ARTIFICIAL SALIVA AND ITS USE IN BIOLOGICAL EXPERIMENTS

Examination of dental materials and their properties at the initial stage of the digestive process requires the development of conditions that mimic the environment of the oral cavity. One of the main components of this area is saliva, where many reactions occur in natural conditions. Human saliva is an important physiological fluid that is essential for the maintenance of good oral health and that of the whole human body, because it is the place where digestion begins and thus it contributes to the supply of those nutrients and health-promoting substances that are essential to the body. Due to the complexity and variability of the composition and the number of individual variable factors influencing saliva, as well as interactions between its components, it is not possible to create a synthetic formula identical to natural saliva. It is difficult to create a perfect chemical and physical model of a natural physiological fluid, which itself has no homogeneous individual composition. Dental materials are also of great importance. Thereby, the preparation of the recipe is based on the typical or average composition of the tested natural saliva samples. At the same time, the use of human saliva in large scale in vitro and in vivo studies is very difficult due to its variable composition and lack of stability outside the oral cavity (1). An additional problem is the colonization of human saliva by bacteria that can lead to changes in its chemical composition, and the sterilization of the liquid may result in the degradation of organic salivary components (2). Thus, the main purpose of using synthetic preparations is to obtain a stable environment and standardized procedures in in vitro tests across a wide range of pharmacological and bioengineering studies (3-5). The basic assumption in creating an artificial saliva is its similarity to natural saliva, in terms of physical characteristics and chemical composition. It is necessary to understand the composition of this bodily fluid as well as the interactions between the elements that compose it and the biological functions it performs.

For this reason, the aim of the study was to review the use of artificial saliva models in the context of stomatological research and bioavailability studies of the biologically active substances.

HUMAN SALIVA-COMPOSITION AND SECRETION

Mucous glands are the epithelial cell products that in evolutionary terms first appeared in Agnatha. They are found in the skin of fish, amphibians and mammals. In mammals these glands are located on the lips and, above all, in the mouth. The major salivary glands, i.e. parotid, submandibular and sublingual, are mainly responsible to produce saliva in the human oral cavity. Each of these produces a different type of saliva, differing in the content of ions and proteins. The parotid gland produces serous secretions, the submandibular-serous and mucous secretions, while the sublingual-mucous and serous secretions (6). The minor salivary glands (palatal, buccal, lingual, lip, molar, tonsil) involved in saliva production, together with secretions produced by the major glands, gingival crevicular fluid and microorganisms, epithelial cells, blood morphotic elements, serous exudate and food residues, generate ‘full saliva’ or ‘oral fluid’ (7, 8).

Depending on the state of stimulation of the glands, their share in saliva production is changed. The submandibular gland provides secretion at 65% at rest, the parotid 20%, the sublingual a further 5%, while 10% is the participation of the minor salivary glands. Stimulated secretory activity depends more than 50% on the parotid gland, 35% on the submandibular gland, and 7 – 8% on the sublingual gland, and to the same extent on the minor salivary glands (7, 9). At rest, about 0.1 to 0.3 mL/min of saliva is secreted, and after stimulation 7 mL/min (7). The average amount of saliva secreted during the day is 1.0 – 1.5 L, which is about 50 mL/hour (9). The normal average pH is in the range of 5.8 to 7.3 and varies depending on the secretory activity of the glands, with a value of about 5.3 at low flow and pH = 7.3 at its peak (7-9).

Human saliva is colorless and exhibits transparency or slight cloudiness, which may be influenced by its composition and the presence of air bubbles. Saliva exhibits only slightly higher viscosity than water, with an average of 1.2 10–3 Pa s. The density of natural saliva varies between 1.0 – 1.1 g/mL (10, 11). Osmolarity of saliva is usually slightly lower than that of plasma (12). The initial effect of the activity of the secretory cells is isotonic saliva, called primary saliva.

At a later stage, the composition changes as it passes through the secretory ducts, than in consequence it becomes hypotonic, the ratio of potassium ions to sodium ions is increased, and it is enriched with additional substances produced by the cells. This is so-called secondary saliva (13).

The main component of saliva is water up to 99.5%, the level of inorganic compounds is ranged from 0.2 to 0.9% and an organic fraction is ranged from 0.4 to 0.6% (10). Saliva contains a wide range of electrolytes such us for example: potassium, sodium, calcium, magnesium and ammonium cations. The anionic group is composed of phosphates, carbonates, chlorides, rhodium and a number of micronutrients (Table 1).

Table 1. The content of inorganic components in saliva according to Lentner (10).

The organic saliva fraction is mostly built by peptides substances occurring in many forms and it contains: histatins, cystatins, statherin, sialin, cathelicidin, defensin, and growth factors: epidermal growth factor (EGF), nerve growth factor (NGF), transforming growth factor alpha (TGF-α). Non-enzymatic proteins include mucins, lactoferrins, proline-rich proteins (PRPs), calprotectin, interferon, albumins and globulins: IgG and IgMg-globulins, and IgA-produced only in the parotid glands (7, 9).

Enzymes contained in saliva are salivary peroxidase, lysozyme and digestive enzymes, e.g. amylase, maltase, lipases, esterases, glucosidases, and proteases (14). In addition to protein substances, saliva contains carbohydrates (mostly in the form of salivary glycoproteins), lipids-phospholipids and cholesterol as well as metabolites of nitrogen compounds, e.g. urea, uric acid and creatinine and amino acids. Vitamins A, B, C and K, as well as numerous hormones-thyroid, sex, and corticosteroids, have also been identified in saliva, which allows it to be used in endocrine diagnostics (15, 16).

All components work together to form a complex multi-task system. Neutralization of the acids and maintenance of the proper pH is regulated by carbonates, phosphates and urea. The most important buffer is carbonic acid/bicarbonate, active mainly in stimulated saliva. Saliva acts as a passive defense barrier through such compounds as peptides, enzymes and immunoglobulins that are anti-infectious. Saliva is supported by mucins responsible for lubrication, protection of the mucous membrane from irritants, and cleansing of the oral cavity by aggregating microorganisms that prevent them from adhering to tissues. The phosphate and calcium ions and also their salts with statherin are responsible for the integrity of the hard tissues of the teeth, by supporting remineralization processes and suppression of demineralization (7, 9).

HUMAN PHYSIOLOGY INFLUENCING THE QUANTITY AND COMPOSITION OF SECRETED SALIVA

The volume and composition of secreted saliva is individual variable and depends on many different factors, including demographic, environmental, physiological (e.g. anatomical variability) and pathological factors (inflammation, disease).

Factors affecting the composition of human saliva are demographic characteristics: e.g. age-the peak of saliva secretion is around the age of 10 and this decreases with age; and sex-higher secretion with higher levels of sodium, calcium and phosphorus is observed in men (12). Other factors affecting the composition and volume of secreted saliva are individual hydration and body nutrition, body posture, lighting conditions, as well as daily and yearly rhythms-for example, the lowest secretion values for the parotid gland occur between June and August (10, 17, 18). Both the flow and saliva composition are regulated by the autonomic nervous system. Parasympathetic stimulation results in an increase in saliva secretion with low organic and inorganic content. Stimulation of α-adrenergic receptors results in low secretion of protein-rich saliva, low in mucin content, which lowers its viscosity. β-adrenergic activation is a signal for the production of mucus saliva with high protein and mucin content (19).

General health, medicines taken, diet, alcohol consumption, and smoking may also have an influence on saliva composition. The body weight and size of the salivary glands of a given individual are not without significance (10). The degree of stimulation of the body, i.e. emotional state, e.g. stress or time after physical activity, is also significant-there is a significant increase in ionic concentration, particularly sodium and -amylase (10, 17, 18).

Temporary decreased secretion may be due to stress, dehydration, physical activity, or smoking (9, 17). A diet containing acidic fruits or hot spices, as well as alcohol consumption increases the secretion of unstimulated saliva (9). Saliva production and composition is also affected by a number of general diseases and their associated pharmacotherapy. Changes in the mouth such as aphtae, necrotizing ulcerative gingivitis (NUG) and oral herpes also increase the flow of saliva. Increased salivation is observed in nausea, vomiting and medical conditions such as cerebral palsy, bulbar palsy, pharyngeal and esophageal cancers, and when taking sympathomimetic drugs. A pathological increase in salivary secretion (sialorrhea) occurs in stomatitis, tooth eruption, and neurological diseases such as epilepsy (10). The factors that reduce the activity of salivary glands include: taking medicines from the group of muscarinic receptor antagonists, sympathomimetic drugs and diuretics, Sjogren’s syndrome, type 2 diabetes mellitus, HIV infection, HCV, as well as dehydration of the body, chemotherapy and postoperative radiotherapy of the head and neck area (20).

One of the tasks of saliva is to protect the mucous membrane of the oral cavity against mechanical, chemical and biological factors. Changes in its quality and quantity may adversely affect an individual’s health, resulting in many adverse effects, such as xerostomia (which can lead to increased incidence of dental caries and periodontal disease), difficulty in articulating speech, dysgeusia, and dysphagia.

The above data show that natural saliva is characterized by a very complex and variable composition, dependent on many factors, so it is not possible to create an exact replica (21, 22).

HISTORY OF THE APPLICATION OF ARTIFICIAL SALIVA IN SCIENTIFIC RESEARCH

Before researchers began working on various formulations of artificial saliva, at the end of the nineteenth century study began on a multistage system that simulated the oral cavity using natural saliva. Over the years, this system has been improved, and in the 1990s this took the form of a computer-controlled multiple artificial mouth (MAM). These systems used saliva derived from donors, which was thus natural, as a result of which plaque development was observed. According to the literature, the MAM system mainly serves to study bacterial colonies, the development of oral diseases, mainly caries, and their effects on human teeth (23). This solves the ethical problems associated with the possible implementation of similar research on the development of human caries, but this system has not been used in oral behavior experiments on a wide variety of materials used in modern dentistry.

The history of research on the use of artificial saliva is reported for the first time by W. Souder and W.T. Sweeney in 1931. They analyzed the toxicity of mercury released from amalgam fillings in the oral cavity. In their experiment, three types of amalgam were placed in a human saliva solution at 37°C for 30 days, and subjected to intense brushing once a day. After a period of one month, solutions were screened to detect the presence of any amalgam particles that could be released from the samples as abrasives-no mercury particles were detected (5). The formula used by the authors was based on an arbitrarily fixed composition of human saliva. These reports gave rise to many years of searching for a model of artificial saliva for use in in vitro tests. In 1937, Greenwood et al. reported results on the retention of stainless steel inserts, showing a new composition for a saliva substitute (24). The latter, after minor modifications, was used by Worner in corrosion studies of amalgam in 1966 (25). After further modifications, this formula was used in subsequent studies on corrosion of alloys and amalgams (26, 27). Innovative research in the development of saliva substitutes was performed by Muhler and Swenson in 1947. They analyzed saliva composition from 11 patients and then developed a consistent formulation based on averaged results (25). In subsequent years, a number of modifications of existing preparations appeared. Of particular note are Fusayama et al. results (28) from 1963, where they modified the unpublished Schwartz formula (29). This introduced a saliva formula, the ‘Fusayama formula’, which in subsequent years was used repeatedly in the analysis of the corrosion of nickel alloys (30), and amalgams (31), galvanic currents between amalgam and gold (32), and the release of fluoride from glass-ionomer cements (33). The next step in the development of saliva substitutes was Darvell’s research conducted in the 1970s. Also based on an analysis of the average human saliva composition, he developed a formula for corrosion studies, but for the first time its components were evaluated for their significant impact on this process to optimize the study. Darvell stated that in the context of oxidation of the amalgam, a solution of artificial saliva should only contain components of the natural product that are associated with the development of corrosion (34).

The reference composition of biological fluids, including saliva, considered the standard in medical research was developed by Lentner in 1983 (10). The author developed a reference saliva composition (Table 1) based on a few hundred samples of stimulated and resting saliva, considering also sex, age and smoking.

Due to the development of technology and the simultaneous increase in interest in dental materials, which resulted in intensive scientific research, many different types of artificial saliva were developed in subsequent years. A detailed study of the composition of artificial saliva can be seen in Gala et al., which comprehensively describes and evaluates sixty substitute formulas (35).

APPLICATION OF ARTIFICIAL SALIVA IN CURRENT BIOLOGICAL TESTS

Due to the profile of the experiment (e.g. medical, dental, analytical, toxicological) the composition of the artificial physiological fluid should be determined. This is due to the multitude of interactions both between the components of the biological fluid and between them and the material. This is particularly important in dental material research, because of the wide range of resources used by dentists in their everyday practice. Therefore, many studies have been devoted to the development of the most complete replication of human saliva (Table 2). Substitute research is intended to determine this composition based on the averaged results of analyses. Due to the effect of so many variables on the salivary composition, it is impossible to create a single model of artificial saliva that is universally applicable. The type of the substitute used is crucial in the preparation phase of the study, as variations in its composition may result in different in vitro and in vivo results (21). Therefore, many artificial saliva variants have been developed in the course of studies, so as to simulate possible variations in the composition of and conditions in the oral cavity (35).

An analysis of research data (2015 – 2017) indicates that many artificial saliva formulations were used by researchers, varying according to the medical experiment conducted. These studies largely related to the assessment of the properties of composite materials (36-40) and metal alloys used in dentistry (41-43) as well as dental ceramics (44, 45). In recent years artificial saliva has been used in studies on impact of laser whitening of enamel (46). There has also been an attempt to assess the duration of the presence of certain substances in the oral environment which may affect the treatment of xerostomia, as well as prolongation of the active time of the drug in the mouth, from which most medicines are removed quite quickly (47, 48).

Table 2. Models of artificial saliva in biological studies.

Dental composites are the main materials used in the treatment of caries and non-carious cervical lesions. For this reason, their longevity and behavior in oral environments undergoing changes under the influence of external factors are important. The presence of these factors in the oral cavity can greatly affect the different properties of dental materials. Changes in the physical and chemical structure of the composites, such as increased surface roughness, favor rapid colonization by bacteria, which can lead to caries and periodontal disease (36). Composite materials have been examined for microhardness changes and surface roughness under the influence of various alcoholic beverages (36). Color variation and erosion and surface roughness have also been monitored in the context of contact with white and red wine (37, 38). An important factor in the use of composite materials is their adhesion to the dentin via adhesive systems. Changes in dentin bond strength in the environment of artificial saliva have been controlled by erosive beverages and enzyme inhibitors (2% chlorhexidine digluconate) (39). Other researchers have focused on the sorption of other substances from the environment and the solubility of composites with different composition (bulk-fill and conventional) (40).

Metal alloys are widely used in such areas of dentistry as prosthetics and orthodontics, and therefore their biocompatibility is very important. In the oral environment, these can undergo various physico-chemical processes such as corrosion, which indicates the need to identify the factors influencing the variability of their structure. Using artificial saliva, corrosion resistance of orthodontic stainless steel archwire (SS), nickel-titanium (Ni-Ti), titanium-molybdenum (TMA) and titanium-molybdenum alloys with implanted ions (L-TMA) has been investigated (41). In the test group, 0.5% NaF was added to the artificial saliva solution, giving it the same concentration as that in pastes for daily use. Other studies have focused on changes in the surface structure and corrosion resistance of cobalt-chromium-molybdenum alloys with or without the addition of nickel at different pH conditions, as well as the same alloy enriched with niobium and zirconium or devoid of these substances (the biological properties of alloys were also controlled in this experiment) (42, 43).

Zirconium oxide, as a highly aesthetic and durable material, is increasingly used to make permanent prosthetic restorations. In recent years, zirconia samples have been anodized in phosphoric acid with or without sodium fluoride, and then tested in the environment of artificial saliva to explain the zirconia corrosion behavior (44). Other studies have evaluated the effect of the construction and finishing of artificially aged zirconium crowns (solid and bilayer (metal/zircon)) to evaluate fracture toughness (45).

Current efforts to achieve the highest aesthetic effect of dentition have resulted in the development of many teeth whitening methods and the need to control these methods in terms of safety for the patient and his orthodontics. In recent years, studies have been conducted using an artificial saliva solution to demonstrate the effect of whitening using an Er, Cr: YSGG laser on the enamel, where the indicators have been microhardness, changes in surface roughness and mineral enamel composition (46).

The problem in the treatment of oral diseases is the short residence time of topical medications, due to the muscle function and the purifying role of saliva. Thus, it has seemed necessary to try to find a substance that could function as a drug-releasing system over a prolonged period of time. In this respect, nanoparticles based on polysaccharides such as chitosan, pectins and alginates have been studied, evaluating their stability in the environment of artificial saliva (48, 49). A common problem, especially in older patients, is xerostomia, or dry mouth, caused by local or general causes. Saliva substitutes are used to reduce the unpleasant experience of patients. The duration of their presence in the oral environment is significant. This has been assessed using polystyrene models and three-dimensional gingival models (47).

Zajac et al. and Muszynska et al. used the artificial saliva model according to Arvidson (1985) in their studies on the effectiveness of the release of biologically active substances such as Zn, Fe, Mg, Cu and organic compounds: indole and phenolic, triterpene saponins (50-53). In these studies, artifical saliva was used as an extraction solution or, as closer to the situation in human physiology, to initiate digestion. A special Gastroel-2014 device has been developed to simulate the conditions of the human digestive tract (peristaltic movements, 37°C-temperature of human body and use of artificial digestive juices for material extraction). In experiments, the artificial saliva was used to study natural resources such as edible fungi, algae and plants. These studies have shown that the amount of biologically active substances (bioelements, especially zinc, iron, copper, magnesium and phenolic compounds, indole, triterpenoid and lutein) in biological material is not identical to their quantity released under human digestive tract conditions, and thus its bioavailability is affected. These results show that tables of active ingredient constituents in drugs and food do not show their actual usefulness and availability to the human body (50-52).

Comparison of the composition of artificial saliva in the discussed models shows it is very diverse, ranging from three components (NaHCO3, NaCl, KCl) up to nine components (artificial saliva according to Gala) (47, 48). In digestion studies in models of the artificial digestive tract, osmotic pH = 6.7 of saliva oscillating at average values during consuming food has been demonstrated (49).

The only component present in all described models of artificial saliva is KCl. The pH of artificial saliva is very different, as it varies from 5.0 to 7.3, which is similar to its physiological lower and upper limits. Applied pH depends on the dental experiment used.

The results indicate that no universal artificial saliva model has been introduced. In clinical studies, it is necessary to use this physiological fluid in a form dependent on the type of experiment or material studied. Modern technology has allowed understanding better the inorganic and rheological functions of saliva. Future models of artificial saliva could contain enhancing both the quality and quantity of saliva for polyfunctional properties of active components which could modulate oral microflora and to promote mineralization and hydration of mouth against xerostomia.

Futhermore, saliva reflects the general health status of the human body and it is easy to collect, therefore it can be used as a non-invasive diagnostic tool (54). The assessment of the parameters of oxidative stress in saliva of Crohn’s disease (CD) revealed that disease activity index (CDAI) was inveresly correlated with SOD in plasma but not saliva (55). Moreover, no significant correlation on SOD and GPx activity both in plasma and saliva were found between CD remission group and the control group (55). These authors concluded that SOD and GPx assays in saliva are not conclusive suggesting that saliva determination in these patients might be not appropriate material for studies in CD individuals (55). On the other hand, in patients with reticular and erosive oral lichen planus, the determination of oxidative status of saliva seems to be very useful (56). In this study, lower levels of GSH and total antioxidant capacity (TAC) were decreased, while the thiobarbituric acid reactive substances (TBARS) were increased in patients who suffered from oral lichen planus cpmpared with control (56). This indicates that saliva can be considered as an important indicator of oxidative stress alterations, especially in patients with oral disorders, but may be less important in patients with more systemic diseases such as CD (55, 56).

Conflict of interests: None declared.

REFERENCES

1. Mandel IR. Relation of saliva and plaque to caries. J Dent Res 1974; 53: 246-266.
2. Leach SA, Higgins JE, Appleton J. Some factors affecting the deposition of material from saliva. Helv Odontol Acta 1973; 17: 7-58.
3. Leung VW, Darvell BW. Artificial saliva for in vitro studies of dental materials. J Dent 1997; 25: 475-484.
4. PN-EN ISO 10993-15, Biologiczna ocena wyrobow medycznych - cz. 15; Identyfikacja i oznaczanie ilosciowe produktow degradacji metali i stopow. 2005.
5. Souder W, Sweeney WT. Is mercury poisonous in dental amalgam restorations? Dent Cosmos 1931; 123: 1145-1152.
6. Dodds MW, Johnson DA, Yeh CK. Health benefits of saliva: a review. J Dent 2005; 3: 223-233.
7. Humphrey SP, Williamson RT. A review of saliva: normal composition, flow, and function. J Prosthet Dent 2001; 85: 162-169.
8. Mamta S, Udita S, Bhasin GK, Rajesh P, Aggarwal SK. Oral fluid: biochemical compositions and functions: A review. J Pharm Biomed Sci 2013; 37: 1932-1941.
9. Almeida PV, Gregio AM, Machado MA, de Lima AA, Azevedo LR. Saliva composition and functions: a comprehensive review. J Contemp Dent Pract 2008; 8: 72-80.
10. Lentner C. Geigy Scientific Tables. Basel, Switzerland: CIBA-Geigy 1983.
11. Davis SS. The rheological properties of saliva. Rheol Acta 1971; 10: 28-35.
12. Cyprysiak G, Tadeusiak W. The use of saliva in medical diagnosis [in Polish]. Nowa Stomatol 2001; 2: 33-36.
13. Nunes S, Alessandro L, Mussavira S, Bindhu OS. Clinical and diagnostic utility of saliva as a non-invasive diagnostic fluid: a systematic review. Biochem Med 2015; 25: 177-192.
14. Nakamura M, Slots J. Salivary enzymes. J Periodontal Res 1983; 18: 559-569.
15. Szydlarska D, Grzesiuk W, Kupstas A, Bar-Andziak E. Saliva as a diagnostic material [in Polish]. Forum Med Rodz 2008; 2: 454-464.
16. Obminski Z, Wojtkowiak M, Stupnicki R, Golec L, Hackney AC. The effect of acceleration stress on salivary cortisol and plasma cortisol and testosterone levels in cadet pilots. J Physiol Pharmacol 1997; 48: 193-200.
17. Chicharro JL, Lucia A, Perez M, Vaquero AF, Urena R. Saliva composition and exercise. Sports Med 1998; 26: 17-27.
18. Paccotti P, Minetto M, Terzolo M, et al. Effects of high-intensity isokinetic exercise on salivary cortisol in athletes with different training schedules: relationships to serum cortisol and lactate. Int J Sports Med. 2005; 26: 747-755.
19. Aps JK, Martens LC. Review: the physiology of saliva and transfer of drugs into saliva. Forensic Sci Int 2005; 150: 119-131.
20. Llena-Puy CL. The role of saliva in maintaining oral health and as an aid to diagnosis. Med Oral Patol Oral Cir Bucal 2006; 11: 449-455.
21. Marques MR, Loebenberg R, Almukainzi M. Simulated biological fluids with possible application in dissolution testing. Dissolut Technol 2011; 18: 15-28.
22. Bjorklund M, Ouwehand AC, Forssten SD. Improved artificial saliva for studying the cariogenic effect of carbohydrates. Curr Microbiol 2011; 63: 46-49.
23. Tang G, Yip HK, Cutress TW, Samaranayake LP. Artificial mouth model systems and their contribution to caries research: a review. J Dent 2003; 31: 161-171.
24. Greenwood JN, Down CH, Worner HK. Investigations of the failure of stainless steel inlay retention posts. Aust J Dent 1937; 41: 73-84.
25. Muhler JC, Swenson HM. Preparation of synthetic saliva from direct analysis of human saliva. J Dent Res 1974: 26: 474.
26. Schoonover IC, Souder W. Corrosion of dental alloys. J Am Dent Assoc 1941; 28: 1278-1291.
27. Habu HA. Polarographic study on the corrosion tendency of dental amalgam. J Nihon Univ Sch Dent 1964; 6: 6-16.
28. Fusayama T, Katayori T, Nomoto S. Corrosion of gold and amalgam placed in contact with each other. J Dent Res 1963; 42: 1183-l197.
29. Swartz ML, Phillips RW Tannir MD. Tarnish of certain dental alloys. J Dent Res 1958: 37; 837-847.
30. Meyer JM, Wirthner JM, Barraud R, Susz CP, Nally JN. Corrosion studies on nickel-based casting alloys. In: Corrosion and Degradation of Implant Materials. Syrett BC, Acharya A (eds). Philadelphia, PA, American Society for Testing and Materials (ASTM) Special Technical Publication no 684, 1979, pp. 295-315.
31. Olsson S, Berglund A, Bergman M. Release of elements due to electrochemical corrosion of dental amalgam. J Dent Res 1994; 73: 33-43.
32. Holland RI. Galvanic currents between gold and amalgam. Scand J Dent Res 1980; 88: 269-272.
33. El Mallakh BF, Sarkar NK. Fluoride release from glass-ionomer cements in de-ionized water and artificial saliva. Dent Mater 1990; 6: 118-122.
34. Darvell BW. The development of an artificial saliva for in vitro amalgam corrosion studies. J Oral Rehabil 1978; 5: 41-49.
35. Gal JY, Fovet Y, Adib-Yadzi M. About a synthetic saliva for in vitro studies. Talanta 2001; 53: 1103-1115.
36. Da Silva MA, Vitti RP, Sinhoreti MA, Consani RL, Silva-Junior JG, Tonholo J. Effect of alcoholic beverages on surface roughness and microhardness of dental composites. Dent Mater J 2016; 35: 621-626.
37. Tanthanuch S, Kukiattrakoon B, Peerasukprasert T, Chanmanee N, Chaisomboonphun P, Rodklai A. The effect of red and white wine on color changes of nanofilled and nanohybrid resin composites. Restor Dent Endod 2016; 41: 130-136.
38. Tantanuch S, Kukiattrakoon B, Peerasukprasert T, Chanmanee N, Chaisomboonphun P, Rodklai A. Surface roughness and erosion of nanohybrid and nanofilled resin composites after immersion in red and white wine. J Conserv Dent 2016; 19: 51-55.
39. von Fraunhofer JA, Rogers MM. Effects of sports drinks and other beverages on dental enamel. Gen Dent 2005; 53: 28-31.
40. Alshalia RZ, Salimc NA, Satterthwaitea JD, Silikasa N. Long-term sorption and solubility of bulk-fill and conventional resin-composites in water and artificial saliva. J Dent 2015; 43: 1511-1518.
41. Pulikkottil VJ, Chidambaram S, Bejoy PU, Femin PK, Parson P, Rishad M. Corrosion resistance of stainless steel, nickel-titanium, titanium molybdenum alloy, and ion-implanted titanium molybdenum alloy archwires in acidic fluoride-containing artificial saliva: an in vitro study. J Pharm Bioallied Sci 2016; 8: S96-S99.
42. Qian C, Wu X, Zhang F, Yu W. Electrochemical impedance investigation of Ni-free Co-Cr-Mo and Co-Cr-Mo-Ni dental casting alloy for partial removable dental prosthesis frameworks. J Prosthet Dent 2016; 116: 112-118.
43. Andrei M, Galateanub B, Hudita A, et al. Electrochemical comparison and biological performance of a new CoCrNbMoZr alloy with commercial CoCrMo alloy. Mater Scie Eng C Mater Biol Appl 2016; 59: 346-355.
44. Romonti DE, Gomez Sanchez AV, Milosev I, Demetrescu J, Cere S. Effect of anodization on the surface characteristics and electrochemical behaviour of zirconium in artificial saliva. Mater Sci Eng C Mater Biol Appl 2016; 62: 458-466.
45. Lameira DP, Silva WA, Silva FA, Souza GM. Fracture strength of aged monolithic and bilayer zirconia-based crowns. BioMed Res Int 2015; 2015: 418641. doi: 10.1155/2015/418641.
46. Dionysopoulos D, Strakas D, Koliniotou-Koumpia E, Koumpia E. Effect of Er,Cr:YSGG laser irradiation on bovine enamel surface during in-office tooth bleaching ex vivo. Odontology 2017; 105: 320-328.
47. Engelhart K, Popescu A, Bernhardt J. Using mid infrared technology as new method for the determination of the dwell time of salivary substitutes on three dimensional gingiva models. BMC Ear Nose Throat Disord 2016; 16: 6. doi: 10.1186/s12901-016-0025-5
48. Pistone S, Goycoolea FM, Young A, Smistad G, Hiorth M. Formulation of polysaccharide-based nanoparticles for local administration into the oral cavity. Eur J Pharm Sci 2017; 96: 381-389.
49. Ablal MA, Kaur JS, Cooper L, et al. The erosive potential of some alcopops using bovine enamel: an in vitro study. J Dent 2009; 37: 835-839.
50. Zajac M, Muszynska B, Kala K, Sikora A, Opoka W. Popular species of edible mushrooms as a good source of zinc released to artificial digestive juices. J Physiol Pharmacol 2015; 66: 763-769.
51. Muszynska B, Kala K, Sulkowska-Ziaja K, Krakowska A, Opoka W. Agaricus bisporus and its in vitro culture as a source of indole compounds released into artificial digestive juices. Food Chem 2016; 199: 509-515.
52. Rojowski J, Zajac M, Muszynska B, Opoka W. Influence of extraction procedure from edible mushroom species Boletus badius on zinc quantity released into simulated gastric fluid. Acta Pol Pharm 2017; 74: 597-602.
53. Arvidson K, Johasson EG. Galvanic current between dental alloys in vitro. Scad J Dent Res 1985; 93: 467-472.
54. Buczko P, Zalewska A, Szarmach I. Saliva and oxidative stress in oral cavity and in some systemic disorders. J Physiol Pharmacol 2016; 66: 3-9.
55. Szczeklik K, Krzysciak W, Domagala-Rodacka R, et al. Alterations in glutathione glutathione peroxidase and superoxide dismutase activities in plasma and saliva in relation to disease activity in patients with Crohn’s disease. J Physiol Pharmacol 2016; 67: 709-715.
56. Darczuk D, Krzysciak W, Vuhouskaya P, et al. Salivary oxidative status in patients with oral lichen planus. J Physiol Pharmacol 2016; 67: 885-894.