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.     

Interactions between collagen chains and fiber formation

The temperature-dependent dissociation of neutral salt-soluble collagen into its component chains was measured in 0.6–1.6 M urea solutions at pH 7.3. The temperature-dependent association of the same radiocactively labeled collagen into fibers was measured in 0–0.4 M urea solutions, pH 7.3. The effect of urea on the temperature, Tm(G), for half dissociation into chains was small, and the value extrapolated to zero urea concentration was 39°C. In contrast, the effect of urea on the temperature, Tm(F), for half association into fibers was large, and the value at zero urea concentration was 30°C. We conclude that while body temperature provides excellent conditions for the matching of collagen chains to form molecules, the conditions are not optimal for the formation of highly ordered fibers. The large effects of 0.1 M urea suggest that other factors in vivo may help to destabilize mismatched molecular association during fiber growth. Alternately this might be facilitated by parts of the extension peptides of procollagen.     

Gel filtration chromatography of triple-helical calf skin collagen

Gel filtration of type I collagen has been of limited use, because at low pH where the protein is not associated it binds to agarose gels, and at neutrality collagen has a tendency to form fibrils. The more porous polyacrylamide-based gels do not interact with collagen but cannot be used at very high flow rates because they are compressible. It was found that these difficulties are surmounted by use of Fractogel TSK HW-65F, a spherical gel made from a weakly hydrophilic vinyl polymer, and use of the buffer system 0.5 m urea, 0.117 m Tris-HCl, pH 7.3, which prevents fibril formation. The solvent has only a slight effect on the thermal stability of collagen, as determined by circular dichroism measurements. The recovery of native collagen, at 25°C, was at least 88% and that of partially unfolded collagen, at 35°C where it is about one-third unfolded, was 98%. The Fractogel TSK gels and the urea, Tris solvent system should be useful for both preparative work and for studies involving interaction of unaggregated type I collagen with smaller molecules at physiological pH.     

Characterization of nuclei in in vitro collagen fibril formation.

Heat precipitation fibril formation in collagen solutions depends upon the prior thermal history of the solution. Collagen solutions were heat precipitated to various extents at 30°C, cooled, and then brought to a second precipitation. Kinetic analysis of the secondary precipitation demonstrated that only the nucleation phase of the precipitation was affected, not the fibril growth phase. Thermal history, or memory, is thus related to the formation of low-temperature-stable nuclei. A range of nuclei sizes is evident, supporting the concept of a homogeneous nucleation process. Schiffs base formation and establishment of cross-linkages play no role in the in vitro nucleation: thiosemicarbazide treated collagen behaves identically to untreated collagen in kinetics of assembly to fibrils. Low-temperature-stable nuclei formed at neutral pH are dissociated in the cold in acetic acid at pH 4. Pronase and pepsin susceptible molecular end regions are important in establishing the low-temperature-stable nuclei. Pronase treatment completely abolishes the acquisition of memory of prior thermal history in collagen solutions. We speculate that biological control mechanisms for fibril formation in vivo relate to specific interactions between non-helical, enzyme susceptible regions on collagen molecules.     

Multinuclear magnetic resonance studies of collagen molecular structure and dynamics

We have measured the percentages of cis and trans Gly-Pro and X-Hyp peptide bonds in thermally unfolded type I collagen. ¹³C-nmr solution spectra show that 16% of the Gly-Pro and 8% of the X-Hyp bonds are cis in unfolded chick calvaria collagen. These results support the hypothesis that cis–trans isomerization is that rate-limiting step in the propagation of the collagen triple helix. We have used multinuclear solid-state nmr to study the molecular dynamics of the collagen backbone in tendon, demineralized bone, and intact bone as a function of temperature, hydration, and pH. These studies show that collagen backbone motions are characterized by a broad distribution of correlation times, τ, covering the range from 10^?4 to 10^?9 s. In the case of nonmineralized collagen, the root-mean-square fluctuations in azimuthal angle, γ_rms, range from ca. 10° when τ ～ 10^?9 s to ca. 30° when τ < 10^?4 s; in the case of bone collagen, γ_rms values are about half as large as those found in nonmineralized collagen. Backbone motions are negligible at temperatures below ?25°C. This is also the case at 22°C when demineralized bone collagen is lyophilized. In contrast, flexibility of hydrated demineralized bone collagen greatly increases as pH is lowered from 7 to 2. The more limited flexibility observed at neutral pH is a consequence of the intermolecular interactions that contribute to fibril organization and strength. However, the fibrils retain significant flexibility at physiological pH, enabling them to distribute stress and dissipate mechanical energy.     

Flexibility of collagen determined from dilute solution viscoelastic measurements

The frequency dependences of the storage and loss shear moduli, G′ and G″, of pronase-treated collagen dissolved in acetate buffer at pH 4.0 were measured at 17.0°C by use of the Birnboim-Schrag multiple lumped resonator apparatus. Some of the solutions contained 70% glycerol. The infinite-dilution moduli were determined and compared with theoretical models for a rigid cylinder and a semiflexible rod. Only the latter could fit the data. A rotational time of 144 μs and a slowest flexural relaxation time of 21 μs, both reduced to water at 20°C, were determined from the fit. The intrinsic viscosity and rotational relaxation time correspond to a semiflexible rod with persistence length of about 170 nm and a Young's modulus of 4 × 10¹⁰ dyn/cm².     

Synthesis and characterization of an ionizable polyhexapeptide collagen model

Poly(Lys(HBr)-Gly-Pro-Pro-Gly-Pro) has been synthesized and studied by circular dichroism (CD) spectroscopy. It is apparently the first polyhexapeptide collagen model reported with an ionizable side chain. The monomer (ε-(p-nitrobenzyloxycarbonyl)-Lys-Gly-Pro-Pro-Gly-Pro-p-nitrophenyl-ester) was prepared by a stepwise strategy employing active esters. Polymerization in N,N-dimethyl formamide, followed by removal of the Lys side chain protection with HBr/acetic acid, gave a polydisperse product. Fractionation was accomplished by gel filtration chromatography. The polydisperse material had a molecular weight (M_r = 5–17,000). High molecular weight fractions from triple helices under concentrated conditions at 2°C. The triple helical structure gives a CD pattern very similar to that of collagen and its triple helical analogs. However, unlike collagen, the polyhexapeptide undergoes spontaneous dissociation at temperatures substantially below the melting temperature from a triple helical form to single chains. This process is promoted at low concentrations, high temperature, neutral pH, and low molecular weight, and is apparently due, in large part, to unfavorable ionic side-chain interactions. In addition to this relatively slow “ionic” dissociation the triple helical polypeptide may be thermally dissociated in a manner similar to collagen. The thermal denaturation is a relatively fast process compared with ionic dissociation. A high molecular weight fraction (3 × M_r = 48,000) was found to melt at 42°C at neutral pH but increased to 54°C at pH 12 where the lysyl side chains are predominantly deprotonated. Furthermore, reconstitution of triple helices appeared to be more readily achieved at high pH. Thus it is concluded that ionic repulsion between side chains causes destabilization of the triple helix and hinders reconstitution.     

Experimental investigation of collagen waviness and orientation in the arterial adventitia using confocal laser scanning microscopy

Mechanical properties of the adventitia are largely determined by the organization of collagen fibers. Measurements on the waviness and orientation of collagen, particularly at the zero-stress state, are necessary to relate the structural organization of collagen to the mechanical response of the adventitia. Using the fluorescence collagen marker CNA38-OG488 and confocal laser scanning microscopy, we imaged collagen fibers in the adventitia of rabbit common carotid arteries ex vivo. The arteries were cut open along their longitudinal axes to get the zero-stress state. We used semi-manual and automatic techniques to measure parameters related to the waviness and orientation of fibers. Our results showed that the straightness parameter (defined as the ratio between the distances of endpoints of a fiber to its length) was distributed with a beta distribution (mean value 0.72, variance 0.028) and did not depend on the mean angle orientation of fibers. Local angular density distributions revealed four axially symmetric families of fibers with mean directions of 0°, 90°, 43° and ?43°, with respect to the axial direction of the artery, and corresponding circular standard deviations of 40°, 47°, 37° and 37°. The distribution of local orientations was shifted to the circumferential direction when measured in arteries at the zero-load state (intact), as compared to arteries at the zero-stress state (cut-open). Information on collagen fiber waviness and orientation, such as obtained in this study, could be used to develop structural models of the adventitia, providing better means for analyzing and understanding the mechanical properties of vascular wall.     

Effect of pH on collagen flexibility determined from dilute solution viscoelastic measurements

The frequency dependences of the storage and loss shear moduli, G′ and G″ of dilute solutions of collagen at pH 7.4, ionic strength approximately 0.2, were measured at 15.0°C by the Birnboim—Schrag multiple-lumped resonator apparatus. By use of two solvents, water and 50% glycerol, the effective reduced frequency range was extended to cover 2.5 logarithmic decades. The intrinsic viscosity and longest (rotational) relaxation time were considerably smaller than those determined at pH 4.0 in an earlier study. At pH 4.0, the behaviour could be modelled by a rodlike molecule with partial flexibility along its entire length and a persistence length of 161 nm with no loose joints. The behaviour at pH 7.4 corresponds approximately to the expectation for a semiflexible rod with two loose joints near the ends and a similar persistence length (169nm) for the centre segment.     

Investigation of molecular motion in collagen using the spin-probe technique.

The spin-probe technique was employed to study molecular motion in collagen and modified collagen samples in the ?160° to +200°C region. The effect of water content in the 0–30-wt. % range, relative to vacuum dried samples, on the electron spin resonance spectrum of the probe was also investigated. The spectra at the lowest temperatures consisted of a broad asymmetric triplet. Narrowing of this triplet above ?40° to ?70°C and the appearance of additional lines in the spectrum, interpreted as due to a narrow triplet, at a temperature dependent on the water content were observed. For samples with low water contents [(0–0.4)%] the broad triplet was that expected for a glassy system up to 150–190°C; for these samples the narrow triplet appears at a temperature above 50°C, its intensity increasing with increasing temperature up to 70–100°C, then decreasing with a further temperature increase. For samples with water contents near 30%, the narrow triplet first completely appears at about 0°C, and reaches relative intensities of 35% at 30°C. The motion taking place in collagen and related samples is discussed in terms of these results.     

Magnetic alignment of collagen during self-assembly

Magnetically induced birefringence is used to monitor the thermally induced self-assembly of collagen fibrils from a solution of molecules. The magnetic torque alone can, at best, only orient the fibrils into planes normal to the field direction. Nevertheless, the gels formed have a high degree of uniaxial alignment, probably due to the additional ordering effects of surface interactions. Thus magnetic orientation is potentially useful in the study of fibrillogenesis and in the production of highly oriented collagen gels.     

Structural changes in cartilage and collagen studied by high temperature Raman spectroscopy

Understanding the high temperature behavior of collagen and collagenous tissue is important for surgical procedures and biomaterials processing for the food, pharmaceutical, and cosmetics industries. One primary event for proteins is thermal denaturation that involves unfolding the polypeptide chains while maintaining the primary structure intact. Collagen in the extracellular matrix of cartilage and other connective tissue is a hierarchical material containing bundles of triple‐helical fibers associated with water and proteoglycan components. Thermal analysis of dehydrated collagen indicates irreversible denaturation at high temperature between 135°C and 200°C, with another reversible event at ～60‐80°C for hydrated samples. We report high temperature Raman spectra for freeze‐dried cartilage samples that show an increase in laser‐excited fluorescence interpreted as conformational changes associated with denaturation above 140°C. Spectra for separated collagen and proteoglycan fractions extracted from cartilage indicate the changes are associated with collagen. The Raman data also show appearance of new features indicating peptide bond hydrolysis at high temperature implying that molecular H₂O is retained within the freeze‐dried tissue. This is confirmed by thermogravimetric analysis that show 5‐7 wt% H₂O remaining within freeze‐dried cartilage that is released progressively upon heating up to 200°C. Spectra obtained after exposure to high temperature and re‐hydration following recovery indicate that the capacity of the denatured collagen to re‐absorb water is reduced. Our results are important for revealing the presence of bound H₂O within the collagen component of connective tissue even after freeze‐drying and its role in denaturation that is accompanied by or perhaps preceded by breakdown of the primary polypeptide structure.     

X-ray diffraction studies on the structure of hydrated collagen

The temperature dependence of the humidity-sensitive spacing, d, related to the lateral packing of collagen molecules was measured for fully hydrated collagen. In the vicinity of 0°C, a sudden change in d was observed, which was reversible with temperature. In the diffraction profile, below 0°C, a set of diffraction peaks identified with the hexagonal crystalline form of ice was observed. With the reduction in water content, the intensity of the set of diffraction peaks decreased and was found to be zero at a water content of 0.38 g/g collagen. These results were considered to be caused by the frozen water in collagen fibril below 0°C. According to the water content dependence of d, it was considered that up to a certain water content water absorbed would be stowed in the intermolecular space of collagen and above that water content water molecules would aggregate to make pools, i. e., extrafibrillar spaces. The unfreezable bound water was considered to be located in the intermolecular space of collagen. Size of the extrafibrillar space, determined from the intensity analysis of a smallangle x-ray scattering pattern, corroborates the speculation that the water showed in the extrafibrillar space is freezable and free. The formation of the hexagonal crystalline form of ice in the extrafibrillar space was considered to cause the sudden change in d at 0°C.     

Scleraldehyde as a stabilizing agent for collagen scaffold preparation

Schizophyllan is a natural polysaccharide, produced by fungi of the genus Schizophyllum. Periodate oxidation specifically cleaves the vicinal glycols in schizophyllan to form their dialdehyde derivatives. The present study investigates the interaction of scleraldehyde with Type I collagen membrane. The formation of the inter and intra interaction between scleraldehyde and the collagen fibres results in significant increase in viscosity of collagen. Crosslinking efficiency of scleraldehyde was found to increase with concentration of scleraldehyde. Scleraldehyde interacted collagen membrane exhibited an increase in thermal stability by 29 °C at pH 8. The gelling time of collagen fibrils was found to decrease with increase in concentration of scleraldehyde due to shift in nucleation centre. Swelling degree of collagen membrane was also found to decrease with increase in concentration of scleraldehyde. Scleraldehyde treated collagen membrane exhibited 93% resistance to collagenase. The modified collagen membrane exhibited non-toxicity towards the fibroblasts cells. The modified collagen membrane by scleraldehyde finds application as a stabilizing agent in scaffold preparation.     

Transformation of the dermal collagen framework under laser heating

The aim of this study was to compare between the changes undergone by the dermal collagen framework when heated by IR laser radiation and by traditional means and to reveal the specific features of the dermal matrix modification under moderate IR laser irradiation. Rabbit skin specimens were heated to 50°C, 55°C, 60°C and 65°C in a calorimeter furnace and with a 1.68‐μm fiber Raman laser. The proportion of the degraded collagen macromolecules was determined by differential scanning calorimetry. Changes in the architectonics of the collagen framework were revealed by using standard, phase‐contrast, polarization optical and scanning electron microscopy techniques. The collagen denaturation and dermal matrix amorphization temperature in the case of laser heating proved to be lower by 10°C than that for heating in the calorimeter furnace. The IR laser treatment of the skin was found to cause a specific low‐temperature (45°C‐50°C) transformation of its collagen framework, with some collagen macromolecules remaining intact. The transformation reduces to the splitting of collagen bundles and distortion of the course of collagen fibers. The denaturation of collagen macromolecules in the case of traditional heating takes its course in a threshold manner, so that their pre‐denaturation morphological changes are insignificant.     

Phase transition in natural collagen]

The state of water protons and peptide chains in natural collagen was investigated by the method of nuclear magnetic resonance. The study of orientation and temperature dependence of collagen PMR spectra allows us to mage a conclusion about the considerable disorder of mutual orientation in the polypeptide chains at the temperature above -4 degrees C. Under these conditions it was observed that natural collagen was near to the liquid-crystal. The phasic transition at -4 degrees C was discovered, the latter was followed by an abrupt change of the character of water proton mobility and by the change of orientation of collagen fibres order. The thermal effect of transition was measured, the latter presents 18k/gr of the whole specimen.     

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.     

Decreased collagen thermal stability as a response to the loss of structural integrity of thyroid cartilage

The amino acid composition, thermal behavior and birefringence properties of thyroid cartilage tissues have been studied. A collagen component in perichondrium consists of type-I and type-II collagens whose fibers form a highly ordered anisotropic structure with a birefringence of 4.75 × 10^?3 and a melting (denaturation) temperature of 65°C. The hyaline constituent, which is visualized as a quasi-anisotropic medium, contains of only type-II collagen, which does not denature in intact tissues at temperatures up to 100°C. However, in tissues whose proteoglycane subsystem is damaged by trypsin, the denaturation of collagen takes place at 60°C. In the integral perichondrium-hyaline system, the temperature of collagen denaturation in the perichondrium reaches 75°C, which indicates the immobilization of collagen in this tissue by the extracellular matrix of the hyaline constituent.     

The state of water on hydrated collagen as studied by pulsed NMR

The spin-lattice relaxation time (T₁) of water adsorbed on collagen fibers was determined at six frequencies and temperatures varying from 25° to ?80°C. Care was taken to eliminate the contributions to the signal of protons other than those in the adsorbed water. Quantitative calculations were made on T₁ and the results were compared with the experimental data. It is suggested that a maximum of about 0.50–0.55 g water per g collagen forms a hydration layer, which cannot be frozen down to ?90°C and exhibits a distribution of motional correlation times. For collagen samples containing a larger quantity of adsorbed water, the additional water molecules behave like ordinary isotropic water, having a single correlation time and a freezing temperature of about ?10°C.     

Stabilization of type I collagen using dialdehyde cellulose

Type I collagen from rat tail tendon (RTT) fibres was crosslinked with dialdehyde cellulose to bring about stabilization of the matrix. Dialdehyde cellulose (DAC) was prepared by periodate oxidation of hydrolyzed cellulose. Autoclaving of DAC resulted in hydrolysis and lower molecular weight oligomeric species. The formation of the crosslinked network between DAC and the collagen fibres has brought about significant thermal and enzymatic stability to collagen. DAC crosslinked collagen fibres exhibited an increase in hydrothermal stability by 20 °C with autoclaved DAC at pH 8. The collagen matrix resulted in an increase in denaturation peak temperature (T_D) and an increase in phase change of activation energy (E_a) and enthalpy change (ΔH) for the shinking process indicating intermolecular crosslinking arising from covalent interactions. Thermal stability and crosslinking efficiency was found to increase with pH and concentration of DAC. DAC treated collagen exhibited 93% resistance to collagenolytic hydrolysis.