**1. Introduction**

#### *1.1. Diabetes Mellitus, Periodontal Disease and Their Interaction*

Diabetes mellitus (DM) is a chronic metabolic disease that alters the physiologic circuit of glucose. If left untreated it can lead to deadly complications such as cardiovascular disease, brain stroke, loss

of sight or renal insu fficiency. In order to enter a cell and to be used for metabolic purposes, glucose requires the presence of insulin. The pancreas produces insulin, but in some situations, it does not produce enough [1]. This is the cause of type I diabetes, also known as "insulin-dependent DM" because patients need insulin administration during treatment. In other situations, the cells may be resistant and insensitive to insulin action, therefore preventing glucose metabolism. This happens in type II diabetes, which is often associated with obesity. The first stages of type II diabetes treatment include diet control, exercise, and antidiabetic medication but may eventually lead to insulin administration as well. Type II DM is the most common type of adult diabetes, being diagnosed in about 90% of diabetes cases [1].

Periodontal disease (PD) is a chronic inflammatory disorder and a worldwide public health challenge. Since the 1960s scientific evidence has been published regarding an association between DM and periodontitis [2]. It is related to many other chronic diseases, such as cardiovascular disease, inflammatory bowel disease, rheumatoid arthritis, respiratory tract infection and Alzheimer's disease, displaying a particular interest in the relationship between oral and systemic health [3–5]. PD is caused by specific oral microorganisms, such as *Porphyromonas gingivalis, Treponema denticola*, *Tannerella forsythia* and *Aggregatibacter actinomycetemcomitans* [6–8], inducing loss of periodontal ligament and alveolar bone, also representing the primary cause of tooth loss [9]. Periodontal impairment is influenced by many risk factors, including alcohol, stress, smoking, heredity, DM and endocrinological changes (pregnancy or menopause); thus, maintaining periodontal health becomes a real challenge [10]. The human oral microbiome is important in the pathogenesis of PD, nutrition being a significant aspect in promoting periodontal homeostasis through antioxidant and immunomodulatory e ffects on bone metabolism [10–12].

Since 2012, the American Diabetes Association has been including the periodontal examination of diabetic patients in its "Standards of Medical Care for Diabetes". This action has been motivated by the fact that PD was o fficially recognized as a complication of DM, together with the five other vascular-derived ones (retinopathy, neuropathy, etc.) [13].

Conversely, DM is also credited with an important influence on the pathogenesis process of certain types of PD, as illustrated by the latest classification of periodontal conditions, issued by the European Federation of Periodontology and the American Academy of Periodontology in 2018 [14]. The bidirectional relationship between the two disorders is currently well documented, opening perspectives of common managemen<sup>t</sup> of diabetic and periodontal patients, in terms of prevention, early diagnosis and integrated treatment protocols [15].

The negative impact that diabetic pathology has on the periodontal status of a ffected patients has been explained by various mechanisms. From a cellular perspective, it seems that the mobility, activity and e fficiency of immune cells, such as polymorphonuclear leukocytes is decreased in a diabetic setting, favoring the aggressive actions of periodontal bacterial pathogens [16,17]. Also, the antibacterial capacity of the saliva and gingival crevicular fluid (GCF) could be downregulated in DM patients, further enhancing the growth of harmful bacteria. In addition to this, periodontal ligament fibroblasts have been shown to decrease chemotaxis when placed in vitro in a hyperglycemic environment [18]. The glucose-rich GCF of DM patients is one such environment, which may explain the di fficult periodontal wound healing and the reduced local host response to bacterial attack, all favoring the onset of periodontal inflammation and damage.

Proinflammatory markers, which drive the inflammatory reaction, are secreted by certain immune cells when they are stimulated by bacterial antigens. It has been shown that the immune cells of DM patients over-react to bacterial antigen stimulation, causing an overproduction of proinflammatory markers. Consequently, a more intense inflammatory periodontal reaction is triggered in DM patients, causing the rapid destruction of periodontal tissues [19,20]. The involved proinflammatory markers include major cytokines, such as interleukin 1β (IL-1β), tumor necrosis factor-alpha (TNFα) and prostaglandin E2 (PGE2), which are all majorly upregulated in DM patients' GCF compared to non-DM

patients. When compared, the GCF levels of PGE2 and IL–1β were higher in DM patients' samples than in non-DM ones, in similar settings of PD inflammation and dissolution.

Poorly controlled diabetes is a key factor in the onset of aggressive and destructive forms of PD [21].

Some studies support the direct connection between high PD prevalence and severity in DM patients [22]. This seems to be especially true for type 2 DM patients, who are more prone to difficulty in glycemic control [23]. Poor glycemic control can also impact the outcome of periodontal treatment. Patients with well-controlled glycemia have been shown to reach similar results after nonsurgical periodontal treatment (scaling and root planning) as those of non-DM patients at a four month recall [24]. In contrast, a less favorable response to treatment can be expected from DM patients with uncontrolled glycemia [25]. Periodontal surgery also delivers similar results in terms of periodontal pocket reduction for well-controlled glycemia DM patients compared to non-diabetic ones [26]. Therefore, favorable results can be expected when treating periodontally compromised DM patients with stable glycemia levels.

It was also found that PD patients with undiagnosed DM exhibit significantly increased glycosylated hemoglobin (HbA1C) serum levels compared to periodontally healthy individuals, and PD was positively correlated with serum levels of (HbA1C) before DM onset [27]. If PD acts as an aggravating cofactor for later DM complications, its treatment may be a way to improve the diabetic status and to stabilize glycemic levels, thereby preventing the onset of dangerous complications.

#### *1.2. Oxidative Stress and Reactive Oxygen Species—Background*

Oxidative stress is a state of imbalance between oxidants and antioxidants in favor of oxidants, leading to harmful effects [28]. Oxidants, also called reactive oxygen species (ROS), include free radicals such as O2• - (superoxide), ONOO - (peroxynitrite) and HO• (hydroxyl) and nonradicals, such as H2O2 (hydrogen peroxide), are products of aerobic cell metabolism by reducing oxygen molecules [29]. There are many sources of ROS, mainly generated by enzymes such as xanthine oxidase, cyclooxygenase, lipooxygenase, myeloperoxidase, cytochrome P450 monooxygenase, uncoupled nitric oxide synthase (NOS), peroxidase and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. They arise intracellularly, extracellularly, or in specific intracellular compartments [30] and are generated by polymorphonuclear lymphocytes through NADPH oxidase [31].

Oxygen-derived free radicals are oxidative agents produced during events such as mitochondrial respiration and phagocytosis, causing post-translational modifications of proteins, with an impact on cell signaling, gene expression and other physiological processes [32]. Low concentrations of ROS enhance antioxidant response by activating a nuclear factor erythroid 2-related factor 2, promoting cell survival [33]. ROS-induced impairment of glycocalyx, cell membranes and junctions contribute to increased permeability and leukocyte and thrombocyte adhesion, with subsequent local activation of inflammation and coagulation, leading to loss of endothelial vasodilation potential and attenuation of vasoconstrictor response [34].

There is also a documented link between oxidative stress, DM and PD, with the oxidative-stress-mediated changes in the inflammatory pathways being possible mechanisms in affecting periodontal tissues [35] (Figure 1). DM and PD involve significant impairment of immune system regulation, while hyperglycemia contributes to advanced glycation end products (AGE) formation and extended levels of proinflammatory cytokines IL-1β, interleukin 6 (IL-6) and TNF-α [36] (Figure 1).

**Figure 1.** The interrelation between vitamin C, diabetes mellitus (DM) and periodontal disease (PD).

#### *1.3. Vitamin C, DM, and PD*

In the 1920s, the forthcoming Nobel laureate Albert Szent-Györgyi from Szeged University, Hungary, identified vitamin C and its role in preventing and treating scurvy [37]. Ascorbic acid (vitamin C) is an essential water-soluble vitamin that cannot be synthesized by the human organism [38,39]. It demands a regular and appropriate intake from natural sources, like citrus fruits, mango, strawberries, kiwi, papaya, green leafy vegetables, tomatoes and broccoli [37], to hamper hypovitaminosis C that is relatively common in Western populations [38,39].

Synthetic vitamin C derived from chemicals is similar to that contained in fruits and vegetables [40]. The main route of administration for ascorbic acid is oral ingestion from food or supplements. Healthy individuals generally need 0.1–0.2 g daily doses. Intravenous administration is used in critically ill patients requiring high doses (1–4 g/day) of this nutrient [41,42]. It is quickly eliminated by the kidneys with a half-life of approximately two hours [41,43].

L-ascorbate, the reduced form of vitamin C, is a physiological antioxidant [44]. Antioxidants are molecules that can donate a hydrogen atom or an electron to a radical, ceasing chain reactions [45] such as metal chelation and protecting cells from radiation damage and the formation of nonradical and nonreactive end products of antioxidant enzymes [28].

Vitamin C improves immune function and facilitates iron absorption, reduction of folic acid derivatives and synthesis of collagen, cortisol, catecholamines and carnitine [46]. Vitamin C also improves the synthesis of prostaglandins PGE1 and PGI2 and nitric oxide (eNO); it has a cytoprotection role, antimutagenic activity, vasodilatory action and inhibitory effect on platelet aggregation, being useful in type 2 DM and high blood pressure [47].

Ascorbic acid deficiency has been associated with stroke, DM, cancer, cardiovascular disease, infectious diseases and sepsis [41].

In type 2 DM the plasma levels of IL-6 and TNF-α are elevated, lipid peroxides are increased and unsaturated fatty acids, especially arachidonic acid (AA) and lipoxin A4 (LXA4), are reduced [48]. Besides, the usefulness of vitamin C in the managemen<sup>t</sup> of type 2 DM is confirmed by a study conducted by Mason et al. [49] which recorded that oral vitamin C (1000 mg daily) reduced hyperglycemia. This action arose after a decrease of plasma isoprostane-F2 in these subjects and indicated that the beneficial action of vitamin C was not only due to its antioxidant property but also to its ability to improve PGE1, PGI2, LXA4 and eNO [49].

Oxidative stress plays an important role in the development of vascular complications in type 2 DM, and the increase of ROS level is due to decreased production of some enzymatic/nonenzymatic antioxidants, i.e., catalase, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), leading to the development of diabetic complications [50]. Free radical formation in type 2 DM is accomplished by nonenzymatic proteins glycation, oxidation of glucose, increased lipid peroxidation, inducing enzyme damage and increased insulin resistance [51]. Insulin signaling is modulated by ROS/RNS (reactive nitrogen species) in two ways: first, in response to insulin, ROS/RNS exerts a physiological function; second, the ROS and RNS pathway negatively regulates insulin signaling, contributing to development of insulin resistance, which is a risk factor for type 2 DM [52].

Oxidative stress and ROS induce complications of DM including coronary heart disease, neuropathy, nephropathy, retinopathy and stroke [50]. Hyperglycemia plays a role in the generation of oxidative stress leading to vascular endothelial dysfunction of patients with DM and, together with dyslipidemia, develops macroangiopathies, which cause oxidative stress leading to atherosclerosis [50]. Vitamin C acts as an antioxidant by detoxification of ROS, hence being an important biomarker of oxidative stress, but, depending on the situation, it might promote toxicity via pro-oxidant formation [51].

Vitamin C plays a key role in maintaining the integrity of the connective tissues, thus of the periodontium. It is a powerful antioxidant, particularly at the intracellular level, being an enzymatic cofactor in metabolic reactions (hydroxylation of proline and lysine needed to stabilize collagen structures during its synthesis) [10].

Regarding the interplay between vitamin C and PD, the results of observational studies are contradictory, depending on the parameter evaluated, with several studies reporting no association between vitamin C and PD [10]. However, Nishida et al. [53] in their study with 12,419 participants identified a dose-dependent relationship between the vitamin C intake and the number of people with PD. Contrary to these results, a relation between vitamin C deficiency and PD was not recorded by other researches [54,55].

Vitamin C intake is necessary to avoid periodontal issues, but when a pathological condition has been established, a supplement with vitamin C is not su fficient to cure periodontal pathology [10]. Also, the e ffect of vitamin C combined with chlorhexidine can prevent and slow down the PD progression [56].

Monea et al. [57], in a case-control study (which included 10 patients with type 2 DM and 8 healthy adults), observed significantly increased malondialdehyde (MDA) levels in periodontal tissues, suggesting increased lipid peroxidation and decreased glutathione tissue levels (GSH), resulting a change of the local defense mechanism. Thus, histological aspects in the periodontal tissues of diabetic subjects confirm the involvement of oxidative stress [57].

In a study with murine models with diabetes, Li et al. [58] found that simultaneous periodontitis and DM synergistically aggravated both local and systemic oxidative lesions, being correlated with more severe periodontal destruction in diabetic periodontitis.

In a study with 10,930 patients, Lee et al. [59] found that in patients with DM between the ages of 30 and 49, there was a significant link between vitamin C intake and periodontitis. In the stratified analysis, the aforementioned association was highlighted among patients with type 2 DM. When there was inadequate vitamin C intake, diabetic subjects were more sensitive to oxidative stress, developing PD. These results pointed out the crucial role of vitamin C in promoting periodontal health among adults [59].

In a prospective cohort study that included 579 men, Dietrich et al. [60] observed that participants with periodontitis had a lower intake of vitamin C, increased risk of DM, higher levels of bleeding, bacterial plaque, loss of attachment and fewer teeth.

Considering the positive association between PD and ischemic heart disease, serum and salivary levels of vitamin C were analyzed in patients su ffering from both conditions and were found to be lower compared to PD patients and healthy individuals [61].

The present study aims to systematically review the available clinical data about the plasmatic and salivary levels of ascorbic acid in patients a ffected by PD and DM and about possible beneficial effects of ascorbic acid supplementation in PD–DM association.
