89 CPC/csDCA/BMP-2 able to induce more dentin formation when compared to the control

groups; 3) is CPC/csDCA/BMP-2 degrading faster when compared to the control groups. The needed sample size was calculated through G*Power software (Erdfelder, Faul, & Buchner, 1996) [32] by using a significance level of 0.05 and 80% power. A standard deviation (SD) of 25% and effect size (d) of 40% were defined based on previous studies related to dentin regeneration [33, 34]. A split-mouth design was used [35]. This usually enhances the power of the study, but the extent of that increase could not be readily estimated. Therefore, the number of animals used was the one obtained in the conservative power calculation. The lower four incisors of each animal were distributed to the four experimental groups (i.e. empty defect, pulp capping with CPC, pulp capping with CPC/csDCA and pulp capping with CPC/csDCA/BMP-2) through a block randomization approach (n = 6 for all the experimental groups, see Table 1). Allocation of the animals to each split-mouth group was also performed through a simple randomization method (i.e. the first animal picked was named as Animal 1, etc.). The study population, used for in vivo direct pulp capping, consisted of 6 healthy, 18 months old, Habsi male goats. At this age the four lower goat incisors are all erupted and have a similar morphology, reducing the inter-group variability [36]. Pulp capping was performed by a single expert operator that was trained in the standardization of the surgical procedure according to previous work [33]. All procedures were performed under general anesthesia induced by intramuscular (IM) injection of ketamine hydrochloride (5 mg/kg) and diazepam (1 mg/kg). To reduce the risk of infection, animals were receiving antibiotics pre-operatively (10 mg/kg intravenously; Amoxicillin®), and post-operatively, at day 1 and day 3 (50 mg/kg intramuscularly; Albipen®). To alleviate pain analgesic Finadyne® (1mg/kg) was given three times a day. Animals were immobilized in a ventral position. The oral tissues were disinfected with 10% Povidone-iodine. Afterward, local anesthesia (Lidocaine 2% with 1:100,000 Epinephrine) was injected around the lower incisors. A cavity was drilled on the vestibular surface of the lower incisor by using a sterile round-shaped bur (1 mm in diameter) mounted on conventional dental handpiece with an appropriate speed and irrigation (Supplementary Information, Figure S1b). A cylindrical defect (about 1 mm in diameter and 2 mm in length) was prepared through the full enamel, dentin and pulp cavity until the dentin on the lingual side was reached (Figure S1c). In this way, pulp tissue along the direction of the defect was also ripped out from the pulp canal (i.e. partial pulpotomy). During the dental procedure, a sterile 0.9% saline solution was used to cool down the drill and to remove the debris. A cotton pellet was placed over the defect for 2-3 minutes to control the pulp hemorrhage until the bleeding was completely controlled.

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Pulp capping was performed using one of the experimental cement pastes inserted in the prepared cavity with a passive application by means of a Dycal® placement instrument (Dentsply Sirona, York, PA). Light pressure was applied using a wet cotton pellet to ensure that the paste would reach the deepest part of the cavity. Also, one group was included leaving the cavities empty (n=6). Due to the chosen experimental groups, the main operator was only partially blinded. Although the cement pastes were prepared by another assistant operator, the difference in color between the pastes (i.e. white color for the CPC and brown color for the CPC/csDCA and the CPC/csDCA/BMP-2) hindered a completely blinded procedure. Still, the main operator was not able to recognize CPC/csDCA from CPC/csDCA/BMP-2 pastes. Two primary outcomes were evaluated during the surgical procedure, i.e. the ability to handle the prepared paste with a specific dental instrument (Dycal® placement instrument), and the ability of the paste to seal the defect. A defined score (i.e. very poor, poor, good, and very good) was attributed by the operator to each specimen. After material placement, all cavities were filled with light cured (LC) glass ionomer cement (GC Fuji II LC® CAPSULE, Cavex) (Figure S1e and S1f). Animals were housed together on a goat farm. Seven weeks post-surgery animals were euthanized by an overdose of Nembutal® (Apharmo, Arnhem, the Netherlands). Lower incisors were extracted and cleaned from the soft tissue. The apical part of the incisors was removed to allow fixative (i.e. 10% Formalin for one week) to flow through the tissues.

2.8. Ex vivo µCT

All specimens (i.e. human and goat teeth from ex vivo study and goat incisors from in vivo study) were wrapped in Parafilm® (SERVA Electrophoresis GmbH, Heidelberg, Germany) and scanned vertically along the direction of the X-ray beam by means of a micro-computer tomography imaging system (Skyscan 1072, Kontich, Belgium). The following parameters were used to get a two-dimensional (2D) reconstruction: X-ray source 100 kV/98 µA, exposure time 3.9 s and 1 mm Aluminium filter. Human molars were scanned using X15 magnification (pixel resolution = 18,88 µm). Goat incisors magnification was set at X25 (pixel resolution = 11,10 µm). Gray values of a defined volume of interest (VOI) were analyzed by CTAnalyser software (version 1.10.1.0, Skyscan) while two-dimensional reconstruction was obtained by DataViewer software (Skyscan 1.5.2.4).

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Table 1. Randomization scheme of the implanted experimental groups and illustration of the

goat lower incisors with individual tooth number.

New dentin formation and cement degradation were both quantified based on µCT acquisitions by using CTAnalyser software (Skyscan). Discrimination between reactionary and reparative dentin was not possible from µCT acquisitions because of imaging resolution limits. Therefore, both kinds of dentin were considered together. Briefly, a cylinder of 1 mm in diameter and 2 mm in height was selected and located in the proximity of the formed dentin

cap. Relative volume was independently quantified using a standardized gray value threshold

and expressed in percentage. For the cement degradation, a cylinder of 2.5 mm in diameter and 1.5 mm in height was selected and placed in proximity of the performed defect. After applying a standardized threshold relative volume was quantified and expressed in percentage. To exclude bias from possible dentin debris left in the defect area after drilling values were normalized by the mean of the dentin-like components volume present in the empty defect group.

2.9. Ex vivo MRI

Prior to the MRI examination, all human and goat teeth specimens (i.e. from ex vivo and in

vivo studies) were embedded in gelatin type A (Sigma-Aldrich) dissolved in MilliQ water

with a final concentration of 5% (wt/v). Thereafter, samples were imaged on an 11.7 Tesla (T) MRI system (Biospec, Bruker) equipped with a proton quadrature volume coil. A zero echo time (ZTE) sequence was employed for the image acquisition. The corresponding scan parameters were repetition time (TR) = 2 ms, flip angle (FA) = 3°, averages = 3, acquisition

Incisor 42 Incisor 41 Incisor 31 Incisor 32

Animal 1 Animal 2 Animal 3 Animal 4 Animal 5 Animal 6

CPC/csDCA CPC/csDCA CPC Empty defect BMP-2

Empty defect CPC/csDCA CPC/csDCA CPC BMP-2

CPC Empty defect CPC/csDCA CPC/csDCA BMP-2

CPC/csDCA CPC Empty defect CPC/csDCA BMP-2 CPC/csDCA CPC/csDCA CPC Empty defect

BMP-2

Empty defect CPC/csDCA CPC/csDCA CPC BMP-2

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time (TA) = 27.34 minutes, matrix size = 256 x 256 x 256 and image resolution = 0.16 mm3. The acquired images were analyzed using OsiriX software (Pixmeo, Bernex, Switzerland). Qualitative evaluation of the MR images was performed to assess signal enhancement, pulp tissue integrity and new dentin formation.

2.10. Histological analysis

After fixation, goat incisors from the in vivo study were dehydrated in a graded series of ethanol (70-100%) and embedded in poly(methyl methacrylate) resin. Cross-sections of approximately 10 µm were prepared along the sagittal direction of the tooth. At least three sections per sample were obtained and stained with Methylene Blue and Basic Fuchsin. Images were acquired using a light microscope (A io Imager Z1, Zeiss, Gӧttingen, Germany) equipped with a digital camera (AxioCam MRc5, Zeiss). Micrographs were obtained at X1, X10 and X20 magnification. Qualitative evaluation of the acquired micrographs was executed independently by three different operators. Tertiary dentin was classified as reactionary dentin or reparative dentin as described elsewhere [6]. Briefly, dentin showing tubular structures in continuity with the pre-existing secondary dentin was classified as reactionary dentin, while atubular calcified scar tissues were classified as reparative dentin.

2.11. Statistical analysis

Statistical analysis was performed by using IBM SPSS Statistic 22.0 software (IBM Corporation, Armonk, NY). Data were reported as mean ± standard deviation. Statistical significant differences in the mechanical properties were investigated by using a Student-t-test with Welch’s correction. Differences in setting times, in gray values, by one-way analysis of variance (ANOVA) with Tukey post hoc test. Differences in dentin formation and cement degradation were analyzed by using a Friedman`s analysis of variance by ranks. All differences were considered significant at p-values <0.05.

3. Results

3.1. Morphological characterization

Scanning electron micrographs of the CPC components (i.e. α-TCP, CMC and PLGA) are reported in the supplementary information (Figure S2). Core-shell DCA particles are shown in Figure 1a. On the external surface of the particles, it was possible to identify apatite-like structures as result of the coating step during the manufacturing process. TEM section images of the csDCA showed finest details on the complex structure of the particles. Specifically, a shell was distinguishable as a dark ring on the external side of the particles, which

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