The accomplishment of developing a truly adhesive bond between a restorative material and the natural tooth structures is the goal of adhesive dentistry. Dentine adhesive systems come into close contact with dental and oral tissue, especially the pulp and gingival cells. Due to this close and long-term contact, adhesives should exhibit a high degree of biocompatibility. Biocompatibility is one of the most important properties of dental materials, and adhesives are no exception. It has been long demonstrated that different components of adhesives can be released. Numerous in vitro investigations have shown that released monomers and other components can cause damage to cultured cells. In addition, many in vivo studies have shown that uncured components which reach the pulpal space cause inflammatory response and tissue disorganization. Only a combination of various in vitro and in vivo tests can provide an overview of the interaction of biomaterials with the host. Therefore, it is necessary on a regular basis to carry out and re-verify the biological compatibility of the increasing number of new dental materials. Adhesives should be biofunctional, protective, and preventive, with health-promoting effects that contribute to a better prognosis for restorative treatments and its biocompatibility.
- dental adhesives
- in vitro assays
- in vivo assays
The use of composite dental filling materials along with adhesive techniques has revolutionized today’s dental practice. Dental adhesive systems are used to improve contact between restorative material and the walls of the prepared cavity of tooth, in order to increase retention as well as to minimize bacterial leakage. Their purpose is based on a twofold adhesion, adhesion to composite fillings, and bonding to enamel and dentine [1–3].
The introduction of the acid-etch technique by Buonocore in 1955 was the genesis of adhesive dentistry. Adhesive systems are constantly being developed with elaborate and complex chemistry. As a result of technological progress, the large number of new materials on the market has been appeared. Adhesive systems are solutions of resin monomers that make the resin-dental substrate interaction achievable. They are composed of monomers with hydrophilic and hydrophobic groups, organic solvents, initiators, inhibitors or stabilizers, and inorganic fillers. The proportional composition and the chemistry of these ingredients have been different between the different classes of adhesives [1, 4]. Dentine adhesives are currently available as three-step, two-step, and single-step systems, depending on how the three basic steps of etching, priming, and bonding to tooth substrate are accomplished. Modern adhesive strategies depend on how adhesive systems interact with the smear layer dissolving it or making it permeable. Consequently, adhesives can be classified into two types: total-etch (also known as etch-and-rinse) and self-etch adhesive systems . The main difference between them is that the etch-and-rinse adhesives use 37% phosphoric acid for pretreatment of hard dental tissues before application of dental bonding agent, whereas the self-etch adhesives do not require a separate etching step, as they contain acidic monomers that simultaneously condition and prime the dental substrate [2, 5]. Etch and rinse adhesive systems are used either through two or three clinical application steps. First step always involves application of the conditioner or acid etchant on both enamel and dentine. According to the number of clinical application steps, self-etch adhesives are divided into two-step and one-step systems .
In order to achieve clinical success, it is necessary for dental adhesives to provide good physical properties. Clinical success of adhesive procedures depends not only on the dental adhesives but also on variables such as substrate condition and composition, clinical technique, restorative material, cavity shape and size, and polymerization quality . However, due to the fact that dental adhesives are in close and extended contact with vital dentine, biocompatibility becomes a property of supreme [5, 7, 8]. The biocompatibility of the adhesive estimated through screening with simple
1.1. Biocompatibility of dental materials
Biomaterial is a substance that is used for a long period within the body with the aim of treating or replacing of tissue, organs, or their functions. Each dental material must be biocompatible or able to function
Biocompatibility is a measure of body's biological response to a material used in specific application. It is a property of a material to interact with its environment. The biological response to a material could be modified if any change occurs in the host or in the application of the material. Therefore, evaluation of any new material intended for human use requires data from systematic testing to ensure that the benefits provided by the final product will exceed any potential risks produced by device materials . Biocompatibility includes the physical, mechanical, and chemical properties of materials, as well as potential cytotoxic, genotoxic, mutagenic, and allergenic effects. According to EN 1441 (European standard for Risk Analysis, 1996), biocompatible material must not be harmful to the host organism . A characteristic of the material to interact with the biological tissue and thus create a stable connection is essential for biocompatibility. The biocompatibility of material is manifested through a number of parameters: (a) cytotoxicity (systemic and topical), (b) genotoxicity, (c) mutagenicity, (d) carcinogenicity, and (e) the immunogenicity .
The toxicity of material is described as the ability of a chemical substance to cause harmful health effects and damage the biological system. It might be local or systemic. Local toxicity occurs at the place of application, as opposed to systemic toxicity where the adverse response occurs distant from its entry point [23, 24]. The term of cytotoxicity is used to describe the cascade of molecular events that cause functional and structural damage to cells. Throughout the years, various assays and protocols were developed to test the cytotoxic effects of biomaterials. The rationale behind doing a cytotoxicity test is to determine how a material sample affects a particular cell type. The primary criteria of these tests are that material must not affect the cell number, cell growth, genetic integrity, membrane integrity, genetic expression, and enzymatic activity of the cells. The cytotoxicity assays are broadly categorized into viability assays, survival assays, metabolic assays, transformation assays, and inflammation assays . The cytotoxicity is related to the damage of the individual cells, for example in cell cultures, and determines the cell viability. There are two modes of cell death, apoptosis (programmed cell death) and necrosis (accidental cell death). They are different significantly in the mechanisms, outcomes, and also in morphology, biochemistry, and biological features. Necrosis occurs as a result of significant damage of the cell, caused by physical or chemical agent. The loss of integrity of the membrane is a hallmark of necrosis. The membrane permeability of damage cell increases, and damage cell cannot control balance of the fluids and ions. It is accompanied by cellular swelling and loses of integrity of organelles. The release of cytoplasmic contents could lead to an inflammatory response in surrounding organs and tissue. Apoptosis could be defined as caspase mediated cell death. Apoptotic cells can be identified based on following morphological features: cytoplasmic and nuclear condensation, chromatin cleavage, formation of apoptotic bodies, maintenance of an intact plasma membrane, exposure of surface molecules targeting cell corpses for phagocytosis and also on the proteolytic activity of certain caspases because these enzymes mediate the process of apoptotic cell death [23, 26, 27]. The primary criteria of these tests are the following: the material must not affect the cell number, cell growth, genetic integrity, membrane integrity, genetic expression, and enzymatic activity of the cells .
Genotoxicity is described as adverse effects on the genomic material and may be caused by DNA damage without direct evidence for mutations . Genotoxic effect of a material can induce changes in the genome that disrupt its integrity or function. Depending on the intensity of that effect, the cell could recover, start neoplastic growth, or die . The long-term exposure to low concentrations of the substance may develop neoplasia and death of the whole organism by the effects on the genetic material. Transfer of genetic damage to the next generation of cells can be avoided by programmed cell death (apoptosis) [9, 28].
Transition of genetic injury to the next generation is called mutagenicity. Mutagenicity and carcinogenicity are not the same entities. Carcinogenicity arises from several mutations, which means that all mutagenic events do not lead to tumour development. Mutagenicity serves as an indicator of ‘possible’ carcinogenicity of substances that damaged DNA. A variety of different methods, mainly
The immunogenicity is the ability of a substance to induce an immune response. An allergic reaction to certain substances can be initiated if the organism was sensitized previously. The dosage that could cause the allergic reaction are generally significantly lower than those that could cause toxic reactions. Immunotoxicological screenings are used to identify the influence of substances or materials to the various components of immune system .
Biomaterials must meet several criteria before they are put in use, and measuring of the biocompatibility of a material is not simple. It is not possible to biologically characterize a material using a single test. Different characteristics can be explored through both
2. Materials and methods
2.1. Blood sampling
Evaluation of the potential genotoxicity of dental adhesive systems and combination of adhesive systems and composite materials were performed on human leukocytes. The donors were two males and four females with ages ranging from 25 to 32. They were not smokers and had not been exposed to any physical or chemical agents that might have interfered with the results of the genotoxicity testing in the 12-month period prior to blood sampling. The donors were acquainted with the purpose of the study and signed permission for the blood samples to be used for scientific purposes. A peripheral blood sample was collected under sterile conditions by venipuncture into heparinized tubes (Becton Dickenson, Plymouth, UK). The study was approved by the Ethical Committee of the School of Dental Medicine, University of Zagreb, Croatia [3, 5].
2.2. Preparation of materials and cell culture treatment
Three dental adhesives were tested: AdheSE (Ivoclar Vivadent, Schaan, Liechtenstein), G-bond (GC, Tokyo, Japan), and Adper Single Bond (3M ESPE, St. Paul, MN, USA), in their polymerized and unpolymerized form. Also were tested aforementioned dental adhesive in polymerized form in combination with composite material from same manufacturers: Tetric EvoCeram (Ivoclar Vivadent, Schaan, Liechtenstein), Gradia Direct Anterior (GC, Tokyo, Japan), and Filtek Z250 (3M ESPE, St. Paul, MN, USA). Six different combinations of two shades of composite resins and adhesives were examined: Gradia Direct Anterior A1 + G-Bond, Gradia Direct Anterior A3.5 + G-Bond, Filtek Z250 A1 + Adper Single Bond, Filtek Z250 A3.5 + Adper Single Bond, Tetric EvoCeram A1 + AdheSE, and Tetric EvoCeram A3.5 + AdheSE.
In order to test the genotoxicity of non-polymerized materials, the dental adhesives were placed in previously weighed bottles (Sartorius BLG10S, Goettingen, Germany). The mass of each dental adhesive was calculated from the difference in weight of empty and full bottles. For each 0.1 g of dental adhesive, 1 ml of saline solution was added (0.9% NaCl, Sigma, St. Louis, MO, USA) for the purpose of elution.
In order to test the genotoxicity of polymerized dental adhesives, each one was polymerized under aseptic condition in accordance with the manufacturer's instructions using an Elipar TriLight halogen curing unit (3M ESPE) from a 2 mm distance for 40 s. To ensure complete polymerization, only two drops of an adhesive were cured at a time. After polymerization, the dental adhesives were weighted, fragmented, and transferred into bottles. We tended to use the same masses of dental adhesive samples (1 g) regardless of whether they were polymerized or non-polymerized. Each dental adhesive eluate was tested after 1 h, 1 day, and 5 days in two different dilutions of eluate (1: 102 and 1: 104) for each time point.
To assess the genotoxicity of polymerized dental adhesives and their compatible dental composites from same manufacturers, the following was conducted: 20 ± 0.2 μl of each adhesive system was placed on the Mylar strip (Contour™ Strips, Ivoclar Vivadent) and photopolymerized by a halogen curing unit Elipar TriLight in standard mode (800 mW/cm2) for 20 s. The adhesives were handled exactly by the manufacturers' instructions. Subsequently, 0.025 ± 0.003 g and 0.05 ± 0.002 g of each composite resin were placed on top of the bonding material, covered with another Mylar strip, and mechanically pressed to obtain a 2 mm thick layer. Resin composite samples (n = 2) were for 40 s. The light curing tip was flush pressed onto the Mylar sheet on top of the composite samples. Thereafter, the polymerized composites were separated from the Mylar sheets, and the samples of each material combination were placed in a plastic sterile tube (Greiner Labortechnik Co., Ltd., Frickenhausen, Germany) with saline solution (0.9% NaCl, Sigma, St. Louis, MO, USA) to be eluted for 1 h, 1 day, 7 days, and 30 days. For each 0.1 g of dental composite materials, 1 ml of saline solution was added for the purpose of elution.
Cultures for cytogenetic testing were set up at the end of the elution period. One millilitre of primary leukocyte culture containing 5.6 × 106 cells was introduced into 9 ml of F-10 HAM's medium (Sigma) without the addition of fetal bovine serum or mitogen. Leukocytes were treated with 1 and 100 μl of eluates obtained from each of the tested dental adhesives to, respectively, simulate final mass concentrations of 0.1 and 10 mg of materials/ml. While composite-adhesive elusion solutions were discarded, and the samples were carefully transferred to a new sterile tube (Greiner Labortechnik Co., Ltd., Frickenhausen, Germany), with added 1 ml of primary leukocyte culture (density of 100 cells/μl) and 5 ml of RPMI 1640 (Gibco-Invitrogen, Carlsbad, CA, USA). After that, leukocytes were treated with 0.025 g and 0.05 g of each of the composite/adhesive combinations to, respectively, simulate final mass concentrations of 4.16 and 8.33 mg of sample/ml. Control leukocyte cultures were exposed for 48 h to a clean medium RPMI 1640 (combination of adhesives and composites) and a saline solution (0.9% NaCl, Sigma). Each DNA damage experiment included also positive control, which was hydrogen peroxide, 60 μl, for 15 min on ice. After the treatment period (48 h at 37°C in a 5% CO2 atmosphere), cultures were centrifuged for 10 min at 70 g, the supernatant was discarded, and cells were transferred into a sterile tube (Nange Nunc International, Naperville, IL, USA). They were resuspended and sampled for vital staining and the comet assay. Each DNA damage experiment included also positive control, which was hydrogen peroxide, 60 μl, for 15 min on ice [3, 5].
2.3. Cytotoxicity testing
Leukocyte viability was tested using the trypan blue exclusion technique . Cell suspension was mixed with 0.4% trypan blue (Sigma) and analysed using an Olympus light microscope (Olympus, Tokyo, Japan) under 100× magnification. For each test group, 1000 leukocytes were analysed by counting unstained (viable) cells. Blue-coloured cells were considered to be nonviable [3, 5].
2.4. Comet assay
The comet assay was carried out under alkaline conditions as described by Singh et al. . All chemicals used to perform the comet assay were obtained from Sigma. Sediment containing leukocytes was suspended in 100 μl of 0.5% low-melting-point agarose. This agarose layer was sandwiched between a layer of 0.6% normal melting point agarose and a top layer of 0.5% low melting point agarose on fully frosted slides. Slides were coded and kept on ice during polymerization of each gel layer. After solidification of the 0.5% agarose layer, slides were immersed in a lysis solution (1% sodium sarcosinate, 2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris-HCl, 1% Triton X-100, and 10% DMSO) at 4°C. After 1 h, the slides were removed from the lysing solution, drained, and placed in an electrophoresis buffer (0.3 M NaOH and 1 mM Na2EDTA, at pH 13) at 4°C for 20 min to allow the DNA to unwind. Electrophoresis was conducted on a horizontal electrophoresis platform in fresh, chilled electrophoresis buffer for 20 min at 300 mA and 19 V. After electrophoresis, slides were neutralized with Tris-HCl buffer (pH 7.5) three times for 5 min each and stained with ethidium bromide (20 μg/ml) for 10 min. Two slides per material per concentration per polymerization form per time point were analysed using an Ortoplan epifluorescence microscope (Leitz, Wetzlar, Germany) at 250× magnification (Figure 1). One hundred comets per slide were analysed by the comet assay II automatic digital analysis system (Perceptive Instruments, Halstead, UK) by measuring the tail length and intensity (% DNA). For the purpose of the analysis, the following were ignored: the edges and eventually damaged parts of the gel as well as debris, superimposed comets, comets of uniform intensity, and comets without a distinct head (i.e., ‘clouds’, ‘hedgehogs’, and ‘ghost cells’) [3, 5].
2.5. Statistical analysis
The comet test results were tested by the Kruskal-Wallis test to determine the statistical significance. The level of significance was set to 0.05. All calculations were performed using the commercial software, Statistica 7.0 (StatSoft, Tulsa, OK, USA).
Before testing, concentrations of the dental adhesives were selected based on the cytotoxicity results. The trypan blue exclusion test showed that the viability was >85% for each material at a dilution of >1:102 and greater than 90% for each material combination at a concentration of 8.33 mg/ml. According to these results, other dilutions and concentrations are considered to be used without any risk. The pH value in the cell culture was always between 7.19 and 7.4, which is, according to the manufacturer, the regular pH value.
The comet test determined the level of primary damage to DNA molecules of leukocytes after treating them with different dilutions of eluates of dental adhesives, depending on the length of time they were rinsed in the saline solution. In the present study for each dilution, elution duration, and polymerization state, 100 comets were analysed. Figures 2 and 3 showed two basic parameters of the comet tail, length, and the intensity of its fluorescence, of leukocytes exposed to a 10−2 and 10−4 elution of polymerized and unpolymerized dental adhesives. None of the tested dental adhesives revealed a statistically significant increase in tail length or tail intensity in treated leukocytes, independent of the applied dilution, elution duration, and polymerization form. Figures 4 and 5 represent the results of comet assay parameters (mean values and standard deviations) in human leukocytes during exposure to tested combinations of adhesive and composite resins in two different shades (A 1 and A 3.5), in concentrations 4.16 mg/ml and 8.33 mg/ml. Obtained results showed statistically higher increase in tail length after 1 day for Tetric EvoCeram in shade A1 for lower concentration (4.16 mg/ml) (15.3 ± 2.52, negative control 14.7 ± 2.85), and after 1 h (15.3 ± 4.70, negative control 14.7 ± 2.85) and 1 day (15.2 ± 9.10, negative control 14.7 ± 2.85) or same material for higher concentration (8.33 mg/ml). Concerning the tail intensity only Gradia Direct in the shade A 3.5 + G-Bond showed statistically higher percentage of DNA in tail in concentration 8.33 mg/ml eluted for 1 day (3.2 ± 5.64, negative control 1.2 ± 3.71) [3, 5].
In vitrobiocompatibility testings of dental adhesives
Contemporary dental adhesive systems are used to improve the contact between composite restorative materials and the walls of the prepared tooth cavity. As these materials come in close and prolonged contact with vital dentine, their influence on pulp cells is critical . Adhesive systems that create a stable relationship with biological tissues and allow both healing and tissue differentiation are considered biocompatible.
The effective toxicity of adhesives and its components is reduced but often not eliminated by the presence of dentine. Adhesive systems are placed on etched dentine (total-etch adhesives) or on cut dentine (self-etch adhesives) that is permeable. Since the available adhesive systems are not able to hermetically seal the deep dentine, in several
The toxicity has been attributed to the release of residual monomers from polymerized adhesive systems due to degradation processes or the incomplete polymerization as well. The degree of conversion of dental bonding systems relies mainly on the wavelength and power intensity of the light curing unit . Polymerization can be inhibited by the presence of oxygen, the presence of intrinsic water from dentine, and the presence of residual solvents in the adhesive . The degree of conversion is an important parameter because it specifies the amount of monomers converted in polymer. Even under ideal conditions, the conversion of monomers to polymers is commonly incomplete, and it is about 55–60% . Unconverted free monomers from polymerized adhesive monomers diluted into saliva after curing as result of degradation may be released into the oral environment where they could produce harmful local effects, as chronic inflammatory reactions of human pulp. Also, it has been demonstrated that they are responsible for many cytotoxic and possible endocrine-disruptive impact. The amount of the monomer release ranges from micrograms to milligrams .
Conventional adhesive systems require acid etching of the surface of enamel and dentine as a separate step before their applications. Application of acids to dentine surface removes the smear layer, opens the dentinal tubules, and decalcifies the intertubular and peritubular dentine . Despite paste apprehension about potential toxicity of acid penetration in dentine tubules and pulp space, the interaction of etchants with dentine is limited to the superficial 1.9–5.8 μm. It is unlikely that the acid is directly responsible for pulp injury. The effect of acid on the dentine is limited by the action of hydroxyapatite and collagen which acts as a baffler . Total etching is considered safe if the length of the remaining dentine is more or equal to 0.5 mm. Therefore, total-etching adhesives are recommended in shallow cavities, located in superficial or sclerotic dentine where the permeability of the dentine is low and the thickness of the remaining dentine is adequate to prevent any adverse effects from diffusing materials [36, 44]. After acid etching, a significant increase in dentine permeability due to smear layer removal and opening of dentinal tubules can facilitate the permeation of resin monomers towards pulp, mainly in deep dentine . About et al.  concluded in their study that dentine-bonding agents do not affect the cytodifferentiation of secondary odontoblasts when the remaining dentine thickness is 0.7 mm and when the materials are properly polymerized. When adhesives are applied in deep cavities, residual monomers might reach the pulp by diffusion, and in etched dentine, this penetration tends to be higher. Odontoblasts are typical pulp cells and are the first cells to be damaged by potentially cytotoxic compounds released from dental materials that diffuse through enamel and dentinal tubules. Injury to the odontoblasts results in loss of the capacity of these cells to secrete reactionary dentine, and secretion of dentine bridge by a new generation of odontoblast-like cells . An evaluation of the cytotoxic effect of total-etch two-step adhesive systems showed that both the acidic and non-acidic components of unpolymerized adhesive resins were responsible for high cytotoxic effects on odontoblast-like cells .
The most widely used self-etching adhesive systems involve two application steps: the conditioning of dentine and enamel with a self-etching primer, followed by the application of an adhesive resin. There are also one component self-etching adhesives, which contain etch, prime, and bond functions in a single solution. For these adhesive systems, the adhesive resin infiltrates to the same depth as the acidic primer exposed the collagen in dentine . Therefore, the use of self-etching adhesives systems is indicated for young, deep, permeable dentine [36, 49]. da Silva et al.  compared the effectiveness and biological compatibility of different generations of dentine adhesives. Their results showed that the one-step self-etch system had the best bond strength performance and was the least toxic to pulp cells, referring to it as a good alternative for specific cases. These results are in accordance with those given by Hashieh et al.  who have shown that one-step dentine-bonding agents were less toxic in cell culture than multistep counterparts. Self-etch adhesives have proved to have less cytotoxicity and better tissue response in histological evaluations than those related to conventional adhesive systems, but at this time they are still harmful to the pulp [51, 52].
Any restorative materials that are placed adjacent to vital pulp can induce biological effects. These effects are controlled by the components that are released from the material and the pulpal response to those components. The composition of the adhesive system plays an important role in the toxic effect it produces, and the choice of adhesive system for clinical use should take into consideration its biocompatibility . The usual composition of bonding agents includes resin monomers, initiators, inhibitors or stabilizers, solvents, and sometimes inorganic fillers . It has been reported that dental adhesives release substances that have biological effects (cytotoxicity, carcinogenicity, mutagenicity, and genotoxicity) and toxic potencies [2, 7]. Acidic and nonacidic components of polymerized and unpolymerized adhesives are considered responsible for the cytotoxic effects on the dentine-pulp system [16, 53, 54]. The resins in the adhesive systems are of interest to many researchers, and its biocompatibility has come under extensive scrutiny [12, 31, 55, 56].
The monomers are found to be cytotoxic in cell cultures and to affect the metabolism of the cells. Bisphenol A-glycidyl methacrylate (Bis-GMA), urethane dimethacrylate (UDMA), hydroxyethyl methacrylate (HEMA), and triethylene glycol dimethacrylate (TEGMA) are considered to be among the most toxic resins [50, 57, 58]. Several
Also, initiators may be released from dental adhesives and have been associated with cytotoxicity, related to their ability to generate free radicals . The cytotoxicity of dentine adhesives may as well be attributed to camphorquinone (CQ), the most commonly used photoactivator [15, 56, 62]. For concentrations higher than 1 mM, CQ caused a significant concentration-dependent increase of intracellular ROS in human pulp fibroblasts (HPF) within 90 min of exposure. The cytotoxicity by CQ can be partly explained by its induction of cell cycle arrest and apoptosis. CQ also inhibited the expression of type I collagen, a major extracellular protein of dental pulp, suggesting the effect of CQ on matrix turnover and pulpal repair. CQ also stimulates prostaglandin E2 (PGE2) and PGF2α production of pulp cells . It has been also documented that the camphorquinone acts not only as a cytotoxic agent but also as a mutagen, and its lixiviation may partly explain why these kinds of resinous products are considered as toxic agents . Another substance known for its toxic, allergenic, and mutagenic effects and present in some dental adhesives is glutaraldehyde, which seems to be harmful by direct contact with mucous tissue, as well as by inhalation of its evaporated form .
A further factor directly related to the biological responses to adhesive systems concerns monomer conversion into polymers, obtained by the polymerization technique. It has been reported that the type of light curing unit and light parameters such as light spectrum and intensity affect the cytotoxic properties of dental adhesives, as well as polymerization time and application mode [66, 73]. All these parameters influence directly the degree of monomer conversion and consequently the release of residual monomers. Ye et al.  evaluated the effect of light irradiance and source on the photopolymerization of three commercial dental adhesives by monitoring the double bond conversion as function of time during and after irradiation. These authors observed that the time for Single Bond with little solvent to reach the conversion plateau was about 20 s, while the time for OneUp Bond F was about 25 s and for Adper Prompt as long as 40 s, and they indicated that the time required to reach the conversion plateau for adhesive polymerization is a valuable information for the dental clinicians. According to Peutzfeld , only 60% of monomers are completely bound in polymerized composite resin. Incomplete conversion results in an increased amount of residual monomer and creates the risk of leaching into the surrounding environment. However, even with sufficient light intensity, a certain amount of unreacted resin monomers may be released, which has a potential impact on the toxicity of the materials . Adhesive/bonding agents cured with light-emitting diode (LED) units demonstrated higher cell survival rates in comparison with those cured with a halogen lamp . Polymerization time can also influence on the cytotoxicity of adhesive systems. For resin-based dental materials, a shorter light-curing duration usually results in a lower degree of conversion, inferior mechanical properties, and a higher cytotoxicity. There is a dramatic difference in the responses of cells to the three conditions of polymerization (light curing for 0, 10, or 40 s) .
Cell culture assays provide a convenient, controllable, and repeatable method to assess the biocompatibility of materials. Increasing public concern regarding the use of animals in biocompatibility evaluation of dental materials has made
In vivobiocompatibility testings of dental adhesives
The material is considered biocompatible when the interaction among a host, a material, and an expected function of material is in harmony. Dental material, furthermore, should not contain any toxic, leachable, and diffusible substances that could be absorbed and cause systemic responses such as teratogenic or carcinogenic effects. The ability of a material to even enter into interaction with biological tissue and create a stable connection is essential to verify its biocompatibility .
In addition to already mentioned
There are conflicting results concerning direct pulp capping trails with adhesives on animals. Therefore, Watts and Peterson  suggested that biocompatibility studies and histological evaluation of the pulp healing after pulp capping need to be performed for each species in particular. So far, investigations were implemented on monkeys, dogs, rats, and cats. Cox et al.  showed in the monkey tooth model that most adhesive systems are biologically compatible with pulp, allowing pulpal wound healing and reparative dentine formation. Study of Nowicka et al.  analysed the pulpo-dentine response after direct capping with self-etch adhesive system. In the majority of the specimens, it was presented inflammatory pulp response with tissue disorganization and lack of dentinal bridge formation. Contrary to this, the teeth in the control group, capped with Ca(OH)2, showed significantly smaller inflammatory pulp response with tissue disorganization and a considerably higher incidence of reparative dentine formation. These results are also in correspondence with those reported from the study of dogs from Koliniotou-Koumpia and Tziafas  and da Silva et al. . They reported a total absence of hard tissue formation after direct pulp capping with dentine adhesive system and presence of chronic inflammatory cell infiltrate, giant cells, and macrophages around the resin globes, morphological cell alteration, and hyalinization of the pulp cells. The pulpal responses to the dentine bonding systems used in the mentioned investigations could be attributed mainly to their acidic nature.
As opposed to animal studies, there are also clinical studies on humans, which are necessary for determining long-term biocompatibility of dental materials. The findings from above mentored studies on animals were similar to those obtained in study in human teeth of Cui et al. . Namely, more pronounced histological changes in pulpal tissue in teeth treated with self-etching adhesive systems were found than in control group treated with Ca(OH)2. Similar results were obtained also by Preira and associates . In their study, the adhesive systems did not stimulate the dental bridge formation even after over 200 days after the pulp capping. On the contrary, the dental bridge healing was observed after 60 days in control group of pulp capping with Ca(OH)2.
There are lots of opposite opinions of various authors about adhesives. Some authors consider that adhesives are safe, accurately biocompatible, and could be used for direct pulp capping on humans [92–94], whereas other researchers consider that adhesives are not suitable for direct pulp capping because of inducing the constant inflammation that does not heal [95–100]. Gwinnett and Tay  observed the pulp response after application of dentine adhesive to etched, deep, and unexposed coronal dentine, and they detected chronic inflammation of the pulp caused by adhesive in dental tubules. These so-called globules of resin in the tubules stimulate an immune reaction to a foreign body and are characterized by the appearance of mononuclear cell infiltration of macrophages or multinuclear giant cells. There has been also observed a degradation and loss of odontoblasts when the remaining dentine thickness was less than 0.3 mm. Furthermore, Hebling et al.  have found a hyaline alteration of extra-cellular matrix associated with oedema and hydropic pulp cell changes. Avles and Sobral  evaluated the biocompatibility of an etch-and-rinse adhesive system based on tertiary butanol applied in deep cavity human teeth with approximately 1 mm of remaining dentine by observing histological changes of the pulp tissue of humans at intervals of 1, 7, 14, and 21 days. They observed mild inflammatory infiltrate, preserved pulp tissue morphology, disorganization of the odontoblast layer in most specimens, as well as absence of bacteria at the intervals of 1, 7, 14, and 21 days.
Types of polymerization units also could be affected on the final cytotoxicity of adhesive system. Spagnuolo et al.  noticed that after lighting with LED units, there was a greater release of reactive oxygen that decreases cell survival than after lighting with halogen units.
There are a lot of studies that show that failure in the pulp capping is attributed to mistakes during operation, for example inadequate haemorrhage, an incomplete polymerization of the material, or usage of poor material properties. It is also known that a blood clot lyses could launch a series of chemotactic peptides that have a role in inflammation process . Recent studies on human teeth have shown irreversible damage of the pulp and odontoblasts, and also chronic inflammatory response with macrophages and mononuclear giant cell infiltration observed over a period of 300 days following restorative procedure. The appearance of these signs indicates the typical immune response to the foreign substance in the organism. This is a result of interaction of resin and its components with plasma proteins, tissues, and connective tissue cells [100, 101]. In the following studies, it has been shown that non-polymerized resin compounds released from dental adhesives cause chronic pulp inflammatory response . The response of the pulp to the dental materials could be modified by bacteria's and their products, and also affect by carious dentine, cavity preparation, and bleeding. The reparative capacity of damaged pulp in those cases is already weaker, and immunological responses are more drastic; therefore, they might contribute to the cytotoxic effects of dentine-bonding agents .
Composite fillings and adhesives contain numerous components that could be released into the environment and cause a biological activity in the organism (cytotoxicity, carcinogenicity, mutagenicity, genotoxicity) [53, 103]. The monomers might be released in the saliva and diffuse into the gingiva, mucosa, salivary glands, and blood where their cytotoxicity or teratogenesis could contribute to the tumour formation which
According to these results, there are lots of concerns about pulp therapy with adhesive systems. Also, the results obtained from animal models cannot be extrapolated to human clinical condition.
Most practitioners purchase materials that are commercially available without any concerns about their biocompatibility. Today, despite the fact that clinicians use an increasing number of materials, most relevant studies concentrate on their physical properties with less emphasis on biological compatibility. There is a large gap between the results published by research laboratories and clinical reports. In order to assess the biocompatibility, clinical studies, as well as
Toxicity of adhesive systems is associated primarily with short-term release occurring during setup time of material, due to insufficient polymerization, but also as long-term release of leachable substances generated by degradation over time . Numerous studies showed that the differential toxicity of the adhesives could be attributed to the different ingredients, the interactions between them, and the degree of resin polymerization. Many studies of applied adhesive systems in direct contact with animal pulp tissue have supported the concept that bacteria, rather than dental materials, promote irritation of pulp tissue . On the contrary, in other studies, it has been shown that those resin-based materials do not seem to be appropriate for use as pulp capping material and that no adhesive system should be applied directly on the pulp tissue [10, 35, 101, 109]. Thus, an adequate dentine barrier prevents or even reduces the amount of monomers and other components capable of causing damage to the pulp-dentine complex [6, 110].
Biocompatibility is one of the most important properties of dental materials, and adhesives are no exception. Future innovation in adhesive systems should seek novel properties like tissue tolerance. Adhesives should be biofunctional, protective, and preventive, with health-promoting effects that contribute to a better prognosis for restorative treatments and its biocompatibility.
According to results obtained from our previous