Ethics approval and consent to participate

Dental pulp-derived mesenchymal stem cells (DPSCs) were isolated from dental pulp tissues of 3 healthy subjects (two males and a female), undergoing third molar extraction. The subjects provided their informed consent, to be included in the study and were naïve for treatments at the time of the medical procedure. The study was approved by the institutional review board ethics committees. Subjects were recruited within a clinical protocol by “Ospedale di Circolo Fondazione Macchi” and approved by the institutional Ethical Committee (protocol n° 0034086, 9-10-2013) according to the Helsinki Declaration of 1975, as revised in 2013.

CD14+ monocytes were isolated from blood samples of healthy-donor volunteers recruited within the protocol n° 463.2021, approved by the IRCCS MultiMedica internal Ethical Committee, according to the Helsinki Declaration of 1975 as revised in 2013.

In vivo studies used male athymic BALB/c nude Crl:CD1-Foxn1nu086 mice (Charles River mice, seven weeks); Mice were housed under standard conditions with a 12-h light/dark cycle and provided ad libitum access to food and water. The experiments were conducted in compliance with the guidelines established by the Italian and European Community (D.L. 2711/92 No.116; 86/609/EEC Directive), adhering to the principles of the 3 Rs (Replacement, Reduction, and Refinement) and carried out within an approved protocol by the institutional ethics committee.

Generation of conditioned media (CM)

DPSC-CMs were prepared following the previously described method, as in [39]. In brief, when the cells at 5th passage reached 70–80% confluence, media were removed, and cells were washed twice with PBS. Cells were incubated in fetal bovine serum (FBS)-free Dulbecco’s Modified Eagle Medium (DMEM), for 72 h in normoxic (21% O2) or hypoxic (2% O2) conditions. The parameters used for hypoxia condition were: 2% O2, 5% CO2, 93% N2. No color changes were observed in culture medium, during the 72 h of starvation, both in normoxic and hypoxic conditions, that together with the proliferation rate (showed in Fig. 1) and cell morphology (Supplementary Fig. 1), confirm a healthy state of DPSCs. The media were then removed and centrifuged at 1000xg for 10 min, to deplete eventual cell debris. To maximize the protein content, collected CMs were concentrated, using the Amicon Ultra 15 mL Centrifugal filter device (Millipore, Darmstadt, Germany) with a 3 kDa cut-off, according to the manufacturer’s instructions. 13 mL of DPSC-CMs were loaded into the tubes and centrifuged at 5000×g for 60 min at 4 °C. The concentrated media were collected, quantified in term of total protein content, and then stored at −80 °C until use for in vivo and in vitro experiments.

Fig. 1

Characterization of DPSCs. DPSCs at different culture passages (P2-P30) were characterized by (A, B) flow cytometry for CD90, CD105, CD73, CD45, EpCAM, CD31 surface markers; (C) real-time PCR for CD44, CD90, CD105, CD45, ALPL, DSPP, p16, p21 gene expression; (D) BrdU assay, for their proliferation rate

Flow cytometry

DPSCs isolated from three donors (see supplementary methods), were seeded in T25 flasks, collected at passages 2, 5, 10, 20, and 30 and characterized by flow cytometry. Briefly, DPSCs were stained for 30 min 4 °C, with the following anti-human monoclonal antibodies (all purchased from BD Biosciences): APC-CD90 (BD clone 5E10), BUV395-CD45 (BD clone HI30), FITC-EpCAM (BD clone EBA-1), PE-CD31 (BD clone WM59), PE-CD73 (BD clone AD2). Viable cells were identified based on doublet exclusion (side scatter area/SSC-A Vs side scatter height/SSC-H) and their morphology (forward scatter area/FSC-A Vs side scatter area/ SSC-A), then interrogated for the fluorescence signals associated to the selected surface antigens.

Following excision from mice, Ultimatrix sponges were filtered, using a 70 μm pore cell strainer (BD Biosciences), using a syringe plugger until complete plug dispersion, to obtain a single cell suspension for flow cytometry analysis. The single cell suspension was stained for 30 min, 4 °C, with the following anti-mouse monoclonal antibodies (all purchased from BD Biosciences): FITC-CD31 (clone: MEC 13.3), BUV-395-CD45 (clone: 30-F11), PE-CF594-F4/80 (clone: T45-2342), BV-421-CD80 (clone: 16-10A1), Alexa Fluor-647-CD206 (clone: MDR5D3). Samples were acquired using a FACS Fortessa × 20 (BD Biosciences), equipped with 5 lasers. Viable cells were identified based on doublet exclusion (side scatter area/SSC-A Vs side scatter height/SSC-H) and their morphology (forward scatter area/FSC-A Vs side scatter area/ SSC-A). Viable cells were used to identify different cell types as follows: CD45− cells (stromal cells), CD45−CD31+ cells (endothelial cells), CD45+ cells (total leukocytes), CD45+F4/80+ cells (total macrophages), CD45+F4/80+CD80+ cells (M1-like macrophages), CD45+F4/80+CD206+ (M2-like macrophages). FACS data were exported as FCS files and analyzed with the FlowJo v10 software (BD Biosciences).

RNA extraction, reverse transcription, and real-time PCR

DPSCs from the three donors were collected at passages 2, 5, 10, 20, and 30 and characterized by qPCR for stem markers CD44, CD90, and CD105, leukocyte marker CD45, differentiation markers Alkaline Phosphatase (ALPL) and Dentin Sialophosphoprotein (DSPP), cell senescence markers p16 and p21. Procedures for RNA extraction, RT and qPCR are detailed in supplementary methods.

HUVECs, following 6 h of stimulation with DPSC-CMs, were collected in trizol reagent, and stored at − 80 °C until further use. The 18S RNA gene was used as a housekeeping gene, and HUVECs or macrophages, cultured in serum-free media, served as the baseline control. Procedures for RNA extraction, RT and qPCR are detailed in supplementary methods.

BrdU assay

To assess the effect of long-term passaging on DPSCs, cell proliferation was evaluated using the kit BrdU cell proliferation ELISA (Roche Life Sciences, Switzerland). DPSCs from the three donors, collected at passages 2, 5, 10, 20, and 30, were used. The experiment was conducted following the manufacturer’s instructions: briefly, 200 cells/well were seeded onto a 96-well plate and incubated for 24 h at 37 °C and 5% CO2. Once cells adhered to the plate, BrdU labelling solution was added and incubated for 24 h. Afterwards, medium was removed and cells were fixed with FixDenat solution for 30 min at RT; anti-BrdU-POD working solution was then added and incubated for 90 min at RT. Wells were then washed three times with washing solution before adding the Substrate solution for 15 min, until color development was sufficient for detection. Afterwards, 1 M H2SO4 was added to each well and the absorbance was measured at 450 nm using the GloMax® Discover Microplate Reader (Promega, Milano, Italy). The same process was repeated for 24 h, 48 h, and 72 h.

In vivo Ultimatrix sponge assay

DPSC-CMs were prepared following the previously described method, as reported in [39].The effects of DPSC-CMs, obtained in normoxia or hypoxia, on angiogenesis in vivo, was tested using the Ultimatrix sponge assay, as in [38]. The procedure for Ultimatrix sponge preparation has been detailed in Supplementary Methods section.

Characterization of DPSC-conditioned media

The DPSC-CMs were characterized, in term of soluble factor content, using the Human Cytokine Array C7 (RayBiotec) (Peachtree Corners, GA, USA), starting from 50 μg of total protein of DPSC-CMs from normoxic or hypoxic conditions and following the manufacturer’s indication and as in [38, 40, 41].

Hemoglobin quantification

Hemoglobin content in excised sponges was determined using the colorimetric Drabkin’s assay. Briefly, sponges were mechanically processed in 300 μL of 1 × PBS (Euroclone). 200 μL of the supernatant were collected ad added to 800 μL of Drabkin’s solution and incubated at room temperature, protected from light, for 20 min. 100 μL of the incubated solution was transferred into a 96 well plate and read at 595 nm, using a SpectraMax plate reader.

Optical microscopy

The formation of new vessels inside the scaffolds was observed by optical microscopy as described in [42]. Samples, fixed in 4% PFA at RT for 2 h, were embedded in paraffin, following sequential dehydration with ethanol (70, 80, 90, 95, 100%), and cut using an RMC-RM3 rotary microtome (TiEsseLab, Milan, Italy). Slides were then stained with hematoxylin and eosin (H&E) solutions, following classical procedures, and finally analyzed. Vessels were counted from five nonconsecutive section (5 µm), considering three-microscope fields per section, using ISCapture software (version 3.6.9.4).

Scanning electron microscopy (SEM) analysis

Samples embedded in paraffin were cut (15–30 μm slices) using an RMC-RM3 rotary microtome (TiEsseLab, Milan, Italy) and placed on slides for SEM observation.

Specimens were dehydrated using a series of ethanol concentrations and dried using hexamethyldisilazane (Sigma Aldrich, Milano, Italy). Subsequently, they were coated with a 10 nm layer of gold using the Emitech K550 system and examined using a Philips SEM-FEG XL-30 electron microscope (Eindhoven, The Netherlands).

Stimulation of endothelial cells with CMs

3 × 105 HUVECs (see supplementary methods for culturing and maintenance) seeded in six well plates, were exposed to 50 μg of total protein from DPSC-CMs, obtained under normoxic and hypoxic conditions, in serum-free media, for 6 h.

Cell migration

The capability of DPSC-CMs (Normox or Hypox) to influence the migration of HUVEC or CD14+ monocytes (see supplementary methods for human CD14+ monocyte isolation) were tested by transwell assay.

For endothelial cell migration, 2 × 104 HUVECs were placed on the upper chamber of 24-well transwell (Corning), with a 10 μm pore filter cut-off coated with 2 μg of human fibronectin (Sigma Aldrich). 50 μg/well of DPSC-CMs, or 1:4 diluted (Normox or Hypox), or the single cytokines IL-6 (50 ng/mL), IL-8 (20 ng/mL), SDF-1 (100 ng/mL), or the IL-6 + IL-8 + SDF-1 combination, were used to induce endothelial cell migration. EBM starvation of complete medium was used as negative and positive internal controls.

For monocyte migration, 25 × 104 CD14+ monocytes were placed on the upper chamber of 24-well transwell (Corning), with a 5 μm pore filter cut-off, respectively, coated with 2 μg of human fibronectin (Sigma Aldrich). 50 μg/well of DPSC-CMs (Normox or Hypox) were used to induce cell migration. RPMI starvation of complete medium was used as negative and positive internal controls.

Transwells were incubated at 37 °C, 5% CO2 for 6 h. Upper chambers were collected, washed in PBS, fixed with 4% PFA for 10 min, at RT, then washed, stained with 10 μg/mL Hoechst 33342 for 15 min, washed in PBS, and finally acquired using a fluorescence microscope (Leica). The number of fluorescent cells, as readout of migration, were counted using the ImageJ software. Tree blind fields for each filter were acquired and summed to estimate the number of migrated cell/filter/conditions.

Tube formation

The ability of DPSC-CMs (Normox or Hypox) to induce a capillary-like network in vitro, was tested by tube formation assay on HUVECs. HUVECs (8 × 104 cells/well) were seeded in a 24-well plate, previously coated with 50 µL of 10 mg/mL polymerized phenol red-free, reduced growth factors Matrigel (BD). Following exposure to 50 µg/mL of DPSC-CMs Normox or DPSC-CMs Hypox, in serum-free EBM medium, HUVECs were incubated at 37 °C, 5% CO2 for 24 h. EBM starvation or complete medium were used as negative and positive internal controls.

The formation of capillary-like structures was detected by microphotographs, using an inverted microscope (Leica). The number of master segments, total master segment length, number of meshes and total mashes area, as readouts of tube formation efficiency, were determined, using ImageJ software and the Angiogenesis Analyzer tool.

Statistical analysis

Results were analyzed using the GraphPad Prism software v10 (GraphPad Prism Inc., San Diego, CA, USA). Data are shown as means ± SEM, Student t-test or One-Way ANOVA, followed by Tukey’s post-hoc test correction. P values ≤ 0.05 were considered statistically significant.