Histologic evaluation of dentin bridge formation by pachymic acid and biodentine in human tooth culture model

Abstract

INTRODUCTION Reactionary or reparative dentinogenesis occurs in response to a mild dental injury, involving either the dentin or the pulpo-dentinal complex. Such situations could be encountered in dental caries, traumatic injuries or during an iatrogenic procedure. In the event of a pulpal exposure, it is necessary to maintain its vitality, by guarding it with an appropriate pulp capping material to enable healing and to maintain its functionality.[1] Calcium hydroxide has long been used as a pulp capping material. However, it has inherent drawbacks, such as undesirable pulp tissue irritation, porous dentin bridge (DB) formation, and deficient sealing ability.[2] With the advent of calcium silicate (CS)-based materials, mineral trioxide aggregate (MTA) has gained importance as a pulp capping material due to its innate ability to promote reparative dentin (RD) formation at a much faster rate and the RD formed is of superior quality compared to calcium hydroxide.[34] MTA induces differentiation of latent stem cells present in the pulp by modulating cytokine production and encouraging mineralization of the newly deposited collagen matrix to form a DB.[5] However, the difficult handling characteristics and long setting time associated with MTA drove researchers worldwide to look for an alternate material. This led to the development of an improved CS-based material, Biodentine (BIO), having properties of reduced setting time, enhanced mechanical properties and sealing ability.[6] Various bioactive compounds in the plant kingdom, especially saprophytic type of fungus, possess a wide range of beneficial effects such as anti-oxidant property, inhibition of pro-inflammatory enzymes and scavenging reactive oxygen species (ROS), thus making them a budding source of bio-pharmacological agents.[7] Pachymic acid (PA) derived from Poria cocos mushroom is known to have anti-cancer, anti-inflammatory and antioxidant properties.[8910] Ríos characterized this fungus and reported its principal components to be triterpenoids and polysaccharides.[11] Lee et al. (2013) stated that PA exhibited potent cyto-protection and induced dentin mineralization against oxidative stress by acting on the cellular expression of heme oxygenase-1 (HO-1).[7] These findings suggest that PA could be a promising pulp capping material. Literature review revealed that PA was assessed only at a cellular level till date, and there are no reports evaluating the ability of this material to induce RD under conditions simulating clinical usage. A novel method to evaluate a biomaterial in the form of entire human tooth cultures was put forth by Téclès et al.[12] This model not only mimics the oral environment, but also serves as a useful tool to evaluate the biomaterials and study the mechanisms underlying dentine repair prior to experiments involving intervention with human subjects. Since this model enables maintenance of a suitable environment for RD formation, the efficacy of a pulp capping material can be closely studied in this set up. It also helps the tooth to be effectively assessed by histo-pathological evaluation, which is considered the gold standard to evaluate tissue and cell specimens, thus providing a direct in-situ analysis at a cellular level.[1314] Hence, the aim of the present study was to comparatively evaluate DB thickness and its continuity, intensity of pulpal inflammation and odontoblast (OD) layer pattern following the use of BIO and PA as pulp capping agents, using entire human tooth culture models, by means of histologic analysis. The null hypothesis was that PA will not induce DB formation in teeth with mechanically exposed pulp in a human tooth culture model. MATERIALS AND METHODS The research protocol was presented to the Institutional Review Board and approval was obtained (SRMDC/IRB/2016/MDS/No. 303). Collection of samples The samples comprised of 40 human maxillary and mandibular premolar or third molar teeth scheduled for orthodontic extraction. Teeth with one-third to two-third root formation and immature root apices alone were selected. Informed consent was obtained from patients and the teeth were atraumatically extracted. Teeth with caries, cracks or fractures were excluded from the study. Immediately after extraction, the periodontal ligament around the roots was removed using sterilized instruments and the samples were transported in Dulbecco's Modified Eagle's Medium (DMEM) (Sigma-Aldrich Chemicals Corp., St. Louis, MO, USA) to carry out the pulp capping procedure. The specimens were randomly allocated to two groups (n = 20) based on the pulp capping agent used, BIO (Group I) and PA (Group II). Preparation of biodentine and pachymic acid BIO (Septodont, Saint Maur des Fossés, France) was mixed according to the manufacturer's instructions. A smooth creamy mix of PA was obtained by mixing PA powder (ChemFaces Biochemical Co., Ltd, Wuhan, China) with distilled water in a ratio of 1:1 using a plastic spatula and a glass slab. Tooth culture The tooth culture model set up was adopted from Téclès et al.[12] The tooth was held atraumatically in a gauze soaked in DMEM solution and a minimal intentional exposure of the pulp (approximately 1 mm2) was made with a sterile round diamond bur (BR-45, Mani Inc., Tochigi, Japan) in a high speed handpiece under sterile water cooling. The capping material was placed at the exposure site with a plastic instrument and condensed lightly with a sterile cotton pellet. While the material set, the samples were suspended in DMEM aided with 5% foetal bovine serum (FBS) and further supplemented with 300 IU/mL penicillin, 300 μg/mL streptomycin and 0.75 μg/mL amphotericin B in such a way that the apex remained within a sterile DMEM-soaked cotton to avoid any desiccation during the preparation procedure. The PCM was overlaid with a thin layer of glass ionomer cement (GIC) (Fuji II LC, GC India Dental Pvt. Ltd., Medak, India). A layer of bonding agent Xeno III (Dentsply DeTrey, GmbH, Konstanz, Germany) was applied on the GIC liner followed by restoration with resin composite (Luxacore Z, DMG America, NJ, USA). The tooth culture model was set up by suspending the samples from a metallic wire (21 Gauge, Konark India Dental Depot, Hyderabad, Telangana, India) bonded to the occlusal composite restoration using a sealant (GC Fuji VII, GC India Dental Pvt. Ltd., Medak, Telangana, India). The samples were cultured in a 5% CO2 incubator (Heracell VIOS 160i, Thermo-Fisher Scientific, Chennai, India) at 37°C for 45 days, with the culture media being replenished every day. At the end of the incubation period, the samples from each group were removed from the set up and taken up for histological analysis. Histological examination The samples were placed in 10% neutral buffered formalin for 3 days to enable fixation of the tissues. Decalcification was carried out in neutral ethylene diamine tetra acetic acid solution for approximately 4 weeks. On an average, 3–4 sections of 4 μm thickness were made from each sample. The specimens were then stained with haemotoxylin-eosin. The histological sections were viewed and analyzed with the help of an optical microscope (Nikon Eclipse E100, Nikon Corporation, Tokyo, Japan). The area of pulpal exposure and surrounding tissues were taken into consideration. The sections were evaluated according to the modified criteria put-forth by Faraco et al.(2004), involving continuity and thickness of the DB, intensity of pulpal inflammation and OD layer pattern as shown in [Table 1].[15] Each section was evaluated using a scale of 1–4, where 1 shows the most intended result and 4 shows the least. The thickness of DB was measured using Adobe imaging software (Version 20.0.1.323, Adobe systems, San Jose, California, USA).Table 1: Scoring criteria for continuity and thickness of dentin bridge, intensity of pulpal inflammation and odontoblast layer patternStatistical analysis The categorical data between the subgroups were evaluated by Pearson Chi-square test for all the parameters. Dunn's post hoc test was used to evaluate multiple pair-wise comparisons between all the groups. P < 0.05 was considered significant for all tests. RESULTS No usable data could be obtained from six samples under both the groups. The remaining 14 samples in each group were taken up for histological evaluation [Figure 1]. While 57.1% of the samples treated with BIO showed an intact DB (Grade-1), a nonuniform DB involving more than 1/2 of area of pulp exposure (grade-2) was most widely expressed in PA-treated samples (57.1%). 50% of samples treated with BIO showed >0.25 mm thick DB (Grade-1). The DB formed was intact and thicker (134 ± 14 μm approximately) in this group. Grade-2 DB thickness (0.1–0.25 mm) was predominant in PA-treated samples (50%). But the DB was nonuniform and its thickness (110 ± 28.6 μm approximately) was significantly lesser than BIO (P < 0.05). 57.1% of samples treated with BIO had no or sparsely located inflammatory cells (Grade-1) and none of the samples showed grade-4 inflammation (>25 cells). But, 35.7% of samples treated with PA showed grade-4 inflammation. 57.1% of samples treated with BIO showed a uniform OD layer (Grade-1). A near-to palisade odontoblastic layer was seen. Comparatively, no OD-like cells were observed in 21.4% of samples treated with PA. Graphical representation of the continuity of DB formed (A) and its thickness (B), intensity of pulpal inflammation (C) and OD layer pattern (D) is given in Figure 2. Under all the four evaluation criteria, BIO was significantly superior when compared to PA (P < 0.05).Figure 1: (a and b) Biodentine showed the presence of a uniform and thick calcific bridge. An intact pulp (p) with few inflammatory cells (arrow) was seen (h and e staining at × 400). (c and d) Pachymic acid showed the presence of a non-uniform CB (arrow) (h and e staining at × 200). (e and f) Pachymic acid showed the presence of a few inflammatory cells (arrow) (h and e staining at × 400)Figure 2: Graphical representation of the continuity of dentin bridge formed (a) and its thickness (b), intensity of pulpal inflammation (c) and odontoblast layer pattern (d) in samples treated with Biodentine and pachymic acidDISCUSSION Various methods including cellular, animal and human models have been used to simulate and study human pulp-dentin complex and its recuperative capacity.[34] In this study, a tooth culture model was used to assess DB formation. The model was infused with a high glucose variant (4,500 mg/L) of DMEM as a nutrient media. It was supplemented with 5% FBS, which aids in in vitro growth of the cells. Furthermore penicillin, streptomycin along with amphotericin B was added, which provided the necessary antibiotic coverage. This culture model was incubated in a favourable atmosphere of 5% CO2 at 37°C, as the culture medium contains sodium bicarbonate buffer system (3.7 g/L) that requires a constant supply of carbon dioxide to maintain the pH.[16] Immature teeth were selected in the present study to enable better diffusion of the medium into the pulp. Dental pulp comprises of diverse cell lines including inflammatory cells, immune cells, and latent pulp cells/progenitor cells which are primarily involved in self-renewal.[17] Upon an insult to the pulp, these progenitor cells are recruited to the wound site. Their growth and differentiation into OD-like cells leads to matrix formation and subsequent mineralization.[18] Thus, it is reasonable to infer that, an innate correspondence exists between host immune system and potent ODs that modulate the differentiation and dentinogenic response. This complex entwined or interrelated mechanism between the inflammatory cells and the OD cells in order to recuperate after an insult requires a harmonious environment that is provided by pulp capping agents.[19] BIO is a second generation pulp capping material, the biological effect of which is supported by various studies. Its potential to increase differentiation of pulp cells to OD -like cells and to promote bio-mineralization is well documented.[62021] Odontoblastic differentiation and uniform DB formation in samples treated with BIO can be attributed to its innate ability to stimulate the release of transforming growth factor-β1. This factor is known to induce differentiation and migration of the stem cells to the target site.[14] The mineralization foci observed surrounding the capping material could be attributed to the increased stimulation of mineralization markers during the pulp response to BIO. Daltoé et al. observed that BIO increased the expression of mineralization markers osteopontin, alkaline phosphatase (ALP) and runt-related transcription factor 2 (RUNX2).[21] This was also supported by a study by Jung et al., who evaluated the mineralization inductive capacity of BIO and stated that it up-regulated the expression of dentin matrix acidic phosphoprotein 1 and dentin sialophosphoprotein, thus explaining its role in formation of RD.[22] Tran et al. observed that well-arranged OD and OD-like cells led to tubular dentin formation over the exposed pulp capped with BIO.[3] Although majority of the samples treated with PA showed grade-2 DB formation, a less thick layer could only be seen in these samples. OD like cells were seen more predominantly adjacent to well-formed calcific bridge. The mineralization potential of PA on OD cells can be evidenced in the literature. Lee et al. showed that PA exhibited anti-inflammatory property and promoted dentin mineralization in oxidatively stressed dental pulp cells via HO-1 pathway. HO-1 is an anti-oxidant response enzyme that plays a pivotal role in cyto-protection against environmental stress, including inflammatory and oxidative stress. It was also shown that presence of PA augmented an increase in ALP activity.[7] Kim et al. evaluated the anti-oxidant property of PA and reported that PA inhibited inflammation and induced mineralization by eliminating ROS. They also showed that the presence of PA increased cell growth and promoted calcium nodule formation. Their observation proved that PA improved the odontoblastic and osteoblastic differentiation. This was attributed to its cytoprotective nature, increased ALP activity and up-regulation of osteogenic markers such as bone morphogenetic proteins 2 and 7 and RUNX2.[23] Based on these findings, it could be hypothesized that reparative dentinogenesis evidenced in the current study by PA could be the result of increased proliferation and differentiation of latent stem cells into OD cells, calcium nodule formation and promoting mineralization. A direct comparison of results of PA-treated samples with any study in the dental literature is not possible as this study is the first of its kind to show PA's ability to form RD. Negm et al. observed that a continuous odontoblastic layer could only be observed at 3 months follow-up.[24] The shorter observation time followed in the present study might not have been sufficient for PA to form a complete DB in the samples. The innate capacity of human immune system is such that, every mineralization phase is usually a sequel to a complex inflammatory cascade. Grade-1 pulpal inflammation was predominant in samples capped with BIO. This could be explained by the ability of BIO to initiate p38-mitogen-activated protein kinases signals, resulting in influencing the transient receptor potential family (TRPA1) responses in OD-like cells. El Karim et al. showed that BIO was able to reduce the tumour necrosis factor-α (TNF-α) induced expression of TRPA1, a channel which enables the signals for pain and inflammation.[25] Grade-2 pulpal inflammation was seen in 28.6% of samples under PA. P. cocos extracts exerted anti-inflammatory effects by reducing the levels of TNF-α, interleukins (IL) 3 and-6. Jeong et al. confirmed the anti-inflammatory potential of PA, by showing that it played a vital role in inhibition of inducible nitric oxide synthase, cyclooxygenase-2, IL-Iβ and TNF-α through inactivation of nuclear factor kappa B signaling pathway.[26] These cellular pathway regulatory actions of PA could be the reasons behind the controlled inflammatory response seen in PA treated samples. These findings suggest that PA could be a potential pulp capping agent. Hence, the null hypothesis was rejected as PA was able to induce RD formation. Though the inflammatory process is helpful in initiating healing and DB formation, it should not extend to necrosis or apoptosis.[27] Ma et al. had shown that natural products like PA possess specific apoptosis-inducing potential in lung cancer cells.[8] This characteristic of PA could have also compromised its reparative dentinogenesis capacity. But, this warrants further investigation. A thorough step by step evaluation is required for a biomaterial to be deemed fit for use in a reparative procedure. Though the tooth culture model enables study of biomaterials in pulp therapy,[28] it has some limitations. The culture model lacks innate circulation, constant CO2 and O2 exchange and therefore, does not simulate the overall in vivo conditions such as the entire inflammatory response and wound resolution. The technique sensitive nature of this model explains the loss of six samples in each group. Further modifications of this technique to improve the circulation of the culture media are required to achieve a closer simulation of the in vivo conditions. Future studies should assess the reparative dentinogenesis potential of PA over longer observation times and using animal models. Since PA is capable of initiating RD formation, this material could further be augmented by the addition of other bioactive compounds to potentiate its bioactivity. Further studies using migration chambers to investigate cell recruitment and controlled inflammatory models should be done using PA.[2029] Any in vitro study testing the reparative capability of a material does not completely simulate the in vivo environment. Hence, caution must be exercised when drawing conclusions to in vivo conditions by using the results of this in vitro study. CONCLUSION Within the limitations of the present in vitro study, it could be concluded that both BIO and PA induced RD formation but the quality of reparative dentinogenesis achieved with BIO was significantly superior to that of PA. Financial support and sponsorship This work was supported by an International Federation of Endodontic Associations (I.F.E.A.) Jean-Marie Laurichesse Research Award Conflicts of interest There are no conflicts of interest.