Dental pulp tissue was collected at the Small Animal Clinic, Veterinary Faculty, University of Ljubljana, from the teeth of two client-owned dogs undergoing clinically indicated mandibular canine tooth extraction of endodontally and periodontally healthy teeth (to treat traumatic malocclusion from linguoversion) under general anesthesia. Dogs (a 4-year-old male Poodle and a 7-month-old male Labrador Retriever) were treated by a board-certified veterinary dentist in accordance with the current state-of-the-art guidelines; no changes to the treatment protocols were made for the purpose of the study. Owners provided written informed consent for the procedures. Immediately after surgical extraction (30), all three teeth were disinfected externally (i.e., briefly rinsed in 2% chlorhexidine), the crowns were sectioned under aseptic conditions to expose the pulp chamber, and pulp tissue was retrieved with a sterile barbed broach and transferred into cold Dulbecco’s phosphate-buffered saline (DPBS; Gibco, Grand Island, NY, USA).

The three isolated dental pulp tissue samples were subsequently washed with DPBS (Gibco, Grand Island, NY, USA), cut into small pieces with a scalpel and incubated overnight at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 0.1% collagenase type II (Sigma–Aldrich, Taufkirchen, Germany). The digested tissue was centrifuged at 240 × g for 4 min, after which the supernatant was discarded. The cell pellet was resuspended in cell culture medium supplemented with DMEM, 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% antibiotic (Penicillin: Streptomycin solution 100X, VWR International, Vienna, Austria). The cell suspension was plated into 6-well plates (TPP, Trasadingen, Switzerland) at passage 0 and cultured at 37 °C in a 5% CO₂ incubator. The cell culture medium was changed every 2–3 days. After reaching 70–90% confluence, the cells were trypsinized and multiplied by seeding into a larger (T75) cell culture flask at passage 1. After a sufficient number of cells were obtained, the cells from passage 1 were frozen at −80 °C in cell freezing medium containing 10% dimethylsulfoxide (Sigma–Aldrich, Taufkirchen, Germany). Thawed cells were seeded at passage 2, multiplied and further processed for the expression of surface markers, differentiation potential, and experimental cell cultures (Table 1). Suspension media cultures were used for MTT and live/dead assays, and conditioned media cultures were used for MTT and live/dead assays, Alizarin Red S staining and gene expression analysis.

Flow cytometry was performed on the untreated cells to evaluate the expression of cell surface markers. Antibodies against the MSC markers CD44, CD90, CD29, and CD34 were applied as previously reported for cDPSCs (CD44⁺/CD90⁺/CD29⁺/CD34⁻) (31, 32). A total of 1 × 10⁶ cells were used. Cells frozen at passage 1 were thawed, seeded at passage 2, multiplied, reseeded and analyzed at passage 3. Following trypsinization, the cells were counted, centrifuged (240 × g for 4 min), and washed twice with DPBS. Cells were stained with the following antibodies for canine adipose-derived mesenchymal stem cells (ADMSCs): allophycocyanin (APC) conjugated against CD44 (antibody clone IM7, 103,012, BioLegend, San Diego, CA, USA), phycoerythrin (PE) conjugated against CD90 (antibody clone YKIX337.217, 12–5,900-42, eBioscience, San Diego, CA, USA), fluorescein isothiocyanate (FITC) conjugated against CD29 (antibody clone MEM-101A, MA1-19566, Thermo Fisher Scientific, Waltham, MA, USA), and CD34 (antibody clone 581, 60013FI, Stemcell Technologies, Cambridge, MA, USA). For antibody titration, 1, 2, 3, 4, 5, and 10 μL of each antiserum per 100 μL of 1 × 10⁶ cells was used. Appropriate dilutions of the antibodies used for staining are shown in Table 2. The cells were then vortexed, incubated at room temperature in the dark for 10 min, washed twice with DPBS, vortexed, and centrifuged again (240 × g for 5 min). The supernatant was subsequently decanted. Finally, the cells were resuspended in 100 μL of DPBS for FACS analysis. The exclusion of nonviable cells was performed by staining cells with propidium iodide solution (Molecular Probes, Eugene, OR, USA). Experimental settings were set up using unstained cells and single-color staining. A minimum of 20,000 events was recorded. The cells were analyzed with a BD FACSAria III flow cytometer (BD Bioscience, Franklin Lakes, NJ, USA). FACSDiva 9.4 software (BD Bioscience) was used for FACS data analysis.

For the determination of differentiation potential, untreated cells were used. Differentiation potential was assessed by inducing cell differentiation into osteocytes and chondrocytes. Cells frozen at passage 1 were thawed, seeded at passage 2, multiplied and reseeded at passage 3 for the differentiation assay. For osteogenic differentiation, 4 × 10⁴ cells were seeded in 12-well plates. After 90–100% confluence was reached, the cell culture medium was removed. Osteogenic medium (StemPro Osteogenesis Differentiation Kit, Gibco, Grand Island, NY, USA) was added, and the medium was changed every 2–3 days. The cell culture medium was added to the wells, which served as a negative control. Osteogenic differentiation was analyzed after 14 days of cultivation using Alizarin Red S staining (Sigma Aldrich, Taufkirchen, Germany) according to the standard procedure. For chondrogenic differentiation, micromass cultures were generated by seeding 5 μL droplets containing 4 × 10⁴ cells into the middle wells of a 12-well plate. After the micromass cultures were cultured for 6 h under high humidity, chondrogenic medium (StemPro Chondrogenesis Differentiation Kit, Gibco, Grand Island, NY, USA) was added to the culture vessels. The cell culture medium was added to the wells, which served as a negative control. The micromass cultures were incubated at 37 °C in an incubator with 5% CO₂ and a humid atmosphere. The medium was changed every 2–3 days. Chondrogenic differentiation was analyzed after 14 days of cultivation using Alcian blue staining (Sigma Aldrich, Taufkirchen, Germany) according to a standard procedure. The differentiated cells were visualized under a light microscope.

Three materials were tested on polystyrene-grown cDPSCs: (1) ProRoot^® MTA (Dentsply Sirona, Johnson City, TN, USA) (MTA), a calcium-silicate endodontic cement commonly used for pulp capping and root-end filling; (2) RS + ™ (GenTech – Genuine Technologies d.o.o., a spin-out of Jožef Stefan Institute, Ljubljana, Slovenia) (RS+), a synthetic bioceramic, calcium trisilicate-based powder with small additions of biocompatible phyllosilicate clay (bentonite) and bioactive amorphous calcium silicate for an enhanced handling, setting, and remineralization response, indicated for root canal repair and sealing; and (3) CellFoam™ (BioChange Ltd., Yokneam, Israel) (CF), a commercially available porous, cell-culture-grade biodegradable scaffold.

We prepared two different media, containing experimental materials, as exposure models:

Each biomaterial was suspended in cell culture medium at an initial concentration of 50 mg/mL (defined as the first dilution, D1). Seven additional twofold serial dilutions were prepared (D1–D8). This powder-in-medium setup simulated the immediate, high-exposure environment that may occur after material placement, which is particularly relevant for calcium silicate-based cements (MTA and RS+), which can transiently release Ca(OH)₂, increase the pH, and directly contact surrounding cells with particulate matter.

Each biomaterial was first suspended in culture medium at 50 mg/mL, shaken overnight at room temperature, and centrifuged the following day at 500 × g for 10 min. After the supernatant was collected, we adjusted its pH to 7.5 to isolate material-specific effects from pH-mediated cytotoxicity. To adjust the pH, we used 1 N HCl, as the buffering components of the culture medium were insufficient to counteract the high alkalinity resulting from Ca(OH)₂ release. The supernatant was then filtered through 0.22 μm syringe filters. The resulting conditioned medium was used for experimental cell culture at four twofold serial dilutions (D1–D4).

An MTT assay was employed for the suspension and conditioned media cultures. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to measure cellular metabolic activity as an indicator of the cytotoxicity of the biomaterials. It is based on the reduction of a yellow tetrazolium salt (MTT) to purple formazan crystals by metabolically active cells. Cells frozen at passage 1 were thawed, seeded at passage 2, multiplied and reseeded at passage 3 for the MTT assay. Cells were seeded in quadruplicate into clear 96-well microtiter plates at a cell density of 10⁴ cells/cm² in a final volume of 100 μL of culture medium. Cells were cultured at 37 °C in a 5% CO₂ incubator for 48 h until they reached 70% confluency. After the incubation period, the cell culture medium was removed, and experimental medium was added. The cells were cultured overnight. The experimental medium was removed, and 10 μL of MTT labeling reagent (at a final concentration of 0.5 mg/mL) was added to 100 μL of DMEM without phenol red (Gibco, Grand Island, NY, USA) in each well. Following 4 h of incubation at 37 °C, in a 5% CO₂ incubator, 100 μL of solubilization buffer was added to each well and incubated overnight at 37 °C in a 5% CO₂ incubator. The next day, the total solubilization of the purple formazan crystals was measured with a Byonoy absorbance reader (Byonoy, Hamburg, Germany). The sample wavelength was set at 562 nm, and the reference wavelength was 650 nm.

A live/dead assay was employed for suspension and conditioned media cultures. Cells frozen at passage 1 were thawed, seeded at passage 2, multiplied and reseeded at passage 3 for the live/dead assay. Cells were seeded at a density of 10,000 cells/cm² into 8-well glass chamber slides (Merck, Darmstadt, Germany) and cultured for 48 h until they reached 70% confluence. After the incubation period, the cell culture medium was removed, and experimental medium was added. The cells were cultured overnight, after which the experimental medium was removed. A live/dead cell imaging kit (488/570) (Thermo Fisher Scientific, Waltham, MA, USA) was added to the cells, which were then incubated for 15 min. The cells were observed under a fluorescence microscope (Nikon Eclipse 80i, Nikon) equipped with a Nikon Digital Sight DS-U2 camera. Images were captured in the NIS-Elements D3.2 Live quality program at 400 × magnification and qualitatively analyzed. To calculate the viability from live and dead cell counts, the 𝑡𝑜𝑡𝑎𝑙 𝑐𝑜𝑢𝑛𝑡 𝑜𝑓 𝑙𝑖𝑣𝑒 𝑐𝑒𝑙𝑙𝑠 and 𝑡𝑜𝑡𝑎𝑙 𝑐𝑜𝑢𝑛𝑡 𝑜𝑓 𝑑𝑒𝑎𝑑 𝑐𝑒𝑙𝑙𝑠 were added to determine the total cell number. Viability was calculated using the following formula:

An Alizarin Red S (ARS) staining assay of cDPSCs differentiated into osteogenic lineages was performed for conditioned media cultures. Cells frozen at passage 1 were thawed, seeded at passage 2, multiplied and reseeded at passage 3 for ARS staining. Cells were first differentiated into osteogenic lineages. For osteogenic differentiation, 15 × 10⁴ cells/cm² were seeded into 6-well plates. At 70% confluency, the cell culture medium was exchanged with experimental medium. After 24 h, the experimental medium was removed. Osteogenic medium (StemPro Osteogenesis Differentiation Kit, Gibco, Grand Island, NY, USA) was added, and the medium was changed every 2–3 days. Osteogenic differentiation was analyzed after 14 days of cultivation using Alizarin Red S staining (Sigma Aldrich, Taufkirchen, Germany) according to the standard procedure. The cells were observed under a fluorescence microscope at 400 × magnification and qualitatively analyzed. In each well, 3 images were taken and processed with the ImageJ program.

Images for the live/dead and ARS staining assays were analyzed with the ImageJ program. Images were captured in the NIS-Elements D3.2 Live quality program. Images were captured at 40 × magnification. For the live/dead assay, 3 images of live cells and 3 images of dead cells were randomly selected from each well and quantitatively analyzed by measuring the areas of green (live) and dead (red) cells in each well and processed with the ImageJ program. In the ImageJ program, images were converted to binary types and then segmented using the DynamicThreshold_1d.class plugin (33), which displayed (max + min)/2 images. The area of particles larger than 100 μm² was measured in each field view, and the total area covered by cells was calculated. For ARS staining, 3 images were randomly selected from each well and quantitatively analyzed by measuring the area of red particles (mineral deposits) in each well, after which the samples were processed with the ImageJ program. In the ImageJ program, the images were processed with background adjustment, separated with Color Deconvolution2 (34), segmented using the DynamicThreshold_1d.class plugin, displayed as (max + min)/2 images, and the red area was measured. Particles larger than 10 μm² were measured in each field view. The total area of red particles was calculated.

RNA was isolated from experimental and control cDPSCs. Cells were detached from the wells with a cell scraper. The cell suspension was removed from the wells and centrifuged at 240 × g for 4 min. The supernatant was discarded, and the pellet was flash-frozen in liquid nitrogen. The cell pellet was then homogenized with a homogenizer (IKA T10 basic, Staufen, Germany) in 350 μL of RLT lysis buffer (Qiagen, Hilden, Germany). Total RNA extraction was carried out with an RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s protocol. The amount of extracted total RNA was measured using a UV spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) at 260/280 nm.

Two-step reverse transcription quantitative polymerase chain reaction (RT–qPCR) for experimental cDPSCs at the third dilution and positive control cDPSCs was performed. First, 2 μg of total RNA from each sample was reverse transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Negative reverse transcription controls were included in each PCR run. All reactions were conducted in a total volume of 20 μL. The conditions for reverse transcription were as suggested in the manufacturer’s protocol: 25 °C for 10 min, 37 °C for 120 min, and 85 °C for 5 min. In the second step, relative quantification was performed using TaqMan Universal PCR Master Mix with UNG (Thermo Fisher Scientific, Waltham, MA, USA) and the TaqMan gene expression assays RunX2 and ALPL. TBP was used as a reference gene (Thermo Fisher). All the qPCR amplifications were conducted in triplicate in a total volume of 20 μL. cDNA (20 ng) was used as a template. Amplification was carried out in 96-well plates with a Light Cycler 96 (Roche Life Science) using the following program: 50 °C for 2 min, 95 °C for 10 min, and 40 cycles at 95 °C for 15 s and 60 °C for 60 s.

Statistical analysis was performed for cells isolated from three dental pulp tissues and grown on standard polystyrene surfaces (Table 1). All the statistical analyses were performed with GraphPad Prism version 9.5.0 for Windows (GraphPad Software, San Diego, CA, USA, www.graphpad.com, accessed on 15 April 2024).

All the data were log-transformed to normalize the data and residuals. The normality and lognormality of the residuals were checked with the Kolmogorov–Smirnov test.

All the data were log-transformed (Y = log(Y)) and normalized to the control samples to account for differences in the control samples (formula: value/baseline). For the MTT, live/dead and ARS staining assays, 2-way ANOVA was performed to analyze the differences between the experimental cell cultures. For qPCR, conditioned media cultures from the third dilution (D3) were used. The efficiency-corrected double delta Ct method was employed to normalize the gene expression values (35). The expression levels of RUNX2, ALPL and MMP13 were compared to the expression levels of RUNX2, ALPL and MMP13 in positive control cDPSCs, and the results were analyzed by one-way ANOVA.

Statistical significance was defined as p < 0.05.

Dental pulp tissue was successfully collected from all three teeth of the two dogs. Under a light microscope, the cells from passage 3 appeared spindle-shaped with a fibroblast-like morphology (Figure 1).

Flow cytometry was performed at passage 3 with conjugated primary antibodies against the positive surface markers CD44-APC, CD90-PE, and CD29-FITC and the negative surface marker CD34-FITC. The cells were positive for CD44, CD90, and CD29 but negative for CD34 (Figure 2).

cDPSCs successfully differentiated into chondrogenic and osteogenic lineages. Chondrogenesis was indicated by the formation of chondrogenic nodules, which were stained blue with Alcian blue (Figure 3A). Mineral deposits in the extracellular matrix stained red with Alizarin Red S indicate osteogenesis (Figure 3B). The corresponding negative controls are shown on the right (Figures 3C,D).

In suspension culture, the MTT absorbance (a proxy for viable cell number) was greater with RS + than with MTA at D1 and greater with CF than with MTA at D1 and D2. CF also yielded higher values than RS + did at D2 and D3 (Figure 4A). No significant differences in MTT absorbance were observed among the groups treated with conditioned media (Figure 4B). In the MTT with the conditioned media, dilutions 2, 3, and 4 were used; the first dilution was omitted to avoid introducing possible artifacts into the analysis because preliminary attempts using this dilution produced inconsistent absorbance values.

In suspension culture, cell viability was greater with CF than with MTA at dilutions D2–D5 and greater with CF than with RS + at D2; RS + also exceeded MTA at D4 and D5 (Figure 5A). In the conditioned media, no statistically significant differences in viability were observed among the groups (Figure 5B).

In the ARS, dilutions 2, 3, and 4 of conditioned media were used; the first dilution was omitted to avoid introducing possible artifacts into the analysis because preliminary attempts using this dilution produced uneven well-to-well staining. There was a difference in ARS staining at D3, where the ARS staining of cDPSCs was greater in cells conditioned with MTA than in cells conditioned with CF (Figure 6). Representative images of ARS staining and the corresponding images processed with ImageJ are shown in Figure 7.

As there was a significant difference in ARS staining on D3, gene expression was evaluated at this dilution. The expression of RUNX2 was lower in cells conditioned with MTA than in cells conditioned with RS + (* p < 0.1) and lower than that in positive control cells (** p < 0.01; Figure 8A). ALPL expression was lower in cells conditioned with MTA than in cells conditioned with RS + (** p < 0.01) and CF (* p < 0.1; Figure 8B). No differences in the expression of MMP13 were observed between the groups (Figure 8C).