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 . Both processes are known to inhibit growth factor down-regulation and increase the infiltration of immunocompetent cells . 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 . The failure of innate-immunity in antigens elimination affects the adaptive-immunity system sustainability . In this condition, macrophages also act as the antigen-presenting cell components, capable of recognizing bacteria and releasing inflammatory mediators . 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 . 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 . The high expression of macrophages and other proinflammatory mediators can be associated with tissue damage occurrence .
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 . The differentiation, activation, and survival ability of an osteoclast is influenced by RANKL and osteoprotegrin produced by M2 macrophage components . Osteoprotegrin is also produced from the release of IL-10 cytokines by Th2 lymphocytes . If RANKL binds more M1 macrophage RANK receptor, then M1 macrophages produces inflammatory mediators to activate the osteoclasts into maturity . On the other hand, if osteoprotegrin can bind more RANKL, fibroblast components can be activated through the production of various mediators growth factor (GF) . 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 .
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 . 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 . Furthermore, lasers act as the stimulators that trigger de-epitheliazation and increase vascular permeability . 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 . 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 .
The periodontitis manifestations depend on response between the host defense tissue with pathogenic bacteria which involves innate and adaptive immunity . 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 .
The response mechanism to pathogenic bacteria in periodontal tissue begins with the response of several leukocyte and endothelial cell infiltrations . 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 . 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:
Lymphocytes are one type of leukocyte cells that play an important role in the body's defense system against antigens, especially and consist of B cells, T cells, and Natural Killer cells (NK-cells). B cells differentiated in the bone marrow and work by producing antibodies, whereas, T cells are differentiated in the thymus gland and act against specific antigens by producing T helper and also T cytotoxic. At the cellular level, if the number of lymphocytes decreases, the body's ability to deal with antigens, such as bacteria, will decrease .
Macrophages are a type of white blood cell and are the adult forms of monocytes that can migrate through blood vessels. Macrophages (macros and phagens) play an important role in the innate immunity system, which works by recognizing foreign antigens with toll-like receptors (TLRs) and consuming foreign antigens (phagocytosis). Macrophages are said to be the main agents that produce metabolic secretions that cause damage to connective tissue to alveolar bone damage . Macrophages play a role in the regulation of inflammation that occurs, depending on their function, macrophages can produce pro or anti-inflammatory mediators.
Fibroblasts are the most common cells found in connective tissue and play a role in the tissue repair process through the synthesis of extracellular matrix components, one of which is the process of rebuilding blood vessels (angiogenesis) . Fibroblasts will be active when the host defense system is damaged and so they will proliferate (fibrogenesis). The processes of fibrogenesis and angiogenesis are regulators for determining the function of macrophages.
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.