All mouse experiments were performed in accordance with approved Institutional Animal Care and Use Committee (IACUC) guidelines, University of Washington. Mice were originally purchased from the Jackson Laboratory. Mice were housed in a specific-pathogen-free (SPF) environment in ventilated cages with filter tops (Allentown Inc., Allentown, NJ, United States). A maximum of 5 mice were housed together in the same cage. Mice were maintained in standard light/dark cycle (6:00 a.m. lights on, 6:00 p.m. lights off), standard food (5058 mouse breeding food), standard temperature (68–79°F), and no environmental restriction. Mice were allocated randomly to control and experimental groups.

Dental pulp tissues were dissected and pooled from 5-day-old neonatal murine teeth. DPSCs were isolated, cultured, expanded, and characterized as previously described (Janebodin et al., 2011). Briefly, dental pulp from lower molar teeth was gently isolated and kept in stem cell media described below. The tissue was washed with phosphate buffer saline (PBS) (HyClone). To release cells, the extracellular matrix was digested with Dispase II (1.2 units/ml), Collagenase IV (2 mg/ml) (Worthington) supplemented with CaCl₂ (2 mM) in PBS for 1 h at 37°C. Then, an equal volume of stem cell media was added to the digested tissue prior to filtering through 70 mm nylon cell strainers (BD Falcon) and centrifuging at 300 × g for 10 min at room temperature. Cells were subsequently resuspended in stem cell media and single cell suspensions were plated at a density of 1000 cells/cm².

Cells were cultured at 37°C under 5% O₂ and 5% CO₂ in stem cell media, containing a final concentration of 60% low-glucose DMEM (Gibco, Invitrogen), 40% MCDB201 (Sigma), 2% fetal calf serum (HyClone), insulin-transferrin-selenium (ITS) (Sigma), linoleic acid with bovine serum albumin (LA-BSA) (Sigma), 10^–9 M dexamethasone (Sigma), 10^–4 M ascorbic acid 2-phosphate (Sigma), 100 units/ml penicillin with 100 mg/ml streptomycin (HyClone), and 1 × 10³ units/ml leukemia-inhibitory factor (LIF-ESGRO, Millipore), 10 ng/mL EGF (Sigma) and 10 ng/mL PDGF-BB (R&D) (Breyer et al., 2006). Once more than 50% cell confluent, they were detached with 0.25% trypsin-EDTA (Invitrogen) and replated at a 1:4 dilution under the same culture condition with fresh media. Differentiation capacity of cultured cells was determined by in vitro multi-differentiation to bone, cartilage and fat cells as previously described (Gronthos et al., 1994; Gregoire, 2001; Dahlin et al., 2014). The osteo-odontogenic media contains serum-free media with 10% FBS, 10 mM β-glycerophosphate, 0.2 mM L-ascorbic acid, and 100 nM dexamethasone. The chondrogenic media contains serum-free high-glucose DMEM, 1% ITS+ premix (BD Biosciences), 50 mg/mL ascorbic acid, 100 nM dexamethasone, supplemented with 10 ng/ml TGF-β3 (Shenandoah Biotech). The adipogenic media contains serum-free media supplemented with 10% horse serum, 100 μM indomethacin (Alfa Aesar), 0.5 mM 3-isobutyl-1-methyl-xanthine (ACROS) and 1 μM dexamethasone.

Murine DPSCs were stably transfected with VEGFR-2 shRNA to silence the expression of VEGFR-2 as per manufacturer’s protocol. Briefly, cells (1000 cells/cm²) were plated and cultured in stem cell media for 24 h. DPSCs were allowed to proliferate until 70% confluent before viral transfection. Then, cells were cultured in stem cell media with polybrene (5 μg/ml) to increase binding between the pseudoviral capsid and the cell membrane. Cells were infected by the VEGFR-2 shRNA lentiviral particles (Santa Cruz Biotechnology, Santa Cruz, CA, United States) with 10:1 of multiplicity of infection (MOI), then mixed gently, and incubated overnight. CopGFP control lentiviral particles (Santa Cruz Biotechnology) were also used as control to evaluate transduction efficiency at the similar MOI used in VEGFR-2 shRNA lentiviral particles. After overnight transduction, infected cells were replaced with stem cell media without polybrene and incubated overnight. To select stable infected cells expressing the shRNA, we expanded cells one more time in stem cell media overnight, and then selected them by exposure to puromycin dihydrochloride (5 μg/ml) (Sigma). The infected cells were cultured in puromycin-containing stem cell media until the resistant cells were identified. The homogeneous population of resistant cells was observed by the expression of Green Fluorescence Protein (GFP) in cells transduced with CopGFP control lentiviral particles. To determine the knockdown efficacy, the level of VEGFR-2 and VEGF-A in both silencing and control groups was determined by mRNA and protein levels through real-time PCR and immunofluorescence analyses.

Cells were fixed with 4% formaldehyde/PBS for 5 min, washed with 1% BSA in 0.1% Triton-X 100/PBS, and stained with primary antibodies as described in Supplementary Table 1 incubated overnight at 4°C. Goat-derived Alexa 594-conjugated secondary antibodies (Invitrogen) were diluted at 1:800 and incubated for 1 h. Cells were stained with 4′, 6-diamine-2-phenylindol (DAPI) at 1:1000 to visualize the nuclei. All antibodies were diluted in 1% BSA in 0.1% Triton-X 100/PBS. We used IgG isotype from the species made for the primary antibody (0.1 ug/ml) (Vector Laboratories Inc., Burlingame, CA, United States), were included for all staining.

VEGFR-2 shRNA treated DPSCs and CopGFP treated DPSCs (2 × 10⁴ cells/cm², n = 4 wells/group) were incubated overnight in stem cell media at 37°C under 5% O₂ and 5% CO₂. After 24 h, each cell type was separately cultured in BMP-2 media, β-glycerophosphate plus ascorbic acid (BGP + Vit. C) media, VEGF media for 21 days to evaluate the osteo-odontogenic differentiation capacity with media change every 3 days. BMP-2 media is serum-free media containing 10% FBS, 100 ng/ml BMP-2 (Shenandoah Biotech), 0.2 mM L-ascorbic acid, and 100 nM dexamethasone (Hu et al., 2004). BGP + Vit. C media is serum-free media containing 10% FBS, 10 mM β-glycerophosphate (CalBiochem), 0.2 mM L-ascorbic acid, and 100 nM dexamethasone (Song et al., 2009). VEGF media is serum-free media containing 10 ng/ml VEGF (R&D) (Forgues et al., 2019). Serum-free media is 60% low-glucose DMEM, 40% MCDB201, ITS, LA-BSA, 10^–9 M dexamethasone, 10^–4 M ascorbic acid 2-phosphate, 100 units/ml penicillin with 100 mg/ml streptomycin. After differentiation, both cell groups were separately divided into 2 subgroups for RNA collection and staining.

After differentiation, cultured cells were stained by ALP staining kit (BioVision) as per manufacturer’s protocol. Briefly, the media were carefully removed from the cultured cell wells. Cells were fixed with 4% formaldehyde/PBS for 15 min at room temperature. Then, 500 μl of wash buffer was gently added and carefully removed using a pipette. 250 μl of ALP Staining Reagent solution was carefully added to completely cover the cells in each well of a 24-well plate. Cells were incubated for 30 min at 37°C before washed gently with 500 μl of wash buffer for 3 times. 300 μl of wash buffer was added and stained cells were imaged using a light microscope.

Cultured cells were stained by Alizarin Red S staining kit (ScienCell Research Laboratories) as per manufacturer’s protocol. Briefly, the media were carefully removed from the cultured cell wells and cells were washed three times with PBS. Cells were fixed with 4% formaldehyde/PBS for 15 min at room temperature. The fixative solution was removed and cells were washed three times with deionized water. The water was removed and 500 μl of 40 mM Alizarin Red S dye was added per well. Cells were incubated at room temperature for 30 min with gentle shaking. The dye was removed and cells were washed five times with deionized water before taking images. For tissue transplants, the sections were deparaffinized and hydrated to 70% ethanol. Then, the tissue sections were fixed with 10% formaldehyde/PBS for 15 min at room temperature before staining following the protocol described above.

Dental pulp stem cells were extracted for total RNA by using the RNeasy Mini kit (Qiagen) according to the manufacturer’s protocol. Quantity and purity of RNA was determined by 260/280 nm absorbance. First-strand cDNA was synthesized from 500 ng of RNA using the High Capacity cDNA synthesis kit (Applied Biosystems) per manufacturer’s protocols using a randomized primer. Real-time PCR primers are included in Supplementary Table 2.

A 20 ng of cDNA for Q-RT-PCR were prepared using the SYBR green PCR master mix (Applied Biosystems). Reactions were processed by the ABI 7900HT PCR system with the following parameters: 50°C/2 min and 95°C/10 min, followed by 40 cycles of 95°C/15 s and 60°C/1 min. Results were analyzed using SDS 2.2 software and relative expression calculated using the comparative Ct method. The threshold cycle (Ct) value for each gene was normalized to the Ct value of GAPDH. The relative mRNA expression, presented as fold change difference, was calculated by the comparative Ct method according to the formula: 2^–ΔΔCt, where ΔCt = Ct target–Ct GAPDH and ΔΔCt = ΔCt target–ΔCt calibrator. Each sample was run in triplicate reactions for each gene. Error bars represent the standard deviation calculated from the triplicate analysis of each sample.

In accordance with approved IACUC protocols, 1 × 10⁶ of VEGFR-2 shRNA treated DPSCs and CopGFP treated DPSCs were separately transplanted into 1-month-old male Rag1 null mice (Jackson Laboratory, Bar Harbor, ME, United States) by dorsal subcutaneous transplantation with hydroxyapatite tricalciumphosphate (HAp/TCP) (Zimmer) (n = 5 mice/cell type). Grafts were harvested after 5 weeks of transplantation. Transplanted tissues samples were fixed with 4% formaldehyde for 2 h then demineralized for 7 days in 10% EDTA at 4°C. Then, the transplants were embedded in paraffin and cut to 5 μm thick sections. Sections were analyzed by H&E, Alizarin Red S and immunohistochemistry staining.

Prior to staining procedures, paraffin-embedded tissue sections were deparaffinized and hydrated to 70% ethanol. Tissues were stained with H&E staining following the manufacturer’s standard protocol. For immunohistochemistry, fixed tissue sections were permeabilized with 1% BSA in 0.1% Triton-X 100 (Sigma)/PBS for 10 min, inhibited endogenous peroxidase activity with 0.3% hydrogen peroxide in methanol for 30 min, and blocked non-specific binding sites with 10% goat or horse serum (Vector Laboratories Inc., Burlingame, CA, United States) for 1 h. Primary antibodies listed in Supplementary Table 1 were used and incubated overnight at 4°C. Stained tissues were incubated with a biotinylated antibody at 1:100 (Vector Laboratories Inc., Burlingame, CA, United States) for 1 h, washed and treated with the Vectastain ABC kit and 3, 3′-diaminobenzidine (DAB) substrate kit according to manufacturer’s protocol (Vector Laboratories Inc., Burlingame, CA, United States).

The number of mice were calculated using power analysis based on previous pilot studies in our laboratory (statistical power of 0.90; p level = 0.05). Statistical analysis of the in vitro studies, histology, and molecular analysis results from three independent experiments were performed by the Student’s t test. Data are presented as mean ± SD. p-values ≤ 0.001, 0.005, 0.05 was considered as statistically significant.

Dental pulp stem cells were isolated from neonatal murine pulp tissue of lower molar teeth. Cell suspension was plated in a low cell density as a single cell deposition. After 2 days of culture in stem cell media, several cell colonies were observed (Figure 1A). Later, larger colonies of cells were seen before cell trypsinization and expansion (Figures 1B,C). Compared to undifferentiated DPSCs (Figure 1D), cultures in specific differentiation media revealed DPSCs differentiation abilities to give rise to osteoblast-like cells (positive staining for BSP and DMP-1, Figures 1E,F), chondrocyte-like cells (positive staining for COL II, Figure 1G), and adipocytes (positive staining with Oil Red O, Figure 1H).

In order to investigate the potential role of VEGFR-2 signaling in osteo-odontogenic differentiation of DPSCs, VEGFR-2 shRNA was utilized to silence VEGFR-2 expression in murine DPSCs. The CopGFP shRNA was also used as a control to determine the efficiency of transfection. We targeted DPSCs with both VEGFR-2 and CopGFP shRNA separately and observed approximately 100% GFP expression in CopGFP (Figures 2A,C) but not in VEGFR-2 shRNA group (Figures 2B,D). CopGFP DPSCs expressed high levels of both VEGFR-2 and VEGF-A (Figures 2E,G,K,L). In contrast, an 8-fold decrease of Vegfr-2 expression was shown in VEGFR-2 shRNA DPSCs compared to the CopGFP control DPSCs (^∗∗∗p < 0.001) (Figure 2K). The gene expression level corresponded to a dramatic decrease in VEGFR-2 immunofluorescence staining of VEGFR-2 shRNA DPSCs (Figures 2F,L). The quantification of the VEGFR-2 fluorescence intensity showed a statistically significant decrease in VEGFR-2 level in VEGFR-2 knockdown DPSCs, compared to normal DPSCs (^∗∗p < 0.005) (Figure 2L). In addition, VEGFR-2 shRNA DPSCs down-regulated VEGF-A gene and protein levels (^∗p < 0.05 and ^∗∗p < 0.005, respectively), compared to the CopGFP control cells (Figures 2H,K,L). The rabbit IgG control (Rb IgG Ctrl) stained in CopGFP control (Figure 2I) and VEGFR-2 shRNA DPSCs (Figure 2J) were completely negative to confirm the specificity of antibodies.

CopGFP DPSCs cultured in BMP-2 media showed strongly positive staining of ALP enzyme (in black) (Figures 3A,C,D). In contrast, VEGFR-2 shRNA DPSCs exhibited significantly decreased ALP staining (Figures 3B,E). Few slightly ALP positive cells in VEGFR-2 shRNA DPSCs were observed (Figure 3F). In addition, abundant mineralized nodules were observed and positively stained Alizarin Red S (in red) in the CopGFP DPSCs (Figures 3G,I) while those nodules were absent in the VEGFR-2 shRNA DPSCs (Figures 3H,K). A cluster of cuboidal cells were seen, indicating osteo-OLCs (Figure 3J). Nevertheless, Alizarin Red S stained slightly positive in the cytoplasmic compartment of VEGFR-2 shRNA DPSCs due to the intracellular calcium contents (Figure 3L). Remarkably, VEGFR-2 shRNA DPSCs maintained a spindle-shaped morphology, resembling an undifferentiated state. Additionally, immunocytochemistry showed certain positive cells of anti-DMP-1 (Figure 4A) and anti-DSP (Figure 4B) staining (in brown) in differentiated CopGFP control DPSCs but slightly stained cells in VEGFR-2 shRNA DPSCs (Figures 4D,E). The rabbit IgG control (Rb IgG Ctrl) stained in CopGFP control (Figure 4C) and VEGFR-2 shRNA DPSCs (Figure 4F) were completely negative to confirm the specificity of antibodies. VEGFR-2 shRNA DPSCs also down-regulated the expression of Dmp-1, Dspp, and Bsp; however, only Dmp-1 and Bsp showed a significant difference compared to the CopGFP DPSCs (^∗p < 0.05) (Figure 4G). Cultured in BGP + Vit. C media, occasional strongly ALP positive cells (Figures 5A,C,E) were seen in the control DPSCs but not in the VEGFR-2 shRNA DPSCs (Figures 5B,D,F). Some Alizarin Red S positive nodules were present in the CopGFP cells whereas those were not detected in the VEGFR-2 shRNA cells (Figure 5M). Similarly, cultured in VEGF media, few ALP positive cells were present in only the CopGFP group (Figures 5G,I,K), not in the VEGFR-2 shRNA group (Figures 5H,J,L). However, there were no mineralized nodules observed in both CopGFP and VEGFR-2 shRNA groups (Figure 5N).

H&E staining revealed the overall morphology of CopGFP DPSC transplants (Figures 6A–C) and VEGFR-2 shRNA DPSC transplants (Figures 6G–I). In vivo transplantation of CopGFP DPSCs demonstrated elongated and polarized cells (Figures 6D–F; arrowheads) running perpendicularly to hydroxyapatite/tricalcium phosphate (HAp/TCP), resembling the morphology of OLCs. In addition, a formation of pulp-like structure was revealed by the presence of loose connective tissue with capillaries containing RBCs (Figures 6D–F) in CopGFP DPSC transplants, mimicking pulp-like tissues in the natural teeth. In contrast, VEGFR-2 shRNA DPSCs transplants lacked OLCs and pulp-like structures. Instead, there was only a formation of disperse collagenous tissue (Figures 6J–L).

In the cross-section of the tooth, Alizarin Red S showed the strongly positive staining of dentin and odontoblasts; it also exhibited the histology of dental pulp which is a vascularized loose connective tissue (Figure 7A). A transplanted tissue generated by the CopGFP DPSCs stained positive for Alizarin Red S and also demonstrated the presence of OLCs (Figures 7B,C; arrowheads) with pulp-like–tissues (Figures 7B,C). In contrast, VEGFR-2 shRNA DPSC transplants showed disorganized tissues faintly positive for Alizarin Red S staining (Figures 7D,E). In the tooth section, anti-DMP-1 and anti-DSP staining was positive in dentin and intense in odontoblasts (Figures 7F,G). Interestingly, the transplanted tissues generated by the control cells also showed strong signal of immunohistochemistry for DMP-1 and DSP proteins in OLCs (Figures 7I,J; arrowheads) and matrix (Figures 7I,J; asterisks). In contrast, we were not able to observe positive DMP-1 and DSP staining in the VEGFR-2 shRNA DPSC transplants (Figures 7L,M). Rb IgG control was used as a negative control (Figures 7H,K,N).