The present study was approved by the Ethics Committee of Sun Yat-sen University [No. ERC-(2017)-34]. Human DPSCs were obtained from healthy pulp tissues harvested from wisdom teeth without caries, which were extracted from 10 donors (both male and female, 22-36 years old) between September, 2018 and March, 2019 at the Hospital of Stomatology, Sun Yat-sen University, Guangzhou, China. Dental pulp tissues were minced and digested for the isolation of DPSCs as previously described (22). DPSCs were cultured in α-MEM with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (MilliporeSigma). HUVECs were purchased from AllCells, LLC and expanded in complete endothelial cell growth medium (EGM; AllCells Biotech Shanghai Co., Ltd.). For the exosome co-culture experiment, 10 µg/ml exosomes were added to the culture medium of HUVECs. All cells were maintained at 37°C in an incubator contained 5% CO₂.

The DPSCs were washed with phosphate-buffered saline (PBS; Biosharp) and incubated in fresh serum-free α-MEM 48 h prior to the isolation of exosomes. The cell culture supernatant was then harvested and subjected to two centrifugation steps (500 × g for 10 min at 25°C; 2,000 × g for 30 min at 25°C) to remove dead cells and cell debris. Exosome pellets were purified from the cleared supernatant by ultracentrifugation at 100,000 × g for 1 h at 4°C. An exosome pellet from about 12×10⁶ cells was resuspended in 100 µl cold PBS. Exosomes were either used immediately or stored at −80°C until use. The exosome protein concentration was quantified using a BCA Protein Assay kit (Jinan Bocai Chemical Technology Co., Ltd.). The expression levels of the exosomal markers, CD9 and CD63 (Affinity Biosciences) were measured using western blot analysis, as described below. Nanoparticle tracking analysis (NTA) was performed to determine the size distribution of exosomes.

The morphology of the exosomes was visualized using TEM. Exosomes suspended in PBS were placed onto formvar/carbon-coated nickel grids and incubated for 30 min at room temperature. Images of the grids were acquired using an H-7650 transmission electron microscope (Hitachi corporation).

All animal experiments were performed in accordance with institutionally approved protocols for animal research (Animal Care and Use Committee of Sun Yat-sen University) (No. SYSU-IACUC-2022-000248). A total of 6 female C57BL/6 mice (8 weeks old; weighing 18-20 g) were purchased from the Guangdong Medical Laboratory Animal Center. Age-matched 8-week-old female mice from the same background were used in the present study. During the experiment, the mice were kept in a specific pathogen-free environment (temperature, 22±2°C; humidity, 55±10%), with free access to food and water. All mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (75 mg/kg) and euthanized by cervical dislocation, while being deeply anesthetized with sodium pentobarbital. Death verification included the absence of heartbeat, breathing or respiration. In the animal experiments, the humane endpoints was reached when the animal lost 20% of its original weight according to predefined criteria (23,24). In the present study, the maximum percentage of body weight loss observed was not >10%. After shaving, a 1×1 cm full-thickness excision skin wound was created with a pair of surgical scissors, on the backs of the mice. After the surgery on day 1, the 6 mice were randomly divided into two groups and were subcutaneously injected with either DPSC-Exos (120 µg per mouse) as the treatment group or an equal volume of PBS (control group) around the wounds at four equally spaced injection sites.

To observe the wound healing process, images of the wounds were captured using a digital camera (iPhone 12, Apple) on days 0, 3, 5, 7, 9, 12 and 14, and the wound healing rates were calculated according to the following formula: Wound healing rate (%)=(A0-An)/A0 ×100, where 'A0' represents the area of the initial wound (t=day 0) and 'An' represents the residual area of the wound on a the certain day (t=day n). The rates of wound closure were quantified using ImageJ software (v.1.52a, National Institutes of Health).

To examine the formation of new blood vessels, the mice were sacrificed on day 14 post-wounding. The skin specimens were harvested and photographed using a stereo-microscope (MZ10F, Lecia Microsystems GmbH), and then analyzed using H&E and immunohistochemical staining. The use of H&E was preferred for viewing cellular and tissue structure details in the wound area. Hematoxylin precisely stains nuclear components which helps to identify inflammation, while eosin stains cytoplasmic components, including collagen and red blood cells (25-27). Immunostaining for CD31 was utilized to identify the endothelial cells of blood vessels in order to quantify the newly formed blood vessels in the wound. CD31, a 130-kDa glycoprotein expressed on endothelial cells, is commonly used as a biomarker for detecting vascular endothelium (28). The tissues were fixed in 4% paraformaldehyde solution, dehydrated with a series of graded ethanol and embedded in paraffin. The sections (10-µm-thick) were stained with H&E (Wuhan Servicebio Technology Co., Ltd.) or angiogenesis-related protein CD31 (1:600, cat. no. GB11063-2, Wuhan Servicebio Technology Co., Ltd.) to evaluate vascularization. For H&E staining, the sections were stained with hematoxylin for 3 min and eosin for 1 min at room temperature. For the immunohistochemical staining of CD31, the sections were incubated with first antibody at 4°C overnight, then followed by incubation with the horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:200, cat. no. G1215-3, Wuhan Servicebio Technology Co., Ltd.) for 1 h at room temperature. After rinsing with PBS, the 3,3'-diaminobenzidine (DAB) Staining kit (Wuhan Servicebio Technology Co., Ltd.) was employed with hematoxylin counterstain. The slides were observed and photographed under an epifluorescence microscope (Zeiss AG). To compare the number of newly formed blood vessels from the different groups, five random fields per section near wound edges were counted using Image-Pro Plus 6 software (Media Cybernetics, Inc.).

To visualize the endocytosis of exosomes by HUVECs, exosomes were stained with PKH26 (MilliporeSigma) according to the manufacturer's instructions. Isolated exosomes were resuspended in 250 µl diluent C, and 1 µl PKH26 was added and immediately mixed by gentle pipetting. The staining was terminated by the addition of an equal volume of exosome-free FBS following incubation for 5 min at room temperature. The mixture was washed twice with PBS by ultracentrifugation (100,000 × g for 1 h at 4°C) and resuspended in 100 µl exosome-free culture medium. The PKH26-labeled exosomes were then added to the HUVECs and incubated for 24 h at 37°C. Following incubation with PKH26-labeled exosomes, the cells were washed with PBS for three times and fixed with 4% paraformaldehyde for 10 min at room temperature, the cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) for 5 min at room temperature. The images of fluorescence-labeled exosome internalization were obtained using a confocal laser scanning microscope (Zeiss AG).

For the Transwell migration assay, Transwell inserts with 8-µm pore filters (Corning, Inc.) were used. The HUVECs (1×10⁴ cells/well) were seeded onto upper chambers containing 200 µl medium supplemented with DPSC-Exos, SB203580 (10 µM; Selleck Chemicals), SB203580 (10 µM) + DPSC-Exos, or PBS. Concurrently, 500 µl cultured medium were added to the bottom chamber and incubated at 37°C for 24 h. The non-migrated cells in the upper chamber were gently removed using a cotton swab and the lower chamber was fixed with 4% paraformaldehyde for 10 min at room temperature, stained with 0.1% crystal violet (MYM Technologies, Ltd.) for 30 min at room temperature, washed with PBS twice, and imaged using an inversion microscope (Zeiss AG). The quantity of migrated cells was counted and analyzed using ImageJ software (v.1.52a, National Institutes of Health).

The HUVECs (2×10⁵ cells/well) were plated in 12-well plates and incubated at 37°C until reaching 90% confluency. The assay was conducted in the absence of serum. A micro-injury was scratched on the monolayer using a 200 µl pipette tip and washed with PBS to remove the debris. The cells were treated with DPSC-Exos, SB203580 (10 µM), SB203580 (10 µM) + DPSC-Exos, or PBS prior to obtaining microscopic images at 0 h. The level of migration was measured by the ratio of closure area to initial wound area (t=0 h) as follows: Migration area (%)=(A0-A6)/A0 ×100, where 'A0' represents the area of initial wound area and 'A6' represents the residual area of wound at the metering point after 6 h (t=6 h). The wound area was quantified using ImageJ software (v.1.52a, National Institutes of Health) to calculate the percentage wound closure and the migration rate.

A Cell Counting Kit-8 (Dojindo Laboratories, Inc.) was used to assess cell proliferation. Concisely, the HUVECs were seeded in 96-well plates (1×10³ cells/well) in EGM supplemented with DPSC-Exos and SB203580 (10 µM). On days 1, 2, 3 and 4, CCK-8 solution was added to HUVECs (10 µl per well) and the cells were incubated at 37°C for 1 h. The absorbance was measured at 450 nm using a microplate reader (BioTek Instruments, Inc.) and the optical density values represented the proliferation level of the HUVECs.

The HUVECs (1×10⁴ cells/well) were seeded in a 96-well plate coated with 50 µl Matrigel (cat. No. 354277, Corning, Inc.) and treated with DPSC-Exos, SB203580 (10 µM), SB203580 (10 µM) + DPSC-Exos, or PBS, respectively. After 6 h of culture incubation at 37°C, gels were observed to examine the image of tube formation. The total tube length and total loops were measured as the mean sum length and loops of capillary-like structures per well using ImageJ software (v.1.52a, National Institutes of Health).

The DPSCs seeded in 75 cm² cell culture flasks were incubated in serum-free α-MEM for 48 h at 37°C and then washed with PBS prior to use. DPSC-Exos were isolated from the culture medium of DPSCs, respectively. Both the DPSCs and DPSC-Exo samples contained three biological replicates. TMT-based quantitative proteomics analysis was conducted by Jingjie PTM BioLab (Hangzhou) Co., Inc. All samples were sonicated on ice in lysis buffer (8 M urea, 1% Protease Inhibitor Cocktail) three times prior to centrifugation (12,000 × g for 10 min) at 4°C to remove the remaining debris. Subsequently, the supernatant was collected. A BCA protein assay (Beyotime Institute of Biotechnology) was performed to determine the protein concentration.

The protein solution was reduced with 5 mM dithiothreitol at 56°C for 30 min, alkylated with 11 mM iodoacetamide (Millipore Sigma) in the dark at room temperature for 15 min, and then diluted with 100 mM tetraethyl ammonium bromide (TEAB; Millipore Sigma) until the urea concentration was <2 M. TEAB is a dissolution buffer for isobaric mass tag labeling experiments (29,30). Trypsin was added to the solution at 1:50 (w/w) trypsin-to-protein mass ratio and incubated at 37°C overnight, and the trypsin-to-protein mass ratio was then changed to 1:100 (w/w). Following trypsin digestion, the peptide was desalted, vacuum-dried, reconstituted in 0.5 M TEAB, and processed using a TMT kit (Thermo Fisher Scientific, Inc.).

A Thermo Betasil C18 column (5 µm particles, 10 mm ID, 250 mm length; Thermo Fisher Scientific, Inc.) was utilized to fractionate tryptic peptides into fractions using high pH reverse-phase HPLC. In brief, peptides were initially separated with a gradient of 8 to 32% acetonitrile (pH 9.0) over a period of 60 min into 60 fractions, and then combined into six fractions before being dried by vacuum centrifugation (300 × g for 2.5 h at 25°C).

For LC-MS/MS analysis, the tryptic peptides were dissolved in solvent A (0.1% formic acid) and separated using a homemade reversed-phase analytical column (15-cm length, 75 µm i.d.). The gradient comprised an increase from 6 to 23% solvent B (0.1% formic acid in 98% acetonitrile) over a period of 26 min, 23 to 35% in 8 min and climbing to 80% in 3 min then holding at 80% for the last 3 min. All processes were performed on an EASY-nLC 1000 UPLC system (Thermo Fisher Scientific, Inc.) at a constant flow rate of 400 nl/min. The peptides were subjected to NSI source and analyzed using tandem mass spectrometry (MS/MS) using the UPLC system coupled online to Q ExactiveTM Plus (Thermo Fisher Scientific, Inc.). Intact peptides were detected in the Orbitrap at 70,000 resolution and selected for MS/MS with the NCE setting as 28, the fragments were then detected at a resolution of 17,500. MS and MS/MS spectra were acquired in a data-dependent manner. The automatic gain control (AGC) was set at 5×10⁴. An electrospray voltage of 2.0 kV was applied. The scan range was set from 350 to 1,800 m/z for full scan and the fixed first mass was set as 100 m/z.

The acquired MS/MS data were processed using Maxquant search engine (v.1.5.2.8, Max Planck Institute of Biochemistry, Germany). Tandem mass spectra were searched against the human UniProt database concatenated with reverse decoy database. Trypsin/P was designated as the cleavage enzyme; up to four missing cleavages were allowed. The mass tolerance for precursor ions was set to 20 ppm (first search) and 5 ppm (main search), and the mass tolerance for fragment ions to 0.02 Da, respectively. The false discovery rate was adjusted to <1% and a minimum score of 40 was set for the identification of modified peptides.

GO annotation proteome was carried out using the UniProt-GOA database (http://www.ebi.ac.uk/GOA/). Proteins were classified by GO annotation into three categories: Biological Process, Cellular Compartment and Molecular Function. InterProScan was used for the functional annotation of identified protein domains based on protein sequence alignment and the InterPro (http://www.ebi.ac.uk/interpro/) domain database. For functional enrichment analysis, the KEGG, http://www.genome.jp/kaas-bin/kaas_main) and GO (http://geneontology.org/) were applied.

A pharmacological p38 MAPK inhibitor (SB203580; Selleck Chemicals), was used to assess the participation of the MAPK signaling pathway in the DPSC-Exo-induced effects on HUVECs. The compound was resuspended to 10 mM in dimethyl sulfoxide (DMSO; MP Biomedicals France) and used at 10 µM. For control treatments (0 µM) 0.1% DMSO was used. For experiments assessing the expression of target proteins using western blot analysis, the HUVECs were plated in six-well plates and pre-treated with 10 µM SB203580 for 6 h. Since SB203580 inhibited p38 MAPK catalytic activity instead of the phosphorylation of p38, the effects of SB203580 on the p38 MAPK pathway were assessed by the expression of hsp27 as a substrate (31).

To determine the participation of the MAPK signaling pathway in the therapeutic effect of DPSC-Exos on cutaneous wound healing in vivo, a total of 12 age-matched 8-week-old female C57BL/6 mice were randomly divided into four groups as follows: PBS + vehicle, PBS + SB203580, DPSC-Exos + vehicle and DPSC-Exos + SB203580 group. After the cutaneous wound had been formed for 6 h, mice in the PBS/DPSC-Exos + SB203580 group were administered an intraperitoneal injection of SB203580 (5 mg/kg). The SB203580 was dissolved in the vehicle (4% DMSO + 30% PEG300 + 5% Tween-80 + 61% ddH₂O) according to the manufacturer's instructions. The dose of SB203580 was selected from a previous study (32).

Total protein was extracted from either DPSCs or HUVECs using RIPA buffer (Jinan Bocai Chemical Technology Co., Ltd.) and quantified using a BCA Protein Assay kit (Jinan Bocai Chemical Technology Co., Ltd.). The protein samples (5 µg per lane) were electrophoresed with 4-20% Bis-Tris sodium dodecyl sulfate polyacrylamide gel and transferred onto a 0.2 µm PVDF membrane (MilliporeSigma). After blocking with 5% (w/v) non-fat milk for 1 h at room temperature, the membranes were incubated with diluted primary antibodies overnight at 4°C. The primary antibodies and concentrations used in the present study were as follows: Phosphorylated (p-)p38 (1:1,000; cat. no. 4511, Cell Signaling Technology, Inc.), p38 (1:1,000; cat. no. 8690, Cell Signaling Technology, Inc.), p-hsp27 (1:1,000; cat. no. AF3080), hsp27 (1:1,000; cat. no. AF6082), cell division control protein 42 (Cdc42; 1:1,000; cat. no. DF6322), β-tubulin (1:8,000; cat. no. AF7011), FYVE, RhoGEF and PH domain containing 5 (FGD5; 1:500; cat. no. DF13013), CD63 (1:1,000; cat. no. AF5117), CD9 (1:1,000; cat. no. AF5139) (all from Affinity Biosciences) and β-actin (1:8,000; cat. no. MA5-11869, Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, the membranes were washed with Tris-buffered saline with Tween-20 (TBST) and incubated with goat anti-rabbit IgG-HRP (1:2,000; cat. no. As006, Asbio Technology, Inc.) as the secondary antibody for 1 h at room temperature. Bands on the blots were visualized using GeneGnome XRQ (Syngene). ImageJ software (v.1.52a, National Institutes of Health) was utilized to quantify the density of the bands.

All experiments were performed with at least three replicates per group and each in vitro experiment was repeated three times. All values are expressed as the mean ± standard deviation (SD). Differences between two groups were evaluated using an unpaired Student's t-test and those between more than two groups were compared using one-way ANOVA with a Bonferroni post hoc test using SPSS 25.0 software (IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.

DPSC-Exos were characterized using TEM, western blot analysis and NTA. TEM images revealed that the vesicles isolated from the culture supernatant of the DPSCs exhibited a spherical morphology (Fig. S1A). Western blot analysis demonstrated that these vesicles were positive for the exosomal surface markers, CD9 and CD63 (Fig. S1B). The results of NTA revealed that the diameters of these nanoparticles ranged from 50-100 nm (Fig. S1C). Taken together, these results confirmed their identity as exosomes.

The present study investigated the effects of DPSC-Exos on cutaneous wound healing using a full-thickness wound model on mice. A significantly more rapid wound closure rate was observed when DPSC-Exos were injected (Fig. 1A and B). Skin images from the underside of the wounds, which were collected 14 days post-wounding demonstrated that the mice treated with the DPSC-Exos exhibited a greater amount of newly formed blood vessels compared to the control group (Fig. 1C). Furthermore, H&E and immunohistochemical staining was performed at the wound site. A considerable amount of capillary-structure blood vessels (Fig. 1D and E) and a higher expression of CD31 in the DPSC-Exo-treated group (Fig. 1F and G) confirmed that the DPSC-Exos enhanced local angiogenesis. These findings suggested that the DPSC-Exos accelerated wound healing in vivo by promoting angiogenesis.

To examine the effects of DPSC-Exos on HUVECs behavior in vitro, the internalization of exosomes into these cells was first monitored. Following 24 h of incubation with PKH26-labeled DPSC-Exos, fluorescence microscopy analysis revealed red fluorescent spots in the cytoplasm around the nucleus of HUVECs, which indicated that the HUVECs were able to take up DPSC-Exos (Fig. 2A). For functional analysis, the present study then assessed how DPSC-Exos modulated HUVEC angiogenesis, including migration, proliferation and tube formation. Transwell migration assay (Fig. 2B and C) and scratch wound assay revealed that the DPSC-Exos significantly increased the motility of HUVECs (Fig. 2D and E). CCK-8 assay was utilized to examine the proliferation of the cells within 4 days of culture time, which revealed that the DPSC-Exos increased the proliferative capacity of the HUVECs (Fig. 2F). DPSC-Exo treatment also improved tube formation in the HUVECs as the total tube length and total loops increased in the DPSC-Exo-treated group (Fig. 2G-I). On the whole, these results indicated that the DPSC-Exos enhanced the migration, proliferation and tube formation ability of HUVECs.

Since the mechanisms involved in the promoting effects of DPSC-Exos on angiogenesis remained unknown, the present study employed TMT-labeled quantitative proteomics analysis to identify the protein expression profiles in DPSC-Exos compared with their parent cells. All proteins and peptides identified and quantified using LC-MS/MS in DPSC-Exos and DPSCs are presented in Table SI. A total of 4,592 proteins were quantified, including 1,159 upregulated and 460 downregulated proteins (Fig. 3A). In addition, a volcano plot illustrating the statistically sizable difference of expressed proteins between DPSC-Exos and DPSCs was constructed (Fig. 3B). A change in differential expression levels exceeding 1.5 was regarded as the change threshold for significance.

To thoroughly determine the proteins obtained in these data, GO analysis and KEGG pathway analysis were performed to classify the biological functions and their characteristics, as well as their molecular interaction networks. The results of GO classification analysis revealed that proteins involved in various biological processes in relation to wound healing, such as positive regulation of cell motility, migration, proliferation, vasculature development and angiogenesis, were markedly increased in the DPSC-Exos (Fig. 3C). KEGG pathway analysis revealed that proteins contained in DPSC-Exos were involved in several pathways, including the MAPK signaling pathway (Fig. 3D and Table SII). As shown in Fig. 3E, tubulin was used as an internal control to normalize the western blot analysis data. The ratios of Cdc42, p38 and p-p38 protein levels to tubulin expression were used for comparisons. HUVECs were treated with DPSC-Exos and a significant increase was found in the expression of Cdc42 and p-p38 (Fig. 3E and F). Cdc42 is required for the activation of p38 and plays a key role in cell migration and proliferation (33,34). Taken together, these findings prompted the hypothesis that the Cdc42/p38 MAPK signaling pathway may play an essential role in DPSC-Exo-induced angiogenesis.

Of note, the proteomics data indicated that the expression of FGD5, a pro-angiogenesis protein, was 2.299-fold higher in the DPSC-Exos than in the DPSCs (Table SIII). FGD5 is known to mediate pro-angiogenic action of vascular endothelial growth factor (VEGF) in human vascular endothelial cells by inducing the activation of Cdc42 (35). In the present study, as shown in Fig. 3G, β-actin was used as an internal control to normalize the western blot analysis data. The ratios of the FGD5 protein level to β-actin expression were used for comparisons. Western blot analysis also verified that FGD5 was enriched in the DPSC-Exos (Fig. 3G), which may be a potential candidate mediating pro-angiogenic function of DPSC-Exos.

To examine whether the p38 MAPK signaling pathway is involved in DPSC-Exo-induced angiogenesis, HUVECs were treated with SB203580 to inhibit the p38 MAPK pathway prior to stimulation with the DPSC-Exos. hsp27 is a terminal substrate of the p38 MAPK pathway (36,37), which is related to the positive regulation of angiogenesis, as well as the positive regulation of blood vessel endothelial cell migration (38). In the present study, as shown in Fig. 4A, tubulin was used as an internal control to normalize the western blot analysis data. The ratios of p38, p-p38, hsp27, p-hsp27 protein levels to tubulin expression were used for comparisons. Once the p38 MAPK signaling pathway was inhibited by SB203580, the protein levels of hsp27 and p-hsp27 were significantly decreased in the HUVECs (Fig. 4A). The ratio of p-p38/p-38 and p-hsp27/hsp27 was also shown (Fig. 4B). Transwell migration assay (Fig. 4C and D) and scratch wound assay (Fig. 4E and F) revealed a decreased capacity of the DPSC-Exos to promote cell migration when the HUVECs were pre-treated with SB203580. As shown by CCK-8 assay, the proliferation of the HUVECs which was promoted by DPSC-Exos was also suppressed by the inhibition of p38 by SB203580 (Fig. 4G). In addition, as shown by the results of tube formation assay, the enhanced tube formation ability of the HUVECs stimulated with the DPSC-Exos was reduced by SB203580 (Fig. 4H-J). These outcomes all confirmed that the p38 MAPK signaling pathway was involved in DPSC-Exo-induced angiogenesis.

The present study also examined the effects of p38 inhibition on the healing process of murine cutaneous wounds. The subcutaneous injection of DPSC-Exos accelerated the cutaneous wound closure rate, while this therapeutic effect was attenuated after the p38 MAPK signaling pathway was blocked by SB203580 (Fig. 5A and B). the wound closure rate in all groups reached almost 100% on day 14; thus, a statistically significant difference was not observed at this time point. On the other hand, wounds in the DPSC-Exos + vehicle group were almost completely closed on day 12, which exhibited a much more rapid healing rate than other three groups. Skin images from the underside of the wounds were collected 14 days post-wounding. Compared with the DPSC-Exos + vehicle group, treatment with SB203580 attenuated the development of newly formed blood vessels, as shown in the DPSC-Exos + SB203580 group (Fig. 5C). The quantification of newly formed blood vessels was performed using H&E and immunohistochemical staining. With the presence of SB203580, the amount of capillary-structure blood vessels (Fig. 5D and E) and the expression of CD31 (Fig. 5F and G) were suppressed in the mice treated with either PBS or DPSC-Exos. The formed vessels and positive rate of CD31 were decreased in the DPSC-Exos + SB203580 group as compared to the DPSC-Exos + vehicle group. Accordingly, these results substantiated that DPSC-Exo-induced angiogenesis was attenuated by the inhibition of the p38 MAPK signaling pathway in vivo.