The separation of both the immature and mature cross-links present in collagen together with the stable cross-links of elastin has been achieved on a single ion-exchange column. This technique avoids the current necessity for two different systems. ion-exchange and high-performance liquid chromatography-fluorescence techniques. The value of the method is illustrated by the comparison of the cross-link contents of ageing bovine skin, from foetal to old age.

The subchondral bone has long been known to thicken in osteoarthritis. However, recent evidence has demonstrated that the turnover of the bone is increased several fold, and further suggests that the thickening occurs prior to degradation of the articular cartilage, indicating that it plays a role in the pathogenesis of osteoarthritis. The mechanical and biochemical properties of the subchondral bone are therefore of particular interest in any attempt to determine the nature of the factors initiating osteoarthritis. We have shown that the subchondral bone collagen of the femoral head possessed a 20-fold increase in turnover, as assessed by procollagen rate of synthesis and metalloproteinase degradation, and a 25% decrease in mineralisation. This increased metabolism and high lysyl hydroxylation leads to narrower and weaker fibres. Additionally the phenotypic expression of the osteoblasts is modified to produce increasing proportions of type I homotrimer in addition to the normal type I heterotrimer, which further reduces the mechanical strength of the bone. Overall, the narrow immature collagen fibres, the reduction in pyrrole cross-linking, decreased mineralisation, and increased amounts of type I homotrimer, all contribute to a weakening of the mechanical properties of the subchondral bone.

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.

Although the mechanical strength of cancellous bone is well known to depend on its apparent density, little is known about the influence of other structural or biochemical parameters. This study specifically investigates the cross-linking of the collagen in human vertebral bone samples and its potential influence on their mechanical behavior. Multiple cylindrical samples were cored vertically in the vertebral bodies of nine subjects (aged 44-88 years). Three spinal levels (T9, T12 or L1, and L4) and three sample sites within a vertebral body (anterior, posterior, and lateral) were used, for a total of 68 samples. The density was measured with peripheral quantitative computed tomography (pQCT) and all cylinders were mechanically tested in compression. After mechanical testing, they were unmounted and used for biochemical analysis. The amount of collagen (wt/wt of bone) and its content in reduced immature cross-links, that is, hydroxylysinonorleucine (HLNL, mol/mol of collagen) and dihydroxylysinornorleucine (DHLNL), as well as stable mature cross-links, that is, hydroxylysyl-pyridinoline (HP), lysyl-pyridinoline (LP), and pyrrole cross-link were determined for each cylinder. None of the biochemical parameters correlated to the density. On multiple linear regression, the prediction of the mechanical properties was improved by combining density data with direct collagen cross-link assessment. The HP/LP ratio appeared as a significant predictor to the strength (r = 0.40; p = 0.001) and stiffness (r = 0.47; p < 0.001) samples with a high HP/LP ratio being stronger and stiffer. Additionally, the ultimate strain correlated to the HP or LP concentration (r = 0.38 or 0.49; p < 0.01). Different subjects had different HP/LP ratios and different HP or LP concentrations in their vertebral bone samples, and the location of origin within a subject had no influence on the concentration. These observations suggest that the nature of the organic matrix in adult vertebral bone is variable and that these variations influence its mechanical competence.

The metabolism and total amount of the collagen of subchondral bone are increased several fold in osteoathritic femurs compared with controls. We now report for the first time that the quality of the collagen is modified by the formation of type I homotrimer. The homotrimer fibre has been reported to possess a reduced mechanical strength and mineralisation in bone. The presence of the latter therefore accounts for narrower disorganised collagen fibres and decreased mineralisation, and a reduction in mechanical stability of the osteoarthritic femoral head. These changes in the subchondral bone are likely to be of considerable importance in the pathogenesis of osteoarthritis.

Little is known regarding the mechanisms that govern the structural organization of cancellous bone. In this study, we compare the nature of the collagen in vertebral cancellous bone with the structural organization of its trabecular network. Cylindrical specimens of cancellous bone from vertebrae were obtained from nine autopsy subjects (ages 46–88). In each subject, eight pairs of corresponding samples were obtained from three levels in the spine and three areas within the vertebral body, leading to a total of 68 pairs of samples. The cylinders from one side were used for morphometry and the classical morphometrical parameters were obtained (BV/TV, bone volume fraction; Tb.Th, trabecular thickness; Tb.N, number; Tb.Sp, trabecular spacing) and strut analysis (TSL, total strut length; Nd, number of nodes; Fe, number of free-ends). The amount of osteoid bone was also quantified. The cylinders from the other side were powdered and used for collagen assessment, including the amount of collagen (% w/w), and its content in immature cross-links; such as hydroxylysinonorleucine (mol/mol of collagen) and dihydroxylysinornorleucine, as well as stable mature cross-links, such as hydroxylysylpyridinoline (HP), lysylpyridinoline (LP), and the pyrrole cross-links. A random regression model was used to explore the correlations. None of the biochemical parameters correlated with the BV/TV except the ratio between immature and mature cross-links (η2 = 0.34, p < 0.05). There was no relationship between the amount of osteoid bone and the cross-link profile. However, the concentration of pyrrole and HP cross-links in the bone samples correlated with the structural organization of its trabeculae, but in an opposite direction. Hence, the pyrrole/HP ratio was a good predictor of Tb.Th, Tb.N, Tb.Sp, and TSL (η2 > 0.65 and p < 0.01) as well as Fe and star marrow space (η2 > 0.45 and p < 0.05). The cylinders from subjects with high pyrrole or low HP in their bone collagen had a relatively thick and simple structure. Those with low pyrrole and high HP had relatively thin trabeculae that were more numerous and spread over a complex network. The relative concentrations of the pyrrole and pyridinoline cross-links appear to reflect the structural organization of the trabeculae.

Estrogen has been shown to stimulate osteoblasts in cell culture and increase bone formation in animal models. Such an anabolic effect of estrogen replacement therapy (ERT) would be beneficial to postmenopausal women with osteoporosis. Hence, we assessed the total collagen content and collagen crosslink maturity in iliac crest bone biopsy from 18 such women before and after 6 years of higher-dose ERT. These results were compared with the serum estradiol level and bone mineral density (BMD). Total collagen content of both cortical and cancellous bone increased, showing a median (95% CI) percent change of 6.7 (0.3–14.2) and 25.6 (13.5–33.8), respectively. Increase in collagen synthesis was supported by a rise in intermediate crosslinks in both cortical and cancellous bone, and mature crosslinks in cortical bone only. At the same time, BMD showed a substantial rise both at the lumbar spine and proximal femur with a median (95% CI) percent change of 28.6 (19.8–37.3) and 14.5 (8.4–20.7), respectively. Serum estradiol and BMD results correlated with cortical bone collagen levels. Our results suggest that long-term higher-dose ERT has a therapeutic role due to its anabolic effect on bone in postmenopausal women with osteoporosis.

Remodelling of the collagen matrix of the myocardium has been implicated in the pathogenesis of dilated cardiomyopathy, a major cause of heart failure in Doberman pinschers. The aim of this study was to characterise the myocardial collagen matrix of Dobermans. In clinically normal Dobermans there was evidence of focal fibrosis. Collagen cross-links were altered in both diseased and clinically normal Doberman myocardium as compared with myocardium from control dogs. Extensive remodelling, in the form of a loss of collagen tethers, increased collagen synthesis and alterations in the collagen cross-links, occurs in diseased Doberman myocardium. Changes in the collagenous matrix are also present in apparently normal Dobermans. These changes are likely to be involved in the progression of the disease and may explain the predisposition of this breed to dilated cardiomyopathy.

Isinglass is widely used commercially to clarify alcoholic beverages by aggregation of the yeast and other insoluble particles. It is derived from swim bladders of tropical fish by solubilisation in organic acids and consists predominantly of the protein collagen. The low content of intermolecular cross-links allows ready dissolution of swim bladder compared to bovine hide which is cross-linked by a high proportion of stable bonds and requires enzymic digestion to solubilise. Isinglass is no longer effective as a clarifying agent if thermally denatured hence the collagenous triple helical structure must be maintained. Thermal denaturation of isinglass occurs at 29 degrees C, compared to 40-41 degrees C for mammalian collagens, primarily due to the lower hydroxyproline content. The hydroxyproline is essential for the formation of H-bonded water-bridges through the hydroxyl group and the peptide chain thereby stabilising the triple helix. Based on the lower enthalpy determined by differential scanning calorimetry we have calculated that the thermally labile domain of the isinglass molecule was 41 residues compared to 66 for mammalian collagen. The fining efficiency was unaffected by pH, chelating agents, detergents and removal of surface proteins from yeast cells. Studies on the mechanism of action of isinglass have shown that higher molecular weight aggregates that increase the length of the collagen molecules (trimers, tetramers, etc.) increase efficiency and that their surface charge are important in the clarification process. By chemical modification, we have shown that blocking positively charged groups had no effect on the fining process, whilst negative charges are clearly essential and that increasing the negative charge by succinylation increases its efficacy. Solutions of bovine hide collagen were shown to be equally effective in refining beers and standard yeast preparations. The higher thermal denaturation temperature, ready availability and reproducibility of bovine collagen preparations gives it considerable advantages over isinglass.

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.

The metabolism of bone collagen has received little attention in relation to age-related loss of bone mass and strength. The aim of the present study was to analyze bone collagen content and metabolism in human bone with respect to age. The material consisted of iliac crest bone biopsies from 94 individuals: 46 women (ages 18–96, mean age 60.8 years) and 48 men (ages 23–92, mean age 59.5 years). Excluded from the study were all individuals with known osteoporotic lumbar vertebral fractures and renal, hepatic, or malignant diseases. Prior to collagen analysis the biopsies were scanned in a pQCT scanner for density assessment and then tested biomechanically. The results showed a decline in apparent bone density with age (P < 0.0001), a decline in maximum stress, Young's modulus, and energy absorption with age (P < 0.001). Concomittantly, there was an age-related decline in the intrinsic collagen content with age (P < 0.001). However, there were no biochemical modifications of the bone collagen during aging. There were no significant differences between women and men in the slopes of the regressions-curves. When multiple regression analyses were performed, only apparent bone density came out as a significant contributor in the correlation to biomechanical properties. Nevertheless, the decrease in bone collagen content with age might indicate an increase in the mineralization degree (probably due to decreased bone turnover) and thereby a change in material properties of bone. In conclusion, the present study has shown that loss of bone mass plays the major role in loss of bone strength. However, there is also a change in bone composition during normal aging, leading to a decrease in collagen content and an increase in the degree of mineralization. At this skeletal site, in a normal population there was no change in the biochemical properties of bone collagen.

An amino acid component, NFC-1, when formed in vitro by the reaction of ribose and protein was shown to comprise a complex mixture of high and low molecular AGE compounds. Two low-molecular-weight components have been successfully isolated and their structure determined. These were alpha NFC-1 [N-delta-(4-oxo-5-dihydroimidazol-2-yl)-L-ornithine] and beta NFC-1 a 4-imidazolon-2-yl derivative existing in three tautomeric forms. These imidazolone compounds have been shown to originate from the reaction of arginine with glyoxal and methylglyoxal, respectively. A third ninhydrin-positive AGE, gamma NFC-1, was shown to be composed of a number of chromatographically similar compounds which have not yet been characterized.

Standardized protocols were developed for use in a detailed investigation into the biomechanical and biochemical properties of a dermal wound healing model in the rat. The use of a rapid freezing method at -80 degrees C minimized the detrimental effects of freezing on the biomechanical properties of the tissue and also allowed for convenient inter-laboratory collaboration to be performed. The methodology described allowed for the simultaneous and reproducible measurement of tensile strength, collagen cross-linking and proteolytic enzyme activity. Increases in the tensile properties of the tissue with time were consistent with an active process of remodelling process as indicated by changes in the cross-link and enzyme profiles. Initially the granulation tissue was comparatively rich in the keto-imine cross-link hydroxylysino-keto-norleucine, which was later replaced by the aldimine cross-link dehydro-hydroxy-lysinonorleucine. The mature cross-link histidino-hydroxy-lysinonorleucine was not observed within the granulation tissue at any stage and was also absent in aged control skin. A peak of matrix metalloproteinase-9 activity was observed at early timepoints (48 hr) and then decreased rapidly to normal levels and is consistent with an acute inflammatory response. In contrast matrix metalloproteinase-2 activity peaked later (3 days) and then decreased gradually, consistent with its role as one of the predominant enzymes involved in the remodelling process. The results described validate the animal model used and emphasize its potential for use in combined biomechanical and biochemical studies of acute wound healing.

The breakdown of intact collagen from bone or hide to produce commercial gelatin results in a polydisperse product with respect to molecular weight. The mechanism of this breakdown and the role of the individual collagen chains in gel formation is not clearly understood.Analysis of these polypeptide chains for molecular weight and inter-chain cross-linking of both commercial gelatin and isolated intact components from purified collagen, and the ability of both to form gels, should provide data on the mode of breakdown and the mechanism of gel formation. We have shown that the isolated pure alpha 1 and beta-components form firm gels, in which a high proportion of triple helix is generated. In contrast, the alpha 2 chain forms weak gels and generates little helix. The highly cross-linked high molecular weight components also produce weak gels, yet generate a high proportion of triple helix. It is possible that the rapid production of triple helices by chains in register due to cross-linking actually reduces network formation, in which multiple chain interactions forming short helices is preferable.

Previous studies have shown that biomechanical analysis of aorta from diabetic subjects reveals a marked increase in stiffness compared to aorta from age-matched control subjects. In the present paper we have proposed that this increased stiffness can be attributed to glycation-induced inter-molecular cross-links based on a direct analysis of the two known glycation cross-links, the fluorescent pentosidine and the non-fluorescent NFC-1. There was a significant difference in the increase in concentration of both cross-links with increasing age for both the intima (p < 0.0025) and the media (p < 0.0005) from the diabetic compared to the control subjects, but no correlation with the mature enzymic cross-link hy droxylysyl-pyridinoline. Finally, we have obtained a significant correlation of stiffness with both glycation cross-links (NFC-1, r = 0.86; p < 0.005 and pentosidine r = 0.75, p < 0.05), but the concentration of NFC-1 is about 50 times greater than that of pentosidine, indicating that it is the major glycation cross-link responsible for the stiffening of the aorta.

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.

Non-enzymic glycation of collagen involves a series of complex reactions ultimately leading to the formation of intermolecular cross-links resulting in changes in its physical properties. During analysis for the fluorescent cross-link pentosidine we identified the presence of an additional component (Cmpd K) in both glucose and ribose incubations. Cmpd K was formed more quickly than pentosidine in glucose incubations and more slowly than pentosidine in ribose incubations. Cmpd K represented 45% of the total fluorescence compared with 15% for pentosidine in glucose incubations and 25% of the total fluorescence compared with 30% for pentosidine in the ribose incubations. Cmpd K is not an artefact of in vitro incubations, as it was shown to be present in dermal tissue from diabetic patients. Subsequent high-resolution h.p.l.c. analysis of glucose-incubated collagen revealed Cmpd K comprise two components (K1 and K2). Further, a similar analysis of Cmpd K from the ribose incubations revealed two different components (K3 and K4). These differences indicate alternative mechanisms for the reactions of glucose and ribose with collagen. The amounts of these fluorescent components and the pentosidine cross-link determined for both glucose and ribose glycation were found to be far too low (about one pentosidine molecules per 200 collagen molecules after 6 months incubation with glucose) to account for the extensive cross-linking responsible for the changes in physical properties, suggesting that a further additional series of cross-links are formed. We have analysed the non-fluorescent high-molecular-mass components and identified a new component that increases with time of in vitro incubation and is present in the skin of diabetic patients. This component is present in sufficient quantities (estimated at one cross-link per two collagen molecules) to account for the changes in physical properties occurring in vitro.

Continuous extension of Dupuytren’s contracture prior to fasciectomy results in a softening of the tissue, allowing straightening of the fingers. The observed change in cross-link profile indicates an increase in newly synthesised collagen due to increased turnover. This was confirmed by demonstration of the increases in levels of the degradative enzymes, the neutral metalloproteinases, collagenase and gelatinase and the acidic cathepsins B and L. Both types of enzyme effectively depolymerize the collagen fibres, albeit by different mechanisms, leading initially to loss of tensile strength and ultimately to solubilization. We suggest that the increase in enzyme activity is generated by tension on the fibroblasts of this metabolically active tissue produced during the continuous extension of the retracted fingers. The weakening of the fibres by degradation and the increase in newly synthezised collagen provide an explanation for the extension of the tissue without trauma.

The incubation of lens capsules with glucose in vitro resulted in changes in the mechanical and thermal properties of type-IV collagen consistent with increased cross-linking. Differential scanning calorimetry (d.s.c.) of fresh lens capsules showed two major peaks at melting temperatures Tm 1 and Tm 2 at approx. 54 degrees C and 90 degrees C, which can be attributed to the denaturation of the triple helix and 7S domains respectively. Glycosylation of lens capsules in vitro for 24 weeks caused an increase in Tm 1 from 54 degrees C to 61 degrees C, while non-glycosylated, control incubated capsules increased to a Tm 1 of 57 degrees C. The higher temperature required to denature the type-IV collagen after incubation in vitro suggested increased intermolecular cross-linking. Glycosylated lens capsules were more brittle than fresh samples, breaking at a maximum strain of 36.8 +/- 1.8% compared with 75.6 +/- 6.3% for the fresh samples. The stress at maximum strain (or ‘strength’) was dramatically reduced from 12.0 to 4.7 N.mm.mg-1 after glycosylation in vitro. The increased constraints within the system leading to loss of strength and increased brittleness suggested not only the presence of more cross-links but a difference in the location of these cross-links compared with the natural lysyl-aldehyde-derived cross-links. The chemical nature of the fluorescent glucose-derived cross-link following glycosylation was determined as pentosidine, at a concentration of 1 pentosidine molecule per 600 collagen molecules after 24 weeks incubation. Pentosidine was also determined in the lens capsules obtained from uncontrolled diabetics at a level of about 1 per 100 collagen molecules. The concentration of these pentosidine cross-links is far too small to account for the observed changes in the thermal and mechanical properties following incubation in vitro, clearly indicating that another as yet undefined, but apparently more important cross-linking mechanism mediated by glucose is taking place.