Keel fractures in the laying hen are the most critical animal welfare issue facing the egg production industry, particularly with the increased use of extensive systems in response to the 2012 EU directive banning conventional battery cages. The current study is aimed at assessing the effects of 2 omega-3 (n3) enhanced diets on bone health, production endpoints, and behavior in free-range laying hens. Data was collected from 2 experiments over 2 laying cycles, each of which compared a (n3) supplemented diet with a control diet. Experiment 1 employed a diet supplemented with a 60: 40 fish oil-linseed mixture (n3: n6 to 1.35) compared with a control diet (n3: n6 to 0.11), whereas the n3 diet in Experiment 2 was supplemented with a 40: 60 fish oil-linseed (n3: n6 to 0.77) compared to the control diet (n3: n6 to 0.11). The n3 enhanced diet of Experiment 1 had a higher n3: n6 ratio, and a greater proportion of n3 in the long chain (C20/22) form (0.41 LC: SC) than that of Experiment 2 (0.12 LC: SC). Although dietary treatment was successful in reducing the frequency of fractures by approximately 27% in Experiment 2, data from Experiment 1 indicated the diet actually induced a greater likelihood of fracture (odds ratio: 1.2) and had substantial production detriment. Reduced keel breakage during Experiment 2 could be related to changes in bone health as n3-supplemented birds demonstrated greater load at failure of the keel, and tibiae and humeri that were more flexible. These results support previous findings that n3-supplemented diets can reduce fracture likely by increasing bone strength, and that this can be achieved without detriment to production. However, our findings suggest diets with excessive quantities of n3, or very high levels of C20/ 22, may experience health and production detriments. Further research is needed to optimize the quantity and type of n3 in terms of bone health and production variables and investigate the potential associated mechanisms.

While it is well established that the composition and organisation of articular cartilage dramatically change during skeletal maturation, relatively little is known about how this impacts the mechanical properties of the tissue.In this study, digital image correlation was first used to quantify spatial deformation within mechanically compressed skeletally immature (4 and 8 week old) and mature (1 and 3 year old) porcine articular cartilage.The compressive modulus of the immature tissue was relatively homogeneous, while the stiffness of mature articular cartilage dramatically increased with depth from the articular surface.Other, well documented, biomechanical characteristics of the tissue also emerged with skeletal maturity, such as strain-softening and a depth-dependent Poisson's ratio.The most significant changes that occurred with age were in the deep zone of the tissue, where an order of magnitude increase in compressive modulus (from 0.97 MPa to 9.4 MPa for low applied strains) was observed from 4 weeks postnatal to skeletal maturity.These temporal increases in compressive stiffness occurred despite a decrease in tissue sulphated glycosaminoglycan content, but were accompanied by increases in tissue collagen content.Furthermore, helium ion microscopy revealed dramatic changes in collagen fibril alignment through the depth of the tissue with skeletal maturity, as well as a fivefold increase in fibril diameter with age.Finally, computational modelling was used to demonstrate how both collagen network reorganisation and collagen stiffening play a key role in determining the final compressive mechanical properties of the tissue.Together these findings provide a unique insight into evolving structure-function relations in articular cartilage.

Objective: To test the hypothesis that heightened advanced glycation endproducts (AGEs) content in cartilage accelerates the progression of spontaneous osteoarthritis (OA) in the Hartley guinea pig (HGP) model.Methods: Twenty-eight male, 3-month-old HGPs were used. Eight were left untreated as a baseline control group and sacrificed at 3 months of age (n = 4) and 9 months of age (n = 4; age-matched controls). The other 20 HGPs received intra-articular knee injections in the right knee whereas the left knees acted as contra-lateral non-injected controls. Injections consisted of 100 mu l phosphate buffered saline (PBS; n = 10) or PBS+2.0.M D-(-)-Ribose (n = 10). Injections were given once weekly for 24 weeks. At the end of the treatment period, the tibiae were fixed with formalin, scanned with microCT for subchondral bone mineral density, and then histological slides were prepared, stained with Safranin-O with Fast Green counter stain and scored using the OARSI-HISTOgp scheme. Cartilage pentosidine (established biomarker for AGES) content, collagen content (% dry mass), glucosaminoglycan GAG-to-collagen ratio (mu g/mu g), GAG-to-DNA ratio and DNA-to-collagen ratio were measured.Results: Pentosidine content increased greatly due to PBS + Ribose injection (P < 0.0001) and reached levels found in cartilage from 80-year-old humans. Surprisingly, mean OARSI-HISTOgp scores for both the injected and contra-lateral controls in the PBS + Ribose group were not detectably different, nor were they different from the mean score for the age-matched control group.Conclusion: AGEs accumulation due to intra-articular ribose-containing injections in the HGP model of spontaneous knee OA did not enhance disease progression. (C) 2012 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

The main objectives of the study were to provide an accurate assessment of current levels of old breaks in end-of-lay hens housed in a variety of system designs and identify the important risk factors. Sixty-seven flocks housed in eight broad subcategories were assessed at the end of the production period. Within each flock, the presence of keel fractures was determined and the tibia, humerus and keel bones dissected for measurement of breaking strength. For each house, variations in internal design and perching provision were categorised and the effective heights of the differing structures recorded. All systems were associated with alarmingly high levels of keel damage although variation in mean prevalence between systems was evident with flocks housed in furnished cages having the lowest prevalence (36 per cent) despite also having significantly weaker bones and flocks housed in all systems equipped with multilevel perches showing the highest levels of damage (over 80 per cent) and the highest severity scores.

Fatigue fracture of cuboidal bones occurs in the human foot as well as the equine carpus. The racehorse provides a naturally-occurring model to study the effects of high-intensity exercise on the morphology and metabolism of cuboidal bones. We studied both the mineral and the collagenous matrix of the third (C(3)) and radial (C(r)) carpal bones of raced and non-raced Thoroughbred (TB) horses. We hypothesised that racehorses would show increases in the mineral component of these bones and post-translational modifications of the collagenous matrix alongside changes in markers of collagen remodelling and bone formation. C(3) and C(r) carpal bones were retrieved from raced TB horses (n=14) and non-raced TB horses (n=11). Standardised proximal-distal sections were taken from each bone and these were sliced transversely to study the proximal-distal differences in bone metabolism from the subchondral plate through to trabecular bone. Histomorphometry and bone mineral density measurements were performed in parallel with biochemical analyses including total collagen, collagen synthesis and cross-links, matrix metalloproteinases-2 and 9 and their inhibitors, calcium and phosphate, and bone alkaline phosphatase. The results of this study show that, while there is a net increase in bone formation in the racehorses, there is additionally an increase in bone collagen synthesis and remodelling, particularly within the trabecular regions of the bone. The increase in bone density would lead to greater stiffness, particularly in the cortical bone, and failure of this 'stiffer' cortical bone may result from its lack of support from the rapidly remodelling and structurally weakened underlying trabecular bone.

The non-enzymic glycation of collagen occurs as its turnover decreases during maturation, with complex carbohydrates accumulating slowly and the end-products of these reactions being permanent. The nature of these advanced glycation end-reaction products (AGEs) can be categorised as: 1) cross-linking: intermolecular cross-linking may occur between two adjacent molecules and involve lysine to lysine or lysine to arginine residues. Several compounds have been characterised. They are believed to be located between the triple helical domains of adjacent molecules in the fibre resulting in major changes of the physical properties, primarily, fibre stiffness, thermal denaturation temperature and enzyme resistance, all of which increase slowly with age but the rate is accelerated in diabetes mellitus due to high glucose levels: 2) side-chain modifications: these changes alter the charge profile of the molecule affecting the interactions within the fibre and if they occur at specific sites can affect the cell-collagen interaction. Modification of arginine within the sites RGD and GFOGER recognised by the two specific integrins (alpha1beta2 and alpha2beta1) for collagen reduce cell interactions during turnover and for platelet interactions (alpha1beta2). These changes can ultimately affect repair of, for example, vascular damage and dermal wound healing in diabetes mellitus. Both types of modification are deleterious to the optimal properties of collagen as a supporting framework structure and as a controlling factor in cell matrix interactions. Glycation during ageing and diabetes is therefore responsible for malfunctioning of the diverse collagenous tissues throughout the body.

The molecular mechanisms involved in the aging of collagen and consequent increase in mechanical strength and stiffness occur in a series of enzymic and non-enzymic intermolecular cross-links. The enzymic mechanism involves divalent aldimine intermolecular cross-links derived from the reaction of aldehydes which then mature to trivalent cross-links and further stabilize the collagen fiber and is now well known. Recent studies have demonstrated that the rate of turnover and level of telopeptide lysyl hydroxylation modifies the nature of the cross-link and hence the mechanical strength of the fiber. The slow turnover of mature collagen subsequently allows accumulation of the products of the adventitious non-enzymic reaction of glucose with the lysines in the triple helix to form glucosyl lysine and its Amadori product, that is, the Maillard reaction. These products are subsequently oxidized to a complex series of advanced glycation end-products, some of which are intermolecular cross-links between the triple helices rendering the fiber too stiff for optimal functioning of the collagen fibers, and consequently of the particular tissue involved. The glycation reactions following maturation are true aging processes, and attempts at their specific inhibition involve competitive inhibition of the Maillard reaction and chemical cleavage of the glycation cross-links. It is clear that the nature of the age-related cross-links and hence tissue strength depends on the rate of turnover of the collagen. An examination of the particular effect of strenuous exercise on the rate of turnover of collagen and hence cross-linking in different tissues could lead to a better understanding of optimal sports training regimes.

OBJECTIVE:To determine the relationship between the dimensions of the distal femoral intercondylar notch (ICN) and the composition and metabolism of the anterior cruciate ligament (ACL) in three dog breeds with different relevant risks to ligament rupture and subsequent osteoarthritis (OA). DESIGN:ICN measurements were obtained from the femurs of Golden Retrievers (high risk), Labrador Retrievers (high risk) and Greyhounds (low risk). Femoral condyle width and height, ICN height and width indices, and notch shape index were measured using Vernier callipers in all dogs. Intact ACLs were obtained from the same dog breeds for a study of the impinged areas and were analysed for collagen content, collagen cross-links, and sulphated glycosaminoglycan (GAG) content, matrix metalloproteinase (MMP)-2 and the tissue inhibitors of metalloproteinases (TIMPs)-1 and -2. RESULTS:Femoral condyle width and height and ICN width indices were significantly greater in the low risk compared to the high risk breeds (P<0.01 for all parameters). In contrast, the pro (P=0.003) and active (P=0.007) forms of MMP-2 and sulphated GAGs (P=0.0002) were significantly greater in the impinged areas of the ACLs of the rupture predisposed breeds. CONCLUSIONS:Impingement by the ICN on the ACLs of the high risk breeds may result in increased collagen remodelling and increased sulphated GAG deposition, causing reduced structural integrity of the ligament. Altered ACL composition may predispose the ligament to increased laxity leading to joint degeneration and OA. This may have a comparative implication for pathogenesis of ACL rupture in humans.

Malondialdehyde, acetaldehyde, acrolein, and 4-hydroxynonenal are all products of fatty acid oxidation found in the fatty streaks of atherosclerotic arteries due to a lack of antioxidants and an increase in glycation products. Previously identified cross-links derived from these molecules have nearly always required more than one molecule of each type, although this is physiologically less likely than a reaction involving a single molecule. Here we provide indirect but strong evidence for a malondialdehyde-derived cross-link requiring just one malondialdehyde molecule to link arginine and lysine, giving 2-ornithinyl-4-methyl(1epsilon-lysyl)1,3-imidazole following a 4-day incubation of albumin with 8 mm malondialdehyde. This cross-link was identified as its partial degradation product Nepsilon-(2-carboxyl,2-aminoethane)-Nepsilon-methanoyl-lysine by NMR and mass spectrometry. Analysis of plasma from treated diabetic patients revealed that one patient levels had as high as 0.46%, 0.67% of their lysine/arginine residues modified by this cross-link, although others had lower levels. Alkaline hydrolysis of serum albumin also revealed two acid-labile malondialdehyde adducts of histidine in significant quantities, the isomers 4- and 2-ethylidene-histidine. These constituted up to 0.93% of the histidines in treated diabetic patients. Although collagen is readily cross-linked by malondialdehyde, none of these particular products could be found in incubations of collagen with malondialdehyde.

Differential scanning calorimetry (DSC) was used to study the thermal stability of native and synthetically cross-linked rat-tail tendon at different levels of hydration, and the results compared with native rat-tail tendon. Three cross-linking agents of different length between functional groups were used: malondialdehyde (MDA), glutaraldehyde and hexamethylene diisocyanate (HMDC). Each yielded the same linear relation between the reciprocal of the denaturation temperature in Kelvin, T(max), and the water volume fraction, epsilon (1/T(max)=0.000731epsilon+0.002451) up to a critical hydration level, the volume fraction of water in the fully hydrated fibre. Thereafter, water was in excess, T(max) was constant and the fibre remained unchanged, no matter how much excess water was added. This T(max) value and the corresponding intrafibrillar volume fraction of water were as follows: 84.1 degrees C and 0.48 for glutaraldehyde treated fibres, 74.1 degrees C and 0.59 for HMDC treated fibres, 69.3 degrees C and 0.64 for MDA treated fibres, and 65.1 degrees C and 0.69 for untreated native fibres. Borohydride reduction of the native enzymic aldimines did not increase the denaturation temperature of the fibres. As all samples yielded the same temperature at the same hydration, the temperature could not be affected by the nature of the cross-link other than through its effect on hydration. Cross-linking therefore caused dehydration of the fibres by drawing the collagen molecules closer together and it was the reduced hydration that caused the increased temperature stability. The cross-linking studied here only reduced the quantity of water between the molecules and did not affect the water in intimate contact with, or bound to, the molecule itself. The enthalpy of denaturation was therefore unaffected by cross-linking. Thus, the "polymer-in-a-box" mechanism of stabilization, previously proposed to explain the effect of dehydration on the thermal properties of native tendon, explained the new data also. In this mechanism, the configurational entropy of the unfolding molecule is reduced by its confinement in the fibre lattice, which shrinks on cross-linking.

1. Collagen characteristics were compared in the tibiotarsus and humerus from 103 females and 38 males aged 68 to 72 weeks from the G6 generation of lines of laying hen selected for resistance or susceptibility to osteoporosis (high and low bone index (BI) lines).2. Selection over the latest generation resulted in further divergence in the breaking strengths of humerus (from 12.3 to 21.8%) and tibia (from 22.3 to 37.3%) in hens. Males also showed line differences in bone strengths.3. Plasma pyridinoline concentration was higher in hens in the low BI line, suggesting a greater rate of bone resorption in this line.4. There were few differences between the lines in collagen and calcium concentrations in humerus and tibiotarsus cortical bone.5. There were no differences between the lines in either sex in reduced immature collagen cross-link content of humerus or tibiotarsus.6. Mature collagen cross-link content was higher in the high BI line in the male humerus but this effect was not apparent in the male tibiotarsus nor in either bone in the females.7. Pyrrolic cross-link contents were higher in the high BI line in the female humerus and tibiotarsus and in the male tibiotarsus.8. Over both lines combined, there were positive correlations between humeral and tibiotarsal pyrrole contents and strengths in females and between tibiotarsal pyrrole content and strength in males.9. It is concluded that an increase in cross-linking, particularly pyrrolic cross-linking, in the collagen matrix contributes in part to the improvement in bone strength in the high BI line.

PURPOSE:The interaction of cancer cells and the extracellular matrix is essential for cancer progression. However, little is known about the influence of cancer on the metabolism of collagen, which is the major constituent of the extracellular matrix. We studied changes in collagen metabolism in prostate cancer with increasing Gleason score and correlated them with clinical parameters and patient survival.MATERIALS AND METHODS:Collagen content and clinical parameters in 3 types of prostatic tissue were compared, including the foci of prostatic cancer, unaffected tissue from the same cancerous prostates and prostatic tissue from patients with no evidence of cancer. In addition, to assess collagen metabolism tissue obtained prospectively from 45 patients undergoing prostatic biopsy was assessed for collagen content, the type and extent of collagen cross-linking, serine proteinase, matrix metalloproteinase and the C-terminal propeptide of type I collagen.RESULTS:With increasing Gleason sum of the cancer collagen content at the focus decreased, while that of surrounding unaffected tissue from the same prostate increased to levels significantly above that from controls with no cancer. Markers of collagen synthesis in the prostate biopsy material were significantly increased in the presence of prostate cancer.CONCLUSIONS:In prostate cancer there are changes in collagen metabolism not only in the cancer focus but also in nearby histologically benign prostatic tissue. These observed changes are related to the Gleason score of the tumor and may represent a host response. Collagen content in the surrounding unaffected tissue may be a predictor of patient survival.

Differential scanning calorimetry has revealed the presence of a new denaturation endotherm at 32 degrees C following UV irradiation of collagen, compared with 39 degrees C for the native triple helix. Kinetic analyses showed that the new peak was a previously unknown intermediate state in the collagen helix-coil transition induced by UV light, and at least 80% of the total collagen was transformed to random chains via this state. Its rate of formation was increased by hydrogen peroxide and inhibited by free radical scavengers. SDS-polyacrylamide gels showed evidence of competing reactions of cross-linking and random primary chain scission. The cross-linking was evident from initial gelling of the collagen solution, but there was no evidence for a dityrosine cross-link. Primary chain scission was confirmed by end group analysis using fluorescamine. Electron microscopy showed that the segment long spacing crystallites formed from the intermediate state were identical to the native molecules. Clearly, collagen can undergo quite extensive damage by cleavage of peptide bonds without disorganizing the triple helical structure. This leads to the formation of a damaged intermediate state prior to degradation of the molecules to short random chains.