Laser Physics, Vol. 15, No. 12, 2005, pp. 1660–1663.

LASER METHODS IN CHEMISTRY, BIOLOGY, AND MEDICINE

Original Text Copyright © 2005 by Astro, Ltd. Copyright © 2005 by MAIK “Nauka /Interperiodica” (Russia).

Changes in Proteoglycan Subsystem of Cartilage as a Result of Infrared-Laser Treatment E. S. Yansen1, *, N. Yu. Ignatieva1, S. V. Averkiev1, A. B. Shekhter2, V. V. Lunin1, and E. N. Sobol3 1 Laboratory

of Catalysis and Gas Electrochemistry, Department of Chemistry, Moscow State University, Vorob’evy gory, Moscow, 119899 Russia 2 Sechenov Moscow Medical Academy, Moscow, 119435 Russia 3 Institute of Laser and Information Technologies, Russian Academy of Sciences, Pionerskaya ul. 2, Troitsk, Moscow oblast, Russia *e-mail: [email protected] Received June 18, 2005

Abstract—Cartilage was exposed to infrared laser treatment of moderate intensity with infrared radiometric temperature control. The time dependences of proteoglycan residue release from cartilage were obtained to the buffer solution and to the solution of proteolytic enzyme tripsin. Histochemical analysis of changes in the cartilage proteoglycan subsystem as a result of infrared laser treatment and tripsin treatment was carried out. It was shown that infrared laser irradiation leads to particular destruction of proteoglycans and proteoglycan aggregates in cartilage and decreases the stability of the proteoglycan subsystem to tripsin.

1. INTRODUCTION Nowadays, infrared laser radiation is widely used in medicine and plastic surgery. In particular, it is applied in the reshaping of nasal septal cartilage. The main phenomenon underlying this application is the stress relaxation in cartilage [1]. Unfortunately, the mechanism of stress relaxation is unknown. Cartilage tissue consists of a number of components with strong interactions between them. The three main components of cartilage are water (75% of tissue wet weight), collagen (15–17% of tissue wet weight), and proteoglycans (6–10% of tissue wet weight). Proteoglycan consists of core protein with glycosaminoglycans attached to it. Glycosaminoglycans are sulfated and acetylated–amidated polysaccharides. In cartilage, proteoglycans are assembled into aggregates bound to a long chain of hyaluronic acid with the use of the G1 domain. This binding is stabilized with the small link protein (M = 45 kDa). There are several types of glycosaminoglycans. The main glycosaminoglycan of cartilage is chondroitin sulfate. This work was performed to study the changes of the proteoglycan subsystem of cartilage as a result of infrared laser irradiation. The changes of the cartilage proteoglycan subsystem were determined by changes in its stability to proteolytic enzyme tripsin. Tripsin cleaves the bonds between arginine and lysine, thus digesting core and link proteins of proteoglycan aggregates. At the same time, it does not affect triple helixes of collagen. The changes in the proteoglycan subsystem can also be determined by the histochemical method. Proteoglycans are negatively charged molecules and can be eas-

ily stained with cationic dyes. One of the commonly used dyes is toluidine blue. 2. MATERIALS AND METHODS Nasal septal cartilages of calves less than one year old were used. Samples with size 20 × 8 × 1 mm (surface area ~160 mm2) were prepared prior to the experiment. Then, part of them was irradiated, and another part was left nonirradiated to use as controls. Cartilage for biochemical investigation was dried, weighed, and stored at –20°C. This method of storage does not affect the native structure and properties of the cartilage matrix [2]. They can be easily restored by simple swilling in a physiological solution. Cartilage for histochemical analysis were fixed in 96% ethanol. Cartilage was irradiated using an infrared laser (Er fiber laser, IRE-Polus, Russia, λ = 1.56 µm). Laser power was varied in the interval of 3–3.5 W. The distance from the laser fiber to the sample of cartilage was 20 mm, and the diameter of the laser spot was ~5 mm. For further investigations, the region of the laser spot was excised. Cartilage surface temperature during irradiation was controlled with an Irtis infrared camera. The heating–cooling curve was obtained for every sample. An example is presented in Fig. 1. It was heated to temperatures of 50, 60, and 70°C (we will call them sample 50°C, sample 60°C, and sample 70°C). The heating time to the same temperature was constant. There were two parts of the experiment. In part one, the proteoglycan subsystem changes as a result of infrared laser treatment. Irradiated and nonirradiated cartilage were fixed in 96% ethanol, stained with paraffin, cut on microtome,

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incubation buffer (concentration 1 mg/ml). The time dependences of proteoglycan residue release from tissue to tripsin solution were obtained. Then, histological analysis of tripsin-treated samples was carried out. The quantity of proteoglycan residues in solution was determined by color reaction with dimethylmethylene blue. This method is described in [3]. Color reagent was prepared by dissolving 16 mg dimethylmethylene blue (Basic Blue 24, Sigma, Germany) in 1 l of water, containing 3.04 g glycine (Helicon, Russia), 2.37 g sodium chloride, and 10 ml 0.95 M hydrochloric acid (pH = 3.0).

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Fig. 1. (a) Dynamics of surface temperature change during the infrared laser heating of cartilage: (1) heating to 50°C, (2) heating to 60°C, (3) heating to 70°C. (b) Temperaturefield distribution during the infrared-laser heating of cartilage: (1) laser optical fiber, (2) cartilage sample, (3) laser spot.

and stained with toluidine blue for histochemical evaluation. The other part of irradiated and nonirradiated cartilage was placed into an incubation buffer. The time dependences of proteoglycan residue release to the buffer were obtained. The incubation buffer was 25 mM EDTA (Quality Biological, United States), 5 mg/ml of streptomycin (Sigma, United States), and 5 units/ml of penicillin (Sigma, United States) in a physiological solution. In part two, the proteoglycan subsystem stability to tripsin was investigated. Irradiated and nonirradiated cartilage were placed into tripsin (Flow Laboratories, Scotland) solution in LASER PHYSICS

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3. RESULTS AND DISCUSSION For part one, the proteoglycan subsystem changes as a result of infrared laser treatment. Figure 2 shows the time dependences of proteoglycan residue release from irradiated and nonirradiated cartilage to the buffer. The rate of release is higher in the case of samples 50°C and samples 60°C than in the control. This shows that destruction of proteoglycan aggregates takes place during infrared laser heating of cartilage. However, this destruction is not complete because, even from the sample 60°C, only 23% of total amount of proteoglycans was released. Histological analysis confirms that the infrared laser irradiation leads to the partial disaggregating of proteoglycan aggregates in cartilage (Fig. 3). Metachromasia is smaller in the irradiated sample of cartilage than in the control. We suppose that the link protein denatures first during laser heating. It is known that it does not possess thermal stability, whereas the half-life period of the G1 domain is 115 min under the heating temperature 80°C [4].

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Fig. 3. Histological pictures for the irradiated and nonirradiated cartilage. (a) Control sample of cartilage. Clearly defined metachromasia of cartilage matrix. Toluidine blue staining. Magnification, × 400. (b) Irradiated sample of cartilage. Maximal heating temperature, 70°C. Partial loss of metachromasia. Toluidine blue staining. Magnification, ×400.

Fig. 4. Histological pictures for the irradiated and nonirradiated cartilage treated with tripsin solution (18 h). (a) Control sample of cartilage. Moderate matrix metachromasia. Toluidine blue staining. Magnification, × 400. (b) Irradiated sample of cartilage. Maximal heating temperature, 70°C. Strong decrease of color intensity and metachromasia. Toluidine blue staining. Magnification, × 400.

For part two, the stability of the proteoglycan subsystem to tripsin was investigated.

less for the sample 60°C, and practically disappears for the sample 70°C. The period of induction can be explained by time, which is necessary for tripsin to destroy the native matrix structure of the control sample. Infrared-laser treatment can, in turn, lead to microchannel formation [5]. These channels can simplify enzyme transport into the tissue and proteoglycan residue transport out of the tissue for the first period of time. The drop of the release rate from sample 60°C to sample 70°C can be related to diffusion limitations. They can appear when heating cartilage to temperatures higher than 60°C. It was shown that collagen-fiber collapse takes place during laser heating [6]. The sorption properties of irradiated and dried cartilages are notably smaller than those of native [7]. This may be related to the decrease in the free internal volume of the tissue as a result of infrared-laser treatment. The bend divides the curve into two parts. We can distinguish two stages of proteoglycan residue release separated in time. The first is the release of proteoglycans destroyed during infrared-laser treatment, and the second is the release of proteoglycans destroyed by

Figure 4 shows histological pictures of cartilage treated by tripsin solution for 18 h. The loss of metachromasia is seen in the irradiated and nonirradiated sample, but it is obviously greater in the irradiated sample. Laser heating of cartilage to temperatures near and higher than 50°C affects the native structure of cartilage matrix, thus reducing its stability to tripsin. In Fig. 5, the time dependences of proteoglycan residue release under the action of tripsin are presented. Let us note the main peculiarities of enzymatic digestion of cartilage proteoglycan subsystem. (1) There is an induction period on the time dependences of controls, and there is no such period for the irradiated samples. (2) The maximum release rate is observed for the sample 60°C. The same was observed in the first part of the experiment. (3) There is a curve bending over for the irradiated samples. It is most obviously seen for the sample 50°C,

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Fig. 5. Time dependences of proteoglycan residue release from cartilage to the tripsin solution: (1) sample 50°C, (2) sample 60°C, (3) sample 70°C.

enzyme in solution. Smoothing of this bend for samples 60 and 70°C shows that the amount of proteoglycans destroyed during irradiation increases with the irradiation temperature. So, for sample 70°C, we can see predominantly the release of proteoglycans destroyed during heating.

We have studied the changes of the proteoglycan subsystem of cartilage as a result of infrared-laser treatment. It was shown that infrared-laser heating leads to the partial destruction of proteoglycans and proteoglycan aggregates in cartilage. ACKNOWLEDGMENTS This research was supported by the Russian Foundation for Basic Research (grant nos. 02-02-16246a and 02-04-16743) and the CRDF (grant no. RP2-5003-MO03). Vol. 15

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1. E. N. Sobol, A. P. Sviridov, A. I. Omelchenko, and V. N. Bagratashvili, in Biotechnology and Genetic Engineering Reviews, Ed. by S. E. Harding (Intercept Ltd., England, 2000), Vol. 17, p. 553. 2. K. M. Meek and A. J. Quantock, Prog. Retin Eye Res. 20 (1), 95 (2001).

4. CONCLUSIONS

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3. R. W. Farndale, D. J. Buttle, and A. J. Barrett, Biochim. Biophys. Acta 883, 173 (1986). 4. T. E. Hardingham, “Cartilage: Aggrecan–Link Protein– Hyaluronan Aggregates,” in Science of Hyaluronan Today. Glycoforum (1998); http://www.glycoforum.gr.jp. 5. E. N. Sobol, A. I. Omel’chenko, M. Mertig, and W. Pompe, Lasers Med. Sci. 15, 15 (2000). 6. K. Hayashi, G. Thabit III, J. J. Bogdanske, et al., Arthroscopy 12 (4), 474 (1996). 7. S. V. Averkiev, N. Y. Ignatieva, V. V. Lunin, and E. N. Sobol, Biofizika 48 (3), 505 (2003).