X-ray diffraction of reconstituted collagen fibers

The X-ray diffraction of fibers reconstituted from purified rat tail tendon collagen has been compared with that of native rat tail tendon. The axial structure is very similar in the two specimens, while the ordered lateral array found in the native state is only poorly reproduced in the reconstituted fiber. Thus, the axial order is determined by the collagen molecules alone, while the native lateral packing may depend, in part at least, on other tissue components.     

Polytonic studies of reconstituted collagen fibers

The tensile force-contraction characteristics of cross-linked reconstituted collagen fibers have been studied for 30 different water-soluble reagents. The data were obtained via a “polytonic” loading technique, which was neither isometric nor isotonic. Certain data were found to fit a master curve, which did not depend strongly on the denaturant. For many denaturants the contractile force appeared to be a maximum at a denaturant concentration corresponding to all the solvent being consumed in denaturant hydration. Also, the maximum contractile force appeared to vary in the same manner as does the hydration number of the individual cations and anions in the solution (i.e., H > Li > Na > K, and I > Br > Cl > F). Several denaturants were found to be as effective as the well-known LiBr and KSCN in contracting the collagen.     

Influence of a lathyric agent and of hypercholesterolemic serum on cell cultures of fetal rabbit aorta]

Cell cultures of foetal rabbit aorta are cultivated with a lathyric agent (beta-amino-propio-nitrile) or with an hypercholesterolemic serum; if morphological features, in these two cases, correspond with modifications observed, in vivo, when adult rabbits are respectively submitted to the same treatment, enzymatic activities of collagen metabolism vary in opposite way. Therefore, the influence of different parameters to be studied on vascular cell functions become easier.     

Oblique banding pattern in collagen fibrils reconstituted in vitro after trypsin treatment.

Collagen fibres from rat tail tendon suspended in small pieces in a solution (pH 7.8) containing 0.5 M CaCl2 were treated with purified bovine trypsin at 20 degrees C for 20 h. After the enzyme treatment collagen from this solution was precipitated out and reconstituted in vitro into native-type fibrils. The banding pattern in these reconstituted fibrils was found to be oblique. This is comparable to that observed recently in fibrils reconstituted from cartilage collagen. On the other hand, normal transverse banding pattern was observed in the fibrils reconstituted in vitro from collagen solution of rat tail tendon which was not pre-treated with trypsin. No significant change was, however, observed in the segment long spacing fibrils precipitated from the enzyme-treated collagen solution. It is possible that the enzyme might affect the mode of organization of tropocollagen molecules during in vitro fibrillogenesis into native-type fibrils either by interacting with the "telopeptide" regions or with the non-collagenous components associated with the native protein and this could probably result into the formation of fibrils with oblique banding pattern.     

Sickle hemoglobin fibers: mechanisms of depolymerization

We examined the depolymerization of hemoglobin (Hb) S fibers in the presence of CO by using photolysis of COHbS to create and isolate individual fibers, then removing photolysis to induce depolymerization. Depolymerization occurs at two sites, fiber ends and fiber sides, with different kinetics and by different mechanisms. At low partial pressure of CO (pCO), end-depolymerization is dominant, proceeding at approximately 1 microm s(-1), whereas at high pCO fibers vanish very rapidly, in much less than one second, by side-depolymerization. Each kind of depolymerization could occur by a ligand-independent path, in which deoxyHb depolymerizes and then is prevented from returning to the polymer by liganding with CO, or by a ligand-dependent path in which CO binds to the polymer inducing dissociation of the newly liganded molecules from it. We find that ligand-independent depolymerization is the dominant path for end-depolymerization and ligand-dependent depolymerization dominates, at least at high pCO, for side-depolymerization. On the basis of our kinetic results and electron micrographs of depolymerizing fibers, we propose a model for side-depolymerization in which a hole is nucleated by cooperative loss of a few molecules from fiber sides, followed by rapid depolymerization from the newly created fiber ends abutting the hole. Potential significance of these results for the pathophysiology of sickle cell disease is discussed.     

The ion-binding characteristics of reconstituted collagen

The ion-binding capacity of highly purified reconstituted calf-skin collagen, and the effects of these ions on the precipitation and solubility of the collagen, were studied with a variety of salt solutions at ionic strength 0·16 and pH7·4. Only a small percentage of the total theoretically available anionic and cationic groups was available for ion-binding. In view of this, it appears that most of the ionizable groups of collagen are involved in intramolecular or intermolecular linkages, or both. Nevertheless, marked differences in the binding of the various ions by collagen were observed. Bivalent cations were bound in extremely small but remarkably similar quantities. In contrast, sodium was bound both in much higher and more variable quantities. Of the anions, pyrophosphate and sulphate were bound in the largest quantities, followed by phosphate, fluoride and chloride, in that order. Despite the minimal uptake by collagen of bivalent cations, they prevented the aggregation of tropocollagen into fibrils, and disaggregated fibrillar collagen. In the presence of multivalent anions, tropocollagen aggregated readily and its fibrillar stability was maintained. On the basis of the imbalance in the binding of ion pairs by the sodium pyrophosphate- and sodium phosphate-treated collagens, it was apparent that a reduced number of side-chain carboxyl groups were dissociated in the presence of these salts.     

Gel beads composed of collagen reconstituted in alginate

Spherical gel beads of collagen/alginate were prepared by discharging droplets of a mixture containing collagen (1.07-1.9 mg/ml) and alginate (1.2-1.5% w/v) into 1.5% w/v CaCl2 solution at 4°C. Collagen in the gel beads was reconstituted by raising the temperature to 37°C after alginate was liquefied by citrate. Scanning electron microscopy of the beads revealed the characteristic fibrous structure of collagen. To demonstrate the application of this new technique in cell culture, GH3 rat pituitary tumor cells were entrapped and grown in the gel beads. The immobilized cells proliferated to a density of 1.95 x 106 cell/ml which is about an order of magnitude higher than that grown in the alginate beads.     

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.     

The effect of ions on the superstructures of reconstituted collagen

The effect of a variety of electrolytes on the stabilization and destabilization of the rod-like superstructures in reconstituted collagen membranes have been studied by small-angle light scattering (SALE). Structural orders at two levels are effected: association–disassociation at the micron level and the helix-coil transitions at the mollecular level. In addition to lyotropic salts, several transition metal ions were also studied. They act to disassociate collagen aggregates by the formation of stable complexes.     

Interaction of bilirubin with reconstituted collagen fibrils.

1. Interaction of bilirubin with collagen fibrils was explored in a two-phase system where collagen was present as an opaque rigid gel composed of striated fibrils, and bilirubin as an aqueous solution. 2. The Ka value of the binding of bilirubin to collagen fibrils is 5.4 X 10(3)M-1. The interaction of bilirubin with collagen fibrils depends on temperature. Below 5 degrees C, the binding is greatly diminished and denaturation of collagen fibril aggregates at 52--53 degrees C into a dissolution state abolishes binding of bilirubin. 3. Salicylate and sulphanilamide do not affect the binding of bilirubin to reconstituted collagen fibrils. 4. Serum albumin (40--80mM), known to reverse the binding of bilirubin to lipids, dissociates only 50% of the bilirubin bound to collagen fibrils. This suggests that sites located on collagen participate in some tight binding of bilirubin and the corresponding binding sites on albumin do not compete with them. 5. Urea (4M) abolishes more than 70% of the binding of bilirubin to collagen. Urea and thermal denaturation studies indicate the importance of conformation and organization of collagen fibrillar aggregates for the binding of bilirubin.     

Influence of fulvic acid on the collagen secretion of bovine chondrocytes in vitro

High concentrations of fulvic acid and selenium deficiency in drinking water are the main causative factors of Kashin-Beck disease, an endemic degenerative chronic osteoarticular disorder found in China. The influence of fulvic acid on collagen secretion was investigated in articular chondrocyte cultures from bovine interphalangeal joints. Collagen secretion in 7-day-old chondrocyte monolayers was determined by measuring [3H]-proline incorporation into collagen macromolecules after a 24-h application in cultured supernatants. Additionally, collagen secretion was measured with a collagen assay based on a dye-binding method of soluble collagens. Both methods showed a dose-dependent increase of collagen secretion after treatment with fulvic acid. The collagen was identified by immunoblotting and immunocytochemistry as type II collagen. Fulvic acid also induced H2O2 production in cartilage cells. After co-incubation with catalase and fulvic acid, the cells secreted the same amount of H2O2 or collagen as the non-treated controls, indicating an influence of H2O2 on collagen secretion. Chondrocytes were then treated directly with H2O2. This led to increased collagen secretion showing a positive correlation with the concentration of H2O2 up to 1 pM H2O2. Larger amounts of H2O2 decreased collagen secretion. Effects of reactive oxygen species, such as lipid peroxidation or lactate dehydrogenase (LDH) release from damaged cells, were not inducable by fulvic acid (<10 ppm). Our results demonstrate a fulvic-acid-induced stimulation of collagen secretion into the supernatant by articular chondrocytes via physiological amounts of H2O2.     

Self-assembly of collagen fibers. Influence of fibrillar alignment and decorin on mechanical properties.

Collagen is the primary structural element in extracellular matrices. In the form of fibers it acts to transmit forces, dissipate energy, and prevent premature mechanical failure in normal tissues. Deformation of collagen fibers involves molecular stretching and slippage, fibrillar slippage, and, ultimately, defibrillation. Our laboratory has developed a process for self-assembly of macroscopic collagen fibers that have structures and mechanical properties similar to rat tail tendon fibers. The purpose of this study is to determine the effects of subfibrillar orientation and decorin incorporation on the mechanical properties of collagen fibers. Self-assembled collagen fibers were stretched 0-50% before cross-linking and then characterized by microscopy and mechanical testing. Results of these studies indicate that fibrillar orientation, packing, and ultimate tensile strength can be increased by stretching. In addition, it is shown that decorin incorporation increases ultimate tensile strength of uncross-linked fibers. Based on the observed results it is hypothesized that decorin facilitates fibrillar slippage during deformation and thereby improves the tensile properties of collagen fibers.