Evaluation of equine articular cartilage degeneration after mechanical impact injury using cationic contrast-enhanced computed tomography.

OBJECTIVE: Cationic agent contrast-enhanced computed tomography (cationic CECT) characterizes articular cartilage ex vivo, however, its capacity to detect post-traumatic injury is unknown. The study objectives were to correlate cationic CECT attenuation with biochemical, mechanical and histological properties of cartilage and morphologic computed tomography (CT) measures of bone, and to determine the ability of cationic CECT to distinguish subtly damaged from normal cartilage in an in vivo equine model. DESIGN: Mechanical impact injury was initiated in equine femoropatellar joints in vivo to establish subtle cartilage degeneration with site-matched controls. Cationic CECT was performed in vivo (clinical) and postmortem (microCT). Articular cartilage was characterized by glycosaminoglycan (GAG) content, biochemical moduli and histological scores. Bone was characterized by volume density (BV/TV) and trabecular number (Tb.N.), thickness (Tb.Th.) and spacing (Tb.Sp.). RESULTS: Cationic CECT attenuation (microCT) of cartilage correlated with GAG (r = 0.74, P < 0.0001), compressive modulus (Eeq) (r = 0.79, P < 0.0001) and safranin-O histological score (r = -0.66, P < 0.0001) of cartilage, and correlated with BV/TV (r = 0.37, P = 0.0005), Tb.N. (r = 0.39, P = 0.0003), Tb.Th. (r = 0.28, P = 0.0095) and Tb.Sp. (r = -0.44, P < 0.0001) of bone. Mean [95% CI] cationic CECT attenuation at the impact site (2215 [1987, 2443] Hounsfield Units [HUs]) was lower than site-matched controls (2836 [2490, 3182] HUs, P = 0.036). Clinical cationic CECT attenuation correlated with GAG (r = 0.23, P = 0.049), Eeq (r = 0.26, P = 0.025) and safranin-O histology score (r = -0.32, P = 0.0046). CONCLUSIONS: Cationic CECT (microCT) reflects articular cartilage properties enabling segregation of subtly degenerated from healthy tissue and also reflects bone morphometric properties on CT. Cationic CECT is capable of characterizing articular cartilage in clinical scanners.

Engineering of hyaline cartilage with a calcified zone using bone marrow stromal cells.

OBJECTIVE: In healthy joints, a zone of calcified cartilage (ZCC) provides the mechanical integration between articular cartilage and subchondral bone. Recapitulation of this architectural feature should serve to resist the constant shear force from the movement of the joint and prevent the delamination of tissue-engineered cartilage. Previous approaches to create the ZCC at the cartilage-substrate interface have relied on strategic use of exogenous scaffolds and adhesives, which are susceptible to failure by degradation and wear. In contrast, we report a successful scaffold-free engineering of ZCC to integrate tissue-engineered cartilage and a porous biodegradable bone substitute, using sheep bone marrow stromal cells (BMSCs) as the cell source for both cartilaginous zones. DESIGN: BMSCs were predifferentiated to chondrocytes, harvested and then grown on a porous calcium polyphosphate substrate in the presence of triiodothyronine (T3). T3 was withdrawn, and additional predifferentiated chondrocytes were placed on top of the construct and grown for 21 days. RESULTS: This protocol yielded two distinct zones: hyaline cartilage that accumulated proteoglycans and collagen type II, and calcified cartilage adjacent to the substrate that additionally accumulated mineral and collagen type X. Constructs with the calcified interface had comparable compressive strength to native sheep osteochondral tissue and higher interfacial shear strength compared to control without a calcified zone. CONCLUSION: This protocol improves on the existing scaffold-free approaches to cartilage tissue engineering by incorporating a calcified zone. Since this protocol employs no xenogeneic material, it will be appropriate for use in preclinical large-animal studies.

Technical note: Three-dimensional imaging of rumen tissue for morphometric analysis using micro-computed tomography.

Rumen development in calves has been evaluated by measuring papillae length, width, and density using microscopy for over 50 yr. Although common in the literature, disadvantages to this method exist, such as large variations in rumen papillae size and shape, small numbers of total papillae being measured, and the time required. The objective of this study was to develop a more effective technique for assessing rumen papillae using micro-computed tomography (micro-CT) and to compare this technique with microscopy. Rumen tissue was collected from the ventral sac of 20 postweaned bull calves at 55 d of age, immediately fixed in 10% neutral buffered formalin for 48 h, and stored in 70% ethanol at 4°C before the contrast enhancement. After evaluation of contrast-enhancement protocols, it was determined that mercury chloride provided the most pronounced contrast for accurate micro-CT imaging based on relative density of the papillae. A 1-cm(2) tissue section from the ventral sac of all bull calves was tensioned on a rapid prototyped curved plastic holder and imaged at 4 5 µm resolution for 56 min using a GE Locus Explore micro-CT (General Electric, Milwaukee, WI). MicroView V2.2 software (General Electric) was used to create a 3-dimensional virtual model of the entire sample. The length and width of papillae were measured 3-dimensionally and compared with measurements of papillae under the light microscope taken from the same region. The length and width measurements using micro-CT (2.47 ± 0.12 and 0.55 ± 0.01 mm) compared with light microscope (2.96 ± 0.03 and 0.86 ± 0.01 mm) were significantly smaller. The difference may reflect a more accurate determination in the base of the rumen tissue with micro-CT or the specificity of mercury chloride to bind only to intact rumen tissue. The mean number of papillae per centimeter squared viewed using micro-CT was 128.5 ± 33.9 with a total surface area of 681.8 ± 112.4 mm(2) and volume of 156 mm(3) per sample. Micro-CT data demonstrated that surface area and volume are positively associated and that papillae length was negatively associated with papillae per centimeter squared and positively associated with total volume of tissue section. This study represents the first time that micro-CT has been being used to assess morphology of rumen tissue. Micro-CT has the potential to improve the accuracy and efficiency of rumen tissue measurements; however, more standardization of each factor involved in tissue preparation, imaging, and location of papillae measurements is required.

Effect of Press-Fit Size on Insertion Mechanics and Cartilage Viability in Human and Ovine Osteochondral Grafts.

OBJECTIVE: The osteochondral allograft procedure uses grafts constructed larger than the recipient site to stabilize the graft, in what is known as the press-fit technique. This research aims to characterize the relationships between press-fit size, insertion forces, and cell viability in ovine and human osteochondral tissue. DESIGN: Human (4 donors) and ovine (5 animals) articular joints were used to harvest osteochondral grafts (4.55 mm diameter, N = 33 Human, N = 35 Ovine) and create recipient sites with grafts constructed to achieve varying degrees of press fit (0.025-0.240 mm). Donor grafts were inserted into recipient sites while insertion forces were measured followed by quantification of chondrocyte viability and histological staining to evaluate the extracellular matrix. RESULTS: Both human and ovine tissues exhibited similar mechanical and cellular responses to changes in press-fit. Insertion forces (Human: 3-169 MPa, Ovine: 36-314 MPa) and cell viability (Human: 16%-89% live, Ovine: 2%-76% live) were correlated to press-fit size for both human (force: r = 0.539, viability: r = -0.729) and ovine (force: r = 0.655, viability: r = -0.714) tissues. In both species, a press-fit above 0.14 mm resulted in reduced cell viability below a level acceptable for transplantation, increased insertion forces, and reduced linear correlation to press-fit size compared to samples with a press-fit below 0.14 mm. CONCLUSIONS: Increasing press-fit size required increased insertion forces and resulted in reduced cell viability. Ovine and human osteochondral tissues responded similarly to impact insertion and varying press-fit size, providing evidence for the use of the ovine model in allograft-related research.

Intra-articular anaesthesia mitigates established pain in experimental osteoarthritis: a preliminary study of gait impulse redistribution as a biomarker of analgesia pharmacodynamics.

OBJECTIVE: Develop a sensitive, functional biomarker of persistent joint pain in a large animal model of experimental osteoarthritis. Evaluate Impulse Ratio as a measure of weight distribution among supporting limbs throughout the early natural history of osteoarthritis and with local anaesthesia and analgesia. DESIGN: The distribution of weight bearing in the trot of 11 skeletally-mature dogs was analyzed before and after unilateral surgical intervention (cranial cruciate transection or distal femoral focal impact). The short-term effects of two analgesic treatments (intra-articular lidocaine and intra-dermal meloxicam) were then evaluated as an index of pain relief based on the redistribution of weight-bearing impulse between normal and injured limbs. RESULTS: Impulse Ratio was able to resolve weight redistribution between limbs in both long-term (weekly for over 400 days) and short-term (15 min intervals) joint evaluations. Joint pain relief from lidocaine administration could be reliably tracked over its brief acting time course. Meloxicam administration resulted in ambiguous results, where average weight bearing in the injured limb did not increase, but the variability of limb use changed transiently and reversibly. CONCLUSION: Joint function and the role of persistent joint pain in the development of osteoarthritis can be investigated effectively and efficiently in a large animal model through the use of Impulse Ratio. Impulse Ratio can be a functionally relevant and sensitive biomarker of locomotion-related joint pain.

Serum non-coding RNAs as biomarkers for osteoarthritis progression after ACL injury.

OBJECTIVE: The aim of this study was to examine serum non-coding RNAs as potential biomarkers for cartilage damage associated with anterior cruciate ligament (ACL) injury. METHODS: Serum was obtained from 80 patients 1 year after surgery for ACL injury and 60 normal donors without overt skeletal injury. Total serum RNA was isolated, small non-coding RNAs profiled by TaqMan array MicroRNA (miRNA) analysis and individual small RNA assays performed by quantitative TaqMan RT-PCR (qPCR). Semi-quantitative magnetic resonance imaging (MRI) analysis was performed using Whole Organ Magnetic Resonance Knee Score (WORMS) scoring for analysis of cartilage damage. RESULTS: Initial TaqMan array miRNA profiling showed an increased serum concentration of a small nucleolar RNA (snoRNA), U48, in five patients with cartilage damage compared with that in five patients without cartilage damage and six normal donors. Independent qPCR analysis of snoRNAs in serum from all patients and normal donors showed a strong association between the serum level of another snoRNA, U38, and cartilage damage in ACL injury patients and together with snoRNA, U48, clear distinction between ACL injury patients and normal donors. CONCLUSION: SnoRNAs U38 and U48 are significantly elevated in the serum of patients developing cartilage damage at 1 year after ACL injury. Serum levels of U38 have the potential to facilitate early diagnosis of patients with cartilage damage after ACL injury. This study suggests serum non-coding RNAs may serve as novel noninvasive biomarkers for the detection and assessment of cartilage damage after ACL injury.

A polarized light microscopy method for accurate and reliable grading of collagen organization in cartilage repair.

OBJECTIVES: Collagen organization, a feature that is critical for cartilage load bearing and durability, is not adequately assessed in cartilage repair tissue by present histological scoring systems. Our objectives were to develop a new polarized light microscopy (PLM) score for collagen organization and to test its reliability. DESIGN: This PLM score uses an ordinal scale of 0-5 to rate the extent that collagen network organization resembles that of young adult hyaline articular cartilage (score of 5) vs a totally disorganized tissue (score of 0). Inter-reader reliability was assessed using Intraclass Correlation Coefficients (ICC) for Agreement, calculated from scores of three trained readers who independently evaluated blinded sections obtained from normal (n=4), degraded (n=2) and repair (n=22) human cartilage biopsies. RESULTS: The PLM score succeeded in distinguishing normal, degraded and repair cartilages, where the latter displayed greater complexity in collagen structure. Excellent inter-reader reproducibility was found with ICCs for Agreement of 0.90 [ICC(2,1)] (lower boundary of the 95% confidence interval is 0.83) and 0.96 [ICC(2,3)] (lower boundary of the 95% confidence interval is 0.94), indicating the reliability of a single reader's scores and the mean of all three readers' scores, respectively. CONCLUSION: This PLM method offers a novel means for systematically evaluating collagen organization in repair cartilage. We propose that it be used to supplement current gold standard histological scoring systems for a more complete assessment of repair tissue quality.

Streaming potential-based arthroscopic device is sensitive to cartilage changes immediately post-impact in an equine cartilage injury model.

Models of post-traumatic osteoarthritis where early degenerative changes can be monitored are valuable for assessing potential therapeutic strategies. Current methods for evaluating cartilage mechanical properties may not capture the low-grade cartilage changes expected at these earlier time points following injury. In this study, an explant model of cartilage injury was used to determine whether streaming potential measurements by manual indentation could detect cartilage changes immediately following mechanical impact and to compare their sensitivity to biomechanical tests. Impacts were delivered ex vivo, at one of three stress levels, to specific positions on isolated adult equine trochlea. Cartilage properties were assessed by streaming potential measurements, made pre- and post-impact using a commercially available arthroscopic device, and by stress relaxation tests in unconfined compression geometry of isolated cartilage disks, providing the streaming potential integral (SPI), fibril modulus (Ef), matrix modulus (Em), and permeability (k). Histological sections were stained with Safranin-O and adjacent unstained sections examined in polarized light microscopy. Impacts were low, 17.3 ± 2.7 MPa (n = 15), medium, 27.8 ± 8.5 MPa (n = 13), or high, 48.7 ± 12.1 MPa (n = 16), and delivered using a custom-built spring-loaded device with a rise time of approximately 1 ms. SPI was significantly reduced after medium (p = 0.006) and high (p<0.001) impacts. Ef, representing collagen network stiffness, was significantly reduced in high impact samples only (p < 0.001 lateral trochlea, p = 0.042 medial trochlea), where permeability also increased (p = 0.003 lateral trochlea, p = 0.007 medial trochlea). Significant (p < 0.05, n = 68) moderate to strong correlations between SPI and Ef (r = 0.857), Em (r = 0.493), log(k) (r = -0.484), and cartilage thickness (r = -0.804) were detected. Effect sizes were higher for SPI than Ef, Em, and k, indicating greater sensitivity of electromechanical measurements to impact injury compared to purely biomechanical parameters. Histological changes due to impact were limited to the presence of superficial zone damage which increased with impact stress. Non-destructive streaming potential measurements were more sensitive to impact-related articular cartilage changes than biomechanical assessment of isolated samples using stress relaxation tests in unconfined compression geometry. Correlations between electromechanical and biomechanical methods further support the relationship between non-destructive electromechanical measurements and intrinsic cartilage properties.

The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the horse.

OBJECTIVE: Equine models of osteoarthritis (OA) have been used to investigate pathogenic pathways of OA and evaluate therapeutic candidates for naturally occurring equine OA which is a significant clinical disease in the horse. This review focuses on the macroscopic and microscopic criteria for assessing naturally occurring OA in the equine metacarpophalangeal joint as well as the osteochondral fragment-exercise model of OA in the equine middle carpal joint. METHODS: A review was conducted of all published OA studies using horses and the most common macroscopic and microscopic scoring systems were summarized. Recommendations regarding methods of OA assessment in the horse have been made based on published studies. RESULTS: A modified Mankin scoring system is recommended for semi-quantitative histological assessment of OA in horses due to its already widespread use and similarity to other scoring systems. Recommendations are also provided for histological scoring of synovitis and macroscopic lesions of OA as well as changes in the calcified cartilage and subchondral bone of naturally occurring OA. CONCLUSIONS: The proposed system for assessment of equine articular tissues provides a useful method to quantify OA change. It is believed that addition of quantitative tracing onto plastic and macroscopic measurement as recently described would be an improvement for overall assessment of articular cartilage change.

Quantitative MR imaging evaluation of the cartilage thickness and subchondral bone area in patients with ACL-reconstructions 7 years after surgery.

OBJECTIVE: To evaluate the cartilage thickness (ThC) and subchondral bone area (tAB) of the operated and contra-lateral non-operated (healthy) knees in patients with anterior cruciate ligament (ACL)-reconstruction 7 years after surgery using a quantitative and regional cartilage MR imaging (qMRI) technique. METHODS: Charts of 410 patients with ACL-reconstructions were retrospectively reviewed. Fifty-two patients (male/female, 28/24; mean age, 33.3 years) were included. Patients underwent KT-1000 testing and qMRI of both knees using coronal fat-saturated 3D spoiled gradient-recalled echo (SPGR) sequences (TR/TE, 44/4 ms) at 1.5 T. Quantitative analyses of ThC and tAB in the femoro-tibial cartilage plates were performed using a subregional approach. In addition, qualitative and quantitative assessment of femoral condyle shapes was performed. t tests with Bonferroni corrections were used for statistical analysis of side-to-side differences between the operated and non-operated knees. RESULTS: KT-1000 testing was abnormal in 3/52 patients (6%). Lateral femoral tAB was significantly lower (-9.2%), and medial tibial tAB was significantly larger (+2%) in the operated vs non-operated knee (P<0.001). Regional and subregional ThC side-to-side differences were less than 0.1mm and, except for the external lateral femoral subregion, they were not statistically significant. Flattened and broader shapes of medial femoral condyles (P<0.001) were found in operated knees. No significant association of presence of cartilage or meniscus lesions at surgery with ThC 7 years post-operatively was found (P=0.06-0.98). CONCLUSION: There is evidence for changes in the tAB and femoral shape 7 years post-ACL-reconstruction, but no side-to-side differences in subregional ThC were found between the operated and contra-lateral non-operated knees.

Role of cartilage collagen fibrils networks in knee joint biomechanics under compression.

Collagen fibrils networks in knee cartilage and menisci change in content and structure from a region to another. While resisting tension, they influence global joint response as well as local strains particularly at short-term periods. To investigate the role of fibrils networks in knee joint mechanics and in particular cartilage response, a novel model of the knee joint is developed that incorporates the cartilage and meniscus fibrils networks as well as depth-dependent properties in cartilage. The joint response under up to 2000N compression is investigated for conditions simulating the absence in cartilage of deep fibrils normal to subchondral bone or superficial fibrils parallel to surface as well as localized split of cartilage at subchondral junction or localized damage to superficial fibrils at loaded areas. Deep vertical fibrils network in cartilage play a crucial role in stiffening (by 10%) global response and protecting cartilage by reducing large strains (from maximum of 102% to 38%), in particular at subchondral junction. Superficial horizontal fibrils protect the tissue mainly from excessive strains at superficial layers (from 27% to 8%). Local cartilage split at base disrupts the normal function of vertical fibrils at the affected areas resulting in higher strains. Deep fibrils, and to a lesser extent superficial fibrils, play dominant mechanical roles in cartilage response under transient compression. Any treatment modality attempting to repair or regenerate cartilage defects involving partial or full thickness osteochondral grafts should account for the crucial role of collagen fibrils networks and the demanding mechanical environment of the tissue.

Cytocompatible gel formation of chitosan-glycerol phosphate solutions supplemented with hydroxyl ethyl cellulose is due to the presence of glyoxal.

To deliver and retain viable repair cells in a surgically prepared cartilage lesion, we previously developed an adhesive in situ-gelling cell carrier by suspending cells in a solution of hydroxyethyl cellulose (HEC), which was then mixed with chitosan-glycerol phosphate to form a chitosan-GP/HEC gel. The purpose of this study was to elucidate the mechanism of gelation to maximally control gel time and viability of encapsulated cells. We analyzed the role of osmolality, pH, gelation temperature, gel shrinkage, and HEC. A chitosan-GP solution at pH 6.8 with cytocompatible osmotic pressure (419 mOsm/kg) was achieved by lowering disodium GP concentration from 370 to 135 mM. This solution was still thermogelling but only at 73 degrees C. We next discovered that glyoxal, a common additive in ether cellulose manufacturing, was responsible for chitosan gelation. Monolayer cells survived and proliferated in up to 1 mM of glyoxal, however only a very narrow range of glyoxal concentration in chitosan-GP/HEC, 0.1-0.15 mM, permitted gel formation, cell survival, and cell proliferation. Chitosan gels containing HEC required slightly less glyoxal to solidify. Chitosan-GP/HEC loaded with viable chondrocytes formed an adhesive seal with ex vivo mosaic arthroplasty defects from sheep knee joints. In mosaic arthroplasty defects of live sheep, bleeding occurred beneath part of the hydrogel carrier, and the gel was cleared after 1 month in vivo. These data indicate that chitosan-GP/HEC is suitable as an adhesive and injectable delivery vehicle for clinical orthopedic applications involving single use treatments that guide acute cartilage repair processes.

Osteochondral defect repair using a novel tissue engineering approach: sheep model study.

Porous calcium polyphosphate (CPP) constructs of desired density were formed by sintering CPP powders. Articular cartilage was formed on these constructs in cell culture over an 8-week period with the resulting cartilage layer forming on the CPP surface and within the near surface pores thereby mechanically anchoring the cartilage to the CPP. The biphasic constructs so formed were implanted in sheep femoral condyle sites and left for short-term periods (3 to 4 months) or longer periods (9 months). Implant fixation within the condyle sites was achieved through bone ingrowth into the inferior CPP pores. The properties and characteristics of the as-in vitro-formed, short- and long-term implanted tissues were compared. The results indicated that such implants might be useful for repair of small subchondral defects.

Contact mechanics of the ovine stifle during simulated early stance in gait. An in vitro study using robotics.

Ovine stifle joint contact pressures and contact areas were measured in vitro using a six degree-of-freedom (DOF) robotic system. The robot generated static joint loads of 1.875 times body weight (BW) compression, 0.15 BW medial shear and 0.625 BW cranial shear at 6.5 degrees of flexion for four specimens, simulating the early stance phase of gait (walking). This condition represents a period of intense loading and was implemented as a worst-case loading scenario for the joint at this gait. We determined that the medial and lateral compartments bore 5.5 +/- 0.9 MPa and 4.4 +/- 1.1 MPa of mean pressure, respectively, on 107.7 +/- 28.7 mm(2) and 60.8 +/- 56.3 mm(2) of area, respectively. The unique contribution of this study is that stifle contact pressures and areas were determined during loading which simulated physiological levels (early stance phase of gait). This information is important to our understanding of the stresses that must be borne by repair tissues/constructs that are implanted into human and animal tibio-femoral joints.

Repair of osteochondral defects with biphasic cartilage-calcium polyphosphate constructs in a sheep model.

There has been interest in developing novel biological treatments to repair focal cartilage defects. We have developed a method of forming biphasic constructs ("osteochondral"-type plug) in vitro consisting of cartilaginous tissue, formed on and anchored to the intended articulation surface of a porous ceramic substrate. The purpose of this study was to evaluate the biochemical and biomechanical properties and morphology of in vitro-formed biphasic constructs 3 and 9 months after implantation into 4mm diameter full thickness osteochondral defects in the trochlear groove of sheep stifles. The implants withstood loading in vivo up to 9 months with evidence of fusion to adjacent native cartilage and fixation by bone ingrowth into the ceramic substrate. The cartilage layer was eroded from those implants that were proud to the joint surface. Control implants (ceramic only) had fibrous tissue on the articulating surface after implantation for 3-4 months. Neither the cellularity nor proteoglycan content of the implanted cartilage, when it remained, changed significantly between 3 and 9 months and the collagen content increased slightly. The elastic equilibrium modulus of the cartilage improved with time with the greatest improvement (10-fold) occurring early during the first 3-4 months after implantation. This study suggests that biphasic constructs may be suitable to repair joint defects as the implants were maintained up to 9 months in sheep. Importantly the mechanical properties of the implanted cartilage improved significantly after implantation suggesting that cartilage can mature in vivo after implantation. The results indicate that further study of this treatment approach is warranted to attempt to overcome the technical surgical difficulties identified in this study.

Mapping of donor and recipient site properties for osteochondral graft reconstruction of subchondral cystic lesions in the equine stifle joint.

REASONS FOR PERFORMING STUDY: To improve osteochondral graft reconstruction of subchondral cystic lesions in the medial and lateral femoral condyles by matching the material properties of donor and recipient sites. OBJECTIVES: To measure biomechanical and biochemical parameters that influence the function and healing of osteochondral grafts used to reconstruct subchondral cystic lesions. HYPOTHESIS: Suitable donor sites are available within the stifle joint for reconstructing the femoral condyles, despite considerable regional property variation. METHODS: Fifty-six osteochondral cores were harvested from 6 distal femurs for initial studies that determined subchondral bone modulus of elasticity and ultimate stress. In a second study, 28 osteochondral cores were harvested from 6 distal femurs to measure cartilage aggregate modulus, thickness and sulphated glycosaminoglycan (sGAG) content. Using micro-CT imaging, subchondral bone mineral density and bone volume fraction were also measured. In both studies 2-dimensional contour plots using a bicubic interpolation method and normalised data were generated to allow visual comparison of joint surface characteristics. Statistical comparisons between donor and recipient site raw data were made using an ANOVA for repeated measures with a post hoc Tukey test. RESULTS: Material properties of cartilage and bone vary considerably over the surface of the stifle joint but the central region of the medial condyle, where subchondral cystic lesions freqdently occur, typically demonstrated bone strength and modulus values of the highest observed. Cartilage thickness and aggregate modulus were highest in the medial femoral condyle and axial aspect of the lateral condyle. CONCLUSIONS: Material properties of the grafts from the trochlear groove and axial aspect of the lateral trochlear ridge were the closest match for those found in the medial condyle, whereas properties of the lateral condyle were most similar to those found in the trochlear groove and axial aspect of the medial trochlear ridge.

Use of apparent transverse quantitative ultrasonography to assess skeletal integrity in layers.

Although there are several techniques currently available to assess skeletal integrity in live birds, few offer important features for application in a commercial setting, offering ease of use and moderate cost. Quantitative ultrasonography (QUS) is an established technique for diagnosis of osteoporosis in humans and horses that has potential application for layers. An OmniSense 700S quantitative ultrasonometer was evaluated for use with Single Comb White Leghorn hens. Humeral QUS values (m/s) were measured in a series of experiments using a total of 144 Shaver White hens. Significant correlations (P < 0.01) were observed among sequential QUS measurements taken on the same bird at 54, 60, and 66 wk of age. At the completion of the studies (66 wk of age), the left and right humeri were excised, cleaned, and rescanned. Postmortem QUS data from left and right humeri were related (R2 = 0.72, P < 0.0001), although future studies may need to consider both sides of the skeleton to account for asymmetry conditions. Ultrasound data collected from live hens at 66 wk of age correlated well with postmortem QUS data (R2 = 0.80, P < 0.0001). Quantitative ultrasonography did not correlate with humeral bone-breaking force measured postmortem. Bones from live hens, surrounded by tissue thicker than 4 mm, could not be read by the QUS probe.

Regional and depth variability of porcine meniscal mechanical properties through biaxial testing.

The menisci in the knee joint undergo complex loading in-vivo resulting in a multidirectional stress distribution. Extensive mechanical testing has been conducted to investigate the tissue properties of the knee meniscus, but the testing conditions do not replicate this complex loading regime. Biaxial testing involves loading tissue along two different directions simultaneously, which more accurately simulates physiologic loading conditions. The purpose of this study was to report mechanical properties of meniscal tissue resulting from biaxial testing, while simultaneously investigating regional variations in properties. Ten left, fresh porcine joints were obtained, and the medial and lateral menisci were harvested from each joint (twenty menisci total). Each menisci was divided into an anterior, middle and posterior region; and three slices (femoral, deep and tibial layers) were obtained from each region. Biaxial and constrained uniaxial testing was performed on each specimen, and Young's moduli were calculated from the resulting stress strain curves. Results illustrated significant differences in regional mechanical properties, with the medial anterior (Young's modulus (E)=11.14 ± 1.10 MPa), lateral anterior (E=11.54 ± 1.10 MPa) and lateral posterior (E=9.0 ± 1.2 MPa) regions exhibiting the highest properties compared to the medial central (E=5.0 ± 1.22 MPa), medial posterior (E=4.16 ± 1.13 MPa) and lateral central (E=5.6 ± 1.20 MPa) regions. Differences with depth were also significant on the lateral meniscus, with the femoral (E=12.7 ± 1.22 MPa) and tibial (E=8.6 ± 1.22 MPa) layers exhibiting the highest Young's moduli. This data may form the basis for future modeling of meniscal tissue, or may aid in the design of synthetic replacement alternatives.

Effect of hoof angle on joint contact area in the equine metacarpophalangeal joint following simulated impact loading ex vivo.

REASONS FOR PERFORMING STUDY: To add to the existing data on impact loading of the metacarpophalangeal (MCP) joint as a precursor to assessing the potential role of impact in joint disease. OBJECTIVES: To examine the effect of impact loading on contact areas of the first phalanx (P1) and proximal sesamoids (PS) with the third metacarpal (McIII) under 3 hoof-strike conditions (toe-first, flat, heel-first). STUDY DESIGN: Randomised, repeated controlled experiment using cadaver material. METHODS: Eight cadaver limbs were subjected to randomised, repeated controlled trials where the hoof was struck by a pendulum impact machine (impact velocity 3.55 m/s) under 3 strike conditions. Data from pressure sensitive film placed over medial and lateral McIII condyles and lateromedially across the dorsal aspect of McIII were quantified: total areas of P1 and PS contact (cm(2) ) at maximum recorded pressure; centroid locations of contact areas relative to the sagittal ridge (cm) and transverse ridge (cm) and dispersion of pixels (cm(4) ) for each McIII condyle (medial/lateral). The effect of the strike conditions on each variable were statistically tested using repeated-measures ANOVA (α = 0.05). RESULTS: Contact area between P1 and McIII condyles fell in well-defined areas bounded by the sagittal and transverse ridge, contact areas from PS were smaller and widely dispersed across McIII palmar border. Ratio of contact area of P1 to PS was 2.83 (P<0001). Hoof strike had no significant effect on contact area (P>0.54) CONCLUSIONS: Contact at impact (primarily from P1 and distally situated on McIII), contrasts with contact areas at midstance from both P1 and PS, symmetrically placed. Under impact, the greatest contact area was on the dorsal aspect of the medial condyle and coincides with the area subjected to the greatest increase in subchondral bone stiffening in joint disease.

A common methylenetetrahydrofolate reductase gene mutation and longevity.

Homozygotes (TT genotype) for the C677T mutation in the gene of methylenetetrahydrofolate reductase (C677T/MTHFR mutation) constitute about 12% of the Caucasian population. They have mild hyperhomocysteinemia which is an established risk factor for cardiovascular disease. If the mutation is associated with premature death its prevalence is expected to be lower in the elderly than in the young. To test this we determined the C677T/MTHFR genotypes in 220 newborn and 222 elderly 80-108-year-old Swedes. In the newborn and elderly, the allele frequency, of the C677T/MTHFR mutation was 29.1 and 27.0% and the mutant homozygote frequency was 10.0 and 9.5%, respectively. In a meta analysis of the present and three previous studies including a total of 1388 elderly and 1415 younger subjects, the odds ratio (OR) representing the likelihood of the TT genotype to attain old age relative to the CC genotype was 0.87 (95% confidence interval (CI), 0.69-1.11) and relative to both the CC and CT genotypes was 0.83 (95% CI, 0.66-1.04). This finding does not suggest that the C677T/MTHFR mutation is a strong risk factor for diseases frequently leading to premature death.