**3.2 Biomarkers determined in saliva**

*Periodontal Disease - Diagnostic and Adjunctive Non-surgical Considerations*

diseases [22].

for oxidative stress [24].

*3.1.8 Telopeptide*

clinical use.

*3.1.9 miRNAs (microRNAs)*

performance of cellular activities [28].

*3.1.10 Other markers of periodontal disease*

susceptibility [21]. In recent years, the inflammatory response has been

bacterial infection and the response of the host to the bacterial challenge, and the disease is modified by environmental factors, acquired risk factors, and genetic

associated with oxidative stress, specifically with reactive oxygen species since it is considered to play a central role in the progression of many inflammatory

Oxidative stress create multiple products in affected tissues, such as reactive oxygen species which are free radicals and other non-radical derivatives which are involved in normal cell metabolism [23], other metabolites can damage DNA such as 8-hydroxy2′-deoxyguanosine (8-OHdG) or 8-oxo-7,8-dihydro-2′ deoxyguanosine (8-oxodG) which are two of the predominant forms of free radicals induced by oxidative lesions. In fact, 8-oxodG has been widely used as a biomarker

Bone resorption is a basic physiological process that is central to the understanding of many key pathologies, with its most common oral manifestation seen as the alveolar bone destruction in periodontitis [25]. The osteoid matrix consists principally of collagen (90%), other smaller proteins, and proteoglycans. The main structural protein of the bone is type I collagen. Consequently, most available bone resorption markers are based on degradation products of type I collagen. According to Koizumi et al. [26], ICTP (telopeptide) is one of the best markers for

Nowadays, RNAs that do not code for protein have taken on great importance because, in addition to maintaining their importance in the determination of cellular phenotypes [27], now they are recognized as dynamic participants in the

It has been mentioned and demonstrated that miRNAs are involved in bone metabolism, in fact, some studies have shown that they are associated with the activator of the nuclear factor receptor kappa-B ligand (RANKL) induced osteoclastogenesis. Within these miRNAs, miR-223 [29] was the first to associate with periodontal tissue, although other miRNAs such as miR-15a, miR-29b, miR-125a, miR-146a, miR148 / 148a, miR-223 and miR-92 have been identified more recently as important in periodontal health and have even been considered potential biomarkers [30].

Other biomarkers have been analyzed to determine periodontitis. One of these is chondroitin sulfate, which is a natural glycosaminoglycan (GAG) present in the extracellular matrix [31]; chondroitin sulfate is recognized for its immunomodulatory effects, such as the reduction of nuclear translocation NF-κB, the decrease in the production of pro-inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α), and the decrease in expression and activity of nitric oxide synthase-2 (NOS-2) and cyclooxygenase-2 (COX-2) [32]. Another molecule that has been proposed as a possible biomarker is MUC-4. Mucins are high-molecular-weight glycoproteins, are involved in diverse biological functions, are members of transmembrane mucin family, and are expressed in airway epithelial cells and body fluids like saliva, tear film, ear fluid, and breast milk [33]. It has been reported that the production of MUC-4 could be regulated by inflammatory cytokines [34].

**22**

Saliva is a seromucous secretion, consisting of 99% water; however, saliva is also composed of glycoproteins, phosphate ions, bicarbonate, sodium, chlorine, fluorine, calcium, and potassium and has a neutral pH [35], which forms a film of liquid consistency that covers the surface of the oral mucosa, with the purpose of lubricating it and keeping it moist among many other characteristics for the maintenance of oral health [36]. The composition of saliva varies from one place to another in the oral cavity of each individual [35]. If there are changes in its composition, there may be significant alterations in deterioration of the health of the host [37].

Due to the described characteristics of saliva, several authors have claimed that these salivary constituents may actually be useful indicators of both local and systemic disorders. These revelations have formed the basis of the field of saliva diagnosis and, therefore, have triggered research that culminated in the identification of saliva-based biomarkers for disorders ranging from cancer to infectious diseases [38]. In addition to the above, saliva has several advantages when compared to other sources for diagnosing diseases since saliva is easily collected and stored and ideal for early detection of disease as it contains specific soluble biological markers [39]. Saliva has been used to diagnose diseases as diverse as autoimmune disorders, cardiovascular diseases, diabetes, HIV, oral cancer, and oral diseases [40].

### *3.2.1 Proteins (cytokines) determined in saliva that could be used as biomarkers*

In our review we observed that there are about 15 works that were dedicated to investigate the possible use of these proteins determined in saliva as biomarkers to determine periodontitis. We can say that of all the works, the majority focuses on comparing healthy groups with periodontitis; only three researches include the group of gingivitis, which indicates that this group should be used more for this type of studies. We need to remember that gingivitis is considered an intermediate stage that may or may not lead to periodontitis [41], and if the patient performs good dental hygiene in combination with the treatment, in general, progression to periodontitis can also be stopped [42].

On the other hand, the cytokine that has been most explored and that better results have given as biomarkers to detect in saliva is the cytokine IL-1β [43–48]; this must be due to the recognized importance of interleukin-1β, as an important mediator in the pathophysiology of periodontitis [49].

However, other cytokines such as IL-6 and IL-2 have also been explored [43, 44, 48, 50]; IL-6 is recognized for playing a role as a pro-inflammatory cytokine acting on bone resorption in the presence of infections [51]. Regarding IL-2, a study that investigated cytokine profiles at different stages of the development of periodontitis found that levels of mRNA for IL-2 were significantly associated with the phase of resolution of the disease [52]. This agrees with reports that IL-2 has been implicated in the stimulation of osteoclasts [53].

Regarding MCP-1, Gupta et al. [54] conducted a study in 45 patients with an average age of 43 years for healthy patients and 41 for patients with periodontitis, with results similar to Nisha et al. [55]. In both studies it was found that the levels of MCP-1 in saliva can be a good biomarker for the development of periodontal disease. One difference between the studies is that Nisha's work included a group of patients with gingivitis, while Gupta's study does not include it.

Regarding the possibility of using prostaglandin E2 (PGE2) as biomarkers in saliva to diagnose gingivitis, Syndergaard et al. [56] conducted a study with 80 participants, 40 without gingivitis and 40 with gingivitis, and found that the levels of PGE2 in the group with gingivitis were significantly higher compared with the

control group. This study reported that PGE2 remained high after prophylaxis. As for other studies conducted with the purpose of comparing the concentrations of PGE2, Sanchez et al. [82] conducted a study in which the population was 74 adult subjects who were grouped according to the progress of the periodontal disease in mild, moderate, and severe; the conclusion was that the levels of PGE2 increase as the severity of the periodontal disease progressed. In addition a high sensitivity and specificity were reported.

When TNF-α is evaluated as a possible biomarker for the diagnosis of periodontitis, we found that there are discrepancies since in some studies, such as Eivazi et al. [57] who conducted a study with one healthy group and another with chronic periodontitis which reported that before and after starting treatment, the concentrations of TNF-α in saliva were higher in the healthy group than in the periodontitis group. In contrary to the results reported by Yue et al. [48], they found that TNF-α concentrations were higher in the saliva of patients with advanced periodontitis than in the saliva of healthy subjects. Yue's study is supported by studies that investigate the loss of the alveolar bone since the concentration of TNF-α in subjects with alveolar resorption is low [58].

Yue et al. [48] found that in the saliva of patients with aggressive periodontitis (AP) have higher levels of IFN-**γ** in the saliva compared to subjects without AP; this decrease was statistically significant throughout the course of treatment (p < 0.05).

#### *3.2.2 Metalloproteinases*

Matrix metalloproteinases (MMPs) are key proteases involved in destructive periodontal diseases. A total of 23 MMPs have been described. These MMPs can be found in periodontal tissues as pro-forms, active forms, complex species, fragmented, and cell-bound species [59]. MMPs are the most important group of proteinases responsible for the degradation of extracellular matrix proteins during periodontitis, and any imbalance between MMPs and their inhibitors can trigger the degradation of the ECM, the basement membrane, and the alveolar bone [60].

In this way and due to the importance of MMPs, several researchers have been dedicated to try to determine if MMPs are opportune as biomarkers. Gursoy et al. [61] did a study with the objective of detecting possible markers of periodontitis in saliva, with high sensitivity and specificity; to determine this, the salivary concentrations of MMP-8, MMP-9, and MMP-13 among others were measured in 230 subjects. The concentrations of MMP-8, MMP-9, and MMP-13 in saliva were higher in subjects with generalized periodontitis than in controls; however, according to the authors, MMP-8 was the only marker capable of differentiating subjects with severe bone loss of those who presented mild bone loss, so they consider that MMP-8 is a strong candidate to detect alveolar bone destruction [61]. These results were corroborated by Rathnayake et al. [46] who found that MMP-8 could be used as a marker of periodontal disease in large patient populations. An interesting fact that they reported is that smokers compared to non-smokers showed slightly lower concentrations of MMP-8.

Another interesting fact regarding MMP-8 by Ebersole et al. [25] in a study that included 30 healthy volunteers and 50 patients diagnosed with chronic periodontitis is that MMP-8 (among others) was investigated as a biomarker associated with inflammatory and destructive processes of periodontal disease and reported that the levels of MMP-8 of patients who have periodontitis are very different from the normal levels found in healthy subjects and showed a particular diagnostic potential.

Morelli et al. [50] examined 168 participants and found higher salivary levels of matrix metalloproteinases, MMP-3, MMP-8, and MMP-9, in diseased groups compared to healthy. In the same year, Miricescu et al. [62] carried out a study where 20

**25**

levels.

*Use of Biomarkers for the Diagnosis of Periodontitis DOI: http://dx.doi.org/10.5772/intechopen.85394*

tis compared to controls.

periodontitis in their patients.

gender implications in periodontitis.

but not with periodontal bone destruction [70].

periodontitis.

*3.2.3 Calcium*

*3.2.4 Phosphorus*

patients were also included with chronic periodontitis and 20 controls and different biomarkers were evaluated including matrix MMP-8, and as a result it was found that the levels of MMP-8 were significantly increased in patients with chronic periodonti-

Ebersole et al. [43] conducted a study that included 65 healthy subjects, 43 subjects with gingivitis, and 101 subjects with periodontitis. In this study, the levels of MMP-8 very similar to the previous studies stood out in a significant way in the group of periodontitis compared to those of gingivitis and healthy subjects. In a more categorical way, Borujeni et al. [63] reported that MMP-8 provides a substantial sensitivity with which physicians can use the test for MMP-8 and thus detect

Similarly, Gupta et al. [64] made an investigation with the objective of establishing MMP-8 as a noninvasive marker for the early diagnosis of chronic periodontitis. The study included 40 subjects who were divided into two groups: 20 healthy subjects and 20 patients with chronic periodontitis. The results of this study demonstrate high concentrations of MMP-8 in individuals with chronic

Already Lira et al. [65] continued to explore the importance of MMP-8 and conducted a study that aimed to evaluate the levels of markers related to innate immunity, the MMP-8 in the saliva from patients with aggressive generalized periodontitis, and patients with gingivitis and healthy. In the saliva, MMP-8 levels were higher in aggressive periodontitis than in healthy patients; in this way it is reaffirmed that MMP-8 can be an important biomarker of periodontitis.

Other researchers such as Virtanen et al. [66] continued to look for other metalloproteinases as potential biomarkers, and some reaffirm that the salivary concentrations of matrix metalloproteinases such as MMP-8 and MMP-9 are slightly higher in patients with periodontitis, although they report that the differences between the groups were not significant. Interestingly, this group reports that MMP-13 values were significantly higher in the group without periodontitis compared to patients with periodontitis and also report that the concentration of MMP-13 may have some

Following with the MMP-8, Mauramo et al. [67] studied whether the levels of MMP-8 in the saliva are associated with periodontitis in 258 subjects. Periodontitis was more frequent among subjects with high levels of MMP-8 in the saliva. The highest levels of salivary MMP-8 were associated with any periodontal diagnosis (mild, moderate, or severe). They concluded that elevated levels of MMP-8 in the

When we search for studies that have explored the detection of calcium present in saliva as a biomarker, we find that while some studies report the usefulness of calcium because the subjects in the periodontitis group had significantly higher levels of salivary calcium than gingivitis and healthy group [68, 69], another work find that high salivary calcium content can be correlated with good dental health

According to Patel et al. [69] study, phosphorus can be considered a biomarker for the diagnosis of sick and healthy periodontal tissues. The study concludes that as the severity of periodontal disease increases, it also increases total phosphorus

saliva are associated with periodontitis in a normal adult population.

*Periodontal Disease - Diagnostic and Adjunctive Non-surgical Considerations*

specificity were reported.

with alveolar resorption is low [58].

*3.2.2 Metalloproteinases*

concentrations of MMP-8.

control group. This study reported that PGE2 remained high after prophylaxis. As for other studies conducted with the purpose of comparing the concentrations of PGE2, Sanchez et al. [82] conducted a study in which the population was 74 adult subjects who were grouped according to the progress of the periodontal disease in mild, moderate, and severe; the conclusion was that the levels of PGE2 increase as the severity of the periodontal disease progressed. In addition a high sensitivity and

When TNF-α is evaluated as a possible biomarker for the diagnosis of periodontitis, we found that there are discrepancies since in some studies, such as Eivazi et al. [57] who conducted a study with one healthy group and another with chronic periodontitis which reported that before and after starting treatment, the concentrations of TNF-α in saliva were higher in the healthy group than in the periodontitis group. In contrary to the results reported by Yue et al. [48], they found that TNF-α concentrations were higher in the saliva of patients with advanced periodontitis than in the saliva of healthy subjects. Yue's study is supported by studies that investigate the loss of the alveolar bone since the concentration of TNF-α in subjects

Yue et al. [48] found that in the saliva of patients with aggressive periodontitis (AP) have higher levels of IFN-**γ** in the saliva compared to subjects without AP; this decrease was statistically significant throughout the course of treatment (p < 0.05).

Matrix metalloproteinases (MMPs) are key proteases involved in destructive periodontal diseases. A total of 23 MMPs have been described. These MMPs can be found in periodontal tissues as pro-forms, active forms, complex species, fragmented, and cell-bound species [59]. MMPs are the most important group of proteinases responsible for the degradation of extracellular matrix proteins during periodontitis, and any imbalance between MMPs and their inhibitors can trigger the degradation of the ECM, the basement membrane, and the alveolar bone [60]. In this way and due to the importance of MMPs, several researchers have been dedicated to try to determine if MMPs are opportune as biomarkers. Gursoy et al. [61] did a study with the objective of detecting possible markers of periodontitis in saliva, with high sensitivity and specificity; to determine this, the salivary concentrations of MMP-8, MMP-9, and MMP-13 among others were measured in 230 subjects. The concentrations of MMP-8, MMP-9, and MMP-13 in saliva were higher in subjects with generalized periodontitis than in controls; however, according to the authors, MMP-8 was the only marker capable of differentiating subjects with severe bone loss of those who presented mild bone loss, so they consider that MMP-8 is a strong candidate to detect alveolar bone destruction [61]. These results were corroborated by Rathnayake et al. [46] who found that MMP-8 could be used as a marker of periodontal disease in large patient populations. An interesting fact that they reported is that smokers compared to non-smokers showed slightly lower

Another interesting fact regarding MMP-8 by Ebersole et al. [25] in a study that included 30 healthy volunteers and 50 patients diagnosed with chronic periodontitis is that MMP-8 (among others) was investigated as a biomarker associated with inflammatory and destructive processes of periodontal disease and reported that the levels of MMP-8 of patients who have periodontitis are very different from the normal levels found in healthy subjects and showed a particular diagnostic potential. Morelli et al. [50] examined 168 participants and found higher salivary levels of matrix metalloproteinases, MMP-3, MMP-8, and MMP-9, in diseased groups compared to healthy. In the same year, Miricescu et al. [62] carried out a study where 20

**24**

patients were also included with chronic periodontitis and 20 controls and different biomarkers were evaluated including matrix MMP-8, and as a result it was found that the levels of MMP-8 were significantly increased in patients with chronic periodontitis compared to controls.

Ebersole et al. [43] conducted a study that included 65 healthy subjects, 43 subjects with gingivitis, and 101 subjects with periodontitis. In this study, the levels of MMP-8 very similar to the previous studies stood out in a significant way in the group of periodontitis compared to those of gingivitis and healthy subjects. In a more categorical way, Borujeni et al. [63] reported that MMP-8 provides a substantial sensitivity with which physicians can use the test for MMP-8 and thus detect periodontitis in their patients.

Similarly, Gupta et al. [64] made an investigation with the objective of establishing MMP-8 as a noninvasive marker for the early diagnosis of chronic periodontitis. The study included 40 subjects who were divided into two groups: 20 healthy subjects and 20 patients with chronic periodontitis. The results of this study demonstrate high concentrations of MMP-8 in individuals with chronic periodontitis.

Already Lira et al. [65] continued to explore the importance of MMP-8 and conducted a study that aimed to evaluate the levels of markers related to innate immunity, the MMP-8 in the saliva from patients with aggressive generalized periodontitis, and patients with gingivitis and healthy. In the saliva, MMP-8 levels were higher in aggressive periodontitis than in healthy patients; in this way it is reaffirmed that MMP-8 can be an important biomarker of periodontitis.

Other researchers such as Virtanen et al. [66] continued to look for other metalloproteinases as potential biomarkers, and some reaffirm that the salivary concentrations of matrix metalloproteinases such as MMP-8 and MMP-9 are slightly higher in patients with periodontitis, although they report that the differences between the groups were not significant. Interestingly, this group reports that MMP-13 values were significantly higher in the group without periodontitis compared to patients with periodontitis and also report that the concentration of MMP-13 may have some gender implications in periodontitis.

Following with the MMP-8, Mauramo et al. [67] studied whether the levels of MMP-8 in the saliva are associated with periodontitis in 258 subjects. Periodontitis was more frequent among subjects with high levels of MMP-8 in the saliva. The highest levels of salivary MMP-8 were associated with any periodontal diagnosis (mild, moderate, or severe). They concluded that elevated levels of MMP-8 in the saliva are associated with periodontitis in a normal adult population.
