The healing process and prognosis depend on rapid intervention and the correct choice of repair material. An intimate seal between the repair filling material and the root canal wall is a significant component that influences treatment results. Traditionally, there were materials used as root-end and perforation repair fillings such as amalgam, EBA, Cavit, and zinc oxide-eugenol which failed to fulfill the required properties. The repair material should be adequate, biocompatible, noncytotoxic, bioactive, antibacterial, and insoluble and should have a bond strength with radicular dentin characteristics.  Mineral trioxide aggregate [MTA] is essentially a classical bio ceramic material that is heavily loaded with metal oxides. It is still the benchmark material for repairing perforations and sealing root-end cavities. It is a highly alkaline biocompatible and bioactive material with prominent sealing features. Nevertheless, several concerns related to MTA have been addressed, such as poor handling, prolonged setting time, discoloration and its availability as a powder/liquid system, which increase the liability of significant material loss [2]. To conquer these issues, bio ceramic materials have been developed.

Bio ceramics are non-toxic, biocompatible, dimensionally, and chemically stable within the biological environment. Recently, premixed bio ceramics have been introduced to gain the advantage of uniform consistency and lack of waste [2]. Neo putty MTA® is a bioactive premixed bio ceramic material with superior handling properties. According to the manufacturer it is characterized by being bioactive, biocompatible, non-cytotoxic, non-genotoxic, initially high in pH [alkaline/basic] and antibacterial.  In addition to that, it shows highest radiopacity in its class, promotes hydroxyapatite formation to support the healing process and it is resin-free so dimensionally stable with no shrinkage to ensure a gap-free seal. Non-Staining so won’t discolor teeth [3].

Therefore, conducting a study to evaluate and compare properties of Neo putty MTA to MTA Angelus as root repair material was thought to be of value.

Our null hypothesis is that blood contamination doesn’t affect properties of Neo putty MTA and MTA Angelus such as marginal adaptation and bioactivity when used as root-end fillings.

Sample Selection

Sixty extracted single rooted maxillary anterior teeth and twenty permanent mandibular first molar teeth. The integrity of the teeth was evaluated using a dental operating microscope to inspect the surface as well as radiographic examination of the canal space.  Inclusion criteria included human sound teeth. Exclusion criteria were as follows: teeth with internal or external resorption, caries, cracks, fractures or with previous endodontic filling were discarded and replaced.

Sample Preparation

Endodontic access cavities were prepared in all teeth using high speed tapered with round diamond stone #12 with air-water coolant. The working length was obtained by subtracting 0.5 mm from the tooth length. The canals were manually instrumented with size #10 and #15 K file until reaching the working length and irrigated with 5 mL of 2.5% sodium hypochlorite. Cleaning and shaping were performed by crown down technique using M-Pro rotary files [3 files system, IMD, China] [#18.09, #20.04, #25.06] then enlarged apically to size #50 K. Patency with K file #15 and irrigation with 5 ml of 2.5% sodium hypochlorite were performed between every file to remove all debris and prevent blockage. A final rinse was performed with 5 mL of 17% EDTA for 1 min to remove the smear layer using a 27-gauge needle. Paper point’s size #50 was used for drying the canals followed by obturation. Gutta percha cones [Meta Biomed, Chungcheongbuk- do, Republic of Korea] of #50/0.04 were used with Adseal resin sealer [Meta Biomed, Chungcheongbuk-do, Republic of Korea] in lateral compaction. After root filling, the coronal space was filled with a temporary filling material and the specimens were kept at 100% humidity and 37°C in an incubator for 1 week to completely set [4].

Root-end resection was done by cutting 3 mm from the apical part of the root perpendicular to its long axis. Root-end cavities were prepared in all teeth under 8x magnification using a dental operating microscope [Labomed, America], using AS3D ultrasonic diamond coated retro-grade tip [Satelec, Cedex, France] attached to an ultrasonic unit [Woodpecker DBA 6, China] at a medium power setting. All cavities were standardized to 3 mm depth and 1 mm diameter.

Sample Classification

• According to method of Evaluation
• Marginal Adaptation [n=20]:

Ten teeth filled with MTA Angelus

• Subgroup A: Five inserted in blood
• Subgroup B: Five inserted in DW

Ten teeth filled with Neoputty MTA

• Subgroup A: Five inserted in blood
• Subgroup B: Five inserted in DW

Bioactivity [n=40]

Twenty teeth filled with MTA Angelus

• Subgroup A: Ten inserted in blood
• Subgroup B: Ten inserted in DW.

Twenty teeth filled with Neputty MTA

• Subgroup A: Ten inserted in blood
• Subgroup B: Ten will be inserted in DW

MTA Angelus

Liquid and powder mixed according to manufacturers’ instructions.The powder was mixed with distilled water in ratio 1:1 powder water ratio until the mixture exhibited wet-like consistency. Pieces of Neoputty MTA were taken from the manufacturer-provided preloaded syringe.

Half of the prepared retro cavities were filled with MTA Angelus and the other half with Neoputty MTA using MTA carrier followed by gentle condensation using pluggers and gutta percha size #40 to make sure complete filling of the cavities to the level of the resection. Shortly after insertion [4-5 minutes] and before the initial setting of the material was complete, all teeth were inserted into tubes [Eppendorf, Hamburg, Germany] containing either containing 1 ml of human blood with sodium citrate added as an anticoagulant or DW allowing the initial setting of the material to take place in the mentioned liquids [5].

Human blood was obtained from one donor from Blood Bank at El-Demerdash Hospital Ain Shams University [Cairo, Egypt]. Blood samples were collected and tested negatively for blood diseases. It was included in the research after donor’s approval. A precision cutting machine was used to cut slices transversely from the mid-root perpendicular to the long axis of the tooth. Root discs 2 mm in thickness with the set material in the form of cylinders in its core were obtained and stored in the tubes containing the HBSS [6]. Twenty discs were used for marginal adaptation testing and forty discs for bioactivity evaluation. In new clean Eppendorf tubes, 1.5 ml of HBSS was injected.

Marginal Adaptation

Examination of the discs under 200X &1500X magnification using a SEM to determine the adaptation of the root-end filling to the dentin walls in micrometers. Several values were collected and the largest gap was recorded for each specimen. Results were obtained and analyzed using IBM SPSS statistical software. Comparison between two materials was performed using two sample t-test.

Bioactivity

Twenty blood-set root discs and 20 dsitill water- set root discs were stored in tubes containing HBSS [Biochrom GmbH, Leonorenstr, Brelin, Germany [7]. Analyses were performed at intervals of 1, 7, and 30 days changing the solution at each interval. For morphological and elemental analysis, SEM connected with EDX unit [Quanta 250 FEG, FEI Company, Oregon, USA] was used. Discs were left to air dry for ten minutes. Discs were mounted on an aluminum stub using carbon sticky pads and inserted in the SEM/EDX unit. Air was expelled to allow analysis of the specimens in vacuum.

Morphological Analysis by SEM

For morphological analysis, the surface was studied at low magnification [x200] to show the full surface of the filling as well as at high magnification [x1500 and x3000] to show the interfacial layer between root dentin and repair material. Samples from each subgroup [blood-set group, and distill water-set group] were tested. The percentage of the surface covered by the formed deposits was measured at day 1, 7 and 30 using Image J software [1.42a/Java 1.6.0-10 image analyzer software].

Elemental Analysis by EDX

For elemental analysis, three areas were selected randomly from the repair-dentin interface of each sample to measure the atomic percentage of calcium and phosphorus at each area as well as other elements like silica, magnesium, carbon and oxygen as seen in Fig.

Statistical Analysis

Statistical analysis was performed using IBM SPSS statistical software. Comparison between two materials was performed using two sample t-test. One-way ANOVA followed by Tukey’s post hoc test was used for intragroup comparison. The data were expressed by mean and standard deviation. Statistical significance was set at 5%.

Marginal Adaptation

The scanning electron microscope of transverse sections of root end filled teeth showed marginal gaps at dentin -rooted filling interface as presented in (Figure 1).

According to mean values, MTA [9.94 ±2.11% in blood and 9.37±1.7 in DW] showed the widest gap in comparison to Neoputty MTA [9.85 ±2.28 on blood and 9.38±2.56 in DW]. Statistically there was no significant difference between two groups [P-value > 0.05]

According to mean values, blood contaminated MTA [9.94 ±2.11%] showed widest gap followed by blood contaminated Neoputty MTA [9.85 ±2.28] while the least gap was present in MTA and Neoputty MTA in DW [9.37 ±1.70% and 9.38 ±2.56% respectively]. Statistically there was no significant difference between both mediums for each material. [P-value > 0.05].

Figure 1: SEM Micrographs Showing Measurements of Largest Gap Distance Between Root-End Filling and Dentin.

Low magnification images of blood-set MTA Angelus at day 1showed irregular restoration margin and the surface of restoration showed voids without any globules while MTA Angelus in DW showed smooth restoration margin and the surface of restoration showed globules. AT DAY 7 and DAY, blood contaminated MTA restoration margin showed smooth restoration margin and globules but were fewer and smaller than MTA in DW. Voids increased with increasing immersing time in blood contaminated samples as presented in (Figure 2).

High magnification images allowed visualization of surface morphology and interfacial deposits at the MTA-dentin interface. At day 1, MTA Angelus in blood, showed no deposition of globules on its surface while MTA Angelus in DW, showed uniform globules of different morphology [spherical and hexagonal patterns] on its surface. MTA Angelus in DW showed higher deposits than blood contaminated group.AT DAY 7 AND DAY 30, blood contaminated MTA showed globules irregular in shape, few and smaller in size in comparison to MTA IN DW as presented in (Figure 2).

Low magnification images of blood-set Neoputty MTA at day 1 showed  irregular restoration margin and the surface of restoration showed voids and few crystals deposition unlike Neoputty in DW which showed smoother restoration margin and the surface of restoration showed higher crystal deposits.At day 7 and day 30:  Blood contaminated Neoputty MTA samples showed regular restoration margin and the surface of restoration showed less voids compared to day 1 and 7 and few crystals deposition in comparison to  Neoputty MTA in DW as presented in (Figure 3).

High magnification images of blood-set Neoputty MTA at day 1 globules irregular in shape, few and smaller in size compared to specimens in DW which showed globules of different morphological patterns [spherical and hexagonal], larger and more uniform. At day 7 and day 30: Blood contaminated Neoputty MTA samples showed globules regular in shape, few and smaller in size but with different morphological patterns [spherical and hexagonal shapes] compared to specimens in DW which showed globules of more morphological patterns [tube like, spherical and hexagonal], larger and more uniform as presented in (Figure 3).

Figure 2: SEM Images of a Specimen from MTA Angelus In Blood. 1, 3, 5 Showing Low Magnification X200 - 2, 4, 6 Showing High Magnification X3000. 1, 2 At Day 1- 3, 4 At Day 7 – 5, 6 At Day 30 - A, Represent Dentin, B, Material, C, Interface.

Figure 3: SEM Images of a Specimen from Neoputty MTA In Blood. 1, 3, 5 Showing Low Magnification X200 - 2, 4, 6 Showing High Magnification X3000. 1, 2 At Day 1- 3, 4 At Day 7 – 5, 6 At Day 30 - A, Represent Dentin, B, Material, C, Interface.

Ca/P ratios [expressed as mean and standard deviation] calculated for each subgroup after 1, 7 and 30 days. Taking into consideration the Ca to Phosphorus atomic ratio of normal stochiometric hydroxyapatite equals 1.67, the Ca/P ratios for MTA Angelus in blood and in DW, were higher than that of the normal stoichiometric Hydroxyapatite at day 1, day 7 and day 30.  There was no statistically significant difference between both materials at day 1 and day 30 but there was statistically significant difference at day 7 as presented in (Table 1 and 2).

The Ca/P ratios for Neoputty MTA in blood and in DW, was higher than that of the normal stoichiometric Hydroxyapatite at day 1. At day 7 the ratio got closer to the Ca/P ratio of normal Hydroxyapatite AT day 30 the ratio got lower to the Ca/P ratio of normal Hydroxyapatite. There was statistically significant difference between both materials at day 1 and day30 but there was no statistically significant difference between them at day 7 as presented in (Table 1 and 2).

Table 1: Mean And Standard Deviation Values of The Calculated Ca/P Ratios at Day 1, 7 And 30 For MTA Angelus And Neoputty in Blood.

Different upper-case letters in the same row indicate statistically significance difference. Different lower-case letters in the same column indicate statistically significance difference.

Table 2: Mean And Standard Deviation Values of The Calculated Ca/P Ratios at Day 1, 7 And 30 For MTA Angelus And Neoputty In DW.

Different upper-case letters in the same row indicate statistically significance difference. Different lower-case letters in the same column indicate statistically significance difference.

* Significant [p<0.05], ns; non-significant [p>0.05].

Endodontic iatrogenic errors can occur during root canal treatment such as root perforation, ledge formation, separated instruments as well as missed canals. These failures can be managed by nonsurgical or surgical procedures.

The healing process and prognosis of any iatrogenic error depend on several factors, one of which is the correct choice of repair material. For example, clinically any material used as root-end filling or as perforation repair filling is subjected to any kind of contamination such as blood or tissue fluids throughout its placement and setting. Therefore, it is important to choose the material least likely to be affected by the environmental field. The properties required adequate seal, biocompatible, bioactive, bacteriostatic, and radiopaque. It should be non-toxic, non-cariogenic, easy to place and non-staining especially when used to repair perforations [7].

Traditionally, there were materials used as root-end and perforation repair fillings such as amalgam, EBA, Cavit, and zinc oxide-eugenol which failed to fulfill the required properties such as they require moisture free environment during placement and setting, evoke inflammatory response at the repair site, and do not adapt well to the dentinal walls resulting in the microleakage [8].

Therefore, Bio ceramics were introduced into the endodontic field.  The potential advantages of bio ceramic materials in endodontics are related to their physico-chemical and biological properties. Bio ceramics are biocompatible, non-toxic, non-shrinking, and usually chemically stable within the biological environment [9]. A further advantage of these materials is their ability to form hydroxyapatite and ultimately create a bond between dentin and the material.

Following the introduction of bio ceramic materials into clinical endodontics, mineral trioxide aggregate [MTA] has become recognized as the gold-standard material for a variety of clinical situations and is perhaps closest to the ideal reparative material, due to its excellent physico-chemical and biological properties. These properties are high pH when unset, is biocompatible and bioactive when set, and provides an excellent seal over time. It has some disadvantages, requires mixing, resulting in considerable waste, is not easy to manipulate, and is difficult to remove from the root canal when set. Clinically, both gray and white MTA stain dentin, presumably due to the heavy metal content of the material [10,11]. Therefore, it is the material of choice for comparison when new materials are introduced in the market.

One of the recently introduced bio ceramic is premixed tricalcium silicate-based repair putty materials characterized by being hydrophilic, easy to use and handle well. The radiopacifying agent and proprietary organic liquid are premixed with a water-miscible carrier to improve handling properties [12].

Neo Putty MTA is one of the new materials in the market. It is premixed and optimized for more efficient handling and placement. Bioactive paste consisting of an extremely fine, inorganic powder of tricalcium/dicalcium silicate in an organic medium its firm, low-tack consistency and bioactivity make it the premier putty for use as retrograde filling materials and perforation repair in endodontics.

To evaluate the properties of such new material in relation to the ideal properties required, the study aimed to compare Neo putty MTA and MTA Angelus regarding marginal adaptation and bioactivity when used as retrograde and perforation repair fillings.

Regarding sample selection, anterior single rooted maxillary teeth were selected for root-end preparation due to large canal so easier to prepare orthograde and retrograde as well as to avoid any curvatures within the root which may have negative effect on the compaction of the materials. Mandibular posterior teeth were chosen for perforation preparation as they are wide mesiodistally so better accessibility, provide wide furcal area, so it was easier to perforate the furcal area as well as often tilted lingually and so the placement of access cavities as it relates the pulp chamber of the idealized occlusal anatomy may not always be relevant, which may lead to furcal perforations [8].

Regarding sample preparation, all samples of anterior and posterior teeth were prepared by one operator for standardization.

Rotary system was used during orthograde preparation of the samples as it is time saving and provide better results regarding centralization within the canal path and shaping of the canal [13]. M-Pro rotary system has been chosen as it is a controlled memory [CM] wire characterized by pre-bending ability, increased flexibility and cyclic fatigue resistance. It has convex triangular cross section to minimize contact with the canal wall and has rounded non cutting tip to avoid overcutting [14]. Irrigation with 5 ml 2.5% sodium hypochlorite between every file and EDTA as a final flush were done to mimic clinical situation. Sodium hypochlorite dissolves organic substances presented in the root canal system such as remaining pulp and necrotic tissue [15]. EDTA removes the smear layer so improve the bonding of resin sealer to root canal wall [16].

Resin based  sealer was used for obturation  because of its reduced solubility, good apical seal, its micro-retention to root canal dentin and better handling as it is prefilled syringe[17]. Cold lateral compaction was chosen due to controlled placement of gutta percha in the canal and easy to standardize [18]. Samples were left for 1 week for complete setting of the filling to avoid deterioration of the quality of the obturating materials during retro cavity preparation.

Regarding retrograde preparation, steps were performed to mimic clinical situations. Root-end resection was done using tapered with round stone [3 mm from the apex] to reduce dentinal tubule exposure, minimize the possibility of microleakage, maximize the remaining tooth tissue and to obtain clean cut resection [19]. Air-water coolant was used during preparation to minimize crack formation. Retro-cavity preparation of depth 3 mm was created using retro tip attached to ultrasonic device instead of round bur as it provides enhanced access to root-ends, more conservative cavities that follow the original path of the root and less risk of lateral perforation [20]. Root-end cavity of depth 3 mm performed to provide a room for retro-filling. These steps were performed under DOM to simulate the procedure of endodontic microsurgery and to ensure clean radicular dentinal walls with no remnants of gutta purcha that could negatively affect the formation of interfacial apatite and the bonding process as well [21-23].All samples were prepared under the same power, setting and minimal pressure for optimum standardization.

Concerning blood needed, there are several alternatives available either natural or substitute such as artificial synthetic blood. To mimic a clinical situation, whole fresh human blood [100 ml] was collected and transported using vacutainer tubes with sodium citrate as an anticoagulant to mimic the same blood components and fluid state [24][25]. Blood was stored at less than 5°C before use to avoid any bacterial contamination, then returned to 37°C at the day of the procedure via incubator to mimic body temperature.

To mimic clinical situation during endodontic microsurgery, a hemorrhagic situation was simulated in contaminated samples by inserting the tooth with the retro-filling material before complete setting in blood contained in Eppendorf tubes [26]. For non-contaminated samples, distilled water was used instead of saline as a control medium so that as much as possible no ions except those in HBSS would bond with the calcium ions in the material and at the same time provide a source of hydration for the setting reaction to take place [27].

To standardize the bulk of filling material releasing calcium ions that will bond with phosphorus ions in surrounding SBF to form apatite, dentin discs of the same dimensions were cut from the end of the resected and retro-filled roots.

Formation of surface apatite is an important requirement for material bonding with adjacent tooth structure and vital bone tissue. SBF allows simulation of the in-vivo conditions were apatite form by interaction of calcium from the material with phosphorus from the surrounding tissue fluid [28]. HBSS is a type of SBF solutions prepared to mimic the ionic concentrations of the human blood plasma [29]. It allows simulation of physiologic tissue fluids and thus the evaluation of in-vitro bioactivity and adaptability of Neoputty MTA as well as MTA Angelus [28,30,31]. It provides a source of phosphorus ions to bond with calcium ions from the filling material.

The first property tested MARGINAL ADAPTATION. Adaptation has been defined as the degree of proximity and interlocking of a filling material to the cavity wall [32]. SEM was used to evaluate the adaptability because it enables investigation of the repair filling adaptation to the radicular dentinal walls in a more accurate way than other techniques [33].

Regarding root-end filling, transverse discs from each subgroup [n=5] of the tested materials were scanned under SEM for measurement of gap distance along root repair material-dentin interface. The samples were examined under 200x to visualize the whole retro-filling material with the dentinal wall appearing all around it and 1500x magnification for better insight into the quality of the marginal adaptation of the tested material. More than one reading was collected and the largest value was recorded [34].

Generally, materials show different degrees of adaptation due to difference in composition of each material, different particle size and the environment at which material is set [35].

Results of adaptation regarding retro-filling revealed the presence of both gap-free regions and gap-containing region at the material-dentine interface, overall Neoputty in blood showed slightly better adaptability in comparison to blood contaminated MTA. Both materials showed similar adaptability when immersed in distilled water, statistically there was no significant difference between the tested materials at root end material– interface.

The good adaptation property of Neoputty MTA may be attributed to differences of ingredients between these two cements. In Neoputty MTA, the decreased size of its constituents and the increase of its powder surface may contribute to faster reaction in comparison to MTA Angelus [36].

Concerning contamination, our results stated that blood contaminated samples [Neoputty MTA and MTA Angelus] showed wider marginal gap distance than non-contaminated samples yet there is no statistically significant difference between both mediums. Blood contamination had limited effect on the property tested due to changes in the composition of both materials in comparison to traditional Portland cement.  For instance, calcium sulfate had been removed from MTA Angelus composition to reduce the setting time as revealed [37]. MTA Angelus is compatible with the human body, has no mutagenic properties, does not cause apoptosis, and has antimicrobial properties and acceptable cytotoxicity, better handling properties and faster setting time. With Neoputty MTA, smaller size of Neoputty particles may aid in enhanced adaptation at the cavity surface and filling interface. Our result was in full agreement with Hemat Mostafa Elsheikh et al who showed that blood contaminated ERRM showed higher mean gap value compared to noncontaminated ERRM yet that there was no statistically significant difference between both mediums [38]. Our results disagree with Salem Milani et al. who reported that exposure to blood during setting has a significant negative effect on marginal adaptation of MTA. Difference in results may be due to different method of sample preparation and method of evaluation [39].

The presence of gaps along the repair- dentin interface for the two materials may be attributed to the technique of preparing and testing the samples. It should be noted that SEM examination is a surface phenomenon and may not represent the adaptation of two surfaces in three dimensions. High vacuum evaporation can cause artifacts such as cracks in hard tissue samples and separation and lifting of the filling material from the surrounding tooth. In addition, there may be expansion or contraction of the tooth and/or filling material. Also, the plane of section and section grinding may influence the outcome of the study. Under SEM, we cannot be sure that the gaps were not artifacts produced by the technique [40,41].

The second property is BIOACTIVITY. Bioactivity as defined in literature, is the ability of the material to interact with the surrounding tissue fluids forming apatite like deposits and therefore bonding with the surrounding living tissues [42,43]. Initially this bond is mechanical; however, it is assumed that with time a diffusion-controlled reaction takes place between apatite layer and the dentinal structure allowing for the formation of a chemical bond [22]. Hence, Neoputty MTA is required to possess such an ability as it is used in close contact with living periapical tissue, therefore allowing stimulation of new bone formation as well as bonding with existing surrounding vital tissues. The ability of Neoputty MTA to dissolve in SBF releasing its cationic components and allowing the deposition of HA on its surface and surroundings was reported very few in literature [44]. Therefore, it was of interest to evaluate the effect of blood contamination on the bioactive properties of Neoputty MTA as well.

Assessment of the in vitro bioactivity is considered an important step preceding and simulating the in vivo behavior of bioactive materials [45]. Precipitation of apatite deposits or even calcium phosphate on biomaterials surface when immersed in SBF has often been considered evidence of their bioactive properties [22,46,47].

In the current study, SEM analysis combined with EDX analysis were used to study the surface morphology and chemical composition of both ion releasing materials with or without blood contamination and monitor the change that takes place in both aspects at different time intervals. SEM and EDX analysis were therefore performed on both groups after 1, 7 and 30-days immersion in HBSS [6,28,43].

SEM was used as a descriptive test for analysis of surface morphology. SEM images at low magnification [x200] and at high magnification [x1500 and x3000] were taken. Low magnification images allowed visualization of the whole retro-filling material with the dentinal wall appearing all around it. This image allowed the calculation of the percentage coverage of surface apatite spherulites in relation to the full surface area of the filling material using Image J software. This was done to compare the amount of surface apatite globules formed with and without blood contamination. It also allowed monitoring the increase in surface apatite coverage within each group over time.

Hydroxyapatite deposits were formed at the interface between the bioceramic material and the dentin. Visualization of the interfacial apatite deposits in both contaminated and non-contaminated conditions and at different time intervals within the same group by high magnification images taken at the Neoputty-dentin interface and MTA-dentine interface were done [22,23].

Both materials in contaminated and non-contaminated samples produced surface precipitates and crystal apatite formation after incubation in HBSS solution as well as at the material-dentin; the formation of an apatite layer was detected on the surface of both tested materials as well as at the material-dentin interface for both materials. This is consistent with findings by several studies [48,49].

Blood-contaminated MTA showed small, and irregular globules. With time, globules showed different morphological patterns [spherical and hexagonal] and increased in size. However, surface porosity increased with increased immersion time.  These results were consistent with the results in their examination of microstructure and chemical analysis of blood-contaminated MTA Angelus [50]. On the other hand, blood contaminated Neoputty MTA, showed irregular crystals on the first day but the globules increased in number and became more regular and spherical   day 7 and day 30. No studies were found in literature evaluating the effect of blood contamination on the bioactive properties of Neoputty MTA to compare.

EDX analysis is a semi-quantitative test, it measures the atomic percentage of different elements on the material surface [28]. In the current study EDX was used to measure the atomic percentage of calcium and phosphorus elements from three different areas along the Neoputty-dentin interface and MTA-dentine interface. The Ca/P atomic ratio was then calculated for each area by dividing the atomic percentage of calcium over that of phosphorus. Average was taken to provide Ca/P atomic ratio for each sample. Comparison of the calculated Ca/P ratio with that of normal stoichiometric hydroxyapatite which is equal to 1.67 was done. Thus, providing a method of tracking the amount of hydroxyapatite formation with or without blood contamination and at different time intervals within each group [6,28].

EDX and elemental analysis of blood-contaminated MTA, Ca/P ratios calculated from EDX analysis were highest at day 1 which decreased with increasing immersion time yet further from ratio of normal HA [1.67].  No significant difference between the three intervals. This could be due to deteriorated calcium ion release or reduced calcium ions bonding with phosphate ions in SBF.

EDX and elemental analysis of MTA in DW, Ca/P ratios calculated from EDX analysis were high at day 1 which increased at day 7 then decreased at day 30 yet further from ratio of normal HA [1.67].  No significant difference between the three intervals. These results are consistent with the results which stated that higher Ca/P ratios indicate calcium precipitation on the material’s surface and lower ratios indicate an incomplete setting reaction and formation of stoichiometric hydroxyapatite [51]. The study showed Ca/P ratios in the MTA cement samples during the first 24 hours, after 7 days, and after 14 days were obtained as 3.84, 8.33, and 2.74%. This can indicate Ca precipitation and ion exchange in the silicate groups during the first 7 days and formation of calcium phosphate after 14 days. No significant difference between the three intervals.

EDX and elemental analysis of blood contaminated Neoputty MTA, Ca/P ratios calculated from EDX analysis were highest at day 1 which decreased greatly with increasing immersion time reaching to a ratio lower than ratio of normal HA [1.67-1.5]. Ca/p ratio decreased from 1.8486 ±0.1049% at day 1 to reach 1.371 ±0.349% at day 30. These values were closely matched to stoichiometric hydroxyapatite [1.67] and human enamel and dentin surfaces [1.50 – 1.70]. There was statistically significant difference between day 1 and day30 but there was no statistically significant difference between them at day 7 and the other two days.

EDX and elemental analysis of Neoputty MTA in DW, Ca/P ratios calculated from EDX analysis were highest at day 1which decreased greatly with increasing immersion time reaching to a ratio lower than ratio of normal HA [1.67-1.5]. Ca/p ratio was decreased from 2.0589 ±0.0948% at day 1 to reach 1.156 ±0.197% at day 30. The ratio at day 1 and day 7 were more comparable to stoichiometric hydroxyapatite [1.67] and human enamel and dentin surfaces [1.50 – 1.70]. However, the ratio at day 30 was far from stoichiometric hydroxyapatite [1.67] and human enamel and dentin surfaces [1.50 – 1.70]. This may be due to the washing out of elements of Neoputty MTA with increased immersion time.

Blood contamination showed a slight effect on bioactivity of Neoputty. This may be due to the presence of the presence of gypsum and aluminum in a relatively high amount in comparison to MTA Angelus. It was reported that presence of gypsum and aluminium fasten the setting reaction of repair material [52].

Calcium to phosphorous ratio in Neoputty MTA was lower in all three intervals and in both mediums in comparison to MTA Angelus. According to the results, there was statistically significant difference between both material at the three intervals and in both mediums. This may be due to smaller particle size and difference in composition compared to MTA Angelus. Neoputty MTA shows similar composition to NeoMTA which has similar composition to the original MTA, but it is ground finer and contain tantalum oxide instead of bismuth oxide [53].

Regarding ion release, the highest values of calcium ions exhibited by MTA Angelus have been associated with its antimicrobial activity and mineralization potential. Since ion release depends on the material’s properties in terms of solubility, setting, and permeability to water [54], the lower release of calcium ions from NeoPutty compared to MTA Angelus could be explained by existing differences in their hydration processes and setting reactions [55]. These results are similar who compared new calcium silicate based cements [NeoPutty versus NeoMTA Plus and MTA] which stated that lower release of Ca2+ from NeoMTA Plus and NeoPutty compared to MTA Angelus could be explained by existing differences in their hydration processes and setting reactions [56].

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