Introduction

Gingivitis is the most prevalent periodontal infection in children and adolescents [1], so today the problem of gingivitis has been identified with a significant long-term social and medical impact on individuals, groups, populations, and social levels in the world, especially in childhood [2]. Gingivitis or gum inflammation is caused by the accumulation of microbial plaque (bacteria) on the tooth surface [3]. It is characterized by halitosis and painless gum bleeding, either spontaneously or during brushing [3]. The gum is one of the components of the periodontal tissue and its diseases can lead to the destruction of the supporting structures of the teeth and alveolar bone [4]. Most of the gum is made up of connective tissue that contains high levels of collagen [5]. In normal healthy gums, collagen makes up around 60% of the total proteins [6]. During the development of gingivitis and periodontitis, significant changes occur in the collagen and non-collagenous proteins of the gums [7], so that 70% of the connective epithelium collagen is lost in the early stages of inflammation [8]. Collagen is composed of different amino acids, the most important of which are glycine, proline, hydroxylysine, and hydroxyproline [5]. Collagen stability is enhanced by hydroxyproline and proline [9, 10].

On the other hand, due to recent advanced technology, various salivary markers have become potentially important in the early diagnosis of oral diseases, and some salivary amino acids play a significant role in predicting periodontal status, including plaque-induced gingivitis [2]. Saliva is an available biological fluid and its biomarkers provide necessary information about oral health. [11]. Factors such as saliva composition and saliva flow rate are very important and necessary for diagnostic, experimental and clinical protocols [12]. Saliva can be extracted from the mucosal surfaces and gingival crevices as well as from the dental surfaces of the oral cavity [11]. Unstimulated saliva is a popular method for examining saliva’s composition. Unstimulated saliva refers to saliva that is passively collected by pouring saliva into the sample container [13].

Several studies have examined the predictive power of hydroxyproline in saliva in the diagnosis of periodontal diseases [14, 15]. These studies have mostly investigated the concentration of hydroxyproline in gingival crevicular fluid (GCF) in adults, for which sampling requires clinical intervention. A few studies conducted in children have measured the amount of hydroxyproline in other body fluids such as urine [16]. In children, oral cavity examination is mostly limited to hard tissue assessment, and soft tissues like gums are given less attention. The short lifespan of primary dentition may have led to a lack of attention to gum health in children. Furthermore, the progression of untreated gingivitis can be accompanied by more serious types of periodontal disease in children. In addition, most children are afraid of receiving dental services and care [17], and traditional diagnostic methods for these diseases include clinical observation, patient history, and measurements such as probe depth assessment and radiography, which are uncomfortable and prone to measurement errors [18]. Therefore, providing simple, convenient diagnostic services without the intervention of health workers in this age group is of particular importance. Therefore, this study investigated the relationship between the concentration of hydroxyproline in unstimulated saliva and the occurrence of gingivitis in 6 to 7-year-old children.

Materials and methods

Results

Discussion

In this case-control study, which was conducted to evaluate and compare the level of salivary hydroxyproline in healthy 6 to 7-year-old children and those with gingivitis, higher levels of salivary hydroxyproline were observed in the group with gingivitis. The results of this study are in agreement with the results of Akalin et al. (1993) [21], Ravi et al. (2021) [14], Koss et al. (2010) [22], and Koss et al. (2009) [23] are consistent. In the study of Ravi et al. (2021) [14] who compared hydroxyproline levels in gingival tissue and GCF samples of healthy people and people with chronic periodontitis, observed a positive correlation between clinical parameters in both groups with hydroxyproline levels and higher concentrations of hydroxyproline reported in GCF and gingival tissue samples of people with chronic periodontitis [14]. Koss et al. (2010) [22] in their study investigated the quantification of inflammation and tissue destruction biomarkers in the GCF to help diagnose the severity of gingival-periodontal disease in adults with gingival-periodontal disease. In that study, total protein increased in gingivitis, moderate and severe periodontitis, and hydroxyproline increased in moderate and severe periodontitis. Finally, they concluded that the levels of GCF biomarkers specific for inflammation and collagen degradation are associated with clinical diagnosis in patients with gingivitis and chronic periodontitis. Also, Koss et al. (2009) [23] in another study that aimed to identify salivary parameters effective in diagnosing different stages of periodontal disease, after examining adults with mild, moderate, or severe chronic periodontitis, concluded that hydroxyproline significantly attention increased in all groups with periodontal diseases compared to healthy people. The study of Akalin et al. (1993) [21] also observed higher gingival hydroxyproline levels in periodontitis samples compared to the control group.

Therefore, it can be concluded that in addition to GCF samples, the level of hydroxyproline in unstimulated saliva samples can also be used as a biomarker in the diagnosis of gingivitis. Because, periodontitis is the primary cause of tooth loss in today’s society [24]. In addition, compared to serum or other saliva-based methods, unstimulated saliva collection is easily performed without any professional assistance [25] and can reduce children’s fear of receiving dental diagnostic services. Also, storage and transportation as well as large sampling make saliva the best research fluid compared to serum or urine [25]. However, to confirm the results of this study, further studies with a larger sample size comparing different severities of periodontitis such as mild, moderate, and severe forms are needed.

On the other hand, considering that the consequences of periodontal diseases observed in adulthood are established in childhood [26, 27], prevention, timely diagnosis and control of gum-related diseases in childhood has a lot of importance [27]. In the present study, the condition of oral and dental health in students with gingivitis was worse than in the non-affected group. So that in the affected group, 21.9% had poor oral hygiene, while this rate was 3.1% in healthy students. In terms of dental care attendance, no difference was observed between the two groups. This result is consistent with the results of other studies [28–30]. Improper oral and dental hygiene and ineffective brushing are known to be the most important factors affecting gum disease in children [30, 31]. Studies conducted in Iran have found that improper oral hygiene (lack of daily brushing), male gender, oral breathing, lower income, family with a higher number of children in the family, mother’s employment status, and mother’s low education level are effective in reducing gum health [28, 29, 32]. Therefore, it is necessary to implement educational programs in intervention methods for children and their parents.

This study has several limitations. The most important limitation is the small sample size, conducting the study in a specific age group (6 to 7 years) and gender (male). Therefore, generalizing the results to other age and gender groups should be done with caution.