CC BY
14
UDC 616.314.18-002-089.873-74-076.5-092.9 https://doi.org/10.26641/2307-0404.2019.4.189606
COMPARATIVE ANALYSIS OF THE EFFECT OF TRICALCIUM SILICATE CEMENT AND MINERAL TRIOXIDE AGGREGATE ON THE CELLULAR COMPOSITION OF THE PULP AFTER THE VITAL AMPUTATION IN EXPERIMENTAL ANIMALS
SE «Dnipropetrovsk medical academy of Health Ministry of Ukraine»1 Department of pediatric dentistry V. Vernadsky str., 9, Dnipro, 49044, Ukraine
SE «Zaporizhzhia medical academy ofpostgraduate education of the Ministry ofHealth of Ukraine» 2
Department of surgical and preventive dentistry
Vintera bul., 20, Zaporizhzhia, 69000, Ukraine
ДЗ «Днтропетровська медична академiя МОЗ Украти» 1
кафедра дитячо'1 стоматологИ
(зав. - д. мед. н., проф. 1.В. Ковач)
вул. В. Вернадського, 9, Днтро, 49044, Украша
ДЗ «Запорiзька медична академiя тслядипломно! освти МОЗ Украти» 2 кафедра хiрургiчноi профшактично'1 стоматологи бул. Вiнтера, 20, Запорiжжя, 69000, Укра'та e-mail: khotimskaya@ukr.net
Цитування: Медичт перспективы. 2019. Т. 24, № 4. С. 145-153 Cited: Medicni perspektivi. 2019;24(4):145-153
Key words: pulpitis, tricalcium silicate, mineral trioxide aggregate, histology, experiment
Ключовi слова: пульпт, трикальцт crnirnm, мтерал триоксид агрегат, гiстологiя, експеримент
Ключевые слова: пульпит, трикальция силикат, минерал триоксид агрегат, гистология, эксперимент
Abstract. Comparative analysis of the effect of tricalcium silicate cement and mineral trioxide aggregate on the cellular composition of the pulp after the vital amputation in experimental animals. Khotimska Yu., Kovach I., Buniatian Kh., Kravchenko L., Varzhapetian S. The purpose of the study was to determine the effect of tricalcium silicate cement on the protective process of pulp in the treatment of reversible pulpitis. There was carried out an experiment on 3-month-old male rabbits to study morphofunctional changes in the pulp tissue with modeling of reversible pulpitis followed by vital amputation with coating of the pulp with tricalcium silicate (8 animals, study group) and mineral trioxide aggregate (MTA) (8 animals, comparison group). In 2 and 6 weeks dental tissues were fixed by 10% formalin solution and after decalcification and done routine procedures, histological sections were made, stained with hematoxylin and eosin. After the application of tricalcium silicate cement in 2 weeks, the odontoblast density changed to 3.92±1.03*103/mm2 from 8.3±1.02*103/mm2 of cell density in the intact group. The number of neutrophils also plays a big role in the regeneration of inflamed pulp. Thus, in 2 weeks the number of neutrophils made up 6.39±2.61x102 p er 1 mm , which is 3 times less than after mineral trioxide aggregate usage - 19.49±2.85x10 per 1 mm2. It was established that the processes of rheological disorders with impaired lymph circulation are replaced by the restoration of the cellular component. In such a way the proposed vital amputation was examined for reversible pulpitis and it revealed similar clinical and pathological effectiveness with a positive regenerative process with formation of a sufficient number of odontoblasts (7.98±1.51*103/mm2) for the subsequent formation of a hard tissue bridge, which consists of collagen fibers in the form of a frame. The main goal of vital amputation procedure is regeneration (proliferation) of the pulp with a short-term signs of alteration and exudation, which is provided by the choice of material for the pulp covering. Thus, after the vital amputation of the pulp, followed by the use of tricalcium silicate cement 6 weeks after, the number of neutrophils was 1.39±0.72x102p er 1 mm2, which is 5 times less than after using mineral trioxide aggregate.
Реферат. Порiвняльний апаш дй трикальцш силжатного цементу та мшерал триоксид агрегату на клггинний склад пульпи шсля проведення в^альноТ пульпотомй в експериментальних тварин. Хоимська Ю., Ковач I., Бунятян Х., Кравченко Л., Варжапетян С. Метою до^дження було визначити вплив трикальцш силжатного цементу на захисний процес пульпи при лжувант зворотнього пульпту. Був проведений експеримент на 3-мiсячних кроликах-самцях для вивчення морфофункцюнальних змт тканини пульпи з моделюванням зворотнього пульпту та подальшою втальною ампутацieю й покриттям трикальцiю
Yu. Khotimska \ I. Kovach \ Kh Buniatian \ L. Kravchenko \ S. Varzhapetian 2
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силжатом (8 тварин, до^джувана група) та мтерал триоксид агрегатом (8 тварин, група порiвняння). Через 2 та 6 тижнiв тканини зуба фiксували 10% формалтом, пся декальцинацИ та рутинних процедур проводили гiстологiчнi зр1зи, як фарбувались гематоксилтом та еозином. Шсля застосування трикальщевого силiкат-ного цементу через 2 тижш щшьмсть одонтобластiв змтилася до 3,92±1,03*103/мм2 з 8,3±1,02* 103/мм2 щiльностi клтин у цшй групi. Юльюсть нейтрофШв також вiдiграе велику роль у регенерацИ запаленог пульпи. Юльюсть нейтрофШв через 2 тижт становила 6,39±2,61х102 на 1 мм2, що в 3 рази менше, мж тсля використання мтерал триоксид агрегату - 19,49±2,85х102 на 1 мм . Встановлено, що процеси реологiчних порушень з порушенням циркуляцп лiмфи замiнюються вiдновленням клтинного компонента. Таким чином, запропонована втальна ампутацiя була обстежена на наявнiсть зворотнього пульпiту та виявила схожу клШчну та патологiчну ефективтсть ¿з позитивним регенеративним процесом 1з утворенням достатньог кiлькостi одонтобластiв (7,98±1,51*103/мм2) для подальшого утворення твердого тканинного мосту, який складаеться з колагенових волокон у виглядi каркаса. Основною метою методики втальног ампутацп е регенерацiя (пролiферацiя) пульпи, з короткочасними ознаками альтерацп й ексудацп, що, звичайно ж, забезпечуеться вибором матерiалу для покриття пульпи. Таким чином, тсля втальног ампутацп пульпи з подальшим застосуванням трикальцт силiкатного цементу через 6 тижнiв юльюсть нейтрофiлiв становила 1,39±0,72х102 на 1 мм2, що в 5 разiв менше, шж тсля використання мтерал триоксид агрегату.
Mature pulp as connective tissue formations is a component of the whole organism. Any change in exogenous and endogenous factors directly affects its histological structure. To date, the most common drug for its treatment is calcium-containing compounds.
However, long-term observations after treatment with calcium hydroxide-based drugs have shown that its effectiveness decreases with the increase of the observation period. All this has become a prerequisite for replacing calcium hydroxide with more modern materials, which are based on mineral trioxide aggregate. It is a biocompatible material that provides high impermeability and regeneration of the soft dental tissue on contact with the cells and periapical tissues [4, 6]. The drug stimulates the formation of dentinal bridges, protecting the exposed pulp much more efficiently than calcium hydroxide. Despite a rather positive effect, it has a number of features that render dentist's work difficult, limiting it to a two-stage treatment modality. To date, of particular interest is the material represented by the compound of tricalcium silicate (TCS), which demonstrates physical and chemical properties similar to the characteristics of some derivatives of Portland cement [8]. From a biological point of view it has excellent compatibility [10] and is capable of causing reactive dentin deposits by stimulating the activity of odontoblasts [9, 11, 12], as well as reparative dentin - due to cell differentiation [3, 5]. The described property may be useful for various treatment options for the vital pulp [7].
The aim of this study was to determine the effects of tricalcium silicate and mineral trioxide aggregate on the course of reversible pulpitis by detection of morpho-functional peculiarities of changes in the pulp tissue.
MATERIALS AND METHODS OF RESEARCH
An experimental investigation on 3-month-old male rabbits in order to study the morphofunctional
changes of the pulp tissue with modeling of reversible pulpitis was performed.
After a general anesthesia (0.4 ml of 5% ketamine per 1 kg of body weight intramuscularly) we performed preparation of hard dental tissues with a micromotor tip with opening of the pulp horn. Half of the pulp was amputated with the following capping with tricalcium silicate (8 animals, study group) and mineral trioxide aggregate (8 animals, comparison group), tooth filling was performed with glass ionomer cement.
After withdrawal of animals from the experiment in 2 and 6 weeks, dental tissues were fixed in 10% formalin, after decalcification in acetic acid and routine proceeding, histological sections were stained with hematoxylin and eosin (H&E), according to van Gieson, Mallory; PAS-reaction was performed. The sections were studied under the microscope "Olympus BX-41" and following interpretation by "Olympus DP - soft version 3.2", which was used for morphometric study. Number of vessel cross-sections per 1 mm2, odontoblasts, x103 per 1 mm2, neutrophils, x102 per 1 mm2, was detected. All values are expressed as means and standard error of the mean for statistical analysis. Statistical comparison was performed using Mann-Whitney test for statistical analysis. The accepted level of significance was p<0.05.
The procedure was done strictly in compliance with the Helsinki Declaration, European Convention for the protection of vertebrate animals (18.03.1986), European Economic Society Council Directive on the Protection of Vertebrate Animals (24.11.1986) after approval of the Regional Ethical Review Board at State Establishment "Dnipropetrovsk Medical Academy" protocol N 2 (01.04.2016).
RESULTS AND DISCUSSION
Clinical trials have shown that MTA and TCS reduce the frequency and severity of serious complications [1]. However, despite a significant amount of research performed by domestic and foreign scientists, the mechanism and degree of action on stimulating activity of odontoblasts after the application of MTA and TCS cement on the tooth pulp after its amputation remains understudied. The search for new methods and materials is extremely important for the treatment of dental pathology [3, 12], especially with an understanding of the pathogenetic background [1, 10].
Thus, calcium silicate material was widely used, and one of the proposed methods for pulp amputation began to be discussed in restorative dentistry. However, the methodology is especially useful and preferable when the tissue of the coronal pulp is inflamed, and the direct coating of the pulp is not suitable in accordance with some works.
Thus, it was found that pulp tissue has high reactive abilities, and direct pulp coating is insufficient to stimulate active regeneration processes with the active formation of a capillary network and new connective tissue elements [8]. Thus, after amputation of a vital pulp, protective mechanisms in the form of an inflammatory process with its resorption, encapsulation of dentin fragments and foci of necrosis against the background of intensive angiogenesis appear two weeks after.
In histological examination of the pulp of the comparison group two weeks after there was revealed reversible pulpitis. Vascular disorders, inflammatory infiltration, changes in the cellular reaction were manifested by infiltration of the affected zone by inflammatory cells with neutrophils x102 per 1 mm2 19.49±2.85 (fig. 1). Morphometric data are presented in table for this and other groups.
2 weeks
0,34
□ Intact group □МТА □TOS
Fig. 1. Neutrophils, x102 per 1 mm2
Layers of epithelial cells are intact, in cells of the basal layer moderately expressed signs of proliferation are found. The radial structure of the dentinal tubules is preserved on the peripheral part of the crown. Small areas with large interglobular spaces and the process of ectasia of the dentinal tubules are noted (fig. 2).
In the central part, in the place of direct contact with the defect, homogenization and mosaicism of
the intertubular dentin and disappearance of the dentinal tubules in it are noted. In rare places, residues of the tubules with the formation of zones of transparent dentin are defined (fig. 3).
In the area of penetration of the defect into the pulp cavity, the area of necrosis with cellular detritus, as well as the foci of neutrophilic infiltrations is determined (fig. 4).
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Fig. 2. Small areas with large interglobular spaces and the process of ectasia of the dentinal tubules are noted. Staining by Mallory x200
Fig. 3. Residues of the tubules. Electron-diffraction photograph, magnification x3510
Fig. 4. Foci of neutrophilic infiltrations. Homogenized vascular walls. Staining with hematoxylin and eosin x200
Odontoblasts of 2.58±1.11 x103 per 1 mm2 are also determined (fig. 5). The processes of odontoblasts are swollen, of various thicknesses with signs of vacuolar hydropic dystrophy. In the area of the neck and root, reactive changes of the pulp predominate, and are characterized by blood- and
lymph flow disorders, including vascular hyperemia before the sludge-phenomenon of red blood cells. In the microvascular bed, erythrocyte aggregation and stasis are observed. Single pre-dentin sites with diapedetic hemorrhages of erythrocytes in the layer of odontoblasts are visualized.
x103 per 1 mm2
□ norm □ MTA DTCS
Fig. 5. Number of odontoblasts after odontotropic materials usage, x103 per 1 mm2
Histological examination of the obtained micro- tricalcium silicate preparation shows the other objects after pulp amputation with the following picture of pulp tissue condition 2 weeks after.
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In general, signs of proliferation are identified. the dentinal tubules around the area of drug The edges of the traumatic defect are even, linear application is saved. Small zones with large inter-without dentin protrusions. The radial structure of globular spaces are noted (fig. 6).
Fig. 6. Small zones with large interglobular spaces. Staining by Van Gieson x200
The enamel-dentin junction is represented by a the form of small tape-like formations are
linear formation, the enamel has the appearance of a visualized. In the area of the dentin-enamel junction,
granular mass. In the zone of the enamel-dentin a slight loosening of enamel beams is observed
junction (staining by Mallory), enamel beams having (fig. 7).
Fig. 7. Loosening of enamel beams is observed. Staining by Van Gieson. x200.
In the central part, in the place of a direct contact with the defect, homogenization and mosaicism of the intertubular dentin and the disappearance of the dentinal tubules in it is noted. Peritubular dentin is impacted, which is characterized by a clear picture of the borders of the tubules. Circum-pulpar dentin is without signs of pathological transformation, only in isolated areas the penetration of reactive pathological changes into the healthy tissue of circum-pulpar dentin is noted.
Cellular composition of the pulp is represented mainly by undifferentiated cells of the fibroblastic lines, fibroblasts, odontoblasts 3.92±1.03x103 per 1 mm2, macrophages; neutrophils 6.39±2.61x102 per 1 mm2 are isolate (fig. 8).
Histological examination of the obtained micro-objects of pulp amputation with the following MTA coating shows signs of a proliferative inflammatory process six weeks after.
Fig. 8. Insignificant number of inflammatory elements.
Moderately pronounced vascular congestion of Small clumps of neutrophils 7.12±0.96 x102 per the microcirculatory bed in the root area with a 1 mm2 are noted (fig. 9). slight swelling in the perivascular space is noted.
6 weeks
0,34
□ Intact group QMTA GTCS
Fig. 9. Neutrophils, x102 per 1 mm2
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Odontoblasts (5.95±1.53x103 per 1 mm2) of root pulp adjacent directly to the damage zone with pronounced signs of dystrophic changes. In the underlying connective tissue a moderately pronounced edema is found. The cytoplasm of macrophages, plasma cells, fibroblasts and endo-theliocytes is pale, odontoblasts with signs of vacuolization.
The histological examination of the obtained micro-objects after pulp amputation with tricalcium
silicate six weeks after shows that cellular elements of the pulp are presented by odontoblasts (7.98±1.51x103 per 1 mm2) in the peripheral layer and in the subodontoblastic and central layers; fibroblasts and sessile macrophages are also revealed in the subodontoblastic and central layers. Hyperemia is less pronounced without hemorrhages; no necrobiotic changes have been revealed. PAS-reaction is evenly positive in connective tissue fibers and vascular membrane of the pulp.
Results of morphometric investigation of dental pulp tissue (M±m)
Intact animals МТА TCS
2 weeks б weeks 2 weeks 6 weeks
Odontoblasts, x103 per 1 mm2 8.3±1.02 2.58±1.11* 5.95±1.53# 3.92±1.03* 7.98±1.51#
Neutrophils, x102 per 1 mm2 0.34±0.03 19.49±2.85* 7.12±0.96# 6.39±2.61* 1.39±0.72#
Notes: * - p<0.05 - index of significance of differences as compared with intact animals; # - p<0.05 - index of significance of differences as compared with data in 2 weeks
The tissue is abundantly vascularized with numerous newly formed microcirculatory beds and with pronounced convoluted and anastomotic lymphatic vessels. Cellular composition of the pulp is represented mainly by undifferentiated cells of the fibroblastic lines, fibroblasts, odontoblasts, macrophages; neutrophils (1.39±0.72x102 per 1 mm2) are isolate.
CONCLUSION
1. Two weeks after the modeling of the reversible pulpitis in the pulp tissue there were found manifestations of protective-adaptive mechanisms in the form of the inflammatory process with its resolution, with necrosis zone being replaced by connective tissue.
2. It was established that after TCS using in 6 weeks the number of neutrophils is 5 times less than
after applying MTA and revealed similar clinical and pathological efficacy with a positive regenerative process with the formation of a bridge of hard tissues, which consists of collagen fibers in the form of a frame.
3. It was established that the number of odontoblasts after the application of TCS is 1.5 times higher in comparison with MTA. Therefore, our results can be useful for TCS in comparison with MTA.
4. Thus, it should be assumed that the use of tricalcium silicate as a material for pulp coating contributes to more active regeneration processes.
Conflict of interests. The author declares that there is no conflict of interest.
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СПИСОК Л1ТЕРАТУРИ
1. Aerobic microflora in the pathogenesis of maxillary sinusitis after the treatment of caries complications / S. Varzhapetian et al. Georgian medical news. 2019. Vol. 289. P. 42-46.
2. Avwioro G. Histochemical Uses of Haemato-xylin. A Review. JPCS. 2011. N 1. P. 24-34.
3. Biocompatibility of New Pulp-capping Materials NeoMTA Plus, MTA Repair HP, and Biodentine on Human Dental Pulp Stem Cells / C. J. Tomas-Catala et al. J. Endod. 2018. Vol. 44, No. 1. P. 126-132. DOI: https://doi.org/10.1016/jjoen.2017.07.017
4. Cell Viability and Tissue Reaction of NeoMTA Plus: An In Vitro and In Vivo Study / L. S. Pinheiro et al. Grecca FS4. J. Endod. 2018. 1 Jun. pii S0099-2399. P. 30169-9. DOI: https://doi.org/10.1016/jjoen.2018.03.007
5. Comparative analysis of the biological effects of the endodontic bioactive cements MTA-Angelus, MTA Repair HP and NeoMTA Plus on human dental pulp stem cells / C. J. Tomas-Catala et al. Int. J. Endod. 2017. Vol. 50. Suppl. 2. P. e63-e72. DOI: https://doi.org/10.1111/iej.12859
6. Guerreiro-Tanomaru M. J., Bosso-Martelo R., Chavez-Andrade G. M., Filho Tanomaru M. J. Solubility, porosity and fluid uptake of calcium silicate-based cements. Torres FFE. Appl Oral Sci. 2018. No. 26. P. e20170465. DOI: https://doi.org/10.1590/1678-7757-2017-0465
7. Influence of Tricalcium silicate on course of traumatic pulpitis / I. Kovach, et al. Georgian medical news. 2018. Vol. 276, No. 3. P. 130-135.
8. Nagas E., Kucukkaya S., Eymirli A., Uyanik M. Effect of Laser-Activated Irrigation on the Push-Out Bond Strength of ProRoot Mineral Trioxide Aggregate and Biodentine in Furcal Perforations. Cehreli ZC2. Photomed Laser Surg. 2017. Vol. 35, No. 4. P. 231-235. DOI: https://doi.org/10.1089/pho.2016.4171
9. Novel Model for Evaluating the Flow of Endo-dontic Materials Using Micro-computed Tomography / M. A. Tanomaru-Filho et al. J. Endod. 2017. Vol. 43, No. 5. P. 796-800.
DOI: https://doi.org/10.1016/jjoen.2016.12.002
10. Övsay E., Kaptan R. F., §ahin F. The Repair of Furcal Perforations in Different Diameters with Bio-dentine, MTA, and IRM Repair Materials: A Laboratory Study Using an E. Faecalis Leakage Model. Biomed Res Int. 2018. 15 Jan. P. 5478796.
DOI: https://doi.org/10.1155/2018/5478796
11. Physicochemical Properties and Volumetric Change of Silicone Bioactive Glass and Calcium Silicate-based Endodontic Sealers / M. Tanomaru-Filho et al. J. Endod. 2017. Vol. 43, No. 12. P. 20972101. DOI: https://doi.org/10.1016/jjoen.2017.07.005
12. The Effect of a Mineralized Bone Graft on the Surface Microhardness of Mineral Trioxide Aggregate and Biodentine / S. Rahimi et al. Iran Endod J. 2018. Vol. 13, No. 1. P. 83-87.
Стаття надшшла до редакцп 12.09.2019
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