*2.2.3 Growth and enlargement of the radicular cyst*

The cyst enlargement is the final stage in radicular cyst pathogenesis. Toller's studies showed that osmosis contributes to the increase in the size of cysts [17]. Ward et al. proved this by simulating the growth of odontogenic cyst by mathematical modeling [18]. This modeling not only confirmed the results of Kubota et al. but also demonstrated that as the cyst became larger, cell proliferation played bigger part than osmotic pressure [19]. Harris and Toller suggested that cyst enlargement depends on epithelial proliferation which continues if inflammatory stimulus is present [20]. **Figure 3** shows large radicular cyst in the upper jaw.

#### **2.3 Gene expression in radicular cysts**

As described previously, among odontogenic cysts with benign pathology, up to 60% of all jaw cysts are radicular cysts, which originate from root canal infection caused by various microorganisms. The consequence of the radicular cyst development is the concomitant resorption of the surrounding bone tissues and periradicular periodontal ligament (PDL; [21]). Studies that have analyzed gene expression in radicular

**63**

**Figure 3.**

*Oral Pathology: Gene Expression in Odontogenic Cysts DOI: http://dx.doi.org/10.5772/intechopen.80555*

cysts have mostly focused on genes that are involved in processes such as bone metabolism, inflammation, and tumorigenesis. Regarding bone metabolism, a gene known as receptor activator of nuclear factor-B ligand (RANKL) has been extensively studied because of its role in bone resorption around the tooth apex. This gene is part of a pathway that activates osteoclasts and is inhibited by a protein called osteoprotegerin (OPG). The role of RANK-RANKL-OPG signaling pathway in radicular cyst pathogenesis is further described in Section 2.3.2. Regarding genes involved in inflammatory processes, studies have analyzed expression of genes that code for chemokines and chemokine receptors that are involved in T helper type 1 (Th1) and Th2 responses that are characterized by the generation of interleukin-2 (IL-2), IL-12, and interferon-c (IFN-c) and by IL-4, IL-5, IL-6, IL-10, and IL-13, respectively [22, 23]. Regarding genes involved in tumorigenesis, *TP53* has been well analyzed in radicular cysts, where it shows low expression. Besides, *TP53*, *PCNA*, *FHIT*, and *Ki67* genes were analyzed

*Large radicular cyst located in the upper jaw on the right side. The appearance of the radicular cyst after mucoperiosteal flap was raised (A). Bone cavity after the cyst enucleation (B). The macroscopic view of the enucleated cyst (C). The histological morphology of radicular cyst with typical cholesterol crystals in the form* 

However, the most extensively studied genes in the pathogenesis of radicular cysts belong to the family of matrix metalloproteinases (MMPs). Their role in the

The family of genes that are most commonly associated with the development of these lesions are matrix metalloproteinases (MMPs), which are metal-dependent endopeptidases that represent the major class of enzymes responsible for extracellular matrix degradation. It has been shown that MMPs have a crucial role in collagen degradation during periodontal tissue destruction [27–29]. Schematic representation of different classes of MMPs and an example of molecular structure is shown in **Figure 4**. Most commonly differentially expressed MMPs in oral diseases are presented in **Table 1**. For example, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, and MMP-13 play a role in the pathogenesis of periapical inflammatory lesions [27–31]. MMP-1 is a crucial enzyme in the initiation of osteoclastic bone

and also showed insignificant changes compared to controls [24–26].

cyst formation and development is described in Section 2.3.1.

*2.3.1 Matrix metalloproteinases (MMPs)*

*of clefts, stained with H&E, 40× magnification (D).*

#### **Figure 3.**

*Gene Expression and Control*

center (**Figure 2**) [16].

**Figure 2.**

*2.2.3 Growth and enlargement of the radicular cyst*

*description of the radicular cyst development is the prevailing theory.*

**2.3 Gene expression in radicular cysts**

independently. The primarily accepted theory proposes that epithelial cells multiply and enclose the surface of connective tissue of an abscess cavity or cavity, which resulted from the breakdown of connective tissue by activity of proteolytic enzymes [16]. The secondary one, which is supported more, states that radicular cyst forms inside of the multiplying epithelial mass in periapical granuloma by cell death in the

*Schematic representation of the development of radicular cyst. Starting from the left, superficial caries forms first, followed by medium and deep caries. Untreated deep caries leads to total pulp inflammation. Subsequently, necrosis of the pulp appears, which becomes infected. The resulting infection of the root canal initiates the epithelial remnants of Hertwig sheath (Malassez epithelial rests) to proliferate. Once the cells proliferate, the epithelial nest is formed. When the epithelial nest reaches the size of 1 cm, the center becomes necrotic leading to the formation of future cystic cavity, which becomes lined with the epithelium. For unknown reasons, this epithelium starts secreting fluid, which is called cystic fluid. These steps lead to the formation of radicular cyst, a round cavity filled with fluid and lined with the epithelium and fibrous connective tissue. This* 

The cyst enlargement is the final stage in radicular cyst pathogenesis. Toller's studies showed that osmosis contributes to the increase in the size of cysts [17]. Ward et al. proved this by simulating the growth of odontogenic cyst by mathematical modeling [18]. This modeling not only confirmed the results of Kubota et al. but also demonstrated that as the cyst became larger, cell proliferation played bigger part than osmotic pressure [19]. Harris and Toller suggested that cyst enlargement depends on epithelial proliferation which continues if inflammatory stimulus is

As described previously, among odontogenic cysts with benign pathology, up to 60% of all jaw cysts are radicular cysts, which originate from root canal infection caused by various microorganisms. The consequence of the radicular cyst development is the concomitant resorption of the surrounding bone tissues and periradicular periodontal ligament (PDL; [21]). Studies that have analyzed gene expression in radicular

present [20]. **Figure 3** shows large radicular cyst in the upper jaw.

**62**

*Large radicular cyst located in the upper jaw on the right side. The appearance of the radicular cyst after mucoperiosteal flap was raised (A). Bone cavity after the cyst enucleation (B). The macroscopic view of the enucleated cyst (C). The histological morphology of radicular cyst with typical cholesterol crystals in the form of clefts, stained with H&E, 40× magnification (D).*

cysts have mostly focused on genes that are involved in processes such as bone metabolism, inflammation, and tumorigenesis. Regarding bone metabolism, a gene known as receptor activator of nuclear factor-B ligand (RANKL) has been extensively studied because of its role in bone resorption around the tooth apex. This gene is part of a pathway that activates osteoclasts and is inhibited by a protein called osteoprotegerin (OPG). The role of RANK-RANKL-OPG signaling pathway in radicular cyst pathogenesis is further described in Section 2.3.2. Regarding genes involved in inflammatory processes, studies have analyzed expression of genes that code for chemokines and chemokine receptors that are involved in T helper type 1 (Th1) and Th2 responses that are characterized by the generation of interleukin-2 (IL-2), IL-12, and interferon-c (IFN-c) and by IL-4, IL-5, IL-6, IL-10, and IL-13, respectively [22, 23]. Regarding genes involved in tumorigenesis, *TP53* has been well analyzed in radicular cysts, where it shows low expression. Besides, *TP53*, *PCNA*, *FHIT*, and *Ki67* genes were analyzed and also showed insignificant changes compared to controls [24–26].

However, the most extensively studied genes in the pathogenesis of radicular cysts belong to the family of matrix metalloproteinases (MMPs). Their role in the cyst formation and development is described in Section 2.3.1.

#### *2.3.1 Matrix metalloproteinases (MMPs)*

The family of genes that are most commonly associated with the development of these lesions are matrix metalloproteinases (MMPs), which are metal-dependent endopeptidases that represent the major class of enzymes responsible for extracellular matrix degradation. It has been shown that MMPs have a crucial role in collagen degradation during periodontal tissue destruction [27–29]. Schematic representation of different classes of MMPs and an example of molecular structure is shown in **Figure 4**. Most commonly differentially expressed MMPs in oral diseases are presented in **Table 1**. For example, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, and MMP-13 play a role in the pathogenesis of periapical inflammatory lesions [27–31]. MMP-1 is a crucial enzyme in the initiation of osteoclastic bone

#### **Figure 4.**

*Schematic classification, domain, and crystal structure of MMPs. Functional classification of MMPs is shown in A. MMPs are classified according to their function into collagenases, gelatinases, stromelysins, membranetype MMPs, matrilysin, enamelin, metalloelastases, and others. Domain structure of collagenases (MMP-1, MMP-8, MMP-13) and gelatinases (MMP-2, MMP-9) is shown in B. The domain structure consists of signal peptide sequence (in blue color, labeled "pro"); prodomain that inhibits the catalytic domain, making it inaccessible to substrates (in pink color, labeled "pre"); catalytic domain that contains zinc atom (in green color, labeled "catalytic"); and hinge domain which links the catalytic and hemopexin domain (in red color, labeled "hemopexin"). Gelatinases also contain fibronectin-like domain repeats which aid in substrate binding (in blue color). Crystal structure of MMP-1 is shown in C.*

resorption [32], because it degrades the collagen of the unmineralized layer found on the surface of the bone. After collagen degradation, the collagen fragments are produced, which function in osteoclast activation [33]. Studies in rats have shown that MMP-1 expression is elevated in the active phase of periapical lesion development [34]. MMP-1 also causes expansion of radicular cysts in humans [35].

MMP-2 and MMP-9 function in the degradation of the extracellular matrix (ECM), particularly during the active stages of lesion development [30], and their expression is increased in those lesions compared to control healthy tissue (gingiva, PDL, or oral mucosa).

MMPs play an important role in normal cellular processes such as tissue growth, bone resorption, and remodeling (wound healing and angiogenesis) [36]. MMPs are secreted in their proenzyme state and require extracellular activation. They are regulated by endogenously secreted inhibitors, called TIMPs (the tissue inhibitor of metalloproteinases). MMPs cleave native, nondenatured collagens with long uninterrupted triple helices and can function as collagenases (**Table 1**) [37]. Thus, the molecular basis of MMP function involves proteolytic cleavage of different substrates and subsequent activation of transforming growth factor-beta (TGF-beta), insulin-like growth factors (IGF), vascular endothelial growth factors (VEGF), and RANKL pathway.

Normal tissues generally show low expression of MMPs. However, during pathological states that require destruction of extracellular matrix, expression of MMPs can be drastically increased [29]. In normal and healthy tissues, components of extracellular matrix are in constant balance between degradation and protein synthesis. It has been shown that elevated MMP levels correlate with nonhealing [38, 39].

In pathological state such as apical periodontitis, MMP expression and secretion are increased, suggesting the direct role of MMPs in tissue remodeling and

**65**

tional processes [43, 45].

prevention and healing process [42].

*Oral Pathology: Gene Expression in Odontogenic Cysts DOI: http://dx.doi.org/10.5772/intechopen.80555*

Collagenase Collagen I,

Collagenase Collagen

Collagenase Collagen I,

Gelatinase Collagen

Gelatinase Collagen I,

MMP-3 Collagen

MMP-1 (collagenase-1, interstitial collagenase)

MMP-8 (collagenase-2, neutrophil collagenase)

MMP-13 (collagenase-3)

MMP-9 (gelatinase B)

MMP-2 (gelatinase A)

**Table 1.**

destruction during lesion development [40–42]. Regulation of their expression is primarily controlled at the transcriptional level even though regulatory mechanisms have still not been fully elucidated [27, 43, 44]. Some of the reasons include the influence of promoter polymorphisms, epigenetic mechanisms, and posttranscrip-

**Name of enzyme Class Substrate Expression in normal tissue and oral disease**

Bone resorption in periapical lesions and human periapical lesions degrades nonmineralized extracellular matrix and stimulates osteoclastogenesis by collagen degradation on bone surface, expression in cystic wall and cystic fluid, pulpitis, squamocellular carcinoma, and normal tissues (stomach and gallbladder)

Chronic pulp inflammation, human periapical lesions, pulpitis, caries, and normal tissues (bone

Chronic pulp inflammation, human periapical lesions, pulpitis, and normal tissue (vagina, lung)

Periodontitis, caries, oral squamocellular carcinoma metastasis, invasiveness and shorter survival, and human periapical lesions

Periodontitis, caries, oral squamocellular carcinoma metastasis, invasiveness and shorter survival, collagen type IV degradation, ameloblastoma, human periapical lesions, and normal tissue (gallbladder, uterine,

Oral squamocellular carcinoma, human periapical lesions, and normal tissue (endometrium,

marrow and spleen)

endometrium, cervix)

salivary gland)

II, III, VII, VIII, and X and gelatin

I, II, III, V, VII, VIII, and X and gelatin

II, III, IV, V, VII, IX, and X

III, IV, V, VII, X, and XI

II, III, IV, V, VII, X, and XI

III, IV, V, VII, IX, X, and XI

*List of MMPs most commonly associated with and expressed in oral disease and healthy tissues.*

Promoter polymorphisms have been detected in MMP promoter regions, suggesting that the changes in MMP expression can predispose individuals to develop periapical inflammatory lesions. Similarly, these MMP promoter polymorphisms can lead to progression of disease [42, 46]. For example, Menezes-Silva et al. investigated genetic predisposition to periapical disease by testing 16 SNP polymorphisms in *MMP2*, *MMP3*, *MMP9*, *MMP13*, *MMP14*, and *TIMP2* genes [42]. They found that polymorphisms in *MMP2* and *MMP3* genes are associated with the development of periapical lesions, suggesting that these markers could assist in

Besides MMP promoter polymorphisms, another regulatory mechanism for MMP expression has been found at the epigenetic level, where methylation of MMP promoters can lead to gene inactivation and subsequent decrease in transcription [47]. Campos et al. have studied the methylation state of *MMP-2* and *MMP-9* in periapical inflammatory lesions [48]. Their results show that *MMP-2* gene was partially methylated in periapical granuloma, radicular cysts, and normal oral mucosa,


#### **Table 1.**

*Gene Expression and Control*

resorption [32], because it degrades the collagen of the unmineralized layer found on the surface of the bone. After collagen degradation, the collagen fragments are produced, which function in osteoclast activation [33]. Studies in rats have shown that MMP-1 expression is elevated in the active phase of periapical lesion development [34]. MMP-1 also causes expansion of radicular cysts in humans [35]. MMP-2 and MMP-9 function in the degradation of the extracellular matrix (ECM), particularly during the active stages of lesion development [30], and their expression is increased in those lesions compared to control healthy tissue (gingiva,

*Schematic classification, domain, and crystal structure of MMPs. Functional classification of MMPs is shown in A. MMPs are classified according to their function into collagenases, gelatinases, stromelysins, membranetype MMPs, matrilysin, enamelin, metalloelastases, and others. Domain structure of collagenases (MMP-1, MMP-8, MMP-13) and gelatinases (MMP-2, MMP-9) is shown in B. The domain structure consists of signal peptide sequence (in blue color, labeled "pro"); prodomain that inhibits the catalytic domain, making it inaccessible to substrates (in pink color, labeled "pre"); catalytic domain that contains zinc atom (in green color, labeled "catalytic"); and hinge domain which links the catalytic and hemopexin domain (in red color, labeled "hemopexin"). Gelatinases also contain fibronectin-like domain repeats which aid in substrate binding (in blue* 

MMPs play an important role in normal cellular processes such as tissue growth, bone resorption, and remodeling (wound healing and angiogenesis) [36]. MMPs are secreted in their proenzyme state and require extracellular activation. They are regulated by endogenously secreted inhibitors, called TIMPs (the tissue inhibitor of metalloproteinases). MMPs cleave native, nondenatured collagens with long uninterrupted triple helices and can function as collagenases (**Table 1**) [37]. Thus, the molecular basis of MMP function involves proteolytic cleavage of different substrates and subsequent activation of transforming growth factor-beta (TGF-beta), insulin-like growth factors (IGF), vascular endothelial growth factors (VEGF), and

Normal tissues generally show low expression of MMPs. However, during pathological states that require destruction of extracellular matrix, expression of MMPs can be drastically increased [29]. In normal and healthy tissues, components of extracellular matrix are in constant balance between degradation and protein synthesis. It has

In pathological state such as apical periodontitis, MMP expression and secretion are increased, suggesting the direct role of MMPs in tissue remodeling and

been shown that elevated MMP levels correlate with nonhealing [38, 39].

**64**

PDL, or oral mucosa).

*color). Crystal structure of MMP-1 is shown in C.*

**Figure 4.**

RANKL pathway.

*List of MMPs most commonly associated with and expressed in oral disease and healthy tissues.*

destruction during lesion development [40–42]. Regulation of their expression is primarily controlled at the transcriptional level even though regulatory mechanisms have still not been fully elucidated [27, 43, 44]. Some of the reasons include the influence of promoter polymorphisms, epigenetic mechanisms, and posttranscriptional processes [43, 45].

Promoter polymorphisms have been detected in MMP promoter regions, suggesting that the changes in MMP expression can predispose individuals to develop periapical inflammatory lesions. Similarly, these MMP promoter polymorphisms can lead to progression of disease [42, 46]. For example, Menezes-Silva et al. investigated genetic predisposition to periapical disease by testing 16 SNP polymorphisms in *MMP2*, *MMP3*, *MMP9*, *MMP13*, *MMP14*, and *TIMP2* genes [42]. They found that polymorphisms in *MMP2* and *MMP3* genes are associated with the development of periapical lesions, suggesting that these markers could assist in prevention and healing process [42].

Besides MMP promoter polymorphisms, another regulatory mechanism for MMP expression has been found at the epigenetic level, where methylation of MMP promoters can lead to gene inactivation and subsequent decrease in transcription [47]. Campos et al. have studied the methylation state of *MMP-2* and *MMP-9* in periapical inflammatory lesions [48]. Their results show that *MMP-2* gene was partially methylated in periapical granuloma, radicular cysts, and normal oral mucosa,

and subsequent association between methylation status and gene expression was not possible [48]. Regarding *MMP-9*, the study found that this gene was more unmethylated in periapical granulomas and radicular cysts than in healthy mucosa, which implies that *MMP-9* mRNA expression is increased and may be epigenetically controlled [48]. Effects of DNA methylation on MMP genes can contribute to individual susceptibility to the development of periapical granuloma and radicular cysts as periapical inflammatory lesions. Moreover, it may also play a role in the patient's response to therapy [48].

Besides MMP studies, other genes such as *FOXP3* have shown interesting results in periapical granulomas and radicular cysts. It has been shown that the *FOXP3* gene promoter methylation was inversely correlated with *FOXP3* transcript levels, suggesting that *FOXP3* may be crucial in determining periapical lesion development [49].

In conclusion, here we presented studies that suggest that genetic predisposition to frequent development of periapical inflammatory lesions could be caused by the presence of polymorphisms in MMP gene promoters or by epigenetic mechanisms such as differential methylation status of MMP genes.

### *2.3.2 RANKL expression*

RANKL-RANK-OPG system discovery has changed our understanding of bone biology (**Figure 5**). This is a signaling system that is crucial for skeletal homeostasis because it maintains the balance between bone resorption by osteoclasts and bone formation by osteoblasts. RANKL is a ligand for its receptor RANK which can be found on osteoclast progenitor cells. After binding, RANK-RANKL system activates NF-κB pathway and upregulation of NFATc1 protein, which is a master regulator of cytokines important for osteoclastogenesis. The disruption of RANKL-RANK system leads to the inhibition of bone resorption. RANKL belongs to the TNF superfamily of proteins. The name RANKL stands for receptor activator of nuclear factor-κB ligand, also known as osteoprotegerin ligand (OPGL), osteoclast differentiation factor (ODF), TNF-related activation-induced cytokine (TRANCE), and TNF ligand superfamily member 11 (TNFSF11) [50, 51]. It is a homotrimeric protein that is membrane bound on osteoblasts and activated T cells. It can also be

#### **Figure 5.**

*RANKL-RANK-OPG system as regulators of bone resorption. Bone metabolism is a dynamic process that balances bone formation and bone resorption. RANKL (ligand) is secreted by osteoblasts and binds to the RANK receptor on osteoclast precursors and activated osteoclasts, which in turn result in bone resorption. The inhibition of this process is mediated by OPG.*

**67**

*Oral Pathology: Gene Expression in Odontogenic Cysts DOI: http://dx.doi.org/10.5772/intechopen.80555*

the affinity of OPG for RANKL.

**3. Odontogenic keratocysts**

secreted by T cells. RANKL is proteolytically cleaved by MMP-3 or MMP-7. Besides T cells and osteoclasts, RANKL expression can be seen in lymph nodes, thymus, mammary glands, spleen, and bone marrow. In tumor cells, RANKL is associated with migration and bone metastasis. Thus, RANKL is a key regulator of bone metabolism, specifically a regulator of osteoclastogenesis and osteoclastoactivation in a normal and pathological states [50, 51]. RANK is a receptor for RANKL and also a member of TNF superfamily. It is a homotrimeric transmembrane protein. Its expression is generally less than RANKL, and high expression is seen in mammary glands and cancer cells. OPG is an inhibitor of RANK-RANKL system. It stands for osteoprotegerin, also known as osteoclastogenesis inhibitory factor (OCIF) or tumor necrosis factor receptor superfamily member 11B, is a cytokine receptor of the TNF receptor superfamily encoded by the *TNFRSF11B* gene. It is a 380 amino acid glycoprotein that is found in soluble form as either monomer or dimer. The OPG dimer is crucial for RANK-RANKL inhibition because OPG dimer increases

RANKL affects the development of the periapical inflammatory lesions by activat-

ing osteoclasts, thus inducing pathological bone resorption [52]. RANKL protein expression was first shown in radicular cysts [53], which colocalized with osteoclasts. Subsequent studies analyzed transcript levels in inflammatory granulomas, which were increased compared to healthy PDL tissue [52]. RANKL expression was found in infiltrating leucocytes, specifically monocytes and dendritic cells, which were shown to be the main cells that secrete this protein. Another study compared RANKL and OPG levels in apical granulomas and radicular cysts [54], finding that both OPG and RANKL expressions were higher in granulomas than in cysts, but their ratio was comparable in these two types of periapical inflammatory lesions. Fukada and colleagues found that RANKL transcript levels were significantly higher in granuloma than in radicular cysts [55]. At the protein level, no difference was observed in RANKL and OPG levels in a study conducted by Fan and colleagues [56]. In a study on endodontically involved disease, RANKL expression was higher in lesions with more intense inflammation, but

the ratio RANKL/OPG in relation to inflammation was not increased [57].

Odontogenic keratocysts (OKCs) represent a rare form of odontogenic cysts which originate from dental lamina remnants or eventually from the basal layer of upper and lower jaw oral epithelium before the odontogenesis ended. Since it was first described in 1876, this form of cysts grabs scientific attention mostly because of its developmental variabilities, histological appearance, and genetic basis [2, 58]. In the past few years, the World Health Organization (WHO) made an attempt to create more appropriate classification of these cysts. Recently, they were considered as keratocystic odontogenic tumors for their aggressive behavior, high mitotic rate, and association with genetic and chromosomal abnormalities. The newest WHO classification reclassifies them again as odontogenic keratocysts because *PTCH1* gene mutations were detected, similarly to other developmental cysts such as dentigerous cyst [59–61]. Despite many classifications, pathologists and surgeons face difficulties in the establishment of proper diagnosis. This is because keratocysts cannot be clinically and radiographically distinguished from other odontogenic cysts. Moreover, it is still debatable what the optimal therapeutic approach is in the treatment of keratocysts in order to prevent recidive, which is a characteristic of this disease [2]. Odontogenic keratocyst is histologically characterized with stratified squamous epithelium, which is five to eight layers thick with palisaded hyperchromatic basal cell layer and "corrugated" parakeratotic epithelial cells on luminal surface [2, 62, 63]. *Gene Expression and Control*

patient's response to therapy [48].

*2.3.2 RANKL expression*

such as differential methylation status of MMP genes.

**66**

**Figure 5.**

*inhibition of this process is mediated by OPG.*

*RANKL-RANK-OPG system as regulators of bone resorption. Bone metabolism is a dynamic process that balances bone formation and bone resorption. RANKL (ligand) is secreted by osteoblasts and binds to the RANK receptor on osteoclast precursors and activated osteoclasts, which in turn result in bone resorption. The* 

and subsequent association between methylation status and gene expression was not possible [48]. Regarding *MMP-9*, the study found that this gene was more unmethylated in periapical granulomas and radicular cysts than in healthy mucosa, which implies that *MMP-9* mRNA expression is increased and may be epigenetically controlled [48]. Effects of DNA methylation on MMP genes can contribute to individual susceptibility to the development of periapical granuloma and radicular cysts as periapical inflammatory lesions. Moreover, it may also play a role in the

Besides MMP studies, other genes such as *FOXP3* have shown interesting results in periapical granulomas and radicular cysts. It has been shown that the *FOXP3* gene promoter methylation was inversely correlated with *FOXP3* transcript levels, suggesting that *FOXP3* may be crucial in determining periapical lesion development [49]. In conclusion, here we presented studies that suggest that genetic predisposition to frequent development of periapical inflammatory lesions could be caused by the presence of polymorphisms in MMP gene promoters or by epigenetic mechanisms

RANKL-RANK-OPG system discovery has changed our understanding of bone biology (**Figure 5**). This is a signaling system that is crucial for skeletal homeostasis because it maintains the balance between bone resorption by osteoclasts and bone formation by osteoblasts. RANKL is a ligand for its receptor RANK which can be found on osteoclast progenitor cells. After binding, RANK-RANKL system activates NF-κB pathway and upregulation of NFATc1 protein, which is a master regulator of cytokines important for osteoclastogenesis. The disruption of RANKL-RANK system leads to the inhibition of bone resorption. RANKL belongs to the TNF superfamily of proteins. The name RANKL stands for receptor activator of nuclear factor-κB ligand, also known as osteoprotegerin ligand (OPGL), osteoclast differentiation factor (ODF), TNF-related activation-induced cytokine (TRANCE), and TNF ligand superfamily member 11 (TNFSF11) [50, 51]. It is a homotrimeric protein that is membrane bound on osteoblasts and activated T cells. It can also be

secreted by T cells. RANKL is proteolytically cleaved by MMP-3 or MMP-7. Besides T cells and osteoclasts, RANKL expression can be seen in lymph nodes, thymus, mammary glands, spleen, and bone marrow. In tumor cells, RANKL is associated with migration and bone metastasis. Thus, RANKL is a key regulator of bone metabolism, specifically a regulator of osteoclastogenesis and osteoclastoactivation in a normal and pathological states [50, 51]. RANK is a receptor for RANKL and also a member of TNF superfamily. It is a homotrimeric transmembrane protein. Its expression is generally less than RANKL, and high expression is seen in mammary glands and cancer cells. OPG is an inhibitor of RANK-RANKL system. It stands for osteoprotegerin, also known as osteoclastogenesis inhibitory factor (OCIF) or tumor necrosis factor receptor superfamily member 11B, is a cytokine receptor of the TNF receptor superfamily encoded by the *TNFRSF11B* gene. It is a 380 amino acid glycoprotein that is found in soluble form as either monomer or dimer. The OPG dimer is crucial for RANK-RANKL inhibition because OPG dimer increases the affinity of OPG for RANKL.

RANKL affects the development of the periapical inflammatory lesions by activating osteoclasts, thus inducing pathological bone resorption [52]. RANKL protein expression was first shown in radicular cysts [53], which colocalized with osteoclasts. Subsequent studies analyzed transcript levels in inflammatory granulomas, which were increased compared to healthy PDL tissue [52]. RANKL expression was found in infiltrating leucocytes, specifically monocytes and dendritic cells, which were shown to be the main cells that secrete this protein. Another study compared RANKL and OPG levels in apical granulomas and radicular cysts [54], finding that both OPG and RANKL expressions were higher in granulomas than in cysts, but their ratio was comparable in these two types of periapical inflammatory lesions. Fukada and colleagues found that RANKL transcript levels were significantly higher in granuloma than in radicular cysts [55]. At the protein level, no difference was observed in RANKL and OPG levels in a study conducted by Fan and colleagues [56]. In a study on endodontically involved disease, RANKL expression was higher in lesions with more intense inflammation, but the ratio RANKL/OPG in relation to inflammation was not increased [57].
