Optimal H2O2 preconditioning to improve BMSCs’ engraftment in wounds healing

The transplantation of bone marrow mesenchymal stem cells (BMSCs) is a promising therapeutic strategy for wound healing. However, the poor migration capacity and low survival rate of transplanted BMSCs in wounds weaken their potential application. The optimal protocol for BMSCs preconditioned with H 2 O 2 was investigated, and the therapeutic ecacy of preconditioned BMSCs in wounds was evaluated. Mouse BMSCs were exposed to various concentrations of H 2 O 2 , and their functions were assessed; The H 2 O 2 -preconditioned BMSCs were transplanted into mice with full-thickness excisional wounds. Wound analysis was performed to assess the transplantation ecacy.


Introduction
Growing evidence indicates that BMSCs based therapy for cutaneous wound healing hold great therapeutic value via differentiating into specialized cell types, producing a large variety of growth factors, and promoting wound closure and angiogenesis [1][2][3]. Therefore, transplantation of BMSCs is a promising therapeutic strategy for wound healing [1]. However, the poor migration capacity and low survival rate of transplanted BMSCs in wounds weaken their potential application.
Preconditioning has become the most powerful and effective cytoprotective strategy for initiating cell survival signaling to counter the rigorous harsh microenvironment [4,5] and enhance the cell migration capacity and survival rate. Reportedly, the injured tissue expresses high level of stromal cell-derived factor (SDF-1), a stem cell homing signal factor which binds to its receptor CXC chemokine receptor 4 (CXCR4) and CXCR7 on cells surface of MSCs and promotes stem cells homing and survival in the injured tissue, respectively [6,7]. However, during in vitro culture expansion, BMSCs lose their CXCR4 receptor [8], and reduce their binding capacity to SDF-1 resulting in [9,10]attenuation of their migration capacity. Moreover, various stress conditions including ex vivo isolation, in vitro expansion of MSCs, and confronting the harsh microenvironment (ischemia, hypoxia, and in ammation) caused oxidative stress injury to BMSCs following engraftment at injured sites, resulting in reducing survival of transplanted BMSCs [11][12][13].
In this study, the optimal concentration of H 2 O 2 on BMSCs preconditioning was rst determined. Then the molecular mechanisms of H 2 O 2 preconditioning BMSCs was explored by focusing on PI3K/AKT pathway due to its role in MSCs survival and migration responding to SDF-1 and oxidative stress [27]. The ultimate therapeutic potential of H 2 O 2− preconditioned BMSCs was evaluated in a mouse full-thickness wound model.

Ethics Statement
Animal studies were performed using 4-week-old male Balb/C mice provided by the Animal Breeding Center of Third Military Medical University. All procedures in the present study were performed in accordance with the "Guide for the Care and Use of Laboratory Animals" (National Institutes of Health

Wound analysis
Wound analysis began at 1 day after wounding. Digital photographs of wounds were taken on days 0, 1, 3, 5, 7, 10, 13 and 15, and analyzed using Image Propus 5.0. The wound closure rate was calculated as follows: [(Area of original wound -Area of actual wound)/Area of original wound] × 100%.
Mice were sacri ced at days 0, 3, 5, 7 and 10 post wounding, and the skin samples were collected for histological and protein analysis. The photos were taken by randomly selecting 5 high-power elds (hpf, × 200), and analyzed using Image Propus 5.0 to calculate the total number of migrated BMSCs with CM-DiI label (red cells) at the wound site.

Microvessel density of the wound area
Microvessel density was determined by immunohistochemical staining with anti-CD31 antibody (Santa Cruz), according to manufacturer's instructions and a method previously described [10].

Statistical analysis
We obtained all the results from at least three independent experiments. All statistical analyses were performed using SPSS13.0 software. Differences between multiple groups were analyzed by analysis of variance (ANOVA), whereas differences between two groups were analyzed using a t-test. A value of p < 0.05 was considered to be statistically signi cant.

Results
3.1. Pretreatment BMSCs with 50 µM H 2 O 2 signi cantly enhanced cell viability/proliferation and migration by augmenting SDF-1/CXCR4/CXCR7 axis To de ne the optimal concentration of H 2 O 2 in preconditioning BMSCs, we selected ve different concentrations between 20-200uM based on previous reports [28] and investigated its effects on cellular properties of viability, proliferation, migration and differentiation. We found that H 2 O 2 at 25 µM and 50 µM signi cantly promoted the proliferation of BMSCs, accompanying by increase in cyclin D1 expression and decrease in p16 expression, whereas 150 and 200 µM H 2 O 2 showed a signi cant inhibitory effect compared with that of the control. 50 µM H 2 O 2 caused a peak cyclin D1 expression and a low p16 expression, which represented the optimal concentration of H 2 O 2 for BMSCs preconditioning in terms of cell proliferation/viability from this study (Fig. 1A, B).
In a scratch assay, BMSCs with 50 µM H 2 O 2 preconditioned showed a signi cantly higher migration capacity compared to that of non-preconditioned BMSCs (Fig. 1C). The Boyden chamber assay was further demonstrated that the number of migrating BMSCs in H 2 O 2 group was much more than in that of the control group, however, the enhanced migration capacity was attenuated by pertreatment with anti-CXCR4 antibody (Fig. 1D, E). Simultaneously, the expression of SDF-1, CXCR4 and CXCR7 in H 2 O 2 group was signi cantly upregulated at 25 µM and 50 µM compared to that of the control (Fig. 1F). These results suggested the 50 µM H 2 O 2 treatment for 12 h could signi cantly enhance the BMSCs viability and migration capabilities, which may be through activating the SDF-1/CXCR4/CXCR7 pathway.
Additionally, we found that the BMSCs still have maintained the multipotent differentiation capacity of stem cells even after pretreated with 50 µM H 2 O 2 for 12 h (Supplement Fig. 1). They could differentiate into adipocytic (Oil red O staining), chondrocytic (Toluidine blue, Alcian blue), as well as osteoblastic (alkaline phosphatase) lineages. This results showed that 50 µM H 2 O 2 treatment did not affected the differentiation capacity of BMSCs.

Optimal H 2 O 2 preconditioning suppressed cell death of BMSCs via mitochondria mediated mechanism
Oxidative stress induces apoptosis through the intrinsic mitochondrial pathway [29]. The ratio of antiapoptotic Bcl-2 and pro-apoptotic Bax (Bcl-2/Bax) are the key to the mitochondrial apoptotic pathway [29]. The ow cytometric (FCM) analysis showed that exposure to 25-100 µM H 2 O 2 did not cause BMSCs apoptosis ( Fig. 2A). However, exposure to 150-500 µM H 2 O 2 induced an obvious increase in apoptotic rate of BMSCs in a dose-dependent manner ( Fig. 2A, B), compared with the control. The results showed that H 2 O 2 exceeding 100 µmol/L might induce oxidative stress thereby leading to cell apoptosis.
Moreover, the result demonstrated that the apoptotic rate of BMSCs was very signi cantly lower in H 2 O 2preconditionded each group than in corresponding high doses of H 2 O 2 groups (Fig. 2C, D). Treatment of BMSCs with 50 µM H 2 O 2 for 12 h resulted in a peak Bcl-2/Bax expression ratio compared to that of the control, but Bax and caspase-3 were not affected (Fig. 3A, B, C). Subsequently, after treatment of BMSCs with 50 µM H 2 O 2 at different times, it was found that when H 2 O 2 treated for 12 h, the Bcl-2/Bax ratio reached a peak, while the expression of Bax and caspase-3 was not affected, however, the two proapoptosis proteins were increased when the action time exceeds 12 h (Fig. 3D,E). The data also con rmed that treatment with 50 µM H 2 O 2 for 12 h might be the optimal pretreatment condition for BMSCs preconditioning in vitro in terms of expression ratio of anti-and pro-apoptotic genes.
In addition to increasing the Bcl-2/Bax expression ratio, BMSCs preconditioned with 50 µM H 2 O 2 for 12 h signi cantly decreased level of cleaved caspase-3 without having effects on caspase-9 and − 3, and the level of cleaved caspase-9 ( Fig. 3F-H). This was in contrast to the effects by 300 µM H 2 O 2 treatment.
These results indicated that preconditioning BMSCs with 50 µM H 2 O 2 for 12 h could suppress oxidative stress-induced activation of mitochondrial apoptosis pathway.

Optimal H 2 O 2 preconditioning activated PI3K/Akt/mTOR signaling while inhibited GSK-3β activity in BMSCs
The PI3K/Akt/mTOR pathway and GSK-3β protein play a vital role in promoting stem cell survival in response to oxidative stress [17][18][19]29]. Exposure to 25-50 µM H 2 O 2 induced an obvious increase in the phosphorylation levels of PI3K, AKT, mTOR and GSK-3β in BMSCs, but they were signi cantly deceased at 200 µM H 2 O 2 , inversely, the GSK-3β expression was up-regulated at higher H 2 O 2 ( Fig. 4A-D). The results indicated that low doses of H 2 O 2 could activate the PI3K/Akt/mTOR pathway and the inhibit GSK-3β activity in BMSCs, whereas high dose of H 2 O 2 -induced oxidative stress inhibited this pathway and enhanced GSK-3β activity. Further, the results showed that 50 µM H 2 O 2 preconditioning could largely prevent 300 µM H 2 O 2 -induced inactivating PI3K/Akt/mTOR pathway and activating GSK-3β. This effect was signi cantly blocked by PI3K inhibitor LY294002 (Fig. 4E), simultaneously, the protective effect of 50 µM H 2 O 2 preconditioning was counteracted by LY294002 with a remarkable increase in the apoptotic rate of BMSCs (Fig. 4F, G). These results showed that the protective effect of 50 µM H 2 O 2 against oxidative stress-induced BMSCs apoptosis was mediated, at least in part, by activation of PI3K/Akt/mTOR pathway and inhibition of GSK-3β activity.

Optimal H 2 O 2 -preconditioning enhanced BMSCs' tissue engraftment and wound healing in mice
In order to further con rm whether H 2 O 2 preconditioning enhanced the target homing e ciency of tail vein infused BMSCs towards wounds and improved the therapeutic potential of BMSCs in wounds, the full thickness wound model in Balb/C mice was established. The number of DiI-labeled BMSCs in the H 2 O 2 -preconditioned group at 1 day and 3 day post-wounding signi cantly increased compared to that of the un-preconditioned BMSCs group. Compared with the rst day post-wounding, the number of BMSCs in the H 2 O 2 -preconditioned group decreased only slightly on the third day, however, the number of BMSCs in the un-preconditioned BMSCs group was signi cantly reduced on the third day (Fig. 5A, B). The result hinted that the H 2 O 2 -preconditioning could not only increase BMSCs migrating and homing toward wounds, but also prolong the residence time of transplanted BMSCs. The wound closure rate was markedly higher in two BMSCs groups than in the PBS control group and was the highest in H 2 O 2preconditioned group from day 5 to day 15 post-wounding (Fig. 5C, D). Therefore, H 2 O 2 preconditioning not only increased migration of BMSCs into wound areas, but also accelerated wound closure. Further, the results showed that the level of VEGF in the two BMSCs groups were signi cantly higher compared to that of the PBS group (Fig. 5E, F), and the microvessel density were the highest in H 2 O 2 -preconditioned group at 5, 7 and 10 day post-wounding (Fig. 5G, H). Similarly, compared with the other two groups, HE staining showed the greatest increase in cell density and blood vessel density in the granulation tissue of H 2 O 2 -preconditioned group at 5 days post-wounding (Supplement Fig. 2). These results suggested that H 2 O 2 -preconditioned BMSCs improved tissue engraftments, and local angiogenesis that resulted in accelerated would healing.

Discussion
Although cell-based therapy plays an important role in the eld of the skin tissue regeneration, low engraftment e ciency and poor survival rate of transplanted stem cells triggered by oxidative stress at the wound tissue limit the therapeutic potential of MSCs [2,3]. Accumulating evidences suggest that H 2 O 2 preconditioning could enhance survival of MSCs [21][22][23]. However, optimal H 2 O 2 concentration, suitable treatment time-course, and the underlying mechanism remained largely undetermined.
Different concentrations of H 2 O 2 profoundly affect biological functions of stem cells [24][25][26]. For a long time, H 2 O 2 was considered deleterious molecules. However, recent studies suggest that H 2 O 2 is necessary for effective self-renewal in stem cells [15,24,30]. In general, moderate levels of H 2 O 2 may function as signals to promote cell proliferation and survival, playing a growth factor-like role in cells. However, severe increase of H 2 O 2 induces senescence and oxidative stress in MSCs [15,26] Simultaneously, we con rmed that 50 µM H 2 O 2 maximized the promotion of SDF-1 and its receptors CXCR4/7 expression, which was also conducive to the enhancement of cell migration and survival, respectively [6,7]. In fact, we observed that 50 µM H 2 O 2 treatment could markedly promote migration capability of BMSCs in vitro, however, the effect was eliminated by CXCR4 antibody, which indicated H 2 O 2 promoting migration of BMSCs through augmenting the SDF-1/CXCR4 axis. Thus, these results con rmed the important effect of H 2 O 2 concentration on biological characteristics of stem cells,and also con rmed that low concentration of H 2 O 2 had a growth factor-like effect to promote stem cell proliferation. Although it has been reported that the concentration of stem cells preconditioned with H 2 O 2 is 25 µM, 50 µM and 100µM [22,23], but there's no su cient basis for deciding which H 2 O 2 concentration to choose, and we believed that 50 µM H 2 O 2 represent the optimal concentration of stem cells preconditioning in our study.
It is well known that oxidative stress induces apoptosis through the intrinsic mitochondrial pathway [28]. The expression ratio of anti-apoptotic Bcl-2 and pro-apoptotic Bax (Bcl-2/Bax) are the key to maintaining mitochondrial membrane stability, preventing mitochondrial depolarization and the release of cytochrome c into the cytoplasm, and thereby inhibiting the mitochondrial apoptotic pathway [28]. High concentrations of H 2 O 2 induced-oxidative stress have been shown to induce mitochondrial injury by reducing mitochondrial membrane potential and decreasing the expression ratio of Bcl-2/Bax, subsequently, initiating apoptosis [29]. In the present study, we similarly con rmed that 50 µM H 2 O 2 could maximize the up-regulation of Bcl-2 and Bcl-2/Bax expression ratio with no changes in pro-apoptotic proteins Bax and cleaved-caspase3 expressions, when using different concentrations of H 2 O 2 to stimulate stem cells for 12 hours. Further, it was found that pretreatment BMSCs for 12 h was the optimal time course when using 50 µM H 2 O 2 to treat BMSCs with different time course, because the Bcl-2 and Bcl-2/Bax expression ratio peaked with no changes in Bax and cleaved-caspase3 expressions. Lastly, we suggested that stem cell preconditioning with 50 µM H 2 O 2 for 12 h is the optimal protocol for resistance to oxidative stress apoptosis of BMSCs through maximizing increasing the Bcl-2/Bax expression ratio,in addition, we also con rmed that 50 µM H 2 O 2 could markedly promote migration capability of BMSCs through maximizing augmenting the SDF-1/CXCR4/7 axis. Further, the results con rmed that the optimal preconditioning protocol (50 µM H 2 O 2 for 12 h) could signi cantly enhance BMSCs survival under oxidative stress and suppress mitochondrial apoptotic pathway through increase the expression ratio of Bcl-2/Bax and inhibition of apoptosis executive proteins cleaved caspase-9 and − 3 expressions. However, LY294002 abolishes of the cytoprotective effect of 50 µM H 2 O 2 preconditioning. The data suggested that the PI3K/Akt pathway was involved in preconditioning-induced cytoprotective effect.
It was reported that the PI3K/Akt pathway has been shown to play a major role in the promotion of cell survival, and prevention of apoptosis in response to oxidative stress [15,20,31]. The activation of PI3K/Akt causes a cascade of a downstream responses from mTOR, Bcl-2, Bax and GSK-3β, which all regulate cellular functions [15,20]. The PI3K/Akt pathway regulates cell migration via activation of mTOR, increases cell survival via Bcl-2/Bax, and increases cell proliferation and against apoptosis via inhibition of GSK-3β [32,33]. Akt directly phosphorylates GSK-3β at Ser9 which negatively regulates its kinase activity [34], phosphorylated GSK-3β, a key molecules inhibits oxidative stress-induced apoptosis through inhibition of the opening of mitochondrial permeability transition pore(mPTP), thereby suppressing cytochrome C release for mitochondria to cytosol, and preventing cell apoptosis [34]. And also, Akt induced GSK-3β phosphorylated leads to the accumulation of β-catenin in the nucleus [33], which is a transcriptional factor that increases the expression of c-Myc to promote cell proliferation through up-regulation of cyclin D1 expression, while inhibiting p16 expression, leading to promoting cell proliferation. In the present study, 25-100 µM H 2 O 2 upregulated the phosphorylated levels of PI3K, Akt, mTOR, and GSK-3β, resulting in activating of PI3K/Akt/mTOR pathway and inhibiting GSK-3β activity, the effect of 50 µM H 2 O 2 is the strongest. These results further demonstrated that low dose of H 2 O 2 possesses growth factor-like characteristics, which could activate the PI3K/Akt pathway. The growth hormones and cytokines are the upstream stimulators of this pathway [16,24]. SDF-1 could promote MSC survival and migration also by the activation of the PI3K/AKT pathway [8,9,[17][18][19][20]. In addition, low dose H 2 O 2 directly activated this pathway through diffusion into the cytoplasm as a second messenger may be another activation pathway [16,24]. Inversely, we con rmed that high concentration of H 2 O 2 (300 µM)induced oxidative stress not only inhibited the PI3K/Akt/mTOR pathway and increased the GSK-3β activity by inhibiting the phosphorylated of these proteins in this pathway, but also signi cantly increased apoptotic rate of BMSCs. Hence, oxidative stress-induced apoptosis was mediated by inactivation of the PI3K/Akt/mTOR pathway and increased GSK-3β activity, which was consistent with literature reports [15,20,31]. However, we found that 50 µM H 2 O 2 preconditioning could reduce the inhibition of PI3K/Akt pathway induced by oxidative stress through promoting the phosphorylation of PI3K, Akt, mTOR, and GSK-3β of this pathway and increasing in Bcl-2 expression in BMSCs, nally, resulting in inhibtion of mitochondrial apoptosis pathway. Therefore, the protective effect of 50 µM H 2 O 2 preconditioning might be dependent on the activation of the PI3K/Akt signaling pathway, contributing inhibition of its downstream target GSK-3β activity and upregulation of anti-apoptotic Bcl-2 expression.
The success of stem-cell therapy depends on the migration and survival of the transplanted cells [35,36]. Therefore, enhancing the migration ability and survival of BMSCs are the key to optimize stem cell therapy in wounds. Based on the vitro experiment, in which we con rmed that 50  Although some studies showed that transplanted MSCs could promote wound healing [37], we observed that the therapeutic effect of H 2 O 2 -preconditioned BMSCs was signi cantly better than that of unpreconditioned BMSCs in terms of increase in the speedy wound closure rate. The higher VEGF levels and the microvessel density in wound healing tissues indicated the critical role of H 2 O 2 -preconditioned BMSCs in elevating angiogenesis for accelerated wound healing. Taken together, these results indicated Page 12/23 that the preconditioning of BMSCs with H 2 O 2 enhanced their therapeutic potential by increasing their engraftment e ciency in wounds, responsive secretion of VEGF, and facilitating neovascularization, thereby improving the quality of wound healing.

Conclusions
This study rst demonstrated that pretreated BMSCs with 50 µM H 2 O 2 for 12 hours was the optimal preconditioning protocol, which was indicated by maximizing activating the pathways of SDF-1/CXCR4 and PI3K/Akt/mTOR and inhibiting GSK-3β activity, thereby playing a cytoprotective role in oxidative stress-induced BMSCs apoptosis. Finally, the data con rmed that BMSCs preconditioned with this protocol could signi cantly improve therapeutic potential of transplanted stem cells, which might represent an attractive intervention strategy to promote wound healing. The data that support the ndings of this study are available from the corresponding author upon request.

Con icts of Interest
The authors declare that they have no con icts of interest.

Figure 4
The protective effect of optimal H2O2 preconditioning is mediated by the activation of PI3K/Akt/mTOR/GSK-3β signaling. (A, B, C, D) Western blot analysis of the expressions of total and phosphorylated PI3K, Akt, mTOR and GSK-3β proteins in BMSCs exposed to various low concentrations of H2O2 for 24 h. Values represent the mean ± SEM (n = 3) *p < 0.05, compared with the control group. (E) Western blot analysis of the expressions for phosphorylated Akt, mTOR, GSK-3β, Bcl-2 and cleaved caspase-3 in H2O2 -preconditioned BMSCs exposed to 300 μmol/L for 24 h after 2 h pre-incubation with 10 μM LY294002. (F, G) The rate of apoptotic cells was determined by flow cytometry. Values represent the mean ± SEM (n = 3). **P<0.01, compared with the control group; ##P<0.01, compared with H2O2 300 μmol/L group. ΔΔP<0.05, compared with the H2O2-proconditioned group.