Combination effect of laser diode for photodynamic therapy with doxycycline on a wistar rat model of periodontitis

Periodontitis is chronic inflammatory disease characterized by progressive damage on the tooth-supporting tissue structure [1]. The damage process depends on the host immune response to the local aggregation of bacteria that causes a variety of biological events that play a role in the achievement of host homeostasis [2]. Damage includes gingival inflammation, erosion of connective tissue attachment, and resorption of the alveolar bone [3]. The resorption in the alveolar bone is mediated by osteoclasts and may cause degradation in the alveolar bone [4]. If such process occurs, it will lead to the disconnection of periodontal ligaments which attached to the alveolar bone, resulting teeth removal from their attachment [5]. Thus, without immediate treatment, this defect will cause more than one tooth loss and even lead to systemic disease.

Periodontitis is known as a disease that is difficult to prevent and cure. Treatment and prevention methods that have been used so far only aim to eliminate bacterial colonies causing infections in the supragingival and subgingival areas [6] so that the tissue can be repaired and regenerated [1, 7]. The mechanical process, either by brushing teeth or the Scale Root Planning (SRP) method, are not effective in removing plaque from the teeth because the probe could not reach a tooth area of > 5 mm [8]. Hence, the use of antibiotics is an effective method for bacterial colonies removal, as stated by the World Health Organization in 2014 [9]. However, long-term application may increase the resistance of bacteria to antibiotics [10]. One alternative treatment modality that would help overcome this problem is photodynamic therapy.

Photodynamic therapy is a non-invasive therapeutic method that utilizes photons of light energy for medical applications [11, 12]. This therapy has been used to treat chronic inflammation and is believed to be an alternative for treating antibiotic-resistant bacteria [9, 13]. The success of the therapy depends on availability of three main components, namely light, photosensitizer, and oxygen [14]. These three components play a vital role in the process of light absorption by photosensitive agents which will activate chemical reactions to produce various reactive oxygen species (ROS) [15, 16]. The various types of ROS produced also play an important role in bacterial photoinactivation since the resulting oxidative effect can inhibit bacterial activity [17] and therefore cause bacterial’s cell death.

Naturally, bacteria produce light sensitive endogenous porphyrin compounds as photosensitizer molecules. Porphyrins produced by bacteria have a very strong absorption in the visible light spectrum, also known as soret peaks located on 405 nm wavelength. The endogenous porphyrin in Porphyromonas gingivalis is dimericiron protoporphyrin IX (μ-oxobishaem). The choice of photosensitizer for photodynamic therapy in dentistry is highly dependent on the light source used. Laser diode produces light with a range of visible and infrared light spectrum, so it can be applied for periodontitis therapy. Diode lasers are bactericidal and able to accelerate the process of coagulation and regeneration of periodontal tissues [18].

Several studies on photodynamic therapy for the bacteria Porphyromonas gingivalis and other periopathogenic bacteria have been reported. The use of blue LED instrumentation and the addition of exogenous photosensitizers with energy density of 16.19 J/cm² caused the inactivation of S. aureus bacteria up to 91% [19]. An exposure of P. gingivalis with 405 nm diode laser and 4 J/cm² energy density inhibits the growth of P. gingivalis bacteria up to 97%, while exposure with energy dose of 8 J/cm² inactivates almost 100% P. gingivalis [20]. These researches have proven that the use of visible light diode laser instrumentation, with or without the addition of photosensitizer, can inactivate periopathogenic bacteria in the photodynamic therapy.

The process of bacterial inactivation in photodynamic therapy involving combined use of lasers and antibiotics [21]. The combination of diode laser with antibiotics has been carried out to increase the effectiveness of bacterial inactivation. Doxycycline, an antibiotic type of bacteriostatic tetracycline, plays a role in reducing the formation of polysaccharides [22]. It has been proven to inhibit collagen-destroying enzymes and suppress the growth rate of the bacteria A. actinomycetemcomitans. Doxycycline 0.1% also acts as an exogenous photosensitizer for its absorbance in 375–780 nm wavelength. The administration of low-dose antibiotics will minimize adverse effects without causing antibiotic resistance in the bacteria [23]. The results of previous studies showed the ability of low dose doxycycline 0.1% as photosensitization agent combined with a diode laser treatment to reduce bacterial biofilms in vitro [24].

This study aimed to determine the effectiveness of 405-nm diode laser photodynamic therapy with an energy density of 8 J/cm², with and without doxycycline 0.1%, in a Wistar rat model of periodontitis with a variation of four therapies on day 1, 3, 5, and 7. The representative factors that can indicate local changes as the effect of laser are histopathological and histomorphometric of periodontal tissue. Histopathology of wounds is a very helpful tool to exclude a malignancy cause, monitor healing progress in the course of treatment, better understand the pathophysiology of non‐healing wounds, assess morphological changes and help with diagnosis. In this study the expression level of histopathological calculated by measuring the number of macrophages, lymphocytes and fibroblasts. Histomorphometry is broadly defined as the measurement of the shape or form of a tissue. Quantitative analysis of bone architecture is achieved using bone histomorphometry which provides valuable information on the amount of bone and its cellular activity. While the expression level of histomorphometric measurement involved calculating the distance between the CEJ-AV.

The diode laser used here has peak specification in 379.81 ± 5.11 nm wavelength. The wavelengths are in accordance with the absorption spectrum range of both Porphyromonas gingivalis bacteria and doxycycline that were used as exogenous photosensitizers in this study. Previous reports have shown doxycycline absorption spectrum within range 375–380 nm [24]. Lasers with specific power and duration of radiation play an important role in the tissue interaction. In our photodynamic therapy, a photochemical interaction of 30.45 mW low laser power was observed. The temperature generated by diode laser during exposure for 5 min was in the range of 31.20 ± 0.06 °C. The temperature tended to fluctuate during exposure; however, still classified as ambient temperature and therefore did not cause photothermal effects. The radiation density was 8 J/cm², and the exposure time was 110 s.

Laser diode produces light spectrum within range of visible and infrared light, making it applicable for periodontitis therapy. Laser treatment given to periodontitis rats have shown antibacterial effects, indicated by the level of macrophage expression which is not significantly different compared to healthy rats. The lymphocyte expression level tended to be higher than the periodontitis group with significant differences observed on the third, fifth, and seventh day. Fibroblasts component increased after the initial administration of therapy, therefore did not differ significantly in the healthy group on the seventh day. Alveolar bone tissue repair observed after administration by fifth day, marked with CEJ-AV distance decrease during fifth and seventh days. First day administration of laser therapy showed that the CEJ-AV distance was not different from the healthy group. Various research results show that the laser acts as a stimulator that triggers the de-epitheliazation process and increases vascular permeability [12]. Both processes are known to inhibit growth factor down-regulation and increase the infiltration of immunocompetent cells [33]. In addition, lasers have been known to trigger the release of various pro and anti-inflammatory mediators such as IL-1, IL-2, IL-6, IL-8, IL-10, IL-1β, TNFα, and G-CSF [32, 34, 35].

There was a significant difference between the healthy rat group and the periodontitis rat group based on calculation in expression level of histopathological analysis. The expression level of macrophages and lymphocytes in the healthy rat group was lower compared to other group. However, the fibroblasts expression level was significantly higher than periodontitis group. Lipopolysaccharides along with other virulence factors are known to have the ability to modulate the host defense system that affects immune down-regulation [25]. The failure of innate-immunity in antigens elimination affects the adaptive-immunity system sustainability [34]. In this condition, macrophages also act as the antigen-presenting cell components, capable of recognizing bacteria and releasing inflammatory mediators [8]. B lymphocytes are involved in bacterial resistance through antigen–antibody reaction, while T lymphocytes mediate host defenses against responses in forming T helper (CD4 + Th) and T cytotoxic (CD8 + cytotoxic) subsets [31]. Failure to overcome the presence of antigens causes the release of various pro-inflammatory mediators, such as IL-1β and TNFα, furthermore, leading to tissue damage [36]. The high expression of macrophages and other proinflammatory mediators can be associated with tissue damage occurrence [5].

Histomorphometric observation results showed a significant increase in the CEJ-AV distance in the healthy group and the periodontitis group. This increase is believed to be the result of bone resorption indicator that occurs in the alveolar bone and become popular method used by many researchers [4, 5, 30].

Bone resorption is one of the physiological responses to prevent bacterial infection using osteoclasts [31]. The differentiation, activation, and survival ability of an osteoclast is influenced by RANKL and osteoprotegrin produced by M₂ macrophage components [32]. Osteoprotegrin is also produced from the release of IL-10 cytokines by Th₂ lymphocytes [34]. If RANKL binds more M1 macrophage RANK receptor, then M1 macrophages produces inflammatory mediators to activate the osteoclasts into maturity [30]. On the other hand, if osteoprotegrin can bind more RANKL, fibroblast components can be activated through the production of various mediators growth factor (GF) [35]. This mediator will activate osteoblasts that influence bone tissue remodeling as well as fibroblasts in the formation of collagen matrix. Alveolar bone tissue that has been repaired is known as woven bond (small bones) and can be associated with a decrease in the CEJ-AV distance [31].

The use of diode lasers in the photodynamic therapy is recognized as a therapeutic modality in the bacterial inactivation process. Various reactive oxygen species are produced in the photophysical process through suitability of hematogenphyrin porphyrin absorption spectrum in P. gingivalis bacteria [36]. In addition to providing an antibacterial effect, this research demonstrates the role of laser as a therapy that affects the host immune system in cases of chronic inflammation, referred to as immunomodulatory therapy. Photodynamic therapy is believed to have no effect on human gingival fibroblast tissue (HGF) through the elimination of photobiological effects [37]. Furthermore, lasers act as the stimulators that trigger de-epitheliazation and increase vascular permeability [38]. Both these processes inhibit growth factor down-regulation and increase the infiltration of immunocompetent cells.

Doxycycline antibiotics given to the periodontitis rats have shown antibacterial effect. This is demonstrated by an increase in the lymphocyte expression and inversely, a decrease in the macrophage expression on day 3, significantly different from periodontitis rat group. Increased fibroblast expression was observed following the administration of doxycycline on day 5 that continued to rise on day 7 with no significant difference in comparison to the healthy rat group. According to the histomorphometric analysis, the CEJ-AV distance following the administration of therapy on day 3 was not different from healthy rats. However, the distance was distinctive compared to periodontitis rats. Meanwhile, we observed increasing CEJ-AV distance until day 7. Moreover, the expression level of each immunocompetent cell indicates tissue repair, especially shown in macrophages cells decrease as well as increasing of fibroblasts number.

Photodynamic therapy with the addition of exogenous photosensitizer elements has been widely used and increased the effectiveness of therapy, especially in the inactivation of bacteria [24, 27]. In addition, the use of low-dose antibiotics combined with photodynamic therapy aim to avoid increasing bacterial resistance to antibiotics [19]. The suitability of doxycycline absorption spectrum causes photosensitizer molecules to be excited, thus generating the type 1 and type 2 photochemical effects. Various free radicals, especially peroxide compounds, have an impact on bacterial death. The diode laser indirectly acts as an immune system modulator. If the presence of antigens can be overcome, macrophage components will undergo lysis so that their quantity depleted. Thus, it can be assumed that the reduction in proinflammatory mediators and the increase in anti-inflammatory mediators induce tissue repair. Increased expression level of fibroblasts that play a role in the new extracellular matrix also supports tissue re-modelling [30].

The periodontitis manifestations depend on response between the host defense tissue with pathogenic bacteria which involves innate and adaptive immunity [34]. Innate immunity is the host's initial defense system against bacteria that works quickly, but has low specificity and diversity. If the bacteria (antigen) activity cannot be resolved, the adaptive immunity response will automatically begin to work. On the other side, the adaptive immunity response is a specific immune response to an antigen, divided into two types of responses, namely cell-mediated immunity (cellular immunity) and humoral immunity. Cellular immunity is a response that works with specific antigens and induces apoptosis, while humoral immunity is a response which regulates antibodies against foreign antigens. The two immune system responses work collaboratively returning the inflamed tissue to homeostasis [39].

The response mechanism to pathogenic bacteria in periodontal tissue begins with the response of several leukocyte and endothelial cell infiltrations [34]. In this phase, the metabolic products of bacteria, such as lipopolysaccharides, induce the tissue to produce cytokines and neuropeptides which cause blood vessels vasodilation. The released neutrophils leave the blood vessels and target the inflamed area in response to chemokines. In the early lesion phase, the number of neutrophils increases in connective tissue and marked by presence of several leukocyte cells such as macrophages, lymphocytes, plasma cells, and mast cells. The transition phase from innate immunity to humoral immunity occurs in the established lesion phase which is dominated by macrophages, plasma cells, and B and T lymphocytes. In addition, in this phase fibroblasts begin to produce collagen which repairs blood vessels. The transition from gingivitis to periodontitis is the last phase (advanced lesion) which is characterized by periodontal pocket erosion that deepens to alveolar bone destruction and observe-able histologically and clinically. In this phase there is an extension of the infiltration process of inflammatory cells which is also characterized by reduced collagen; as well as increase in number of lymphocytes, plasma cells, and macrophages that play an active role in chronic inflammation. Inflammation is the first physiological response mechanism as a host defense system against local bacterial aggregation in tissue [34]. Inflammation can be described by means of inflammation, resolution, and healing that involve several components of the immune system that interact to protect periodontal tissue. Some of these important components can be explained by their roles and functions as follows:

In this study, researchers had limited information about the antibacterial effects produced through photodynamic therapy using diode lasers combined with doxycycline photosensitizers. Several studies in the use of photodynamic therapy that have been performed on rat with periodontitis have used more specific measurement methods, such as radiological features for measuring the CEJ-AV distance. In addition, specific pro/anti-inflammatory cytokines were used for determining the extent of tissue damage such as IL-1β, IL-6, PGE₂, TNFα and repair of VEGF, HGF, COL-1A tissues have been conducted, although a different therapeutic method was used.

The results showed that the combination treatment of doxycycline 0.1% and laser exposure yielded immunomodulatory effects. The expression level of macrophage cells decreased from day 1 (5.00 ± 1.00) to day 7 (2.00 ± 0.00) and the highest level of lymphocyte cells was on day 3 (11.67 ± 3.21). The healing process that is mediated by the expression level of fibroblast is similar to that in healthy rats, especially on day 5 of the therapy (34.00 ± 3.61). The histomorphological analysis performed by measuring the distance between CEJ-AV bone resulting 538.27 ± 88.94 µm, showed that the combination of doxycycline and laser therapy exerted healing effect in rat models of periodontitis on day 7. Based on immunocompetent cells and the histomorphological analysis that measured the macrophage cells, lymphocyte cells, fibroblast and CEJ-AV distance, it can be concluded that the combination of doxycycline and laser therapy provide a healing effect in rats models of periodontitis.