Methods to evaluate and strategies to improve the biocompatibility of dental materials and operative techniques

Abstract

Objective

The general aim of this article is to describe the state-of-the-art of biocompatibility testing for dental materials, and present new strategies for improving operative dentistry techniques and the biocompatibility of dental materials as they relate to their interaction with the dentin–pulp complex.

Methods

The literature was reviewed focusing on articles related to biocompatibilty testing, the dentin–pulp complex and new strategies and materials for operative dentistry. For this purpose, the PubMed database as well as 118 articles published in English from 1939 to 2014 were searched. Data concerning types of biological tests and standardization of in vitro and in vivo protocols employed to evaluate the cytotoxicity and biocompatibility of dental materials were also searched from the US Food and Drug Administration (FDA), International Standards Organization (ISO) and American National Standards Institute (ANSI).

Results

While there is an ongoing search for feasible strategies in the molecular approach to direct the repair or regeneration of structures that form the oral tissues, it is necessary for professionals to master the clinical therapies available at present. In turn, these techniques must be applied based on knowledge of the morphological and physiological characteristics of the tissues involved, as well as the physical, mechanical and biologic properties of the biomaterials recommended for each specific situation. Thus, particularly within modern esthetic restorative dentistry, the use of minimally invasive operative techniques associated with the use of dental materials with excellent properties and scientifically proved by means of clinical and laboratory studies must be a routine for dentists. This professional and responsible attitude will certainly result in greater possibility of achieving clinical success, benefiting patients and dentists themselves.

Significance

This article provides a general and critical view of the relations that permeate the interaction between dental materials and the dentin–pulp complex, and establish real possibilities and strategies that favor biocompatibility of the present and new products used in Dentistry, which will certainly benefit clinicians and their patients.

Introduction

There has been much discussion about new proposals for pulp tissue regeneration therapy. For several decades researchers have related that the acquisition of more in-depth knowledge of molecular biology and the mechanisms involved in the process of repair and regeneration of the dentin–pulp complex may direct new treatment strategies for pulps submitted to various types of aggressions, such as contamination, trauma, cytotoxicity of dental material components, and thermal injuries. This has appeared to become more evident and possible by means of the recent advancements in knowledge of the functions and activities of pulpal stem cells and signaling molecules present in dental tissues . However, applying this body of knowledge and the promising results obtained in laboratory researches to clinical situations has been a great challenge.

The incessant search for new knowledge that direct future therapies associated with a more biological approach that allows and/or stimulates pulp repair has, up to a certain point, led to neglect in evaluating the dentin–pulp complex response to the different clinical procedures being used at present. This becomes evident when new dental materials and operative techniques are recommended for certain therapies without these having been scientifically evaluated, so that they are effectively shown to be safe for all patients. Proof of this was the recommendation, about 15 years ago, to etch the exposed pulp tissue with acidic agents and proceed with direct pulp capping with bonding agents . This type of therapy, in spite of having been scientifically demystified some years ago , has been the focus of some roundtable discussions even today, in which some consider this therapy a promising clinical procedure capable of stimulating the repair of exposed pulps . In addition, one modality of an esthetic clinical procedure that presents limited evaluation with respect to possible damage to the dentin–pulp complex is dental bleaching. This type of therapy has been widely used all over the world, even knowing that around 70–80% of the patients submitted to dental bleaching have related some type of pain or discomfort during and/or post-treatment . What would be the reason for this undesirable side effect caused by bleaching vital teeth? Why does post-bleaching pain regress and disappear within some days? Would it not be interesting to research this clinical procedure and understand the possible damage caused to the dentin–pulp complex by the different techniques and products used in dental bleaching?

Logically, researches must be conducted to establish a certain therapy as being safe for patients and for dentists themselves. From then on, it may be recommended for clinical application, with this maxim being true for any new dental material or new recommendation for treatment in the health area. This is the guideline of International Organizations, such as the US Food and Drug Administration (FDA) and International Organization of Standardization (ISO), which determine the performance of rational tests in vitro , in animals and usage/clinical tests, which must be adapted to present days . Undoubtedly, the development and the constant search for knowledge within the topic of tissue repair are relevant as a whole, and certainly will, in the very near future, direct important modalities of therapy for the dentin–pulp complex. Therefore, the main objective of this article is to provide a general and critical view of the relations that permeate the interaction between dental materials and the dentin–pulp complex, and establish real possibilities and strategies that favor biocompatibility of the present and new products used in Dentistry, which will certainly benefit clinicians and their patients.

Biocompatibility tests for dental materials

In 1970, Autian proposed a sequence of studies to determine the safety of the clinical use of new materials. The purpose of conducting in vitro researches, followed by investigations in animals, and finally, clinical studies is to evaluate the biocompatibility of new materials in an ethical and financially feasible manner, and at every level, eliminate materials that present greater cytotoxic potential. Nevertheless, results obtained in vitro may diverge from those observed in animals and humans. Therefore, the great challenge to laboratory tests at present, whether in vitro or in animals, is to seek greater approximation to clinical reality (for further details, we recommend reading the work of Wataha and Bayne ).

In vitro studies

Analysis of the biocompatibility of materials in vitro occurs outside of live organisms, using cell cultures or constituents. These tests allow the incorporation of strategies to increase the proximity to clinical practice, such as for example, the use of physical barriers, such as enamel and/or dentin discs. The evolution of these methodologies allows for evaluation of cell function and viability, gene expression, the expression of proteins and inflammatory mediators, reactive oxygen species, type of cell death, cell morphology, among others . Furthermore, in vitro studies enable greater reproducibility, speed, low cost, ease in determining control groups and avoid legal and ethical conflicts, such as submitting animals or humans to pain, suffering and possible risks in general .

The laboratory methodologies established some decades ago, and which continue be used nowadays, have been extensively criticized. For this reason, the in vitro research models have evolved significantly with the aim of being better able to mimic in vivo conditions .

The insertion of models that use a dentin barrier has been recognized as an important evolution of in vitro tests of dental materials biocompatibility, providing greater similarity of the experimental conditions to those observed in vivo . Thus, the use of the artificial pulp chamber (APC) is an effective experimental model for performing different laboratory protocols for the purpose of evaluating new materials and their application techniques ( Fig. 1 ). For this in vitro model, dentin and/or enamel discs, which may be selected and standardized according to the object understudy, are inserted into APCs. In this protocol, the pulpal surface of the disk is kept in contact with the culture medium, and the occlusal surface remains exposed, so that the materials and/or technique can be applied and evaluated . Particularly for in vitro tests in which dentin discs are used, an important advantage inherent to this methodology is that the samples, which may be obtained from human teeth or those of animals, may also be selected and standardized as regards thickness and permeability, factors that are directly related to the diffusion of toxic components related from the experimental materials under analysis .

Fig. 1
Schematic representation of an artificial pulp chamber.

The use of APCs also enables different stages of clinical procedures to be performed, such as acid etching and the application of adhesive systems before the application of restorative materials, among other techniques . Moreover, it is also possible to perform tests with light polymerizable and chemically setting materials concomitantly with contact of the materials with the dentin substrate, and contact of the latter with the cell lineage to be evaluated . Other models of APCs allow the application of intra-pulpal pressure, which may influence the conditions of application and solubility of the test materials, as well as the transdental diffusion of their components.

Another important parameter that should be taken into consideration for the development of in vitro studies, which must as far as possible, approximate and mimic the clinical conditions, is the selection of cell lineages that must be relevant to the analysis of the dental materials . For the evaluation of dental materials and techniques for the application of new products indicated for use on different dental tissues, it is important to select cells that present an odontoblastic phenotype, because in the teeth of mammals this type of cell is organized in layers to line the coronal and root dentin internally ( Fig. 2 a and b). Within this context, the odontoblasts are the first pulp cells to come into contact with the components of dental materials capable of diffusing through the dentin and/or enamel/dentin. Therefore, cells of an odontoblastic lineage, such as the MDPC-23 cells, which were obtain from rat teeth by Hanks et al. , have been widely used for laboratory tests of new materials and techniques . For these tests, undifferentiated pulp cells (OD-21) ; primary cultures of human pulp cells , or undifferentiated cells from human pulp have also been used, which may more safely indicate the possible effects of the tested materials .

Fig. 2
(a) MDPC-23 cells are attached to dentin substrate on which they were cultured; SEM × 500. (b) High magnification of Fig. 2 a. Note the morphology of the pulp cells which exhibit a number of short and thin cytoplasmic processes; SEM × 1000.

It should also be pointed out that a large portion of the in vitro cytotoxicity/biocompatibility studies have been developed with the use of monolayer cell culture models . However, the 3D culture models, in which cells are cultivated in specific types of collagen matrix (scaffolds), appear to provide more favorable conditions for the morphological and phenotypical expression of the cells, and this experimental model may also be used for the direct and indirect evaluation of the biologic effects of new dental materials and techniques .

Another factor that contributes to the efficacy of the in vitro cytotoxicity/biocompatibility tests for dental materials is the selection of the tests to be performed , which may vary according to the type of product to be tested and the following levels of response one wishes to obtain: cytotoxicity, the induction of an inflammatory responses, biostimulation or cell differentiation capacity, among other important cellular functions for the homeostasis and repair of the dentin–pulp complex.

When considering cytotoxicity tests, the most widely used methods are those that determine cell viability, particularly by means of analyzing the mitochondrial activity of the cells exposed to the materials and/or their isolated components . Other tests in turn, evaluate the occurrence of cell death and whether this was due to necrosis or apoptosis . These analyses may contribute to understanding the intensity of the toxic effects of dental materials, as those that induce apoptosis are considered less aggressive when compared with materials that induce cell death by necrosis . Another parameter that may also be evaluated is the production of reactive oxygen species, which is directly related to the induction of cell lesion, and may be detected by means of fluorescent probes .

The application of cell metabolism tests may indicate stimulation or inhibition of cell differentiation, such as protein production and gene and protein expression of specific molecules, such as Collagen Type I and alkaline phosphatase, responsible for the formation and mineralization of the dentin matrix, respectively . The protocol for analyzing the production of mineralized nodules may also be used to determine the induction or inhibition of the mineralization capacity of cells in culture after contact with different materials and/or their components . On the other hand, laboratory methods that evaluate inflammatory cytokine expression by these cells in culture may indicate the possible in vivo induction of an inflammatory response in the pulp tissue .

In addition to quantitative tests, the effects of dental material on cell cultures may also be evaluated in a qualitative manner, such as in complementary analyses of cells by scanning electron microscopy . By means of this method, it is possible to verify morphological changes caused by the materials and/or their isolated components, as well as to determine whether there was a reduction or increase in the population of cells adhered to a substrate after they had been exposed to the products being tested ( Fig. 3 a and b).

Fig. 3
(a) Pulp horn of a human sound tooth. Note that a continuous odontoblast layer (arrows) is underlying the dentin (D); H/E, 64×. (b) Detail of the dentin–pulp complex. Note the tubular dentin (D), predentin (PD), odontoblast layer (OL), cell-free zone (vertical arrow) and the cell-rich zone (horizontal arrows). H/E, 125×.

A third determinant factor for in vitro cytotoxicity/biocompatibility studies of dental materials is the selection of adequate concentrations and volumes of these products and their components, which must be close to those of clinical conditions .

In general, in spite of the limited clinical significance of scientific data obtained by in vitro researches, it is important to point out that the results of laboratory studies, if evaluated attentively and judiciously, may direct future in vivo investigations to be conducted in animals, and contribute significantly to the understanding of the data observed in clinical studies .

In vivo studies (in animals)

The use of animals for research is a controversial subject and has been the target of broad discussions, mainly of an ethical nature . Nevertheless, these studies may provide more relevant scientific data than those observed in vitro and enable the evaluation of important parameters, such as the interaction of the material with blood, chronic inflammatory responses and bone regeneration . Researches using laboratory animals present lower costs than clinical studies and may be satisfactorily controlled. Nevertheless, the results obtained in these tests may be influenced by the species, age and gender of the animal used in the studies and cannot automatically be extrapolated to clinical situations in humans . Moreover, interpretation of the responses observed is complex, since various events occur simultaneously, such as the trauma generated by the applications of the test materials in contact with the animal’s tissues and possible local infections.

Studies in animals may, in a preliminary manner, determine the safety of using the material being tested, and predict its clinical success in a specific function . In the first case, some specific parameters, such as dosage and administration pathway do not represent circumstances of clinical relevance; however, the main proposal is to expose the animal to extreme conditions that will allow the safety of the material being analyzed to be determined. Whereas, in the second case, the tests attempt to reproduce, in the best possible manner, the conditions found in clinical reality, such as size, dosage, method of application, presence of physical barriers, such as dentin and/or dentin/enamel, as well as the composition and manipulation of the materials .

Another difficulty found with regard to the use of animals for research is in determining adequate control groups that favor interpretation of the results, with the lowest possible number of biases. In addition, statistical analysis of the responses characterizes a great challenge, since there may be problems with respect to the correct definition of independent variables.

Clinical studies

Clinical studies are characterized by the application of the experimental materials in human volunteers and are considered the “gold standard” for the evaluation of properties and performance of dental materials . These types of studies may present various experimental protocols that vary as regards cost and the difficulty of conducting them . However, the large majority of clinical studies have sought to evaluate the mechanical properties of materials, so that biocompatibility would not be their main focus . In general, clinical studies are more expensive, take longer, are more difficult to control and interpret the results, particularly when compared with researches developed in animals and in vitro tests. In addition, these studies using human beings face strong ethical barriers .

Among the different types of clinical studies, there are retrospective, cross-sectional and prospective studies. Longitudinal or prospective studies are more representative with regard to determination of the biological performance of a material, and there are strategies that can be used to increase their reliability, such as blinding, randomization, placebo groups and strategies to minimize biases . After the treatment, the patients are followed-up over the course of time, which allows data collection. Nevertheless, in general, these types of studies are expensive, require a long period for their finalization and may be influenced by the operator’s skill, which may be far above or below the clinical average .

Correlation between biocompatibility tests

Unfortunately, it is frequently not possible to obtain strong correlation between laboratory observations and the set of clinical properties in the short or long term . This occurs for various reasons, among them the fact that laboratory tests are static, and on the other hand, analysis of clinical properties involves dynamic observations, such as change in the material over the course of time which, in spite of also occurring in vitro , does not necessarily reflect the same conditions as those observed in vivo .

In spite of the challenges found in correlating in vitro studies in animals and clinical studies, the regulatory agencies recognize the importance of laboratory studies in the evaluation of the biocompatibility of dental materials . As previously mentioned, these tests must be as close as possible to the clinically relevant conditions, which range from the use of physical barriers through to the selection of the cell type to be used and its condition of exposure to the products being tested. For studies in animals, the choice of species, site of application of the experimental material and time intervals of evaluation must also be taken into consideration .

It is known that the most efficient manner of evaluating the biocompatibility of a material is by means of the association of the results of in vitro tests with those obtained in animals and clinical tests . Nevertheless, the manner in which this association of the scientific data should occur continues to be the area of many discussions and a challenge to be overcome, because the sequence of tests does not always present good correlation, particularly due to the limitations of laboratory tests in reproducing clinical conditions . However, in vitro test are most useful for the initial determination of the cytotoxic potential of dental materials and for investigating specific mechanisms of the cell response to products and therapies.

In a recent literature review, Wataha proposed a new strategy for the use of laboratory and clinical tests, in which in vitro studies would mainly be used for the evaluation and characterization of the constituents released by the materials and their dangers and risks, since the majority of the undesirable effects are caused by toxic substances related from these products. Tests in animals would have the main functions of complementing the in vitro tests and evaluate the risks and dangers of new products, determining their safe application. Thus, these tests may determine the main risks of the material (cytotoxicity, genotoxicity, carcinogenicity, etc. ), so that from these initial data, safer clinical tests may be performed.

Biocompatibility tests for dental materials

In 1970, Autian proposed a sequence of studies to determine the safety of the clinical use of new materials. The purpose of conducting in vitro researches, followed by investigations in animals, and finally, clinical studies is to evaluate the biocompatibility of new materials in an ethical and financially feasible manner, and at every level, eliminate materials that present greater cytotoxic potential. Nevertheless, results obtained in vitro may diverge from those observed in animals and humans. Therefore, the great challenge to laboratory tests at present, whether in vitro or in animals, is to seek greater approximation to clinical reality (for further details, we recommend reading the work of Wataha and Bayne ).

In vitro studies

Analysis of the biocompatibility of materials in vitro occurs outside of live organisms, using cell cultures or constituents. These tests allow the incorporation of strategies to increase the proximity to clinical practice, such as for example, the use of physical barriers, such as enamel and/or dentin discs. The evolution of these methodologies allows for evaluation of cell function and viability, gene expression, the expression of proteins and inflammatory mediators, reactive oxygen species, type of cell death, cell morphology, among others . Furthermore, in vitro studies enable greater reproducibility, speed, low cost, ease in determining control groups and avoid legal and ethical conflicts, such as submitting animals or humans to pain, suffering and possible risks in general .

The laboratory methodologies established some decades ago, and which continue be used nowadays, have been extensively criticized. For this reason, the in vitro research models have evolved significantly with the aim of being better able to mimic in vivo conditions .

The insertion of models that use a dentin barrier has been recognized as an important evolution of in vitro tests of dental materials biocompatibility, providing greater similarity of the experimental conditions to those observed in vivo . Thus, the use of the artificial pulp chamber (APC) is an effective experimental model for performing different laboratory protocols for the purpose of evaluating new materials and their application techniques ( Fig. 1 ). For this in vitro model, dentin and/or enamel discs, which may be selected and standardized according to the object understudy, are inserted into APCs. In this protocol, the pulpal surface of the disk is kept in contact with the culture medium, and the occlusal surface remains exposed, so that the materials and/or technique can be applied and evaluated . Particularly for in vitro tests in which dentin discs are used, an important advantage inherent to this methodology is that the samples, which may be obtained from human teeth or those of animals, may also be selected and standardized as regards thickness and permeability, factors that are directly related to the diffusion of toxic components related from the experimental materials under analysis .

Fig. 1
Schematic representation of an artificial pulp chamber.

The use of APCs also enables different stages of clinical procedures to be performed, such as acid etching and the application of adhesive systems before the application of restorative materials, among other techniques . Moreover, it is also possible to perform tests with light polymerizable and chemically setting materials concomitantly with contact of the materials with the dentin substrate, and contact of the latter with the cell lineage to be evaluated . Other models of APCs allow the application of intra-pulpal pressure, which may influence the conditions of application and solubility of the test materials, as well as the transdental diffusion of their components.

Another important parameter that should be taken into consideration for the development of in vitro studies, which must as far as possible, approximate and mimic the clinical conditions, is the selection of cell lineages that must be relevant to the analysis of the dental materials . For the evaluation of dental materials and techniques for the application of new products indicated for use on different dental tissues, it is important to select cells that present an odontoblastic phenotype, because in the teeth of mammals this type of cell is organized in layers to line the coronal and root dentin internally ( Fig. 2 a and b). Within this context, the odontoblasts are the first pulp cells to come into contact with the components of dental materials capable of diffusing through the dentin and/or enamel/dentin. Therefore, cells of an odontoblastic lineage, such as the MDPC-23 cells, which were obtain from rat teeth by Hanks et al. , have been widely used for laboratory tests of new materials and techniques . For these tests, undifferentiated pulp cells (OD-21) ; primary cultures of human pulp cells , or undifferentiated cells from human pulp have also been used, which may more safely indicate the possible effects of the tested materials .

Fig. 2
(a) MDPC-23 cells are attached to dentin substrate on which they were cultured; SEM × 500. (b) High magnification of Fig. 2 a. Note the morphology of the pulp cells which exhibit a number of short and thin cytoplasmic processes; SEM × 1000.

It should also be pointed out that a large portion of the in vitro cytotoxicity/biocompatibility studies have been developed with the use of monolayer cell culture models . However, the 3D culture models, in which cells are cultivated in specific types of collagen matrix (scaffolds), appear to provide more favorable conditions for the morphological and phenotypical expression of the cells, and this experimental model may also be used for the direct and indirect evaluation of the biologic effects of new dental materials and techniques .

Another factor that contributes to the efficacy of the in vitro cytotoxicity/biocompatibility tests for dental materials is the selection of the tests to be performed , which may vary according to the type of product to be tested and the following levels of response one wishes to obtain: cytotoxicity, the induction of an inflammatory responses, biostimulation or cell differentiation capacity, among other important cellular functions for the homeostasis and repair of the dentin–pulp complex.

When considering cytotoxicity tests, the most widely used methods are those that determine cell viability, particularly by means of analyzing the mitochondrial activity of the cells exposed to the materials and/or their isolated components . Other tests in turn, evaluate the occurrence of cell death and whether this was due to necrosis or apoptosis . These analyses may contribute to understanding the intensity of the toxic effects of dental materials, as those that induce apoptosis are considered less aggressive when compared with materials that induce cell death by necrosis . Another parameter that may also be evaluated is the production of reactive oxygen species, which is directly related to the induction of cell lesion, and may be detected by means of fluorescent probes .

The application of cell metabolism tests may indicate stimulation or inhibition of cell differentiation, such as protein production and gene and protein expression of specific molecules, such as Collagen Type I and alkaline phosphatase, responsible for the formation and mineralization of the dentin matrix, respectively . The protocol for analyzing the production of mineralized nodules may also be used to determine the induction or inhibition of the mineralization capacity of cells in culture after contact with different materials and/or their components . On the other hand, laboratory methods that evaluate inflammatory cytokine expression by these cells in culture may indicate the possible in vivo induction of an inflammatory response in the pulp tissue .

In addition to quantitative tests, the effects of dental material on cell cultures may also be evaluated in a qualitative manner, such as in complementary analyses of cells by scanning electron microscopy . By means of this method, it is possible to verify morphological changes caused by the materials and/or their isolated components, as well as to determine whether there was a reduction or increase in the population of cells adhered to a substrate after they had been exposed to the products being tested ( Fig. 3 a and b).

Fig. 3
(a) Pulp horn of a human sound tooth. Note that a continuous odontoblast layer (arrows) is underlying the dentin (D); H/E, 64×. (b) Detail of the dentin–pulp complex. Note the tubular dentin (D), predentin (PD), odontoblast layer (OL), cell-free zone (vertical arrow) and the cell-rich zone (horizontal arrows). H/E, 125×.

A third determinant factor for in vitro cytotoxicity/biocompatibility studies of dental materials is the selection of adequate concentrations and volumes of these products and their components, which must be close to those of clinical conditions .

In general, in spite of the limited clinical significance of scientific data obtained by in vitro researches, it is important to point out that the results of laboratory studies, if evaluated attentively and judiciously, may direct future in vivo investigations to be conducted in animals, and contribute significantly to the understanding of the data observed in clinical studies .

In vivo studies (in animals)

The use of animals for research is a controversial subject and has been the target of broad discussions, mainly of an ethical nature . Nevertheless, these studies may provide more relevant scientific data than those observed in vitro and enable the evaluation of important parameters, such as the interaction of the material with blood, chronic inflammatory responses and bone regeneration . Researches using laboratory animals present lower costs than clinical studies and may be satisfactorily controlled. Nevertheless, the results obtained in these tests may be influenced by the species, age and gender of the animal used in the studies and cannot automatically be extrapolated to clinical situations in humans . Moreover, interpretation of the responses observed is complex, since various events occur simultaneously, such as the trauma generated by the applications of the test materials in contact with the animal’s tissues and possible local infections.

Studies in animals may, in a preliminary manner, determine the safety of using the material being tested, and predict its clinical success in a specific function . In the first case, some specific parameters, such as dosage and administration pathway do not represent circumstances of clinical relevance; however, the main proposal is to expose the animal to extreme conditions that will allow the safety of the material being analyzed to be determined. Whereas, in the second case, the tests attempt to reproduce, in the best possible manner, the conditions found in clinical reality, such as size, dosage, method of application, presence of physical barriers, such as dentin and/or dentin/enamel, as well as the composition and manipulation of the materials .

Another difficulty found with regard to the use of animals for research is in determining adequate control groups that favor interpretation of the results, with the lowest possible number of biases. In addition, statistical analysis of the responses characterizes a great challenge, since there may be problems with respect to the correct definition of independent variables.

Clinical studies

Clinical studies are characterized by the application of the experimental materials in human volunteers and are considered the “gold standard” for the evaluation of properties and performance of dental materials . These types of studies may present various experimental protocols that vary as regards cost and the difficulty of conducting them . However, the large majority of clinical studies have sought to evaluate the mechanical properties of materials, so that biocompatibility would not be their main focus . In general, clinical studies are more expensive, take longer, are more difficult to control and interpret the results, particularly when compared with researches developed in animals and in vitro tests. In addition, these studies using human beings face strong ethical barriers .

Among the different types of clinical studies, there are retrospective, cross-sectional and prospective studies. Longitudinal or prospective studies are more representative with regard to determination of the biological performance of a material, and there are strategies that can be used to increase their reliability, such as blinding, randomization, placebo groups and strategies to minimize biases . After the treatment, the patients are followed-up over the course of time, which allows data collection. Nevertheless, in general, these types of studies are expensive, require a long period for their finalization and may be influenced by the operator’s skill, which may be far above or below the clinical average .

Correlation between biocompatibility tests

Unfortunately, it is frequently not possible to obtain strong correlation between laboratory observations and the set of clinical properties in the short or long term . This occurs for various reasons, among them the fact that laboratory tests are static, and on the other hand, analysis of clinical properties involves dynamic observations, such as change in the material over the course of time which, in spite of also occurring in vitro , does not necessarily reflect the same conditions as those observed in vivo .

In spite of the challenges found in correlating in vitro studies in animals and clinical studies, the regulatory agencies recognize the importance of laboratory studies in the evaluation of the biocompatibility of dental materials . As previously mentioned, these tests must be as close as possible to the clinically relevant conditions, which range from the use of physical barriers through to the selection of the cell type to be used and its condition of exposure to the products being tested. For studies in animals, the choice of species, site of application of the experimental material and time intervals of evaluation must also be taken into consideration .

It is known that the most efficient manner of evaluating the biocompatibility of a material is by means of the association of the results of in vitro tests with those obtained in animals and clinical tests . Nevertheless, the manner in which this association of the scientific data should occur continues to be the area of many discussions and a challenge to be overcome, because the sequence of tests does not always present good correlation, particularly due to the limitations of laboratory tests in reproducing clinical conditions . However, in vitro test are most useful for the initial determination of the cytotoxic potential of dental materials and for investigating specific mechanisms of the cell response to products and therapies.

In a recent literature review, Wataha proposed a new strategy for the use of laboratory and clinical tests, in which in vitro studies would mainly be used for the evaluation and characterization of the constituents released by the materials and their dangers and risks, since the majority of the undesirable effects are caused by toxic substances related from these products. Tests in animals would have the main functions of complementing the in vitro tests and evaluate the risks and dangers of new products, determining their safe application. Thus, these tests may determine the main risks of the material (cytotoxicity, genotoxicity, carcinogenicity, etc. ), so that from these initial data, safer clinical tests may be performed.

Dentin–pulp complex

Although dentin and pulp present distinct structural characteristics, these tissues arising from the same embryonic origin are intimately related and interdependent, so that both are recognized as a functional unit, denominated the dentin–pulp complex (DPC) . Therefore, the development of strategies with the aim of increasing the biocompatibility of new dental materials and clinical procedures, maintaining the functional activity and vitality of the DPC, are directly related to knowledge of the biology and physiology of these tissues, and understanding of their inter-relations.

The DPC is a highly dynamic structure, capable of adapting to different stimuli that may trigger an inflammatory reaction, either associated with the synthesis and deposition of dentin matrix, or not . The main link of communication between the dentin and pulp are the odontoblasts, which are responsible for the deposition and maintenance of dentin and are also involved in transmission of sensory stimuli in the DPC and cellular defense against pathogens . These highly specialized cells, which present the main nucleus and organelles located in the cell body, are organized in a layer at the periphery of the pulp tissue, so that they internally line the coronal and radicular dentin. Cytoplasmic prolongations originated from the odontoblasts may be found within the dentinal tubules. Thus, a cavity preparation in dentin may lead to cutting off many odontoblast prolongations, which determines that this operative procedure must be judiciously and very carefully performed. The organization of odontoblasts at the periphery of the pulp and the presence of effective junctional complexes among these cells, make the odontoblastic layer act as a “semipermeable barrier” that allows only the exudation of interstitial fluid and low molecular weight proteins into the tubules, forming the dentinal fluid; thus, dentin physiology provides this tissue with two basic characteristics: permeability and humidity .

It is known that the diameter and quantity of tubules per area of dentin increase according to proximity to the pulp, which results in deep dentin presenting greater permeability, and consequently being more humid than superficial dentin . These morphological differences of dentin have a direct relationship with the strategy to be defined for the maintenance of viability and homeostasis of the DPC, when different operative procedures are performed. Dentin permeability regulates the rate of diffusion of toxic products (bacterial or those released from dental materials) in the direction of the pulp tissue, which may determine the pattern and intensity of tissue response. It has also been demonstrated that the humidity present in dentin contributes to the resilience of this tissue, which is important for the distribution of stress resulting from masticatory forces . Nevertheless, this characteristic of dentin may have adverse effects on the dental materials applied to this tubular and humid tissue . Therefore, restorative procedures to be applied to exposed deep dentin tissue have become a challenge, particularly with regard to the advance of Adhesive Restorative Dentistry . In order to go more deeply into knowledge of dentin tissue, we suggest reading the work of Tjärderhane et al. .

In addition to the primordial function of the odontoblasts in the synthesis and deposition of dentin matrix, it is known that these specialized pulp cells also act in the modulation of immune and inflammatory pulp responses, and are therefore considered the first line of defense of the DPC . It has been demonstrated that the odontoblasts, which have receptors for the recognition of molecular patterns associated with pathogens, are capable of expressing inflammatory cytokines and chemokines , as well as proangiogenic mediators and others involved with the maturation and chemotaxis of dendritic cells . Therefore, certain stimuli applied on dentin tissue have a direct repercussion on the subjacent odontoblasts, which may trigger a tissue defense response with intensity directly related to the intensity of the aggression.

The inflammatory response is considered the initial and integral phase of the DPC reparative and regenerative process . According to the degree and persistence of the inflammatory process generated, the tissue response triggered may result in significant pulp damage . Aggressions of light intensity result in an increase in the regulation of dentinogenesis by the subjacent primary odontoblasts (reactionary dentinogenesis). However, when the injury is moderate or intense, resulting in the death of the primary odontoblasts that line the dentin internally, the reactionary condition becomes more complex, involving the recruitment of undifferentiated mesenchymal cells from the pulp and their differentiation into odontoblast-like cells, which begin to synthesize and deposit dentin matrix (reparative dentinogenesis) with an amorphous, sometimes atubular characteristic . Consequently, the clinical use of dental materials or application of operative techniques that result in intense pulp cell death, followed by the deposition of reparative dentin, do not characterize acceptable and safe clinical procedures. This is because, in addition to rupture in the equilibrium of the dentin/pulp connection by the death of the primary odontoblasts, loss of their cytoplasmic prolongations and intra-tubular nerve endings, the recruitment of a large number of undifferentiated mesenchymal cells from the pulp also occurs, resulting in the reduction in cellularity and consequent early aging of the pulp tissue ( Fig. 4 a and b).

Nov 25, 2017 | Posted by in Dental Materials | Comments Off on Methods to evaluate and strategies to improve the biocompatibility of dental materials and operative techniques
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