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Year : 2021  |  Volume : 33  |  Issue : 2  |  Page : 86-91

Evaluation of calcium hydroxide incorporated with niobium pentoxide as a direct pulp capping agent – A preliminary ex vivo tooth culture model analysis

Department of Conservative Dentistry and Endodontics, SRM Dental College, Ramapuram; SRM Institute of Science and Technology, Ramapuram Campus, Chennai, Tamil Nadu, India

Date of Submission13-Jan-2021
Date of Decision02-Mar-2021
Date of Acceptance15-Apr-2021
Date of Web Publication11-Jun-2021

Correspondence Address:
Dr. Sekar Mahalaxmi
Department of Conservative Dentistry and Endodontics, SRM Dental College, Ramapuram, SRM Institute of Science and Technology, Ramapuram Campus, Chennai, Tamil Nadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/endo.endo_22_21

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Aims: To assess the effect of addition of niobium pentoxide (NP) to calcium hydroxide (CH) when used as a pulp capping agent. The aim was to evaluate the material characteristics and hydration potential of CH incorporated with NP at 15 days in vitro followed by the evaluation of dentin bridge formation in an ex vivo tooth culture model.
Materials and Method: Two groups, CH (Dycal) and NPCH (5wt% NP added to Dycal prior to mixing) were mixed and the set cement was evaluated under scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS) and X-ray diffraction (XRD). NPCH was also used for direct pulp capping in two freshly extracted human mandibular third molars for dentin bridge evaluation. The teeth were incubated in Dulbecco modified eagle's medium for 15 days, following which the teeth were sectioned and analyzed for hard tissue formation under SEM.
Results: SEM analysis of the modified material revealed interlacing network with stronger attachment of the agglomerated CH particles and reactionary by-products formed due to the interaction of CH and NP. EDS analysis showed presence of calcium and oxygen; with Group 2 showing additional presence of niobium and phosphate. XRD showed higher intensity peaks with NPCH than CH. The tooth culture model showed distinct hard tissue formation.
Conclusion: Within the limitations of this preliminary study, it can be concluded that the incorporation of NP into CH is a viable alternative to CH for direct pulp capping procedures.

Keywords: Calcium hydroxide, ex vivo tooth culture model, niobium pentoxide, pulp capping, reparative dentine

How to cite this article:
Kritika S, Mahalaxmi S. Evaluation of calcium hydroxide incorporated with niobium pentoxide as a direct pulp capping agent – A preliminary ex vivo tooth culture model analysis. Endodontology 2021;33:86-91

How to cite this URL:
Kritika S, Mahalaxmi S. Evaluation of calcium hydroxide incorporated with niobium pentoxide as a direct pulp capping agent – A preliminary ex vivo tooth culture model analysis. Endodontology [serial online] 2021 [cited 2021 Oct 18];33:86-91. Available from: https://www.endodontologyonweb.org/text.asp?2021/33/2/86/318130

  Introduction Top

Repair and regeneration of lost tooth structure is the core concept in today's research. Pulp capping procedures preserve the vitality of the pulp, eliminate the contaminated tissue and induce reparative dentine formation. The remaining dentine thickness after the exposure to external stimuli (i.e., caries, trauma, or iatrogenic errors) warrants indirect or direct pulp capping therapy. The American Association of Endodontists states that direct pulp capping is performed using a dental material, directly on teeth with mechanical or traumatic pulpal exposure. In recent years, researchers' focus on inventing a biomaterial which can induce reparative dentinogenesis or repair of diseased or inflamed pulp.

Calcium hydroxide (CH) is a “gold standard” pulp capping agent which has been employed for several decades.[1],[2],[3] CH acts by the release of bioactive molecules including the growth factors such as tumor necrosis factor-beta 1 and bone morphogenetic protein from the dentine matrix, which promote remineralization of the lost dentine and accelerate pulpal repair.[4],[5],[6] However, it has inherent drawbacks such as increased solubility, porosity, degradation over time, presence of tunnel defects in the newly formed dentine bridge and decreased mechanical properties cause inflammation and necrosis of the pulp.[7],[8],[9],[10]

Numerous pulp capping materials has evolved in the past 2 decades, amongst which the advent of calcium silicate-based biomaterials resulted in better and more homogeneous reparative dentine formation. Mineral trioxide aggregate (MTA) and Biodentine exhibited excellent biocompatibility and superior sealing ability. However, these also have several disadvantages such as longer setting time, difficulty in manipulation, requiring specialized delivery systems that restrict their clinical use.[1],[8],[11],[12],[13],[14] Further, though MTA and Biodentine have been advocated instead of CH, the setting reaction of both results in the formation of CH, hence the inherent disadvantages of CH remain.[15]

Niobium pentoxide (NP) is a transition metal with enriched biomedical applications due to its increased biocompatibility, resistance to corrosion and enhanced mechanical properties.[16] Studies illustrate the intense bioactivity of NP by hydroxyapatite crystal growth when it comes in contact with human saliva.[17] It is chemically stable in the oral environment, exists in a monoclinic state and is less prone to degradation.[18] Viapiana et al.[19] proved that the addition of NP decreased the solubility quotient, improved the setting time and flow ability of calcium silicate-based endodontic sealer.

As there is no literature evidence on the incorporation of NP into CH pulp capping agent, the present study was aimed to evaluate the material characteristics and hydration potential of CH incorporated with NPCH at 15 days in vitro and in an ex vivo tooth culture model.

  Materials and Method Top

The experiment was conducted in two phases; the initial in vitro characterization of the combination of NP with CH was analyzed using scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD) analysis. The ability of NPCH to form a dentine bridge was evaluated using a tooth culture model.

Preparation of the samples

NP (Sisco Research Laboratories, Mumbai, India) was procured in powder form and CH (Dycal, Dentsply, Milford, USA) available in two paste form were used for the present study.

Equal proportions of the two pastes of Dycal were dispensed on a nonabsorbent pad and mixed using a plastic spatula, according to the manufacturer's instructions (Group 1– CH). Five wt% of NP was mixed with similar proportions of Dycal (Group 2– NPCH) to obtain an uniform mix. The mix were filled into circular teflon molds of 6 mm × 4 mm dimensions and covered with mylar strips. The methodology for the in vitro characterization of the samples and ex vivo experimentation was adopted from the article published by Camilleri et al.[20]

In vitro material characterization

Once set, the prepared specimens were immersed in 5 mL of freshly prepared hanks balanced salt solution (HBSS, Chenchemicals, India) and incubated at 37°C for 14 days. The HBSS contains 8.0 g/L sodium chloride, 0.06 g/L anhydrous potassium dihydrogen phosphate, 0.35 g/L sodium bicarbonate, 0.4 g/L potassium chloride, 0.05 g/L anhydrous disodium hydrogen phosphate and 1 g/L D-glucose. On the 15th day, the samples were removed from HBSS, vacuum desiccated and subjected to material characterization.

Surface characterization

The hydrated specimens (n = 2 in each group) were subjected to gold sputter coating and scanning electron microscopic analysis (F E I Quanta FEG-200 h-SEM, Thermo Fisher, USA) was carried out at magnifications of ×4000, ×8000, ×15000, ×30000 and ×60000 for surface characterization. The degree of hydration and release of the microstructural components (reaction by-products) was evaluated using SEM and EDS.

Material activity

The hydrated specimens (n = 1 in each group) were dried, crushed into a fine powder using a mortar and pestle and subjected to XRD analysis (XRD 3003TT, GE Inspection Technologies, USA), where the diffractometer used CuK radiation ((λ = 1.54 Å) at 40 KV power, 80 mA current and recorded the peaks in the range of 10°–70° at 2 theta degree scale.

Ex vivo experimentation of direct pulp capping using tooth culture model

Institutional Review Board approval was obtained (SRMU/MandHS/SRMDC/2020/S/027). A pilot study of tooth model evaluating the hard tissue formation of 2 freshly extracted impacted, immature human mandibular third molars, indicated for extraction were collected after informed consent from the patients. The age of the patients was 19 and 21 years. After extraction, the tooth was immediately stored at 4°C in Dulbecco modified eagle's medium (DMEM) (HiMedia, Einhausen, Germany) supplemented with 300 IU/mL penicillin, 300 μg/mL streptomycin and 0.75 μg amphotericin B. The immature teeth with wide open apices were held in position with a locking tissue forceps at the level of CEJ without causing any disturbance to the root surface. The teeth were individually cleaned using 0.2% aqueous chlorhexidine solution for 10 s to remove the blood stains and impurities covering the root surface, following which it was neutralized by rinsing in 5 mL of phosphate buffered saline of pH 7.4 for 30 s. Immediately thereafter, the immature tooth was held using a sterile absorbent cotton swab soaked in supplemented DMEM solution which was reloaded every 1 min to avoid any desiccation. Cavity preparation was done using a large round diamond bur (BR 31, Mani, India) mounted on a high speed handpiece with sterile water coolant. The cavity was extended deep until an evident pulpal exposure was seen. Subsequently, the bleeding in the exposure site was controlled and gently air-dried. The experimental NP incorporated CH was placed over the pulp. Once set, the cavity was restored with composite resin restoration (Tetric N Ceram, Ivoclar Vivadent, Liechenstein). Further, the roots were suspended in the supplemented DMEM solution and incubated at 37°C for 14 days, with the medium being replenished every day. On the 15th day, the tooth sample was radiographed to evaluate the hard tissue formation, following which the teeth were demineralized and mounted in paraffin wax and sectioned longitudinally using a hard tissue microtome (Leica SP 1600, Heidelberger, Germany) into 200 micron thick slices with relation to our area of interest. The excess material was washed out during this process. The ultrathin slices were visualized under scanning electron microscope (Carl Zeiss 2500, Hallbergmoos, Germany) at various magnifications of ×25, ×600, and ×2000 and the images were recorded. The SEM of reparative dentine was analyzed at 3 points at 3 different places on the thickest portion. The thickness and width of the newly formed reparative dentine were measured and averaged.

Statistical analysis

In the present study, qualitative analysis using SEM and XRD was done and therefore statistical analysis was not warranted.

  Results Top

In vitro material characterization

Surface characterization

The hydrated specimens of Group 1 (CH) depict the agglomerated hydrated particles of CH with interspersed reactionary byproducts and presence of voids. On the other hand, the hydrated specimens of Group 2 (NPCH) showed a characteristic interlacing network with stronger attachment of the agglomerated CH particles and reactionary by-products formed due to the interaction of CH and NP [Figure 1]. The EDS analysis of both the groups showed presence of calcium and oxygen; with Group 2 showing additional presence of niobium and phosphate.
Figure 1: Scanning electron micrographs of calcium hydroxide (a-e) and niobium pentoxide calcium hydroxide (f-j) after immersion in hanks balanced salt solution at 370 C for 15 days at ×4000, ×8000, ×15000, ×30000 and ×60000

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Material activity

The XRD pattern of Group 1 revealed that the intensity of the peaks is less when compared to Group 2, which showed high intensity peaks [Figure 2].
Figure 2: X-ray diffraction peaks of calcium hydroxide (black) and niobium pentoxide calcium hydroxide (red)

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Ex vivo experimentation of direct pulp capping using tooth culture model

SEM analysis of the pulpal exposure site revealed a distinguishable hard tissue formation; the average thickness of the dentine bridge being 1.97 mm to 2.83 mm in thickness and 3.43 mm wide. At ×600 magnification, when qualitatively assessed a line of separation between the newly formed hard tissue structure and the native dentine was observed. The layer by layer deposition of the hard tissue was noted at ×1000 magnification. SEM image of ×2000 magnification showed that in the topmost layer there was a migration and adhesion of hard tissue deposits over the newly formed hard tissue [Figure 3].
Figure 3: Scanning electron micrographs of hard tissue formed at ×25 (a). Line of separation between the newly formed hard tissue structure and the native dentine at ×600 (b). Layer by layer deposition of the hard tissue (c). Migration and adhesion of hard tissue deposits over the newly formed hard tissue (d)

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  Discussion Top

Researchers postulated that the exact mechanism of action of calcium silicate cements in reparative dentinogenesis is not fully understood.[21] On hydration, MTA releases CH as a by-product which stimulates reparative dentine formation. Camilleri et al.[15] interpreted that set MTA can be considered as CH in a silicate matrix. Since MTA has poor handling properties, any material that can improve the properties of CH as a pulp capping material is warranted. Therefore to overcome this impediment, the present study was designed to modify the Dycal CH with the incorporation of NP.

NP is a metal oxide which when incorporated as a filler proved to increase the radiopacity, microhardness and rate of polymerization of the adhesive resin.[22] Literature reports reveal that the use of 5 wt% NP improved the radiopacity of the material and is equivalent to that of enamel.[23] Collares et al.[24] proved that 5% NP stimulates the phosphate deposition over the resin surface. Therefore, a concentration of 5 wt% NP was chosen for the present study.

NP stimulates the calcium phosphate from the fluids and exhibits its bioactivity by phosphate deposition even after 7 days of immersion in simulated body fluid (SBF).[17] The results of the present study revealed that on SEM analysis of the hydrated specimens, NPCH acted as a filler and minimized the voids formed in the final set cement resulting in a more strengthened network. In addition, on visual examination, it was evident that the stored hydrated NPCH immersed in HBSS solution had minimal disintegration/solubility when compared to CH. Therefore, it could be inferred that the NP played a vital role in yielding a strong matrix for the CH to resist degradation over time. A characteristic release of calcium and phosphate in the EDS analysis was observed, which provides the necessary environment for calcium and phosphate deposition over time. The local ion-rich alkaline environment provided by CH could possibly favor the apatite formation and matrix phosphorylation.

Miyazaki et al.[25] proved that NP induces apatite crystal deposition which occurs by the formation of Nb-OH bond when it comes in contact with SBF. Further, the increased crystallization of CH was evidenced in this study as the incorporation of NP into CH intensified the peaks when compared to CH group when subjected to XRD analysis.

In order to evaluate the microstructural changes and material activity of CH and NPCH, the present in vitro parameters were analyzed following the hydration of specimens by immersing the materials in HBSS to mimic the oral environment. Thereafter, the experimental pulp capping agent was tested in the ex vivo human tooth culture model. Previous literature studies succinctly describe the beneficial effects of the tooth culture model as it reproduces the in vivo pulp capping conditions ex vivo.[20],[26],[27] The ex vivo model clearly simulates the in vivo conditions where the activation of progenitor cells occurs, which differentiates into the odontoblast-like cells and stimulates the process of reparative dentine formation.[28]

Studies show that the use of CH ensues a thin interrupted dentine bridge formation at 15 days, following which at 30 days tunnel defects with porosity in the tubular pattern has been noticed.[29] Conversely in this study, the classical illustration of the dense newly formed hard tissue barrier which appears to be the reparative dentine was clearly visualized in the SEM images at 15 days. This could possibly be due to the addition of NP to CH that enhanced the bioactivity by calcium and phosphate deposition thus propagating the formation of apatite crystals, and decreased the degradation over time resulting in the expedited synthesis and secretion of the reparative dentine layer. Based on the previous studies by Camilleri et al.[20] and Téclès et al.,[26] the present study on the tooth culture model was carried out for 15 days. Silva et al.[30] incorporated NP into calcium silicate cements and demonstrated the fibroblast proliferation and decreased inflammatory response, proving it to be biocompatible. Therefore it can be inferred that the disadvantages of CH such as increased degradation and solubility resulting in microleakage, tunnel defects in the dentine bridge and poor mechanical properties can be compensated by the incorporation of NP which would renew and re-establish CH as the material of choice for direct pulp capping procedures.

The limitations of this preliminary study include smaller sample size and short-term follow up. Further studies are underway to extensively analyze the structural changes in the material and its application on tooth structure using histological and immunohistochemistry methods.

  Conclusion Top

Within the limitations of the present study, it can be concluded that CH incorporated with NP can serve as a promising alternative to conventional CH as it showed superior properties than the CH when hydrated and also exhibited a homogeneous reparative dentine formation.

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Conflicts of interest

There are no conflicts of interest.

  References Top

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