tibia. Anterior/posterior forces were modulated to create incongruous specimens that either maintained a stable articulation between the talus and distal tibia or developed gross instability during motion. Real-time contact stresses were measured using a custom-designed ankle stress transducer at 132 Hz. Contact stress data were differentiated using a central-differencing formula to calculate transient contact stress directional gradients over the entire ankle articulation. RESULTS: Transient 95th percentile contact stress directional gradient values increased by 30 and 100%, respectively, in stable-incongruous and unstable-incongruous conditions compared to intact conditions. Compared to stable-incongruous conditions, transient contact stress directional gradients increased by 60% in unstable-incongruous conditions. CONCLUSIONS: Instability resulted in greater percentage increases in transient contact stress directional gradients compared to incongruity. Pathologic increases in contact stress directional gradients potentially play an important role in the etiology of post-traumatic arthritis.

The effect of accuracy of implantation on range of movement of the Scandinavian Total Ankle Replacement.

When performing the Scandinavian Total Ankle Replacement (STAR), the positioning of the talar component and the selection of mobile-bearing thickness are critical. A biomechanical experiment was undertaken to establish the effects of these variables on the range of movement (ROM) of the ankle. Six cadaver ankles containing a specially-modified STAR prosthesis were subjected to ROM determination, under weight-bearing conditions, while monitoring the strain in the peri-ankle ligaments. Each specimen was tested with the talar component positions in neutral, as well as 3 and 6 mm of anterior and posterior displacement. The sequence was repeated with an anatomical bearing thickness, as well as at 2 mm reduced and increased thicknesses. The movement limits were defined as 10% strain in any ligament, bearing lift-off from the talar component or limitations of the hardware. Both anterior talar component displacement and bearing thickness reduction caused a decrease in plantar flexion, which was associated with bearing lift-off. With increased bearing thickness, posterior displacement of the talar component decreased plantar flexion, whereas anterior displacement decreased dorsiflexion.

Significance of caveolin-1 and matrix metalloproteinase 14 gene expression in canine mammary tumours.

Canine mammary tumours (CMTs) are the most common neoplasms affecting female dogs. There is an urgent need for molecular biomarkers that can detect early stages of the disease in order to improve accuracy of CMT diagnosis. The aim of this study was to examine whether caveolin-1 (Cav-1) and matrix metalloproteinase 14 (MMP14) are associated with CMT histological malignancy and invasion. Sixty-five benign and malignant CMT samples and six normal canine mammary glands were analysed using quantitative reverse transcription-polymerase chain reaction. Cav-1 and MMP14 genes were highly expressed in CMT tissues compared to normal tissues. Cav-1 especially was overexpressed in malignant and invasive CMT tissues. When a CMT cell line was cultured on fluorescent gelatin-coated coverslips, localisation of Cav-1 was observed at invadopodia-mediated degradation sites of the gelatin matrix. These findings suggest that Cav-1 may be involved in CMT invasion and that the markers may be useful for estimating CMT malignancy.

Influence of the interosseous talocalcaneal ligament injury on stability of the ankle-subtalar joint complex--a cadaveric experimental study.

The present study aims to clarify the influence of the interosseous talocalcaneal ligament (ITCL) injury associated with injury to the lateral ankle ligaments on the ankle-subtalar joint complex motion under conditions of physiologic loading. We conducted mechanical tests using five fresh cadaveric lower extremities. Each specimen was mounted in the loading device and an axial cyclic load from 9.8 to 686 N was applied. Three-dimensional rotations of the ankle and the subtalar joint were measured simultaneously by a linkage electric goniometer. Mechanical tests were repeated after sectioning of the anterior talofibular ligament (ATFL), and again after additional sectioning of the ITCL. In the intact condition, the ankle and the subtalar joints rotated consistently with increase of the load. The predominant rotations were plantar flexion and adduction at the ankle joint, with some eversion demonstrated at the subtalar joint. Although ATFL sectioning did not significantly change the motion of the two joints, additional sectioning of the ITCL significantly increased adduction and total rotation of the ankle joint. The present study demonstrated that a combined injury of the ATFL and the ITCL can induce anterolateral rotatory instability of the ankle joint under conditions of axial loading.

Acute inversion injury of the ankle: magnetic resonance imaging and clinical outcomes.

This study was undertaken to compare the clinical and magnetic resonance imaging results of 24 patients who had sustained ligament injuries after acute inversion injury of the ankle. On magnetic resonance imaging, the following lesions were detected: anterior talofibular ligament tear in 23 patients, calcaneofibular ligament lesion in 15, posterior talofibular ligament lesion in 11, interosseous talocalcaneal ligament lesion in 13, cervical ligament lesion in 12, and deltoid ligament lesion in 8. Compared with the clinical outcome at the follow-up study, there was a statistically significant relationship between interosseous talocalcaneal ligament lesion and each of giving way, pain, and limitation of ankle motion; between cervical ligament lesion and both giving way and pain; and between deltoid ligament lesion and giving way (P < 0.05).

The microtubule-associated protein astrin transgenesis rescues spermatogenesis and renal function in hypogonadic (hgn/hgn) rats.

Male hypogonadic (hgn/hgn) rats show male sterility, reduced female fertility, progressive renal insufficiency and body growth retardation. These defects are associated with loss-of-function mutation of astrin and appear to be related to organ hypoplasia resulting from abnormal cell proliferation and increased cell death during embryonic and early postnatal development. As targeted disruption of mouse spag5 (astrin ortholog) has been reported to show no phenotype, we performed rescue experiments based on the introduction of rat astrin cDNA transgene into hgn/hgn rats to determine whether astrin is actually necessary for the establishment of normal male fertility and renal function. Astrin transgenic (Tg) rats were mated with hgn/+ rats of the HGN strain, and Tg-hgn/+ rats were then crossed to obtain Tg-hgn/hgn. Tg-hgn/hgn males showed recovery of body growth, fertility and renal function. Testis size was smaller in these transgenic animals than normal controls, but showed an increase by 16.5-fold compared with hgn/hgn males. Spermatogenesis occurred in Tg-hgn/hgn testes, and their accessory reproductive organs were of approximately normal size. hgn/hgn males show hypergonadotropic hypogonadism. Increased testosterone and decreased LH levels in Tg-hgn/hgn serum indicated the recovery of Leydig cells' function. Tg-hgn/hgn males showed normal reproductive behaviour, and their mating with Tg-hgn/hgn females produced pups in normal litter size. Their renal sizes and glomerular numbers showed complete recovery, and renal function assayed by biochemical parameters was normal. These results indicated that the transgene is functional in the testis and kidney development as well as body growth. In conclusion, astrin is necessary for the establishment of normal size (cell number) and function of the testis and kidney in rats.

MTBP plays a crucial role in mitotic progression and chromosome segregation.

Murine double minute 2 (MDM2) binding protein (MTBP) has been implicated in tumor cell proliferation, but the underlying mechanisms remain unclear. The results of MTBP expression analysis during cell cycle progression demonstrated that MTBP protein was rapidly degraded during mitosis. Immunofluorescence studies revealed that a portion of MTBP was localized at the kinetochores during prometaphase. MTBP overexpression delayed mitotic progression from nuclear envelope breakdown (NEB) to anaphase onset and induced abnormal chromosome segregation such as lagging chromosomes, chromosome bridges, and multipolar chromosome segregation. Conversely, MTBP downmodulation caused an abbreviated metaphase and insufficient mitotic arrest, resulting in abnormal chromosome segregation, aneuploidy, decreased cell proliferation, senescence, and cell death, similar to that of Mad2 (mitotic arrest-deficient 2) downmodulation. Furthermore, MTBP downmodulation inhibited the accumulation of Mad1 and Mad2, but not BubR1 (budding uninhibited by benzimidazoles related 1), on the kinetochores, whereas MTBP overexpression inhibited the release of Mad2 from the metaphase kinetochores. These results may imply that MTBP has an important role in recruiting and/or retaining the Mad1/Mad2 complex at the kinetochores during prometaphase, but its degradation is required for silencing the mitotic checkpoint. Together, this study indicates that MTBP has a crucial role in proper mitotic progression and faithful chromosome segregation, providing new insights into regulation of the mitotic checkpoint.

Mtbp haploinsufficiency in mice increases tumor metastasis.

Mdm2 inhibits the function of the p53 tumor suppressor. Mdm2 is overexpressed in many tumors with wild-type p53 suggesting an alternate mechanism of loss of p53 activity in tumors. An Mdm2-binding protein (MTBP) was identified using a yeast two-hybrid screen. In tissue culture, MTBP inhibits Mdm2 self-ubiquitination, leading to stabilization of Mdm2 and increased degradation of p53. To address the role of MTBP in the regulation of the p53 pathway in vivo, we deleted the Mtbp gene in mice. Homozygous disruption of Mtbp resulted in early embryonic lethality, which was not rescued by loss of p53. Mtbp+/- mice were not tumor prone. When mice were sensitized for tumor development by p53 heterozygosity, we found that the Mtbp+/-p53+/- mice developed significantly more metastatic tumors (18.2%) as compared to p53+/- mice (2.6%). Results of in vitro migration and invasion assays support the in vivo findings. Downmodulation of Mtbp in osteosarcoma cells derived from p53+/- mice resulted in increased invasiveness, and overexpression of Mtbp in Mtbp+/-p53+/- osteosarcoma cells inhibited invasiveness. These results suggest that MTBP is a metastasis suppressor. These results advance our understanding of the cellular roles of MTBP and raise the possibility that MTBP is a novel therapeutic target for metastasis.

Stance-phase aggregate contact stress and contact stress gradient changes resulting from articular surface stepoffs in human cadaveric ankles.

OBJECTIVE: Determine how stepoff incongruities of the distal tibia affect aggregate (whole-cycle) contact stresses and contact stress gradients for a complete motion cycle in human cadaveric ankles. METHOD: Ten human cadaveric ankles were subjected to quasiphysiologic forces during stance-phase range of motion. Each specimen was loaded intact, with anatomic reduction of the anterolateral quarter of the distal tibia, and with increasing stepoffs of the anterolateral fragment up to 4.0mm. Transient contact stresses were measured using a custom-built, real-time stress transducer that sampled stresses at 132Hz at 1472 separate foci (sensels). Aggregate stresses were calculated by summing the sequential transient stress values multiplied by the transient sampling duration for the complete motion cycle at each sensel. Transient contact stress gradients were calculated at each sensel using a central-differencing formula applied to adjacent transient stress measurements. Aggregate contact stress gradients were calculated by vector summation of sequential transient stress gradients multiplied by the sampling duration. RESULTS: Compared to the intact configuration, anatomic reduction of the fragment caused minimal changes in aggregate contact stresses and stress gradients (30% increase compared to intact values). In contrast, stepoffs caused substantial increases (200% increase compared to intact values) in peak and mean whole-cycle stresses and gradients. CONCLUSIONS: Aggregate contact stresses and stress gradients quantify loading history for the complete motion cycle. Incongruity-associated changes in aggregate stresses and gradients are a surrogate for "accumulated" damage over a motion cycle in stepoff specimens. These loading abnormalities may be important determinants of posttraumatic arthritis.

The effect of agility ankle prosthesis misalignment on the peri-ankle ligaments.

In the Agility total ankle replacement system, motion is constrained by the implant's articulating surfaces and the peri-ankle ligaments. The effects of plausibly occurring implant malpositioning on peri-ankle ligament functional extension during walking were explored in this study. The intent was to determine whether certain ligaments could serve as guides to assist in proper component positioning at implantation. Using a cadaver preparation with simulated physiologic motion and loading, we monitored change of ligament length of the anterior talofibular, posterior talofibular, calcaneofibular, and tibiocalcaneal ligaments resulting from controlled malpositioning of the tibial component relative to a neutral position. During a simulated walking cycle, effects of mediolateral and anterior/posterior translation, internal and external rotation, inversion and eversion, and elevation of the component were evaluated. In all cases, tibial component displacement from the neutral position caused atypical length change in one or more of the peri-ankle ligaments. In particular, anterior/posterior displacement significantly changed the lengthening behavior of all four tested ligaments. The anterior talofibular ligament was sensitive to transverse plane displacements, whereas the tibiocalcaneal ligament was sensitive to coronal plane displacements. For the Agility prosthesis, these two ligaments seem to be sensitive guides for tibial component positioning at implantation.