The changing role of the superficial region in determining the dynamic compressive properties of articular cartilage during postnatal development.

OBJECTIVE: To explore how changes to the superficial region (SR) of articular cartilage during skeletal development impact its functional properties. It was hypothesised that a functional superficial region is not present in skeletally immature articular cartilage, and removal of this zone of the tissue would only negatively impact the dynamic modulus of the tissue with the attainment of skeletal maturity. METHODS: Porcine osteochondral cores were mechanically tested statically and dynamically with and without their respective superficial regions in confined and unconfined compression at different stages of postnatal development and maturation. A novel combination of histological, biochemical and imaging techniques were utilised to accurately describe changes to the superficial region during postnatal development. RESULTS: Articular cartilage was found to become stiffer and less permeable with age. The confined and unconfined dynamic modulus significantly decreased after removal of the superficial region in skeletally mature cartilage, whilst no significant change was observed in the 4 week old tissue. Biochemical analysis revealed a significant decrease in overall sGAG content with age (as % dry weight), whilst collagen content significantly increased with age, although the composition of the superficial region relative to the remainder of the tissue did not significantly change with age. Helium ion microscopy (HIM) revealed dramatic changes to the organization of the superficial region with age. CONCLUSIONS: The findings demonstrate that the superficial region of articular cartilage undergoes dramatic structural adaptation with age, which in turn plays a key role in determining the dynamic compressive properties of the tissue.

Postnatal changes to the mechanical properties of articular cartilage are driven by the evolution of its collagen network.

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.

The effects of long (C20/22) and short (C18) chain omega-3 fatty acids on keel bone fractures, bone biomechanics, behavior, and egg production in free-range laying hens.

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.

Enhanced levels of non-enzymatic glycation and pentosidine crosslinking in spontaneous osteoarthritis progression.

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 µl 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 sub-chondral 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 (µg/µg), 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.

Regulation of osteoarthritis by omega-3 (n-3) polyunsaturated fatty acids in a naturally occurring model of disease.

OBJECTIVE: To examine effects of high omega-3 (n-3) polyunsaturated fatty acid (PUFA) diets on development of osteoarthritis (OA) in a spontaneous guinea pig model, and to further characterise pathogenesis in this model. Modern diets low in n-3 PUFAs have been linked with increases in inflammatory disorders, possibly including OA. However, n-3 is also thought to increases bone density, which is a possible contributing factor in OA. Therefore we aim to determine the net influence of n-3 in disease development. METHOD: OA-prone Dunkin-Hartley (DH) Guinea pigs were compared with OA-resistant Bristol Strain-2s (BS2) each fed a standard or an n-3 diet from 10 to 30 weeks (10/group). We examined cartilage and subchondral bone pathology by histology, and biochemistry, including collagen cross-links, matrix metalloproteinases (MMPs), alkaline phosphatase, glycosaminoglycan (GAG), and denatured type II collagen. RESULTS: Dietary n-3 reduced disease in OA-prone animals. Most cartilage parameters were modified by n-3 diet towards those seen in the non-pathological BS2 strain - significantly active MMP-2, lysyl-pyridinoline and total collagen cross-links - the only exception being pro MMP-9 which was lower in the BS2, yet increased with n-3. GAG content was higher and denatured type II lower in the n-3 group. Subchondral bone parameters in the DH n-3 group also changed towards those seen in the non-pathological strain, significantly calcium:phosphate ratios and epiphyseal bone density. CONCLUSION: Dietary n-3 PUFA reduced OA in the prone strain, and most disease markers were modified towards those of the non-OA strain, though not all significantly so. Omega-3 did not increase markers of pathology in either strain.

High-intensity exercise induces structural, compositional and metabolic changes in cuboidal bones--findings from an equine athlete model.

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.

An efficient method for the isolation of intramuscular collagen.

Ultrasonic fragmentation of the myofibrils and subsequent solubilisation in buffer has been used to isolate intramuscular collagen (IMC) in high yield and purity. The method is superior to previously reported techniques in providing both a high yield of collagen and intact fibres. The material obtained is suitable for both physical and biochemical analysis in attempts to demonstrate its role in determining the tenderness of meat.

Collagen cross-linking in porcine m. longissimus lumborum: Absence of a relationship with variation in texture at pork weight.

The determination of all currently known intermolecular cross-links present in intramuscular collagen of porcine m. longissimus lumborum is described in relation to the texture of the meat as determined both objectively by instrumentation and subjectively by sensory panel. The variation in texture observed in the m. longissimus lumborum of pork weight pigs has been shown to be unrelated to the total collagen content or to the nature of the collagen intermolecular cross-links. We have also demonstrated a considerable error in the colorimetric method for quantitation of hydroxyproline when determining the very low values of collagen present in pig meat. During this study we have established a sound protocol for the determination of all the known cross-links in intramuscular collagen of meat from any meat animal species.

Influence of housing system and design on bone strength and keel bone fractures in laying hens.

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.

The effects of the Maillard reaction on the physical properties and cell interactions of collagen.

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.

Distal femoral intercondylar notch dimensions and their relationship to composition and metabolism of the canine anterior cruciate ligament.

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.

Enzymic and non-enzymic cross-linking mechanisms in relation to turnover of collagen: relevance to aging and exercise.

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.

Mechanical and chemical properties of the skin and its collagen from lean and obese-hyperglycaemic (ob/ob) mice.

We have compared the mechanical and chemical properties of the skin and its collagen from 24-week-old obese-hyperglycaemic (ob/ob) and lean mice. The skin from obese mice was mechanically weaker and generated a lower hydrothermal isometric force. However, there were no significant differences from lean mice in the type of reducible cross-links in the collagen or in its solubility, although it contained more reducible cross-links and glycosylated lysine. The total amount of skin collagen was similar in obese and lean mice from 3 to 24 weeks of age but the skin surface area was 60% greater in 24-week-old obese mice. When corrected for collagen content the tensile strength of skin from obese mice was greater than that from lean mice and we suppose that the weakness of obese mouse skin is caused by a failure of collagen deposition to match the increase in skin surface area as the animals become obese.