Epithelial Proliferation
The stimuli for epithelial proliferation have been discussed previously, but in an established cyst LPS plays a less important role, and epithelial growth is sustained by cytokines and growth factors resulting from the inflammatory response (Table 3.2) (Bernardini et al. 2015 ; Silva et al. 2007 ; Lin et al. 2007 ; Nair 2004 ). However, as the cyst matures and inflammation subsides, the rate of cell proliferation may be relatively low. Soluk Tekkeşın et al. (2012a) showed that only about 1.0% of cells were positive for Ki‐67 in the epithelial lining of radicular cysts, compared to 2.0% or more in odontogenic keratocysts. They also found that radicular cysts showed higher levels of the pro‐apoptotic protein Bax and low levels of bcl‐2, suggesting that low levels of proliferation are accompanied by high levels of apoptosis. Others have identified apoptotic markers in radicular cysts and have suggested that this contributes to their slow growth (Loreto et al. 2013 ). As the cyst continues to expand, this slow proliferation and high levels of apoptosis probably lead to the thinning of the epithelial lining that is seen in long‐standing and residual cysts (Figure 3.11).
Figure 3.11 A long‐standing radicular cyst. The cyst wall has become fibrous with little inflammation and the epithelial lining is relatively thin and regular. Note the considerable cellular and proteinaceous debris in the lumen (top left).
Degradation of the Connective Tissues and Bone Resorption
The next crucial element of cyst growth is degradation of the connective tissues. Among the most important and widely studied factors are the matrix metalloproteinases (MMPs). These are a large family of calcium‐dependent and zinc‐containing proteases, capable of degrading a wide range of extracellular matrix proteins. A number of studies have shown expression of MMPs in odontogenic cysts, including the gelatinases (MMP‐2 and MMP‐9; Teronen et al. 1995a ; Kubota et al. 2000 ; D'addazio et al. 2014 ; Alvares et al. 2017 ; Andrade et al. 2017 ) and collagenases (MMP‐1, MMP‐8, and MMP‐13; Teronen et al. 1995b ; Lin et al. 1997 ; Wahlgren et al. 2001 ; D'addazio et al. 2014 ; Andrade et al. 2017 ), suggesting a role for these enzymes in cyst growth and development. Furthermore, MMP activity increases with the intensity of inflammation and is often more prominent in periapical granulomas than in established cysts (Lin et al. 1997 ; Andrade et al. 2017 ). In keeping with this, inflammatory cytokines, especially IL‐1, up‐regulate active MMP‐9 in odontogenic cysts (Kubota et al. 2000 ) and increased numbers of mast cells are associated with MMP activation in odontogenic cysts (Teronen et al. 1996 ; Rodini et al. 2008 ; Andrade et al. 2017 ). Inflammation in radicular cysts is also associated with secretion of neutrophil collagenase (MMP‐8) and with increased expression of plasminogen activator (Tsai et al. 2004 ), which indirectly forms plasmin that may also activate MMPs.
All odontogenic cysts are encased in the bone of the jaws, and therefore any expansion must be associated with bone resorption. Normal bone undergoes continuous remodelling controlled by a number of factors, the most important of which are receptor activator of nuclear factor kappa B (RANK), its ligand (RANKL), and osteoprotegerin (OPG, also known as osteoclastogenesis inhibitory factor). These form the RANKL/RANK/OPG system, which is primarily responsible for bone homeostasis (Graves et al. 2011 ; Kular et al. 2012 ). RANK is expressed on the surface of osteoclasts and their precursor cells and when bound by RANKL promotes osteoclast formation and maturation. OPG secreted by osteoblasts acts as a decoy receptor and binds RANKL to prevent activation by RANK. RANKL is primarily cell bound on the surface of osteoblasts and in normal bone osteoblast–osteoclast interactions is the main method of bone turnover regulation. In pathological conditions, however, RANKL may be secreted by osteoblasts and a number of other cell types, including Th1 lymphocytes, endothelial cells, and epithelial cells, and may function as a soluble receptor. It should also be noted that RANKL is the only cytokine that can stimulate osteoclastogenesis, although a number of other factors support recruitment of osteoclast precursors, or promote osteoclast maturation and activation (Table 3.2).
Perturbation of the RANKL/RANK/OPG system is involved in all pathological conditions associated with bone resorption, and many studies have now provided evidence for its role in increased osteoclast activity in many odontogenic lesions, including radicular cysts (Tay et al. 2004 ; Menezes et al. 2006 ; da Silva et al. 2008; de Moraes et al. 2011 ; Graves et al. 2011 ; Soluk Tekkaşin et al. 2011 ; Belibasakis et al. 2013 ).
Tay et al. (2004 ) were the first to demonstrate that RANKL was expressed in osteolytic lesions of the jaws, including radicular cysts. RANKL was expressed in the fibrous wall of radicular cysts, and its association with osteoclast recruitment was confirmed by the demonstration of tartrate‐resistant acid phosphatase (TRAP) and calcitonin‐receptor positive osteoclasts adjacent to the RANKL‐positive cells. In a more detailed analysis, Menezes et al. (2006 ) confirmed these findings, but were also able to demonstrate that RANKL and OPG were expressed by a range of cell types, including PMNs, lymphocytes, macrophages, endothelial cells, and the epithelial lining of radicular cysts. They found that RANKL was more highly expressed than OPG and that expression was greater in cysts than granulomas. Other studies have shown similar levels of expression, but have also shown, as would be expected, that RANKL expression is not specific to radicular cysts and is also found at similar or even greater levels in dentigerous cysts, odontogenic keratocysts, and ameloblastomas (Tay et al. 2004 ; Menezes et al. 2006 ; de Moraes et al. 2011 ; Soluk Tekkaşin et al. 2011 ). It is likely that the level of expression at any moment in time will be related to the degree of ‘maturation’ of the cyst or to the extent of inflammation. Kawashima et al. (2007 ) studied the kinetics of RANKL, RANK, and OPG expression in experimentally induced periapical lesions in rats. They showed that all three factors peaked at between two and three weeks, but that the greatest increase was in RANKL, with the highest RANKL/OPG ratios at this time. They also found that RANKL was expressed in a range of cell types, but these were close to the alveolar bone and were associated with activated osteoclasts. The up‐regulation of the RANKL/RANK/OPG system also correlated to increased expression of pro‐inflammatory cytokines, known to be able to activate RANKL, including IL‐1α and IL‐1β. Similar data have been presented in a number of subsequent studies (reviewed in da Silva et al. 2008 ; Graves et al. 2011 ; Belibasakis et al. 2013 ), confirming the role of inflammatory cytokines and the close association between the inflammatory response and bone resorption.
The most important cytokines involved in the process are indicated in Table 3.2, and include IL‐1α (which was originally called osteoclast‐activating factor, OAF), IL‐6, and TNF‐α. A range of chemokines are also responsible for chemotaxis of osteoclast precursors and differentiation of osteoclasts, including CXCL8/IL‐8, which has been mentioned previously as an important initiator of the process, since it is up‐regulated by LPS and also chemotactic to PMNs. Prostaglandins have also been shown to be important mediators of bone remodelling and may act to stimulate both bone deposition and resorption. There are a number of prostaglandins, but PGE2 is particularly important in bone resorption, since it stimulates expression of RANKL and inhibits OPG on osteoblasts (Blackwell et al. 2010 ). Early studies by Harris and his research group (Harris and Goldhaber 1973 ; Harris et al. 1973 ; Harris 1978 ; Meghji et al. 1989 ) were the first to demonstrate that cultures of cyst walls had potent bone‐resorbing activity, and identified prostaglandins as a key factor. Matejka et al. (1985a ,b , 1986 ) confirmed these findings and also demonstrated that the granulation tissue and the inflammatory cells in the wall of radicular cysts were the main source of PGE2. Further studies from Harris's group (Bando et al. 1993 ; Meghji et al. 1996 ) reinforced these findings and also showed that IL‐1 and IL‐6 were the predominant cytokines with bone‐resorbing activities.