The present study represented that bacterially mediated LPS generation results in redox and inflammatory imbalance in patients with radicular cyst. This results in enhanced production of nitric oxide by up-regulating i-NOS activity and matrix metalloproteinases (MMPs) playing key role in the development of radicular cyst and its progression to peri-apical region. The higher levels of NO provoke disturbance in sRANKL and OPG ratio resulting in osteoclast maturation. The periapical bone remodeling depends also on raised AGEs levels and over-expression of RAGE due to oxidative stress. The reduced OPG levels cause hyperactivation of sRANKL leading to stimulation of NF-kB signaling cascade in peri-alveolar tissue leading to proliferation of RC to adjacent structures of the involved tooth. The bacterial growth can be affected by oral hygiene, cyst localization, and cyst fluid color. It has been observed that bacterial expansion was higher in mandible cysts as compared to maxillary cysts and from straw-colored fluids highest bacterial isolates were recovered relatively to whitish-colored fluids (Mustafa, 2013). The prime causative factors involved in the endurance of species are the provision of nutrients, oxygen and PH levels. RC is poly-microbial thus, the obligatory anaerobic bacteria including Prevotella buccae, Fusobacterium nucleatum etc. depend on serum proteins and proteins from the degradation of pulpal tissue resulting in acidic environment favoring bacterial proliferation (Figdor and Gulabivala, 2008).
Lipopolysaccharide (LPS) produced by bacteria results in oxidative stress and inflammatory mediators (IL-1, IL-6 and TNF-α) generation which mediate induction of i-NOS activity in the epithelial cells of RC as shown in Fig. 1 and Table 3 (LPS Vs IL-1, r = 0.648**, LPS Vs TNF-α, r = 0.741** and LPS Vs i-NOS, r = 0.568*). They also up-regulate the expression of keratinocyte growth factor (KGF) and insulin-like growth factor (IGF) resulting in stimulation of rests of Malassez to form RC, a direct sequel of periapical granuloma (Lin et al., 2007). The enhanced i-NOS expression results in increased NO levels which have a dual function, one is an immunomodulatory role via blocking the activity of NFkB, activator protein-1 (AP-1) and zinc finger transcription factor such as sp1 inhibit Th1 response (Kroncke et al., 1998). The other effect of raised NO is that it combines with superoxide anion to form peroxynitrite (ONOO−) which ultimately leads to lipid peroxidation of prostaglandin E-2 (PGE-2) resulting in malondialdehyde (MDA) and isoprostane production thus, playing role in periapical bone resorption (NO Vs MDA, r = 0.711** and NO Vs isoprostanes, r = 0.679*). Inducible NOS dependent NO synthesis has a distinctive role in cellular mechanisms such as the mediation of apoptosis, hampering mitochondrial respiration and control of oxidative phosphorylation (Festjens et al., 2006). The acidic PH, reactive oxygen species, inflammatory markers and LPS dependent NO production from macrophages results in production of matrix metalloproteinase-1 (MMP-1) by the host cells (LPS Vs MMP-1, r = 0.457* and NO Vs MMP-1, r = 0.615*) leading to ECM degradation by stimulating osteoclastogenesis mediating progression of RC to alveolar bone. The present study also indicates that the higher levels of MMP-2, MMP-9 and MMP-13 have a significant role in the development and proliferation of inflammatory bone resorption and soft periodontal tissue degradation. Sun et al. also suggested similar findings that MMPs are thought to be involved in excessive pre-osteoclast recruitment and relocation.
The levels of pro-oxidants derived end-products were higher and that of antioxidants were lower in radicular patients according to the present study. A recent study also validated the findings of the present study showing that the anti-oxidants levels (specifically SOD and GSH) and inflammatory mediators were reduced before treatment in periodontal disease and reverted to normal after non-surgical therapy. As the pocket depth increases non-surgical treatment alone cannot hamper the disease thus, surgical intervention must be required to eliminate the inflammation reservoirs properly (Shah et al., 2022). The resultant ROS production triggers redox-dependent transcription factors like NFkB and AP-1 which mediate tissue damage and aggravation of inflammation. The AGE-RAGE complex is up-regulated by enhanced ROS and inflammatory mediators production due to protein carbonylation resulting in the activation of NFkB signaling pathway (A Ajith et al., 2016). Thus, the higher levels of AGEs and over-expression of RAGE may be involved in the progression of RC to peri-radicular tissue by exacerbating intracellular inflammatory pathways and invasion into the periapical bone. RAGE-deficient mice demonstrated enhanced bone mineral density and bone mass by decreasing bone resorptive activity. The association between i-NOS and RAGE to explicate inflammatory reactions and penetration of RAGE expressing cells surrounding i-NOS producing cells has been evident (Zhou et al., 2006). The current study also implicates a positive relationship between AGEs and NO and AGES and sRANKL (AGEs Vs NO, r = 0.715** and AGEs Vs sRANKL, r = 0.783**).
Reactive oxygen species may have an osteolytic impact by blocking bone formation via hampering osteoblast maturation and enhancing osteoclast differentiation through the induction of sRANKL by the activation of monocytes and macrophages (Dezerega et al., 2012). Thus, the balance between sRANKL and OPG which is normally in favor of OPG blocks sRANKL by binding to it and making it unable to bind with RANK present on osteoclasts, shifts towards sRANKL in RC mediating its osteoclastogenic effects. sRANKL stimulates TNF receptor-associated factor-6 (TRAF-6), c-Fos and calcium signaling cascade are responsible for activation of nuclear factor of activated T cells c-1 (NFATc-1) has a dual effect i.e., the key switch for osteoclastic activity and also regulates the maturation and proper functioning of lymphocytes. The expression of TRAF-6 is also dependent on i-NOS activity and positively correlated with the levels of NO thus, having its role in osteoclast activation (Jianbo et al., 2021). Conversely, other reports proposed that NO inhibits bone resorption by inhibiting leukocyte migration and increasing OPG levels (Yan et al., 2021). Silva et al. also observed that in i-NOS knockout mice the expression of RANK was up-regulated while that of OPG was decreased thus the higher NO levels may be beneficial in reducing the RC progression towards periapical bone. The present study is in the favor of the concept that increased NO levels may have a role in bone mass reduction as it has a negative correlation with OPG levels and a positive correlation with sRANKL (NO Vs OPG, r = -0.568* and NO Vs sRANKL, r = 0.648*).
Lipopolysaccharide (LPS) recognizes CD14 and TLR4 receptors in peri-alveolar tissue which participate in the initiation of immuno-inflammatory reaction, playing a major role in the development of periapical lesion and ultimately bone resorption by the activation and mobilization of osteoclasts (Han et al., 2023). This mobilization is dependent on chemokine stromal cell derived factor-1α (SDF-1α) which binds to its receptors CXCR4, largely expressed on osteoclast precursors (He et al., 2023). A series of molecules called suppressors of cytokine signaling (SOCS) mediate the enhancement of pro-inflammatory cytokine genes controlling bone resorption (Petean et al., 2022). It has been demonstrated that SOCS-1 mRNA is inversely correlated to sRANKL gene expression. IL-1 and TNF-α both are the major role players in the activation and differentiation of osteoclasts as dendritic cells and monocytes are the main source of sRANKL in peri-alveolar lesions (IL-1 Vs sRANKL, r = 0.801*** and TNF-α Vs sRANKL, r = 0.709***). It has been also revealed that the co-administration of IL-1 and TNF-α has a synergistic impact on expression of sRANKL but negatively affect OPG expression (Lim et al., 2023). P. gingivalis stimulates T-cells to synthesize TNF-α converting enzyme (TACE), a metalloproteinase having highest specificity for shedding sRANKL resulting in odontoclast production and dentine resorption (Zhang et al., 2021). The higher sRANKL levels (7.7 Vs 4.9) and lower OPG levels (8.1 Vs 9.2) were observed in patients with periodontitis showing that reduced OPG levels may be due to decreased angiogenesis as OPG production is related to blood vessels (Crotti et al., 2004). Thus, the development of radicular cyst and associated periapical osteo-invasion as a result of crosstalk among pro-oxidants, pro-inflammatory mediators and bone remodeling markers can be impeded by employing TIMPs and inhibition of sRANKL activity.
LPS released from bacterial cell wall act as mitogen on epithelial rests of Malassez in the periapical periodontium stimulating the polymorphonuclear cells, macrophages and natural killer cells to release pro-inflammatory mediators and cytokines and a variety of ROS (Fig. 1.). The cascade of intracellular reactions triggered by LPS, excessive ROS production and AGE RAGE association result in increased levels of NEMO and MAPKs which promotes NF-kB activation. This NF-kB now translocates to the nucleus which potentiates the release of pro-inflammatory cytokines, increases matrix degradation by upregulating MMPs levels, and promotes maturation, immigration, and endurance of osteoclasts thus enhancing bone resorption contributing to cyst expansion. The activity of iNOS is also augmented by NF-kB which enhances the production of NO and nitrotyrosine, allowing massive production of LPS from bacteria and lipid peroxidation. The resultant LPS further activates the whole cycle of NF-kB, NO and LPS synthesis thus causes massive bone resorption and cyst expansion into periapical bone.