Novel Molecule Nell-1 Promotes the Angiogenic Differentiation of Dental Pulp Stem Cells

Introduction This work aimed to reveal the crucial role of Nell-1 in the angiogenic differentiation of human dental pulp stem cells (DPSCs) alone or co-cultured with human umbilical vein endothelial cell (HUVECs) in vitro and whether this molecule is involved in the pulp exposure model in vivo. Methods Immunofluorescence was conducted to ascertain the location of Nell-1 on DPSCs, HUVECs, and normal rat dental tissues. RT-PCR, Western blot, and ELISA were performed to observe the expression levels of angiogenic markers and determine the angiogenic differentiation of Nell-1 on DPSCs alone or co-cultured with HUVECs, as well as in vitro tube formation assay. Blood vessel number for all groups was observed and compared using immunohistochemistry by establishing a rat pulp exposure model. Results Nell-1 is highly expressed in the nucleus of DPSCs and HUVECs and is co-expressed with angiogenic markers in normal rat pulp tissues. Hence, Nell-1 can promote the angiogenic marker expression in DPSCs alone and co-cultured with other cells and can enhance angiogenesis in vitro as well as in the pulp exposure model. Conclusion Nell-1 may play a positive role in the angiogenic differentiation of DPSCs.


INTRODUCTION
Nel-like molecule-1 (Nell-1) is a novel signaling molecule, with a crucial positive regulatory role in chondrogenesis and osteogenesis (Li C. et al., 2019); however, its involvement in pulp angiogenesis, which is an important part of pulp regeneration, remains poorly studied. Vasculogenesis is the differentiation of endothelial cells to form blood vessels during embryonic development, while angiogenesis means new vessels sprouting from pre-existing vasculature (Chung and Ferrara, 2011), both of them are mediated by angiogenic growth factors (Rombouts et al., 2017). Among which, vascular endothelial growth factor (VEGF) is a strong regulator of physiological and pathological angiogenesis during embryogenesis and pathological angiogenesis associated with tumors (Ferrara et al., 2003) and alveolar bone process morphogenesis . VEGF receptor (VEGFR) has a potential part in mediating the biological effects of VEGF, leading to homodimerization and autophosphorylation when VEGF dimers bind to VEGFR-1 and VEGFR-2 (Ferrara, 2002). Activating VEGFR-2 (Flk-1) could induce angiogenesis and increase vascular permeability, mitogenesis, and chemotaxis in endothelial cells.
Nell-1 encodes a secreted protein (Zhang et al., 2010) and was discovered by Ting et al. (1999), when they accidentally operated for the surgical correction of unilateral coronal synostosis. In our previous study, Nell-1 shows spatiotemporal expression patterns during murine tooth (Tang et al., 2013) and is mainly expressed in the odontoblasts, pulp fibroblasts, and endothelial cells of the blood vessels in human teeth (Tang et al., 2013;Liu et al., 2016). Fahmy-Garcia et al. (2018) confirmed that Nell-1 can enhance the migration of mesenchymal stem cells (MSCs) and the angiogenesis of HUVECs. DPSCs form a dentin/pulp-like complex (Gronthos et al., 2000) with neurallike cells (Stevens et al., 2008;Gonmanee et al., 2018;Li D. et al., 2019), endotheliocytes (d' Aquino et al., 2007), and vascular tissues (Karbanova et al., 2011) and have a predominant proangiogenic influence compared with dental follicle precursor cells (FSCs) (Hilkens et al., 2014). These data indicate that DPSCs are a promising population of stem cells that could achieve angiogenesis. The potential of Nell-1 to induce the angiogenetic differentiation of DPSCs is of great interest.
The survival rate of regenerating vascular dependent tissues could be increased when MSCs are co-transplanted with hematopoietic stem cells (Moioli et al., 2008). Endothelial cells (ECs) are a potential source of neovascularization during tissue regeneration (Moioli et al., 2008). Angiogenesis highly occurs between stem cells and endothelial cells through synergistic effect or direct cell contact (Aguirre et al., 2010;Allen et al., 2011;Rao et al., 2012;Kang et al., 2013). Several studies also confirmed that the co-culture of HUVECs with stem cells from the apical papilla (SCAPs) or DPSCs can enhance the angiogenic potential (Dissanayaka et al., 2012;Liu et al., 2018). Whether Nell-1 is directly involved in the angiogenetic differentiation of DPSCs co-cultured with HUVECs must be explored.

Isolation, Culture, and Identification of DPSCs and Co-culture of DPSCs With HUVECs
Third molars were acquired after obtaining informed consent from each patient (15-25 years of age, male and female) who underwent routine extraction with no caries or periodontal diseases. The extracted teeth were washed with PBS and cut with fissure in sterile conditions. The acquired pulp tissues were digested with 3% I-type collagenase (Solarbio, Beijing, China), and the dental tissues were transported in 25 cm 2 cell culture flasks. HUVECs and its specific endothelial cell medium (ECM) were acquired from ScienCell company (San Diego, United States) (Leopold et al., 2019). Each cell type was used at passages 3-5 in all experiments.
Human DPSCs at passage 3 were collected. The cells were tested for MSC markers CD90, CD44 and CD105, and hematopoietic stem markers CD34 and CD45 by using flow cytometric (CytoFLEX, CA, United States) with PBS as the negative control. Osteogenic and adipogenic differentiation assays were performed on the DPSCs to detect their multi-lineage differentiation ability. The cells were stained and examined under an inverted microscope after 21 days. The experiments group was grown in osteogenic differentiation medium (α-MEM containing 10%FBS, 0.01 nmol/l dexamethasone, 10 mmol/l β-glycerophosphate, and 50 mg/mL ascorbic acid) (Sigma, St Louis, MO, United States) or adipogenic differentiation medium (Pythonbio, China), and the control group was treated similarly to the above cell culture.
DPSCs and HUVECs were mixed directly at a 1:1 number ratio with new mixed medium prepared by mixing the α-MEM (containing 10% FBS) with ECM at 1:1 ratio.

Western Blot
The cells were planked similarly to PCR, lysed with RIPA buffer containing 1% PMSF (Solarbio, Beijing, China) for 30 min. Cellular proteins were isolated and quantified by a BCA kit (Solarbio, Beijing, China), separated on 10% polyacrylamide gels, and transferred onto the polyvinylidene difluoride membranes (Millipore, Billerica, United States). Following washing with TBST, the membranes were incubated with primary antibodies including Flk-1 antibody (1:1,000 dilution; Novus, NB200-208, United States) and GAPDH antibody (1:10,000 dilution; Proteintech, China) overnight at 4 • C. Then, the membranes were incubated with secondary antibodies for 2 h at RT and then washed again. The results were analyzed by Image J software.

ELISA
Quantikine ELISA kit (Dakewe, Shenzhen, China) was used to measure VEGF expression, and the process was similar to that in RNA extraction. Cell supernatants were collected at 1, 2, 3, 7, and

In vitro Tube Formation Assay
To investigate which minimum concentration of Nell-1 could promote formation of endothelial tubules and a blood vessel network when DPSCs were co-cultured with HUVECs, Matrigel angiogenesis assay was carried out. Co-culture groups (HUVECs: DPSCs, 1:1) were cultured in 24-well plates in mixed medium (containing 0.5%FBS) with 0, 50, 100, and 200 ng/mL Nell-1 at 6 h. Images were observed under an inverted phasecontrast microscope.

Tooth Sample Collection and Preparation
All animal experimental protocols were performed in accordance with the guidelines of the Institutional Review Board of School of Stomatology, Shandong University. Twelve Wistar rats were randomly categorized into three groups: (1) collagen group, pulp cavity was covered with collagen sponge (BIOT Biology, China) soaked with PBS; (2) Nell-1 group, pulp cavity was covered with collagen sponge soaked with 700 ng/mL Nell-1; and (3) normal teeth group, upper molars did not receive cavities or any other treatments. The occlusal surface of noncarious upper first molars was selected to establish the rat pulpitis model with a #35 K-file under anesthesia. Afterward, the cavities were sealed with glass ions (VOCO, Ionofil Molar, Germany) as the bottom material and with resin (3MESPE, Filtek Z350 XT, United States) for the collagen and Nell-1 groups. After 1 week, the rats were sacrificed. The experimental teeth were fixed in 4% paraformaldehyde for 24 h, demineralized with 10% ethylenediaminetetraacetic acid solution, and cut with a blade (5 um serial sections). The sections were processed for histological and immunohistochemical examinations.

Statistical Analyses
Student's t-test or one-way ANOVA was used to compare statistical significance between various treatments and respective controls by using GraphPad Prism software. All experiments were repeated three times from independent samples from different donors. Data were expressed as mean ± standard deviation. P < 0.05 was considered statistically significant.

Isolation and Characterization of DPSCs
The primary cells were successfully isolated from pulp tissues and displayed a long spindle shape with adherent growth (Figure 1A). Alizarin red and oil red O staining showed that DPSCs possess strong osteo/dentinogenic and adipogenic potential (Figures 1B-E). Flow cytometry results showed that DPSCs are positive for CD90, CD44, and CD105 but negative for CD34, CD45 (Figures 1F-J).

Nell-1 Promoted the Angiogenesis-Related Gene and Protein Expression in DPSCs
Angiogenic markers including VEGF and Flk-1 were detected by qRT-PCR. In DPSC group, 50 ng/mL Nell-1 remarkably promoted VEGF and Flk-1 expression compared with those in the control in days 3, 7, and 14 (apart from Flk-1 in day 14) (Figures 2A,B). The gene expression levels of VEGF in DPSC group increased in days 1 and 2 compared with those in the control group; however, no statistical significance was found.
Western blot analysis was performed to detect relative protein expression. In the experiment group, the angiogenesis-related protein expression was increased in days 3 and 7, but no significant difference was observed in days 1, 2, and 14 compared with those in the control group (Figures 2C,D).

Nell-1 Promoted the Angiogenesis-Related Gene and Protein Expression and the Formation of Vessel-Like Structures in the Co-culture Group
On the basis of the VEGF expression levels, 50 ng/mL Nell-1 significantly promoted the angiogenetic differentiation in the experiment group compared with that in the control in days 3 and 7, but no statistical significance was observed in days 1, 2, and 14 ( Figure 3A). Flk-1 expression level was increased by 50 ng/mL Nell-1 treatment compared with that in the control in days 2 and 7 ( Figure 3B). However, these levels decreased significantly in days 3 and 14.
Western blot analysis and ELISA were performed to investigate Flk-1 and VEGF protein expression, respectively. In the experiment group, the angiogenesis-related protein Flk-1 was increased in days 3, 7, and 14, but no significant difference was found in days 1 and 2 compared with that in the control group (Figures 3C,D). The concentration of VEGF protein in supernatants secreted by the co-culture group was increased by 50 ng/mL Nell-1 compared with that in the control group in days 3, 7, and 14 (Figures 3E,F).
Compared with control group, co-culture group seeded on Matrigel with 50 and 100 ng/mL Nell-1 formed more vessel-like structures, whereas no significant difference was observed in 200 ng/mL Nell-1 (Figure 4).

Nell-1 Distribution in DPSCs, HUVECs, and Normal Pulp Tissues
Cell immunofluorescence assay displayed that Nell-1 is mainly expressed in the nucleus of DPSCs and HUVECs (Figures 5A-H). HE staining and immunofluorescence were conducted to present the structure of normal pulp tissues and the distribution of Nell-1.
Double immunofluorescence of pulp tissue sections was used to observe the distribution of Nell-1, VEGF, and Flk-1 in the dental pulp (Figures 5I-R). High expression of Nell-1, VEGF, and Flk-1 was found in the odontoblasts, pulp fibroblasts, and endothelial cells of the blood vessels of the dental pulp. The merged picture showed that Nell-1 is co-expressed with VEGF and Flk-1.
of CD31 and CD34 revealed vascular lumens in pulp tissues. The collagen group had higher amount of inflammatory cell infiltration but lower numbers of blood vessels around cavities than the Nell-1 group (Figures 6E,F,K,L). Both groups had more inflammatory cell infiltration and fewer blood vessels than normal teeth group (Figures 6D-L). In addition, no significant difference in the area away from the cavities was found among the three groups (Figures 6G-I).

DISCUSSION
DPSCs are regarded as great stem cell sources in different generation fields because they could be easily isolated by a noninvasive method and without moral concern (Lee et al., 2019;Yamada et al., 2019). Direct co-culture of DPSCs and HUVECs was applied in this study according to the previous studies which has showed greater expression of angiogenic markers compared with DPSCs alone (Dissanayaka et al., 2012).
In this study, Nell-1 upregulated VEGF and Flk-1, which are responsible for proangiogenic properties. Flk-1 protein expression was downregulated in the co-culture group at day 3 when Nell-1 was added; however, the mechanisms are still unclear. Cell immunofluorescence staining revealed that Nell-1 is expressed in the nucleus of DPSCs and HUVECs and coexpressed with VEGF and Flk-1 in normal pulp tissues. This finding indicates that Nell-1 may have a synergistic or similar effect with VEGF and Flk-1 on promoting angiogenesis. The in vitro tube formation assay also confirmed Nell-1 effects on promoting angiogenesis.
The survival rate of inflamed pulp tissue is related to the surrounding angiogenesis (Saghiri et al., 2015). Immunohistochemical staining of CD31 and CD34 indicated that the number of blood vessels in the Nell-1 group is higher than that in the collagen group. This phenomenon can be explained as follows. In vitro experiment revealed that Nell-1 may promote angiogenesis by increasing the expression of VEGF and Flk-1 in DPSCs. Considering the co-expression of Nell-1 and angiogenetic markers, we speculate that Nell-1 may have a synergistic or similar effect to VEGF and Flk-1.
Pulp regeneration mainly includes blood vessels, nerves, and dentin. In an adult tooth, each large myelinated nerve fiber bundle surrounds a single arteriole in the root canals and pulp chambers (Steiniger et al., 2013). During early tooth formation, FIGURE 3 | Angiogenesis-related gene and protein expression in the co-culture group. (A) VEGF gene expression was improved by 50 ng/mL Nell-1 treatment in days 3 and 7. (B) Flk-1 gene expression was improved by 50 ng/mL Nell-1 treatment in days 2 and 7, but was decreased in day 3 and 14. (C,D) Flk-1 protein expression was increased by 50 and 100 ng/mL Nell-1 treatment in days 3, 7, and 14. (E,F) VEGF protein expression was increased by 50 ng/mL Nell-1 treatment in days 3, 7, and 14. Data are shown as the mean ± SD. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
tooth innervation occurs through vasculogenesis (Shadad et al., 2019). In this study, we found that Nell-1 can enhance the expression of proteins and angiogenetic genes as early as day 2, which was almost the same rate as that of the odontoblastic and neural-like differentiation of DPSCs (Liu et al., 2016;Han et al., 2019). Subsequent experiments will be conducted to confirm the relevant mechanisms. Nell-1 application may be a promising strategy in dental pulp regeneration field since its contribution to dentin formation, neurogenesis and angiogenesis (Liu et al., 2016;Han et al., 2019). Xuan et al. (2018) confirmed that human deciduous autologous tooth stem cells can regenerate the whole dental pulp and have a promising effect on pulp regeneration; however, the mechanisms are still unclear. The present study investigated   (C) Collagen group. HE staining showed the cavities, inflamed tissue, and normal structure of rat pulp tissues. The staining of CD31 (D-I) and CD34 (J-L) revealed the blood vessels in pulp tissues. Nell-1 induction group had higher number of blood vessels around cavities than the collagen group, and both of them had fewer blood vessels than negative control group (D-F,J-L). There was no significant difference in the area away from the cavities between three groups (G-I). d, dentin; p, dental pulp; bd, blood vessel. the effect of Nell-1 on pulp angiogenesis and found that this molecule may promote the angiogenic differentiation of DPSCs, thus further confirming its role in pulp regeneration.

CONCLUSION
Nell-1 is highly expressed in the nucleus of DPSCs and HUVECs and co-expressed with angiogenetic markers in normal pulp tissues. This molecule could enhance the angiogenic differentiation of DPSCs in vitro and in vivo and thus may be a promising drug for the regeneration of the whole pulp.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Institutional Review Board of School of Stomatology (No. R20180801). The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Institutional Review Board of School of Stomatology (No. D20180801).

AUTHOR CONTRIBUTIONS
XW and QW designed, supervised, and funded the study. ML performed the experiments and prepared the manuscript. QH, JW, and HZ performed the animal experiment. XB and YC performed the analysis. CY collected the clinical sample. All authors were actively involved with their work on this manuscript. All authors read and approved the final manuscript.