**Recent Advance in Biomaterials, Clinical Dentistry and Dental Diseases 2.0**

Editors

**Naji Kharouf Davide Mancino Salvatore Sauro Louis Hardan**

Basel ' Beijing ' Wuhan ' Barcelona ' Belgrade ' Novi Sad ' Cluj ' Manchester

*Editors* Naji Kharouf Department of Biomaterials and Bioengineering University of Strasbourg Strasbourg France

Davide Mancino Department of Biomaterials and Bioengineering University of Strasbourg Strasbourg France

Salvatore Sauro Dental Biomaterials and Minimally Invasive Dentistry Universidad CEU-Cardenal Herrera Valencia Spain

Louis Hardan Department of Restorative Dentistry Saint-Joseph University Beirut Lebanon

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Bioengineering* (ISSN 2306-5354) (available at: www.mdpi.com/journal/bioengineering/special issues/A4YZ96DJ8R).

For citation purposes, cite each article independently as indicated on the article page online and as indicated below:

Lastname, A.A.; Lastname, B.B. Article Title. *Journal Name* **Year**, *Volume Number*, Page Range.

**ISBN 978-3-0365-9653-2 (Hbk) ISBN 978-3-0365-9652-5 (PDF) doi.org/10.3390/books978-3-0365-9652-5**

© 2023 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license. The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons Attribution-NonCommercial-NoDerivs (CC BY-NC-ND) license.

 Prof Kharouf has been working in dental biomaterials, endodontic treatment, coronal restorative techniques, bioactive molecules, microsurgical skeels, and dental morphology since 2018 (H-Index: 14, 2024). He has been a speaker at several international conferences. He has published, in collaboration with international researchers, more than 68 scientific papers in international peer-review journals with a high impact factor in the dental and biomaterials field. He has a Certificate in Oral Implantology, a Master's degree in Biomaterials for Health, a Microsurgical Diploma, an Esthetic of Smile Diploma, a PhD in Endodontics, a post-doc in Dental materials, and finally an H.D.R. in dental materials modified with graphene. He is a lecturer and monitor in several dental courses at Strasbourg University, and he also supervises Master's, PhD, and post-doc students.

## **Davide Mancino**

Professor Mancino graduated in Dentistry and Dental Prosthetics from the University of Palermo in 2001. In 2011, he successfully completed a Master's degree in Micro-Edodontics at the University of Turin. In 2019, he received a PhD in Biomaterials (chemistry and physics) from Strasbourg University. He is currently a Professor of Endodontics at the University of Strasbourg and has been a researcher at the Inserm Research Laboratory since 2014.

He is the director of the Diploma in Clinical and Surgical Microendodontics at the University of Strasbourg.

He is the author of several international publications in the field of endodontics, a speaker at national and international congresses, and a certified trainer in France, where he organizes several training courses in endodontics.

#### **Salvatore Sauro**

Dr Salvatore Sauro is currently a Professor in Dental Biomaterials and Minimally Invasive Dentistry at the "*Departamento de Odontolog´ıa, Facultad de Ciencias de la Salud, Universidad CEU-Cardenal Herrera*", coordinator of "Dental Research", and Director (Principal Investigator) of the research group "In Situ Dental Tissues Engineering and Minimally Invasive Therapeutic Adhesive Rehabilitation". He is also a Visiting Senior Lecturer at the Faculty of Dentistry, King's College London Dental Institute (KCLDI), Visiting Professor at the School of Dentistry, Moscow, Sechenov University of Moscow (Russia), Visiting Professor at the School of Dentistry, University of Federico II of Napoli, as well as University Aldo Moro, Bari. He is also an Honorary Professor at the Dental School, University of Hong Kong. Dr. Sauro has been working in dental biomaterials and preventive and minimally invasive dentistry research for more than 15 years (H-Index: 46 2024) and he has published, in collaboration with internationally renowned researchers, more than 150 articles in international peer-review journals with a high impact factor in the dental and biomaterials field, along with more several research abstracts and lectures at international conferences.

#### **Louis Hardan**

Louis Hardan is a full professor at the Department of Esthetic and Restorative Dentistry, School of Dentistry, Saint-Joseph University, Beirut, Lebanon, and owns a private practice in his hometown of Byblos. He is the former Head of the Restorative and Esthetic Department at Saint-Joseph University, Beirut, Lebanon (2013–2019), has been a Director of a Master's program since 2019, and a Coordinator of the Prosthetic and Esthetic Master's program since 2021. He graduated in Dentistry in 1989 and continued his post-doctoral education at Saint-Joseph University; he obtained a certificate for Basic Science in 1993, completed his specialization in Restorative and Esthetic Dentistry in 1995, and completed his PhD in Oral Biology and Materials in 2009. He was an active member of the Lebanese Dental Association (LDA) board (2006–2009) and was assigned to be the General Secretary of the LDA in 2009. He has been the Country Chairperson for Lebanon of the European Society of Cosmetic Dentistry (ESCD) since 2020, is ESCD certified, an ESCD board member, and has been the International Affairs Chairperson since 2021. He was the winner of the Academic Award of the ESCD during the Oscar Night Ceremony of the 20th ESCD Anniversary in 2023. He is the inventor of Smile Lite MDP (Smile Line, Switzerland), a device which takes high-quality dental pictures with a mobile phone (winner of the best of class technology award for 2017 in the USA and winner of the CEDE 2022 in Poland), and Posterior Misura (LM instrument), an instrument for direct posterior composites. Prof. Hardan has many publications in many indexed and high-impact-factor international journals, chapters in international dental books, and is the author of a book entitled *Protocols for Mobile Dental Photography with Auxiliary Lighting* (Quintessence Publishing, 2020) and has given several international lectures and courses on dental adhesion, esthetic and restorative dentistry, and mobile dental photography.

## **Preface**

The dental biomaterials field is developing very quickly. The dental market needs biomaterials which are biocompatible and bioactive. The bioactivity of dental material means that the material could ensure a biological effect or simulate this reaction. These bioactive biomaterials are used in all dental fields to enhance antibacterial activity and ameliorate the healing process.

> **Naji Kharouf, Davide Mancino, Salvatore Sauro, and Louis Hardan** *Editors*

## *Article* **Effectiveness of Two Endodontic Instruments in Calcium Silicate-Based Sealer Retreatment**

**Antoun Farrayeh 1 , Samar Akil 1 , Ammar Eid 1 , Valentina Macaluso 2 , Davide Mancino 3,4,5 , Youssef Haïkel 3,4,5 and Naji Kharouf 4,5,\***

**Abstract:** The objective of the present in vitro work was to investigate the effectiveness and time required for the removal of calcium silicate-based sealer using two rotary retreatment systems. Sixty extracted, single-canal, lower premolars were used. After obturation using the single-cone technique with calcium silicate-based sealer, samples were divided into four groups according to the technique of desobturation: Group 1 (G1): D-Race; Group 2 (G2): D-Race followed by the use of XP–Endo Finisher R; Group 3 (G3): Protaper Universal Retreatment; and Group 4 (G4): Protaper Universal Retreatment followed by the use of XP–Endo Finisher R. Cone beam computed tomography (CBCT) images were used to calculate the remaining filling materials at the middle and apical thirds. Times required to perform each method were recorded. Scanning electron microscopy (SEM) and digital microscopy were used to evaluate the remaining filling materials. Data were statistically analyzed using the *t*-test and one way ANOVA on ranks tests. No statistically significant difference was found between G1 and G3 after CBCT observations (*p* > 0.05). Xp-Endo Finisher R significantly increased the ability to remove materials regardless of the initially used retreatment system (*p* < 0.05). Statistically significant longer time was found in G3 and G4 compared to G1 and G2, respectively (*p* < 0.05), to reach the full working length. No retreatment system was able to totally remove the calcium silicate-based sealer from the root canal at the middle and apical thirds (*p* > 0.05). Digital microscopy demonstrated that the residual materials were the remaining sealers on the canal walls. SEM showed the mineral depositions of calcium silicate materials onto the canal walls and into the dentinal tubules. However, that calcium silicate materials provide mineral deposition into the dentinal tubules might indicate that the traditional irrigants could not be sufficient to remove calcium silicate-based materials from the root canal, and other agents should be used to make retreatment considerably easier.

**Keywords:** root canal retreatment; calcium silicate-based cement; ProTaper Universal Retreatment; D-Race

#### **1. Introduction**

Non-surgical endodontic retreatment is the first option after the failure of conventional endodontic treatment [1]. It leads to elimination of the microorganisms that are responsible for persistent infections [2,3]. Bacteria such as *Enterococcus faecalis* remain after primary endodontic treatment in areas that were unreachable by instrumentation and irrigation [4,5]. It is impossible to totally remove the filling material from the root canal during retreatment using conventional instruments and techniques due to their anatomical complexity, especially in oval canals [6,7]. The residual microorganisms and filling materials could affect the final retreatment outcome [8]. In addition, the main purpose of endodontic retreatment

**Citation:** Farrayeh, A.; Akil, S.; Eid, A.; Macaluso, V.; Mancino, D.; Haïkel, Y.; Kharouf, N. Effectiveness of Two Endodontic Instruments in Calcium Silicate-Based Sealer Retreatment. *Bioengineering* **2023**, *10*, 362. https://doi.org/10.3390/ bioengineering10030362

Academic Editors: Chengfei Zhang and Elena A. Jones

Received: 5 March 2023 Revised: 13 March 2023 Accepted: 14 March 2023 Published: 15 March 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

is to retrieve healthy periapical tissue. It aims to regain access to the apical region through total removal of filling materials [9].

Removal of gutta-percha can be accomplished using hand files, rotary and reciprocating systems, ultrasonic methods, lasers, and chemical solvents [10–14]. Mechanized protocols were proposed to be supplementary steps after the initial removal of filling materials, including the use of Self-Adjusting File (SAF) instruments (ReDent, Ra'anana, Israel) [15], sonic and ultrasonic tips [7], the XP-endo Finisher (FKG Dentaire, La Chauxde-Fonds, Switzerland), and the XP-endo Finisher R (FKG Dentaire, La Chaux-de-Fonds, Switzerland), with the main cause of removing the remaining materials from the root canal system.

The use of nickel-titanium (NiTi) rotary retreatment systems is an efficient and safe approach to remove filling materials during root canal retreatment [16]. In addition, several dental companies have introduced NiTi rotary retreatment systems on the dental market. The ProTaper Universal Retreatment system (Dentsply Maillefer, Ballaigues, Switzerland) includes three files—D1, D2 and D3—to remove filling materials from the root canal. These files have a convex triangular cross-section identical to the ProTaper shaping and finishing instruments [17].

The D-RaCe retreatment system (FKG Dentaire, La-Chaux-de Fonds, Switzerland) contains two instruments—DR1 (30/0.10) and DR2 (25/0.04)—for cleaning of the coronal third and to reach the working length (WL), respectively. Both instruments possess an active working tip to promote the initial penetration into the root canal filling material [18].

The XP-endo Finisher R (XPF-R) (FKG Dentaire, Switzerland) is a non-tapered file fabricated with NiTi MaxWire alloy (Martensite-Austenite Electropolish Flex). At temperatures less than 30 ◦C, this file is straight (martensitic phase "M-phase"), while the placement of this file inside the root canal at body temperature can transform it into an austenitic phase. In this phase, the file takes on a spoon shape in the last 10 mm, with a depth of roughly 1.5 mm. During entry of this file into the root canal, its austenite phase conversion and shape memory enhance its effectiveness in displacing and touching root-obturation materials. Therefore, this profile allows the instrument to attain irregular zones without modifying the original shape of the canal [1,8,19].

Various chemical compositions have been used in endodontic sealers to obturate the root canal with a gutta-percha point. These materials include zinc oxide-eugenol, gutta-percha flow, epoxy-resin, and calcium silicate cements [20]. Calcium silicate (CS) materials, called bioceramics, are considered a breakthrough in endodontic treatment due to their advantageous biocompatibility, antibacterial activity, appropriate filling ability, and good physicochemical properties. Their setting reaction could generate hydroxyapatite on their surfaces and cause tags in the dentinal tubules [21,22]. Different forms of calcium silicate sealers were introduced as powder-liquid and premixed products [23]. Premixed sealers demonstrated greater filling ability and easier application than conventional sealer. Ceraseal (Metabiomed, Korea) is a premixed calcium silicate sealer that demonstrated high obturation quality and appropriate biological, mechanical, and physicochemical properties [23]. The main disadvantage of calcium silicate materials is difficulty with their retreatment, especially at the apical third, and their removal requires both mechanical and chemical procedures [21].

Various dental companies have developed NiTi removal systems such as Mtwo retreatment (VDW, Munich, Germany), R-Endo, and Remover (Micro-Mega, Besançon, France). Hassan et al. studied the retreatment efficiency of R-endo and Xp-endo shaper files in root canals obturated using a bioceramic root canal sealer. The results showed that Xp-endo shaper files were significantly more effective in removing obturation materials. However, neither system totally removed the remaining materials from the coronal, middle, and apical thirds [24]. Donnermeyer et al. studied the retreatability of three calcium silicate sealers and one epoxy resin sealer with four different root canal instruments. They reported that engine-driven NiTi instruments were better suited to remove root canal fillings than stainless steel Hedström files [25].

Until now, there has been no validated protocol for the desobturation of calcium silicate material from the root canal in the literature; thus, research studies are still needed to enhance the efficacy of instruments and solvents in bioceramic retreatment.

The objective of this study was to evaluate the effectiveness of two retreatment instruments with or without a supplementary activation step in the removal of calcium silicate-based sealer. The null hypothesis was that there is no difference between the different instruments and the supplementary cleaning step regarding the quality of calcium silicate retreatment.

## **2. Materials and Methods**

#### *2.1. Teeth Preparation*

After approval by the ethics committee of Damascus University, Damascus, Syria (protocol n. 3215-2020), 63 extracted single canal lower premolars without caries and previous endodontic treatment were selected for the present study. The exclusion criteria were teeth with resorption, incomplete apices, severe curvatures, and cracks. The canal curvature was measured as described in a previous study [26]; thus, teeth with a maximum canal curvature of 15◦ were selected. Root surfaces were cleaned, and all soft tissue and calculus were removed mechanically with periodontal curettes. Afterwards, the teeth were rinsed and then kept in 0.9% NaCl at 4 ◦C.

After preparation of the access cavity using diamond burs and ultrasonic tips, a size 10 stainless steel K-file (Mani, Takenzawa, Japan) was inserted to reach the apical foramen. All endodontic steps were performed under 4.5× magnification using Q-Optics Loupes (Q-Optics, Duncanville, TX, USA). To standardize the samples, all tooth crowns were sectioned to reach a root length of 17 mm and a working length (WL) of 16 mm. The same operator prepared all canals using ProTaper Next instruments (Dentsply Sirona, Konstanz, Germany) up to an X2 file at the WL with an electric motor (VDW, Munich, Germany) at a speed of 300 rpm and 3 Ncm of torque. After the use of each instrument, the canals were irrigated with 1 mL of 2.5% NaOCl using a 30-gauge needle. After shaping procedures, the smear layer was eliminated using 5 mL of 17% EDTA and 5 mL of 2.5% NaOCl for final irrigation, and then paper points were used to dry the canal. The single-cone technique was used to obturate the root canal with calcium silicate-based sealer (Ceraseal, Metabiomed, Cheongju, Republic of Korea). The intracanal tip was inserted into the coronal third of the canal, and the sealer was dispensed into the canal. The gutta-percha cone was then painted with the calcium silicate material and slowly inserted into the canal to the appropriate length. Finally, to remove the extra gutta-percha, a hot plugger was used to remove it. The access cavities were filled with a temporary filling material (MD-Temp, Metabiomed, Republic of Korea). The samples were then stored in phosphate-buffered saline (PBS10×, Dominique Dutscher, Bernolsheim, France) for 28 days to insure an appropriate setting of the filling material.

### *2.2. Non-surgical Root Canal Retreatment*

After the storage period, coronal access was performed using size 3 and 2 files at 2000 rpm with Peeso drills (Mani, Takenzawa, Japan). The samples were randomly divided into four equal groups (n = 15) as follows:

7- to 8-mm lengthwise movements up to the WL in a brushing action against the root canal walls. canal with no rotation. Subsequently, the instrument was activated for 1 min using slow and gentle 7‐ to 8‐mm lengthwise movements up to the WL in a brushing action against the root canal walls.

• Group 3 (G3): ProTaper Universal Retreatment files (Figure 1) were used at a speed of 300 rpm with 3 Ncm of torque. A ProTaper D1 file (30/0.09) was used to prepare the coronal third of the canal. At the middle and apical thirds, D2 (25/0.08) and D3 (20/0.07) files were used to remove the filling materials. Group 3 (G3): ProTaper Universal Retreatment files (Figure 1) were used at a speed of 300 rpm with 3 Ncm of torque. A ProTaper D1 file (30/0.09) was used to prepare the coronal third of the canal. At the middle and apical thirds, D2 (25/0.08) and D3 (20/0.07) files were used to remove the filling materials.

 Group 2 (G2): the same procedure as in G1 was used with an additional step. The XP–Endo Finisher R file (XPF‐R) (Figure 1) was used as a supplementary file following the initial retreatment procedures. The XPF‐R file was used following the manufacturer's instructions at a torque of 1 Ncm and a speed of 800 rpm. A contra angle handpiece was used with the instrument. The XPF‐R file was placed into the

*Bioengineering* **2023**, *10*, x FOR PEER REVIEW 4 of 11

• Group 4 (G4): the same procedure as in G3 was used with an additional step. The XP– Endo Finisher R file (XPF-E) (Figure 1) was applied as a supplementary file according the retreatment procedures. Group 4 (G4): the same procedure as in G3 was used with an additional step. The XP–Endo Finisher R file (XPF‐E) (Figure 1) was applied as a supplementary file according the retreatment procedures.

**Figure 1.** SEM images of the tips of (**a**–**c**) the ProTaper Universal Retreatment D1, D2 and D3, (**d**,**e**) the D‐Race DR1 and DR2; and (**f**) the XP–Endo Finisher R file. **Figure 1.** SEM images of the tips of (**a**–**c**) the ProTaper Universal Retreatment D1, D2 and D3, (**d**,**e**) the D-Race DR1 and DR2; and (**f**) the XP–Endo Finisher R file.

During instrumentation procedures, irrigation was performed with 2 mL of 2.5% NaOCl. All procedures were performed in an incubator at 37 °C. All steps were completed by the same operator. All errors during treatment, including instrument fracture, canal ledges, and blockages, were recorded. In case of a fractured instrument, the tooth was discarded and replaced with another one (three teeth were replaced due to procedural errors). Final irrigation was performed using 5 mL of 17% EDTA and 5 mL of 2.5% NaOCl. After the final irrigation step, the canals were dried with paper points. Two procedural times were recorded to obtain the total working time; including the time needed to attain the WL (T1) and the time needed to remove the gutta‐percha (T2). The retreatment procedure was considered complete once the instrument flutes or the irrigation solution had no more residue from the calcium silicate‐based sealer or gutta percha material [27]. During instrumentation procedures, irrigation was performed with 2 mL of 2.5% NaOCl. All procedures were performed in an incubator at 37 ◦C. All steps were completed by the same operator. All errors during treatment, including instrument fracture, canal ledges, and blockages, were recorded. In case of a fractured instrument, the tooth was discarded and replaced with another one (three teeth were replaced due to procedural errors). Final irrigation was performed using 5 mL of 17% EDTA and 5 mL of 2.5% NaOCl. After the final irrigation step, the canals were dried with paper points. Two procedural times were recorded to obtain the total working time; including the time needed to attain the WL (T1) and the time needed to remove the gutta-percha (T2). The retreatment procedure was considered complete once the instrument flutes or the irrigation solution had no more residue from the calcium silicate-based sealer or gutta percha material [27].

#### *2.3. Remaining Filling Materials Observations 2.3. Remaining Filling Materials Observations*

#### 2.3.1. Cone Beam Computed Tomography (CBCT) and Micro*‐*computed Tomography 2.3.1. Cone Beam Computed Tomography (CBCT) and Micro-computed Tomography (µCt)

(μCt) CBCT scans were obtained using a PaX‐i3D Green unit (Vatech, Hwaseong‐si, Republic of Korea). The samples were exposed to 90 kV and 10.2 mA with an FOV of 5 × 5 cm and an isotropic resolution of 0.1 mm, with 12.57 s of exposure time. The artifacts created by the radiopaque root fillings were eliminated using inbuilt software. The images were evaluated in cross‐sections plans, and the percentages of remaining materials at the apical and middle thirds were calculated using the inbuilt software. Finally, the CBCT CBCT scans were obtained using a PaX-i3D Green unit (Vatech, Hwaseong-si, Republic of Korea). The samples were exposed to 90 kV and 10.2 mA with an FOV of 5 × 5 cm and an isotropic resolution of 0.1 mm, with 12.57 s of exposure time. The artifacts created by the radiopaque root fillings were eliminated using inbuilt software. The images were evaluated in cross-sections plans, and the percentages of remaining materials at the apical and middle thirds were calculated using the inbuilt software. Finally, the CBCT cross-sections were analyzed, and scores were obtained for all CBCT images by two observers.

After the CBCT observations, the most meaningful sample from each group was analyzed using µCT (IRIS, Inviscan, http://www.inviscan.fr/product\_iris\_pet\_ct.html accessed date: 15 December 2022) to show and evaluate the void percentages in them. The acquisition settings were 2000 projection (60 × 60 × 60 µm 3 voxel size) and 80 kVp. The images were visualized and manipulated using the Mimics Innovation Suite, version 24 (Materialise, Louvain, Belgium).

#### 2.3.2. Digital Microscopy

After CBCT observations, the samples were prepared by creating two shallow longitudinal grooves in the buccolingual direction by a diamond bur. The grooves were created following the canal morphology and curvature. These grooves did not enter into the canal space. Each sample was split by a chisel and mallet to investigate the internal walls [28]. The internal walls of the root canal for each sample were investigated using a digital microscope (KEYENCE, Osaka, Japan) at 100× magnification to analyze the nature of the residual filling materials (gutta-percha and/or sealer), which could not be observed through CBCT analysis.

#### 2.3.3. Scanning Electron Microscopy (SEM)

After digital macroscopy observations, the samples were dried in a graded series of ethanol (50, 70, 95, and 100%) for 10 min each. Finally, these samples were mounted on SEM stubs sputter-coated with gold–palladium (20/80) using a Hummer JR sputtering device (Technics, San Jose, California, USA). The samples were observed at different magnifications (100×−20.000×) with a working distance of 10 mm and a 10-kV acceleration voltage of the electrons through a scanning electron microscope (SEM, Quanta 250 FEG scanning electron microscope, FEI Company, Eindhoven, the Netherlands) [29].

#### *2.4. Statistical Analysis*

Statistical analysis was performed using SPSS software (version 17, SPSS, Chicago, IL, USA). The normality was verified with the Shapiro–Wilk test. However, when the normality test was not passed, an analysis of the variance on ranks, along with a multiple comparison procedure (Tukey's test), was performed to determine whether significant differences existed in the remaining material values between the different retreatments. The lapse to complete the retreatment of all groups was determined using the *t*-test, and the chi-square test was used to determine whether significant differences existed in the re-establishing of the working length. In all tests, a statistical significance level of α = 0.05 was adopted.

#### **3. Results**

#### *3.1. Ability to Reach WL and Required Time*

The WL was re-established in 93.34%, 86.67%, 80%, and 86.67% of teeth for G1, G2, G3, and G4 respectively (*p* > 0.05) (Figure 2). Statistically longer time values were recorded for the groups in which Protaper Universal Retreatment was used compared to the groups in which D-Race was used regardless of the use of XP–Endo Finisher R (Table 1). *Bioengineering* **2023**, *10*, x FOR PEER REVIEW 6 of 11

**Figure 2.** μCt images of the samples after retreatment procedures. (**a**,**b**) G1: D‐Race files; (**c**,**d**) G2: D‐Race and XP–Endo Finisher R; (**e**,**f**) G3: ProTaper Universal Retreatment; (**g**,**h**) G4: ProTaper Universal Retreatment and XP–Endo Finisher R file. **Figure 2.** µCt images of the samples after retreatment procedures. (**a**,**b**) G1: D-Race files; (**c**,**d**) G2: D-Race and XP–Endo Finisher R; (**e**,**f**) G3: ProTaper Universal Retreatment; (**g**,**h**) G4: ProTaper Universal Retreatment and XP–Endo Finisher R file.

**Table 1.** Time required to re‐establish the working length in the different groups. Different superscripted letters (a–d) indicate significant differences between the different groups (*p* < 0.05). Group 1 (G1): D‐Race; Group 2 (G2): D‐Race followed by the use of XP–Endo Finisher R; Group 3 (G3): Protaper Universal Retreatment; and Group 4 (G4): Protaper Universal Retreatment followed

**G1 G2 G3 G4**

Time (s) 214 ± 13 a 269 ± 28 b 304 ± 34 c 362 ± 35 d a < c and b < d

No significant differences were found for the remaining material percentages between G1 and G3 (*p* > 0.05). The use of XP–Endo Finisher R (G2 and G4) demonstrated statistically greater efficacy in the removal of remaining materials than the other groups

**Table 2.** Residual material percentages after all retreatment techniques. Different superscripted letters (a–d) indicate significant differences between the different groups (*p* < 0.05). Group 1 (G1): D‐Race; Group 2 (G2): D‐Race followed by the use of XP–Endo Finisher R; Group 3 (G3): Protaper Universal Retreatment; and Group 4 (G4) Protaper Universal Retreatment followed by the use of

**G1 G2 G3 G4**

The use of CBCT did not reveal the nature of the remaining materials; thus, digital microscopy was used to investigate this finding. However, the most common residual materials were the calcium silicate sealers with small amounts of gutta‐percha residue, as

Middle (%) 12 ± 6 a 8.4 ± 4.1 b 16.4 ± 11.1 a 8.8 ± 3.8 b b < a Apical (%) 14 ± 7 a 4.5 ± 0.6 b 15.4 ± 10.6 a 6.5 ± 6.4 b b < a

**Statistical Analysis (***p* **< 0.05)**

**Statistical Analysis (***p* **< 0.05)**

by the use of XP–Endo Finisher R.

*3.2. CBCT and μCt Evaluations*

XP–Endo Finisher R.

shown in Figure 3.

*3.3. Digital Microscope Observations*

(G1 and G3) (*p* < 0.05) (Figure 2 and Table 2).

**Table 1.** Time required to re-establish the working length in the different groups. Different superscripted letters (a–d) indicate significant differences between the different groups (*p* < 0.05). Group 1 (G1): D-Race; Group 2 (G2): D-Race followed by the use of XP–Endo Finisher R; Group 3 (G3): Protaper Universal Retreatment; and Group 4 (G4): Protaper Universal Retreatment followed by the use of XP–Endo Finisher R.

#### *3.2. CBCT and µCt Evaluations*

No significant differences were found for the remaining material percentages between G1 and G3 (*p* > 0.05). The use of XP–Endo Finisher R (G2 and G4) demonstrated statistically greater efficacy in the removal of remaining materials than the other groups (G1 and G3) (*p* < 0.05) (Figure 2 and Table 2).

**Table 2.** Residual material percentages after all retreatment techniques. Different superscripted letters (a–d) indicate significant differences between the different groups (*p* < 0.05). Group 1 (G1): D-Race; Group 2 (G2): D-Race followed by the use of XP–Endo Finisher R; Group 3 (G3): Protaper Universal Retreatment; and Group 4 (G4) Protaper Universal Retreatment followed by the use of XP–Endo Finisher R.

#### *3.3. Digital Microscope Observations*

The use of CBCT did not reveal the nature of the remaining materials; thus, digital microscopy was used to investigate this finding. However, the most common residual materials were the calcium silicate sealers with small amounts of gutta-percha residue, as shown in Figure 3. *Bioengineering* **2023**, *10*, x FOR PEER REVIEW 7 of 11

**Figure 3.** Digital microscope images demonstrate the effectiveness of the retreatment technique and the nature of the residual materials in (**a**,**b**) the root canal at (**c**,**d**) the middle and (**e**,**f**) apical thirds (black arrows). **Figure 3.** Digital microscope images demonstrate the effectiveness of the retreatment technique and the nature of the residual materials in (**a**,**b**) the root canal at (**c**,**d**) the middle and (**e**,**f**) apical thirds (black arrows).

#### *3.4. SEM Observations 3.4. SEM Observations*

mineral components (white arrows).

**4. Discussion**

SEM was used to investigate the quality of smear layer and filling material removal after instrumentation and final irrigation using EDTA and NaOCl. Most dentinal tubules SEM was used to investigate the quality of smear layer and filling material removal after instrumentation and final irrigation using EDTA and NaOCl. Most dentinal tubules in

in all of the groups at the middle and apical thirds were not opened after the final irrigation (Figure 4a,b), while only a few zones in G2 and G4 demonstrated partially

material, and this layer was not eliminated using the different protocols (Figure 4e). This layer covers the entrance of the dentinal tubules (Figure 4e) and prevented complete cleaning of the root canal system. After the immersion period, calcium silicate‐based materials ensure the mineral deposition in the dentinal tubules, which could totally close them (Figure 4f). Therefore, the cleaning and opening of the dentinal tubules in root canals

**Figure 4.** SEM images demonstrate (**a**,**b**) closed dentinal tubules; (**c**,**d**) partially opened dentinal tubules; (**e**) remaining calcium silicate sealer on the root canal walls; (**f**) dentinal tubules filled with

Root canal treatments usually fail due to persistent periapical disorders after treatment [30]. Moreover, coronal leakage, periodical caries, necrotic tissue, tooth cracks and fractures, and bacterial biofilms could lead to treatment failure [31]. In addition, these etiological factors should be eliminated to establish adequate periapical recovery; thus,

could be very difficult after the use of bioceramic material.

all of the groups at the middle and apical thirds were not opened after the final irrigation (Figure 4a,b), while only a few zones in G2 and G4 demonstrated partially opened dentinal tubules (Figure 4c,d). Some zones were covered totally with sealer material, and this layer was not eliminated using the different protocols (Figure 4e). This layer covers the entrance of the dentinal tubules (Figure 4e) and prevented complete cleaning of the root canal system. After the immersion period, calcium silicate-based materials ensure the mineral deposition in the dentinal tubules, which could totally close them (Figure 4f). Therefore, the cleaning and opening of the dentinal tubules in root canals could be very difficult after the use of bioceramic material. in all of the groups at the middle and apical thirds were not opened after the final irrigation (Figure 4a,b), while only a few zones in G2 and G4 demonstrated partially opened dentinal tubules (Figure 4c,d). Some zones were covered totally with sealer material, and this layer was not eliminated using the different protocols (Figure 4e). This layer covers the entrance of the dentinal tubules (Figure 4e) and prevented complete cleaning of the root canal system. After the immersion period, calcium silicate‐based materials ensure the mineral deposition in the dentinal tubules, which could totally close them (Figure 4f). Therefore, the cleaning and opening of the dentinal tubules in root canals could be very difficult after the use of bioceramic material.

**Figure 3.** Digital microscope images demonstrate the effectiveness of the retreatment technique and the nature of the residual materials in (**a**,**b**) the root canal at (**c**,**d**) the middle and (**e**,**f**) apical thirds

SEM was used to investigate the quality of smear layer and filling material removal after instrumentation and final irrigation using EDTA and NaOCl. Most dentinal tubules

*Bioengineering* **2023**, *10*, x FOR PEER REVIEW 7 of 11

**Figure 4.** SEM images demonstrate (**a**,**b**) closed dentinal tubules; (**c**,**d**) partially opened dentinal tubules; (**e**) remaining calcium silicate sealer on the root canal walls; (**f**) dentinal tubules filled with mineral components (white arrows). **Figure 4.** SEM images demonstrate (**a**,**b**) closed dentinal tubules; (**c**,**d**) partially opened dentinal tubules; (**e**) remaining calcium silicate sealer on the root canal walls; (**f**) dentinal tubules filled with mineral components (white arrows).

#### **4. Discussion 4. Discussion**

(black arrows).

*3.4. SEM Observations*

Root canal treatments usually fail due to persistent periapical disorders after treatment [30]. Moreover, coronal leakage, periodical caries, necrotic tissue, tooth cracks and fractures, and bacterial biofilms could lead to treatment failure [31]. In addition, these etiological factors should be eliminated to establish adequate periapical recovery; thus, Root canal treatments usually fail due to persistent periapical disorders after treatment [30]. Moreover, coronal leakage, periodical caries, necrotic tissue, tooth cracks and fractures, and bacterial biofilms could lead to treatment failure [31]. In addition, these etiological factors should be eliminated to establish adequate periapical recovery; thus, establishing patency and working length in retreatment cases could significantly provide for better periapical healing outcomes [32]. However, retreatment procedures are not always possible due to several factors, including root canal anatomy and resistant filling materials [21,27,33,34].

Currently, calcium silicate materials are used frequently in endodontic practice due to their great biological, mechanical, and physicochemical properties [35,36]. Ceraseal sealer, which was used in the present study, is one of these calcium silicate endodontic products that has demonstrated an alkaline pH and release of Ca2+ ions [23]. Therefore, these properties might enhance and provide for the mineralization process and the formation of hydroxyapatite tags in the dentinal tubules [21,23,29,35].

In the present in vitro study, SEM observations, digital analysis, and CBCT investigations demonstrated that there is no retreatment system that could totally eliminate the remaining filling materials and completely open the dentinal tubules. The results demonstrated that there are no statistically significant differences between the two rotary systems (G1 and G3) regarding the efficacy of filling material removal from the root canal at the middle and apical thirds (*p* > 0.05), while the supplementary cleaning step (XPF-R in G2 and G4) enhanced statistically the efficacy of retreatment (*p* < 0.05). These findings suggest that supplementary steps are able to decrease the amount of remaining filling materials. Moreover, the tested techniques in this study showed that they cannot provide for complete removal of the filling materials from the root canal system. Therefore the null hypothesis must be partially rejected.

The findings of the present work demonstrated that retreatment with the D-Race system required less time compared to ProTaper Universal Retreatment files, which might be attributed to the greater efficacy of D-Race files in removing gutta-percha, as well as two instruments being used in the D-Race system compared to three instruments being used in the ProTaper Universal Retreatment. Regarding the amount of remaining filling materials after retreatment, no statistically significant differences were found between the two systems: D-RaCe and ProTaper rotary instruments (*p* > 0.05). In contrast, the addition of a supplementary cleaning step using XPF-R (G2 and G4) demonstrated statistically higher cleaning efficiency results in all root segments compared to the two initial retreatment systems without this supplementary step (*p* < 0.05). This finding could be attributed to XPF-R metallurgy. The manufacture and improvement of these files depend on the shape-memory of its alloy. The file is straight in its M-phase, which is composed in cooled conditions. When the file is subjected to body temperature, its shape transforms into the A-phase. The shape in this phase permits the file to clean the zones that are otherwise inaccessible with regular instruments [37,38]. Therefore, novel instruments, systems, and supplementary steps and techniques are required for remaining filling material removal, especially in ovalshaped root canals, as well as in filled canals with bioceramic materials [28,39,40]. Removal of root filling materials during endodontic retreatment was previously evaluated using stereomicroscopy, scanning electron microscopy, digital microscopy, and x-ray radiography. Some studies used CBCT for the assessment of remaining filling materials. CBCT provides three-dimensional (3D) images on the axial, coronal, and axial planes, and it is capable of visualizing the root canal system and analyzing the quality of cleaning in extracted teeth [10,41].

Digital images demonstrated that most remaining materials in the root canals after retreatment procedures are the residues of calcium silicate sealers, which are stuck on the dentinal walls (Figure 3). Kaloustian et al. [28] used a digital microscope to investigate the amount of the remaining materials after different retreatment methods. Previous studies have reported that gutta-percha material could be eliminated mechanically using instruments and chemically by the application of different solvents [10–14]. In contrast, calcium silicate sealers and cements are very difficult in retreatment due to their high compressive strength, appropriate interactions with dental tissues, and mineralization reactions [20,21,23,29,35,36,42]. A previous study demonstrated that the use of special acids, such as formic acid, is the best route to retreat teeth filled with calcium silicate-based sealer [43].

Various studies have demonstrated that the use of EDTA and NaOCl as irrigants could provide for smear layer removal in permanent and primary teeth [44,45]. In contrast, SEM images in the present study rarely showed dentinal zones with opened dentinal tubules and an eliminated smear layer. Most observations showed debris in the root canal, closed dentinal tubules, remaining calcium silicate particles, and their mineralogical reactions in the dentinal tubules, which are near the principal root canal. In accordance, a previous study demonstrated that the use of calcium silicate-based material by orthograde obturation of the root canals could provide favorable conditions for bacterial entombment by intratubular mineralization [22]. Therefore, plugging the dentinal tubules with calcium silicate materials and/or their mineralogical reactions could close totally the dentinal tubules; thus, special solvent should be used to open them. In contrast, this plugging could enclose the remaining bacteria in the dentinal tubules and kill these microorganisms over time [22].

Finally, the findings of the present study are in accordance with previous studies reporting that retreatment with calcium silicate materials is considered a big challenge in endodontic treatment [21,34,46,47]. These materials could be considered to result in real blockage of the apical foramen, preventing patency of the root canal and the reestablishing of the WL, which are considered to be important outcomes. Further research is needed to evaluate the effects of different acids and solvents accompanying mechanical process and activation in retreatment with calcium silicate materials in straight and curved root canals. Moreover, the mineralization process of the different calcium silicate materials and

the time needed to totally close the dentinal tubules will be further investigated. All the samples were observed using CBCT, while fewer samples were analyzed with µCt, which was used to obtain a higher resolution of data that were process using Mimics Innovation Suite software, version 24 (Materialise, Louvain, Belgium). This fact could be considered a limitation of the present work.

#### **5. Conclusions**

Within the limitations of the present in vitro study, none of the used systems was able to completely remove the CS material from the root canal. No significant difference was found between the two retreatment systems. Supplementary cleaning steps are effective tools to enhance the cleanliness of the root canal after a retreatment procedure. The traditional irrigants (EDTA and NaOCl) could not completely the open dentinal tubules after an obturation procedure using CS materials.

**Author Contributions:** Conceptualization, S.A.; methodology, A.F.; software, A.F.; validation, S.A.; formal analysis, A.F.; investigation, A.E.; resources, A.F.; data curation, A.F.; writing—original draft preparation, A.F.; writing—review and editing, N.K., D.M., V.M., A.E. and A.F.; visualization, N.K. and Y.H.; supervision, S.A.; project administration, S.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** This in vitro study was conducted according to the guidelines of the Declaration of Helsinki and approved by the ethics committee of Damascus University (protocol no. 3215-2020).

**Informed Consent Statement:** Each patient was informed of the study's procedure and objectives, and his or her non-opposition to the use of his or her tooth was recorded in his or her chart by the oral surgeon.

**Data Availability Statement:** The data presented in this study are available on request from the first author.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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## *Article* **The Influence of Carious Lesion and Bleeding Time on the Success of Partial Pulpotomy in Permanent Molars with Irreversible Pulpitis: A Prospective Study**

**Rami Zen Aldeen 1 , Ossama Aljabban 1 , Ahmad Almanadili 2 , Saleh Alkurdi 3 , Ammar Eid 1 , Davide Mancino 4,5,6 , Youssef Haikel 4,5,6,\* and Naji Kharouf 4,5,\***

**Abstract:** This prospective study aimed to evaluate the success rate of partial pulpotomy using mineral trioxide aggregate (MTA), in permanent molars with symptomatic irreversible pulpitis. Moreover, this study aimed to investigate the effect of carious lesion depth and activity and bleeding time on the outcome of partial pulpotomy. Forty permanent molars with deep and extremely deep carious lesions clinically diagnosed with symptomatic irreversible pulpitis were included. The status of the carious lesion was evaluated clinically and radiographically to determine its activity (rapidly or slowly progressing) and depth (deep or extremely deep). A partial pulpotomy was performed and MTA was used. Clinical and radiographic analysis were performed at 3, 6 and 12 months. Chi-square analysis and Fisher's exact test were used. Scanning electron microscope and energy dispersive X-rays were used to investigate the crystalline structures and their chemical composition onto MTA surfaces after immersion in several conditions. The partial pulpotomy was 88.9% successful, with no significant difference in outcome between deep and extremely deep carious lesions (*p* = 0.22) or between rapidly and slowly progressing lesions (*p* = 0.18). Nevertheless, all failed cases were associated with rapidly progressing lesions and extremely deep lesions. All failures occurred when the bleeding time was more than 3 min (*p* = 0.10). Different crystalline structures were detected on MTA surfaces, with higher calcium percentages in PBS conditions. Within the limitations of the present study, favorable results demonstrated that MTA might be recommended as a suitable agent for partial pulpotomy in permanent molars with irreversible pulpitis. The depth and activity of the carious lesion as well as the bleeding time are important factors in the success of partial pulpotomy treatment. The prolonged bleeding time and the extremely deep rapidly progressing caries could be related with the failure cases in partial pulpotomy treatment of irreversible pulpitis.

**Keywords:** carious lesion activity; irreversible pulpitis; partial pulpotomy; vital pulp therapy; mineral trioxide aggregate

## **1. Introduction**

Numerous chemical, thermal, microbiological and traumatic factors can cause inflammation in the dental pulp [1]. Traditionally, in the reversible stage, the tooth could be

**Citation:** Aldeen, R.Z.; Aljabban, O.; Almanadili, A.; Alkurdi, S.; Eid, A.; Mancino, D.; Haikel, Y.; Kharouf, N. The Influence of Carious Lesion and Bleeding Time on the Success of Partial Pulpotomy in Permanent Molars with Irreversible Pulpitis: A Prospective Study. *Bioengineering* **2023**, *10*, 700. https://doi.org/ 10.3390/bioengineering10060700

Academic Editors: Chengfei Zhang and Elena A. Jones

Received: 22 May 2023 Revised: 3 June 2023 Accepted: 6 June 2023 Published: 8 June 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

healed by the elimination of the stimulus whilst, in the irreversible stage, the pulp tissue is so damaged that it is impossible to be recovered, and thus, root canal treatment (RCT) is recommended [2].

Several clinical signs could be considered as indicators of irreversible pulpits, such as severe pre-operative pain that is spontaneous or long-lasting and is accompanied by a deep carious lesion [2]. However, several histological studies demonstrated that inflammatory changes in pulp tissues are limited to the coronal region adjacent to the carious lesion, while the residual parts of the coronal pulp tissue remain normal and uninflamed [3,4]. These histological findings, along with recent improvements in the understanding of the pulp reparative processes [5], as well as developments in the field of bioactive materials, such as calcium silicate-based materials and their biological effects [6–8], have made it possible to change the concept of root canal treatment and to adopt more conservative treatment strategies such as vital pulp therapy (VPT) for the management of pulpitis in mature permanent teeth [9,10].

VPT is a minimally invasive, biologically based treatment aimed at preserving the vitality of the entire dental pulp or a portion of it by sealing the pulp wound with bioactive material after removing the infected tissue [11,12]. VPT represents a group of therapeutic strategies, including indirect pulp capping, direct pulp capping, partial pulpotomy, and full pulpotomy [13]. Several clinical studies over the last two decades have demonstrated that full and partial pulpotomy could be a promising biologically based treatment alternative to RCT for the management of carious mature teeth, even with symptoms of irreversible pulpitis [14–16]. Nevertheless, the medical literature is divided over whether or not a partial pulpotomy could be an indication for irreversible pulpitis management in mature permanent teeth, and more clinical studies are still required [17,18].

The success of the VPT is dependent upon an accurate assessment of the inflammatory status of the dental pulp. Due to the limited link between clinical signs and symptoms and the histological state of the pulp, this assessment is, unfortunately, more predictive than accurate in clinical practice [4,19]. Therefore, to limit the predictive diagnosis in teeth presenting with a carious lesion, it may be essential to pay attention to additional criteria such as the depth of penetration and activity state of the carious lesion [20]. It has suggested that the depth and activity of the carious lesion may be an important clinical measure for the regenerative potential of the pulp tissue and the degree of pulp inflammation and may influence the outcome of pulp exposure treatment [21]. Interestingly, data on carious lesion penetration and activity concerning VPT, including partial or full pulpotomy, are seldom described in the literature. In addition, the relation between depth and activity of carious lesion, and bleeding time with the success of partial pulpotomy treatment should be clarified. Furthermore, the appearance of the pulp tissues under the amputation level after partial or full pulpotomy, as well as the time needed to control bleeding, can ensure valuable information about the degree and extent of inflammation within the pulp tissues and determine the most appropriate treatment type and prognosis [9].

Calcium silicate materials, called bioceramics, are used in dentistry, especially in endodontic treatment due to their physicochemical, mechanical and biological effects [22–24]. PD-MTA White (Produits dentaires, Vevey, Switzerland) is one of the calcium silicate cements which is used in dental practice due to its thin hydrophilic particles, biological activities and short setting time (15 min) [25]. However, there is no previous study in the literature which investigates the effectiveness of PD-MTA on the dental pulp in term of VPT. Moreover, the crystallographic reactions of this material after contact with saliva, blood and phosphate-buffered solution have never been analyzed.

The aim of the present prospective study was to investigate the influence of the carious lesion depth and activity as well as the bleeding time on the partial pulpotomy using PD-MTA in permanent molars with irreversible pulpitis after one year of follow-up. The null hypothesis was that there is no impact of the carious lesion depth, activity and the bleeding time on the partial pulpotomy success in permanent molars with irreversible pulpitis after one year. Moreover, it aimed to evaluate the crystallographic reaction of MTA after exposure to artificial saliva, blood and phosphate-buffered solution (PBS) by using scanning electron microscope and energy-dispersive X-rays.

## **2. Materials and Methods**

### *2.1. Ethical Considerations*

The study protocol was approved by the Scientific Research and Postgraduate Board of Damascus University Ethics Committee, Syria (UDDS-1819-07052018/SRC-1450). The patients signed assent and informed consent forms. Patients were offered a full pulpotomy or root canal treatment in case of treatment failure.

#### *2.2. Study Design and Sample Size Calculation*

The study was a prospective longitudinal single-arm clinical investigation of the predictability of partial pulpotomy with MTA in permanent molars exhibiting symptoms and signs of irreversible pulpitis. The sample size was determined using a sample-size calculation program, based on the teeth numbers included in previous studies which investigated both the outcome of full and partial pulpotomy in teeth which have reversible and irreversible pulpitis [15,26,27]. Sample size was calculated using G\*Power <IP_ADDRESS> software (Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany) based on a previous study [15] in order to have 95% power, an alpha error probability of 0.05 and degree of freedom at 1 which has been concluded from a total sample size of 51 teeth.

#### *2.3. Participants*

Sixty-four patients aged from 18 to 65 years old attending the Restorative and Endodontic Dentistry Department, Faculty of Dentistry, Damascus University were enrolled. One permanent molar per patient was included.

### *2.4. Inclusion and Exclusion Criteria*

Inclusion and exclusion criteria were summarized in Table 1.

**Table 1.** Inclusion and exclusion criteria.

#### *2.5. Patient Assessment and Operative Procedure*

Before the operation, a medical/dental history and chief complaint were recorded as part of the clinical assessment. Afterward, the periodontal tissues, tooth mobility, and possibility for restoration were evaluated. In addition, the pulp response was assessed using the Endo Ice cold sensibility test (Coltene, Altstätten, Switzerland), and the pre-apical tissues were evaluated using the percussion and papulation tests. A preoperative periapical radiograph was performed using a digital sensor (Vatech, Ezsensor HD, Saul, Republic of Korea) with a film holder (Dentsply, Elgin, IL, USA) to evaluate the condition of the periapical and furcation areas. In addition, a bitewing radiograph was taken to assess the depth of penetration of the carious lesion (Figure 1a,b). The carious lesions were later divided, according to the depth of penetration within the dentine as shown radiographically, into deep (Figure 1a) (caries reaching the inner quarter of dentine but with a zone of hard or firm dentine between caries and the pulp) or extremely deep (Figure 1b) (caries penetrating the entire thickness of the dentine with certain pulp exposure) [13,28]. Later, radiographs were evaluated independently by the endodontist and a second examinator, for the depth of the lesion as a deep or extremely deep case. After that, a local anesthesia was applied using 2% lidocaine with epinephrine 1/80,000 (Scandonest; Septodont, Saint-Maur-des-fosses Cedex, France) and a rubber dam was applied. The cavity was prepared, under a dental operating microscope (Labomed, Los Angeles, CA, USA), by using a diamond bur (EX-41, Dia burs-Mani, Tochigi, Japan) with a water-cooled high-speed handpiece. Caries at the lateral walls of the cavity and only the superficial part of the demineralized dentin was removed with a sharp excavator, followed by rinsing of the cavity with saline. After drying the cavity using a sterile cotton pellet, the carious lesion was carefully examined to determine its activity. The operator assessed the level of caries activity based on the color and surface texture (moisture and consistency) of the carious dentine. The operator determined the demineralized dentine color by comparing the clinical situation with photographs of the five typical dentine color classes, which are light yellow, yellow, light brown, dark brown, or black [29]. The consistency of the dentine was categorized as soft dentine (when it can be excavated with minimum resistance using hand instruments), firm dentine (when it was resistant to excavation using manual instruments), and hard dentine (when it was resistant to probe penetration) [13]. Surface humidity was determined by inserting a probe into the carious tissue; if the tissue oozed moisture, it was categorized as wet, and if it did not, it was classified as dry [30]. Accordingly, demineralized dentin with a light yellow/yellow color and a soft/moist surface texture was classified as a rapidly progressing lesion (Figure 1c), while demineralized dentin with a light brown/brown color and a firm/dry surface texture was classified as a slowly progressing lesion (Figure 1d).

After that, the gross caries was removed in a non-selective manner with a round bur and a slow handpiece (Figure 2d). After the pulp was exposed (Figure 2d), a carbide bur on a high-speed handpiece was used to eliminate around 2 to 3 mm of coronal pulp tissues under water-cooling (Figure 2e). Following the rinsing of the pulp with 2.5% NaOCl [31] for one minute, the state of the pulp tissues below the amputation level was thoroughly evaluated under magnification. If the pulp tissues' appearance was normal with no sign of infection or degradation, hemostasis was attained by applying a cotton pellet drenched in 2.5% NaOCl to the wound pulp. Hemostasis was controlled every minute for up to 6 min [26]. The time to control bleeding was recorded and subsequently divided into two categories as follows: bleeding time between 1 and 3 min, and bleeding time between 4 and 6 min. Finally, mineral trioxide aggregate (MTA) (PD-MTA, Produits Dentaires SA, Verey, Switzerland) was prepared following the manufacturer's instructions and added gradually over the fresh pulp wound and surrounding dentine to a thickness of 2–3 mm using the MapOne carrier (MapOne system, Produits Dentaires SA, Verey, Switzerland) (Figure 2f). The MTA was coated with a sterile, moist cotton pellet immersed in saline for 15 min to provide primary hydration. After that, a layer of resin-modified glass-ionomer cement ''RMGIC" (Fuji II LC; GC Corp, Tokyo, Japan) was placed on the MTA (Figure 2g) [32]. An universal adhesive system (Tetric-N Bond, Ivoclar Vivadent) was applied and covered by

*Bioengineering* **2023**, *10*, x FOR PEER REVIEW 5 of 15

using the MapOne carrier (MapOne system, Produits Dentaires SA, Verey, Switzerland) (Figure 2f). The MTA was coated with a sterile, moist cotton pellet immersed in saline for 15 min to provide primary hydration. After that, a layer of resin-modified glass-ionomer

direct composite restorative resin (Tetric-Ceram, Ivoclar Vivadent) (Figure 2h) [33]. All the clinical steps are summarized in Figure 3. cement ''RMGIC'' (Fuji II LC; GC Corp, Tokyo, Japan) was placed on the MTA (Figure 2g) [32]. An universal adhesive system (Tetric-N Bond, Ivoclar Vivadent) was applied and covered by direct composite restorative resin (Tetric-Ceram, Ivoclar Vivadent) (Figure 2h) [33]. All the clinical steps are summarized in Figure 3.

**Figure 1.** (**a**) Bitewing preoperative radiograph for first mandibular molar with deep carious lesion shows that the lesion involves the inner quarter of dentine with a radio-dense zone (hard or firm dentine) between caries and the pulp. (**b**) Bitewing preoperative radiograph for second mandibular molar with extremely deep carious lesion shows the lesion penetrating the entire thickness of the dentine with a radio-dense zone located within the pulp chamber indicative of tertiary dentine. (**c**) Intraoral image shows a rapidly progressing lesion and the yellow color of the demineralized dentine with soft and wet appearance of the surface texture. (**d**) Intraoral image shows a slowly progressing lesion and the brown color of the demineralized dentine with firm/dry appearance of the surface texture. **Figure 1.** (**a**) Bitewing preoperative radiograph for first mandibular molar with deep carious lesion shows that the lesion involves the inner quarter of dentine with a radio-dense zone (hard or firm dentine) between caries and the pulp. (**b**) Bitewing preoperative radiograph for second mandibular molar with extremely deep carious lesion shows the lesion penetrating the entire thickness of the dentine with a radio-dense zone located within the pulp chamber indicative of tertiary dentine. (**c**) Intraoral image shows a rapidly progressing lesion and the yellow color of the demineralized dentine with soft and wet appearance of the surface texture. (**d**) Intraoral image shows a slowly progressing lesion and the brown color of the demineralized dentine with firm/dry appearance of the surface texture. *Bioengineering* **2023**, *10*, x FOR PEER REVIEW 6 of 15

**Figure 2.** Mandibular right first molar presented with symptomatic irreversible pulpitis treated with partial pulpotomy using PD-MTA. (**a**) Clinical and radiographic examinations revealed deep carious lesion confined at a proximal surface with no evidence of dentine exposure, reflecting a close lesion environment. (**b**) Rubber dam application. (**c**) After removal of the undermined enamel and the exposition of the demineralized dentine. (**d**) Pulp exposure after non-selective caries removal. (**e**) Partial pulpotomy by removing of 2–3 mm of pulp tissues under exposure and hemostasis achieved. Note that the pulp tissues under level of amputation showed a normal appearance, texture, and color. (**f**) PD-MTA application as capping material. (**g**) Base of resin-modified glass–ionomer cement placed above MTA material. (**h**) Composite resin restoration. (**i**) Postoperative bitewing radiograph. **Figure 2.** Mandibular right first molar presented with symptomatic irreversible pulpitis treated with partial pulpotomy using PD-MTA. (**a**) Clinical and radiographic examinations revealed deep carious lesion confined at a proximal surface with no evidence of dentine exposure, reflecting a close lesion environment. (**b**) Rubber dam application. (**c**) After removal of the undermined enamel and the exposition of the demineralized dentine. (**d**) Pulp exposure after non-selective caries removal. (**e**) Partial pulpotomy by removing of 2–3 mm of pulp tissues under exposure and hemostasis achieved. Note that the pulp tissues under level of amputation showed a normal appearance, texture, and color. (**f**) PD-MTA application as capping material. (**g**) Base of resin-modified glass–ionomer cement placed above MTA material. (**h**) Composite resin restoration. (**i**) Postoperative bitewing radiograph.

**Figure 3.** Schematic graph presents the different steps of clinical procedure in partial pulpotomy.

**Figure 2.** Mandibular right first molar presented with symptomatic irreversible pulpitis treated with partial pulpotomy using PD-MTA. (**a**) Clinical and radiographic examinations revealed deep carious lesion confined at a proximal surface with no evidence of dentine exposure, reflecting a close lesion environment. (**b**) Rubber dam application. (**c**) After removal of the undermined enamel and the exposition of the demineralized dentine. (**d**) Pulp exposure after non-selective caries removal. (**e**) Partial pulpotomy by removing of 2–3 mm of pulp tissues under exposure and hemostasis achieved. Note that the pulp tissues under level of amputation showed a normal appearance, texture, and color. (**f**) PD-MTA application as capping material. (**g**) Base of resin-modified glass–ionomer cement placed above MTA material. (**h**) Composite resin restoration. (**i**) Postoperative bitewing

**Figure 3.** Schematic graph presents the different steps of clinical procedure in partial pulpotomy. **Figure 3.** Schematic graph presents the different steps of clinical procedure in partial pulpotomy.

#### *2.6. Outcome Evaluation*

radiograph.

Three follow-up appointments (3, 6, and 12 months) were determined. At each time point, participants were clinically and radiographically examined by an endodontist who was blinded about the depth and activity of caries and the bleeding time. Different clinical parameters including the absence and presence of clinical signs, a vitality pulp test, a periodontal examination, and a percussion test were recorded. Periapical and bitewing radiographs were taken to evaluate any pathological changes at the periapical or furcal area and to detect dentinal bridge formation. The radiographs were later evaluated independently by two blinded examiners; the accuracy between the examiners was investigated by repeating the evaluation of the images after one week [34]. After one year, the teeth were classified as successful or failed treatments. To be classified as retaining overall success, the tested tooth should have both clinical and radiographic success. Treatment was considered successful when the tooth responded positively to a cold test within normal limits, there were no signs of pulpitis, there was no abnormal mobility or fistula, and there was no evidence of an apical radiolucency or internal and/or external root resorption.

#### *2.7. Scanning Electron Microscope (SEM) and Energy Dispersive X-ray Analysis (EDX)*

After the end of the follow-up (12 months), 9 samples of the same MTA material were prepared using Teflon molds (height: 3.8 mm/diameter: 3 mm). The samples were put at 37 ◦C for 48 h to achieve a good setting time [12]. Each of the three samples were stored in 50 mL of human blood, phosphate-buffered saline (PBS10x, Dominique Dutscher, Bernolsheim, France) or artificial saliva (Serlabo Technologies, Entraigues-sur-la-Sorgue, France) at 37 ◦C for 7 days. After the immersion time, the samples were gently rinsed with distilled water for 3 min and were sputter-coated with gold–palladium (20/80) using a Hummer JR sputtering device (Technics, San Jose, CA, USA). After that, the surface of each sample was investigated using SEM (Quanta 250 FEG scanning electron microscope "FEI Company, Eindhoven, The Netherlands"; 10 kV acceleration voltage of the electrons) and studied at a magnification of 1000× and 4000× for morphological evaluations and mineralization changes through SEM. Moreover, EDX analyses were performed for 30 s at 10 mm of distance in order to investigate the chemical composition.

#### *2.8. Statistical Analyses*

The SPSS 24.0 software (SPSS Science, Chicago, IL, USA) was used to perform the statistical analyses. The level of significant difference was at α = 0.05. Fisher's exact test and the Chi-square test were used to assess the influence of the depth and activity of a carious lesion and the bleeding time on the outcome of treatment. The preoperative caries depth assessment inter-observer agreement and the postoperative intra-observer reproducibility and inter-observer agreement in terms of any pathological changes in the periapical or furcal area were assessed using Cohen's Kappa coefficient.

#### **3. Results**

Sixty-four patients were assessed for eligibility, having presented with signs symptomatic of irreversible pulpitis in molar teeth. Fourteen participants were excluded from the study due to refusal to participate (five patients) or not meeting inclusion criteria (nine patients). Fifty patients (50 teeth, one tooth per patient) were enrolled to be treated by partial pulpotomy treatment. Ten patients were subsequently excluded intraoperatively, three cases due to uncontrolled bleeding and seven cases due to extension of infected tissue to the root canal orifices or beyond. These patients were treated either with full pulpotomy or with RCT. Finally, forty teeth were included and treated in this study. Four participants could not attend the follow-ups, resulting in an overall recall rate of 90% (36/40) (Figure 4). *Bioengineering* **2023**, *10*, x FOR PEER REVIEW 8 of 15

**Figure 4.** Study flow diagram. **Figure 4.** Study flow diagram.

extremely deep carious lesions (*p* > 0.05).

min bleeding time (*p* > 0.05) (Table 2).

carious lesion depth, and bleeding time.

Activity of carious lesion

Carious lesion depth

(33.3%).

The patients consisted of 13 males and 23 females, aged 18–65 years old (32.75 ± 10.7 years old). The included cases consisted of twenty teeth (55.5%) and sixteen teeth (44.5%) with deep and extremely deep caries, respectively. The carious lesion was actively pro-The patients consisted of 13 males and 23 females, aged 18–65 years old (32.75 ± 10.7 years old). The included cases consisted of twenty teeth (55.5%) and sixteen teeth (44.5%) with deep and extremely deep caries, respectively. The carious lesion was actively progressing in twenty-four cases (66.7%) compared to twelve slowly progressing lesions (33.3%).

gressing in twenty-four cases (66.7%) compared to twelve slowly progressing lesions The mean bleeding time was 3.80 ± 1.47 min; fifteen teeth (41.7%) needed time between 1–3 min to achieve hemostasis, while twenty-one teeth (53.3%) needed time between 4–6 min to control the bleeding.

The mean bleeding time was 3.80 ± 1.47 min; fifteen teeth (41.7%) needed time between 1–3 min to achieve hemostasis, while twenty-one teeth (53.3%) needed time between 4–6 min to control the bleeding. The success rate for partial pulpotomy managing irreversible pulpitis was 88.9% after one year of follow-up, and failure was observed in four cases. Early failure occurred The success rate for partial pulpotomy managing irreversible pulpitis was 88.9% after one year of follow-up, and failure was observed in four cases. Early failure occurred within three months in three cases, while late failure was observed after 12 months in one case.  However, no statistically significant

*n* **(%) Success (%) Failure (%)**

Rapid progression 24 (66.7) 20 (83.3) 4 (16.7) 0.18 Slow progression 12 (33.3) 12 (100) 0 (0)

Deep 20 (55.5) 19 (95) 1 (5) 0.221 Extremely deep 16 (44.5) 13 (81.3) 3 (18.7)

*p***-Value**

within three months in three cases, while late failure was observed after 12 months in one

significant difference was observed (*p* > 0.05). Regarding caries depth, only one failure case was related to a deep lesion, whilst the other three failure cases were associated with

**Table 2.** Fisher's exact test results to compare the outcome according to the activity of carious lesion,

Bleeding time 1–3 min 15 (41.7) 15 (100) 0 (0) 0.102 4–6 min 21 (58.3) 17 (81) 4 (19)

The results were subdivided by both depth and activity of carious lesions into four categories: rapidly progressing deep (*n* = 14), rapidly progressing extremely deep (*n* = 10), slowly progressing deep (*n* = 6), and slowly progressing extremely deep (*n* = 6). Three cases of rapidly progressing extremely deep lesions and one case of rapidly progressing

**Variables Overall Outcome**

As for bleeding time, it was noted that all failure cases were associated with a bleeding time ranging between 4 and 6 min, whilst no failure was detected for cases with 1–3 difference was observed (*p* > 0.05). Regarding caries depth, only one failure case was related to a deep lesion, whilst the other three failure cases were associated with extremely deep carious lesions (*p* > 0.05).

As for bleeding time, it was noted that all failure cases were associated with a bleeding time ranging between 4 and 6 min, whilst no failure was detected for cases with 1–3 min bleeding time (*p* > 0.05) (Table 2).

**Table 2.** Fisher's exact test results to compare the outcome according to the activity of carious lesion, carious lesion depth, and bleeding time.

The results were subdivided by both depth and activity of carious lesions into four categories: rapidly progressing deep (*n* = 14), rapidly progressing extremely deep (*n* = 10), slowly progressing deep (*n* = 6), and slowly progressing extremely deep (*n* = 6). Three cases of rapidly progressing extremely deep lesions and one case of rapidly progressing deep lesions failed, whereas neither slowly progressing deep lesions nor slowly progressing extremely deep lesions failed (*p* > 0.05) (Table 3).

**Table 3.** Chi-Square test results to compare the outcome according to depth/activity of carious lesion.

The two examinations showed a good level of accordance in investigating caries depth (κ = 0.8). When assessing the periapical and furcal areas, the Kappa value for the interobserver accordance was 0.89, and for the intra-observer reproducibility, the Kappa values were 0.95 and 0.92 for the 1st and 2nd observers, respectively.

SEM micrographs showed the reaction of MTA surfaces after 7 days of immersion in blood, PBS and artificial saliva at 37 ◦C. The crystalline textures of the MTA in the three conditions are demonstrated in Figure 5. Different crystalline appearances were detected.  MTA surfaces exposed to saliva did not show any crystalline structures. EDX analysis for MTA surfaces after 7 days presented different % of Ca, Si and P among the three conditions. Higher Ca mass percentages were detected on MTA surfaces in PBS condition compared to blood and saliva.

**Figure 5.** Scanning electron micrographs and Energy dispersive x-ray for Mineral Trioxide Aggregate surfaces in contact with saliva, PBS and blood at ×1000 and ×4000 magnifications. Different calcium percentages were detected by using EDX analysis (Black arrows). **Figure 5.** Scanning electron micrographs and Energy dispersive x-ray for Mineral Trioxide Aggregate surfaces in contact with saliva, PBS and blood at ×1000 and ×4000 magnifications. Different calcium percentages were detected by using EDX analysis (Black arrows).

deep lesions failed, whereas neither slowly progressing deep lesions nor slowly progress-

**Table 3.** Chi-Square test results to compare the outcome according to depth/activity of carious le-

Rapidly progressing deep 14 (38.88) 13 (92.9) 1 (7.1)

Slowly progressing, deep 6 (16.66) 6 (100) 0(0)

Slowly progressing, extremely deep 6 (16.66) 6 (100) 0(0)

values were 0.95 and 0.92 for the 1st and 2nd observers, respectively.

Rapidly progressing, extremely deep 10 (27.8) 7 (70) 3 (30) 0.149

**State of Carious Lesion** *n* **(%) Success (%) Failure (%)** *p***-Value**

The two examinations showed a good level of accordance in investigating caries depth (κ = 0.8). When assessing the periapical and furcal areas, the Kappa value for the inter-observer accordance was 0.89, and for the intra-observer reproducibility, the Kappa

SEM micrographs showed the reaction of MTA surfaces after 7 days of immersion in blood, PBS and artificial saliva at 37 °C. The crystalline textures of the MTA in the three conditions are demonstrated in Figure 5. Different crystalline appearances were detected.  MTA surfaces exposed to saliva did not show any crystalline structures. EDX analysis for MTA surfaces after 7 days presented different % of Ca, Si and P among the three conditions. Higher Ca mass percentages were detected on MTA surfaces in PBS condition compared to blood and saliva.

**Overall Outcome**

ing extremely deep lesions failed (*p* > 0.05) (Table 3).

sion.

#### **4. Discussion**

Partial pulpotomy is described as the removal of 2–3 mm of pulp tissue at the exposure site, followed by sealing of the pulp wound with bioactive material [13]. This procedure differs from pulp capping in that it removes the superficial layer of infected or inflammatory tissue and the accompanying biofilms [26]. In addition, it preserves a significant portion of the coronal pulp compared to a full pulpotomy, which removes the entire coronal tissue up to the canal orifices [12]. Therefore, partial pulpotomy could be the most conservative and predictable treatment for carious pulp exposure.

The success rate in the present study was 88.9% after one year of follow-up. Several clinical studies over the last 10 years have shown success for the partial pulpotomy procedure in treating carious pulp exposure in adult and young permanent teeth, even when there are symptoms and signs of irreversible pulpitis [15,16,26]. In accordance with the results of the present study, Uesrichai et al. noted a 90% success rate for partial pulpotomy in the management of irreversible pulpitis in mature and young teeth in children aged 6 to 18 years [16]. Another study showed a success rate of 90% after 12 months partial pulpotomy procedure on complete developed permanent teeth within a sample of reversible and irreversible pulpitis [26]. A longer follow-up study (2 years) performed on partial pulpotomy in complete developed adult teeth with symptomatic irreversible pulpitis showed a slightly decrease in the success rate to 80% [15]. In accordance with the previous study of Taha and Khazali [15], the current study relied on standardizing the initial diagnosis of the dental pulp and limiting it to irreversible pulpitis in adult mature permanent molars to analyze the partial pulpotomy procedure in such cases more precisely. In addition, the originality of the current study was to associate the effect of carious lesion activity and depth, as well as the use of PD-MTA on the success of a partial pulpotomy procedure, which was never previously investigated.

No statistically significant relation was found between the failure with deep or extremely deep lesions (*p* > 0.05). Therefore, the null hypothesis must be accepted. Only one failure case was related to a deep lesion, whilst three failure cases were associated with extremely deep carious lesions. This results could be explained by the fact that pulpitis will be present at the early stage of the carious process, and the severe inflammatory response and the significant levels of inflammatory cells are not identified in the pulp until caries have progressed to within around 0.5 mm near the pulp [35]. It was found that bacteria were mainly present in the primary dentine in deep carious lesions. Extremely deep lesions, on the other hand, were linked to pulp-reaching microorganisms, as well as inflammatory infiltration and subsequent partial necrosis [20]. Therefore, the majority of failure cases in the current study were associated with extremely deep lesions rather than deep ones. In accordance, Careddu and Duncan [26] noted that all failure cases in their study, which were treated with a partial pulpotomy using Biodentine as capping material, were in the extremely-deep caries group.

Ten patients were excluded intraoperatively due to either the extension of infected tissue to the root canal orifices or beyond, as confirmed by careful examination under magnification and illumination, or uncontrolled bleeding that may indicate advanced pulpal inflammation [36]. In addition, these patients had extremely deep caries. In accordance, a previous histological study [20] revealed that more than half of extremely deep caries were associated with inflammatory infiltrates that affected the complete coronal pulp and the most coronal part of the radicular pulp, which renders partial pulpotomy inappropriate in such cases. As there was no significant difference between deep and extremely deep lesions, extremely deep carious lesions can still be treated with partial pulpotomy if a magnified examination of the underlying tissues is performed to determine the extent of inflamed and infected tissues within the dental pulp.

No failure cases were detected after one year for slowly progressing lesions, whilst all failure cases were associated with actively progressing lesions. In addition, all cases presented with slowly progressing lesions were successful even if they had extremely deep penetration, while 30% of extremely deep lesions and 7.1% of deep lesions failed if they rapidly progressed. These findings could be due to rapidly progressing lesions being associated with the presence of heavily cariogenic biofilm, while the cariogenic biofilms are significantly diminished in slowly progressing lesions [20,30]. Moreover, a slowly progressing lesion with limited cariogenic biofilms is less likely to transmit stimuli into the pulp, which is associated with a low-intensity inflammatory response favorable for healing and repair [37]. Therefore, we can conclude that the rapidly progressing and extremely deep caries associated with clinically diagnosed irreversible pulpitis have less chance of being treated with a partial pulpotomy, even though no statistically significant difference was observed in treatment outcomes according to both depth and activity of the carious lesion.

It is commonly agreed that avoiding pulpal exposure and selectively removing carious tissue is the optimal strategy for the management of a deep carious lesion [38]. In the current study, all teeth were clinically judged to have irreversible pulpitis, necessitating the total or partial removal of coronal pulp tissue [14,15]. Therefore, regardless of the depth of caries, they were chosen to have the carious tissues removed non-selectively rather than selectively. Notably, the majority of included deep lesions were proximal caries, in which the undermined enamel maintains a closed ecosystem and a rapid progression rate. These carious lesion characteristics may be associated with no or less prominent tertiary dentine formation, and bacterial invasion of the pulp may be evident even before caries reaches the pulp radiographically [39]. Moreover, pulpitis may be induced by a highly acidogenic environment in such lesions even before the pulp is exposed [28,40]. This may explain the occurrence of irreversible pulpitis in a significant part of cases included in this study despite their presenting with deep lesions without reaching the pulp radiographically, and it highlights once again the importance of evaluating the activity of the carious lesion in addition to its depth to determine the most appropriate treatment.

All failed cases in the present study occurred when the bleeding time was more than 3 min. Consistently, the present results showed no significant difference in the success rates of partial pulpotomy with the difference in the time needed for hemostasis (*p* > 0.05). In accordance, several recent studies demonstrated that there was no marked association between bleeding time and the outcome of pulpotomy treatments [18,26]. The control of bleeding is essential for the success of VPT, as the profuse, difficult-to-stop pulp bleeding could be an indicator of advanced pulp inflammation [36]. However, the mechanical and chemical elimination of the infectious challenge at the pulp wound by partial pulpotomy and NaOCl lavage may make the bleeding time a less significant factor in the treatment outcome [31]. Moreover, the local anesthesia which was applied using 2% lidocaine with epinephrine 1/80,000 in the present study could play an important role on the bleeding time. Chu et al. demonstrated that the use of local anesthesia based on lidocaine and epinephrine decreases the pulpal blood flow and may protect the dental pulp by attenuating the increase in pulpal blood flow caused by tooth preparation [41]. Moreover, it is known that the infiltration anesthesia is used successfully in the maxillary teeth but is less effective in the mandibular molar regions due to the density of bone [42]; therefore, block anesthesia was used in the present study for the mandibular molar regions.

In order to clarify the outcomes of the use of MTA in partial pulpotomy, when this biomaterial is in direct contact with pulpal tissue, blood and dentinal fluids, SEM analyses were performed on a MTA surface after immersion in PBS, blood and artificial saliva at 37 ◦C for 7 days. PBS was utilized to simulate the in vivo dental tissue fluids [43] to assess the mineral development that could take place on the material surfaces. SEM images showed crystallite formation on the material surfaces in the cases of PBS and blood conditions, whilst no mineralization process and no notable changes were demonstrated for MTA surfaces after immersion for 7 days in artificial saliva. Therefore, MTA is a bioactive material which could promote the remineralization process. In addition, calcium silicate-based materials are capable of making calcium hydroxide and calcium silicate hydrate when it is in a humid medium [22,24,44]. Lower calcium peaks were found for the MTA surfaces immersed in blood and artificial saliva, whilst higher calcium peeks were detected for MTA surfaces exposed to PBS. These findings could be related to the different environmental conditions and the dissolution of calcium hydroxide because of hydration [45]. These outcomes are similar to the results of a previous study which was conducted on MTA ProRoot [45]. Moreover, in the present study, RMGIC was used on MTA in order to have the optimal bond strength with the final restoration (composite) as described previously [46,47]. In addition, several studies have also shown that the MTA's physical properties might be affected by the acidic environment that existed before composite buildup [48,49]. Therefore, RMDIC was used to have an optimal coronal sealing ability, which is related to the long success of VPT.

Some limitations were detected in the present study. For example, because the study was designed to investigate the effect of the depth and activity of the carious lesion, there was no control group to which the patients could be assigned randomly. Moreover, PD-MTA as a powder–liquid bioceramic material should be carefully mixed following the manufacturer's instructions in order to avoid errors during manual mixing, which could alter the physicochemical properties of this material [22]. Moreover, the irrigant which was used, NaOCl, is known for its high cytoxicity, which could lead to severe tissue damage if it is used in high concentrations [50,51]; in contrast, 2.5% NaOCl has a high efficacity as a lavage solution due to the unique capability of NaOCl to selectively dissolve necrotic soft tissue, thus reducing the necessity to mechanically remove infected tissues [31]. In addition, radiographic monitoring was crucial to follow the treatment results; however, the indication to take radiographs in clinical practice is limited to reasonable requests in order to decrease patients' exposure to ionizing radiation [52]. Further studies with higher numbers of included cases and longer follow-up periods should be performed to investigate the relation between the activity of a carious lesion and the success of partial pulpotomy treatment. In addition, the carious lesion depth was estimated on a bitewing radiograph. Although there was a good inter-observer agreement (0.8), it was still challenging to identify the exact depth of caries on a two-dimensional radiograph. Finally, future studies should consider using cone-beam computed tomography (CBCT) as a more precise technique for this purpose. The color, moisture, and surface texture of the carious tissues were also used as indications of carious lesion activity. These indicators, however, are somewhat subjective, and a more reliable method is required.

### **5. Conclusions**

Within the limitations of the present study, favorable results demonstrated that MTA might be recommended as suitable agent for partial pulpotomy in permanent molars with irreversible pulpitis, with an 88.9% success rate after one year. It could be concluded that the depth and activity of the carious lesion as well as the bleeding time are important factors in the success of partial pulpotomy treatment. The prolonged bleeding time and the rapidly progressing extremely deep lesions could be related to the failure cases in partial pulpotomy treatment of irreversible pulpitis.

**Author Contributions:** Conceptualization, R.Z.A. and O.A.; methodology, R.Z.A.; software, R.Z.A.; validation, R.Z.A.; formal analysis, R.Z.A. and S.A.; investigation, R.Z.A.; resources, R.Z.A.; data curation, R.Z.A.; writing—original draft preparation, R.Z.A., A.E., A.A. and O.A.; writing—review and editing, N.K., Y.H. and D.M.; visualization, R.Z.A.; supervision, O.A.; project administration, O.A.; funding acquisition, R.Z.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Scientific Research and Postgraduate Board of Damascus University Ethics Committee, Syria (UDDS-1819-07052018/SRC-1450).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Data are available from the first author<EMAIL_ADDRESS>(R.Z.A.)).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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## *Article* **Physicochemical and Antibacterial Properties of Bioactive Retrograde Filling Materials**

**Tarek Ashi 1,2, Davide Mancino 1,2,3 , Louis Hardan 4 , Rim Bourgi 4 , Jihed Zghal 5,6, Valentina Macaluso 7 , Sharif Al-Ashkar 8 , Sleman Alkhouri 9 , Youssef Haikel 1,2,3,† and Naji Kharouf 1,2,\* ,†**

	- 92410 Ville d'Avray, France

**Abstract:** The purpose of the present study was to evaluate the physicochemical properties and antibacterial activity of three calcium silicate cements. Mineral trioxide aggregate (MTA Biorep "BR"), Biodentine (BD) and Well-Root PT (WR) materials were investigated using scanning electron microscopy (SEM) at 24, 72 and 168 h of immersion in phosphate buffered saline (PBS). The antibacterial activity against *Enterococcus faecalis* (*E. faecalis*), the solubility, roughness, pH changes and water contact angle were also analyzed. All results were statistically analyzed using a one-way analysis of variance test. Statistically significant lower pH was detected for BD than WR and BR (*p* < 0.05). No statistical difference was found among the three materials for the efficacy of kill against *E. faecalis* (*p* > 0.05). Good antibacterial activity was observed (kill 50% of *bacteria*) after 24 h of contact. The wettability and the roughness of BR were higher than for the other cements (*p* < 0.05). BD was more soluble than WR and BR (*p* < 0.05). In conclusion, the use of bioceramic cements as retrograde materials may play an important role in controlling bacterial growth and in the development of calcium phosphate surface layer to support healing. Moreover, the premixed cement was easier to use than powder–liquid cement.

**Keywords:** calcium silicate cement; retrograde materials; premixed cement; powder–liquid cement

## **1. Introduction**

The success of surgical endodontic treatment requires root-end filling materials that are easy to use, biocompatible, stable and economical [1–3]. The goal is to seal the apex hermetically and prevent microorganisms from entering the root canal [4,5].

Retrograde root-end filling materials have included zinc oxide eugenol cements, amalgam, glass ionomer and resins which have failed to meet the ideal requirements of root-end filling treatment [6,7].

Calcium silicate cement materials, colloquially denoted as "Bioceramic", in both forms, sealer [8] or thicker mixture [9], are considered as the ideal endodontic material for retrograde treatment due to their excellent physicochemical and biological properties [10–15],

**Citation:** Ashi, T.; Mancino, D.; Hardan, L.; Bourgi, R.; Zghal, J.; Macaluso, V.; Al-Ashkar, S.; Alkhouri, S.; Haikel, Y.; Kharouf, N. Physicochemical and Antibacterial Properties of Bioactive Retrograde Filling Materials. *Bioengineering* **2022**, *9*, 624. https://doi.org/10.3390/ bioengineering9110624

Academic Editor: Chengfei Zhang

Received: 8 October 2022 Accepted: 26 October 2022 Published: 28 October 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

including biocompatibility and stability. These inorganic and non-corrosive ceramic cements contain tricalciums silicate and various radiopaque powders [16].

Mineral Trioxide aggregate (MTA) was the original calcium silicate cement introduced for endodontic treatment in 1993 and it is considered as the gold-standard material for various endodontic applications [10]. Other tricalcium silicate-based products have been developed, improvements on the original Portland cement invention [10]. MTA Biorep (Itena Clinical, Paris, France) is a powder–liquid product containing calcium silicate cement and calcium tungstate. Its water-based liquid, containing an organic plasticizer, improves the handling and plasticity [10].

Biodentine™ (Septodont, Saint-Maur-des-fossés, France) is a calcium silicate cement material and it has higher strength than other similar products [17]. This product consists of a powder–liquid material, where the liquid contains calcium chloride with an admixture of polycarboxylate [17]. MTA Biorep and Biodentine cements are indicated for several endodontic treatments, including pulpotomy, pulp capping, resorption, apicoectomy and open apex [9,10,18–21].

Some bioceramic cements require manual mixing and handling, powder–liquid systems, which require certain skills [16,22]. In addition, any change in the powder–liquid ratio or the mixing could affect and alter the physicochemical properties of these cements [8,23]. Premixed cements have been introduced to avoid errors during manual mixing. These premixed materials do not require any preparation before clinical application [8,16]. As mentioned in previous studies [8,16], these premixed materials are advantageous for some clinicians in the handling.

Well-Root™ PT is a novel premixed calcium aluminosilicate cement delivered in capsules for direct clinical use [24]. No information was found in the literature on the antibacterial activity and the physicochemical properties of this cement.

The purpose of the present research was to investigate the physicochemical properties and the antibacterial activity of three calcium silicate cements. The hypothesis concerned whether there would be antibacterial and physicochemical differences between the three tested materials.

#### **2. Materials and Methods**

#### *2.1. Materials*

MTA Biorep "BR" (Itena Clinical, Paris, France), Biodentine™ "BD" (Septodont, Saint-Maur-des-fossés, France) and Well-Root™ PT "WR" (Vericom, Gangwon-Do, Korea) were used in the present study, following the manufacturer's instructions (Table 1). All specimens were conserved in the dark in a container at 37 ◦C and 95% relative humidity for 48 h until completely set [25].

#### *2.2. pH Measurements of the Aqueous Solution in Contact with the Cement*

Five samples of each group were prepared using Teflon molds (3.8 mm in high and 3 mm in diameter). Each sample was put in contact with 10 mL distilled water at 37 ◦C. A pH meter, "CyberScan pH 510" (Thermo Scientific, Waltham, Massachusetts, USA), was used to measure the pH of water at 3, 24, 72 and 168 h. Before each pH test, the calibration of pH meter was performed using standard solutions at pH 10, 4 and 7 (Hanna Instruments, Lingolsheim, France). Distilled water was used to rinse and eliminate the previous solution from the pH meter electrode.

#### *2.3. Solubility*

Five samples (2 mm in height and 20 mm in diameter) of each material were analyzed following the method of a previous study [26]. The samples were weighed using a digital system, then the disks were immersed for 24 h in 50 mL of water at 37 ◦C. The samples were removed from distilled water and then dried at 37 ◦C for 24 h. Finally, the samples were weighed again to obtain the final weight. The solubility was defined from the difference in mass between the final and the initial weight.

**Table 1.** Manufacturer and manipulation of the tested materials.

#### *2.4. Scanning Electron Microscope (SEM) of Crystallites Creation*

Twelve samples for each material were created (3.8 mm in high and 3 mm in diameter. After the setting time, as described in Section 2.1, three samples from each group were stored in hermetic boxes and kept in dry condition. The remaining samples (9 samples) from each group were put in 10 mL of phosphate-buffered saline (PBS10×, Dominique Dutscher, Bernolsheim, France) at 37 ◦C. After 24, 72 and 168 h in PBS, 3 samples for each period were washed with distilled water for 5 min, sputter-coated with gold–palladium (20/80) [27], then, analyzed using an SEM (FEI Company, Eindhoven, The Netherlands, 10 kV) at a magnification of 5000×. Energy Dispersive X-ray (EDX) analysis was used during an acquisition time of 1 min and a working length of 10 mm to attain the spectrum of chemical elements present on the surface.

#### *2.5. Roughness and Water Sorption Tests*

Five samples from each product were created using Teflon molds (10 mm in diameter and 2 mm in height). After the setting time, as described in Section 2.1, the samples were kept in dry in the fume hood overnight. The roughness of each surface was measured using a 3D digital profilometer (Keyence, Osaka, Japan) at 2500× magnification. The average roughness (Sa) was calculated using software (Keyence 7000 VHX, Osaka, Japan).

After measuring the surface roughness, on the same samples, a contact angle device (Biolin Scientific, Espoo, Finland) was used to observe the infiltration time of a 5 µL droplet of water into the material surface. A movie was recorded to track the profile and the absorption time of the water droplet.

#### *2.6. Antimicrobial Activity*

Brain Heart Infusion medium (BHI) (Darmstadt, Germany) was used to culture *Enterococcus faecalis* (*E. faecalis*, ATCC 29212). The turbidity was adjusted to OD600 (nm) = 0.3. A direct contact test (DCT) was performed to investigate the antibacterial activity of the three products against *E. faecalis*. Triplicate samples were placed in 24-well culture plates. One milliliter of the bacterial medium was put to each well and incubated anaerobically for 24 h at 37 ◦C (constant stirring at 450 rpm). The bacterial medium without the cement materials was used as the control group. After 24 h, 10-fold serial dilutions up to 106 in BHI were performed on each specimen. One hundred microliters of each diluted medium was added onto a BHI agar plate, homogeneously spread and incubated at 37 ◦C for 24 h. Manual CFU/mL (colony forming units/mL) counting was measured the *E. faecalis* concentration.

## *2.7. Statistical Analysis*

The results of pH, solubility, roughness and antibacterial activity were statistically analyzed using the Kruskal–Wallis test along with the Tukey Test. SigmaPlot release 11.2 (Systat Software, Inc., San Jose, CA, USA) was used with a statistical significance was set at α = 0.05.

### **3. Results**

#### *3.1. pH Measurements*

The pH of the solution in contact with the three cements over 7 days is shown in Figure 1. All three cements were alkaline for the solution for up to 72 h. BR and WR demonstrated statistically higher pH than BD at all time points (3, 24, 72 and 168 h) (*p* < 0.05). No significance difference was found between BR and WR (*p* > 0.05). *Bioengineering* **2022**, *9*, x FOR PEER REVIEW 5 of 11 *Bioengineering* **2022**, *9*, x FOR PEER REVIEW 5 of 11

**Figure 1.** pH changes for the three products after 3, 24, 72 and 168 h of contact with water. \* *p* < 0.05. **Figure 1.** pH changes for the three products after 3, 24, 72 and 168 h of contact with water. \* *p* < 0.05. **Figure 1.** pH changes for the three products after 3, 24, 72 and 168 h of contact with water. \* *p* < 0.05.

#### *3.2. Solubility 3.2. Solubility*

*3.2. Solubility* The mean and standard deviation of solubility (wt.%) values are presented in Figure 2. The mean and standard deviation of solubility (wt.%) values are presented in Figure 2. BD was more soluble than BR and WR at 24 h (*p* < 0.05). The mean and standard deviation of solubility (wt.%) values are presented in Figure 2. BD was more soluble than BR and WR at 24 h (*p* < 0.05).

**Figure 2.** Solubility percentages of the different products after aging in water for 24 h at 37 °C. \* *p* < 0.05. **Figure 2.** Solubility percentages of the different products after aging in water for 24 h at 37 °C. \* *p* < 0.05. **Figure 2.** Solubility percentages of the different products after aging in water for 24 h at 37 ◦C. \* *p* < 0.05.

The crystalline structures of the three cements are shown in Figures 3 and 4. All three cements had crystalline deposits after immersion in PBS at 37 °C. At each immersion period (24, 72 and 168 h), different crystalline appearances were observed. At 24 and 72 h,

The crystalline structures of the three cements are shown in Figures 3 and 4. All three cements had crystalline deposits after immersion in PBS at 37 °C. At each immersion period (24, 72 and 168 h), different crystalline appearances were observed. At 24 and 72 h,

WR had elongated crystals, BD and BR had globular and cubic crystals (Figure 3).

*3.3. Scanning Electron Microscope (SEM)*

*3.3. Scanning Electron Microscope (SEM)*

#### *3.3. Scanning Electron Microscope (SEM)*

The crystalline structures of the three cements are shown in Figures 3 and 4. All three cements had crystalline deposits after immersion in PBS at 37 ◦C. At each immersion period (24, 72 and 168 h), different crystalline appearances were observed. At 24 and 72 h, WR had elongated crystals, BD and BR had globular and cubic crystals (Figure 3). *Bioengineering* **2022**, *9*, x FOR PEER REVIEW 6 of 11

**Figure 3.** Scanning electron microscope images (5000× magnification) demonstrate the morphological changes of each material at 24 and 72 h of immersion in phosphate-buffered solution at 37 °C. **Figure 3.** Scanning electron microscope images (5000× magnification) demonstrate the morphological changes of each material at 24 and 72 h of immersion in phosphate-buffered solution at 37 ◦C. P and Si among the three materials. Other chemical elements were detected on WR (Zr) and BR (Al) surfaces.

**Figure 4.** Scanning electron microscope images (5000× magnification) demonstrate the morphological changes and EDX spectrums of each material at 168 h of immersion in phosphate-buffered solution at 37 °C. **Figure 4.** Scanning electron microscope images (5000× magnification) demonstrate the morphological changes and EDX spectrums of each material at 168 h of immersion in phosphate-buffered solution at 37 ◦C.

**Figure 4.** Scanning electron microscope images (5000× magnification) demonstrate the morpholog-

lution at 37 °C.

After 168 h, BR and BD showed cubic crystals. The cubic crystals of BR were larger (8–10 µm) than for BD (3–6 µm). WR had globular and elongated crystal features at 168 h. EDX analysis for the three cements after 168 h in PBS showed different percentages of Ca, P and Si among the three materials. Other chemical elements were detected on WR (Zr) and BR (Al) surfaces.

#### *3.4. Roughness and Water Sorption Tests*

BR demonstrated the highest hydrophilicity for a 5 µL of a drop of distilled water compared to BD and WR. Contact angles of 15◦ and 9◦ for BD and WR, respectively, were investigated after 10 s (Table 2). Whereas, the water sorption in the BR surface was faster (<10 s) than the other cement surfaces. The contact angle of the drop in contact with BR surface after 10 s was 0◦ (Figure 5). All tested cement surfaces were analyzed using KEYENCE 7000 VHX to measure the roughness of these surfaces. In addition, rougher surfaces were obtained for BR and BD compared with WR surfaces (*p* < 0.05) (Table 2 and Figure 5). *Bioengineering* **2022**, *9*, x FOR PEER REVIEW 7 of 11

> **Table 2.** Contact angles of 5 µL of distilled water on the different material surfaces after 10 s of deposition. Mean and standard deviations of the roughness (Sa) of the tested materials. Superscript letters a, b, c and x, y, z indicate statistical significance (*p* < 0.05). *3.4. Roughness and Water Sorption Tests* BR demonstrated the highest hydrophilicity for a 5 µL of a drop of distilled water com-

**Figure 5.** Contact angles of 5 µL of water drop on the different cement surfaces after 10 s. Digital micrographs of the different surfaces using KEYENCE 7000 VHX demonstrate the roughness of each material. **Figure 5.** Contact angles of 5 µL of water drop on the different cement surfaces after 10 s. Digital micrographs of the different surfaces using KEYENCE 7000 VHX demonstrate the roughness of each material.

#### **Table 2.** Contact angles of 5 µL of distilled water on the different material surfaces after 10 s of *3.5. Antimicrobial Activity*

*3.5. Antimicrobial Activity*

deposition. Mean and standard deviations of the roughness (Sa) of the tested materials. Superscript letters a, b, c and x, y, z indicate statistical significance (*p* < 0.05). **Test\Materials Biodentine MTA Biorep Well-Root PT Statistical**  Bacterial growth was significantly inhibited with the three cements. No significant difference was found among them for the efficiency against *E.faecalis* (*p* > 0.05). The three cements killed about 50% of the *bacteria* after 24 h, versus the control (*p* < 0.05) (Figure 6).

Roughness (Sa) 0.7 ± 0.05 a 0.9 ± 0.2 b 0.3 ± 0.02 c *p* < 0.05

difference was found among them for the efficiency against *E.faecalis* (*p* > 0.05). The three cements killed about 50% of the *bacteria* after 24 h, versus the control (*p* < 0.05) (Figure 6).

**Significance**

y 8.9 ± 0.4 z *p* < 0.05

Contact angle (°) 15.2 ± 3.5 x 0

**Figure 6.** Number of colony-forming units/mL of *Enterococcus faecalis* in contact with Biodentine **Figure 6.** Number of colony-forming units/mL of *Enterococcus faecalis* in contact with Biodentine (BD), MTA Biorep (BR) and Well-Root PT (WR) after 24 h at 37 ◦C in anaerobic conditions. \* *p* < 0.05.

(BD), MTA Biorep (BR) and Well-Root PT (WR) after 24 h at 37 °C in anaerobic conditions. \* *p* < 0.05**.**

#### **4. Discussion**

**4. Discussion** Since their introduction in the dental market, calcium silicate cement materials have attained popularity due to their excellent physicochemical, biological and mechanical properties and their positive outcomes in clinical applications [9,28]. Calcium silicate cement products are the ideal dentine repair materials for various endodontic applications [2,3,17,20,29]. A number of investigations have been conducted to determine the differ-Since their introduction in the dental market, calcium silicate cement materials have attained popularity due to their excellent physicochemical, biological and mechanical properties and their positive outcomes in clinical applications [9,28]. Calcium silicate cement products are the ideal dentine repair materials for various endodontic applications [2,3,17,20,29]. A number of investigations have been conducted to determine the differences among the products as retrograde bioactive material.

ences among the products as retrograde bioactive material. Our present in vitro study comparing BR, BD and WR showed significant antibacterial activity and formation of crystals on their surfaces after immersion in PBS. Therefore, Our present in vitro study comparing BR, BD and WR showed significant antibacterial activity and formation of crystals on their surfaces after immersion in PBS. Therefore, the null hypothesis was partially rejected.

the null hypothesis was partially rejected. Alkaline pH was detected with the three materials (Figure 1), but BD had a lower pH than WR and BR (*p* < 0.05). The alkalinity is key to the antibacterial activity and healing process [8,30–32]. Kharouf et al. [10] measured a high alkaline pH with MTA Biorep. Oliveira et al. [33] measured a lower pH for BD (pH = around 6–7) after 24 h than the one attained our study (pH = around 9–10), whilst Hassan et al. [34] measured higher pH values (pH = around 11–12) for BD after 24 h. The differences may be related to the methods of exposing the materials. The premixed bioceramic cement (WR) created a similar pH to Alkaline pH was detected with the three materials (Figure 1), but BD had a lower pH than WR and BR (*p* < 0.05). The alkalinity is key to the antibacterial activity and healing process [8,30–32]. Kharouf et al. [10] measured a high alkaline pH with MTA Biorep. Oliveira et al. [33] measured a lower pH for BD (pH = around 6–7) after 24 h than the one attained our study (pH = around 9–10), whilst Hassan et al. [34] measured higher pH values (pH = around 11–12) for BD after 24 h. The differences may be related to the methods of exposing the materials. The premixed bioceramic cement (WR) created a similar pH to the powder–liquid BR cement.

the powder–liquid BR cement. In the present study, the solubility of the three cements did not exceed the 3% mass after 24 h in distilled water; however, the ISO 6876 was not used. The results of the present study agree with those of the Al-Sherbiny study [35] for BD results and with the study of Queiroz et al. [36] for MTA Repair HP. The premixed bioceramic (WR) had a solubility similar to that of the other two cements (Figure 2). Solubility is important because if it is high, voids and gaps may be formed, which would be a pathway for the microorganisms to re-infect the root canal system [8,27]. BD demonstrated lower pH values than the other products, but the solubility of BD was higher. Weckwerth et al. [37] noted that a higher solubility does not always correlate with higher pH. The cement may release other com-In the present study, the solubility of the three cements did not exceed the 3% mass after 24 h in distilled water; however, the ISO 6876 was not used. The results of the present study agree with those of the Al-Sherbiny study [35] for BD results and with the study of Queiroz et al. [36] for MTA Repair HP. The premixed bioceramic (WR) had a solubility similar to that of the other two cements (Figure 2). Solubility is important because if it is high, voids and gaps may be formed, which would be a pathway for the microorganisms to re-infect the root canal system [8,27]. BD demonstrated lower pH values than the other products, but the solubility of BD was higher. Weckwerth et al. [37] noted that a higher solubility does not always correlate with higher pH. The cement may release other components, which do not have any effect on pH changes and the liberation of these components increases the solubility of this material.

ponents, which do not have any effect on pH changes and the liberation of these components increases the solubility of this material. The direct contact test was used in this in vitro study to evaluate the antibacterial activity of the different cements. The agar contact test was not used, because in our previous study [8], we noted that these cements infiltrate the agar plates and hide the inhibition zones. *E. faecalis* was used in our experiment because this Gram-positive facultative anaerobe microorganism is the most predominant *bacterium* found in root canal infections and failure [8,38–41]. No significant differences were found among the capacity of killing bacteria of the The direct contact test was used in this in vitro study to evaluate the antibacterial activity of the different cements. The agar contact test was not used, because in our previous study [8], we noted that these cements infiltrate the agar plates and hide the inhibition zones. *E. faecalis* was used in our experiment because this Gram-positive facultative anaerobe microorganism is the most predominant *bacterium* found in root canal infections and failure [8,38–41]. No significant differences were found among the capacity of killing bacteria of the three cements (*p* > 0.05). All the materials demonstrated high potential of killing

bacteria after 24 h (kill around 50%) compared to the control group (bacterial medium). The antibacterial activity of these cements comes from the high alkaline pH [8,10,16,42,43].

All the three cements had different crystalline features (Figures 3 and 4) after immersion in PBS. Cubic crystals were observed on BR and BD samples after 7 d of immersion in PBS (Figure 4). The crystallites of BD were more numerous and smaller than BR crystallites. Elongated crystals were observed on WR surfaces. Yoo et al. [44] showed the importance of biomineralization to entomb the microorganisms in dentinal tubules, since the elimination of 100% of bacteria from the root canal system is impossible [45,46]. EDX analysis showed different chemical compositions of formed crystals onto each cement. Ca, Si and P were detected on the surfaces of the three materials, which reflects the reactions between calcium silicate and PBS. Zr presented onto WR and Al onto BR surface due to the initial composition which contain Zirconium oxide and Tricalcium aluminate, respectively. Al was not detected on WR surface which is a calcium aluminosilicate compound. Therefore, EDX could be considered as a qualitative method and the composition of these crystals could not be identified without X-ray diffraction analysis, which could be considered as limitation of this in vitro study.

A contact angle test was used to determine the capacity of absorption of 5 µL drops of distilled water. This test is an indicator of the wetting behavior of a solid material (cement) and a liquid (water). Contact angle measurement is affected by the surface roughness [47] and the chemical surface composition [48]. The roughnesss of WR surface was less than that of BR and BD (*p* < 0.05), which could be related to the particles size of each cement. After 10 s, the 5 µL drop was totally absorbed by the surface of BR which had the higher roughness values compared to BD and WR (*p* < 0.05). Whatever the considered wetting model (Wenzel or Cassie–Baxter) [48], the higher roughness and hydrophilic surface would increase the adhesion, protein adsorption and the cellular attachment, and provide a superior biocompatibility [48–50]. In contrast, a decrease in cell proliferation and growth could be related to a critical roughness ration, where the elastic energy of the cell hinders the insertion of the cells into surface trenches, where cells install over the tips of the rough surfaces leading to only point-contact, which minimizes cell–surface interaction [51].

Further studies are required to investigate the cytotoxicity, the setting time, the flowability, calcium ions releasing and the filling ability of the novel premixed cement.

#### **5. Conclusions**

Within the limitations of the present study, the three calcium silicate cement products, MTA Biorep, Biodentine and Well-Root PT, had a high antibacterial activity, formation of phosphate crystal in PBS alkaline and had comparable solubility. The premixed format was more convenient as a retrograde agent.

**Author Contributions:** Conceptualization, N.K.; methodology, T.A.; software, J.Z.; validation, L.H., R.B., S.A., Y.H. and S.A.-A.; formal analysis, V.M. and J.Z.; investigation, T.A.; resources, T.A.; data curation, T.A.; writing—original draft preparation, D.M., N.K. and T.A.; writing—review and editing, N.K. and T.A.; funding acquisition, N.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** We acknowledge the INSERM UMR\_S 1121 laboratory for providing the experimental setups.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**

## *Review* **Peptides in Dentistry: A Scoping Review**

**Louis Hardan 1,† , Jean Claude Abou Chedid 2,†, Rim Bourgi 1,3 , Carlos Enrique Cuevas-Suárez 4,\* , Monika Lukomska-Szymanska 5 , Vincenzo Tosco 6 , Ana Josefina Monjarás-Ávila 4 , Massa Jabra 7 , Fouad Salloum-Yared 8 , Naji Kharouf 3,9,\* , Davide Mancino 3,9,10 and Youssef Haikel 3,9,10,\***

**Abstract:** Currently, it remains unclear which specific peptides could be appropriate for applications in different fields of dentistry. The aim of this scoping review was to scan the contemporary scientific papers related to the types, uses and applications of peptides in dentistry at the moment. Literature database searches were performed in the following databases: PubMed/MEDLINE, Scopus, Web of Science, Embase, and Scielo. A total of 133 articles involving the use of peptides in dentistry-related applications were included. The studies involved experimental designs in animals, microorganisms, or cells; clinical trials were also identified within this review. Most of the applications of peptides included caries management, implant osseointegration, guided tissue regeneration, vital pulp therapy, antimicrobial activity, enamel remineralization, periodontal therapy, the surface modification of tooth implants, and the modification of other restorative materials such as dental adhesives and denture base resins. The in vitro and in vivo studies included in this review suggested that peptides may have beneficial effects for treating early carious lesions, promoting cell adhesion, enhancing the adhesion strength of dental implants, and in tissue engineering as healthy promotors of the periodontium and antimicrobial agents. The lack of clinical trials should be highlighted, leaving a wide space available for the investigation of peptides in dentistry.

**Keywords:** antimicrobial; osseointegration; surface modification; tissue engineering

## **1. Introduction**

Dental plaques contain over 750 different bacterial species, which are the major reason for dental caries, with streptococci being the most predominantly present. These bacteria, due to the production of acids, can demineralize and affect mineralized tooth tissues [1]. Different additives and biomaterials were used in dental treatments in order to eliminate and decrease the number of bacteria in the oral cavity and teeth tissues. Some dental materials, such as calcium silicate-based products, have been introduced in the dental market due to their antibacterial, antioxidant and remineralization properties [2]. Other solutions

**Citation:** Hardan, L.; Chedid, J.C.A.; Bourgi, R.; Cuevas-Suárez, C.E.; Lukomska-Szymanska, M.; Tosco, V.; Monjarás-Ávila, A.J.; Jabra, M.; Salloum-Yared, F.; Kharouf, N.; et al. Peptides in Dentistry: A Scoping Review. *Bioengineering* **2023**, *10*, 214. https://doi.org/10.3390/ bioengineering10020214

Academic Editors: Francesca Scalera and Chengfei Zhang

Received: 18 January 2023 Revised: 2 February 2023 Accepted: 3 February 2023 Published: 6 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

60126 Ancona, Italy

that have antibacterial effects are used to clean the root canal and kill resistant bacteria in the root canal system [3]. Even though they sometimes display high cytotoxicity [4,5], these materials are still currently used in dentistry.

It should be remembered that a peptide is expressed as a short polymer of amino acids (AA) [6]. According to the description of diverse authors in the literature, sizes of peptides may vary from <20, <50, to <100 [7–10]. The use of peptides has been paid attention to over the last two decades [6,11]. These peptides were used in various dental fields such as in endodontic treatment, coronal restoration, caries management, bone and dental tissue remineralization and in the modification of dental materials in order to promote the biological effects of these materials in the oral environment [6,12].

In the last periods, over 7000 native peptides (NP) have been considered by means of significant human physiological functions [13]. These peptides have functions by way of cell-penetrating, cell adhesion motifs, tumor-homing peptides, neuropeptides, structural peptides, peptide hormones, antimicrobial peptides, peptide tags, matrix metalloprotease substrates, growth factors, amyloid peptides, and erstwhile diverse NPs [10].

Nevertheless, one should state that NPs are frequently not truthfully appropriate for therapeutic usage since they have intrinsic drawbacks, including their poor physical and chemical stability, low oral bioavailability, short flowing plasma half-life, and quick removal from the circulation through the kidneys and the liver [9,13,14]. It is well described that peptides such as insulin and adrenocorticotrophic hormone were used for human therapeutic purposes in the first half of the 20th century [15]. Later on, synthetic oxytocin and vasopressin arrived in clinical use in the 1950s with the chemical elucidation of the sequences of these peptides [16].

Lately, pharmaceutical manufacturing has amplified the consideration of novel therapeutic peptides, persistently touching clinical claims [9,17]. By 2018, more than 60 peptides were approved by the Food and Drug Administration (FDA), and more than 600 were undergoing preclinical and clinical examinations [9,18]. With the current elaborations of solid-phase peptide synthesis, the production of therapeutic synthetic peptides (SP) has become achievable [9]. Accordingly, innovative synthetic approaches permit the modulation of pharmacokinetic assets and focus on specificity through AAs, the integration of non-natural AAs, backbone adjustments, and the peptide conjugates refining solubility or prolonging the half-life [8,13,14].

It is recognized that human dental masses, once fashioned, cannot be biologically replaced or repaired, and their multifaceted conformations require diverse approaches for regeneration [6]. However, it is unclear in the literature which specific peptides could be effective for applications in different fields of dentistry. Thus, the aim of this scoping review was to map the contemporary scientific papers related to the use and applications of peptides in dentistry at present.

#### **2. Materials and Methods**

The present scoping review has been described according to the PRISMA extension for scoping reviews guideline [19]. The review protocol was registered at Open Science Framework, and it is available at https://osf.io/up6ty (accessed on 18 December 2022). The systematic search was performed according to the following parameters: (i) population: peer-reviewed articles; (ii) intervention: use of natural or synthetic peptides; (iii) comparison: other substances or treatments; (iv) outcome: dental applications, (v) study design: in vitro or in vivo articles. The general question of the review was as follows: what scientific applications of products based on peptides are being used for dental applications?

#### *2.1. Information Sources and Search*

The literature database search was performed by two independent reviewers (RB and CECS) until September 2022. The search was carried out in the following databases: PubMed/MEDLINE, Scopus, Web of Science, Embase, and Scielo. The search strategy was first defined for the MEDLINE database using a controlled vocabulary and free keywords

(Table 1). The MEDLINE search strategy was then adapted to other electronic databases. The reviewers also hand-searched the reference lists of the included articles to identify additional manuscripts.

**Table 1.** Search strategy used in the MEDLINE database.

(Peptide) OR (Polypeptides) OR (Polypeptide) AND (Materials, Dental) OR (Dental Material) OR (Material, Dental)

## *2.2. Selection Process and Data Collection Process*

After running the search strategy, a reference management program was used (End-Note X9, Clarivate Analytics, Philadelphia, PA, USA) to store the files of all databases. Then, duplicate articles were removed, followed by manual removal after the organization of titles in alphabetical order. All studies were initially scanned for relevance by title followed by abstract using an online software program (Rayyan, Qatar Computing Research Institute, HBKU, Doha, Qatar). The titles and abstracts of the articles were screened according to the following inclusion criterium: in vitro or in vivo studies that evaluated or reported the use of peptides for dental applications. The search was carried out on documents published in any language without restrictions on their date of publication. Reviews, case reports, case series, pilot studies, and conference abstracts were excluded. If the review authors were not sure about the eligibility of any study, it was kept for the next phase. All phases were carried out by two independent reviewers (RB and CECS) to check whether they met the inclusion criteria. The same two reviewers summarized and categorized the data using a standardized form. The information collected included the type of study, the peptide used, the application proposed and the main results.

#### **3. Results**