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Dentinogenic Activity of Biodentine in Deep Cavities of Miniature Swine Teeth
Basic Research—Biology
Dentinogenic Activity of Biodentine in Deep Cavities of Miniature Swine Teeth Christina Tziafa, DDS, MSc,* Eugenia Koliniotou-Koumpia, DDS, PhD,* Seraphim Papadimitriou, DDS, PhD,† and Dimitrios Tziafas, DDS, PhD‡ Abstract Introduction: The aim of the present study was to evaluate comparatively the bioactivity potential of a calcium silicate–based material (Biodentine; Septodont, SaintMaur-des-fosses Cedex, France) after the restoration of deep dentinal cavities of miniature swine teeth with or without the application of a calcium hydroxide–containing pulp protective base (Dycal; Caulk Lab, Milford, DE). Methods: Thirty-three permanent teeth (premolars, canines, and incisors) of 3 miniature swine were used. Class V cavities were prepared on the buccal surface of teeth. The cavities were restored with Biodentine in the presence (control group) or absence (experimental group) of a Dycal protective base. The pulpal tissue responses were histologically and histomorphometrically assessed at postoperative periods of 3 and 8 weeks. Three specimens were further evaluated with scanning electron microscopy. The maximum thickness of the postoperatively formed mineralized matrix beneath the cavity floor was measured. Data were statistically analyzed by the Kruskal-Wallis and the Mann-Whitney U tests. Results: A bacterial staining reaction along the cavity walls or intense inflammatory infiltration in the pulp was not detected in any of the specimens. A continuous zone of the postoperatively formed mineralized matrix mostly of atubular structure with scattered defects and cellular inclusions and occasionally followed by a thin zone with tubular morphology was detected in all specimens of the control group and 13 of 18 experimental group teeth. In the remaining teeth of the experimental group, a separate zone composed of the osteotypic mineralized matrix and soft tissues was noted between the circumpulpal and the newly formed matrix. Scanning electron microscopy confirmed the fibrous structural morphology of the tertiary dentin. A significantly higher rate of the postoperatively formed mineralized matrix had been formed in the teeth of the experimental group in both periods of 3 and 8 weeks (P < .01). Conclusions: The present investigation indicates that under the present experimental conditions
tertiary dentin with occasional intermediate formation of osteodentin is observed after the application of Biodentine in the presence or absence of a Dycal protective base. The thickness of the tertiary dentine zone was significantly higher in the absence of Dycal. (J Endod 2015;-:1–6)
Key Words Biodentine, calcium hydroxide, calcium silicate, Dycal, miniature swine teeth, pulp capping materials, pulp protection, tertiary dentin
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t has been recognized that local dentinal injuries in nonexposed cavities such as carious lesions, physical trauma, tooth grinding, restorative materials, and so on affect the vitality and function of the pulp (1–3). With milder injury in which the inflammation is minimal and in the absence of bacteria in the affected dentin area (4), the insult is overcome, the pulp-dentinal complex progressively returns to pulp healing and pulp vitality, and dentinogenic function is preserved (5). Up-regulation of survived odontoblast biosynthetic activity or replacement of lost odontoblasts by newly differentiated odontoblastlike cells has been described as a part of pulp tissue repair, leading to reactionary or reparative dentin formation, respectively (6). Peritubular dentin formation may also be noted in the affected dentin zone (2, 7, 8). As a result of the increased odontoblast activity, the permeability of affected dentin is reduced (9), leading to effective long-term protection of the pulp. In today’s clinical practice, the restoration of dentinal cavities without pulp exposure is a safe and predictable method of treatment (3) despite the fact that data from high-quality randomized control clinical trials are rare (10, 11). The remaining dentin thickness has been widely recognized as the main factor that determines the long-term success of the treatment in the absence of bacteria, whereas material toxicity also has been implicated with pulpal injury and subsequent pulp healing (3, 12). Thus, it has been recognized that in cases of deep dentinal cavities pulp protection is required to protect the pulp-dentin complex from materials’ chemical injury and further bacterial invasion. For many years, calcium hydroxide–containing cements and glass ionomers have been considered as materials of choice for pulp protection in deep dentinal cavities (13). Recently, a number of new formulas of calcium silicate–based materials have been introduced as restorative materials. Among them, Biodentine (Septodont, Saint-Maurdes-fosses Cedex, France) has been tested as a dentin substitute. Biodentine is composed of a powder component (tricalcium silicate, calcium carbonate, and zirconium oxide) and a liquid component (calcium chloride in water). In in vitro studies, Biodentine showed good physicochemical properties and interfacial characteristics with the dentin (14–16). Biocompatibility and interesting biological properties
From the *Department of Operative Dentistry; †Department of Clinical Sciences, Veterinary School; and ‡Department of Endodontology, School of Dentistry, Aristotle University of Thessaloniki, Thessaloniki, Greece. Address requests for reprints to Prof Dimitrios Tziafas, Department of Endodontology, School of Dentistry, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2015.03.018
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Transdentinal Dentinogenic Effects of Capping Materials
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Basic Research—Biology related to pulpal repair and odontoblastlike cell differentiation have been repeatedly reported with Biodentine in vitro (17–21) and in direct pulp capping situations in vivo (22–24). According to the manufacturer’s instructions, Biodentine is indicated for use in permanent deep dentinal cavity restorations and temporary enamel restorations for periods less than 6 months. However, the safety of its use in deep dentinal cavities without any measure for pulp protection has not been investigated, whereas the ability of Biodentine to induce transdentinal dentinogenic effects remains unclear. The aim of the present study was to evaluate comparatively the bioactivity potential of Biodentine after the restoration of deep dentinal cavities of miniature swine teeth with or without the use of a calcium hydroxide–containing pulp protective base.
Materials and Methods Three healthy miniature swine, all 18 months of age, with intact dentitions were used for the present experimental work. The experimental study was performed in accordance with the Ethical Guidelines of the Aristotle University of Thessaloniki (European Communities Directive of 24 November 1986-86/609/EEC) for the care of animals in experimental procedures and approved by the Ethical Committee of the School of Dentistry, Aristotle University of Thessaloniki, Thessaloniki, Greece. All measures were taken to minimize pain or discomfort of the animals. Each animal was sedated with an intramuscular injection of 1 mg/ kg xylazine. General anesthesia was induced with an intramuscular injection of 6 mg/kg thiopentone. Before the beginning of all experimental procedures, the trachea was intubated, and general anesthesia was maintained using halothane (1.5%–2.5%) in oxygen delivered through a semiclosed breathing circuit.
Experimental Procedures Twelve premolars, 8 canines, and 13 incisors of both jaws were selected for experimentation. Preoperative radiographic examination showed fully developed roots in all teeth. Teeth were scaled and polished with a rubber cup on the day of the operative procedures, isolated with rubber dams, and cleaned with an iodine solution (5%), whereas saliva was controlled with high-speed evacuation. Class V cavities (approximately 2.00 mm wide, 2.00 mm long, and 2.0 mm deep) were prepared on the buccal surface of the teeth using a tungsten carbide pear-shaped bur (ISO #330 L; SS White, Lakewood, NJ) at ultrahigh speed with copious water spray. The active tip of the bur was limited to 1.4 mm. The preparations were cut 0.5–1 mm above the free gingiva, parallel to the cementoenamel junction. The cavities were washed with sterile saline, dried with dry cotton pellets, and treated immediately. The cavities were randomly assigned to 2 groups, scheduled for 2 observation periods each, and treated as follows: 1. Control group: 12 teeth (4 premolars, 4 canines, and 4 incisors) were divided equally into 2 subgroups and treated with the Ca(OH)2-base material Dycal (Caulk Lab, Milford, DE) for periods of 3 or 8 weeks. 2. Experimental group: 21 teeth (8 premolars, 4 canines, and 9 incisors) were treated with Biodentine for periods of 3 weeks (4 premolars, 2 canines, and 6 incisors) or 8 weeks (4 premolars, 2 canines, and 3 incisors). In all cases, the manufacturers’ instructions for restorative procedures and recommended times were followed strictly. Both Biodentine and Dycal were applied in 1-mm-thick layers, whereas all the cavities were further restored with Biodentine. At the termination of the exper2
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imental periods, teeth were radiographically examined, and the animals were sacrificed by using an overdose of pentobarbital sodium. Jaws were fixed in 10% neutral-buffered formalin solution for 2 weeks and demineralized using Morse’s solution (20% formic acid + 10% sodium citrate) for 2 months. Finally, 30 teeth (12 from the control and 18 from the experimental group) were embedded in paraffin and serially sectioned at 7-mm thickness. The 3 remaining teeth (1 premolar and 2 incisors) treated with Biodentine for 3 weeks were processed for scanning electron microscopic analysis. After fixation of the specimens in 10% buffered formalin, the teeth were split with a guided vertical crown fracture through the cavity floor, and soft tissues were mechanically removed. The specimens were immersed in 5% sodium hypochlorite for 4 hours and dehydrated in alcohol. The interfaces primary dentin with Biodentine and postoperatively formed mineralized matrix were examined using a JEOL JSM-840A scanning electron microscope (JEOL, Tokyo, Japan). Coded specimens were used to avoid possible bias. All sections coming through the cavity floor were stained either with Mayer hematoxylin-eosin stain or using a modified Brown and Brenn technique and analyzed twice by 2 independent observers.
Histologic Assessment The pulpal inflammatory and reparative tissue responses to the combined effects of cavity preparation and restoration were evaluated histologically according to the following criteria. Inflammatory Cell Response. Inflammatory cell infiltration of the pulp tissue was classified as minimal or intense, representing the absence/presence of a few scattered inflammatory cells or the presence of moderate to heavy inflammatory cell infiltration, respectively. Infection. The presence of stained bacteria along the cavity walls/ within the cut dentinal tubules was characterized as dentin-positive bacterial detection. Tertiary Dentin Formation. The presence of a continuous zone of the postoperatively formed mineralized matrix beneath the cavity floor of atubular or tubular form was evaluated as tertiary dentin formation, regardless of the presence of a few scattered defects and cellular inclusions. The presence of mineralized matrix of an osteotypic form was characterized as osteodentin. All stained sections were further evaluated histometrically; the remaining dentin thickness (RDT) was measured between the cavity floor and the line of interface between primary dentin and the postoperatively formed mineralized matrix, and the minimum RDT was recorded for every specimen. Also, the maximum thickness of the postoperatively formed mineralized tissue was estimated for every specimen of all groups as follows. The maximum thickness of the mineralized tissue zone was measured for each section. The average of the 5 maximum values was calculated as the maximum mineralized matrix thickness for each specimen. Interobservation variation was not noticed in the histologic evaluation. Variation in histometric analysis, ranging from 8–25 mm, was noticed in measurements of maximum thickness of tertiary dentin formation in 7 specimens. In these cases, the average score was finally recorded.
Statistical Analysis For the comparison of the pulp reactions regarding the maximum thickness of postoperatively formed mineralized tissue formation, Kruskal-Wallis and Mann-Whitney U tests were conducted. A 2-way analysis of variance test was conducted to compare the natural logtransformed values of the minimum RDT between experimental and control groups. The significance level was set at P < .05. JOE — Volume -, Number -, - 2015
Basic Research—Biology TABLE 1. Mean Values and Standard Deviations (SD) of the Minimum Remaining Dentin Thickness (RDT) in Teeth of the Control and Experimental Groups Treatment
N
Mean value of RDT ± SD in mm
Biodentine + Dycal (experimental) Biodentine (control)
12 18
394 143 378 122
Groups are not significantly different (P = .75).
Results The minimum RDT was compared among the control and experimental groups to evaluate if it randomly distributed across the control and experimental groups. In Table 1, the mean values of the minimum RDT in each group of teeth are presented. No statistical difference was observed among the groups (P = .75). No positive bacterial reaction was identified along the dentin cavity walls or within the cut dentinal tubules. Partial/total pulp necrosis or the presence of moderate to heavy inflammatory cell infiltration was not detected in any of the specimens. The presence of a few scattered cells was found in 2 and 3 three-week specimens of the control and experimental groups, respectively. In light microscopy, all specimens showed a continuous zone of the postoperatively formed mineralized matrix (Figs. 1A–G and 2A– D). All 12 teeth treated with Dycal for both observation periods and 13 of 18 teeth treated with Biodentine (7 of 3-week and 6 of 8-week periods) showed the same homogenous structure of tertiary dentin formation with only a few scattered defects and tunnels mostly detected at the firstly deposited zone (Fig. 1). The tertiary dentin was of atubular structure, with only limited short dentinal tubules associated with the odontoblast layer. In the remaining 5 teeth, a separate zone composed of the osteotypic mineralized matrix and soft tissues was noted between
the primary and the tertiary dentin (Fig. 2). Results concerning the maximum thickness of the postoperatively formed mineralized matrix are presented in Table 2. No relationship between the tooth type or the minimum RDT and the maximum thickness of hard tissue bridge was detected. Data submitted to a statistical test showed that a significantly higher rate of the mineralized matrix had been formed in teeth in the experimental group in both periods of 3 and 8 weeks (P < .01). Significant differences in the maximum thickness of hard tissue were also found between the observation periods in both the control and experimental groups. Teeth examined with scanning electron microscopy confirmed the following structural characteristics of tertiary dentin described previously (Fig. 2): a fibrous matrix with limited defects in the firstly deposited matrix, showing the characteristic tubular surface morphology of tertiary dentin.
Discussion Histologic testing of dental materials to evaluate their transdentinal effects on dental pulp includes investigations in animals (monkeys, pigs, dogs, bovines, ferrets, and rats) and extracted human teeth. Human studies are most appropriate when investigating dentin-pulp reactions to various materials and commercially available formulas because of possible animal species’ variations in the anatomy and physiology of the dentin-pulp complex. Tooth organ or tooth slice culture systems can be also used to evaluate specific effects of dental materials (25) but with limitations in controlling all the multifactorial effects of operative and postoperative events on the pulp-dentin complex (12). Obviously, because it is a need to protect patients from possible hazards presented by newly introduced dental materials, animal usage tests are still necessary. However, alternative methodology for the preclinical assessment of dental materials needs to be developed to replace, partly at least, animal experimentation (ISO 7405, 1995).
Figure 1. Light and scanning electron microscopy of nonexposed cavities of a miniature swine tooth after the application of Biodentine (A and magnification in B) with or (C and magnification in D, E and magnification in F and G) without the protective Dycal base for periods (C–F) 3 weeks and (A, B, and G) 8 weeks. The continuous zone of postoperatively formed tertiary dentin with a few scattered small defects (stars), which is followed by a thin zone of tubular morphology (arrows) can be seen. (F) The fibrous form of the postoperative deposited matrix can be seen in the scanning electron microscopic micrograph. d, primary dentin; td, tertiary dentin. Hematoxylin-eosin; original magnification (A and C) 40, (B and D) 100, (E) 30, and (F) 400.
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Basic Research—Biology
Figure 2. Light microscopy of nonexposed cavities of a miniature swine tooth after the application of Biodentine without the protective Dycal base for periods of (A and magnification in B) 3 and (C and magnification in D) 8 weeks. Osteodentin with soft tissue interposed between the primary dentin and the postoperatively formed mineralized matrix can be seen. d, primary dentin; td, tertiary dentin; od, osteodentin. Hematoxylin-eosin; original magnification (A and C) 40 and (B and D) 100.
Operative parameters that affect the dynamics of the dentin-pulp complex and pulp responses to materials placed in a dentinal cavity have attracted much attention (9). Cavity preparation and restoration procedures have been recognized as potential sources of pulpal injury (5, 12). Leaking bacterial invasion and RDT have been shown to be the most important parameters (26–29). The numbers of odontoblasts surviving the injury caused by the cavity cutting and restoring procedures and the tertiary dentin deposition have been strongly correlated with RDT (3, 27, 28). The presence of inflammation at the dental pulp underlying a mechanically prepared cavity has also been correlated with the RDT (29) and the bacterial leakage around dental restorations (4). Intense inflammation or tissue necrosis was not observed in the present investigation. Furthermore, positive bacterial staining reaction was not detected along the cut cavity walls and within the cut dentinal tubules in any of the specimens, indicating that the bacterial leaking had been well controlled around restorations in the present experimental conditions (30). Biodentine was selected for 3- and 8-week cavity restoration according to the material’s indications specified by the manufacturer that it can be used as a restorative material for periods less than 6 months. Glass ionomer was not selected as a restorative material in the present study because of the undetermined setting reactions in the complex application of acidic glass ionomer with alkalinic capping materials and their possible interference with material activity (31). Thus, taking into consideration that the application of 2 materials was performed in cavities with a similar minimal effect on pulp status and the RDT randomly distributed across control and experimental groups, as proven statistically, it is reasonable to assume that the results can be attributed to the different effect of ma-
terial used in contact with the cavity floor and not to other operative parameters. The biosynthetic activity of the odontoblast layer subjacent to the cavity floor appears to be maintained subjacent to deep cavities exposed to both materials. The mineralized tissue formed in response to calcium silicate–based material Biodentine showed quantitative and in a number of teeth qualitative differences when compared with that found in response to the calcium hydroxide–based material. Calcium hydroxide–based materials have represented for more than 4 decades the gold standard as the materials of choice for vital pulp protection and therapy (3, 32, 33). In the present study, all teeth treated with Biodentine showed a significantly higher rate of the mineralized matrix in both periods of 3 and 8 weeks. In qualitative terms, the same structure of the mineralized matrix zone was found in teeth treated either with Biodentine or Dycal. It was mainly composed from an atubular matrix with rare cellular inclusions and tunnels and a thin zone of tubular matrix produced in a pre–dentinlike form. The firstly deposited mineralized matrix under Biodentine was expectedly more irregular in nature with defects because of its faster deposition by stimulated odontoblasts. The fact that in a number of specimens treated with Biodentine osteotypic mineralized and soft tissue was found between the primary and tertiary dentin indicate that high upregulation of biosynthetic activity of odontoblasts or underlying pulpal cells may not be excluded. These findings are in line with our previous experiments in exposed pulps of miniature swine teeth in which a thick zone of the osteodentinal matrix was consistently observed beneath the capping material after 3 and 8 weeks (24). These data obtained from a miniature swine tooth model indicate that Biodentine is associated with higher thickness of the postoperatively formed mineralized matrix in
TABLE 2. Mean Values and Standard Deviations of the Maximum Thickness of the Postoperatively Formed Mineralized Matrix beneath the Floor of the Cavities in Teeth of the Control and Experimental Groups Maximum thickness of mineralized matrix zone in mm Treatment
3 weeks Postop
8 weeks Postop
Biodentine + Dycal (experimental) Biodentine (control)
32 6 (P: 33, A: 27) 95 34 (P: 104, A: 81)
76 14 (P: 81, A: 68) 142 21 (P: 144, A: 123)
Mean values of maximum thickness for premolars (P) and incisors/canines (A) are seen in parentheses. Differences between the 2 groups of teeth for each observation period and between 2 observation periods are significantly different.
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Basic Research—Biology comparison with other capping materials used as controls. The fact that Biodentine is occasionally related with an atypic formation of pulp calcification might be attributed to the young age of the animals, but further research is required. In general, dentinogenic effects of Biodentine might be further evaluated on the basis of its ability to induce the desired pattern of tertiary dentin formation. The reparative events occurring during tertiary dentinogenesis require tissue-specific signaling molecules. The bioactive molecules bound within the dentinal matrix and released during injury may contribute to the up-regulation of odontoblast biosynthetic activity (34). Recently, the p38-mitogen-activated protein kinase (p38MAPK) pathway, which has been reported to up-regulate secretory activity of odontoblast and pulp cells, has been shown to be activated with the dentin bioactive molecules, bacteria, and their components (35, 36). In the frame of these molecular signaling mechanisms, stimulation of tertiary dentinogenesis by dental materials should be hypothesized. The formation of an appropriate environment in the pulp-dentin complex because of the material alkalinity has been considered as a critical influence favoring expression of the dentinogenic secretory activity (3). The role of released hydroxyl ions has been suggested for pulp repair after the application of calcium hydroxide–based materials in both direct and indirect pulp capping applications (32–34, 37) and cannot be excluded for calcium silicate– based materials. Genes encoding for activin, bone morphogenetic proteins, and transforming growth factor betas and their receptors have been expressed in the pulp-dentin complex in health and disease (38–40). The expression of ligands, genes, and receptors for growth factors (particularly of the transforming growth factor beta superfamily) is more evident in carious pulp cells compared with intact teeth (41–43), and this increased expression may mirror important regulative roles during dental pulp repair. Experimentally, the osteogenic protein 1, dentin matrix components, and transforming growth factor beta1 induced transdentinal effects (8, 26, 44). The release of growth factors, which are detected in the dentin matrix both in an active and latent form, has been suggested for calcium hydroxide–based materials (6) and may not be excluded for the calcium silicate– based materials. The stimulatory role of Biodentine in inducing molecular reparative events has been shown. In a recent study, Biodentine showed in vitro down-regulation of osteoblastic markers and up-regulation of mature odontoblastic markers (18). Extracellular signal-regulated kinase pathway, expression of substrate adhesion molecules, the down-regulation of alkaline phosphatase, and the up-regulation of osteocalcin messenger RNA have been induced by Biodentine (18, 45). In conclusion, this study indicates that under the present experimental conditions, tertiary dentin is observed after the restoration of deep dentinal cavities with Biodentine in the presence or absence of a Dycal protective base. The thickness of the tertiary dentin zone was significantly higher in the absence of Dycal, whereas an intermediate osteodentin zone was also observed in this group of teeth. Within the limits of this investigation, it can be assumed that the application of Biodentine in direct contact with the deep cavity floor provides significantly higher stimulatory activity in inducing tertiary dentin formation in comparison with the application of Dycal. The occasional nonspecific effects of Biodentine in stimulating osteodentin formation need to be investigated further.
Acknowledgments This research was cofinanced by the EU (ESF) and Hellenic national funds, Program ‘‘Education and Lifelong Learning’’ (NSRF) JOE — Volume -, Number -, - 2015
Research Funding Program: ‘‘ARISTEIA’’/‘‘EXCELLENCE’’ (grant no. 1904), and Septodont, France. The authors deny any conflicts of interest related to this study.
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39. Gu K, Smoke RH, Rutherford D. Expression of genes for bone morphogenetic proteins and receptors in human dental pulp. Arch Oral Biol 1996;41:919–23. 40. Smith AJ, Matthews JB, Hall RC. Transforming growth factor b1 (TGF-b1) in dentin matrix. Ligand activation and receptor expression. Eur J Oral Sci 1998;106(suppl 1):179–84. 41. Mc Lachlan JL, Smith AJ, Sloan AJ, Cooper PR. Gene expression analysis in cells of the dentin-pulp complex in healthy and carious teeth. Arch Oral Biol 2003;48: 273–83. 42. Sloan AJ, Perry H, Matthews JB, Smith AJ. Transforming growth factor-b isoform expression in mature human molar teeth. Histochem J 2000;32:247–52. 43. Piattelli A, Rubini C, Fioroni M, et al. Transforming growth factor–beta 1 (TGFbeta1) expression in normal healthy pulps and in those with irreversible pulpitis. Int Endod J 2004;37:114–9. 44. Rutherford RB, Spanberg L, Tucker M, Charette M. Transdentinal stimulation of reparative dentine formation by OP-1 in monkeys. Arch Oral Biol 1995;40: 681–3. 45. Du R, Wu T, Liu W, et al. Role of the extracellular signal-regulated kinase ½ pathway in driving tricalcium silicate-induced proliferation and biomineralization of human dental pulp cells in vitro. J Endod 2013;39:1023–9.
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Dentinogenic Activity of Biodentine in Deep Cavities of Miniature Swine Teeth Christina Tziafa, DDS, MSc,* Eugenia Koliniotou-Koumpia, DDS, PhD,* Seraphim Papadimitriou, DDS, PhD,† and Dimitrios Tziafas, DDS, PhD‡ Abstract Introduction: The aim of the present study was to evaluate comparatively the bioactivity potential of a calcium silicate–based material (Biodentine; Septodont, SaintMaur-des-fosses Cedex, France) after the restoration of deep dentinal cavities of miniature swine teeth with or without the application of a calcium hydroxide–containing pulp protective base (Dycal; Caulk Lab, Milford, DE). Methods: Thirty-three permanent teeth (premolars, canines, and incisors) of 3 miniature swine were used. Class V cavities were prepared on the buccal surface of teeth. The cavities were restored with Biodentine in the presence (control group) or absence (experimental group) of a Dycal protective base. The pulpal tissue responses were histologically and histomorphometrically assessed at postoperative periods of 3 and 8 weeks. Three specimens were further evaluated with scanning electron microscopy. The maximum thickness of the postoperatively formed mineralized matrix beneath the cavity floor was measured. Data were statistically analyzed by the Kruskal-Wallis and the Mann-Whitney U tests. Results: A bacterial staining reaction along the cavity walls or intense inflammatory infiltration in the pulp was not detected in any of the specimens. A continuous zone of the postoperatively formed mineralized matrix mostly of atubular structure with scattered defects and cellular inclusions and occasionally followed by a thin zone with tubular morphology was detected in all specimens of the control group and 13 of 18 experimental group teeth. In the remaining teeth of the experimental group, a separate zone composed of the osteotypic mineralized matrix and soft tissues was noted between the circumpulpal and the newly formed matrix. Scanning electron microscopy confirmed the fibrous structural morphology of the tertiary dentin. A significantly higher rate of the postoperatively formed mineralized matrix had been formed in the teeth of the experimental group in both periods of 3 and 8 weeks (P < .01). Conclusions: The present investigation indicates that under the present experimental conditions
tertiary dentin with occasional intermediate formation of osteodentin is observed after the application of Biodentine in the presence or absence of a Dycal protective base. The thickness of the tertiary dentine zone was significantly higher in the absence of Dycal. (J Endod 2015;-:1–6)
Key Words Biodentine, calcium hydroxide, calcium silicate, Dycal, miniature swine teeth, pulp capping materials, pulp protection, tertiary dentin
I
t has been recognized that local dentinal injuries in nonexposed cavities such as carious lesions, physical trauma, tooth grinding, restorative materials, and so on affect the vitality and function of the pulp (1–3). With milder injury in which the inflammation is minimal and in the absence of bacteria in the affected dentin area (4), the insult is overcome, the pulp-dentinal complex progressively returns to pulp healing and pulp vitality, and dentinogenic function is preserved (5). Up-regulation of survived odontoblast biosynthetic activity or replacement of lost odontoblasts by newly differentiated odontoblastlike cells has been described as a part of pulp tissue repair, leading to reactionary or reparative dentin formation, respectively (6). Peritubular dentin formation may also be noted in the affected dentin zone (2, 7, 8). As a result of the increased odontoblast activity, the permeability of affected dentin is reduced (9), leading to effective long-term protection of the pulp. In today’s clinical practice, the restoration of dentinal cavities without pulp exposure is a safe and predictable method of treatment (3) despite the fact that data from high-quality randomized control clinical trials are rare (10, 11). The remaining dentin thickness has been widely recognized as the main factor that determines the long-term success of the treatment in the absence of bacteria, whereas material toxicity also has been implicated with pulpal injury and subsequent pulp healing (3, 12). Thus, it has been recognized that in cases of deep dentinal cavities pulp protection is required to protect the pulp-dentin complex from materials’ chemical injury and further bacterial invasion. For many years, calcium hydroxide–containing cements and glass ionomers have been considered as materials of choice for pulp protection in deep dentinal cavities (13). Recently, a number of new formulas of calcium silicate–based materials have been introduced as restorative materials. Among them, Biodentine (Septodont, Saint-Maurdes-fosses Cedex, France) has been tested as a dentin substitute. Biodentine is composed of a powder component (tricalcium silicate, calcium carbonate, and zirconium oxide) and a liquid component (calcium chloride in water). In in vitro studies, Biodentine showed good physicochemical properties and interfacial characteristics with the dentin (14–16). Biocompatibility and interesting biological properties
From the *Department of Operative Dentistry; †Department of Clinical Sciences, Veterinary School; and ‡Department of Endodontology, School of Dentistry, Aristotle University of Thessaloniki, Thessaloniki, Greece. Address requests for reprints to Prof Dimitrios Tziafas, Department of Endodontology, School of Dentistry, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2015.03.018
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Transdentinal Dentinogenic Effects of Capping Materials
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Basic Research—Biology related to pulpal repair and odontoblastlike cell differentiation have been repeatedly reported with Biodentine in vitro (17–21) and in direct pulp capping situations in vivo (22–24). According to the manufacturer’s instructions, Biodentine is indicated for use in permanent deep dentinal cavity restorations and temporary enamel restorations for periods less than 6 months. However, the safety of its use in deep dentinal cavities without any measure for pulp protection has not been investigated, whereas the ability of Biodentine to induce transdentinal dentinogenic effects remains unclear. The aim of the present study was to evaluate comparatively the bioactivity potential of Biodentine after the restoration of deep dentinal cavities of miniature swine teeth with or without the use of a calcium hydroxide–containing pulp protective base.
Materials and Methods Three healthy miniature swine, all 18 months of age, with intact dentitions were used for the present experimental work. The experimental study was performed in accordance with the Ethical Guidelines of the Aristotle University of Thessaloniki (European Communities Directive of 24 November 1986-86/609/EEC) for the care of animals in experimental procedures and approved by the Ethical Committee of the School of Dentistry, Aristotle University of Thessaloniki, Thessaloniki, Greece. All measures were taken to minimize pain or discomfort of the animals. Each animal was sedated with an intramuscular injection of 1 mg/ kg xylazine. General anesthesia was induced with an intramuscular injection of 6 mg/kg thiopentone. Before the beginning of all experimental procedures, the trachea was intubated, and general anesthesia was maintained using halothane (1.5%–2.5%) in oxygen delivered through a semiclosed breathing circuit.
Experimental Procedures Twelve premolars, 8 canines, and 13 incisors of both jaws were selected for experimentation. Preoperative radiographic examination showed fully developed roots in all teeth. Teeth were scaled and polished with a rubber cup on the day of the operative procedures, isolated with rubber dams, and cleaned with an iodine solution (5%), whereas saliva was controlled with high-speed evacuation. Class V cavities (approximately 2.00 mm wide, 2.00 mm long, and 2.0 mm deep) were prepared on the buccal surface of the teeth using a tungsten carbide pear-shaped bur (ISO #330 L; SS White, Lakewood, NJ) at ultrahigh speed with copious water spray. The active tip of the bur was limited to 1.4 mm. The preparations were cut 0.5–1 mm above the free gingiva, parallel to the cementoenamel junction. The cavities were washed with sterile saline, dried with dry cotton pellets, and treated immediately. The cavities were randomly assigned to 2 groups, scheduled for 2 observation periods each, and treated as follows: 1. Control group: 12 teeth (4 premolars, 4 canines, and 4 incisors) were divided equally into 2 subgroups and treated with the Ca(OH)2-base material Dycal (Caulk Lab, Milford, DE) for periods of 3 or 8 weeks. 2. Experimental group: 21 teeth (8 premolars, 4 canines, and 9 incisors) were treated with Biodentine for periods of 3 weeks (4 premolars, 2 canines, and 6 incisors) or 8 weeks (4 premolars, 2 canines, and 3 incisors). In all cases, the manufacturers’ instructions for restorative procedures and recommended times were followed strictly. Both Biodentine and Dycal were applied in 1-mm-thick layers, whereas all the cavities were further restored with Biodentine. At the termination of the exper2
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imental periods, teeth were radiographically examined, and the animals were sacrificed by using an overdose of pentobarbital sodium. Jaws were fixed in 10% neutral-buffered formalin solution for 2 weeks and demineralized using Morse’s solution (20% formic acid + 10% sodium citrate) for 2 months. Finally, 30 teeth (12 from the control and 18 from the experimental group) were embedded in paraffin and serially sectioned at 7-mm thickness. The 3 remaining teeth (1 premolar and 2 incisors) treated with Biodentine for 3 weeks were processed for scanning electron microscopic analysis. After fixation of the specimens in 10% buffered formalin, the teeth were split with a guided vertical crown fracture through the cavity floor, and soft tissues were mechanically removed. The specimens were immersed in 5% sodium hypochlorite for 4 hours and dehydrated in alcohol. The interfaces primary dentin with Biodentine and postoperatively formed mineralized matrix were examined using a JEOL JSM-840A scanning electron microscope (JEOL, Tokyo, Japan). Coded specimens were used to avoid possible bias. All sections coming through the cavity floor were stained either with Mayer hematoxylin-eosin stain or using a modified Brown and Brenn technique and analyzed twice by 2 independent observers.
Histologic Assessment The pulpal inflammatory and reparative tissue responses to the combined effects of cavity preparation and restoration were evaluated histologically according to the following criteria. Inflammatory Cell Response. Inflammatory cell infiltration of the pulp tissue was classified as minimal or intense, representing the absence/presence of a few scattered inflammatory cells or the presence of moderate to heavy inflammatory cell infiltration, respectively. Infection. The presence of stained bacteria along the cavity walls/ within the cut dentinal tubules was characterized as dentin-positive bacterial detection. Tertiary Dentin Formation. The presence of a continuous zone of the postoperatively formed mineralized matrix beneath the cavity floor of atubular or tubular form was evaluated as tertiary dentin formation, regardless of the presence of a few scattered defects and cellular inclusions. The presence of mineralized matrix of an osteotypic form was characterized as osteodentin. All stained sections were further evaluated histometrically; the remaining dentin thickness (RDT) was measured between the cavity floor and the line of interface between primary dentin and the postoperatively formed mineralized matrix, and the minimum RDT was recorded for every specimen. Also, the maximum thickness of the postoperatively formed mineralized tissue was estimated for every specimen of all groups as follows. The maximum thickness of the mineralized tissue zone was measured for each section. The average of the 5 maximum values was calculated as the maximum mineralized matrix thickness for each specimen. Interobservation variation was not noticed in the histologic evaluation. Variation in histometric analysis, ranging from 8–25 mm, was noticed in measurements of maximum thickness of tertiary dentin formation in 7 specimens. In these cases, the average score was finally recorded.
Statistical Analysis For the comparison of the pulp reactions regarding the maximum thickness of postoperatively formed mineralized tissue formation, Kruskal-Wallis and Mann-Whitney U tests were conducted. A 2-way analysis of variance test was conducted to compare the natural logtransformed values of the minimum RDT between experimental and control groups. The significance level was set at P < .05. JOE — Volume -, Number -, - 2015
Basic Research—Biology TABLE 1. Mean Values and Standard Deviations (SD) of the Minimum Remaining Dentin Thickness (RDT) in Teeth of the Control and Experimental Groups Treatment
N
Mean value of RDT ± SD in mm
Biodentine + Dycal (experimental) Biodentine (control)
12 18
394 143 378 122
Groups are not significantly different (P = .75).
Results The minimum RDT was compared among the control and experimental groups to evaluate if it randomly distributed across the control and experimental groups. In Table 1, the mean values of the minimum RDT in each group of teeth are presented. No statistical difference was observed among the groups (P = .75). No positive bacterial reaction was identified along the dentin cavity walls or within the cut dentinal tubules. Partial/total pulp necrosis or the presence of moderate to heavy inflammatory cell infiltration was not detected in any of the specimens. The presence of a few scattered cells was found in 2 and 3 three-week specimens of the control and experimental groups, respectively. In light microscopy, all specimens showed a continuous zone of the postoperatively formed mineralized matrix (Figs. 1A–G and 2A– D). All 12 teeth treated with Dycal for both observation periods and 13 of 18 teeth treated with Biodentine (7 of 3-week and 6 of 8-week periods) showed the same homogenous structure of tertiary dentin formation with only a few scattered defects and tunnels mostly detected at the firstly deposited zone (Fig. 1). The tertiary dentin was of atubular structure, with only limited short dentinal tubules associated with the odontoblast layer. In the remaining 5 teeth, a separate zone composed of the osteotypic mineralized matrix and soft tissues was noted between
the primary and the tertiary dentin (Fig. 2). Results concerning the maximum thickness of the postoperatively formed mineralized matrix are presented in Table 2. No relationship between the tooth type or the minimum RDT and the maximum thickness of hard tissue bridge was detected. Data submitted to a statistical test showed that a significantly higher rate of the mineralized matrix had been formed in teeth in the experimental group in both periods of 3 and 8 weeks (P < .01). Significant differences in the maximum thickness of hard tissue were also found between the observation periods in both the control and experimental groups. Teeth examined with scanning electron microscopy confirmed the following structural characteristics of tertiary dentin described previously (Fig. 2): a fibrous matrix with limited defects in the firstly deposited matrix, showing the characteristic tubular surface morphology of tertiary dentin.
Discussion Histologic testing of dental materials to evaluate their transdentinal effects on dental pulp includes investigations in animals (monkeys, pigs, dogs, bovines, ferrets, and rats) and extracted human teeth. Human studies are most appropriate when investigating dentin-pulp reactions to various materials and commercially available formulas because of possible animal species’ variations in the anatomy and physiology of the dentin-pulp complex. Tooth organ or tooth slice culture systems can be also used to evaluate specific effects of dental materials (25) but with limitations in controlling all the multifactorial effects of operative and postoperative events on the pulp-dentin complex (12). Obviously, because it is a need to protect patients from possible hazards presented by newly introduced dental materials, animal usage tests are still necessary. However, alternative methodology for the preclinical assessment of dental materials needs to be developed to replace, partly at least, animal experimentation (ISO 7405, 1995).
Figure 1. Light and scanning electron microscopy of nonexposed cavities of a miniature swine tooth after the application of Biodentine (A and magnification in B) with or (C and magnification in D, E and magnification in F and G) without the protective Dycal base for periods (C–F) 3 weeks and (A, B, and G) 8 weeks. The continuous zone of postoperatively formed tertiary dentin with a few scattered small defects (stars), which is followed by a thin zone of tubular morphology (arrows) can be seen. (F) The fibrous form of the postoperative deposited matrix can be seen in the scanning electron microscopic micrograph. d, primary dentin; td, tertiary dentin. Hematoxylin-eosin; original magnification (A and C) 40, (B and D) 100, (E) 30, and (F) 400.
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Basic Research—Biology
Figure 2. Light microscopy of nonexposed cavities of a miniature swine tooth after the application of Biodentine without the protective Dycal base for periods of (A and magnification in B) 3 and (C and magnification in D) 8 weeks. Osteodentin with soft tissue interposed between the primary dentin and the postoperatively formed mineralized matrix can be seen. d, primary dentin; td, tertiary dentin; od, osteodentin. Hematoxylin-eosin; original magnification (A and C) 40 and (B and D) 100.
Operative parameters that affect the dynamics of the dentin-pulp complex and pulp responses to materials placed in a dentinal cavity have attracted much attention (9). Cavity preparation and restoration procedures have been recognized as potential sources of pulpal injury (5, 12). Leaking bacterial invasion and RDT have been shown to be the most important parameters (26–29). The numbers of odontoblasts surviving the injury caused by the cavity cutting and restoring procedures and the tertiary dentin deposition have been strongly correlated with RDT (3, 27, 28). The presence of inflammation at the dental pulp underlying a mechanically prepared cavity has also been correlated with the RDT (29) and the bacterial leakage around dental restorations (4). Intense inflammation or tissue necrosis was not observed in the present investigation. Furthermore, positive bacterial staining reaction was not detected along the cut cavity walls and within the cut dentinal tubules in any of the specimens, indicating that the bacterial leaking had been well controlled around restorations in the present experimental conditions (30). Biodentine was selected for 3- and 8-week cavity restoration according to the material’s indications specified by the manufacturer that it can be used as a restorative material for periods less than 6 months. Glass ionomer was not selected as a restorative material in the present study because of the undetermined setting reactions in the complex application of acidic glass ionomer with alkalinic capping materials and their possible interference with material activity (31). Thus, taking into consideration that the application of 2 materials was performed in cavities with a similar minimal effect on pulp status and the RDT randomly distributed across control and experimental groups, as proven statistically, it is reasonable to assume that the results can be attributed to the different effect of ma-
terial used in contact with the cavity floor and not to other operative parameters. The biosynthetic activity of the odontoblast layer subjacent to the cavity floor appears to be maintained subjacent to deep cavities exposed to both materials. The mineralized tissue formed in response to calcium silicate–based material Biodentine showed quantitative and in a number of teeth qualitative differences when compared with that found in response to the calcium hydroxide–based material. Calcium hydroxide–based materials have represented for more than 4 decades the gold standard as the materials of choice for vital pulp protection and therapy (3, 32, 33). In the present study, all teeth treated with Biodentine showed a significantly higher rate of the mineralized matrix in both periods of 3 and 8 weeks. In qualitative terms, the same structure of the mineralized matrix zone was found in teeth treated either with Biodentine or Dycal. It was mainly composed from an atubular matrix with rare cellular inclusions and tunnels and a thin zone of tubular matrix produced in a pre–dentinlike form. The firstly deposited mineralized matrix under Biodentine was expectedly more irregular in nature with defects because of its faster deposition by stimulated odontoblasts. The fact that in a number of specimens treated with Biodentine osteotypic mineralized and soft tissue was found between the primary and tertiary dentin indicate that high upregulation of biosynthetic activity of odontoblasts or underlying pulpal cells may not be excluded. These findings are in line with our previous experiments in exposed pulps of miniature swine teeth in which a thick zone of the osteodentinal matrix was consistently observed beneath the capping material after 3 and 8 weeks (24). These data obtained from a miniature swine tooth model indicate that Biodentine is associated with higher thickness of the postoperatively formed mineralized matrix in
TABLE 2. Mean Values and Standard Deviations of the Maximum Thickness of the Postoperatively Formed Mineralized Matrix beneath the Floor of the Cavities in Teeth of the Control and Experimental Groups Maximum thickness of mineralized matrix zone in mm Treatment
3 weeks Postop
8 weeks Postop
Biodentine + Dycal (experimental) Biodentine (control)
32 6 (P: 33, A: 27) 95 34 (P: 104, A: 81)
76 14 (P: 81, A: 68) 142 21 (P: 144, A: 123)
Mean values of maximum thickness for premolars (P) and incisors/canines (A) are seen in parentheses. Differences between the 2 groups of teeth for each observation period and between 2 observation periods are significantly different.
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Basic Research—Biology comparison with other capping materials used as controls. The fact that Biodentine is occasionally related with an atypic formation of pulp calcification might be attributed to the young age of the animals, but further research is required. In general, dentinogenic effects of Biodentine might be further evaluated on the basis of its ability to induce the desired pattern of tertiary dentin formation. The reparative events occurring during tertiary dentinogenesis require tissue-specific signaling molecules. The bioactive molecules bound within the dentinal matrix and released during injury may contribute to the up-regulation of odontoblast biosynthetic activity (34). Recently, the p38-mitogen-activated protein kinase (p38MAPK) pathway, which has been reported to up-regulate secretory activity of odontoblast and pulp cells, has been shown to be activated with the dentin bioactive molecules, bacteria, and their components (35, 36). In the frame of these molecular signaling mechanisms, stimulation of tertiary dentinogenesis by dental materials should be hypothesized. The formation of an appropriate environment in the pulp-dentin complex because of the material alkalinity has been considered as a critical influence favoring expression of the dentinogenic secretory activity (3). The role of released hydroxyl ions has been suggested for pulp repair after the application of calcium hydroxide–based materials in both direct and indirect pulp capping applications (32–34, 37) and cannot be excluded for calcium silicate– based materials. Genes encoding for activin, bone morphogenetic proteins, and transforming growth factor betas and their receptors have been expressed in the pulp-dentin complex in health and disease (38–40). The expression of ligands, genes, and receptors for growth factors (particularly of the transforming growth factor beta superfamily) is more evident in carious pulp cells compared with intact teeth (41–43), and this increased expression may mirror important regulative roles during dental pulp repair. Experimentally, the osteogenic protein 1, dentin matrix components, and transforming growth factor beta1 induced transdentinal effects (8, 26, 44). The release of growth factors, which are detected in the dentin matrix both in an active and latent form, has been suggested for calcium hydroxide–based materials (6) and may not be excluded for the calcium silicate– based materials. The stimulatory role of Biodentine in inducing molecular reparative events has been shown. In a recent study, Biodentine showed in vitro down-regulation of osteoblastic markers and up-regulation of mature odontoblastic markers (18). Extracellular signal-regulated kinase pathway, expression of substrate adhesion molecules, the down-regulation of alkaline phosphatase, and the up-regulation of osteocalcin messenger RNA have been induced by Biodentine (18, 45). In conclusion, this study indicates that under the present experimental conditions, tertiary dentin is observed after the restoration of deep dentinal cavities with Biodentine in the presence or absence of a Dycal protective base. The thickness of the tertiary dentin zone was significantly higher in the absence of Dycal, whereas an intermediate osteodentin zone was also observed in this group of teeth. Within the limits of this investigation, it can be assumed that the application of Biodentine in direct contact with the deep cavity floor provides significantly higher stimulatory activity in inducing tertiary dentin formation in comparison with the application of Dycal. The occasional nonspecific effects of Biodentine in stimulating osteodentin formation need to be investigated further.
Acknowledgments This research was cofinanced by the EU (ESF) and Hellenic national funds, Program ‘‘Education and Lifelong Learning’’ (NSRF) JOE — Volume -, Number -, - 2015
Research Funding Program: ‘‘ARISTEIA’’/‘‘EXCELLENCE’’ (grant no. 1904), and Septodont, France. The authors deny any conflicts of interest related to this study.
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