Thermodynamic characteristics of collagen fibrils reconstructed in vitro at different temperatures and concentrations

The results of a calorimetric study of type I collagen fibrillogenesis were analyzed. The dependence of the half-width of the temperature transition of a collagen solution on the concentration and temperature of collagen formation was studied. It was demonstrated that, by varying temperature and collagen concentration, one can regulate the density of packing and dimensions of cooperative fibril blocks. At temperatures below the physiological level (25 degrees C and 30 degrees C), and a relatively low concentration of collagen (0.3 mg/ml), fibrils with the lowest density of packing are formed. The degree of order does not change as the collagen concentration increases twofold but grows as the concentration increases fourfold. It was shown that, at the physiological temperature (35 degrees C), fibrils with a dense packing of molecules are formed at all collagen concentrations studied. The value of fibril formation enthalpy is minimal at a temperature of 35 degrees C, pH 7.2, an ionic strength of 0.17 M and a concentration of 1.2 mg/ml. Based on the results obtained, a conclusion was made that the packing density of fibrils formed at physiological temperature does not depend on collagen concentration over the concentration range of 0.3 - 1.2 mg/ml.     

Morphological characterization of tendon development during chick embryogenesis: measurement of birefringence retardation

Morphological observations and physical measurement of (I) birefringence retardation, (2) mean fibre profile width, and (3) cell volume fraction were used to characterize chick hind limb extensor tendon development. Observations were made at days 7, 10, 14 and 17 embryologic and 1-1.5 post-hatching. Microanatomical observations illustrated a sequential development of tendon microanatomy consisting of (1) a uniaxial cellular framework with discontinuous collagen fibril bundles present in day 7 embryos; (2) a continuous network of birefringent collagen fibres, and early evidence of tendon fasciculation and crimp development by embryonic day 10; and (3) completion of the basic cytoarchitecture of tendon observed at day 14 of embryogenesis. These observations suggest that collagen deposition in tendon involves first a longitudinal and then a lateral organization of tendon fibroblasts. Associated with the progressive anatomical development of tendon was an increase in birefringence retardation, mean collagen fibre profile width, and a decrease in the cell volume fraction. Birefringence retardation per unit thickness, however, did not change. This suggested that the fibril packing density of the fibres remained constant, although the fibres were observed to increase in size. These results indicate that collagen fibrillogenesis in vivo can be quantitatively studied by measurement of the birefringence retardation using polarized light.     

Preliminary X-ray crystallographic data on phospholipase C from Bacillus cereus.

Low-angle X-ray diffraction shows that, despite the well-defined regular axially projected structure, there is no long-range lateral order in the packing of molecules in native (undried) or dried elastoidin spicules from the fin rays of the spurhound Squalus acanthias. The equatorial intensity distribution of the X-ray diffraction pattern from native elastoidin indicates a molecular diameter of 1.1 nm and a packing fraction for the structure projected on to a plane perpendicular to the spicule (fibril) axis of 0.31 (the value for tendon is much higher at around 0.6). Density measurements support this interpretation. When the spicule dries the packing fraction increases to 0.43 but there is still no long-range order in the structure. The X-ray diffraction patterns provide no convincing evidence for any microfibrils or subfibrils in elastoidin. Gel electrophoresis shows that the three chains in the elastoidin molecule are identical. The low packing fraction for collagen molecules in elastoidin explains the difference in appearance between electron micrographs of negatively stained elastoidin and tendon collagen. In elastoidin, but not in tendon collagen, an appreciable proportion of the stain is able to penetrate between the collagen molecules.     

Effects of carcass maturity on meat quality characteristics of beef semitendinosus muscle for chinese native yellow steers

This work was designed to study the effects of carcass maturity on meat quality characteristics and intramuscular connective tissue of beef semitendinosus muscle from Chinese native Yellow steers. Chemical determinations, histological and mechanical measurements were performed on the raw and cooked meat at 4 days post mortem. In raw meat, intramuscular fat, collagen solubility, mechanical strength and transition temperature of intramuscular connective tissue increased (P < 0.05) with carcass maturity before body maturation, whilst moisture, total collagen, fibre diameter decreased after body maturation. Warner-Bratzlar shear force (WBSF) of cooked meat increased with maturity before body maturation due to the muscle atrophy, and thus the decline of moisture content and the increase of cooking losses. After body maturation, the increase of WBSF was neutralised by the increase of intramuscular fat, the decrease of total collagen and the elongation of sarcomere length.     

Multiple denaturational transitions in fibrillar collagen

The heat denaturation of pepsinized bovine nonfibrillar and fibrillar collagen was studied by differential scanning calorimetry. For fibrillar preparations that had been rapidly precipitated with stirring at low ionic strength, then resuspended at physiological ionic strength, multiple denaturational transitions were observed. At heating rates of 10°C/min, melting endotherms occurred at about 44, 50, 53, and 57°C. Fibrillar collagen that was slowly gelled without stirring at physiological ionic strength exhibited a similar series of endotherms, but the lower melting transitions were less conspicuous. In contrast, nonfibrillar bovine collagen in acidic solution showed only a single denaturational transition at 40°C. Nonfibrillar solutions at pH 7, to which inhibitors of fibrillogenesis were added, showed a major endotherm as high as 46°C. These results suggest that reconstituted fibrillar collagen contains a heterogeneous fibril population, possibly including molecules in a nonfibrillar state. It was proposed that the multiple melting endotherms of such preparations were due to sequential melting of molecular and fibril classes, each with a distinct melting temperature. The fibrillar classes may represent three or more types of banded and nonbanded species that differ from each other in packing order, collagen concentration, and possibly also in fibril width and level of cross-linking.     

Structure of type I and type III heterotypic collagen fibrils: an X-ray diffraction study

The molecular packing arrangement within collagen fibrils has a significant effect on the tensile properties of tissues. To date, most studies have focused on homotypic fibrils composed of type I collagen. This study investigates the packing of type I/III collagen molecules in heterotypic fibrils of colonic submucosa using a combination of X-ray diffraction data, molecular model building, and simulated X-ray diffraction fibre diagrams. A model comprising a 70-nm-diameter D- (approximately 65 nm) axial periodic structure containing type I and type III collagen chains was constructed from amino acid scattering factors organised in a liquid-like lateral packing arrangement simulated using a classical Lennard-Jones potential. The models that gave the most accurate correspondence with diffraction data revealed that the structure of the fibril involves liquid-like lateral packing combined with a constant helical inclination angle for molecules throughout the fibril. Combinations of type I:type III scattering factors in a ratio of 4:1 gave a reasonable correspondence with the meridional diffraction series. The attenuation of the meridional intensities may be explained by a blurring of the electron density profile of the D period caused by nonspecific or random interactions between collagen types I and III in the heterotypic fibril.     

X-ray diffraction evidence of collagen molecular packing and cross-linking in fibrils of rat tendon observed by synchrotron radiation.

X-ray diffraction patterns of fibres from 90 day (mature) rat-tail tendons were investigated using synchrotron radiation. The specimens were kept isometric at their corresponding in vitro rest length, and effects of pH and ionic strength were studied during short X-ray exposures. The results indicate that fibrils, equilibrated in physiological Ringer prior to exposure, have segregated lateral regions of well ordered collagen molecular packing. Lowering the ionic strength or the pH to 4.0 causes an order/disorder transition during which the fibril crystallinity decreases. At pH 3.5 a dramatic increase in the lateral swelling was observed. This effect was absent for fibres pretreated with sodium borohydride. The results are interpreted on the basis of cross-linking phenomena whereby the aldimine cross-link seems to be a controlling component of the lateral packing arrangement of collagen molecules.     

Ossification score is a better indicator of maturity related changes in eating quality than animal age

Ossification score and animal age are both used as proxies for maturity-related collagen crosslinking and consequently decreases in beef tenderness. Ossification score is strongly influenced by the hormonal status of the animal and may therefore better reflect physiological maturity and consequently eating quality. As part of a broader cross-European study, local consumers scored 18 different muscle types cooked in three ways from 482 carcasses with ages ranging from 590 to 6135 days and ossification scores ranging from 110 to 590. The data were studied across three different maturity ranges; the complete range of maturities, a lesser range and a more mature range. The lesser maturity group consisted of carcasses having either an ossification score of 200 or less or an age of 987 days or less with the remainder in the greater maturity group. The three different maturity ranges were analysed separately with a linear mixed effects model. Across all the data, and for the greater maturity group, animal age had a greater magnitude of effect on eating quality than ossification score. This is likely due to a loss of sensitivity in mature carcasses where ossification approached and even reached the maximum value. In contrast, age had no relationship with eating quality for the lesser maturity group, leaving ossification score as the more appropriate measure. Therefore ossification score is more appropriate for most commercial beef carcasses, however it is inadequate for carcasses with greater maturity such as cull cows. Both measures may therefore be required in models to predict eating quality over populations with a wide range in maturity.     

The role of the alpha2 chain in the stabilization of the collagen type I heterotrimer: a study of the type I homotrimer in oim mouse tissues

We have previously reported that the fragility of skin, tendon and bone from the oim mouse is related to a significant reduction in the intermolecular cross-linking. The oim mutation is unlikely to affect the efficacy of the lysyl oxidase, suggesting that the defect is in the molecule and fibre. We have therefore investigated the integrity of both the oim collagen molecules and the fibre by differential scanning calorimetry.The denaturation temperature of the oim molecule in solution and the fibre from tail tendon were found to be higher than the wild-type by 2.6deg.C and 1.9deg.C, respectively. With the loss of the alpha2 chain, the hydroxyproline content of the homotrimer is higher than the heterotrimer, which may account for the increase.There is a small decrease in the enthalpy of the oim fibres but it is not significant, suggesting that the amount of disorder of the triple-helical molecules and of the fibres is small and involves only a small part of the total bond energy holding the helical structure together. The difference in denaturation temperature of the skin collagen molecules (t(m)) and fibres (t(d)) is significantly lower for the oim tissues, 19.9deg.C against 23.1deg.C, indicating reduced molecular interactions and hence packing of the molecules in the fibre. Computation of the volume fraction of the water revealed that the interaxial separation of the oim fibres was indeed greater, increasing from 19.6A to 21.0A. This difference of 1.4A, equivalent to a C-C bond, would certainly decrease the ability of the telopeptide aldehyde to interact with the epsilon -amino group from an adjacent molecule and form a cross-link. We suggest, therefore, that the reduction of the cross-linking is due to increased water content of the fibre rather than a distortion of the molecular structure.The higher hydrophobicity of the alpha2 chain appears to play a role in the stabilisation of heterotrimeric type I collagen, possibly by increasing the hydrophobic interactions between the heterotrimeric molecules, thereby reducing the water content and increasing the binding of the molecules in the fibre.     

Formation of collagen type I fibrils in vitro

The assembly of collagen fibrils as a function of temperature and collagen concentration was studied. It was shown that temperature increases from 25 to 35 degrees C, the degree of ordering of collagen fibrils increases 1.5-fold at collagen concentration above 1 mg/ml and 2-fold at low collagen concentration. A maximum ordering of fibril structure occurs under conditions close to physiological (T approximately 35 degrees C and collagen concentration 1.2 mg/ml). As temperature is elevated from 30 to 35 degrees C, the packing of collagen molecules in fibrils becomes more ordered: the values of enthalpy and entropy of the transition of fibrils from the native to a disordered state decrease at all collagen concentrations used. At high collagen concentration, the dimensions of cooperative blocks in fibrils formed at 25 and 30 degrees C coincide with those of cooperative blocks of monomeric collagen in solution. Upon increasing the temperature to 35 degrees C, the dimensions of cooperative blocks increase.     

Turbidimetric and morphological studies of type I collagen fibre self assembly in vitro and the influence of fibronectin

Collagen undergoes several stages of self assembly including turbidimetric lag, growth and plateau steps. The later stages of type I collagen self assembly were studied by turbidity—time measurements, low angle laser light scattering and by determination of the birefringence retardation of collagen fibres formed in vitro. These studies were conducted in the presence and absence of fibronectin to evaluate the effect of fibronectin on the kinetics and extent of type I collagen fibrillogenesis. The results of these studies indicate that the collagen fibres observed at the end of the lag phase appear to be identical to fibres seen in the growth phase of turbidity—time curves based on fibre diameter and birefringence retardation measurements. Birefringence retardation measurements suggest that the diffracting unit may be the collagen fibril and that the volume fraction of fibrils in fibres is about 0.95 using a model developed for a series of parallel ellipsoids. Morphological observations suggest that the distribution of fibre diameters formed in vitro during the growth phase is narrow and appears to be independent of time with only the mass of collagen in fibres increasing during the growth phase. During the growth phase, layers of parallel fibres are formed with alternating layers appearing almost orthogonal. In the presence of fibronectin the mechanism of fibre formation appeared unchanged. It was concluded that fibronectin appeared to modify the kinetics of self assembly by preventing collisions between collagen molecules.     

Ageing of the human corneal stroma: structural and biochemical changes

High and low angle X-ray diffraction patterns from the corneal stroma give information about the mean intermolecular spacing of the collagen molecules and the mean interfibrillar spacing of the collagen fibrils, respectively. X-ray data were collected, using a high intensity synchrotron source, from human corneas and sclera at approximately physiological hydration. The spacings were measured as a function of tissue age. Between birth and 90 years there is an increase in the cross-sectional area associated with each molecule in corneal collagen from approx. 3.04 nm² to 3.46 nm², and an increase in scleral collagen from approx. 2.65 nm² to 3.19 nm². These changes may be due to an increase in the extent of non-enzymatic cross-linking between collagen molecules over the age range. We have investigated this possibility by measuring collagen glycation using the thiobarbituric acid assay and the subsequent advanced glycation end-products (AGEs) using fluorescence emission. The results obtained have shown an age-related increase in glycation and AGEs in both tissues. We have also demonstrated a decrease in the interfibrillar spacing of corneal collagen with increasing age which may be related to changes in the proteoglycan composition of the interfibrillar matrix.     

Fibrillogenesis in dense collagen solutions: a physicochemical study

Fibrillogenesis, the formation of collagen fibrils, is a key factor in connective tissue morphogenesis. To understand to what extent cells influence this process, we systematically studied the physicochemistry of the self-assembly of type I collagen molecules into fibrils in vitro. We report that fibrillogenesis in solutions of type I collagen, in a high concentration range close to that of living tissues (40-300 mg/ml), yields strong gels over wide pH and ionic strength ranges. Structures of gels were described by combining microscopic observations (transmission electron microscopy) with small- and wide-angle X-ray scattering analysis, and the influence of concentration, pH, and ionic strength on the fibril size and organization was evaluated. The typical cross-striated pattern and the corresponding small-angle X-ray scattering 67-nm diffraction peaks were visible in all conditions in the pH 6 to pH 12 range. In reference conditions (pH 7.4, ionic strength = 150 mM, 20 °C), collagen concentration greatly influences the overall macroscopic structure of the resultant fibrillar gels, as well as the morphology and structure of the fibrils themselves. At a given collagen concentration, increasing the ionic strength from 24 to 261 mM produces larger fibrils until the system becomes biphasic. We also show that fibrils can form in acidic medium (pH ∼ 2.5) at very high collagen concentrations, beyond 150 mg/ml, which suggests a possible cholesteric-to-smectic phase transition. This set of data demonstrates how simple physicochemical parameters determine the molecular organization of collagen. Such an in vitro model allows us to study the intricate process of fibrillogenesis in conditions of molecular packing close to that which occurs in biological tissue morphogenesis.     

A generalized packing model for type I collagen

Only tail tendon (TT) collagen has a sharp X-ray diffraction pattern, so that packing models for the equatorial arrangement of molecules in collagen fibrils have been developed primarily for TT collagen. A more general structure is developed applicable to all type I collagen tissues. Comparison of water content-equatorial diffraction spacing plots of several collagens shows all have essentially the same dry state diffraction spacing but differ as water content increases. TT collagen has the least spacing and the sharpest pattern. The interplanar spacing of the Hulmes-Miller quasi-hexagonal model for TT collagen was used to calculate the intermolecular spacing, which matched the observed diffraction spacing for bone matrix collagen. It is inferred that wet bone matrix collagen packs in a rectangular pattern because of the interaction between the many intermolecular crosslinks and the water absorbed on the collagen molecules. This argument also indicates that TT collagen packs into a quasi-hexagonal scheme because there are fewer intermolecular crosslinks than in bone matrix collagen.     

Ageing of the human corneal stroma: structural and biochemical changes.

High and low angle X-ray diffraction patterns from the corneal stroma give information about the mean intermolecular spacing of the collagen molecules and the mean interfibrillar spacing of the collagen fibrils, respectively. X-ray data were collected, using a high intensity synchrotron source, from human corneas and sclera at approximately physiological hydration. The spacings were measured as a function of tissue age. Between birth and 90 years there is an increase in the cross-sectional area associated with each molecule in corneal collagen from approx. 3.04 nm2 to 3.46 nm2, and an increase in scleral collagen from approx. 2.65 nm2 to 3.19 nm2. These changes may be due to an increase in the extent of non-enzymic cross-linking between collagen molecules over the age range. We have investigated this possibility by measuring collagen glycation using the thiobarbituric acid assay and the subsequent advanced glycation end-products (AGEs) using fluorescence emission. The results obtained have shown an age-related increase in glycation and AGEs in both tissues. We have also demonstrated a decrease in the interfibrillar spacing of corneal collagen with increasing age which may be related to changes in the proteoglycan composition of the interfibrillar matrix.     

The intermolecular space of reconstituted collagen fibrils

The extent, geometry and heterogeneity of the intermolecular space of hydrated, purified and reconstituted steer skin collagen fibrils has been characterized. The extent of the space has been assessed experimentally by an X-ray diffraction method and a new physical chemical technique, and found to be 1.14 ml per gram collagen. A theoretical model relating the intermolecular space to X-ray diffraction parameters has been presented, and this suggests that the geometry of the intermolecular space arises from a near-hexagonal packing of the collagen molecules. On the basis of an assumed microfibrillar packing model and a geometric construction of the shape of a collagen molecule, the distribution of the space within reconstituted collagen fibrils has been characterized as follows: 0.13 ml of the intermolecular space/g collagen can be attributed to the helical groove of the collagen molecules per se and 1.01 ml/g is interstitial; 0.66 ml/g is present in the form of “pores” (hexagonally-closed packed spaces), whereas 0.48 ml/g is present in the form of “holes” (hexagonal volume defects); 0.73 ml/g of the intermolecular space is associated with a region of the collagen fibrils where holes are localized and 0.41 ml/g is attributable to the regions of the fibril in which pores only are present.     

Collagen fibril formation in vitro at nearly physiological temperatures

Fibril formation by collagen from piglet skin was studied at temperatures of 28–39°C. Collagen fibrils obtained in this temperature range differ in the degree of ordering. Electron microscopy shows that fibrils of minimal diameter are formed at physiological pH, ionic strength (PBS), and temperature. The greater diameter of fibrils formed at 34.5°C is due to enhanced collagen hydration. Fibril diameter at 38.5°C is increased because of cooperative unfolding of the triple helix and weaker binding between collagen molecules. The optimal temperature for fibrillogenesis appears to be 36.5°C, and such fibrils are most similar to those observed in vivo.     

The effects of productivity and seasonality on life history: comparing age at maturity among moose (Alces alces) populations

The productivity hypothesis in respect of an animal species’ geographical range predicts that whereas higher productivity at the equatorial periphery of a species’ range favours superior competitors, lower productivity at the centre of a species’ range favours high reproduction and reduced competitive traits. I test whether life‐history patterns follow this hypothesis, using demographic data from 15 Canadian moose (Alces alces) populations. Two models are contrasted; the first assumes that intraspecific variation in age at maturity is explained proximately by density and juvenile mortality. Age at maturity was found to increase with decreasing juvenile mortality (P = 0.01) and increasing density (P = 0.006). To test the productivity hypothesis, the second model additionally included primary productivity and seasonality as geographical explanatory variables that would ultimately influence age at maturity via juvenile mortality and density. Path analysis indicated that including productivity and seasonality improved the model predictions of variation in age at maturity (R_a² 0.56 and 0.85). In bivariate comparisons, seasonality was negatively associated (P = 0.01) with age at maturity. In the best model, however, primary productivity was the environmental variable that explained 25% of the variance in age at maturity, and forest cover replaced seasonality as an explanatory variable. The positive association between primary productivity and age at maturity is consistent with the productivity hypothesis. Relative to populations that lived at the centre of the species’ range (51°N), moose populations living in relatively high productivity and low seasonality environments (equatorial periphery of species’ range; 48°N) experienced less juvenile mortality, more variable year‐to‐year density, higher relative density and slower life history (slower growth rate, later age at maturity, lower fecundity).     

Growth, size and age at maturity of the agile frog (Rana dalmatina) in an Iberian Peninsula population

The mean age of a population of agile frogs (Rana dalmatina) from the Iberian Peninsula was estimated using mark and recapture and skeletochronology. Life-history parameters, including growth rate, body length, age and size at maturity, sexual dimorphism and longevity, were studied. The regression between age and snout-vent length (SVL) was highly significant in both sexes. Males reached sexual maturity at two years of age, although sometimes they can reach it at only one year of age. The average SVL at maturity was 51.75 mm (standard error (SE) = 0.71; n = 45). Females reached sexual maturity at two years of age with an average SVL of 62.14 mm (SE = 2.20; n = 14). A subset of the female population reached sexual maturity at three years of age. Growth was rapid until sexual maturity was reached. There was an overlap of SVL between different age classes. Growth was continuous, fulfilling the conditions of Von Bertalanffy's model. The growth coefficient (K) was 0.840 in males and 0.625 in females. The maximum SVL was greater in females (73.00 mm) than in males (59.50 mm). Sexual dimorphism was significantly biased towards females in all age classes. The maximum longevity observed was 6 years in females and 8 years in males. Management strategies for agile frogs should take into account factors such as these life-history characteristics.     

A synchrotron radiation X-ray diffraction study of the crystallinity of tendon fibres

The equatorial diffraction pattern of tendon collagen fibres was measured during short successive exposures at different lengths using a double focusing X-ray synchrotron radiation camera with film and with an area detector. Similarly, patterns from thin fibres from premature rats were recorded. The patterns unambiguously illustrate the relationship between fibre crystallinity and the age of the animal. Further, the results indicate that in the initial part of the linear region of the stiffness-versus-length curve, the collagen fibres are characterized by a quasihexagonal arrangement of collagen molecules, whereas at the end of this region, the molecular arrangement becomes hexagonal.