Nanostructured endodontic materials mixed with

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Nov 29, 2018 - MPCA1 and ALBO-MPCA2 were high alkaline, while in case of MTA. + they were ... Biodentine (Septodont, St Maure des Foss'es, France),.
Journal of Materials Science: Materials in Medicine _#####################_ https://doi.org/10.1007/s10856-018-6200-z

ENGINEERING AND NANO-ENGINEERING APPROACHES FOR MEDICAL DEVICES Original Research

Nanostructured endodontic materials mixed with different radiocontrast agents—biocompatibility study Bojana Ćetenović1 Božana Čolović1 Saša Vasilijić2 Bogomir Prokić3 Snežana Pašalić1 Vukoman Jokanović1 Zvezdana Tepavčević4 Dejan Marković4 ●













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Received: 12 June 2018 / Accepted: 29 November 2018 © Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract The aim of this study was to investigate the biocompatibility of nanostructured materials based on highly active calcium silicates mixed with different radiocontrast agents in comparison to MTA+ using in vitro and in vivo model. Morphology of materials’ samples was analyzed using SEM while the phase compositions were identified by XRD. pH values of materials’ suspensions were conducted by pH-meter. The cytotoxicity of materials’ solutions was tested by MTT test (100, 50, 25 and 12.5 mg/ml). LDH and 3H-thymidine assay were utilized for biocompatibility investigations of materials’ eluates (24 h, 7 day and 21 day). Eighteen Guinea pigs were used for intramuscular implantation, as teflon tubes with freshly prepared materials were placed into intramuscular pockets. All samples were composed of round and needle-like particles equally distributed with Ca/Si ratio ~2.7 at%, with the presence of hydrated calcium silicate phases. The pH values of ALBOMPCA1 and ALBO-MPCA2 were high alkaline, while in case of MTA+ they were lower and continuously declined (p < 0.05). Investigated materials didn’t exhibit dose-dependent effect on metabolic activity of L929 cells (p > 0.05). Significant differences in the percentage of cytotoxicity between diluted and undiluted extracts between all tested materials after 24 h and 7 day were noticed (p < 0.05). Increase in L929 cells proliferation was noticed in case of undiluted eluates of ALBOMPCA1 and ALBO-MPCA2 after 7 day (p < 0.05). There were no statistically significant differences in the intensity of inflammatory response between investigated materials and control group after 60 day (p > 0.05). Evaluation of biocompatibility of both ALBO-MPCA1 and ALBO-MPCA2 indicate their potential clinical use.

Supplementary information The online version of this article (https:// doi.org/10.1007/s10856-018-6200-z) contains supplementary material, which is available to authorized users. * Bojana Ćetenović [email protected] 1

2

Vinca Institute of Nuclear Sciences, Mike P. Alasa 12–14, Belgrade 11001, Serbia Institute for Medical Research, Military Medical Academy, Faculty of Medicine, University of Defense, Crnotravska 17,

Belgrade 11000, Serbia 3

Faculty of Veterinary Medicine, University of Belgrade, Bulevar oslobodjenja 18, Belgrade 11000, Serbia

4

School of Dental Medicine, University of Belgrade, Dr. Subotica 11, Belgrade 11000, Serbia

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Graphical Abstract

2 Materials and methods 1 Introduction

2.1 Materials synthesis

Revised definition of material’s biocompatibility was introduced by Williams in 2008 as materials’ ability to perform the appropriate function in the body without causing any undesirable local or systemic reaction of the host, and thereby generating an adequate cellular and tissue response in a specific situation [1]. The optimization of the host response to a foreign body presence was the main goal of materials’ development during the last decades [2, 3]. The first bioactive material based on calcium silicates presented by Abedi and Ingle [4], and Torabinejad et al. [5], named as mineral trioxide aggregate (MTA), initially was recommended as the material of choice for retrograde cavities. MTA represents a mixture of 75% refined Portland cement (PC), 20% Bi2O3 and 5% gypsum, with traces of SiO2, CaO, MgO, Na2SO4 and K2SO4 [6–8]. Chemical modifications of calcium silicates based materials, in terms of aluminium and bismuth absence as potentially compromised biocompatibility elements-Bioaggregate (Innovative BioCeramix, Vancouver, Canada) or calcium carbonate incorporation in order to decrease setting timeBiodentine (Septodont, St Maure des Foss’es, France), were lately introduced [9, 10]. In attempt to find a new formulation which would overcome limitations of MTA, a new endodontic material based on calcium silicates (mineral polyoxide carbonate aggregate (ALBO-MPCA)) was synthesized recently [11]. It has shown significant decrease of setting time due to fast hydration of highly active calcium silicate phases and a good biocompatibility so far [11]. The aim of this study was to investigate the biocompatibility of new endodontic materials ALBO-MPCA1 and ALBO-MPCA2 based on highly active calcium silicates mixed with different radiocontrast agents in comparison to MTA+ at in vitro and in vivo conditions.

To obtain final formulations of new endodontic materials ALBO-MPCA1 and ALBO-MPCA2, it was necessary to synthesize calcium silicate phases (2β-CaSiO4 (β-C2S) and Ca3SiO5 (C3S)) and calcite (CaCO3). For the synthesis of calcium silicate phases the stoichiometric quantities of CaCl2·5H2O (Merck, Germany) (35.59 g) and silica sol obtained by hydrothermal treatment (15 g of 30% sol solution) with ratio of C3S:C2S = 2:1, were used to obtain silicate active phase. To provide the production of a small amount (3.01%) of active tricalcium aluminate phase 4.55 g of Al(C2H3O2) (Alfa Aesar, Germany) was added to this mixture. Then, 71.3 g of NH4NO3 (Fluka, Germany), as oxidation agent, and 53.51 g of C6H8O7·H2O (Alfa Aesar, Germany), as fuel during combustion reaction, were added to the mixture, followed with drying at 80 °C to obtain a gel and then drying at 150 °C to remove water. Temperature increase up to 180 °C led to the ignition of the gel which was swelling into foam and undergoing a strong self-propagating combustion reaction. After thermal treatment the sample was very quickly dried using Cu plates, to provide low crystallinity and high reactivity of obtained β-C2S and C3S phases. The resulting black powder was further treated at 650 °C for 4 h to obtain the desired product with small crystallite sizes. At the end, the powder was milled during a few minutes to obtain calcium silicate phases for the use in final cement mixtures. For the production of CaCO3, 5 mmol of CaCl2·4H2O (Sigma-Aldrich, USA) was dissolved in 50 ml of ethylene glycol (Sigma-Aldrich, USA) and sonicated at 40 °C (Elmasonic S30H). Then, 10 mmol of NaHCO3 (SigmaAldrich, USA), dispersed in 50 ml of ethylene glycol, was added drop wise and continually stirred. The obtained dispersion was heated at 40 °C for 30 min, upon which calcite was separated from a solution by centrifugation (9000 rpm, 30 min), washed several times with mixture of water and

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ethanol (1:4) and finally with water. Sulfonyl dodecyl sulfate (0.5%) was added as an anti agglomeration agent. The obtained nanoparticles were sonicated for 30 min and vigorously stirred for 5 h. The resulting powder, after drying at 120 °C for 5 h, was heated at 500 °C for 1 h, to obtain pure calcite phase. Bi2O3 was obtained by the calcination of Bi(NO3)3 (Kemika, Croatia) at the temperature of 450 °C during 20 h, while BaSO4 was purchased from Merck, Germany. A new endodontic materials ALBO-MPCA1 and ALBO-MPCA2 were made by mixing calcium silicate phases (C3S and βC2S) with CaCO3 and Bi2O3, or BaSO4 respectively, in the ratio 1:2:2 [11]. Mineral trioxide aggregate (MTA+, Cercamed, Stalowa Wola-Poland) was utilized as control material.

2.2 Characterization of materials’ specimens Morphology of investigated materials’ powders (ALBOMPCA1, ALBO-MPCA2 and MTA+) was assessed using the scanning electron microscopy (SEM, JEOL, JSM-5300, Tokyo, Japan). After being dried at 110 °C and vaporized with a thin layer of gold, the materials’ specimens were processed in the chamber of the instrument at a voltage of 30 kV. Energy dispersive analysis (EDS) was used to determine Ca/Si ratio and chemical homogeneity of the phases. The phase compositions before and after materials’ 7-day hydration were detected by X-ray diffractometer (XRD, Philips PW 1050, Almelo, Netherlands).

2.3 pH measurements Assessment of pH values of materials’ suspensions (50 mg/ ml) after 1, 3 and 24 h was conducted by pH-meter (pHvision Microcomputer 6071, JENCO Electronics Ltd., Linkou Shiang, Taiwan), previously calibrated (Carlo Erba, Italy). The test solutions were shaken for 30 min on the vibrator, and then centrifuged for 15 min at 4000 rpm/min. Measurements of pH values for each sample were repeated three times.

medium was sampled, high speed centrifuged and kept at −20 °C until using.

2.5 Cell culture For in vitro cytotoxicity testings, mouse fibroblasts L929 cell line (ATCC, Rockville, USA) was used. Before performing the assays, the cells were cultured in T25cm2 cell culture flasks (Sarstedt) in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (PAA Laboratories Vienna, Austria), 50 μM 2- mercaptoethanol, 2 mM L-glutamine (Sigma-Aldrich, Munich, Germany) and 100 IU/ml penicillin/streptomycin (Galenika, Belgrade, Serbia) at 37 °C in a humidified atmosphere. After reaching approximately 80% confluency, cells were detached by 0.25% Trypsine (Sigma-Aldrich), washed with Phosphate Buffered Saline (Sigma-Aldrich) and counted in 0.4% Trypan Blue dye (Sigma-Aldrich) using haemocytometer (Improved Neubauer’s chamber, BOECO, Humburg, Germany). The cells from the 3th to the 5th passage with viability greater than 90% were used for the further assays. All assays were performed in 96-well microplates (Sarstedt) where 1 × 104 cells per well were seeded 18–24 h earlier.

2.6 MTT assay The cytotoxicity of materials’ solutions was tested in the following concentrations: 100, 50, 25 and 12.5 mg/mL. Fresh solutions were prepared by dissolving the materials in RPMI medium and then centrifuged for 15 min at 3000 rpm/ min. After initial overnight cultivation of L929 cells, the culture medium was removed and 100 µl of fresh materials’ solutions was added and incubated at 37 °C, in an atmosphere with 5% CO2 for 24 h. After that, 10 μl of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) stock solution (5 mg/ml) was added per well. The cells were additionally incubated for 4 h, and 100 µl of 10% sodium dodecyl sulfate (SDS) in 0.01 M HCl (Serva, Heidelberg, Germany) was added. The percentage of the cell metabolic activity (% M) was calculated based on the formula [13]:

2.4 Conditioning of materials’ samples %M ¼

Freshly mixed materials were prepared and placed into plastic molds (R = 5 mm, h = 5 mm). After complete setting and sterilization with UV light, the samples (n = 4 per material) were incubated in 6-well plates (Sarstedt, Nümbrecht, Germany) in RPMI medium (Sigma-Aldrich, Munich, Germany), in atmosphere with 5% CO2 (Binder GmbH, Tuttlingen, Germany) at 37 °C, for 24 h, 7 days and 21 days. The samples were conditioned according to the ISO standard [12], and total surface to volume ratio was approximately 43.96 mm2/ml. After conditioning, the

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OD cell culutre with samples  OD samples without cell culutre  100 OD cell culutre without samples  OD control medium

ð1Þ After 24 h an optical density was quantified spectrophotometrically on microplate reader (DV990/BV6, Roma, Italy) at a wavelength of 570 nm.

2.7 LDH assay The cytotoxicity of 50% diluted and undiluted materials’ eluates (24 h, 7 day and 21 day) was investigated. After

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initial overnight cultivation of L929 cells, the culture medium was removed and the cells were incubated for 24 h at 37 °C in an atmosphere with 5% CO2 in the presence of materials eluates. The cells cultivated without samples and with 1% Triton X-100 (Sigma) were used as negative and positive control, respectively. Triton X-100 was used in order to achieve maximal release of cytosolic LDH. The amount of released LDH was determined using a commercial ADVIA system (ADVIA 1800, Clinical Chemistry System, Siemens). The LDH assay was conducted in triplicate, while the percentage of the cell cytotoxicity (% C) was calculated based on the formula: LDH concentration in cell culture with samplesLDH concentration in cell culture without samples %C ¼ LDH concentration in culture with Triton X100LDH concentration in cell culture without samples 100

ð2Þ

2.8 Thymidine incorporation assay The degree of cell proliferation was estimated based on cell labeling with a radionuclide 3H-thymidine. After initial overnight cultivation of L929 cells and the removal of culture medium, 100 µl of undiluted or 50% diluted materials’ eluates conditioned for 24 h, 7 days and 21 days were added in cell triplicates. The cells were additionally cultivated in a humidified atmosphere for 24 h at 37 °C. For last 8 h cells were pulsed with [3H] thymidine (1 µCi/well; Amersham, Amersham, UK), and then harvested onto glass fiber filters. The incorporation of the radionuclide was measured further by β-scintillation counter (LKB-1219; Rackbeta, Turku, Finland). The radioactivity of the samples was presented as counts per minute (CPM), proportional to the amount of 3H-thymidine incorporated into the DNA. The index of cell proliferation was calculated by the following formula: %P ¼

CPM of cell culture with sample ¼ 100 CPM of cell culture without sample

ð3Þ

2.9 Experimental design for intramuscular implantation Experimental procedures in vivo were performed with the approval of the local Ethics Committee (Protocol No. 36/7, 20/02/2013) and in accordance to the National Institutes of health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978). The experiment procedure was carried out on eighteen male, 4 months old Guinea pigs, with an average weight of about 1.5 kg, under general dissociative anesthesia. Freshly prepared materials were placed into sterile teflon tubes (10 mm of length, inner diameter of 1 mm), so that one end always remained empty serving as negative control. After

the back of the skin was shaved and cleaned with povidone iodine, a 2.5 cm long medial incision was made, followed by a sharp dissection of the muscle longissimus fascia. Using blunt dissection, four intramuscular pockets were formed, in which teflon tubes were placed. The operative area was sutured by resorbable sutures (SofsilkTM 4-0, SynetureR, England). By using intracardial injection of sodium pentobarbital (Pentobarbital sodium salt, SigmaAldrich Chemie GmbH, Steinheim, Germany), animals were sacrificed after 15, 30 and 60 days. Tissue samples along with teflon tubes were collected, fixed in 10% formalin, and 5 μm serial tissue sections were stained with standard hematoxylin-eosin (HE) dye. Histological analysis was conducted in four separate areas using a light microscope (Model Lambda LQT 2; ATTO Instruments Co, Hong Kong, China). The intensity of inflammatory reaction was categorized as Grade 0- absence of inflammatory cells (none), Grade 1less than 25 inflammatory cells (minimal), Grade 2- from 26 to 50 inflammatory cells (mild), Grade 3- from 51 to 75 inflammatory cells (moderate) and Grade 4- more than 75 inflammatory cells (severe). The extension of inflammatory reaction was assessed using the following scale: Grade 0absence of inflammatory cells, Grade 1- inflammatory cells close to the investigated material, Grade 2- inflammatory cells present deeper in the tissue and Grade 3- tissue completely infiltrated with inflammatory cells or necrotic tissue. The presence of fibrous capsule was classified as: Grade 0- absence of fibrous capsule, Grade 1- minimal layer of fibrous capsule, Grade 2- thin layer of fibrous capsule, Grade 3- thick layer of fibrous capsule and Grade 4- large fibrous capsule; as well as absence or presence of Giant cells, Grade 0 and Grade 1, respectively.

2.10 Statistical analysis The statistical аnаlysis wаs carried out using two-way ANOVA Repeаted Meаsures test (post hoc Tukey’s test) and Kruskal-Wallis test (post hoc Dunn’s test). The level of significаnce wаs set аt p < 0.05, аnd the dаtа were processed using the stаtisticаl softwаre IBM SPSS (IBM SPSS 20, IBM Corporation, New Orchard Road Armonk, New York, SAD).

3 Results 3.1 Physico-chemical characterization of the materials All investigated materials were composed of a mixture of round and needle-like particles (1–5 µm in diameter) equally distributed (Fig. 1a–c). Analysis of elemental

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Fig. 1 a SEM micrograph of fresh powder (ALBO-MPCA1, magnication 5000×), b SEM micrograph of fresh powder (ALBO-MPCA2, magnication 5000×), c SEM micrograph of fresh powder (MTA+, magnication 1000×), d X-ray diffractogram of unhydrated and

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hydrated ALBO-MPCA1, e X-ray diffractogram of unhydrated and hydrated ALBO-MPCA2, f X-ray diffractogram of unhydrated and hydrated MTA+. C3S- tricalcium silicate, C2S- dicalcium silicate, Ccalcite, Bi- Bi2O3, Ba- BaSO4, T- tobermorite, P- portlandite

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Table 1 EDS analysis of investigated materials (wt%) Materials Element

ALBO-MPCA2

ALBO-MPCA1

MTA+

O

46.35

43.32

47.78

Ca

33.72

34.91

32.07

Si

4.13

4.75

5.38

C

5.12

8.39

4.31

Bi

7.30

0.00

6.51

Al

0.88

1.41

1.56

Fe

0.00

0.00

0.76

Mg

0.00

0.80

0.00

Ba

0.00

3.95

0.00

S

2.50

2.47

0.00

P

0.00

0.00

Total

100

1.00

100

100

Table 2 Kinetics of pH values of fresh materials over 24 h Time Materials

1h

3h

24 h

ALBO-MPCA1 ALBO-MPCA2 MTA+

11.52 ± 0.01a,*,# 11.80 ± 0.29*,† 10.68 ± 0.01b,c,#,†

11.67 ± 0.03*,# 11.77 ± 0.04*,† 9.09 ± 0.01b,d,#,†

12.01 ± 0.01a,*,# 11.91 ± 0.01*,† 8.26 ± 0.03c,d,#,†

Symbols indicate statistical differences between materials at one test period (p < 0.05) Lower case letters indicate statistical differences within one material at different test periods (p < 0.05)

composition by EDS (Table 1) showed that Ca/Si ratio was approximately 2.7 atomic% for all investigated materials, which corresponds to C3S:β-C2S = 2:1. XRD analysis after 7 days hydration showed the presence of hydrated calcium silicate phase (tobermorite) mixed with calcium hydroxide (portlandite) but in smaller quantities, while calcite, Bi2O3 and BaSO4) remained almost the same as before hydration (Fig. 1d–f).

3.2 pH measurements The pH values of fresh materials ALBO-MPCA1 and ALBO-MPCA2 were higher than 11.2 respecting all observation periods (p > 0.05, Table 2). In the case of MTA + , pH values were generally lower in comparison to the two other tested materials, and continuously declined, but were still alkaline (p < 0.05, Table 2).

3.3 In vitro biocompatibility analysis The investigated materials did not exhibit dose-dependent effect on metabolic activity. An increase of metabolic activity of cells respecting all tested concentrations of

Fig. 2 Metabolic activity of cells (%) following the exposure to fresh materials. Asterisks indicate statistical differences between materials at one test period (p < 0.05). Lower case letters indicate statistical differences within one material at different test periods (p < 0.05)

MTA+ was noticed (p > 0.05, Fig. 2). The same trend characterized ALBO-MPCA1 and ALBO-MPCA2, with the exception of maximum tested concentration (p > 0.05, Fig. 2). No significant differences in metabolic activity of cells between ALBO-MPCA1 and ALBO-MPCA2 was noticed (p > 0.05, Fig. 2). Following the extraction time, the percentage of cytotoxicity decreased, except in the case of 50% diluted eluates of ALBO-MPCA2, but this difference was not greater than 6% (Fig. 3a, b). Significant differences in the percentage of cytotoxicity between diluted and undiluted eluates between all tested materials after a 24 h and 7 day were noticed (p < 0.05). The percentage of cell proliferation decreased with the extraction time and was constant in case of 50% diluted eluates of MTA+ and ALBO-MPCA2 after 7 day. The increase in the percentage of cell proliferation was noticed in the case of undiluted eluates of ALBO-MPCA1 and ALBOMPCA2 after 7 day. There were no statistically significant differences in the percentage of cell proliferation between undiluted and 50% diluted eluates of the same material regardless of the observation time (p > 0.05, Table 3).

3.4 In vivo biocompatibility analysis Fifteen days after implantation, at the site of ALBO-MPCA1 and MTA+ implantation, the presence of moderate inflammation was noticed, while the intense inflammation was evident in case of ALBO-MPCA2 samples (Table 4, Fig. 4a, d, g). Following 30-day observation period, ALBO-MPCA1 and MTA+ samples exhibited mild inflammation, while histopathological findings differed significantly in case of ALBO-MPCA2 samples (Table 4, Fig. 4b, e, h). After the experimental period of 60-day, thin fibrous capsules were

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Fig. 3 a Cytotoxicity (%) following the exposure to diluted (50%) eluates of set materials, b Cytotoxicity (%) following the exposure to undiluted (100%) eluates of set materials. Asterisks indicate statistical differences between materials at one test period (p < 0.05)

formed around all samples, almost without signs of inflammation (Table 4, Fig. 4c, f, i). Statistically significant differences in the intensity of inflammatory response between the tested materials and the control group after 60 day were not registered (p > 0.05, Table 4). Analyzing the extension of inflammatory reaction and thickness of the fibrous capsule good signs of tissue recovery could be observed (Table 4). No statistically significant differences respecting the presence of Giant cells were observed in the case of all investigated materials (p > 0.05, Table 4).

4 Discussion XRD analysis after hydration indicated the presence of predominant tobermorite phase confirming the occurrence

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of hydration processes. While active calcium silicate phases exhibited transformation, barite phase (hydrated Bi2O3 and BaSO4) remained almost unchanged, non-influencing hydration mechanism significantly [7, 14, 15], but potentially influencing on materials’ biocompatibility. According to the previous studies, Bi2O3 may affect the hydrated phase, by forming a calcium silicate bismuth hydrate (C-SH-Bi), and reducing the precipitation of Ca(OH)2, thus influencing the material’s bioactivity [15, 16], but this phase was not found in the case of our analysis. Furthermore, the release of free bismuth may generate undesirable effects regarding the material’s biocompatibility. SEM and EDS analyses of investigated materials suggested a homogeneous distribution of the phases in the samples which confirmed that the synthesized materials are adequately designed. Moreover, it was found that Si/Ca molar ratio has an influence on cell attachment, cell proliferation and cell migration, as more cells with AC stress fibers were observed on surfaces with higher Si content [17, 18]. These reports validate the impact of Si on cell adhesion, along as Ca ions enhance osteoblast chemotaxis and proliferation, as well as the differentiation by increasing the expression of cells’ markers [19]. Synthesized materials, organized at three hierarchical levels (agglomerates, crystallites and nanoparticles), as presented in our study, have potentially high biological value, as the size of agglomerates are not equivalent to the channels in the cell membrane, and thus are nondestructive, and nanoelements allow their distinct activity after placing in the vital tissues due to rapid setting. Moreover, particles smaller than the diameter of dentinal tubules (