Systemic Injection of Substance P Promotes Murine Calvarial Repair Through Mobilizing Endogenous Mesenchymal Stem Cells

Craniofacial defect is a critical problem in dental clinic, which has a tremendous impact on patients’ quality of life. Mesenchymal stem cell-based therapy has emerged as a promising approach for tissue defect repair. However, reduced survival after mesenchymal stem cells (MSCs) transplantation remains as a major problem in this area, which hampers the outcome of regeneration. Recently, the mechanism to mobilize endogenous MSCs for tissue regeneration has received increasing attentions, as it does not require exogenous cell transplantation. The primary goal of this study was to confirm the role of intravenous substance P in mobilizing endogenous CD45−CD11b−CD29+ MSCs in critical-sized bone defect animals and to investigate the effects of substance P on calvarial bone repair. Flow cytometry analyses revealed that intravenous substance P promoted the mobilization of endogenous CD45−CD11b−CD29+ MSCs after bone defect. In addition, Micro-CT showed that intravenous substance P improved the outcomes of calvarial bone repair. Furthermore, we discovered that systemic injection of substance P attenuated inflammation and enhanced the survival of the local-transplanted GFP+ MSCs. Our findings suggested that substance P together with its mobilized CD45−CD11b−CD29+ MSCs helped improve calvarial defect repair through regulating inflammatory conditions and promoting the survival of local-transplanted cells.

In this study, we identified whether systemic delivery of substance P can promote endogenous MSCs mobilization and homing in mice with calvarial defects. Next, we evaluated the inflammation state through analyses of pro-inflammatory cytokine expression in both injury sites and peripheral circulation. Finally, we tested the capacity of systemic-injected substance P in promoting calvarial defect repair. Here, we demonstrated that systemic delivery of substance P could promote CD45 − CD11b − CD29 + MSCs mobilization and calvarial defect repair. In addition, our study indicated the potential role of systemic-injected substance P in regulating inflammation during bone healing process.

Results
Substance P mobilized CD45 − CD11b − CD29 + cells. Previous studies had reported that substance P had a strong mobilization effect on the endogenous CD45 − CD11b − CD29 + cell population at early stage in corneal burn injury models. These cells all expressed similar molecular markers with BMSCs and had multipotent differentiation capacities at early passages 12 .
To identify the function of systemically injected substance P in the bone defect model, we established the calvarial critical-sized defect model with a diameter of 5 mm and gave each mouse a systemic injection of substance P (5 nmol/kg) through the tail vein. 3 days after surgery, we collected 1 ml peripheral blood and counted CD45 − CD11b − CD29 + cells using flow cytometry. The absolute numbers of CD45 − CD11b − CD29 + cells in peripheral blood from the other three groups (injured or i.v.substance P) were significantly higher than that of the uninjured group (Fig. 1a,b) (P < 0.01). This indicated that both calvarial injury and i.v.substance P could promote the enrichment of CD45 − CD11b − CD29 + cells in peripheral blood. Meanwhile, no significant differences were observed between uninjured + i.v.substance P group and calvarial injured +i.v.substance P group (P > 0.05) (Fig. 1b). Both groups exhibited a larger number of cells than the calvarial injured group (P < 0.01) (Fig. 1b), indicating that the motivation capability of i.v.substance P was much greater than injury itself.
To confirm that i.v.substance P helps motivate the CD45 − CD11b − CD29 + cell population specifically, we measured the relative number of this cell population together with the total cell number in collected peripheral blood (Fig. 1c,d). The percentage of CD45 − CD11b − CD29 + cells in peripheral blood of the calvarial injured group (0.5% ± 0.1) was significantly higher than that of the uninjured group (0.267% ± 0.12) (P < 0.05) (Fig. 1c). There was no significant difference between the uninjured +i.v.substance P group (0.867% ± 0.05) and the calvarial injured +i.v.substance P group (0.967% ± 0.12) (P > 0.05), and both groups had a larger number than that in the calvarial injured and uninjured groups (P < 0.01) (Fig. 1c). In addition, there was no significant difference in the total cell number of peripheral blood among the four groups (P > 0.05) (Fig. 1d).
We also tested the in vitro role of substance P in proliferation of CD45 − CD11b − CD29 + cell population using cell counting and CCK-8 ( Fig. 2a-c). The statistic analyses have shown that substance P stimulation could increase the proliferation of these mobilized CD45 − CD11b − CD29 + cells.
Intravenous substance P controls inflammatory state both systemically and locally in calvarial injured mice. Substance P plays an important role in neurogenic inflammation and can promote the infiltration of inflammatory cells 13 . Also, substance P can stimulate the secretion of TNF-α from mononuclear-macrophage. To evaluate the inflammatory state of the calvarial injured animal after systemic injection of substance P, we carried out ELISA of the peripheral blood, western blot, and RT-PCR of the tissue within the primary defect areas 2 weeks after the surgery.
First of all, ELISA analyses of peripheral blood have revealed that the inflammation was attenuated with decreased concentration of pro-inflammatory cytokine IFN-γ and TNF-α in peripheral circulation after systemic injection of MSCs or substance P (Fig. 3a,b) (P < 0.05). It has also been reported that TSG-6 can be secreted by intravenous MSCs 14 , which is able to abort the early inflammatory response through the modulation of nuclear factor NF-κB signaling in resident macrophages 15 . In the current study, ELISA analyses have demonstrated that the TSG-6 expression levels in peripheral circulation were significantly higher in groups using intravenous MSCs or substance P than that of the other groups ( Fig. 3c) (P < 0.01).
Secondly, western blot analyses of tissue within the primary defect areas have shown that the expression of IFN-γ and TNF-α was significantly reduced after systemic application of MSCs or substance P (Fig. 3d,e) (P < 0.05). RT-PCR analyses of primary injured tissues further confirmed that the corresponding mRNA expressions of IFN-γ and TNF-α were also inhibited in i.v.GFP + MSC and GFP + MSC +i.v.substance P groups (Fig. 3f) (P < 0.05). Additionally, both the mRNA and corresponding protein expression of TSG-6 at the local injury site were enhanced after intravenous MSCs or substance P injection, as shown by RT-PCR and western blot, respectively (Fig. 3e,f) (P < 0.01). Interestingly, the TSG-6 expression level in i.v.GFP + MSC group was similar to that in GFP + MSC +i.v.substance P group (Fig. 3e) (P > 0.05), suggesting that substance P-mobilized CD29 + cells, like intravenous exogenous MSCs, could also secrete TSG-6 to help control inflammation.
Recent research has also demonstrated that IFN-γ can inhibit the osteogenesis of exogenous bone marrow MSCs through downregulation of Runx2 pathway and synergistically enhance TNF-α-induced cell apoptosis 9 . Considering that the survival and osteogenic capability of MSCs are essential to bone repair within our calvarial defect models, we wonder whether the Runx2 pathway in defect areas could be modulated by i.v.substance P or i.v.MSCs. To address this critical question, we evaluated the mRNA and protein expression of Runx2 within the primary injury site. The results showed that Runx2 expression increased in GFP + MSC + i.v.substance P group compared with those in other groups (Fig. 3e,f) (P < 0.01). This indicated that the survival and osteogenic capacity of MSCs within the defect in GFP + MSC +i.v.substance P group was higher than those in other groups, which was potentially due to substance P-mediated inflammatory states.
To further confirm the effect of substance P-mediated inflammation on survival of transplanted GFP + MSCs, we checked the GFP signal using real-time in vivo GFP fluorescence imaging (Fig. 4a-d). The GFP signal was significantly higher in GFP + MSC + i.v.substance P group than in GFP + MSCs-scaffold group (Fig. 4a- suggesting that i.v.substance P might enhance the survival of GFP + MSCs within the defect areas. We also compared the mRNA and protein expression of GFP in defect areas among groups ( Fig. 4e-g). The expression of GFP was only detected within groups using GFP + MSCs-seeded scaffold, confirming that intravenous exogenous GFP + MSCs could hardly reach the injury site 16 . The statistical analyses indicated that the GFP expression was significantly higher in GFP + MSC + i.v.substance P group than in GFP + MSCs-scaffold group (Fig. 4f,g) (P < 0.05), which was consistent with real-time in vivo GFP fluorescence imaging data.
All the above results suggested that intravenous substance P could control the inflammatory state both locally and systemically in the calvarial critical-sized defect model.  Intravenous substance P promoted calvarial injury repair. Considering that substance P can mobilize CD29 + cells, which demonstrate multipotent differentiation capacity and can control the inflammatory state after injury, we wonder whether intravenous substance P can promote bone repair in calvarial defects. The hematoxylin-eosin staining showed that there were more bone-like structures formed within the primary defect areas in GFP + MSC + i.v.substance P group compared with other groups (Fig. 5). Consistently, the reconstructed three-dimensional images of Micro CT showed that the remaining defect area after repair was much smaller in GFP + MSC + i.v.substance P group than that of the other groups (Fig. 6a), which suggested that more new bones were formed in GFP + MSC + i.v.substance P group. Further analyses of the bone parameters from Micro CT images suggested that both systemic injection of MSCs and substance P enhanced calvarial bone repair while intravenous substance P brought about better effects than intravenous MSCs in calvarial defect mice ( Fig. 6b-g) (P < 0.05).

Discussion
Recently, researchers have revealed that substance P, as a damage-inducible factor, is a powerful factor for mobilizing endogenous MSCs in corneal burn injury 12 . In our study, we confirmed the ability of substance P in endogenous MSCs mobilization at the early stage of bone injury. These results suggest that substance P may provide a promising candidate for mobilizing endogenous MSCs in different kinds of injury models. Based on previous literature, these substance P-mobilized MSCs may be derived from the bone marrow 12 . Unfortunately, with current techniques, we cannot demonstrate their definite origin. However, we provided some evidence regarding the potential origin of these mobilized cells. On one hand, the molecular markers of substance P mobilized cells are similar to those of BMSCs derived from the bone marrow 12  that substance P could stimulate the proliferation of those mobilized cells, which was consistent with the effect of substance P on BMSCs 12 . Taking all these together, we speculate that these mobilized cells may be derived from the bone marrow, which may contribute to clavarial bone repairing.  Figure 3. The inflammatory state after intravenous substance P. C57BL/6 wide-type mice were divided into four groups: blank scaffold group, i.v.GFP + MSCs group, GFP + MSCs-scaffold group, GFP + MSCs + i.v.substance P group. 2 weeks post surgery, 1 ml of peripheral blood was harvested from each group for ELISA analysis (n = 6 for each group) and the tissues of the calvarial defect areas were collected for western blot and RT-PCR analysis (n = 3 for each group). Inflammatory state has been evidenced to be critical for tissue regeneration. Previous studies have focused on the pro-inflammatory roles of substance P as a damage-inducible factor. It has also been reported that substance P can promote the infiltration of inflammatory cells and stimulate secretion of TNF-α from mononuclear-macrophage 13 . In the mouse liver injury model, the inflammation of mice liver was attenuated after administration of NK-1R (a receptor of substance P) antagonists and levels of TNF-α and IFN-γ in serum  were also reduced 17 . To identify the effects of intravenous substance P on inflammatory status of our calvarial defect animals, we assessed the pro-inflammatory protein levels both locally and systemically. Interestingly, in the current study, the inflammation was significantly attenuated by systemic infusion of substance P. Previous studies have shown that exogenous substance P has a short half-life while endogenous injured tissue-derived substance P has mostly regressed by Day 3 after injury 12,18 . In this way, the alleviated inflammatory state at 2 weeks  after surgery could be due to indirect roles of substance P rather than direct roles. Considering that substance P can mobilize endogenous MSCs, which have immune-regulatory properties 19 , we hypothesized that substance P-mobilized endogenous MSCs may orchestrate the inflammatory properties of substance P itself and help regulate inflammatory responses after calvarial defects. In support of this notion, we also evaluated the TSG-6 expression both systemically and locally. ELISA data showed that anti-inflammatory protein TSG-6 in peripheral blood was induced after intravenous substance P injection. In accordance with this result, TSG-6 expression within primary defect site was also increased. Previous studies have reported that TSG-6 could be derived from MSCs in peripheral blood 14 . Therefore, it is possible that increased TSG-6 in this study could be secreted by substance P-mobilized MSCs, which might contribute to indirect effect of substance P in controlling inflammation.
To boost bone repair after injury, we also combined the systemic injection of substance P with local-transplanted MSCs. We have found that intravenous substance P might improve the survival of local-transplanted MSCs. Considering the immune-regulatory effects of substance P-mobilized endogenous MSCs both locally and systemically, it is possible that systemic infusion of substance P can promote local-transplanted MSCs survival through its mobilized endogenous MSCs. However, more experiment, like in vitro cell experiment, is needed to fully elucidate the underlying mechanisms.
In addition, the bone parameters from Micro CT analysis revealed that the new bone formation was enhanced after systemic application of MSCs or substance P. Interestingly, the outcome in GFP + MSC + i.v.substance P group was even better than that in i.v. GFP + MSC group, which might be explained by pro-inflammatory T cell inhibition on exogenous MSCs 9 .
All these results suggest that intravenous substance P can promote bone repair. Similar to our findings, another group has recently promoted bone repair through local application of substance P 20 . However, in our model, we highlighted the combination of systemic infusion of substance P with local-transplanted MSCs in bone repair, which not only took advantage of endogenous MSCs, but also promoted the survival of local-transplanted MSCs. Moreover, we observed that the Runx2 expression within the defect sites, which was associated with osteogenesis of mesenchymal stem cells 9 , was enhanced after intravenous substance P injection, suggesting that systemic infusion of substance P together with its mobilized MSCs could promote osteogenesis of local MSCs in the bone defect animals. All these above results indicate that i.v.substance P can effectively promote bone repair in the calvarial defect mice.
Collectively, our study highlights the effects of substance P in bone repair through mobilizing endogenous MSCs and also indicates the possible roles of intravenous substance P in regulating inflammatory conditions in bone defects. In addition, the combination of intravenous substance P and local-transplanted MSCs treatment can effectively promote the osteogenesis of MSCs and boost calvarial bone repair.

Methods
Animal experiments. For the first part of animal experiments, twelve C57BL/6 wide-type mice (10-weekold) were randomly divided into four groups: uninjured, calvarial injured, uninjured + i.v.substance P and calvarial injured + i.v.substance P groups. We established the calvarial critical-sized defect model with a diameter of 5 mm. In uninjured + i.v.substance P and calvarial injured + i.v.substance P groups, each mouse was given a systemic injection of substance P (5 nmol/kg, dissolved in phosphate-buffered saline) through the tail vein. 3 days after surgery, we collected 1 ml peripheral blood and counted CD45 − CD11b − CD29 + cells using flow cytometry.
For the second part of animal experiments, thirty C57BL/6 wide-type mice (10-week-old) were randomly divided into five groups: control, blank scaffold, i.v.GFP + MSCs, GFP + MSCs-scaffold and GFP + MSCs + i.v.substance P groups. We established the calvarial critical-sized defect model with a diameter of 5 mm. In blank scaffold group, we put the 5 × 5 mm 2 gelatin sponge within calvarial defect area. In i.v.GFP + MSCs group, we gave each mouse a systemic injection of 5 × 10 6 GFP + MSCs (dissolved in 200 μl phosphate-buffered saline) through its tail vein. In GFP + MSCs-scaffold group, the 5 × 5 mm 2 gelatin sponge seeded with GFP + MSCs was set in calvarial defect area of each mouse. In GFP + MSCs + i.v.substance P groups, we put one GFP + MSCs-seeded scaffold in calvarial defect area and gave each mouse a systemic injection of substance P (5 nmol/kg, dissolved in phosphate-buffered saline) through the tail vein. Flow cytometry. 3 days after surgery, 1 ml peripheral blood was harvested from calvarial injured mice (uninjured, calvarial injured, uninjured + i.v.substance P and calvarial injured + i.v.substance P groups). Each sample from an individual mouse was separately prepared and incubated with the antibodies CD29 (BioLegend, San Diego, CA), CD11b (BioLegend, San Diego, CA), CD45 (BioLegend, San Diego, CA) for 30 minutes at 4 °C before flow cytometry analysis. FlowJo was used for flow cytometric analyses.
CCK8 was also carried out. We seeded 4 × 10 3 CD29 + cells in each well of 96-well plate in 100 μl of culture medium and pre-incubated the plate for 24 hours at 37 °C and 5% CO 2 . Then the cells were treated with 1 × 10 −10 mol/l, 1 × 10 −8 mol/ and 1 × 10 −6 mol/l of substance P in three experiment groups respectively. After 48 hours of treatment, 10 μl of CCK8 solution (Dojindo, Tabaru, Japan) was added to each well of the plate. After 2-hour incubation at 37 °C and 5% CO2, the optical density (OD) value of each well was measured at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).