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The aim of the study was to develop a diagnostic method for the quantitative determination of the main components of cartilage tissue of various types based on multivariate IR spectral analysis and verification of data using classical chemical analysis., Materials and Methods: Cartilages of the nasal septum, knee joint, rib, and nucleus pulposus of the intervertebral disc, as well as trypsinized and defective cartilage samples, were examined as samples. The IR spectra of the cartilage samples, as well as calibration mixtures of collagen and chondroitin sulfate, were obtained. The IR spectra were collected using the attenuated total reflectance techniques, and their processing was performed using the TQ Analyst software and the principal component regression calibration technique. Based on calibration dependence, the K sp coefficient was determined as the ratio of the mass fractions of collagen and chondroitin sulfate. Its value was compared with the value of K chem , equal to the ratio of the mass fractions of collagen and chondroitin sulfate, obtained using the classical chemical analysis of these substances., Results: The IR spectra of cartilage tissues are a superposition of the IR spectra of collagen and chondroitin sulfate and qualitatively reflect their composition. A change in the ratio between the relative intensities of the characteristic bands of compounds in the IR spectrum is obvious only with a significant change in the content of these compounds in cartilage. This change occurs after trypsinization, when K sp increases from 0.88±0.05 ( K chem ~0.8) to 4.55. The use of a calibration model with a complete analysis of the cartilage IR spectrum made it possible to determine the difference in the ratio of the main components in the matrix of different samples in the absence of obvious changes in the IR spectra. Thus, a statistically significant decrease in the content of chondroitin sulfate in degraded articular cartilage ( K sp =4.4±1.8; K chem ~5.5) was shown compared with intact samples ( K sp =2.8±1.1; K chem ~2.6)., Conclusion: IR spectrometric express analysis of cartilage tissue employing the principal component regression method allows a correct determination of the ratio of the main components in the cartilage matrix, those of collagen and glycosaminoglycans. The proposed technique includes one measurement, does not require prolonged and laborious sample preparation, does not require long, multi-stage and laborious chemical manipulations to determine each of the components, and makes it possible to determine the features and changes in the composition for a large set of samples of cartilage tissue of different types. In future, this approach can be used for non-invasive diagnostics of cartilage tissue., Competing Interests: No conflicts of interest are declared.

The aim of the study was to design a construct based on a nasal septal cartilage plate providing required cell differentiation in different layers to replace a deep osteochondral defect and develop an algorithm of chemical and physical effect sequence to create non-immunogenic two-layer porous structure with requisite elasto-mechanical properties., Materials and Methods: The plates derived from porcine nasal septal hyaline cartilage covered by perichondrium were multi-stage treated including freezing, equilibrating in a hypotonic saline solution (type I specimens); trypsinization, point IR-laser effect, re-trypsinization (type II specimens); a stabilizing effect of crosslinking agents - glyceraldehyde/ribose in an acidic medium - washing (type III specimens).For all type specimens:there were established stability parameters (collagen denaturation temperature using a thermal analysis; and Young's modulus using a mechanical analysis);there were determined morphological characteristics using light and polarization microscopy with classical staining and nonlinear optical microscopy in second-harmonic generation mode., Results: Thermal, mechanical, and morphological properties in type I specimens slightly differed from those of the initial nasoseptal system. A considerable part of cells had destroyed membranes.In type II specimens, thermal stability of collagen frame was significantly lower; Young's modulus decreased more than fourfold compared to type I specimens. Collagen structure of hyaline cartilage appeared to be disarranged, although the morphological differences of the hyaline part and perichondrium preserved. The construct matrix was almost completely decellularized. Successive exposure to laser radiation and trypsin resulted in the formation of partial holes in the matrix, ~100 μm in diameter.In type III specimens, both the thermal stability of the collagen frame and Young's modulus (E) increased. Glyceraldehyde was more effective than ribose, E having reached the value typical for intact hyaline cartilage. Collagen fibers in type III specimens were thicker than in type I and II specimens. The morphological differences of the hyaline part and perichondrium and partial holes were preserved., Conclusion: Due to sequential treatment by salts, trypsin, IR-laser radiation, and nontoxic crosslinking agents, nasal septal cartilage plate forms porous acellular construction consisting of two layers formed by type I (from perichondrium) and type II (from hyaline part) collagen fibers. In the present construction, stability, mechanical properties, and size of the partial holes can be assigned for cell colonization. It enables to use the construction to replace articular cartilage defects., Competing Interests: Conflicts of interest. The authors have no conflicts of interest related to the present study.

Endovenous laser ablation is an effective and minimally invasive alternative to surgical removal of incompetent veins. However, many controversies concerning optimal laser parameters usage in this procedure still remain. The purpose of this experimental study was to assess the adequate parameters required for vein wall destruction and to evaluate the role of fiber pullback velocity on vessel wall degradation. Varicose vein segments were treated with 1470-nm diode laser 3 to 9.5 W in power. The fiber moved through the vein at a velocity of 0.7 or 1.5 mm/s; the applied linear endovenous energy density (LEED) was 40-95 J/cm. The temperature of the vein surface in the course of laser irradiation was controlled by IR thermography. The intact collagen in treated vein specimens was studied by differential scanning calorimetry. The increase in the surface temperature with applied energy was found to be about three times slower for the pullback velocity of 0.7 mm/s than that of 1.5 mm/s. The collagen in the tissue was totally denatured in the case of the surface temperature of about 91 °C. The critical values of LEED ensured complete degradation of vein wall were of 53 and 71.5 J/cm for velocities of 1.5 and 0.7 mm/s, respectively. Our experimental study supports the conception that it is laser power and pullback velocity rather than LEED value that determine the temperature as well the collagen framework degradation and therefore the thermal response of procedure.

Objective: to perform a comparative morphological study of biocompatibility, biodegradation, and tissue response to implantation of collagen matrices (scaffolds) for tissue engineering in urology and other areas of medicine., Material and Methods: Nine matrix types, such as porous materials reconstructed from collagen solution; a collagen sponge-vicryl mesh composite; decellularized and freeze-dried bovine, equine, and fish dermis; small intestinal submucosa, decellularized bovine dura mater; and decellularized human femoral artery, were implanted subcutaneously in 225 rats. The tissues at the implantation site were investigated for a period of 5 to 90 days. Classical histology and nonlinear optical microscopy (NLOM) were applied., Results: The investigations showed no rejection of all the collagen materials. The period of matrix bioresorption varied from 10 days for collagen sponges to 2 months for decellularized and freeze-dried vessels and vicryl meshes. Collagen was prone to macrophage resorption and enzymatic lysis, being replaced by granulation tissue and then fibrous tissue, followed by its involution. NLOM allowed the investigators to study the number, density, interposition, and spatial organization of collagen structures in the matrices and adjacent tissues, and their change over time during implantation., Conclusion: The performed investigation could recommend three matrices: hybrid collagen/vicryl composite; decellularized bovine dermis; and decellularized porcine small intestinal submucosa, which are most adequate for tissue engineering in urology. These and other collagen matrices may be used in different areas of regenerative medicine.

Background and Objective: To examine the possibilities of laser thermoplasty of whole costal cartilages for correction the human congenital chest wall deformities., Study Design/materials and Methods: Ex vivo the samples of porcine costal cartilages were heated with lasers of differing wavelengths, including a 0.97-μm diode laser, a 1.56-μm erbium-doped quartz fiber laser, and a 1.68-μm fiber Raman laser. The dynamics of the temperature fields and the degradation of collagen in the laser-affected regions of samples were determined by using, respectively, thermometry, trypsin treatment, and light microscopy. Ex vivo the whole mechanically deformed costal cartilages of pigs were treated by laser radiation with wavelength 1.68-μm. The changes of cartilage shape were recorded at certain intervals over a 24-hour period by photographing them in a fixed position with a digital camera., Results: Treatment of costal cartilage samples from 5 to 11 mm in thickness by laser radiation with 0.97, 1.56, and 1.68 μm wavelengths showed that the 1.68-μm radiation could produce the necessary nonuniform bulk heating of the exposed sample. The altered shape of costal cartilage proved to remain stable after treatment when the laser irradiation settings used provided for the heating of a broad region within the tissue to temperatures about 80°C., Conclusion: This study demonstrates the possibilities of laser thermoplasty of whole costal cartilages for treatment of human congenital chest wall deformities. The development of novel approaches based on laser cartilage engineering techniques will enable to treat the human congenital chest wall deformities., (© 2014 Wiley Periodicals, Inc.)

The effects of non-ablative infrared (IR) laser treatment of collagenous tissue have been commonly interpreted in terms of collagen denaturation spread over the laser-heated tissue area. In this work, the existing model is refined to account for the recently reported laser-treated tissue heterogeneity and complex collagen degradation pattern using comprehensive optical imaging and calorimetry toolkits. Patella ligament (PL) provided a simple model of type I collagen tissue containing its full structural content from triple-helix molecules to gross architecture. PL ex vivo was subjected to IR laser treatments (laser spot, 1.6 mm) of equal dose, where the tissue temperature reached the collagen denaturation temperature of 60 ± 2°C at the laser spot epicenterin the first regime, and was limited to 67 ± 2°C in the second regime. The collagen network was analyzed versus distance from the epicenter. Experimental characterization of the collagenous tissue at all structural levels included cross-polarization optical coherence tomography, nonlinear optical microscopy, light microscopy/histology, and differential scanning calorimetry. Regressive rearrangement of the PL collagen network was found to spread well outside the laser spot epicenter (>2 mm) and was accompanied by multilevel hierarchical reorganization of collagen. Four zones of distinct optical and morphological properties were identified, all elliptical in shape, and elongated in the direction perpendicular to the PL long axis. Although the collagen transformation into a random-coil molecular structure was occasionally observed, it was mechanical integrity of the supramolecular structures that was primarily compromised. We found that the structural rearrangement of the collagen network related primarily to the heat-induced thermo-mechanical effects rather than molecular unfolding. The current body of evidence supports the notion that the supramolecular collagen structure suffered degradation of various degrees, which gave rise to the observed zonal character of the laser-treated lesion.

The purpose of this study was to characterize essential changes in the structure of annulus fibrosus (AF) after hydrothermal and infrared (IR) laser treatment and to correlate these results with alterations in tissue state. Polarization-sensitive optical coherence tomography imaging was used to measure collagen birefringence in AF. Differential scanning calorimetry was used as a complementary technique, providing detailed information on thermodynamic processes in the tissue. Birefringence, peak of the denaturation endotherm, and the enthalpy of denaturation (DeltaHm) were determined before and after hydrothermal heat treatment (85 degrees C for 15 min) and non-ablative Er:glass fiber laser exposures on AF in the whole disk (vertebrae-disk-vertebrae complex). Our data have demonstrated quantitative differences between results of laser and hydrothermal heating. Birefringence did not disappear and DeltaHm did not change after treatment in the water bath, but loss of birefringence and a decrease in the enthalpy did occur after laser exposure. These results could be explained by the photomechanical effect of laser irradiation. We suggest that thermo-mechanical stress played a dominant role in the disruption of the collagen network of AF under non-homogeneous laser heating.