Exosomes Derived From Stem Cells From Apical Papilla Promote Craniofacial Soft Tissue Regeneration Through Enhancing Cdc42-Mediated Vascularization

Background: Reconstruction of complex critical-size defects (CSD) in craniofacial region is a major challenge, and the soft tissue regeneration is crucial in determining the therapeutic outcome of craniofacial CSD. Stem cells from apical papilla (SCAP) are neural crest-derived mesenchymal stem cells (MSCs) which are homologous to craniofacial tissue, and represent a promising source for craniofacial tissue regeneration. Exosomes, which contained compound bioactive contents, are the key factors of stem cell paracrine action. However, the roles of exosomes derived from SCAP (SCAP-Exo) in tissue regeneration are not fully understood. Here, we explored the effects and underlying mechanisms of SCAP-Exo on CSD in maxillofacial soft tissue. Methods: SCAP-Exo were isolated and identied by transmission electron microscopy and nanoparticle tracking analysis. The effects of SCAP-Exo on wound healing and vascularisation were detected by measuring wound area, histological and immunouorescence analysis in the palate gingiva CSD of mice. Real-time live cell imaging and functional assays were used to assess the effects of SCAP-Exo on the biological functions of endothelial cells (ECs). Furthermore, the molecular mechanisms of SCAP-Exo mediated ECs angiogenesis in vitro was tested by immunouorescence staining, Western blot and Pull-Down assays. Finally, in vivo experiments were carried out to verify whether SCAP-Exo could affect the vascularisation and wound healing through Cdc42. Results: We showed that SCAP-Exo promoted tissue regeneration of palatal gingiva CSD by enhancing vascularisation in the early phase in vivo, and also indicated SCAP-Exo improved the angiogenic capacity of endothelial cells (ECs) in vitro. Mechanistically, SCAP-Exo elevated cell migration by improving cytoskeletal reorganization of ECs via cell division cycle 42 (Cdc42) signalling. Furthermore, we revealed that SCAP-Exo transferred Cdc42 into the cytoplasm of ECs, and the Cdc42 protein could be reused directly by the recipient ECs, which resulted in the activation of 3, days post-wounding compared to SCAP-Exo group. H&E staining showed that the thickness of connective tissue (yellow segment) in SCAPsiCdc42-Exo group was thinner than that in SCAP-Exo group at 7 days post-wounding. Scale bar = 200 μm. c The histological views showed less newly formed blood vessels containing red blood cells in the gingiva wound of SCAPsiCdc42-Exo group at 1, 3 days post-wounding when compared with SCAP-Exo group. Scale bar = 50 μm. d Immunouorescence staining and quantication showed that the percentage of CD31 positive area (red) in SCAPsiCdc42-Exo group was lower than that in SCAP-Exo group at 1, 3 days post-wounding. The epidermis and connective tissue were separated by the white dotted line in the images. Slides were counterstained with DAPI (blue). Scale bar 50 μm. n in each group.

and tissue regeneration, and adequate blood vessel formation could supply su cient oxygen, nutrients, and eliminate metabolic waste [5]. Therefore, vascularization in the early phase of wound healing plays a critical role for the regeneration of craniofacial soft tissue [6]. The strategy based on bioactive factors derived from mesenchymal stem cells (MSCs) has been addressed as a promising approach for regenerative medicine [7]. Exosome, one of the most important excellular microvehicles secreted from stem cells, contain lots of cytoplasmic contents including proteins, peptides, RNA, DNA and so on [8].
Increasing evidence demonstrates that exosomes have similar functions to donor MSCs, which regulate signal transcription and protein expression to mediate cellular functions of recipient cells [9]. In the process of tissue regeneration, exogenous MSCs are located around the endothelial cells (ECs), and promote angiogenesis by secreting exosome [10,11]. In addition, compared to MSCs, exosomes may have a superior safety pro le and can be steadily stored without losing cellular functions, which successfully overcome some of the major challengers related to cell based approach of regenerative medicine.
Stem cells from apical papilla (SCAP), as one type of neural crest-derived MSCs, are homologous to craniofacial region, which have been identi ed as a promising stem cell source for tissue engineering with high self-renewal and multi-lineage differentiation [12]. Since they are isolated from the highly vascularized developing apical tissue of immature permanent teeth, SCAP may have excellent properties for the promotion of angiogenesis [13]. Even in the stress microenvironment lacking oxygen, serum and glucose, SCAP also maintain biological activity and secrete a large amount of pro-angiogenesis growth factors, while the secretion of anti-angiogenesis growth factors is reduced to promote the ECs angiogenesis [14]. What's more, SCAP enhance vascularization to improve pulp-like tissue regeneration in vivo and emphasize the promising role in tissue engineering [15]. However, the effects and underlying mechanisms of the exosomes derived from SCAP (SCAP-Exo) in tissue regeneration are unclear.
In this study, we explored the role and potential application of SCAP-Exo for craniofacial soft tissue regeneration in vivo. Our ndings suggested that SCAP-Exo improved cell migration of ECs and angiogenic capacity via cell division cycle 42 (Cdc42)-dependent cytoskeletal reorganization, which resulted in the promotion of tissue regeneration of palatal gingiva CSD. To the best of our knowledge, this is the rst study in which SCAP-Exo can be used as a cell-free approach to optimize soft tissue regeneration in the clinic.

Materials And Methods
Animals C57BL/6J mice and BALB/c nude mice were purchased from Vital River Laboratory Animals Technology (Beijing, China). All animal experiment protocols (2018029) were approved by the Institutional Animal Care and Use Committee of China Medical University.

SCAP-Exo isolation and identi cation
SCAP were cultured with exosome-free medium for 48 h. The culture supernatant was collected and centrifuged at 4 °C and 3,000 × g for 20 min, 20,000 × g for 30 min, and 120,000 × g for 2 h in an ultracentrifuge (Beckman Optima L-100XP, USA). Exosomes were resuspended in sterile PBS and stored at -80 °C. SCAP-Exo were observed by transmission electron microscopy (TEM) (H-800, Hitachi, Japan). A nanoparticle tracer analyser (ZetaView, Germany) was used to measure the size of particles. Exosomal surface markers including CD9, CD63, and Alix were detected by western blot.

SCAP-Exo in wound healing of CSD in the palatal gingiva
The wound model was identical to a previous study [16]. Brie y, a 2-mm diameter punch was used to make CSD in the palatal gingiva of C57BL/6J mice (n = 5). SCAP-Exo or PBS (as the control) was injected into the wounds locally. Mice were sacri ced after 7 days, xed in 4% paraformaldehyde and decalci ed with 10% ethylenediaminetetraacetic acid solution. The samples were embedded in para n, sectioned and stained with haematoxylin and eosin (H&E). In addition, the samples were embedded in optimal cutting temperature compound and sectioned. The frozen sections were stained with immuno uorescent CD31, an endothelial marker of micro-vessels, and the CD31 positive area was analysed using the Image J software (1.50i, National Institutes of Health, Bethesda, MD, USA).

Real-time live-cell imaging (RT-LCI)
Human umbilical vein endothelial cells (HUVECs) were seeded into a 35-mm dish (81158, Ibidi, Germany) and stained with FM TM 4-64FX. SCAP-Exo were labelled with PKH-67 and added to the HUVECs. The process of HUVECs taking up SCAP-Exo was observed under the laser confocal microscope (ECLIPSE Ti2, Nikon, Japan) for 30 min continuously.

Western blot analysis
Proteins (20 μg) were loaded onto a 12% sodium dodecyl sulphate-polyacrylamide gel for electrophoresis, and then transferred to polyvinylidene di uoride membranes. The membranes were exposed to the appropriate primary and secondary antibodies. Finally, the bands were revealed using an Odyssey CLx instrument (LI-COR, Lincoln, NE, USA). The density of the bands was measured with Image J to quantify protein expression.

Tube formation assay
Matrigel (50 μL) (#356234, BD Biosciences, San Jose, CA) was precoated in each well of a 96-well plate and polymerized at 37 °C. HUVECs and SCAP-Exo-pretreated HUVECs were seeded at a density of 1.5 × 10 4 cells/well and cultured for 8 h. Photos of tube formation were taken by a stereoscopic microscope (ECLIPSE TE2000-S, Nikon, Japan). The indexes of tube formation were analysed using Image J.

Matrigel plug assay
Matrigel (200 μL) (#356231, BD Biosciences, San Jose, CA) was mixed with SCAP-Exo or PBS on ice. The mixtures were injected subcutaneously into the dorsum of BALB/c nude mice (n = 5). After 14 days, the matrigel plugs were extracted. H&E staining was used and the number of vessels in the matrigel was counted.

Cell proliferation assay
Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8) and Ki-67 staining assay. HUVECs were seeded into 96-well plates at a density of 2,000 cells/well, and cultured with SCAP-Exo. The plates were incubated for 24, 48, and 72 h. CCK-8 solution (Dojindo, Kumamoto, Japan) was added and incubated in the dark. The absorbance of each well was measured at 450 nm using a microplate reader (Tecan, Salzburg, Austria). In addition, HUVECs (2 × 10 4 /well) were seeded on glass coverslips placed inside a 12-well plate and cultured to the logarithmic phase. Thereafter, cells were xed and stained with Ki-67 immuno uorescent antibody. The number of Ki-67-positive cells was indicated as a percentage of the total cell number.

Cell migration assay
Cell migration was measured using the transwell cell migration and scratch wound healing assay.
HUVECs were seeded into the upper transwell insert of a 24-well plate at a density of 1 × 10 4 cells/well. SCAP-Exo were added into the lower chamber and incubated for 24 h. Thereafter, cells in the transwell chamber were removed. After staining with crystal violet, the number of cells migrating below the transwell layer was counted. Moreover, HUVECs (5 × 10 5 /well) were seeded into a 6-well plate and a scratch in the cells was made with a 200 μL sterile tip. The serum-free medium containing SCAP-Exo was then replaced. After 0, 12, and 24 h, the boundaries of the scratches were recorded and the wound closure rates were measured and calculated using Image J.

Pull-Down assay
RhoA/Rac1/Cdc42 Activation Assay Combo Biochem Kit (Cytoskeleton, Denver, CO, USA) was used following the manufacturer's instructions. Brie y, the equivalent protein amounts of lysate were added to a pre-determined amount of rhotekin-RBD (for RhoA activation) or PAK-PBD beads (for Rac1 and Cdc42 activation) and incubated at 4 °C on a rotator for 1 h. Next, the beads were centrifuged and washed. The bead pellets were resuspended with 20 μL loading buffer and boiled. The samples were then analysed by western blot.

F-actin immuno uorescence staining
HUVECs were xed for 30 min and stained with ActinGreen TM 488 at 4 °C for 30 min. Pseudopodia formation was observed by uorescence microscopy (ECLIPSE 80i, Nikon, Japan). We counted the number of lopodia and used Image J software to quantitatively analyze the length of lopodia.

Plasmid transfection and fluorescence co-localization
The Cdc42-EGFP or Cdc42-mCherry fusion protein expression plasmids were transfected into SCAP to extract SCAP Cdc42-EGFP -Exo or SCAP Cdc42-mCherry -Exo. SCAP Cdc42-EGFP -Exo was added to HUVECs and passaged to the 6 th passage. SCAP Cdc42-mCherry -Exo was added to HUVECs overnight. Cells were incubated with Cdc42 primary antibody and uorescent secondary antibody. The co-localization of Cdc42 and Cdc42-mCherry was observed by confocal microscopy (ECLIPSE Ti2, Nikon).

Statistical analysis
All data were recorded as the mean ± standard deviation (SD). Comparisons between two groups were analysed using an independent two-tailed Student's t test, and comparisons between more than two groups were analysed using one-way analysis of variance (ANOVA) with SPSS 20.0 (SPSS Inc., Chicago, IL, USA). A value of P < 0.05 was statistically signi cant.

SCAP-Exo promoted vascularization to accelerate tissue regeneration of palatal gingiva
To explore the roles of SCAP-Exo on craniofacial soft tissue regeneration, we rstly isolated microvesicles from the culture supernatant of SCAP. The micro-vesicles exhibited a bilayer membrane and a cup-plate-shaped structure under TEM (Fig. S1a). The nanoparticle tracking analysis showed that the micro-vesicles with diameter at 120.1 nm accounted for 97.2% of nanoparticles, and the mean diameter of micro-vesicles was 139.2 ± 62.5 nm (Fig. S1b). Western blot analysis showed that the micro-vesicles expressed the exosomal markers Alix, CD9 and CD63, and failed to express calnexin (Fig. S1c). Therefore, following the minimal information for studies of extracellular vesicles guidelines [17], we identi ed the micro-vesicles isolated from SCAP as exosomes.
We locally infused SCAP-Exo into CSD in the palatal gingiva of mice and analysed the therapeutic affects at 1, 3, and 7 days (Fig. 1a). We found that the wound area of the palatal gingiva was signi cantly reduced in the SCAP-Exo infusion group at 3 and 7 days post-wounding compared to that in the controls (Fig. 1b). By H&E staining, the newly formed and integral epidermis and connective tissues were observed in the SCAP-Exo group at 7 days post-wounding, while the formation of epidermis and connective tissues were markedly delayed in the controls (Fig. 1b). We further focused on vascularization in the early phase of healing of the palatal gingiva CSD. H&E staining showed that there were signi cantly more newly formed blood vessels in the gingiva wounds in the SCAP-Exo group than in the controls at 1 and 3 days post-wounding (Fig. 1c). Immunostaining con rmed that the percentage of the CD31 positive area was signi cantly increased in the SCAP-Exo group at 1 and 3 days post-wounding (Fig. 1d). Therefore, our data indicated that SCAP-Exo promoted vascularization in the early phase of healing and accelerated tissue regeneration of the palatal gingiva.

SCAP-Exo improved the angiogenic capacity of HUVECs
To determine the effects of SCAP-Exo on angiogenesis, we used SCAP-Exo to treat HUVECs in vitro. RT-LCI showed that SCAP-Exo were endocytosed into the cytoplasm of HUVECs (Fig. S2a-e). Based on the analysis of the X-T, Y-T and X-Y-Z image axes, we observed the entire process of SCAP-Exo taken into HUVECs ( Fig. S2f-h). To select an optimal concentration of SCAP-Exo, different doses (5-20 μg/mL) of SCAP-Exo were added to HUVECs. We found that SCAP-Exo increased the expression of angiogenic protein in HUVECs in a dose-dependent manner (Fig. S3). Therefore, we used 20 μg/mL SCAP-Exo in subsequent experiments.
The expression levels of CD31 in HUVECs were markedly upregulated in the SCAP-Exo treated group, when compared to those in the non-treated controls by western blot (Fig. 2a). The in vitro matrigel tube formation assay indicated that SCAP-Exo-treated HUVECs had an elevated capacity of forming vascular lumen compared to that of the controls, as shown by increased total tube length, total meshes, total branches, total nodes and total junctions (Fig. 2b). Furthermore, we examined the effects of SCAP-Exo on blood vessel formation ex vitro. We found that in the SCAP-Exo group, there were more newly formed blood vessels in the implanted matrigel compared to that in the controls (Fig. 2c). H&E staining also showed signi cantly increased formation of vascular lumen structure with increased red blood cells in the SCAP-Exo group compared to those in the controls (Fig. 2d). These data suggested that SCAP-Exo improved the angiogenic capacity of ECs in vitro.

SCAP-Exo mediated cell migration contributing to HUVEC angiogenesis
It is well known that cell migration and cell proliferation play important roles in angiogenesis of ECs [18]. To clarify how SCAP-Exo improved the angiogenic capacity of HUVECs, we rst tested the cell proliferation of HUVECs in both the SCAP-Exo and control groups. We found that there was no signi cant change in the proliferation rate of HUVECs in the SCAP-Exo group when compared to that in the controls, as assessed by CCK-8 assay and Ki-67 staining (Fig. 3a, b). However, the transwell cell migration assay showed that the number of migrated cells in the SCAP-Exo-treated HUVECs was higher than that in the non-treated HUVECs (Fig. 3c). The wound healing rate of HUVECs in the SCAP-Exo group was also signi cantly increased at 12 and 24 h, compared to that in the controls (Fig. 3d). These experimental data indicated that SCAP-Exo upregulated cell migration of ECs to promote angiogenesis.
SCAP-Exo improved cell migration of HUVECs via Cdc42-mediated cytoskeletal reorganization The reorganization of the actin cytoskeleton and pseudopodia formation provides a driving force for cell migration [19]. Accordingly, F-actin immuno uorescence staining showed that the actin cytoskeleton and newly formed lopodium were increased in the cytoplasm of HUVECs treated with SCAP-Exo when compared to those in untreated HUVECs, as indicated by the upregulation of the number of lopodia per cell and lopodia length (Fig. 4a). The Rho GTPase family acts as a molecular switch to regulate cytoskeletal reorganization, contributing to cell migration, including RhoA, Rac1 and Cdc42 [20]. Therefore, we tested the expression of Rho GTPases in both the SCAP-Exo-treated HUVECs and the controls. Interestingly, the Pull-Down assay and western blot showed that the expression levels of total Cdc42 and Cdc42-GTP were elevated in SCAP-Exo-treated HUVECs when compared to those in the nontreated HUVECs, while there was no signi cant difference in the expression levels of RhoA and Rac1 (Fig.  4b).
To determine the role of Cdc42 in SCAP-Exo-mediated EC migration, we used Cdc42 siRNA and a Cdc42 inhibitor (ML141) to downregulate Cdc42 expression in SCAP-Exo. We showed that the total Cdc42 and Cdc42-GTP expression levels of HUVECs were not changed with the treatment of SCAP siCdc42 -Exo or SCAP ML141 -Exo, when compared with those in non-treated HUVECs (Fig. 4c). Moreover, we found that SCAP siCdc42 -Exo or SCAP ML141 -Exo induced signi cantly decreased number and length of lopodia of HUVECs compared with the SCAP vehicle -Exo-treated HUVECs (Fig. 4d). Moreover, the scratch wound healing assay showed that the knockdown of Cdc42 in SCAP-Exo blocked the SCAP vehicle -Exo-mediated elevation of cell migration in HUVECs (Fig. 4e). These data indicated that SCAP-Exo improved cytoskeletal reorganization to contribute to cell migration of ECs via Cdc42 signalling.
To explore the detailed mechanism of SCAP-Exo-induced Cdc42 expression in HUVECs, we used western blot analysis to examine the protein levels of Cdc42 in SCAP and SCAP-Exo. We found that Cdc42 was steadily expressed in SCAP and SCAP-Exo derived from different SCAP populations, and Cdc42 had higher expression level in SCAP-Exo than that in SCAP (Fig. 4f). Furthermore, we transfected SCAP with a Cdc42 enhanced green uorescent protein (Cdc42-EGFP) plasmid and isolated SCAP Cdc42-EGFP -Exo. We used SCAP Cdc42-EGFP -Exo to treat HUVECs and found that the Cdc42-EGFP protein derived from SCAP-Exo was transferred to HUVECs and expressed continuously in the cytoplasm of passage 0 (P0), P3 and P6 HUVECs (Fig. 4g). In addition, we produced SCAP Cdc42-mCherry -Exo by transfecting SCAP with a Cdc42-mCherry u orescent protein plasmid and then used SCAP Cdc42-mCherry -Exo to treat HUVECs. Laser confocal microscope showed the co-localization of Cdc42-mCherry (red) and Cdc42 (green) in the cytoplasm of SCAP-Exo-treated HUVECs (Fig. 4h). Taken together, these experimental data indicated that SCAP-Exo elevated cell migration by the transfer of Cdc42 protein to improve the cytoskeletal reorganization of ECs.

SCAP-Exo facilitated tissue regeneration of palatal gingiva via Cdc42-mediated vascularization
We wondered whether the Cdc42 derived from SCAP-Exo contributed to the accelerated tissue regeneration of the palatal gingiva, so we produced Cdc42 knockdown SCAP-Exo (SCAP siCdc42 -Exo) and infused SCAP siCdc42 -Exo or SCAP-Exo locally into the gingival wounds (Fig. 5a). We found that gingival healing was signi cantly delayed in the SCAP siCdc42 -Exo group at 3 and 7 days post-wounding compared to the SCAP-Exo group (Fig. 5b). H&E staining showed that in the SCAP siCdc42 -Exo group, newly formed epidermis and a thin layer of connective tissue was observed at 7 days post-wounding, while in the SCAP-Exo group, the epidermis tissue was intact and continuous, and the connective tissues were markedly thickened (Fig. 5b). We further veri ed vascularization in the early phase of wound healing in both the SCAP siCdc42 -Exo and SCAP-Exo groups. H&E staining showed that the SCAP siCdc42 -Exo group exhibited less newly formed blood vessels than in the SCAP-Exo group at 1 and 3 days post-wounding (Fig. 5c). Immunostaining con rmed that knockdown of Cdc42 in SCAP-Exo blocked the SCAP-Exo-mediated upregulation of vascularization in the early phase, as indicated by the decreased percentage of CD31 positive area in the SCAP siCdc42 -Exo group at 1 and 3 days post-wounding compared to that in the SCAP-Exo group (Fig. 5d). Therefore, our data indicated that SCAP-Exo facilitated tissue regeneration of palatal gingiva via Cdc42-mediated vascularization.

Discussion
Angiogenesis is the formation and remodelling of new blood vessels and capillaries from the growth of existing blood vessels, which plays a crucial role in wound healing [21]. In the process of wound healing, rapid and su cient vascularization not only provides oxygen and nutrients to the surviving cells, but also eliminates necrotic substances and controls infections [22]. Thus, the stimulation of vascularization in the early phase is the most important factor for the therapeutic effects of MSC-based tissue engineering. SCAP are the postnatal population of epidermal neural crest stem cells, which have a high capability to promote angiogenesis when compared with bone marrow mesenchymal stem cells (BMMSCs) [23].
Meanwhile, the neural crest-derived MSCs carry neurovascular factors such as vascular endothelial growth factor, platelet derived growth factor, and brain-derived neurotrophic factor, which mediate the angiogenic process to improve tissue regeneration and treat ischemic diseases [24][25][26]. Exosomes have similar cellular properties to the parent cell [27]. Here, we showed that the local infusion of SCAP-Exo promoted new blood vessel formation at 1 and 3 days post-wounding, and accelerated the healing of CSD in the palatal gingiva. Furthermore, we had a novel nding that SCAP secreted a large amount of exosomes compared to BMMSCs (data no shown). Therefore, as the important paracrine factor secreted from SCAP, SCAP-Exo have an excellent effect on promoting angiogenesis. Moreover, we pursued a cellfree approach, nding inspiration from the consensus that SCAP-Exo have the advantages of low immune rejection, high stability, ease of accessing the wound surface and no vascular obstruction [28][29][30][31]. Thus, these data provide a new option for tissue engineering of craniofacial region in the clinic.
In the process of blood vessel formation, the proliferation and cell migration of ECs interact with angiogenic factors and result in the formation of new capillaries. Thereafter, capillaries are covered by smooth muscle cells to form mature blood vessels with viscoelasticity and contractility [18]. Here, we showed originally that SCAP-Exo specially increased the cell migration of ECs, contributing to ECs angiogenesis, while there were no marked effects on the cell proliferation of ECs. Cytoskeletal reorganization and pseudopodia formation play critical roles in cell migration [19]. Additionally, the formation of lopodia in SCAP-Exo-treated ECs was elevated when compared to that in non-treated ECs, which con rmed the promotive effects of SCAP-Exo on cell migration. Several extracellular signals are involved in modulating the activity of micro lament-binding proteins to re-organize the cytoskeleton. The family of Rho GTPases, as the key downstream target, is currently believed to promote cytoskeletal organization [20]. Cdc42, Rac1 and RhoA are the main members of the Rho GTPase family, which play different roles in cytoskeletal reorganization [32]. Therefore, we examined the expression levels of the family of Rho GTPases and found that only Cdc42 signalling were signi cantly upregulated in SCAP-Exotreated ECs compared to those in the controls. The activation of Cdc42 leads to the combination of WASP and Arp2/3 complex by binding to the GTPase binding domain of WASP, and directly regulates the polymerization of globular actin, resulting in the formation of brous Actin (F-actin), Which causes the cytoplasmic membrane to protrude outwards and form lopodia [33,34]. Filopodia formation is the initial step of cell movement, and is helpful to determine the direction of cell migration [35]. Here, we revealed a potential mechanism that infusion of SCAP-Exo promoted the cell migration of ECs by activating Cdc42, leading to accelerated healing of CSD in the palatal gingiva through enhancing vascularization.
Multiple data have demonstrated that exosomes are able to mediate the cellular functions of receptor cells [9]. The underlying mechanisms are complicated, but may include releasing the contents to the recipient cell by endocytosis or direct combination with the molecule receptor of the recipient cell [36][37][38].
Here, we showed that SCAP-Exo were internalized into the cytoplasm of HUVECs. We further used the colocalization and continued passage experiment to indicate that Cdc42 derived from SCAP-Exo was directly transferred into HUVECs and reused by HUVECs. Our previous study revealed that MSC transplantation rescues impaired BMMSCs and the osteoporotic phenotype of MRL/lpr mice, via reusing the donor MSC-Exo derived Fas protein [39]. Additionally, it is worth mentioning that increased amount of Cdc 42 protein was enriched in SCAP-Exo when compared with SCAP, which suggested SCAP-Exo might have a superior promotion on angiogenesis. Cdc42, a kind of Rho GTPase, continuously switches between the active GTP-binding state and the inactive GDP-binding state; the GTP situation can allow downstream signalling activation [40]. Interestingly, we found that not only the total Cdc42, but the Cdc42-GTP expression levels were also signi cantly increased in SCAP-Exo-treated ECs. In addition to the total Cdc42-mediated GDP-GTP switch, guanine nucleotide exchange factors (GEFs), guanine nucleotide activating proteins (GAPs) and guanine dissociation inhibitors (GDIs) may contribute to active GTPbinding state of ECs [41]. The detailed mechanism of the induction of the active GTP binding state requires further investigation.

Conclusion
In summary, this study showed that local infusion of SCAP-Exo accelerated tissue regeneration of the palatal gingiva CSD by promoting vascularization in the early phase. Mechanistically, SCAP-Exo improved cell migration through enhancing cytoskeletal reorganization of ECs by directly transferring Cdc42 into the cytoplasm of recipient ECs. These data suggested SCAP-Exo could present a superior promotion on angiogenesis and provide a new strategy for using SCAP-Exo as a cell-free approach to optimize tissue regeneration in the clinic.

Consent for publication Not applicable.
Competing interests The authors declare that they have no competing interests. showed that the percentage of CD31 positive area (red) in SCAP-Exo group was higher than that in control group at 1, 3 days post-wounding. The epidermis and connective tissues were separated by the white dotted line in the images. Slides were counterstained with DAPI (blue). Scale bar = 50 μm. n = 5 in each group. NS: P > 0.05, **P < 0.01, ***P < 0.001; Error bars: mean ± SD. SCAP-Exo improved the angiogenic capacity of HUVECs. a Western blot analysis showed that SCAP-Exo treatment upregulated the expression levels of CD31 in HUVECs. b In vitro tube formation assay showed that the untreated HUVECs were scattered and formed less lumens, while SCAP-Exo treated HUVECs formed complete lumens. The data including total tube length, total meshes, total branches, total nodes and total junctions were increased in SCAP-Exo-treated HUVECs when compared with untreated HUVECs.
Scale bar = 100 μm. c Representative images of matrigel plugs showed that there were more newly formed blood vessels in SCAP-Exo group when compared with control group. d HE staining showed that there were more vascular lumens (yellow arrow) containing red blood cells in SCAP-Exo group compared to control group. Scale bar = 50 μm. n = 5 in each group. *P < 0.05, **P < 0.01, ***P < 0.001, Error bars: mean ± SD. SCAP-Exo mediated cell migration contributing to HUVECs angiogenesis. a CCK-8 assay showed that SCAP-Exo treatment had no effect on the proliferation rate of HUVECs when compared with control group. b Ki-67 staining assay showed the percentage of Ki-67 positive cells (red) in SCAP-Exo group was not different from that in control group. Slides were counterstained with DAPI (blue) and F-actin (green).
Scale bar = 100 μm. c Transwell cell migration assay showed that SCAP-Exo treatment upregulated the cell motility of HUVECs compared to control group. Scale bar = 200 μm. d Representative images of scratch wound healing assay showed that the wound healing rate in SCAP-Exo group was increased at 12 h and 24 h when compared with control group. Scale bar = 100 μm. n = 5 in each group. NS: P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001; Error bars: mean ± SD. SCAP-Exo improved cell migration of HUVECs via Cdc42-mediated cytoskeletal reorganization. a F-actin immuno uorescence staining showed that the actin cytoskeleton and lopodia formation (green) was obviously increased in SCAP-Exo-treated HUVECs compared to control HUVECs. The number of lopodia for per cell and the lopodia length of SCAP-Exo-treated HUVECs was higher than that of control HUVECs.
Scale bar = 50 μm. b Western blot and Pull-down assay showed that the expression levels of Cdc42-GTP and Cdc42 were elevated in SCAP-Exo treated HUVECs, while the expression of RhoA and Rac1 had no signi cant changes. c The expression levels of Cdc42 and Cdc42-GTP were signi cantly upregulated in SCAPvehicle-Exo-treated HUVECs compared to control HUVECs, while the SCAPsiCdc42-Exo and SCAPML141-Exo did not increase the expression levels of Cdc42 and Cdc42-GTP in HUVECs. d F-actin immuno uorescence staining showed that the actin cytoskeleton and the number and length of lopodia of HUVECs were increased in SCAPvehicle-Exo group compared to control group, while the SCAPsiCdc42-Exo or SCAPML141-Exo treated HUVECs were decreased compared to SCAPvehicle-Exo group. Scale bar = 50 μm. e Representative images of scratch wound healing assay showed that the HUVECs migration in SCAPvehicle-Exo group was higher than that in control group, while the migration ability of HUVECs in SCAPsiCdc42-Exo or SCAPML141-Exo group were reduced when compared with SCAPvehicle-Exo group at 24 h. Scale bar = 100 μm. f Western blot showed that Cdc42 was continuously expressed in SCAP and SCAP-Exo derived from different populations. g Immuno uorescence staining showed that Cdc42-EGFPlabeled protein derived from SCAP-Exo (green) expressed steadily in the cytoplasm of HUVECs in the 0, 3th and 6th passage. Scale bar = 100 μm. h Laser confocal microscope image showed the co-localization of SCAP-Exo derived Cdc42-mCherry (red) and Cdc42 (green) in the cytoplasm of HUVECs. Slides were counterstained with DAPI (blue). Scale bar = 50 μm. n = 5 in each group. **P < 0.01, ***P < 0.001; Error bars: mean ± SD.