In vitro non-enzymatic ribation reduces post-yield strain accommodation in cortical bone.

Non-enzymatic glycation (NEG) and advanced glycation endproducts (AGEs) may contribute to bone fragility in various diseases, ageing, and other conditions by modifying bone collagen and causing degraded mechanical properties. In this study, we sought to further understand how collagen modification in an in vitro non-enzymatic ribation model leads to loss of cortical bone toughness. Previous in vitro studies using non-enzymatic ribation reported loss of ductility in the cortical bone. Increased crosslinking is most commonly blamed for these changes; however, some studies report positive correlations between measures of total collagen crosslinking and work-to-fracture/toughness measurements whilst correlations between general NEG and measures of ductility are often negative. Fifteen bone beam triplets were cut from bovine metatarsi. Each provided one native non-incubated control, one incubated control and one ribated specimen. Incubation involved simulated body fluid±ribose for fourteen days at 37°C. Pentosidine and pyridinoline crosslinks were measured using HPLC. Three-point bending tests quantified mechanical properties. Fracture surfaces were examined using scanning electron microscopy. The effects of ribation on bone collagen molecular stability and intermolecular connectivity were investigated using differential scanning calorimetry and hydrothermal isometric tension testing. Ribation caused increased non-enzymatic collagen modification and pentosidine content (16mmol/mol collagen) and inferior post-yield mechanical behaviour, especially post-yield strain and flexural toughness. Fracture surfaces were smoother with less collagen fibril deformation or tearing than observed in controls. In the ribated group only, pentosidine content and thermomechanical measures of crosslinking were positively correlated with measures of strain accommodation and energy absorption before failure. Non-enzymatic ribation and the resulting modifications reduce cortical bone pseudo-plasticity through a reduced capacity for post-yield strain accommodation. However, the positive correlations we have found suggest that increased crosslinking may not provide a complete explanation for this embrittlement.

Reduced bone breakage and increased bone strength in free range laying hens fed omega-3 polyunsaturated fatty acid supplemented diets.

INTRODUCTION: The omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) are the immediate precursors to a number of important mediators of immunity, inflammation and bone function, with products of omega-6 generally thought to promote inflammation and favour bone resorption. Western diets generally provide a 10 to 20-fold deficit in omega-3 PUFAs compared with omega-6, and this is thought to have contributed to the marked rise in incidence of disorders of modern human societies, such as heart disease, colitis and perhaps osteoporosis. Many of our food production animals, fed on grains rich in omega-6, are also exposed to a dietary deficit in omega-3, with perhaps similar health consequences. Bone fragility due to osteoporotic changes in laying hens is a major economic and welfare problem, with our recent estimates of breakage rates indicating up to 95% of free range hens suffer breaks during lay. METHODS: Free range hens housed in full scale commercial systems were provided diets supplemented with omega-3 alpha linolenic acid, and the skeletal benefits were investigated by comparison to standard diets rich in omega-6. RESULTS: There was a significant 40-60% reduction in keel bone breakage rate, and a corresponding reduction in breakage severity in the omega-3 supplemented hens. There was significantly greater bone density and bone mineral content, alongside increases in total bone and trabecular volumes. The mechanical properties of the omega-3 supplemented hens were improved, with strength, energy to break and stiffness demonstrating significant increases. Alkaline phosphatase (an osteoblast marker) and tartrate-resistant acid phosphatase (an osteoclast marker) both showed significant increases with the omega-3 diets, indicating enhanced bone turnover. This was corroborated by the significantly lower levels of the mature collagen crosslinks, hydroxylysyl pyridinoline, lysyl pyridinoline and histidinohydroxy-lysinonorleucine, with a corresponding significant shift in the mature:immature crosslink ratio. CONCLUSIONS: The improved skeletal health in laying hens corresponds to as many as 68million fewer hens suffering keel fractures in the EU each year. The biomechanical and biochemical evidence suggests that increased bone turnover has enhanced the bone mechanical properties, and that this may suggest potential benefits for human osteoporosis.

Fibroblast growth factor 2 and transforming growth factor ß1 induce precocious maturation of articular cartilage.

OBJECTIVE: We have discovered that a combination of fibroblast growth factor 2 and transforming growth factor ß1 induce profound morphologic changes in immature articular cartilage. The purpose of this study was to test the hypothesis that these changes represent accelerated postnatal maturation. METHODS: Histochemical and biochemical assays were used to confirm the nature of the morphologic changes that accompany growth factor stimulation of immature bovine articular cartilage explants in serum-free culture medium. Growth factor-induced apoptosis, cellular proliferation, and changes in the collagen network were also quantitatively analyzed. RESULTS: Growth factor stimulation resulted in rapid resorption from the basal aspect of immature cartilage explants that was simultaneously opposed by cellular proliferation from the apical aspect driven from a pool of chondroprogenitor cells we have previously described. Maturation-dependent changes in tissue stiffness, collagen crosslinking, and collagen fibril architecture as well as differentiation of the extracellular matrix into distinct pericellular, territorial, and interterritorial domains were all present in growth factor-stimulated cartilage samples and absent in control samples. CONCLUSION: Our data demonstrate that it is possible to significantly enhance the maturation of cartilage tissue using specific growth factor stimulation. This may have applications in transplantation therapy or in the treatment of diseased cartilage, through phenotype modulation of osteoarthritic chondrocytes in order to stimulate growth and maturation of cartilage repair tissue.

Changes in collagen with aging maintain molecular stability after overload: evidence from an in vitro tendon model.

Soft tissue injuries are poorly understood at the molecular level. Previous work using differential scanning calorimetry (DSC) has shown that tendon collagen becomes less thermally stable with rupture. However, most soft tissue injuries do not result in complete tissue rupture but in damaging fiber overextension. Covalent crosslinking, which increases with animal maturity and age, plays an important role in collagenous fiber mechanics. It is also a determinant of tissue strength and is hypothesized to inhibit the loss of thermal stability of collagen due to mechanical damage. Controlled overextension without rupture was investigated to determine if overextension was sufficient to reduce the thermal stability of collagen in the bovine tail tendon (BTT) model and to examine the effects of aging on the phenomenon. Baseline data from DSC and hydrothermal isometric tension (HIT) techniques were compared between two groups: steers aged 24-30 months (young group), and skeletally mature bulls and oxen aged greater than five years (old group). Covalent crosslinks were quantified by ion exchange chromatography. Overextension resulted in reduced collagen thermal stability in the BTT model. The Young specimens, showing detectably lower tissue thermomechanical competence, lost more thermal stability with overextension than did the old specimens. The effect on old specimens, while smaller, was detectable. Multiple overextension cycles increased the loss of stability in the young group. Compositional differences in covalent crosslinking corresponded with tissue thermomechanical competence and therefore inversely with the loss of thermal stability. HIT testing gave thermal denaturation temperatures similar to those measured with DSC. The thermal stability of collagen was reduced by overextension of the tendon--without tissue rupture--and this effect was amplified by increased cycles of overextension. Increased tissue thermomechanical competence with aging seemed to mitigate the loss of collagen stability due to mechanical overextension. Surprisingly, the higher tissue thermomechanical competence did not directly correlate with the concentration of endogenous enzymatically derived covalent crosslinking on a mole per mole of collagen basis. It did, however, correlate with the percentage of mature and thermally stable crosslinks. Compositional changes in fibrous collagens that occur with aging affect fibrous collagen mechanics and partially determine the nature of mechanical damage at the intermolecular level. As techniques develop and improve, this new information may lead to important future studies concerning improved detection, prediction, and modeling of mechanical damage at much finer levels of tissue hierarchy than currently possible.