Exosomal Mir-21 Derived from Umbilical Cord Mesenchymal Stem Cells Promotes Angiogenesis by Activating SPRY1/PI3K/AKT Pathway and Contributes New Bone Formation in a Rat Model.


 Background: Delayed atrophic healing and non-union remain the most difficult types of complications to treat and can lead to numerous re-operations and high socioeconomic cost. Exosome derived from MSCs is a promising strategy in the treatment of fracture. However, it’s still not fully clear in the underlying mechanisms. Methods: In this study, we first elucidated the underlying mechanism of umbilical cord MSC (uMSC)-Exo-induced angiogenesis in bone regeneration and fracture repair. RT-PCR, luciferase reporter, western blot and immunofluorescence in situ hybridization assay were performed to identify the expression of related genes and pathways in uMSC-Exo. Proliferation, tube formation and transwell migration assay were used to evaluated the effects of uMSC-Exo in vitro. A rat calvarial defect model was used to evaluated the effects of uMSC-Exo in vivo.Results: We found that, miR-21 was the most abundant miRNA in the uMSC-Exos. In vivo and in vitro experiments demonstrated that, the uMSC-Exo could promote the migration, angiogenic and capacity of HUVECs via miR-21, inhibition of miR-21 could attenuate the effects of uMSC-Exos. For mechanical study, we found that, exosomal miR-21 derived from uMSCs activates PI3K/AKT signalling by targeting SPRY1 in HUVECs, SPRY1 knockdown could promote the PI3K/AKT activation and HUVEC proliferation, migration and angiogenesis. In addition, exosomal miR-21 derived from uMSCs enhanced local microvascular network formation and bone regeneration in vivo. ConclusionIn: In conclusion, we firstly reported that uMSC-derived exosomes as a ready-made regenerative medicine therapy to bone healing.


Introduction
Effective treatment for fracture remains challenging, and poor or impaired recovery from fracture is an increasingly severe problem in an ageing society [1,2]. In approximately 5-10% of cases, fracture union is delayed or compromised and can lead to numerous re-operations and high socioeconomic costs [3,4].
Throughout the processes of bone regeneration and fracture healing, angiogenesis is indispensable [5].
The effects of drug intervention on the regeneration of intractable sequestrum or atrophic callus are not adequate by reason of poorly vascularized tissue surrounding the haematoma at the fracture site [5,6]. Thus, factors of angiogenesis, which is the growth of new vasculature, may be promising therapeutic targets for fracture healing and crucial to the development of more-e cacious biological strategies.
Evidence has shown that, application of mesenchymal stem cells (MSCs) could be a potential therapeutic strategy to induce bone regeneration [7,8]. Nevertheless, it remains limitary for the clinical use of stem cells due to many risk factors, including undesirable immune reaction, thrombosis, and even tumour formation [9,10]. Various studies have shown that exosomes derived from MSCs plays an important role in clinical therapy, which are emerging as components of a cell-free regenerative medicinal approach to tissue repair because of their stem cell-like pro-regenerative properties and the prospect of circumventing some of the drawbacks of MSCs use [11,12]. The results from our previous study demonstrated that umbilical cord mesenchymal stem cells-derived exosomes (uMSC-Exos) promoted angiogenesis in a rat femur fracture model [13]. The pro-angiogenic function of endothelial cells has been induced, and several pro-angiogenic factors, such as HIF-1α, have been upregulated. These results are consistent with those of several recent studies [14][15][16]. But the underlying mechanisms of uMSC-Exo-promoted angiogenesis in bone healing are not fully understood.
Exosomes are reported as nano-carriers ranging from 40-100 nm in diameter, which contains a various of genetic information, such as mRNA and miRNA. Among all the molecules possibly contained within exosomes. A growing body of evidence indicates that miRNAs in exosomes regulate the cell activity [17]. For instance, uMSC-derived exosomes promoted wound healing while preventing scar formation via the intercellular transfer of speci c miRNAs, such as, miR-21, miR-23a, miR-125b, and miR-145 [18]. To further investigate the exosome-related cell-cell communication between endothelial cells and uMSCs, we analyzed the critical miRNAs of uMSC-Exos and evaluated their roles in endothelial cell biological function. In addition, the target genes and molecular signalling pathways in recipient cells were revealed. Ultimately, we veri ed the hypothesized molecular mechanisms and demonstrated the possible optimal therapeutic effects of uMSC-Exos application in a cranial defect model.

Materials And Methods
uMSC and HUVEC cultures uMSCs were cultured in α-MEM culture medium added by 15% FBS and 4% penicillin and streptomycin (Gibco, Grand Island, USA). The human umbilical cord vein endothelial cells (HUVECs) were separated and harvested as previously described [1]. The cell culture protocols were authorized through Ethical Committee of the Navy Medical University. Brie y, uMSC were obtained from 3 healthy donors respectively and were collected to get uMSC-Exos.

Quantitative real-time PCR
Total RNA of HUVECs transfected with uMSC-Exo-anti-miR-NC, uMSC-Exo-anti-miR-21 and uMSC-Exo was isolated by TRIzol solution followed by the informations of manufacturer. The concentration of the total RNA solution were measured by a commercial kit (Thermo Fisher Scienti c). Then, we reversed the rststrand cDNA via 2 µg RNA with a cDNA commercial kit (Thermo). Quantitative real-time PCR (qRT-PCR) was conducted with a 25-µL reaction volume (Detailed steps are visible in the supplementary material).
Puri cation and identi cation of exosomes derived from uMSCs Firstly, the uMSCs were seeded at 1.5×10 5 cells in a T25 polystyrene culture asks (Corning) with 5 mL of α-MEM culture medium for 24 hours adherence. After that, the asks were washed for 3 times by PBS and the culture medium with exo-free-FBS [3] was added in them. After 48 h, the freshly collected supernatant was centrifuged at 1500×g for 5 min at room temperature and then 10000×g for half an hour at 4 ℃ to remove the impurities. Exosomes were precipitated by an ExoQuick-TC kit (ExoQuick-TC) followed by the manufacturer's instructions. The identity of the exosomes was determined or con rmed by their binding a nity for CD63, CD81, and CD9 antibodies (Abcam, USA), which were reported to be speci c exosome biomarkers [4,5].
Immuno uorescence in situ hybridization assay of miR-21 To perform immuno uorescence in situ hybridization assays, HUVECs were treated with UEFS and uMSC-Exo-miR-21, seeded on coverslips and xed by 4% PFA at room temperature for 5 min. After that, the coverslips were treated with blocking reagents (Sigma-Aldrich, USA) for 15 min. Next, a uorescent probe for miR-21 and the scramble control was hybridized with the HUVEC slides according to well-established consecutive procedures. After two hours of incubation, the hybridization was stopped via stopping buffer (Sigma-Aldrich, USA) and the nuclei were counterstained with DAPI. After washing three times, the slides were dehydrated and photographed under an immuno uorescence microscope at 200× magni cation.

Luciferase reporter assay
We predicted the potential binding sites of miR-21 to get the fragments sequences on the biological prediction website (http://www.microrna.org), and the SPRY1 was proved as a target gene to miR-21. The chemical synthesis was performed, after that, luciferase reporter plasmid were cotransfected into HEK293 cells. Cells were incubated for 36 hours in a 12-well plate. After transfection, the cells were examined by a commercial dual-luciferase reporter assay kit (Nanjing, Jiangsu, China), and the luciferase activity was detected.

Western blotting
HUVECs treated with uMSC-Exo-anti-miR-NC, uMSC-Exo-anti-miR-21 or uMSC-Exo were washed with icecold PBS three times after the culture medium was removed. The total protein content was extracted from the HUVECs by highly concentrated RIPA lysis buffer and detected by the BCA protein assay kit. 10 µg of total protein was added to SDS-PAGE and transferred into a PVDF membrane. A 5% non-fat milk solution prepared with 1×TBST was utilized to block the PVDF membrane for one hour at 4 ℃. Then, the PVDF membrane was incubated with the following speci c primary antibodies for 1 h at room temperature Proliferation assay: To put it simply, 1×10 5 HUVEC cells/well were incubated in a 24-well plates and treated with uMSC-Exos (10 µg/well), uMSC-Exo-anti-miR-NC and uMSC-Exo-anti-miR-21. A group of HUVECs with the same volume of PBS served as the control group. At different points in time, 15 µL of CCK-8 solution (Kyushu Island, Japan) was put into the well. Cells were incubated at room temperature for 2 hours. The absorbance of cells was detected at 450 nm by a microplate reader.
Tube formation assay (in vitro angiogenesis) Cryopreserved HUVECs were seeded in a tissue culture ask. A commercial tube formation assay kit (Cell Biolabs, USA) was used to assess in vitro angiogenesis within 18 h of cell seeding. Thawed ECM gel was added to a plate and cultured for 1 hour. After that, HUVECs in suspension were added to the solidi ed ECM gel and cultured for 12 h at room temperature. We added uMSC-Exo-anti-miR-NC, uMSC-Exo-anti-miR-21, uMSC-Exo (100 µg/mL; 200 µg/well) to the HUVEC tube formation system. We examined and imaged the tube formation at 12 h and 24 h using a light microscope in a high magni cation eld.

Scratch test
A total of 2×10 5 HUVECs were put into a 12-well plate and incubated until con uent. Using pipette tip, we made a straight scratch in the monolayer to simulate a wound. The HUVECs were transfected with PBS, uMSC-Exo-anti-miR-NC, uMSC-Exo-anti-miR-21, and uMSC-Exo (100 µg/mL; 200 µg/well). After 0 h, 6 h, 12 h and 24 h of incubation, the width of the wound was photographed and cell migration was evaluated using an optical microscope (Leica, Germany). The width of the wound at 0 h was set as a negative control. Moreover, the wound width at each time point was normalized to that at 0 h and compared to the width measured at other time points. The assay was repeated at least three times.

Rat calvarial defect model
All surgical procedures were performed under general anaesthesia using intraperitoneal injection of chloral hydrate (4%, 9 mL/kg body weight), and post-operative analgesic care was ensured with the administration of tramadol. All operations were performed under sterile conditions. Classical porous β-TCP scaffolds (5-mm diameter and 2-mm depth) with an average pore size of 500 µm and 75% porosity and a HyStem-HP hydrogel (catalogue: GS315, Glycosan BioSystems, Salt Lake City, UT, USA) were used as the exosome carriers. Aliquots of 200 µg of MSC-Exos with or without overexpressed miR-21 (uMSC-Exo group and uMSC-Exo-miR-21 + group, respectively) were dropped onto each scaffold under sterile conditions and lyophilized for at least 4 h, and the scaffold generated with phosphate-buffered saline was used as a negative control group (blank group). The scaffolds were implanted into calvarial defects in adult male Wistar rats (weighing 400-450 g) as previously described [15]. All surgeries were performed according to a protocol approved by the Institutional Animal Care Committee at the Navy Medical University.

Micro-CT analysis
The rats were sacri ced successively at predetermined time points over 6 weeks. The cranium of each rat was obtained and xed in a 4% paraformaldehyde solution before further analysis. The vascularity was detected using a micro-CT-based method as described previously [13] (Detailed steps are visible in the supplementary material). The defect area was analysed to calculate the percentage of new bone volume (BV), tissue volume (TV), BV/TV, vessel volume (VV), and bone mineral density (BMD) (n = 3).

Histological analysis.
Specimens and intact surrounding tissues were further processed to obtain 6 µm para n-embedded sections. Haematoxylin/eosin (HE) staining was conducted for histological assessments. SPRY1, VEGFA and CD31 expression was analyzed by immunohistochemistry with the appropriate antibodies (Abcam, USA). Ultimately, all the sections were observed with a light microscope (Leica, Germany), and then the levels of VEGFA, CD31 and SPRY1 expression were analyzed in each sample via Image J software.

Statistical analysis
Each assay in this study was performed in triplicate. All experimental data were statistically analysed by SPSS software and presented as the mean ± SD. A two-sided Student's t-test was performed to compare the differences between two groups. The difference was considered as signi cant when the p value was less than 0.05 or 0.01.

Results
1. miR-21 was the most abundant miRNA in the uMSC-Exos and was shuttled directly from uMSCs to HUVECs via exosomes.
Accumulating evidence demonstrates that exosomes can fuse with the target cell membrane to deliver various messengers, such as miRNA, proteins or lipids, thereby facilitating signal cross talk between different kinds of cell types to control multiple target genes [19]. Although the miRNA pro les of some typical cancer-derived exosomes have been thoroughly illustrated [20], the exosome miRNA signatures in uMSCs and other cells have not been fully elucidated and need to be investigated further. To characterize the uMSC-derived exosomal miRNAs, we analysed the expression levels of microRNAs in uMSC-Exos via high-throughput sequencing patterns with a HEK293-Exos as controls. The microRNA expression patterns in uMSCs were then analysed using a GEO data set (GSE46989, http://www.ncbi.nih.gov/geo/). We demonstrated that the speci c miRNA signature in uMSC-Exos that was completely different from that of HEK293-Exos and uMSCs. The miR-21 was expressed at the highest level among the total miRNAs in the uMSC-Exos ( Figure 1A, B). Based on the top 10 expression of miRNAs in the uMSCs and uMSC-Exos, we found that miR-21-5p, miR-100-5p, miR-125b-5p, and let-7f-5p were also highly expressed in the uMSCs, with miR-21 accounting for the highest proportion in both the maternal cells and the exosomes ( Figure  1C, D).
To verify our conjecture, qRT-PCR was used to evaluate the 10 most abundant miRNAs in uMSC-Exos and their pre-miRNAs expression levels in the HUVECs treated with uMSC-Exos or HEK293-Exos for 48 h. Only the expression of miR-21-5p in the uMSC-Exo group was signi cantly increased compared with that of the other miRNAs, while the pre-miRNAs were not affected ( Figure 1E, F). This result con rmed that an amount of mature miR-21 su cient to perform biological functions was transported into the HUVECs by the uMSC-Exos. Exosomes have been shown to perform biological roles similar to that of their maternal cells. Moreover, the paracrine function of uMSCs is the most critical in the eld of tissue regeneration [11]. Therefore, we hypothesized that the role of uMSCs may be associated with the secretion of exosomes that deliver miR-21 to target cells.
To investigate whether exosomes mediate miR transfer, the expression of miR-21 was measured after MSCs were treated with 10 μM GW4869 (an exosome release inhibitor) for 48 h in conditioned medium (CM). GW4869 is a cell-permeable symmetrical dihydroimidazole-amide compound that acts as a potent, speci c, non-competitive inhibitor of membrane neutral sphingomyelinase (nSMase) which has been reported to markedly reduce exosome release [21,22]. As shown in Figure 1G, the levels of miR-21 in the CM collected from MSCs treated with GW4869 were signi cantly decreased compared with those in the CM obtained from control uMSCs. In addition, the expression of miR-21 in HUVECs treated with GW4869-CM for 48 h was also signi cantly decreased compared to that of HUVECs treated with uMSC-CM ( Figure  1H), indicating that exosomes mediated the miRNA transport between the uMSCs and HUVECs. To improve the visibility of the results, uMSC-Exos uorescently stained with PKH67 were cultured with HUVECs for 48 h. In addition, FAM was conjugated to miR-21 (miR-21-FAM) in the uMSC-Exos to trace miR-21 by in situ hybridization. The immuno uorescence showed that the green exosomes were concentrated mainly near the nuclei of the HUVECs and the location of the ectogenic miR-21 (red spots) was consistent with that of the exosomes. However, exosome and miRNA components were not detected in the uMSC-Exo-free supernatant (UEFS) ( Figure 1I).
All these results indicated that miR-21 might be contained in the exosomes excreted by the uMSCs, a nding that supports our hypothesis that uMSCs deliver miR-21 directly to the target cell for biological function.

Preparation and identi cation of uMSC-Exos with inhibited miR-21
To validate the critical roles of miRNAs, inhibition experiments are more illustrative than overexpression experiments. We developed a strategy to stably inhibit miRNAs inside the uMSC-Exos. The uMSCs were transfected with antagomir RNA-21 to block miR-21 (uMSC-Exo-anti-miR-21) or a scrambled antagomir as a negative control (uMSC-Exo-anti-miR-NC). The exosomes were then extracted using a kit, and the qRT-PCR results demonstrated that the uMSC-Exo-anti-miR-21 group had a signi cantly downregulated level of miR-21 and the level was not changed in the control groups ( Figure 2A).
The exosomes in the three groups were successfully precipitated by an ExoQuick-TC kit (ExoQuick-TC, System Biosciences) according to the manufacturer's instructions. The morphology of the puri ed exosomes was observed by using transmission electron microscopy (TEM). Whether exposed to the inhibitor or not, all exosomes had a saucer-like shape with a diameter ranging from 40 to 100 nm ( Figure  2B). The diameter of the exosomes was determined with a NanoSight LM10 instrument (NanoSight, Amesbury, U.K., http://www.nanosight.com) ( Figure 2C). The markers of the exosomes, namely, CD9, CD63 and CD81, were also detected by Western blotting, and the results showed that compared with the amounts in the UEFS, the CD9, CD63 and CD81 levels were enriched in the exosome samples ( Figure 2D). This nding indicates that the remodelling and extraction of the uMSC-Exos were effective and reliable.

Effects of exosomal miR-21 on the cellular functions of HUVECs
We evaluated the role of miR-21 in uMSC-Exo by in vitro and in vivo experiments. All phenotypic experiments were conducted with four groups: The uMSC-Exo-anti-miR-NC, uMSC-Exo-anti-miR-21, uMSC-Exo groups and a blank group (HUVECs with PBS). Previous reports demonstrated that miR-21 has an effect on cancer cell proliferation [6]. However, the mechanism of uMSC exosomal miR-21 in HUVEC proliferation remains unknown. The CCK-8 analysis showed that exosome stimulation resulted in a signi cant increase in HUVEC proliferation, although the effect was reduced by the miR-21 inhibitor ( Figure 3A).
To determine whether exosomal miR-21 modulates cell migration, HUVECs were incubated with PBS, uMSC-Exo-anti-miR-NC, uMSC-Exo-anti-miR-21 or uMSC-Exo and the monolayer was disrupted with a straight scratch. After 12 h and 24 h, the wound width in the culture of each group was photographed ( Figure 3B). We observed that at the same time point (12 h or 24 h), the migration rate of HUVECs in the uMSC-Exo-anti-miR-21 group was signi cantly lower than that of the other two groups (uMSC-Exo and uMSC-Exo-anti-miR-NC) and similar to that of the blank control group. Furthermore, we conducted a Transwell assay, and the results showed that the number of migrating HUVECs was reduced by approximately one-half after the treatment with exosomal miR-21 inhibitor ( Figure 3C). These results indicated that the loss of miR21 uMSC-Exo no longer enhanced the migration capacity of HUVECs.
The tube formation assay was performed to obtain direct evidence of the angiogenic function of miR-21 in HUVECs. The results showed that there were newly growing branch points and tube lengths in the uMSC-Exo group and uMSC-Exo-anti-miR-NC group that formed in a time-dependent manner while the tube formation in the group with inhibited miR-21 was weak ( Figure 4A). Thus, these results directly demonstrated the angiogenic role of uMSC-Exos in HUVECs, which could be suppressed by the miR-21 inhibitor.
The role of miR-21 derived from uMSC-Exos in promoting angiogenesis was veri ed by an in ovo angiogenesis assay. Eight-day-old embryonated chicken eggs were used for different treatments once a day. Two days after treatment, the CAM was assessed for changes in the number and length of blood vessels. The data showed that uMSC-Exo-anti-miR-21 was able to impair in ovo neovascularization, and the value was two-fold less than that of the other two control groups as assessed on the tenth day. In addition, on the 12th day, the density of the vessels increased compared to that observed in previous days, although the inhibition effect was most obvious in the experimental group ( Figure 4B). This nding is consistent with our in vitro test results showing that exosomes with inhibited miR-21 have a signi cantly reduced effect on angiogenesis.

Exosomal miR-21 derived from uMSCs activates PI3K/AKT signalling by targeting SPRY1 in HUVECs
VEGFA has been reported to have an essential role in HUVEC-mediated angiogenesis [7]. Various types of evidence veri ed that VEGF increases the vascular density through SPRY1 and that SPRY1 negatively regulates angiogenesis. SPRY1 was predicted to be a potential target gene of miR-21 by the microRNA (http://www.microrna.org/) and TargetScan (http://www.targetscan.org/) databases. However, whether uMSC-Exo-miR-21 plays a critical role through SPRY1 in HUVEC-mediated angiogenesis has not been reported, and the signalling pathways are also unknown. In this study, the luciferase reporter assay results showed that miR-21-5p can bind to the 3'-UTR of SPRY1 and suppress the transcription of SPRY1 when the miR-21 overexpression vector was transfected into HEK293 cells ( Figure 5A, B).
An inhibitory effect of SPRY1 on angiogenesis, which is induced by PI3K/AKT activation in cancer cells, has been demonstrated [23]. Thus, we used the method of SPRY1 knockdown to identify the downstream pathway in HUVECs. The qRT-PCR results indicated that the mRNA level of SPRY1 increased dramatically by approximately two-fold when uMSC-Exo-anti-miR-21 was used to interfere with HUVECs, and no difference was observed between the other two groups ( Figure 5C). The mRNA level of the downstream gene P13K showed the opposite trend under the same conditions, although the changes in the total AKT levels were not apparent. The miR-21 inhibitor markedly decreased the upregulated mRNA levels of HIF-1α and VEGFA (genes related to vascularization) induced by exosomes T (Figure 5D). These results were consistent with those of the Western blot assay ( Figure 5E). Notably, uMSC-Exo and uMSC-Exo-anti-miR21 induced signi cant increases in the phosphorylation of Akt while the total AKT remained unchanged.. Hence, we suggested that uMSC-Exo-miR-21 suppressed the expression of SPRY1 and promoted the hyperactivation of AKT (p-AKT), leading to increased angiogenesis by regulating the SPRY1/PI3K/AKT signalling axis in the HUVECs. Thus, the activation of SPRY1/PI3K/AKT pathways may be the underlying mechanism by which miR-21 containing uMSC-Exos enhance angiogenesis.

SPRY1 knockdown increases PI3K/AKT activation and HUVEC proliferation, migration and angiogenesis.
To con rm the key role of SPRY1 in angiogenesis and assess whether knocking down the expression of SPRY1 can achieve similar effects as uMSCs-Exo on angiogenesis, we used siRNA to inhibit the expression of SPRY1 in HUVECs. First, we examined the inhibitory e ciency of these siRNAs by qRT-PCR and Western blot ( Figure 6A, B), and the most effective siRNA (siSPRY1 #1) was used for the following functional assays. Next, HUVECs were transfected with SPRY1 siRNAs#1 in culture for 24 h. PI3K/AKT activation and the target genes (VEGFA and HIF-α) were determined by qRT-PCR and Western blot. As expected, we observed an increased level of PI3k, AKT, VEGFA and HIF-1α in the uMSC-Exo+siRNA-SPRY1 #1 group. (Figure 6C, D) Because SPRY1 was found to decrease PI3K/AKT activation, we then tested whether SPRY1 knockdown truly stimulates HUVEC-induced angiogenesis. To determine the extent of HUVEC migration, the scratch experiment showed that at 12 h and 24 h, the HUVECs in the uMSC-Exo+siRNA-SPRY1 #1 group migrated at a faster rate, although the number of cells that migrated increased in both groups ( Figure 6E). Similarly, the tube formation data showed that after SPRY1 was silenced, the number of branches and blood vessels more than doubled ( Figure 6F).
Taken together, the data from our in vitro functional assays on HUVECs suggested that SPRY1 knockdown increased PI3K/AKT activation and HUVEC proliferation, migration and angiogenesis.

Exosomal miR-21 derived from uMSCs enhanced local microvascular network formation and bone regeneration in vivo
To study the in uence of exosomal miR-21 on bone formation in vivo, the rat cranial bone defect model was carried out for a duration of 6 weeks. In particular, to evaluate the potential therapeutic role of regulated exosomes in bone healing, we developed an experimental group with exosomes that overexpressed miR-21 (uMSC-Exo-miR-21+) and compared the ndings with those of the uMSC-Exo group and the negative control (NC) group.
Then, micro-CT and histological analyses were used to evaluate the extent of calvarial defect repair in the three groups ( Figure 7A). Similar to our previous study results in a femur fracture model, the administration of uMSC-Exos led to an obvious increase in the amount of new bone formation compared with that in the NC group. Moreover, compared to both the uMSC-Exo and the control groups, treatment with uMSC-Exos with overexpressed miR-21 led to a signi cant increase in the bone volume (BV), BV/TV and bone mineral density (BMD). Vascular growth within the bone callus was evaluated by imaging of contrast-perfused, decalci ed specimens. The vessel volume was remarkably increased in the uMSC-Exo-miR-21+ group.
H&E staining was used to observe the microscopic bone and soft tissues surrounding the calvarial defect 6 weeks post-operation ( Figure 7B). Histological observations at low magni cation (40×) revealed that the implanted scaffolds were covered with abundant cells and tissue in both the uMSC-Exo and uMSC-Exo-miR-21+ groups, although a greater amount of callus tissue was observed on the scaffolds in the inner spaces of the uMSC-Exo-miR-21+ group. Moreover, the immunohistochemistry assay results showed that signi cantly more CD31-positive blood vessels formed in the uMSC-Exo-miR-21+ group. Compared with that in the control and uMSC-Exo groups, the expression of VEGFA in the new callus tissue was upregulated in the uMSC-Exo-miR-21+ group. Unsurprisingly, the expression of SPRY1 in the uMSC-Exo-miR-21+ group was decreased. These results were consistent with the results from our cell experiments, implying that exosomal miR-21 delivery stimulated greater vessel formation within callus regions and enhanced bone formation in the defect space.

Discussion
Our primary study con rmed that exosomes derived from uMSCs are bene cial to fracture healing in both a cell culture model and a well-established clinically relevant rat model of fracture. Interestingly, we found that in the process of promoting fracture healing, but not in regulating osteoblast proliferation, uMSC-Exos played a more important angiogenic role [13]. In the eld of fracture healing, one of the most critical factors for union is su cient blood supply at the fracture site [2]. Angiogenesis is observed before osteogenesis and continues throughout the entire healing process [2,24]. Thus, promoting angiogenesis immediately after trauma is urgent and even critical. Therefore, in this paper, we provide the demonstration of the mechanism underlying the ability of uMSC-Exos to promote angiogenesis.
As intercellular communication is required for various physiological and pathological processes, exosomes can be readily isolated from MSCs of various origin, and these exosomes carry biologically active molecules (protein, microRNA, and DNA) that can be transferred to target cells to exert therapeutic effects [17]. Current research has indicated that the transfer of miRNAs delivered by exosomes to recipient cells is a novel mechanism [25]. Herein, we analysed the global expression of miRNAs in uMSC-Exos via high-throughput sequencing and miR-21 was found in greater abundance in both the uMSC-Exos and uMSCs groups than it was in the control group. Moreover, the expression of miR-21 in the HUVECs was signi cantly increased following treatment with uMSC-Exos. The immuno uorescence results indicated that exosomal miR-21 was indeed transferred to the HUVECs. As reported in the literature, miR-21 is frequently overexpressed in human cancers and acts as an oncogene, and it also plays a regulatory role in endothelial cell proliferation and migration and in uences angiogenesis by interacting with hypoxia inducible factor 1 alpha (HIF-1α) or other factors[26-28]. Thus, in this study, we hypothesize that exosomal miR-21, the most abundant miRNA in the uMSC-Exos, was the main transmitter of cellular information.
To con rm the role of exosomal miR-21 in this process, we transfected an inhibitor of miR-21 into uMSCs, extracted the exosomes, and determined the expression level of miR-21 in these exosomes. The expression decreased correspondingly, and subsequent tests showed a trend of proliferation and migration suppression when the HUVECs were co-cultivated with uMSC-Exo with a miR-21 inhibitor.
Finally, the pro-angiogenesis function of HUVECs was also con rmed, and the results showed that exosomal miR-21 from uMSCs played a crucial angiogenic role in the HUVECs. However, details on the downstream pathway remain to be provided.
Bioinformatics analyses and luciferase assays were used to con rm that miR-21 binds directly to the 3′-UTR of the SPRY1 mRNA and inhibits the transcription of SPRY1. SPRY1 was previously described as a potent angiogenesis inhibitor in endothelial cells [29] and was found to inhibit the ERK/MAPK signalling induced by bFGF and VEGF [30][31][32]. Recent studies have shown that miR-21 contributes to the proliferation, migration, and apoptosis of cancer cells with the concomitant degradation of SPRY1 [33,34]. However, rarely is focused on the mechanism of miR-21 targeting of SPRY1 during angiogenesis or fracture healing. Our qRT-PCR and Western blot data showed that the mRNA level of SPRY1 increased dramatically when uMSC-Exos with miR-21 inhibitor were transfected into HUVECs. The mRNA expression of VEGFA and HIF-1α and the phosphorylation of AKT in the HUVECs were signi cantly decreased. It is reported that VEGF plays an important role in the endothelial cell mitosis and blood vessels fusion, thereby promoting neovascularization. HIF-1 exhibits enhanced stability under hypoxic conditions and regulates VEGF expression through transcriptional activation [29]. AKT is a wellcharacterized target of PI3K, and AKT phosphorylation is essential for the activation of the PI3K/AKT pathway [35,36]. In the context of angiogenesis, accumulating evidence from a number of studies has shown that the PI3K/AKT signalling pathway plays a key role in ischaemic injury and the expression of multiple angiogenic molecules, such as VEGF and SDF-1, may be upregulated in this pathway [31,37,38].
To con rm that angiogenesis depends on SPRY1, we also used siRNA to inhibit the expression of SPRY1 in HUVECs. The qRT-PCR and Western blot analysis results showed that SPRY1 siRNA signi cantly inhibited the expression of HIF-1α and VEGF as well as PI3K/AKT pathway components. In addition, a series of results on cell function suggested that the angiogenic ability of HUVECs had been restricted. In accordance with these ndings, the miR-21-dependent regulation of endothelial cell activity and angiogenesis were regulated by the activation of PI3K/AKT signalling upon downregulation of SPRY1. The calvarial defect model is commonly used for studying bone formation [15]. To make our experimental results more convincing, we studied micro-vessels and bone formation in vivo. The role of exosomal miR-21 was evaluated by testing its ability in calvarial defect repair in a rat model. The restoration of blood ow is critical in bone formation not only for the transport of oxygen and nutrient but also for the initiation of ossi cation and recruitment of MSCs [2,39]. Therefore, it was not surprising that the uMSC-Exo-induced formation of micro-vessel networks promoted bone healing in the calvarial defect, which has been observed in the previously studied femur fracture model [13]. In the current model, newly formed vessels in the defect area were observed more clearly through three-dimensional images of the radiopaque contrast-lled vascular network. Moreover, the results from the in vivo study not only veri ed our hypothesis that a SPRY1-dependant mechanism involving exosomal-miR-21 induces angiogenesis but also indicated that the trends of signi cant in vivo microvascular remodelling and osseous regeneration lend substantial credibility to the utilization of uMSC-Exos and exosomal miR-21 in the development of future therapeutic tissue remodelling strategies.
In this research, we demonstrates a potential role for exosomal miR-21, additional studies are expected to determine the overall importance of exosomal miR-21 compared to the wider secretome and identify the deeper interactional mechanisms underlying exosome-related angiogenesis and osteogenesis.

Conclusions
Taken together, the ndings from our current study indicate that the speci c miR-21, which is shuttled directly from uMSCs to HUVECs via exosomes, regulates HUVEC proliferation, migration and tube formation through the activation of the PI3K/AKT pathway and induction of HIF-1α and VEGFA expression by suppressing the target gene SPRY1. Furthermore, a cranial defect model was utilized to verify this pro-angiogenic phenomenon and its impact on bone formation (Figure 8). Overall, the data presented herein provide the rst evidence of a novel mechanism of exosomal miR-21 in inducing angiogenesis, which may provide a basis for prospective therapeutic approaches in the treatment of atrophic delayed or non-union fracture in the future.

Declarations
Ethical Approval and Consent to participate:   At the same time point (12 h or 24 h), although the number of migrating cells in the uMSC-Exo-anti-miR-21 group was increased, the rate was signi cantly slower than that of the other two groups (uMSc-Exo and uMEC-Exo-anti-miR-NC groups). (C) Transwell experiments showed that the number of migrating HUVECs was reduced by approximately one-half after exposure to exosome inhibitors. Data are presented as the means±SD; *p<0.05.

Figure 4
Page 23/28 Effects of uMSC-Exo-anti-miR-21 on the pro-angiogenesis of the HUVECs (A) Tube formation assay performed to determine the extent of in vitro angiogenesis, and the results showed growing branch points and tube elongation in the uMSC-Exo group and uMSC-Exo-anti-miR-NC group as well as in the uMSC-Exo-anti-miR-21 group and blank group in a time-dependent manner. Compared with that of the other two groups, the uMSC-Exo group treated with inhibitors of miR-21 and the blank group had the lowest growth rates. (B) Chicken chorioallantoic membrane (CAM) assay results showed that uMSC-Exo-anti-miR-21 was able to impair in ovo neovascularization, which was decreased two-fold compared to that of the other two groups on the tenth day. In addition, at the following time points, it continued to increase at a lower rate than the two control groups. Data are presented as the means±SD; *p<0.05.    Schematic representation of proposed uMSC-Exo mechanism of action in bone repair and regeneration.
The exosomal miR-21-mediated pro-angiogenesis roles in HUVECs and promoted blood vessels and bone formation in a cranial defect model.