rus
Issue #1/2019
S. V. Tarasenko, A.B. Shekhter, V. P. Minaev, E. A. Morozova, A. M. Gutorova, A. N. Zhuravlev
0.97 And 1.94 Mkm Wavelength Laser Radiation Effect On The Oral Mucous Membrane Regeneration In The Experiment
0.97 And 1.94 Mkm Wavelength Laser Radiation Effect On The Oral Mucous Membrane Regeneration In The Experiment
The article presents data on the peculiarities of the processes of regeneration of wounds of the oral mucous membrane of laboratory animals after exposure to laser radiation with a wavelength of 1.94 µm and 0.97 µm at various radiation powers. It is known that oral mucous membrane exposed to laser radiation is prone to coagulation necrosis and the subsequent inflammatory reaction of the tissue with destruction of the epithelium, mucous membrane and partially muscular tissue. It has been established that tissue regeneration and wound defect healing slows down with increasing power.
DOI: 10.22184/1993-7296.FRos.2019.13.1.108.116
DOI: 10.22184/1993-7296.FRos.2019.13.1.108.116
INTRODUCTION
The wide distribution of lasers in surgical dentistry is due to the following positive properties of laser radiation: tissue asepsis during dissection, reliable hemostasis and lymphostasis, pronounced anesthetic, ablastic effects, anti-inflammatory effect by reducing vascular wall permeability, stimulation of metabolic processes and regeneration processes, increased tissue oxygen content, which speeds up the regeneration and prevents the formation of coarse scars, reducing the likelihood of postoperative complications [1, 2, 5, 7–11]. However, there are only a few reports presenting the data of a comparative analysis of tissue regeneration using different laser systems [2]. Tissue regeneration is an adjustable, cascade and multicomponent morphofunctional process [3]. The reasonable choice of a low-impact alteration instrument for surgical intervention allows creating optimal conditions for the regeneration of tissues located in the intervention zone. Surgical laser technologies are being actively introduced into clinical practice, which, thanks to precise power control and other parameters, create a minimal zone of thermal damage. The prospect of optimizing the course of the wound process is a regulating effect on the balance between surgical alteration, stimulation of regeneration and control of inflammation [1].
Reparative regeneration is a genetically determined process; however, different tissues and organs differ significantly in the degree of scar development. The healing process of skin wounds is significantly different from the healing process of wounds of the oral mucous membrane. Establishing the reasons for this may lead to the development of new methods to control the course of reparative regeneration processes in various parts of the body, including the oral cavity [6, 12–14].
The emergence of domestic portable, reliable and cost-efficient LSP-"IRE-Polyus" devices based on fiber and semiconductor lasers that generate radiation with a wavelength of 1.94 and 0.97 microns, differently impacting on the soft tissues, expands the arsenal of doctors, gives them new opportunities. It should be borne in mind that the laser with a wavelength of 0.97 is a diode one, and the laser with a wavelength of 1.94 is a fiber one. The radiation of laser diodes with fiber output is radiation by means of special welded elements, which is reduced to a single fiber, from which it is fed through the connector to the working optical fiber. The developed technologies allow introducing into the device a portion of activated fiber with fiber analogs of mirrors that form a fiber laser. In this case, it becomes possible to obtain laser radiation with different wavelengths. In fact, such a device is a coil of optical fiber with laser diodes welded to it and, due to the properties of the fiber to retain light, it does not need to be adjusted and is not afraid of external mechanical effects up to the magnitude leading to the destruction of the fiber. No access of dust and moisture is possible inside the fiber [4].
Therefore, to implement the possibilities of using these lasers in the clinic of surgical dentistry, it is necessary to study the characteristics of the results of such an impact, determine the optimal operating modes for oral mucous membrane.
The purpose of the research is to experimentally study the features of reparative processes of the oral mucous membrane when laser radiation is altered by repetitively pulsed lasers with a wavelength of 0.97 µm and 1.94 µm of various powers.
MATERIALS AND RESEARCH METHODS
The experimental study was performed at the Department of Surgical Dentistry of the Institute of Dentistry of FSAEI of HE Sechenov First Moscow State Medical University of the Ministry of Health of Russia (Sechenov University). The study was single-center, prospective (the study groups were formed prior to the beginning of the experiment), randomized (random samples) and comparative.
The experiment involved 18 rabbits of the chinchilla breed, males weighing 3.5–4.0 kg, average weight 3.7 ± 0.2 kg, at the age of 6 months, kept in vivarium conditions, according to the rules of laboratory practice when conducting preclinical studies in the Russian Federation (GOST R50258–92, GOST 351000.3–96 and 51000.4–96).
Biopsy specimens of the oral mucous membrane were studied using pulsed-periodic lasers with wavelengths of 0.97 µm and 1.94 µm with a radiation power of 1.2 W, 1.5 W and 1.8 W.
Before surgery, the animals were weighed and the number of injected drugs was calculated. Premedication and anesthesia were carried out with the combination of Ronitar (3 mg / kg animal body weight) and Zoletil (5 mg / kg animal body weight), adequate for surgical intervention. The drugs were used once, intramuscularly. Under surgery conditions, in compliance with the aseptic and antiseptic rules, a defect on the cheek mucosa of the animals was formed with laser radiation with wavelengths of 1.94 and 0.97 µm in a pulse-periodic mode of operation with a pulse duration of 400 ms, a pause duration of 500 ms. The laser fiber was positioned at a distance of 1 mm from the mucous membrane surface, the exposure time was 5 seconds. The control was the mucous membrane of the right (intact) cheek.
All animals were divided into 6 study groups. The animals of the 1st group had the wound defect formed by the radiation of LSP-"IRE-Polus" (1.94 µm / 5 W)" laser with a pulse duration of 400 ms, a pause of 500 ms, and a radiation power of 1.2 W. The animals of the 2nd group had the wound defect formed by the same laser radiation, but with increased power up to 1.5 W. The animals of the 3rd group had the wound defect formed when the power increased to 1.8 W. In the 4th group, the wound defect in animals was formed by the radiation of "LSP – "IRE-Polyus (0.97 µm / 10 W)" laser with a pulse duration of 400 ms, 500 ms and an output power of 1.2 W. In animals of the 5th group, the wound defect was formed by the same laser radiation, but with a power of 1.5 W. In animals of the 6th group, during the formation of a wound defect, the power was increased to 1.8 W.
The animals were removed from the experiment by excessive administration of hexanal on the 3rd, 7th, 14th day after the operation. Gross specimens of the mucous membrane were sent for histological examination.
RESULTS AND DISCUSSION
It was found that when the mucous membrane was exposed to laser radiation with a wavelength of 1.94 µm, 1.2 W for 3 days, all animals in the affected area have a wound defect with a destroyed epithelium (Fig. 1). The surface of the defect is covered with fibrin, neutrophilic infiltration is noted with an admixture of macrophages and lymphocytes. The vessels are full-blooded, there are single diapedemic hemorrhages. A fibrinous-leukocyte layer is formed on the surface of the wound, and under it a partially necrotic muscular membrane with neutrophilic-eosinophilic infiltration. When the mucous membrane was exposed to a laser with a wavelength of 0.97 µm, a power of 1.2 W for 3 days in the region of alteration, destruction of the epithelium, areas of coagulation necrosis in the mucosa and muscle tissue, but to a lesser extent, are also observed (Fig. 2).
When the mucous membrane was exposed to a laser with a wavelength of 1.94 µm, a power of 1.5 W, for 3 days, the epithelium is absent in the area of operation, the center of coagulation necrosis of muscle tissue is detected (Fig. 3). The stroma between necrotic muscle fibers is infiltrated with neutrophils and eosinophils, the vessels are dilated and full-blooded. When exposed to a laser with a wavelength of 0.97 µm and a power of 1.5 W, the epithelium undergoes destruction in the area of laser radiation, necrotic and inflammatory changes are less pronounced than in animals exposed to a laser with a wavelength of 1.94 µm (Fig. 4).
On day 3 after alteration of the mucous membrane by laser radiation with a wavelength of 1.94 µm and a power of 1.8 W, the size of the coagulative necrosis of muscle tissue is larger in size than with a laser power of 1.5 W. A vascular thrombosis, neutrophilic infiltration, with an admixture of lymphocytes and macrophages and the beginning proliferation of fibroblasts (Fig. 5) are noted around the center of necrosis. With an increase in laser power of 0.97 µm to 1.8 W in all animals, large areas of muscle necrosis with pronounced neutrophilic infiltration are observed in the alteration area (Fig. 6).
On day 7, using a 1.94 µm laser, with a power of 1.2 W, the defect is epithelialized only from the edges, neutrophilic eosinophilic infiltration is maintained in the fibrous-scar tissue. When using a laser with a wavelength of 0.97 µm and a power of 1.2 W for 7 days, unlike the group of animals that used a laser with a wavelength of 1.94 µm, all animals show epithelialization in the area of impact, the epithelium is thicker than in the intact areas (Fig. 7). When using a laser with a wavelength of 1.94 µm and a power of 1.2 W, a similar picture was observed on day 14, when the wound defect was completely epithelized (Fig. 8).
When the mucous membrane was exposed to a laser with a wavelength of 1.94 µm, a power of 1.5 W for 7 days, the area of necrosis is reduced and is replaced by granulation tissue consisting of cords of proliferating fibroblasts, collagen fibers and numerous vessels. Moderate neutrophilic-eosinophilic infiltration is maintained. When the laser power is 1.5 W with a wavelength of 0.97 µm on day 7, the defects are epithelized in all animals, fibrous-scar tissue is formed under the epithelium.
On day 7, when using a laser with a wavelength of 1.94 µm, a power of 1.8 W, foci of coagulation necrosis of the underlying muscles are maintained. The underlying muscle plate is almost completely replaced by scar-fibrous tissue, partially covered with regenerating epithelium. Fibrous tissue consists of collagen fibers and fibroblasts.
On day 14, with a laser exposure power of 1.94 µm 1.5 W, all animals have complete epithelialization of the wound surface, fibrous-scar tissue is located under it, and inflammatory infiltration is maintained.
With a laser wavelength of 1.94 µm a power of 1.8 W on day 14 epithelization of the defect is completed. Under the epithelium, there is a large field of scar tissue with inflammatory infiltration.
On day 14, when the mucous membrane was exposed to a laser with a wavelength of 0.97 µm, a power of 1.2 W, the epithelium is mature and differentiated. When using the radiation power of a 1.5 W laser with a wavelength of 0.97 µm all animals show a layer of connective tissue under the epithelial defect with moderate lymph macrophage infiltration, proliferation of fibroblasts and longitudinal arrangement of collagen fibers. With an increase in power up to 1.8 W, the laser defects in all animals are epithelialized, the epithelium is mature and well differentiated. Under the epithelium, scar tissue without noticeable inflammatory infiltration, resorption of muscle fibers is clearly visible.
In order to establish differences in the dynamics of the wound process of the soft tissues of the oral cavity, the depth of thermal damage to the tissues and the length of the zone of coagulation necrosis were studied. It was established that the average depth of coagulation necrosis on day 3 when exposing to a laser with a wavelength of 0.97 µm varied from 623.5 ± 79.3 µm to 995.7 ± 55.7 µm and power from 1.2 to 1.8 W, when using a laser with a wavelength of 1.94 µm and a power of 1.2 to 1.8 W, the average depth of coagulation necrosis on day 3 was from 645.3 ± 28.9 µm to 1035 ± 80.2 µm. On day 3, the smallest average depth of coagulation necrosis was observed with a power of 1.2 W by a laser with a wavelength of 0.97 µm – 623.5 ± 79.3 µm, the highest one with an impact power of 1.8 W by a laser with a wavelength of 1.94 µm – 1035 ± 80.2 microns. When the mucous membrane of the oral cavity of a rabbit was exposed to IRE-Polus (0.97 micron / 10 W) laser with a power of 1.5 W and 1.8 W compared to IRE-Polyus (1.94 micron / 5 W) laser, reliable differences in the depth of coagulation necrosis of the wound on day 3 (766.3 ± 67.8 µm and 851.8 ± 41.6 µm) and (995.7 ± 55.7 µm and 1035 ± 80.2 µm), respectively, with radiation power 1, 2 W differences were not significant (623.5 ± 79.3 µm and 645.33 ± 28.9 µm) (Table).
From the data in Table 1, it follows that the depth of coagulation necrosis varied, depending on the laser power, both when exposed to a laser λ = 0.97 µm, and when using a laser λ = 1.94 µm.
CONCLUSION
Thus, after laser alteration of the mucous membrane of experimental animals, tissue regeneration occurs. The effect of laser radiation on the mucous membrane of the mouth causes coagulation necrosis and the subsequent inflammatory tissue reaction with destruction of the epithelium, mucous membrane and partly muscle tissue. The intensity of these processes is directly proportional to the power of the laser radiation. Tissue regeneration and wound defect healing slows down as power increases. Laser radiation with a wavelength of 0.97 µm causes a change in tissue of lower intensity than radiation with a wavelength of 1.94 µm. The speed of the regeneration processes is also higher.
The wide distribution of lasers in surgical dentistry is due to the following positive properties of laser radiation: tissue asepsis during dissection, reliable hemostasis and lymphostasis, pronounced anesthetic, ablastic effects, anti-inflammatory effect by reducing vascular wall permeability, stimulation of metabolic processes and regeneration processes, increased tissue oxygen content, which speeds up the regeneration and prevents the formation of coarse scars, reducing the likelihood of postoperative complications [1, 2, 5, 7–11]. However, there are only a few reports presenting the data of a comparative analysis of tissue regeneration using different laser systems [2]. Tissue regeneration is an adjustable, cascade and multicomponent morphofunctional process [3]. The reasonable choice of a low-impact alteration instrument for surgical intervention allows creating optimal conditions for the regeneration of tissues located in the intervention zone. Surgical laser technologies are being actively introduced into clinical practice, which, thanks to precise power control and other parameters, create a minimal zone of thermal damage. The prospect of optimizing the course of the wound process is a regulating effect on the balance between surgical alteration, stimulation of regeneration and control of inflammation [1].
Reparative regeneration is a genetically determined process; however, different tissues and organs differ significantly in the degree of scar development. The healing process of skin wounds is significantly different from the healing process of wounds of the oral mucous membrane. Establishing the reasons for this may lead to the development of new methods to control the course of reparative regeneration processes in various parts of the body, including the oral cavity [6, 12–14].
The emergence of domestic portable, reliable and cost-efficient LSP-"IRE-Polyus" devices based on fiber and semiconductor lasers that generate radiation with a wavelength of 1.94 and 0.97 microns, differently impacting on the soft tissues, expands the arsenal of doctors, gives them new opportunities. It should be borne in mind that the laser with a wavelength of 0.97 is a diode one, and the laser with a wavelength of 1.94 is a fiber one. The radiation of laser diodes with fiber output is radiation by means of special welded elements, which is reduced to a single fiber, from which it is fed through the connector to the working optical fiber. The developed technologies allow introducing into the device a portion of activated fiber with fiber analogs of mirrors that form a fiber laser. In this case, it becomes possible to obtain laser radiation with different wavelengths. In fact, such a device is a coil of optical fiber with laser diodes welded to it and, due to the properties of the fiber to retain light, it does not need to be adjusted and is not afraid of external mechanical effects up to the magnitude leading to the destruction of the fiber. No access of dust and moisture is possible inside the fiber [4].
Therefore, to implement the possibilities of using these lasers in the clinic of surgical dentistry, it is necessary to study the characteristics of the results of such an impact, determine the optimal operating modes for oral mucous membrane.
The purpose of the research is to experimentally study the features of reparative processes of the oral mucous membrane when laser radiation is altered by repetitively pulsed lasers with a wavelength of 0.97 µm and 1.94 µm of various powers.
MATERIALS AND RESEARCH METHODS
The experimental study was performed at the Department of Surgical Dentistry of the Institute of Dentistry of FSAEI of HE Sechenov First Moscow State Medical University of the Ministry of Health of Russia (Sechenov University). The study was single-center, prospective (the study groups were formed prior to the beginning of the experiment), randomized (random samples) and comparative.
The experiment involved 18 rabbits of the chinchilla breed, males weighing 3.5–4.0 kg, average weight 3.7 ± 0.2 kg, at the age of 6 months, kept in vivarium conditions, according to the rules of laboratory practice when conducting preclinical studies in the Russian Federation (GOST R50258–92, GOST 351000.3–96 and 51000.4–96).
Biopsy specimens of the oral mucous membrane were studied using pulsed-periodic lasers with wavelengths of 0.97 µm and 1.94 µm with a radiation power of 1.2 W, 1.5 W and 1.8 W.
Before surgery, the animals were weighed and the number of injected drugs was calculated. Premedication and anesthesia were carried out with the combination of Ronitar (3 mg / kg animal body weight) and Zoletil (5 mg / kg animal body weight), adequate for surgical intervention. The drugs were used once, intramuscularly. Under surgery conditions, in compliance with the aseptic and antiseptic rules, a defect on the cheek mucosa of the animals was formed with laser radiation with wavelengths of 1.94 and 0.97 µm in a pulse-periodic mode of operation with a pulse duration of 400 ms, a pause duration of 500 ms. The laser fiber was positioned at a distance of 1 mm from the mucous membrane surface, the exposure time was 5 seconds. The control was the mucous membrane of the right (intact) cheek.
All animals were divided into 6 study groups. The animals of the 1st group had the wound defect formed by the radiation of LSP-"IRE-Polus" (1.94 µm / 5 W)" laser with a pulse duration of 400 ms, a pause of 500 ms, and a radiation power of 1.2 W. The animals of the 2nd group had the wound defect formed by the same laser radiation, but with increased power up to 1.5 W. The animals of the 3rd group had the wound defect formed when the power increased to 1.8 W. In the 4th group, the wound defect in animals was formed by the radiation of "LSP – "IRE-Polyus (0.97 µm / 10 W)" laser with a pulse duration of 400 ms, 500 ms and an output power of 1.2 W. In animals of the 5th group, the wound defect was formed by the same laser radiation, but with a power of 1.5 W. In animals of the 6th group, during the formation of a wound defect, the power was increased to 1.8 W.
The animals were removed from the experiment by excessive administration of hexanal on the 3rd, 7th, 14th day after the operation. Gross specimens of the mucous membrane were sent for histological examination.
RESULTS AND DISCUSSION
It was found that when the mucous membrane was exposed to laser radiation with a wavelength of 1.94 µm, 1.2 W for 3 days, all animals in the affected area have a wound defect with a destroyed epithelium (Fig. 1). The surface of the defect is covered with fibrin, neutrophilic infiltration is noted with an admixture of macrophages and lymphocytes. The vessels are full-blooded, there are single diapedemic hemorrhages. A fibrinous-leukocyte layer is formed on the surface of the wound, and under it a partially necrotic muscular membrane with neutrophilic-eosinophilic infiltration. When the mucous membrane was exposed to a laser with a wavelength of 0.97 µm, a power of 1.2 W for 3 days in the region of alteration, destruction of the epithelium, areas of coagulation necrosis in the mucosa and muscle tissue, but to a lesser extent, are also observed (Fig. 2).
When the mucous membrane was exposed to a laser with a wavelength of 1.94 µm, a power of 1.5 W, for 3 days, the epithelium is absent in the area of operation, the center of coagulation necrosis of muscle tissue is detected (Fig. 3). The stroma between necrotic muscle fibers is infiltrated with neutrophils and eosinophils, the vessels are dilated and full-blooded. When exposed to a laser with a wavelength of 0.97 µm and a power of 1.5 W, the epithelium undergoes destruction in the area of laser radiation, necrotic and inflammatory changes are less pronounced than in animals exposed to a laser with a wavelength of 1.94 µm (Fig. 4).
On day 3 after alteration of the mucous membrane by laser radiation with a wavelength of 1.94 µm and a power of 1.8 W, the size of the coagulative necrosis of muscle tissue is larger in size than with a laser power of 1.5 W. A vascular thrombosis, neutrophilic infiltration, with an admixture of lymphocytes and macrophages and the beginning proliferation of fibroblasts (Fig. 5) are noted around the center of necrosis. With an increase in laser power of 0.97 µm to 1.8 W in all animals, large areas of muscle necrosis with pronounced neutrophilic infiltration are observed in the alteration area (Fig. 6).
On day 7, using a 1.94 µm laser, with a power of 1.2 W, the defect is epithelialized only from the edges, neutrophilic eosinophilic infiltration is maintained in the fibrous-scar tissue. When using a laser with a wavelength of 0.97 µm and a power of 1.2 W for 7 days, unlike the group of animals that used a laser with a wavelength of 1.94 µm, all animals show epithelialization in the area of impact, the epithelium is thicker than in the intact areas (Fig. 7). When using a laser with a wavelength of 1.94 µm and a power of 1.2 W, a similar picture was observed on day 14, when the wound defect was completely epithelized (Fig. 8).
When the mucous membrane was exposed to a laser with a wavelength of 1.94 µm, a power of 1.5 W for 7 days, the area of necrosis is reduced and is replaced by granulation tissue consisting of cords of proliferating fibroblasts, collagen fibers and numerous vessels. Moderate neutrophilic-eosinophilic infiltration is maintained. When the laser power is 1.5 W with a wavelength of 0.97 µm on day 7, the defects are epithelized in all animals, fibrous-scar tissue is formed under the epithelium.
On day 7, when using a laser with a wavelength of 1.94 µm, a power of 1.8 W, foci of coagulation necrosis of the underlying muscles are maintained. The underlying muscle plate is almost completely replaced by scar-fibrous tissue, partially covered with regenerating epithelium. Fibrous tissue consists of collagen fibers and fibroblasts.
On day 14, with a laser exposure power of 1.94 µm 1.5 W, all animals have complete epithelialization of the wound surface, fibrous-scar tissue is located under it, and inflammatory infiltration is maintained.
With a laser wavelength of 1.94 µm a power of 1.8 W on day 14 epithelization of the defect is completed. Under the epithelium, there is a large field of scar tissue with inflammatory infiltration.
On day 14, when the mucous membrane was exposed to a laser with a wavelength of 0.97 µm, a power of 1.2 W, the epithelium is mature and differentiated. When using the radiation power of a 1.5 W laser with a wavelength of 0.97 µm all animals show a layer of connective tissue under the epithelial defect with moderate lymph macrophage infiltration, proliferation of fibroblasts and longitudinal arrangement of collagen fibers. With an increase in power up to 1.8 W, the laser defects in all animals are epithelialized, the epithelium is mature and well differentiated. Under the epithelium, scar tissue without noticeable inflammatory infiltration, resorption of muscle fibers is clearly visible.
In order to establish differences in the dynamics of the wound process of the soft tissues of the oral cavity, the depth of thermal damage to the tissues and the length of the zone of coagulation necrosis were studied. It was established that the average depth of coagulation necrosis on day 3 when exposing to a laser with a wavelength of 0.97 µm varied from 623.5 ± 79.3 µm to 995.7 ± 55.7 µm and power from 1.2 to 1.8 W, when using a laser with a wavelength of 1.94 µm and a power of 1.2 to 1.8 W, the average depth of coagulation necrosis on day 3 was from 645.3 ± 28.9 µm to 1035 ± 80.2 µm. On day 3, the smallest average depth of coagulation necrosis was observed with a power of 1.2 W by a laser with a wavelength of 0.97 µm – 623.5 ± 79.3 µm, the highest one with an impact power of 1.8 W by a laser with a wavelength of 1.94 µm – 1035 ± 80.2 microns. When the mucous membrane of the oral cavity of a rabbit was exposed to IRE-Polus (0.97 micron / 10 W) laser with a power of 1.5 W and 1.8 W compared to IRE-Polyus (1.94 micron / 5 W) laser, reliable differences in the depth of coagulation necrosis of the wound on day 3 (766.3 ± 67.8 µm and 851.8 ± 41.6 µm) and (995.7 ± 55.7 µm and 1035 ± 80.2 µm), respectively, with radiation power 1, 2 W differences were not significant (623.5 ± 79.3 µm and 645.33 ± 28.9 µm) (Table).
From the data in Table 1, it follows that the depth of coagulation necrosis varied, depending on the laser power, both when exposed to a laser λ = 0.97 µm, and when using a laser λ = 1.94 µm.
CONCLUSION
Thus, after laser alteration of the mucous membrane of experimental animals, tissue regeneration occurs. The effect of laser radiation on the mucous membrane of the mouth causes coagulation necrosis and the subsequent inflammatory tissue reaction with destruction of the epithelium, mucous membrane and partly muscle tissue. The intensity of these processes is directly proportional to the power of the laser radiation. Tissue regeneration and wound defect healing slows down as power increases. Laser radiation with a wavelength of 0.97 µm causes a change in tissue of lower intensity than radiation with a wavelength of 1.94 µm. The speed of the regeneration processes is also higher.
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