2.1

Ex-vivo human tooth-culture model

2.1.1

Teeth sampling and culturing

The samples were gathered after approval by the Commission for Medical Ethics of KU Leuven (file number S54254) and following informed consent from the donors. Fully impacted human third molars with their roots only partially formed, were collected immediately after extraction from three healthy young patients (15–17 years). A total of 9 teeth were used in this experiment. In the surgery room, the teeth were placed immediately after extraction in 50 ml centrifuge tubes (VWR, Leuven, Belgium), containing Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Merelbeke, Belgium) supplemented with 10% Fetal Bovine Serum (FBS; Gibco), 1% penicillin-streptomycin (Gibco) and 1% amphotericin B (Gibco) (tooth-culture medium). The samples were brought to the cell-culture room within 4 h to proceed with the cell-culture experiments. From each patient, one tooth was kept as negative control (n = 3; exposure without capping), while the other teeth were capped with PPL_hCSC (n = 3) or Biodentine (n = 3) and kept in tooth-culture medium for 4 wk, as described below.

Once in the cell-culture room, all teeth were rinsed with 70% ethanol (Hydral 70, VWR) for 1 min, followed by rinsing with sterile Phosphate Buffered Saline (PBS; Sigma Aldrich, St. Louis, MO, USA) for 1 min, upon which they were then placed in tooth-culture medium until further processed. The periodontal ligament was removed with a sterile #15 scalpel blade (Swann Morton, Sheffield, UK). After cleaning, the teeth were handled with sterile gauzes (Yibon Medical, Kuurne, Belgium) and soaked in tooth-culture medium to avoid desiccation.

2.1.2

Ex-vivo human tooth-culture pulp-capping assay

Nine teeth from three different patients were gathered and handled as described above. Once the teeth were cleaned, a pulp-capping procedure was performed in sterile conditions and with the aid of 2.8× magnification, similarly as done clinically. For the pulp-capping assay, we followed the protocol described by Téclès et al. [ ]. Briefly, a class-I cavity (approx. 4 × 4 × 4 mm) was cut using a sterile bur (1.1 mm in diameter; Endo Access Bur Size 1, A 0164 300 001 00, Dentsply Sirona, Ballaigues, Switzerland) at high speed under copious irrigation with sterile saline (Fresenius Kabi, Bad Homburg, Germany). The pulp was exposed with a round carbide bur (1.0 mm in diameter; H1SE.205.010, Komet, Lemgo, Germany) at low speed with abundant irrigation. Afterwards, the cavity was cleaned with sterile saline and gently dried with sterile cotton pellets.

The teeth were divided into three groups depending on the pulp-capping procedure carried out: (1) application of the experimental phosphopullulan-functionalized hCSC ‘(PPL_hCSC’; n = 3; GC), (2) application of the commercial hCSC Biodentine (n = 3; Septodont), or (3) exposure without capping as negative control (n = 3). The composition and application mode of the two pulp-tissue engineering agents investigated are detailed in Table 1 .

PPL_hCSC and Biodentine were applied in a 2−3 mm layer following the manufacturers’ recommendations and gently compacted with sterile cotton pellets. The cavity was restored with glass-ionomer cement (Fuji II LC Capsules, GC). Next, flowable composite (G-aenial Flo, GC) was applied on the occlusal surface, in which a sterilized stainless steel orthodontic wire (M form; ORMCO, Orange, CA, USA) was seated, followed by 40-s light-curing of the flowable composite using a light-curing unit with a light output of 1200 mW/cm ² (Bluephase 20i, Ivoclar Vivadent, Schaan, Liechtenstein). The teeth were immediately hanged using the wire in separate wells of 24-well culture plates (Costar, Cambridge, MA, USA), each containing 1.5 ml of tooth-culture medium to ensure generous exposure of the pulp tissue to the medium. The medium was refreshed every day and after four weeks the wire was removed and the teeth immediately placed in 4% PFA for two weeks to properly fix the tissue.

2.1.3

Ex-vivo human tooth-culture model – histology

The chemically fixed teeth were demineralized (4–6 weeks) with 10% formic acid (Chem-Lab Analytical, Zedelgem, Belgium) with the decalcifying solution refreshed every 3 days. The decalcification endpoint was determined by dental radiography (MINRAY, Soredex, Tuusula, Finland) and visual/tactile evaluation. After decalcification, the glass ionomer and composite restorations were manually removed, upon which the teeth were subsequently immersed in water for 24 h and then dehydrated in ascending concentrations of ethanol (70% for 12 h, 80%, 95% and 100% for 2 h each), followed by xylene (VWR) clearance. The teeth were next immersed in liquid paraffin (56 °C melting point; Paraclean, Klinipath, Duiven, The Netherlands) for 24 h prior to being embedded in paraffin blocks. Serial paraffin sections were cut with a thickness of 5−7 μm using a microtome (Microm HM 360 Microtome, Hyland Scientific, Stanwood, WA, USA), this from the level where the interaction of the pulp-capping agent and the pulp tissue first appeared until any interaction was no longer visible. Every six sequential sections, two sections were randomly selected for staining. One section was stained with Gill’s III hematoxylin (Leica Microsystems, Diegem, Belgium) and with 1% aqueous eosin solution (Leica Microsystems), while the other section was stained with Gram-Twort staining to detect the presence of bacteria. The stained sections were examined using light microscopy (Axio Imager M2, Carl Zeiss Microscopy, Jena, Germany). The formation/presence of a fibrous band and mineralized tissue around the exposed area was evaluated by two independent researchers.

2.2

Animal experimentation

2.2.1

Minipigs

All experimental protocols complied with the ARRIVE guidelines and were approved by the Ethical Committee for Animal Experimentation of KU Leuven under the file number P016/2015. The procedures were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and EU Directive 2010/63/EU for animal experiments. Three female Göttingen minipigs (Ellegaard, Dalmose, Denmark) at the age of 33–35 months and with a weight of 44−60 kg and intact permanent dentitions were group-housed on wood shavings with ad libitum access to water; they were fed twice a day with a minipig special diet (Welzijnskorrelminivarkens, AVEVE, Wilsele, Belgium).

2.2.2

Direct pulp capping

A pre-operative computed tomography (CT) scan (SOMATOM Force, Siemens Healthcare, Erlangen, Germany) of the mandible and maxilla of each minipig was taken in order to confirm that all teeth were in a healthy state and the roots were fully developed. Direct pulp capping was carried out at two times, resulting in a 7-day and 70-day post-operative observation period, as recommended by the ISO 7045-2008 standard [ ]. Twenty permanent teeth (5 incisors, 2 canines, 8 premolars and 5 molars) on the left side were capped in the first phase (70-day post-capping observation). Sixty-three days later, 20 permanent teeth (7 incisors, 2 canines, 6 premolars and 5 molars) on the right side received a pulp-capping treatment (7-day post-capping observation).

Animals were anesthetized with a combination of xylazine (1 mg/kg; 2% Xyl-M, VMD, Arendonk, Belgium) and zolazepam/tiletamine (2.5−3 mg/kg; Zoletil 100, Virbac, Fort Worth, TX, USA). After endotracheal intubation, general anesthesia was maintained with isoflurane (1–1.5%); then, the animals were ventilated with a tidal volume of 8−10 ml/kg at a frequency of 10–12 times per min to preserve normocapnea. Prior to direct pulp capping, the teeth were ultrasonically cleaned (MiniMaster Ultrasonic Scaler; Electro Medical Systems (EMS), Nyon, Switzerland), polished, and disinfected with 10% povidone iodine (iso-Betadine Dermicum, Meda Pharma, Belgium). Under local anesthesia using 2% lidocaine (1 ml per tooth), class-V butt-joint cavities were prepared on the buccal surface of incisors, canines and premolars, whereas class-I cavities were cut on the occlusal surface of molars. The cavities were prepared using a sterile round diamond bur (1.1 mm in diameter; Endo Access Bur size 1, Dentsply Sirona) at ultra-high speed with copious sterile saline irrigation. Subsequently, the pulp tissue was mechanically exposed using a sterile round carbide bur (1.0 mm in diameter; H1SE.205.010, Komet) mounted on a dental hand-piece at high speed under sterile saline (Fresenius Kabi) cooling. The cavities were rinsed with sterile saline (Fresenius Kabi) and hemorrhage was controlled using sterile wet cotton pellets with light pressure, upon which the cavities were dried with sterile dry cotton pellets.

All teeth receiving pulp-capping treatment were randomly divided into 2 groups. For PPL_hCSC, 12 teeth consisting of 3 incisors, 1 canine, 5 premolars and 3 molars were selected. For the commercial reference cement Biodentine (Septodont), 8 teeth (2 incisors, 1 canine, 3 premolars and 2 molars) were chosen. The pulp-capping agents were applied with a thickness of approximately 2 mm. PPL_hCSC was prepared by activating the capsule followed by mixing for 10 s with a capsule mixer (RotoMix Capsule Mixer, 3 M Oral Care, Seefeld, Germany). For Biodentine (Septodont), 5 drops of liquid were added to the powder capsule, upon which the capsule was immediately mixed for 30 s with the mixer (Rotomix, 3 M Oral Care). Once the pulp-capping agents were placed following the manufacturers’ recommendations and gently compacted with sterile cotton pellets, a layer of resin-based glass-ionomer cement (Fuji II LC Capsule, GC) was applied. The cavities were finally restored with a universal adhesive (G-Premio Bond, GC) and a light-curing composite (G-aenial Posterior, GC). During restorative procedures, humidity was controlled with sterile cotton rolls in combination with oral suction. Soft diet was provided post-operatively and no anti-inflammatory medication appeared necessary for any of the three animals.

Sixty-three days after the first phase, the same dental procedures were performed in the teeth on the right side. The animals were euthanized 7 days after the second phase using an overdose of barbiturates (400 mg/ml; Euthasol, Le Vet, Oudewater, The Netherlands). The mandible and maxilla were simultaneously fixed by immersion in 4% paraformaldehyde solution (VWR) via a closed head-only perfusion system with inflow through the carotid artery and outflow via the jugular vein [ ]. The teeth were removed individually from the jaw using an electric saw (HB 8894, HEBU Medical, Tuttlingen, Germany) under copious water irrigation and afterwards immediately fixed in 30 ml of 4% paraformaldehyde solution (VWR) at 4 °C for 2 weeks.

2.2.3

Micro-computed tomography (μCT)

All teeth were scanned by means of a μCT scanner (Skyscan 1172, Bruker MicroCT, Kontich, Belgium) using the following parameters: 100 kV/100 μA X-ray source, 0.5 mm aluminum filter, pixel size of 9.92 μm, averaging frame of 7, rotation step of 0.3°, random movement of 50 and 360° rotation around the vertical axis. The raw data were reconstructed using NRecon v.1.6.9.18 software (Bruker MicroCT), using the same parameters for smoothing, beam hardening and ring artifact corrections.

2.2.3.1

Continuity of mineralized tissue

For exposed pulps capped for 70 days, the presence of radiodensity change in the pulp tissue was qualitatively documented using the Dataviewer software v. 1.5.1.2 (Bruker MicroCT). The radiopaque layer underneath the exposure site, if existed, was further identified as mineralized tissue or not, this using the corresponding histology of the teeth as a reference. The continuity of mineralized tissue was classified as ‘none’, ‘partial’ or ‘complete’, based on the extent of mineralized tissue compared to that of the exposure site.

2.2.3.2

Three-dimensional bridge segmentation and porosity analysis

We used CTAn v.1.13 software (Bruker MicroCT) to define different volumes of interest (VOIs) for each structural feature (dentin, dentin bridge and pulp tissue/pores) by manual definition of the regions of interest (ROIs) using the full-axial slices dataset. Dataviewer software and the corresponding histology were used as support references. An automatic thresholding method (Otsu 2D) was applied for segmenting dentin and the dentin bridge from the pulp tissue/pores. CTVox software (Bruker MicroCT) was used to render the volume and to merge the different structural features on the 3D models.

2.2.4

Animal experimentation – histology

After μCT scanning, the chemically fixed teeth were processed for paraffin-section histology and evaluated using light microscopy (Axio Imager M2, Carl Zeiss Microscopy), as described above for the ex-vivo human tooth-culture pulp-capping assay. The inflammatory response of the exposed pulp as well as the nature of newly formed mineralized tissue were evaluated based on the criteria detailed below.

2.2.4.1

Inflammatory response

The grade of inflammatory response in the pulp tissue, being capped with each pulp-capping agent, was calculated according to the grading scale of inflammatory changes described in the ISO 7045-2008 standard [ ]:

GRADE 0: no inflammation;

GRADE 1: mild inflammation – scattered inflammatory cells in the pulp tissue adjacent to the pulp exposure;

GRADE 2: moderate inflammation – inflammatory cells with small focal groupings in the pulp tissue adjacent to the pulp exposure;

GRADE 3: severe inflammation – extensive inflammatory cell infiltration in the pulp tissue adjacent to the pulp exposure;

GRADE 4: abscess formation or extended inflammatory cell infiltration not only limited to the pulp tissue adjacent to the pulp exposure.

2.2.4.2

Nature of mineralized tissue

The formation/continuity of mineralized tissue was previously assessed by μCT. The nature of mineralized tissue was defined as either ‘immature’, ‘maturing’ or ‘mature’ on basis of the presence of reparative dentin with well-aligned dentin tubules as well as a layer of well-arranged odontoblast-like cells.

2.2.4.3

Bacterial staining

Gram-Twort staining was performed for bacterial recognition.

2.3

Statistical analysis

For the presence of mineralized tissue in the ex-vivo human tooth-culture model among the three different groups, the non-parametric Kruskal–Wallis test was used (Bonferroni correction applied), this at a significance level of p < 0.05. The occurrence of inflammatory response as well as mineralized tissue formation induced by the different pulp-capping agents in vivo were evaluated by two independent observers. In case of disagreement, re-evaluation was done and the more severe evaluation was chosen. The data were statistically compared using the Fisher’s Exact test or Chi-square test of independence, this at a significance level of p < 0.05.