|Year : 2021 | Volume
| 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
Selvakumar Kritika, Sekar Mahalaxmi
Department of Conservative Dentistry and Endodontics, SRM Dental College, Ramapuram; SRM Institute of Science and Technology, Ramapuram Campus, Chennai, Tamil Nadu, India
|Date of Submission||13-Jan-2021|
|Date of Decision||02-Mar-2021|
|Date of Acceptance||15-Apr-2021|
|Date of Web Publication||11-Jun-2021|
Dr. Sekar Mahalaxmi
Department of Conservative Dentistry and Endodontics, SRM Dental College, Ramapuram, SRM Institute of Science and Technology, Ramapuram Campus, Chennai, Tamil Nadu
Source of Support: None, Conflict of Interest: None
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 24];33:86-91. Available from: https://www.endodontologyonweb.org/text.asp?2021/33/2/86/318130
| Introduction|| |
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.,, 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.,, 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.,,,
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.,,,,, 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.
Niobium pentoxide (NP) is a transition metal with enriched biomedical applications due to its increased biocompatibility, resistance to corrosion and enhanced mechanical properties. Studies illustrate the intense bioactivity of NP by hydroxyapatite crystal growth when it comes in contact with human saliva. It is chemically stable in the oral environment, exists in a monoclinic state and is less prone to degradation. Viapiana et al. 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|| |
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.
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.
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.
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.
In the present study, qualitative analysis using SEM and XRD was done and therefore statistical analysis was not warranted.
| Results|| |
In vitro material 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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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|| |
Researchers postulated that the exact mechanism of action of calcium silicate cements in reparative dentinogenesis is not fully understood. On hydration, MTA releases CH as a by-product which stimulates reparative dentine formation. Camilleri et al. 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. Literature reports reveal that the use of 5 wt% NP improved the radiopacity of the material and is equivalent to that of enamel. Collares et al. 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). 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. 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.,, 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.
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. 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. and Téclès et al., the present study on the tooth culture model was carried out for 15 days. Silva et al. 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|| |
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.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Iwamoto CE, Adachi E, Pameijer CH, Barnes D, Romberg EE, Jefferies S. Clinical and histological evaluation of white ProRoot MTA in direct pulp capping. Am J Dent 2006;19:85-90.
Tuna D, Olmez A. Clinical long-term evaluation of MTA as a direct pulp capping material in primary teeth. Int Endod J 2008;41:273-8.
Qudeimat MA, Barrieshi-Nusair KM, Owais AI. Calcium hydroxide vs mineral trioxide aggregates for partial pulpotomy of permanent molars with deep caries. Eur Arch Paediatr Dent 2007;8:99-104.
Barthel CR, Levin LG, Reisner HM, Trope M. TNF-alpha release in monocytes after exposure to calcium hydroxide treated Escherichia coli LPS. Int Endod J 1997;30:155-9.
Graham L, Cooper PR, Cassidy N, Nor JE, Sloan AJ, Smith AJ. The effect of calcium hydroxide on solubilisation of bio-active dentine matrix components. Biomaterials 2006;27:2865-73.
Zhang W, Walboomers XF, Jansen JA. The formation of tertiary dentin after pulp capping with a calcium phosphate cement, loaded with PLGA microparticles containing TGF-beta1. J Biomed Mater Res A 2008;85:439-44.
Nowicka A, Wilk G, Lipski M, Kołecki J, Buczkowska-Radlińska J. Tomographic evaluation of reparative dentin formation after direct pulp capping with Ca (OH) 2, MTA, biodentine, and dentin bonding system in human teeth. J Endod 2015;41:1234-40.
Aeinehchi M, Eslami B, Ghanbariha M, Saffar AS. Mineral trioxide aggregate (MTA) and calcium hydroxide as pulp-capping agents in human teeth: A preliminary report. Int Endod J 2003;36:225-31.
Nair PN, Duncan HF, Pitt Ford TR, Luder HU. Histological, ultrastructural and quantitative investigations on the response of healthy human pulps to experimental capping with mineral trioxide aggregate: A randomized controlled trial. Int Endod J 2008;41:128-50.
Cox CF, Sübay RK, Ostro E, Suzuki S, Suzuki SH. Tunnel defects in dentin bridges: Their formation following direct pulp capping. Oper Dent 1996;21:4-11.
Parirokh M, Asgary S, Eghbal MJ, Kakoei S, Samiee M. A comparative study of using a combination of calcium chloride and mineral trioxide aggregate as the pulp-capping agent on dogs' teeth. J Endod 2011;37:786-8.
Dominguez MS, Witherspoon DE, Gutmann JL, Opperman LA. Histological and scanning electron microscopy assessment of various vital pulp-therapy materials. J Endod 2003;29:324-33.
Asgary S, Eghbal MJ, Parirokh M, Ghanavati F, Rahimi H. A comparative study of histologic response to different pulp capping materials and a novel endodontic cement. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;106:609-14.
Tran XV, Gorin C, Willig C, Baroukh B, Pellat B, Decup F, et al
. Effect of a calcium-silicate-based restorative cement on pulp repair. J Dent Res 2012;91:1166-71.
Camilleri J, Montesin FE, Brady K, Sweeney R, Curtis RV, Ford TR. The constitution of mineral trioxide aggregate. Dent Mater 2005;21:297-303.
Challa VS, Mali S, Misra RD. Reduced toxicity and superior cellular response of preosteoblasts to Ti-6Al-7Nb alloy and comparison with Ti-6Al-4V. J Biomed Mater Res A 2013;101:2083-9.
Karlinsey RL, Hara AT, Yi K, Duhn CW. Bioactivity of novel self-assembled crystalline Nb2O5 microstructures in simulated and human salivas. Biomed Mater 2006;1:16-23.
Khan AM, Suzuki H, Nomura Y, Taira M, Wakasa K, Shintani H, et al
. Characterization of inorganic fillers in visible-light-cured dental composite resins. J Oral Rehabil. 1992;19:361-70.
Viapiana R, Flumignan DL, Guerreiro-Tanomaru JM, Camilleri J, Tanomaru-Filho M. Physicochemical and mechanical properties of zirconium oxide and niobium oxide modified Portland cement-based experimental endodontic sealers. Int Endod J 2014;47:437-48.
Camilleri J, Laurent P, About I. Hydration of Biodentine, Theracal LC, and a prototype tricalcium silicate-based dentin replacement material after pulp capping in entire tooth cultures. J Endod 2014;40:1846-54.
Min KS, Park HJ, Lee SK, Park SH, Hong CU, Kim HW, et al
. Effect of mineral trioxide aggregate on dentin bridge formation and expression of dentin sialoprotein and heme oxygenase-1 in human dental pulp. J Endod 2008;34:666-70.
Leitune VC, Collares FM, Takimi A, de Lima GB, Petzhold CL, Bergmann CP, et al
. Niobium pentoxide as a novel filler for dental adhesive resin. J Dent 2013;41:106-13.
Garcia IM, Leitune VC, Balbinot GS, Samuel SM, Collares FM. Influence of niobium pentoxide addition on the properties of glass ionomer cements. Acta Biomater Odontol Scand 2016;2:138-43.
Collares FM, Portella FF, da Silva Fraga GC, Semeunka SM, Almeida LD, da Rosa Santos E, et al. Mineral deposition at dental adhesive resin containing niobium pentoxide. Appl Adhes Sci 2014;2:1-6.
Miyazaki T, Kim Hm, Kokubo T, Ohtsuki C, Nakamura T. Apatite-forming ability of niobium oxide gels in a simulated body fluid. J Ceram Soc 2001;109:929-33.
Téclès O, Laurent P, Aubut V, About I. Human tooth culture: A study model for reparative dentinogenesis and direct pulp capping materials biocompatibility. J Biomed Mater Res B Appl Biomater 2008;85:180-7.
Abdul Sahib N, Al-Dahan Z, Al-Hijazi A. Expression of TGF-β1 by pulp tissue of human permanent and primary teeth capped by biodentine. J Nat Sci Res 2015;5:21-7.
Téclès O, Laurent P, Zygouritsas S, Burger AS, Camps J, Dejou J, et al
. Activation of human dental pulp progenitor/stem cells in response to odontoblast injury. Arch Oral Biol 2005;50:103-8.
Swarup SJ, Rao A, Boaz K, Srikant N, Shenoy R. Pulpal response to nano hydroxyapatite, mineral trioxide aggregate and calcium hydroxide when used as a direct pulp capping agent: An in vivo
study. J Clin Pediatr Dent 2014;38:201-6.
Silva GF, Guerreiro-Tanomaru JM, da Fonseca TS, Bernardi MI, Sasso-Cerri E, Tanomaru-Filho M, et al
. Zirconium oxide and niobium oxide used as radiopacifiers in a calcium silicate-based material stimulate fibroblast proliferation and collagen formation. Int Endod J 2017;50:e95-108.
[Figure 1], [Figure 2], [Figure 3]