N. Yu. Ignat’eva

Determination of hydroxyproline in tissues and the evaluation of the collagen content of the tissues

The concentrations of hydroxyproline (an amino acid specific of collagen) in a number of connective tissues were determined. Two procedures were compared. In one of them, amino acids were preseparated by chromatography and then determined on a standard amino acid analyzer. In the other procedure, hydroxyproline was selectively oxidized without amino acid separation and determined by a spectrophotometric reaction with Ehrlich’s reagent. Data obtained for purified collagen preparations in accordance with the two procedures were consistent with each other. The results can be somewhat different in unpurified preparations and tissues because of the presence of polysaccharide components in the tissues.

The effect of uniaxial tension on the stability of collagen fibers under the conditions of nonuniform laser heating

Collagen degradation caused by IR laser irradiation in ligament tissues was studied by thermal analysis and cross-polarization optical coherent tomography. It was found that, at 60°C, laser-induced modification of the quasi-crystalline packing of ordered collagen fibers occurred without the helix-coil molecular conformation transition. It was shown that, for uniaxial tension of ligaments, laser irradiation caused serious distortions in the structure of collagen and increased the fraction of macromolecules in the random coil state. It was assumed that the thermomechanical effect of laser treatment during laser heating played an important role.

Effect of Venous Wall Immobilization on the Thermal Degradation of Collagen

The results from a comparative study of the thermal denaturation of collagen in the venous walls of reference samples and samples with varicose disease are presented. Changes in the organization of collagen network of the tissue matrix are detected via thermal analysis and multiphoton microscopy with recording of the second harmonic generation (SHG). It is established that the collagen network of venous walls degrades in varicose disease. It is shown that the disordering of the tertiary structure of collagen molecules is reflected in a 40% drop in the enthalpy of protein denaturation compared to reference (ΔHD = 12.4 ± 4.9 J/g dry residue). The disorganization of fiber structures is recorded on SHG images. It is shown that upon the hydrothermal heating of sequestered samples of venous walls, the complete degradation of the tissue network occurs at 75°C. However, it is noted that upon the mechanical immobilization of samples of both types, the stability of collagen increases and complete denaturation is observed at temperatures above 84°C. It is suggested that the number of available conformations of polypeptide chains in the random coil state falls under tension, lowering ΔSD and raising the temperature of the denaturation of protein.

Effect of supramolecular organization of a cartilaginous tissue on thermal stability of collagen II

The thermal stability of collagen II in various cartilaginous tissues was studied. It was found that heating a tissue of nucleus pulposus results in collagen II melting within a temperature range of 60–70°C; an intact tissue of hyaline cartilage (of nasal septum and cartilage endplates) is a thermally stable system, where collagen II is not denatured completely up to 100°C. It was found that partial destruction of glycosaminoglycans in hyaline cartilage leads to an increase in the degree of denaturation of collagen II upon heating, although a significant fraction remains unchanged. It was shown that electrostatic interactions of proteoglycans and collagen only slightly affect the thermal stability of collagen II in the tissues. Evidently, proteoglycan aggregates play a key role: they create topological hindrances for moving polypeptide chains, thereby reducing the configurational entropy of collagen macromolecules in the state of a random coil.

Changes in the physicochemical characteristics of rabbit sclera upon scleral reinforcement

Biochemical analysis and differential scanning calorimetry demonstrated that the connective tissue (capsule) formed around a reinforcing scleroplastic implant is similar to intact sclera, its main component being type I collagen organized in perfect fibrils with cross-linking sufficient for normal thermomechanical properties. DSC also revealed a fraction of collagen with heat-labile ‘immature’ cross-links around implants containing a stimulatory plant product Panaxel, which suggested high synthetic activity of fibroblasts.

Features of Laser-Induced Modification of Sclera and Cornea

Results are presented from studying collagen degradation in scleral and corneal tissues upon CW IR laser irradiation at a wavelength of 1.56 μm and powers of 1.4 and 7 W. The exposure time is such that the radiometrically determined maximum tissue temperature ranges from 55 to 70°C. It is shown that at a power of 1.4 W, laser-induced collagen denaturation in sclera and cornea starts at 55 and 60°C, respectively. When the power is increased to 7 W, the temperature of denaturation onset rises by 2°C. These findings are discussed from the viewpoint of the joint photothermal and photomechanical effects of laser irradiation.

Modifying the collagen framework of costal cartilage under the impact of UV and a flavin mononucleotide

Modifications of the matrix of the tissue of costal cartilage under the impact of UV (λ = 365 nm) and a flavin mononucleotide (FMN) is studied. The changes in the macroscopic properties of the tissue are detected by means of differential scanning calorimetry and under the conditions of uniaxial compression during mechanical testing. The endothermic effects of the denaturation of the collagen framework of the tissue and the Young’s modulus are determined. It is shown that the presence of a flavin mononucleotide in the interstitial fluid leads lowers the temperature of collagen denaturation by 2.5°С and doubles the Young’s modulus. It is found that the temperature of denaturation and the Young’s modulus grow gradually after treating the tissue with the UV radiation, and their values ultimately exceed by far the corresponding values for intact samples. It is concluded that the obtained data indicate the possibility of stabilizing the framework of the matrix of costal cartilage under the impact of UV radiation and a flavin mononucleotide.

Thermal and non-thermal effects of nonablative IR laser irradiation of the collagen of annulus fibrosus tissues

Collagen modifications in the tissues of an annulus fibrosus under moderate-intensity IR laser irradiation at various pulse durations and frequencies were studied. Changes at various levels of the ordered structural hierarchy of collagen in tissues were detected by differential scanning calorimetry, multiphoton microscopy with second harmonic generation, and cross-polarized optical coherence tomography. The obtained data confirm the photothermal and photomechanical effect of IR laser action on fibrous structures. Details of these effects (mechanical defect formation or protein denaturation) were determined, and the greatest tissue damage was demonstrated to be generated by their combined action.

Increase in scleral collagen stability during glycosylation with threose in vitro

A systematic study of changes in the physicochemical characteristics of scleral collagen in the course of glycosylation by threose, including their dependence on the time changes of transverse cross-linking, was performed. Glycosylation by threose leads to a significant increase in heat, proteolytic, and biomechanical stability of collagen in the scleral tissue and has been shown to be a useful approach for stabilizing scleral collagen. It was found that a fraction of collagen with a reduced denaturation temperature is, apparently, an intermediate in the reaction of glycosylation by threose. The most likely reason for its occurrence is the elongation of the side chains of amino acid residues of the protein in the early stages.

Laser-induced modification of collagen in nucleus pulpose gel

The thermal behavior of collagen in intact and IR laser irradiated nucleus pulpose tissues was studied. The enthalpy of denaturation of the intact tissue was determined (53.1 ± 1.2 J/g of collagen). The denaturation of collagen during laser heating at temperatures below the characteristic melting point occurred under the influence of the photomechanical component of laser irradiation.