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Journal of Extracellular Vesicles logoLink to Journal of Extracellular Vesicles
. 2024 Jul 4;13(7):e12473. doi: 10.1002/jev2.12473

A new subtype of artificial cell‐derived vesicles from dental pulp stem cells with the bioequivalence and higher acquisition efficiency compared to extracellular vesicles

Xingxiang Duan 1, Rui Zhang 1, Huixian Feng 4, Heng Zhou 1, Yu Luo 1, Wei Xiong 1, Junyi Li 1, Yan He 2,3,✉, Qingsong Ye 1,4,5,✉
PMCID: PMC11223992  PMID: 38965648

Abstract

Extracellular vesicles (EVs) derived from dental pulp stem cells (DPSC) have been shown an excellent efficacy in a variety of disease models. However, current production methods fail to meet the needs of clinical treatment. In this study, we present an innovative approach to substantially enhance the production of ‘Artificial Cell‐Derived Vesicles (ACDVs)’ by extracting and purifying the contents released by the DPSC lysate, namely intracellular vesicles. Comparative analysis was performed between ACDVs and those obtained through ultracentrifugation. The ACDVs extracted from the cell lysate meet the general standard of EVs and have similar protein secretion profile. The new ACDVs also significantly promoted wound healing, increased or decreased collagen regeneration, and reduced the production of inflammatory factors as the EVs. More importantly, the extraction efficiency is improved by 16 times compared with the EVs extracted using ultracentrifuge method. With its impressive attributes, this new subtype of ACDVs emerge as a prospective candidate for the future clinical applications in regenerative medicine.

Keywords: artificial cell‐derived vesicles, burns, dental pulp stem cells lysate, extracellular vesicles, intracellular vesicles

1. INTRODUCTION

Extracellular vesicles (EVs) are bilayer lipid vesicles released from intracellular nanovesicles that participate in intercellular communications. They play an important role in many a processes in tissue regeneration and early diagnosis of diseases (Kalluri & LeBleu, 2020). EVs derived from stem cells have great therapeutic potential in regenerative therapy of various tissues (Li et al., 2021; Zhou et al., 2022). Despite that EVs are highly heterogeneous in size, loaded contents and origin, they have been thoroughly studied. Many methods have been developed for the isolation and extraction of EVs (Chen et al., 2021; Jiang et al., 2020).

Traditionally, EVs could be extracted by ultracentrifugation, size exclusion chromatography and polymer‐based precipitation. These methods can handle large volume samples, yet neither could guarantee high yields nor absolute purity. Some methods are intricate, costly and necessitate specialized instruments. For instance, ultracentrifugation poses a risk of inducing mechanical damage to EVs under high centrifugal force, compromising the bioactivity and morphological integrity (Zhang et al., 2020).

Novel isolation methods are imperative to achieve high yield while maintaining the bioactivity and structural integrity of EVs (Shi et al., 2021; Wang, Li, et al., 2022; Wen et al., 2022). EVs can be studied effectively only on the basis of high‐quality products. Therefore, selecting appropriate separation technology serves as a crucial prerequisite in EVs research. In our previous study, we identified numerous vesicles in DPSC lysate, demonstrating EVs‐like therapeutic efficacy in photoaging models (Duan et al., 2023). This paper presents a method using ultracentrifugation to obtain high purity artificial cell‐Derived vesicles (ACDVs) from DPSC lysate, identified as a premature EVs. Compared ACDVs with EVs extracted by normal ultracentrifugation, the consistency of general properties and functions was proved.

The incidence of burns remains high and can cause severe disability or death. Most burns are primarily caused by thermal injuries, including fire and explosions. Skin grafting is one of the crucial therapeutic approaches to reduce the mortality rates of severe burn patients. However, finding suitable skin sources remains challenging (Mahmood et al., 2019). In recent years, nanotechnology has exhibited immense potential in revolutionizing the treatment of severe burns. Many nanodrug delivery systems have been created and are being applied in the areas of burn infection and skin repair (Anamizu & Tabata, 2019; Dong et al., 2020; Shafiee et al., 2021). Extensive research has demonstrated that the nanocarriers have some advantages: (1) excellent compatibility with the burned skin, maintaining a favourable microenvironment of wound repair; (2) accurate release of drugs around burned area and higher drug retention rate and (3) controlled drug release and prolonged effectiveness of time. Microvesicles released by MSCs have unique abilities in suppressing inflammation and promoting wound‐healing to modulate the immune response after injury by stimulating the release of anti‐inflammatory factors and inhibiting pro‐inflammatory cytokines (Chen et al., 2024). Therefore, it has become a new treatment strategy for severe burns. Here, we synergistically harness the potential of nanomaterial carriers and DPSC microvesicles to better facilitate the recovery of burn patients.

2. MATERIALS AND METHODS

2.1. Isolation, culture and identification of DPSCs

Human third molars collected from volunteers (16–30 years old) were used for DPSC isolation and this study with consent and permission in written. All procedures in this study were approved by the Ethics Committee of Renmin Hospital of Wuhan University (Approval Number: WDRY‐2022‐K025, Wuhan, China). Extracted teeth were transferred to the laboratory in Dulbecco's phosphate‐buffered saline (DPBS; Gibco, USA) complemented with 100 U/mL penicillin‐G (Servicebio, China) and 100 mg/mL streptomycin (Servicebio, China) at 4°C within 6 h for further process. Tooth was crashed by a hydraulic forceps to expose dental pulps. Then these pulp tissues were carefully separated from the teeth, minced and digested in a mixture of 4 mg/mL neutral protease (Roche, Basel, Switzerland) and 3 mg/mL collagenase type I (Sigma‐Aldrich, St. Louis, MO, USA) for 30 min at 37°C. Minimum Essential Medium α (α‐MEM; Gibco, USA) supplemented with 20% fetal bovine serum (FBS; Gibco, USA) was used to terminate this process. Tissue was centrifuged and cultured maintained in an atmosphere of 5% CO2 at 37°C till the cell emerged and reached 80% confluence. The culture was maintained in α‐MEM supplemented with 10% FBS, penicillin and streptomycin and passaged every 3–4 day. Cells in passages 5–7 were used for this study.

To determine cell surface markers, cells were suspended in flow cytometry staining buffer (Invitrogen, USA). 1 × 106 cells were then incubated at 4°C for 60 min with FITC‐anti‐human CD44 (1:50, Thermo, USA), FITC‐anti‐human CD45 (1:40, Thermo, USA) and FITC‐anti‐human CD73 (1:25, Proteintech, China). After three washes with staining buffer, the cells were suspended in 200 µL staining buffer and examined with a CytoFLEX flow cytometer (Beckman Coulter, CA, USA).

2.2. Isolation and characterization of the new ACDVs and EVs

At 90% confluency, DPSCs were harvested using trypsin‐EDTA (Gibco, USA) and centrifuged three times in DPBS to remove residual trypsin‐EDTA and culture media components. To harvest ACDVs, first cell membrane osmotic rupture was achieved as follows, 1 mL of deionized H2O was added to 1 × 107 cells and incubated for 30 min. Then this cellular suspension was subjected to three freeze‐thaw cycles at 80°C to further dissociate the lysed cell sediments, followed by 30 min of centrifugation at 4000 × g. The supernatants were then purified by two cycles of ultracentrifugation in PBS (100,000 × g, 70 min, Thermo Fisher Scientifc, Sorvall WX 80+) and the deposited ACDVs was re‐suspended in PBS.

According to previously reported methods (Wang et al., 2024), EVs were isolated and purified from cell culture medium. Briefly, serum‐free cell culture medium was collected and centrifuged at 2000 × g for 10 min to remove cellular debris. The supernatants were then collected and centrifuged at 10,000 × g for 30 min, and ultracentrifuged twice at 100,000 × g for 70 min. The resuspension was sterilized with a 0.22 µm filter disc. EVs was re‐suspended with PBS.

All centrifugation procedures were conducted below 4°C. Stocke suspension of ACDVs and EVs were preserved at −80°C for no longer than 4 weeks. A nanoparticle tracking analysis was performed to determine the size and concentration using ZetaView PMX 110 (Particle Metrix, Germany).

TEM was used to observe the microstructure of ACDVs and EVs. Sample solution was pipetted on a carbon‐coated grid and dried for 10 min at room temperature. Then the grids were washed with DPBS and stained for 1 min with uranyl oxalate at a concentration of 2% (w/v). Samples were imaged with TEM (HITACHI, HT7700, Japan).

Protein concentrations were determined using a BCA protein assay kit (Beyotime, China) to determine the degradation rate of ACDVs and EVs at 37°C for 48 h and −80°C for 4 m, respectively. Western blot was performed to identify the specific markers contained in ACDVs and EVs. ACDVs and EVs were mixed with 1 mM phenylmethanesulfonyl fluoride (PMSF, Beyotime, China). A total of 30 mg protein was loaded onto a 10%–15% SDS‐PAGE gel for each sample. PVDF membranes were incubated with primary Alix, TSG101, CD9, CD63, GM130, IL‐1β, IL‐10, CD31 (1:1000, Abcam, USA) VEGF (1:500, Servicebio, CN) and β‐actin (1:5000, Servicebio, CN) overnight at 4°C, after being blocked with protein free rapid blocking buffer (Epizyme, China) for 15 min at room temperature.

The comparison of ACDVs and EVs was performed using a human cytokines array (GSH‐GF‐1, RayBiotech, USA) according to the manufacturer's instructions.

2.3. Mouse dermal fibroblasts (MDFs) and human umbilical vein endothelial cells (HUVECs) isolation and culture

The Laboratory Animal Welfare Ethics Committee of Renmin Hospital and Wuhan University approved all animal procedures (Approval Number: WDRM20211002, Wuhan, China). As previously described, MDFs were isolated from the dorsal dermis of newborn littermates 24–48 h after birth. Every 4–5 days, the cells were maintained in Dulbecco's modified Eagle medium (DMEM, Gibco, USA) supplemented with 10% FBS, penicillin, and streptomycin in a 5% CO2 atmosphere at 37°C. For this study, cells from passage two or three were used.

The HUVECs were purchased from the ATCC and cultured in endothelial cell medium (ECM) (Sciencell, USA), according to the instructions. The medium was refreshed every 3 days, and the cells were passaged once they were 80%–90% confluent.

2.4. Cell proliferation assay

For the cell counting kit‐8 (CCK‐8; MCE, USA) assay, HUVECs were seeded in a 96‐well plate at 2 × 103 cells/well. After incubation with different concentrations (0, 5, 10, 20 and 40 µg/mL) of EVs and ACDVs for 12, 24, 48 and 72 h. Then, a 10 µL solution of CCK‐8 was added to each well, and the cells were incubated at 37°C for 1 h. The absorbance of various groups was measured at 450 nm using an enzyme‐labelling device (PerkinElmer EnSight, USA).

2.5. Cell migration assay

The migratory capacity of treated and untreated MDFs was studied using a transwell assay. Polycarbonate membrane Transwell inserts with a pore size of 8 µm (BIOFIL, TCS‐003‐024) were applied in the study. Briefly, 5 × 104 MDFs suspended in serum‐free DMEM medium were seeded into the upper chamber, whereas 600 µL DMEM medium containing 20 µg/mL EVs or ACDVs were added to the lower chamber. After 24 h incubation, the upper chamber was washed three times with PBS and then fixed with 4% paraformaldehyde for 30 min. Non‐migrating cells over the membrane were carefully wiped off with a cotton swab from the inside of the upper chamber. The membrane was dried and the cells on the outside of bottom were stained with crystal violet staining solution (Beyotime Biotechnology, C0121) for 15 min. Migrated cells were the stained and visualized under a standard bright field microscope, and a total of 5–6 microscope fields/well were randomly selected for quantification.

In the scratch assay, HUEVCs were seeded into six‐well plates and cultured to 90% confluence. Then a cell free gap was created with a pipette tip scraping over the cells. Images of the re‐growth of the cells were obtained at 0, 12 and 24 h via a microscope. The size of cell free areas was measured by ImageJ.

2.6. Tube formation assay

First, 50 µL/well of Matrigel was plated in the 96‐well plate on ice with pre‐cooled pipet tips. Then, the plate was placed in a 37°C incubator for 1 h to allow gelation. HUVECs (1 × 104 cells/well) were seeded into the 96‐well plate and treated with 100 µL DMEM added 20 µg/mL of EVs, ACDVs or the same volume PBS. After 3 and 6 h, an optical microscope was used to capture the tube formation images, which were analyzed using ImageJ. The quantitative parameters included the total vessel length, vessel percentage area, and total number of junctions. ImageJ was used to convert the tube formation images into a binarized image to show the contours, paths and nodes of blood vessels.

2.7. Synthesis and characterization of temperature‐sensitive hydrogel dressing

0.6 mg Glucose Oxidase (GOX) was added to the 2‐methylimidazole (MIM) solutions (1.15 mmol in 0.5 mL ddH2O) incubated for 30 min at room temperature while shaking. Then zinc nitrate hexahydrate ((Zn (NO3)2 ·6H2O, 0.032 mmol in 0.5 mL ddH2O) was added and incubated for 1 h at room temperature with shaking. The ZIF‐8@GOX was separated by centrifugation (10 min at 12,000 g) and then suspended and rinsed three times in ddH2O to eliminate any residual material.

Pluronic F127 (PF127) powder was stirred overnight in normal saline to make 15% solutions at 4°C. Then ZIF‐8@GOX was added into the above PF127 solutions to form a final synthesis of PF127@ZIF‐8@GOX (PZG). To functionalize PZG, EVs or ACDVs (1 mg/mL) was mixed with ZIF‐8@GOX and suspended in PF127 to get final hydrogel dressing materials of PZG@EVs and PZG@ACDVs.

The microstructure of synthetic particles and dressing materials was examined by the TEM (HITACHI, HT7700, Japan) and FESEM (Zeiss SIGMA, UK).

2.8. Animals model of 3RD degree burn on skin

All animal procedures were approved by the Laboratory Animal Welfare Ethics Committee of Renmin Hospital of Wuhan University (Approval Number: WDRM20211002, Wuhan, China). The BALB/c mice were anesthetized with isoflurane, and the hair on the back was shaved. After anesthetization, full‐thickness skin scald wounds were created on the dorsal side with a 100°C metal rods. The mice were randomly divided into four groups: CON, PZG, PZG@EVs, PZG@ACDVs. CON: control, no intervention was applied on the wound; PZG: dressing hydrogel that was used to load EVs or ACDVs in this study; PZG@EVs, dressing hydrogel loaded with 1 mg/mL EVs; PZG@ACDVs, dressing hydrogel loaded with 1 mg/mL ACDVs. The hydrogel was re‐applied on the wound surface every 2 days until 14 days, and the wound areas were photographed and calculated by ImageJ. After 14 days of treatment, the mice were euthanized, and wound tissues were collected for histopathological and immune analyses.

2.9. In vivo imaging study

DiR‐labelled EVs and ACDVs were mixed with PZG on the back wounding of mice. The IVIS Spectrum imaging system (PerkinElmer EnSight, USA) was used to capture images of each mouse at 0, 1, 2, 3, 7 and 14 days after treatment.

2.10. Metabolic cage study

To study the impact of PZG@EVs and PZG@ACDVs on the general metabolism of injured animals, real time change of locomotor activity, oxygen consumption and carbon dioxide production were recorded with a LabMaster system (TSE Systems). Mice were individually housed in metabolic cage immediately after the removal of scab, data were collected for 11.5 days and analyzed. Light and feeding conditions were maintained as usual.

2.11. Histological and immunofluorescence analyses

After 14 days of treatment, H&E and Masson staining were used to assess the epidermal thickness and collagen deposition around the wounds. Western Blot was applied to study the inflammation status of the skin tissue. Immunofluorescence staining was used to evaluate the expression of CD31 and VEGF at the wound sites. Nuclei were stained with DAPI. Microscopic images were collected and analyzed using ImageJ.

2.12. Statistical analysis

Statistical analyses were conducted utilizing Prism 8.0 (Graphpad Software, California, USA). The difference was considered significant if it was p < 0.05. Unpaired student's t‐test was utilized for statistical comparisons between two groups, and ANOVA was utilized to analyze multiple groups.

3. RESULTS

3.1. Extraction and characterization of DPSC

A standardized protocol has been developed for the extraction of ACDVs from DPSCs (Figure 1). Medical sources of waste teeth are premium cell extraction reserves. DPSCs were isolated from pulp of teeth of medical origin (Figure 2a). Notably, DPSCs derived from young dental pulp exhibit greater vitality compared to those from aging dental pulp, rendering their extraction relatively straightforward. After the pulp tissue has been collagenase and fully digested, it takes approximately 5–7 days for cells (P0) to germinate from the debris and form dense colonies. By the second generation, DPSCs showed typical fibroblast‐like morphology (Figure 2b). These cells were characterized by flow cytometry and showed positive expression of CD44 and CD73 and negative expression of CD45 and CD34 (Figure 2c,d). These are typical molecular phenotypes of mesenchymal stem cells (Jing et al., 2022). The above experimental results proved that the extracted cells were DPSCs. As shown in Figure 2e, TEM results indicated that there were many ACDVs, pre‐mature EVs, in the cell ready to be released. According to our study, both EVs and ACDVs degrade easily at 37°C, about 80% would degrade in 48 h (p > 0.05). In contrast, low temperature could well preserve both EVs and ACDVs from degradation, only about 20% of degradation in 4 months at −80°C (Figure 2f,g).

FIGURE 1.

FIGURE 1

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Standardized procedure for the extraction of EVs and ACDVs.

FIGURE 2.

FIGURE 2

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Extraction and characterization of DPSC. (a) Digital photographs of extracted teeth and dental pulp tissue. (b) Morphology of primary culture expanded DPSC at passage 0 and passage 2, scale bar: 200 µm. (c) Expression of surface antigens (CD44∖CD34∖CD45) of DPSC determined by immunofluorescence, scale bar: 100 µm. (d) Expression of surface antigens (CD45∖CD44∖CD73) of DPSC determined by flow cytometry. (e) Representative TEM image of DPSC, red arrow: unsecreted EVs, scale bar: 500 nm. (f) Experimental schematic. EVs or ACDVs were stored at 37°C and −80°C). (g) Degradation of EVs and ACDVs at 37°C and −80°C (n = 3/group, ns p > 0.05).

3.2. Isolation and characterization of the new subtype of ACDVs and EVs

Comparing the extraction methods of EVs and ACDVs, the extraction of ACDVs was 16 times more efficient than that of EVs. ACDVs represented higher production yield and product purity. According to our study, one cell‐culture dish (∅ = 15 cm) of DPSCs at 100% confluency could yield to ∼0.1 mg ACDVs, while it required at least 16 dishes of DPSCs to achieve the same amount of EVs (Zheng et al., 2022). One milligram ACDVs contains 5.26 × 1011 particles and 1 mg EVs contains 7.80 × 1011 particles (Figure 3a).

FIGURE 3.

FIGURE 3

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Characterization of DPSC lysate. (a) Schematic illustration of cells required for 0.1 mg ACDVs and EVs was harvested. (b) Representative TEM image of ACDVs and EVs, scale bar: 200 nm. (c) The expression of specific markers of EVs (Alix, TSG101, CD9, CD63 and GM130) in the ACDVs, EVs and DPSC‐CM by western blot analysis. (d) The diameter of ACDVs and EVs determined by a nanoparticle tracking analysis. (e) Cytokine array of ACDVs and EVs by densitometric analysis (n = 3/group, *p < 0.05).

Besides above varied features, we found that ACDVs and EVs were consistent in multiple dimensions of EVs, which echoed previous studies (Bakhtyar et al., 2018; Banakh et al., 2023; Li et al., 2016). TEM, Western Blotting and Nanoparticle tracking analysis were used as the general criteria for EV identification. Our studies did not show significant difference between EVs and ACDVs. TEM results revealed that ACDVs and EVs exhibited sphere‐shaped morphologies with a lipid bilayer., Nanoparticle tracking analysis revealed that the average diameter of ACDVs and EVs were 108.6 ± 17.5 and 103.9 ± 10.9 nm. The marker expressions of ACDVs and EVs were verified by Western Blotting. Both ACDVs and EVs were positive in expression of Alix, TSG101, CD9 and CD63, and negative in expression of GM130 (Camões et al., 2022) (Figure 3b‐d). We also screened 40 cell growth factors in ACDVs and EVs by cytokines array (Wang, Cao, et al., 2022b). As shown in Figure 3e, ACDVs and EVs have similar protein secretion profiles. BDNF, bFGF, EGF‐R,HGF, PDGF‐AA and SCF‐R in ACDVs were found significantly higher in ACDVs than in EVs, and IGFBP‐1 and VEGF‐A in EVs were significantly higher in EVs than in ACDVs.

3.3. New ACDVs promoted cellular proliferation, migration and vessel formation

All concentrations of ACDVs showed an obvious effect on promoting the proliferation of HUVECs’ until 24 h compared with the control. There was no significant difference between EVs and ACDVs in cell proliferation. And 20 and 40 µg/mL of ACDVs depicted no difference in cell proliferation as well (Figure 4a). Fibroblasts are involved in the inflammatory, proliferative and remodelling stages of wound healing, and have significant effects on wound contraction and skin tissue remodelling (Bian et al., 2022; Pu et al., 2019). It can effectively accelerate wound healing by promoting fibroblast migration.

FIGURE 4.

FIGURE 4

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(a) The HUVECs proliferation in each group determined by CCK‐8 assay (n = 3/group). (b) The schematic illustration of MDFs transwell chemotaxis upon action of ACDVs and EVs and the comparison of effects of ACDVs and EVs on the migration of MDFs, scale bar: 100 µm. (c) Migrated MDFs counted in different groups (n = 3/group, ***p < 0.001). (d) Representative images of the wound closure of HUVECs in different groups by cell scratch assay at different time points. (e) Statistical analysis of migration area (%) in scratch assay (n = 3/group, ***p < 0.001). (f) Immunofluorescent staining for CD31 of HUVECs, respectively. Scale bar: 100 µm. (g) Statistical analysis of intensity of CD31 positive cells in different treatment groups. (h) Tube formation of HUVECs with different treatment. Scale bar: 100 µm. (i) Quantitative analysis of tube‐like structures formation. (n = 3/group, ***p < 0.001).

Compared with plain DMEM medium, ACDVs supplemented DMEM attracted twice more MDFs over 24 h culture (Figure 4b,c). Similar effect was reported previously (Banakh et al., 2023; Sovkova et al., 2018). According to the scratch assay (Figure 4d), comparing with the control, ACDVs formulation showed prominent effect on the migration of fibroblasts early on 12 h after the scratch was done. And this effect was amplified till at 48 h post scratch (Figure 4e).

The results of immunofluorescence study and tube formation assay showed that ACDVs effectively enhanced the expression of CD31 in HUVECs. The same results were found in the EVs intervention group (Figure 4f,g). ACDVs stimulated more capillary‐like tube structures and branches on HUVECs than the control (Figure 4h,i).

These results indicated that ACDVs had a strong impact on promoting angiogenesis. ACDVs formulation showed a slightly stronger impact on cellular proliferation, migration and vessel formation than EVs formulation (p > 0.05, Figure 4).

3.4. PZG@ACDVs kept porous structure and PZG showed mild analgesic‐like effect on individual

According to our previous work (Albashari et al., 2023), we adopted 20% PF‐127 hydrogel to synthesize PZG in this study, which maintained its transition temperature at 37°C (Figure 5a). ZIF‐8 was proved in our previous work being an outstanding nano‐carrier that could provide a reliable control release of drugs (Duan et al., 2023). In this study, we loaded ZIF‐8 with GOX, an antibiotic compound (Dai et al., 2024; Deng et al., 2022; Tian et al., 2023) to offer extra protection over the wound. Compared with ZIF‐8 alone, when loaded with GOX, the TEM images of nanoparticles revealed a lightly larger and furrier outline (Figure 5b). Observed from SEM image, PZG showed the typical porous structures of hydrogel and the addition of ACDVs or EVs did not alter this important micro‐architecture of the hydrogel (Figure 5c). This feature could guarantee a good permeability and compound transportation, which is conducive during the healing of a massive skin wounds.

FIGURE 5.

FIGURE 5

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(a) PF127 hydrogel can be switched from liquid at room temperature to solid at 37°C. (b) The TEM images of the ZIF‐8 and ZIF‐8@GOX sample. (c) The SEM images of the PZG, PZG@ACDVs and PZG@EVs sample. (d) Activity from metabolic cages recordings in CON group and PZG group (n = 3/group, 11.5 days recording). (e) RER from metabolic cages recordings in CON group and PZG (n = 3/group, 11.5 days recording). (f) O2 depletion from metabolic cages recordings in CON group and PZG group (n = 3/group, 11.5 days recording).

We noticed that PZG might have mild analgesic‐like effect. From data in Figure 5d‐f, we found that within 11.5 days post burn, mice in the control group were significantly more active while their RER and oxygen consumption did not differ from the one in PZG group. The size of wound area between the control and PZG was similar (Figure 6d).

FIGURE 6.

FIGURE 6

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(a) In vivo imaging photographs of back wound in mice cover with DiR labelled PZG@ACDVs or PZG@EVs over 14 days. (b) Paradigm of burn model preparation and treatment by PBS, PZG, PZG@EVs and PZG@ACDVs in mice (male, age 3 months old, n  =  4‐5/group). (c) Representative photos exhibit the process of wound closure on every other day for 14 days. (d) Quantitative analysis of wound area per group (n = 4‐5/group, *p < 0.05, **p < 0.05). (e) Western blot of IL‐1β, IL‐10, VEGF, CD31 and β‐actin. (f) The relative expression of IL‐1β, IL‐10, VEGF, CD31 (n = 3/group, *p < 0.05, **p < 0.01, ***p < 0.001).

3.5. PZG@ACDVs accelerated skin wound healing after burn on mice

Figure 6a showed that ACDVs labelled with DiR, bright orange in in vivo imaging, was confined in the defect only. There was no signal in any other parts of the body. In vivo imaging photos disclosed the distribution of ACDVs during a 14‐day treatment regime where PZG@ACDVs was applied every other day, seven times in total (Figure 6b).

Till day 4 post burn, the speed of recovery started to differ among groups. Comparing the wound size between day 0 and day 14, defect area of PZG@ACDVs shrank to 1/6 of the original, and defect of the control and PZG shrank to 1/3 of the original (Figure 6c,d). The healing effect of PZG@ACDVs did not vary from the effect provided by PZG@EVs.

We evaluated the status of skin tissue on day 14. Results from western blot and histoimmumochemical staining showed that the level of anti‐inflammatory factor (IL‐10) and angiogenesis factors (VEGF and CD31) was much higher and the level of inflammatory factor IL‐1β was much lower in PZG@ACDVs groups, when compared with those of the control and PZG groups (Figures 6e,f and 7c,d,f,g). There was no difference between PZG@ACDVs and PZG@EVs.

FIGURE 7.

FIGURE 7

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(a) Representative photomicrographs of each group H&E‐stained wounds on day 14, Scale bar: 100 µm or 20 µm. (b) Representative photomicrographs of each group Masson‐stained wounds on day 14, Scale bar: 100 or 20 µm. (c) Representative photomicrographs of each group immumohistochemical‐stained (CD31) wounds on day 14, Scale bar: 100 or 20 µm. (d) Representative photomicrographs of each group immumohistochemical‐stained (IL‐1β) wounds on day 14, Scale bar: 100 or 20 µm. (e) Quantitative analysis of collagen synthesis (n = 3/group). (f) Quantitative analysis of inflammatory biomarker CD31 (n = 3/group). (g) Quantitative analysis of inflammatory biomarker IL‐1β (n = 3/group, * p < 0.05, ** p < 0.01, *** p < 0.001, ns., no significant differences were found between groups).

On day 14, compared to the control and PZG, PZG@ACDVs treated wounds had more granulation tissue filling the defects, more collagen production and elastic fibre regeneration (Figure 7a,b,e). PZG@ACDVs also showed better results in vascular regeneration (Figure 7c) with the highest CD31+ cell count (Figure 7f). There was no significant difference in the IL‐1β expressions among the control group and PZG group. While the IL‐1β expressions of the PZG@EVs group and PZG@ACDVs group were significantly lower than that of the control group (Figure 7d,g).

4. DISCUSSION

EVs is an important component of MSC paracrine nanoparticle products and is considered as a promising drug due to its strong regenerative potential, good biocompatibility and safety (Shi et al., 2021; Wang, He, et al., 2019). However, due to the limited production of cellular EVs, the production of sufficient quantities of EVs to meet clinical needs has been a challenge. It has been reported that some strategies, such as induced hypoxia and mechanical pressure stimulation, can promote the secretion of EVs, but their clinical transformation may be limited due to the difficulty in the application of techniques (He et al., 2018; Sun et al., 2022). To solve this dilemma, we developed a new protocol to collect ACDVs, and assessed its characteristics and tissue regeneration efficacy in full layer skin defect caused by burn. The method of ACDVs collection is simple, efficient and reliable. Comparing with EVs, the production yield of ACDVs has been greatly improved. According to MISEV2023 (Welsh et al., 2024), Compare with the other types of ACDVs, ACDVs derived from cell lysate is novel in two aspects. First, the production process, conventional ACDVs are produced via extrusion process which involves a pressing force on the cells; while ACDVs derived from cell lysate is pressure free and produced through membrane lysis. Second, the acquisition efficiencies in the total number of particles as well as protein content among EVs, ACDVs derived from cell lysate and conventional ACDVs were compared. In our study, there were approximately 10 times more particles in ACDVs than EVs and 16 times more protein content in ACDVs than that of EVs, meaning each ACDVs particle contains more protein content compare with matures EVs. The literature reported that the conventional ACDVs acquired by extrusion exhibited a ∼140‐fold increase in the total number of particles but only a ∼20‐fold increase in protein content compared to EVs (Jiang et al., 2024; Sun et al., 2022), meaning each ACDV particle on average contains only one seventh of protein content compares to EVs. This might be attribute to the extrusion process of the conventional ACDVs, the broken cell membranes form new empty vesicles/debris due to the action of extrusion force. In this respect, ACDVs derived from cell lysate is a nature‐derived intact vesicles while the extruded ACDVs are manufactured under pressure. So, we think the ACDVs derived from cell lysate is a new subtype of ACDVs. The new subtype of ACDVs come from the cells without through the process of cellular secretion, thus we can also named it as intracellular vescicles.

EVs are intracellular multivesicular bodies containing luminal vesicles that fuse with the cell membrane through the double invagination of the plasma membrane, and the intracellular substances are released into the body in the form of vesicles (Kim et al., 2021; Yerneni et al., 2022). In our study, we confirmed traditionally, EVs was collected from cell culture medium, also known as conditioned medium. However, EVs are easy to degrade, consistent with our findings where over 80% of EVs was degraded at body temperature within 48 h. This might explain the higher yield of ACDVs in our study. Our protocol by‐passed the exposure of EVs in cell culture. In our study, we observed that our new ACDVs had similar vesicle shape as EVs under TEM. The number distribution of the two vesicles was consistent with the definition of EVs and expressed TSG101 and other EVs surface markers.

In our study, once collected, ACDVs faded as fast as EVs at body temperature. Some reported that low temperature (−80°C) could preserve the quality of EVs (Gelibter et al., 2022; Gorgens et al., 2022). In our study, we managed to maintain 80% of ACDVs and EVs after 4 months storage at −80°C. No studies have reported to solve the problem of EVs degrading at this stage of serum‐free culture‐medium collection and our strategy can avoid this unresolved problem (Ali et al., 2022).

Many studies have reported that EVs have an excellent therapeutic effect in various disease models such as brain ischemia injury, skin photoaging and cartilage injury (Feng et al., 2021; Hu et al., 2019). In our previous studies, DPSC lysate showed significantly anti‐skin‐aging properties and the potential to prevent and treat cutaneous aging (Duan et al., 2023). In this study, we improved our protocol to isolate ACDVs out from the DPSC lysate. In the first part of this paper, we proved that there was no difference in physiological characteristics between ACDVs and EVs, and ACDVs contained several times more proliferation‐related proteins than EVs (Li et al., 2022; Lim et al., 2020). Our protocol could render a much higher production of ACDVs. In the later part of this paper, we assessed the application of ACDVs in vitro and in vivo, and we proved that ACDVs has stronger ability to promote cellular proliferation and migration, full layer skin re‐growth. ACDVs were as good as EVs at skin tissue regeneration if not significantly better.

The care of burn patients and wound repair is a very complex problem in the medical field. The treatment of burn patients has improved over the past few decades, but the healing of burn patients is still slow. Burn patients are prone to be lack of immune response and delayed wound healing, leading to poor prognosis. When severely burn damaged, the body is under a lot stress including prolonger inflammatory status, high level of inflammatory factors and ROS (Surowiecka et al., 2022). These pathological reactions further delay wound healing in burn patients. At present, the application of nanocarriers and mesenchymal stem cell products has made great progress in regenerative medicine, and treatment of burns has great room for improvement (Song et al., 2023; Wang, Cao, et al., 2022a). Nanocarriers can be individually designed to adapt to different wound conditions, making them suitable for tissue re‐growth in various scenarios (Wang, Lu, et al., 2019). PZG, temperature‐sensitive, is a suitable wound dressing for skin injury. Through LabMaster monitoring system, we found that animal who carried PZG appeared calmer than the ones without, while animals from neither groups were under a lot stress or pain according to their RER and oxygen consumption during 11.5 days post burn. At the same time, PZG can promote wound healing, increase collagen regeneration and reduce stress response in mice, but it failed to inhibit the expression of inflammation and promote vascular regeneration. In contrast, PZG@ACDVs served a very well job on skin re‐growth and provided an anti‐inflammatory and angiogenic micro‐environment for wounded animal and localized around the wound only. ACDVs and EVs have similar protein secretion profiles, but ACDVs showed better efficacy in the treatment of burn models. Interestingly, protein chip results showed that VEGF content was higher in EVs, but ACDVs was still superior to EVs in terms of vascular regeneration ability. From these results, ACDVs is more suitable in the treatment of burns from the aspects of efficacy and application prospects.

Over all, we proved that ACDVs extracted from DPSCs has excellent anti‐inflammatory regulation, immune regulation and the ability to promote angiogenesis and wound healing, and can play a promoting role in various stages of wound healing (Figure 8). The combination of nanocarriers and ACDVs is undoubtedly a suitable solution. In clinical practice, large quantity of such biological components is often required in critical size defect or systematic application. The high yield of ACDVs, simple collection protocol and low temperature preservation technique can greatly reduce production cost and promote the manufacturing efficiency. Compared with EVs, ACDVs is undoubtedly more suitable for clinical application.

FIGURE 8.

FIGURE 8

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Schematic diagram of PZG@ACDVs to repair third degree burns. ACDVs can regulate inflammation, promote vascular regeneration and promote wound healing as well as EVs. In addition, the application of PZG can also reduce discomfort in mice, by Figdraw.

5. CONCLUSION

The extraction of ACDVs from DPSC lysate is a novel method with convenience and efficiency. This paper demonstrated that the new subtype of ACDVs is equivalent to EVs in skin regeneration. Its yield is 16 time higher than that of traditionally collected EVs’. DPSCs, as a type of mesenchymal stem cell source, raise less ethical concerns. DPSC‐derived ACDVs can be standardized for mass production to solve the bottleneck of the clinical application of EVs and has a promising application prospect.

Early treatment is very important for the recovery of burn wound. However, clinically MSCs‐based therapy is rarely used in the inflammatory phase of the burn. ACDVs extracted from MSCs avoids the potential risks of direct use of MSCs. For this reason, it is imperative to develop and produce standardized MSCs extracts for clinical applications. Last, the combined use of hydrogel, nanocarrier and intracellular vesicles will bring more benefits to burn patients.

AUTHOR CONTRIBUTIONS

Qingsong Ye: Conceptualization (lead); Methodology (lead); data curation (equal); funding acquisition (lead); project administration (lead). Xingxiang Duan: Conceptualization (lead); data curation (lead); formal analysis (lead); methodology (lead); project administration (lead); resources (lead); software (lead); validation (lead); visualization (lead); writing—original draft (lead); writing—review and editing (supporting). Rui Zhang: Data curation (supporting); formal analysis (supporting); funding acquisition (supporting); methodology (supporting); project administration (supporting); resources (equal); software (equal); validation (supporting); writing—original draft (supporting). Huixian feng: Formal analysis (equal); validation (equal); visualization (lead). Heng Zhou: Formal analysis (equal); software (lead); validation (lead). Yu Luo: Methodology (supporting); software (lead). Wei Xiong: Data curation (equal); software (equal). Junyi Li: Data curation (equal); Formal analysis (equal). Yan He: Conceptualization (supporting); funding acquisition (lead); methodology (equal); resources (equal); writing—review and editing (lead).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

ACKNOWLEDGEMENTS

This work was supported by the key Project of Ministry of Science and Technology China (2022YFC2504200 from Qingsong Ye), Key Research and Development Project of Hubei Province (2022BCA029 from Qingsong Ye) and Chutian Researcher Project (X22020024 from Yan He).

Duan, X. , Zhang, R. , Feng, H. , Zhou, H. , Luo, Y. , Xiong, W. , Li, J. , He, Y. , & Ye, Q. (2024). A new subtype of artificial cell‐derived vesicles from dental pulp stem cells with the bioequivalence and higher acquisition efficiency compared to extracellular vesicles. Journal of Extracellular Vesicles, 13, e12473. 10.1002/jev2.12473

Xingxiang Duan and Rui Zhang contributed equally to this work.

Contributor Information

Yan He, Email: helen-1101@hotmail.com.

Qingsong Ye, Email: qingsongye@hotmail.com.

REFERENCES

  1. Albashari, A. A. , He, Y. , Luo, Y. , Duan, X. , Ali, J. , Li, M. , Fu, D. , Xiang, Y. , Peng, Y. , Li, S. , Luo, L. , Zan, X. , Kumeria, T. , & Ye, Q. (2023). Local spinal cord injury treatment using a dental pulp stem cell encapsulated H(2) S releasing multifunctional injectable hydrogel. Advanced Healthcare Materials, 13, e2302286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ali, M. , Namjoshi, S. , Benson, H A. E. , Kumeria, T. , & Mohammed, Y. (2022). Skin biomechanics: Breaking the dermal barriers with microneedles. Nano TransMed, 1, e9130002. [Google Scholar]
  3. Anamizu, M. , & Tabata, Y. (2019). Design of injectable hydrogels of gelatin and alginate with ferric ions for cell transplantation. Acta Biomaterialia, 100, 184–190. [DOI] [PubMed] [Google Scholar]
  4. Bakhtyar, N. , Jeschke, M. G. , Herer, E. , Sheikholeslam, M. , & Amini‐Nik, S. (2018). Exosomes from acellular Wharton's jelly of the human umbilical cord promotes skin wound healing. Stem Cell Research & Therapy, 9, 193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Banakh, I. , Rahman, M. M. , Arellano, C. L. , Marks, D. C. , Mukherjee, S. , Gargett, C. E. , Cleland, H. , & Akbarzadeh, S. (2023). Platelet lysate can support the development of a 3D‐engineered skin for clinical application. Cell and Tissue Research, 391, 173–188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bian, D. , Wu, Y. , Song, G. , Azizi, R. , & Zamani, A. (2022). The application of mesenchymal stromal cells (MSCs) and their derivative Exosome in skin wound healing: A comprehensive review. Stem Cell Research & Therapy, 13, 24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Camões, S. P. , Bulut, O. , Yazar, V. , Gaspar, M. M. , Simões, S. , Ferreira, R. , Vitorino, R. , Santos, J. M. , Gursel, I. , & Miranda, J. P. (2022). 3D‐MSCs A151 ODN‐loaded exosomes are immunomodulatory and reveal a proteomic cargo that sustains wound resolution. Journal of Advanced Research, 41, 113–128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen, J. , Zhao, X. , Qiao, L. , Huang, Y. , Yang, Y. , Chu, D. , & Guo, B. (2024). Multifunctional on‐demand removability hydrogel dressing based on in situ formed AgNPs, silk microfibers and hydrazide hyaluronic acid for burn wound healing. Advanced Healthcare Materials, 13, e2303157. [DOI] [PubMed] [Google Scholar]
  9. Chen, Y. , Zhu, Q. , Cheng, L. , Wang, Y. , Li, M. , Yang, Q. , Hu, L. , Lou, D. , Li, J. , Dong, X. , Lee, L. P. , & Liu, F. (2021). Exosome detection via the ultrafast‐isolation system: ExoDUS. Nature Methods, 18, 212–218. [DOI] [PubMed] [Google Scholar]
  10. Dai, W. , Shu, R. , Yang, F. , Li, B. , Johnson, H. M. , Yu, S. , Yang, H. , Chan, Y. K. , Yang, W. , Bai, D. , & Deng, Y. (2024). Engineered bio‐heterojunction confers extra‐ and intracellular bacterial ferroptosis and hunger‐triggered cell protection for diabetic wound repair. Advanced Materials, 36, e2305277. [DOI] [PubMed] [Google Scholar]
  11. Deng, M. , Zhang, M. , Huang, R. , Li, H. , Lv, W. , Lin, X. , Huang, R. , & Wang, Y. (2022). Diabetes immunity‐modulated multifunctional hydrogel with cascade enzyme catalytic activity for bacterial wound treatment. Biomaterials, 289, 121790. [DOI] [PubMed] [Google Scholar]
  12. Dong, Y. , Cui, M. , Qu, J. , Wang, X. , Hyung Kwon, S. , Barrera, J. , Elvassore, N. , & Gurtner, G C. (2020). Conformable hyaluronic acid hydrogel delivers adipose‐derived stem cells and promotes regeneration of burn injury. Acta Biomaterialia, 108, 56–66. [DOI] [PubMed] [Google Scholar]
  13. Duan, X. , Luo, Y. , Zhang, R. , Zhou, H. , Xiong, W. , Li, R. , Huang, Z. , Luo, L. , Rong, S. , Li, M. , He, Y. , & Ye, Q. (2023). ZIF‐8 as a protein delivery system enhances the application of dental pulp stem cell lysate in anti‐photoaging therapy. Materials Today Advances, 17, 100336. [Google Scholar]
  14. Feng, K. , Xie, X. , Yuan, J. , Gong, L. , Zhu, Z. , Zhang, J. , Li, H. , Yang, Y. , & Wang, Y. (2021). Reversing the surface charge of MSC‐derived small extracellular vesicles by epsilonPL‐PEG‐DSPE for enhanced osteoarthritis treatment. Journal of Extracellular Vesicles, 10, e12160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gelibter, S. , Marostica, G. , Mandelli, A. , Siciliani, S. , Podini, P. , Finardi, A. , & Furlan, R. (2022). The impact of storage on extracellular vesicles: A systematic study. Journal of Extracellular Vesicles, 11, e12162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gorgens, A. , Corso, G. , Hagey, D. W. , Jawad Wiklander, R. , Gustafsson, M. O. , Felldin, U. , Lee, Y. , Bostancioglu, R. B. , Sork, H. , Liang, X. , Zheng, W. , Mohammad, D. K. , van de Wakker, S. I. , Vader, P. , Zickler, A. M. , Mamand, D. R. , Ma, L. , Holme, M. N. , Stevens, M. M. , & El Andaloussi, S. (2022). Identification of storage conditions stabilizing extracellular vesicles preparations. Journal of Extracellular Vesicles, 11, e12238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. He, C. , Zheng, S. , Luo, Y. , & Wang, B. (2018). Exosome theranostics: Biology and translational medicine. Theranostics, 8, 237–255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hu, S. , Li, Z. , Cores, J. , Huang, K. , Su, T. , Dinh, P. U. , & Cheng, K. (2019). Needle‐free injection of exosomes derived from human dermal fibroblast spheroids ameliorates skin photoaging. ACS Nano, 13, 11273–11282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jiang, W. , Zhan, Y. , Zhang, Y. , Sun, D. , Zhang, G. , Wang, Z. , Chen, L. , & Sun, J. (2024). Synergistic large segmental bone repair by 3D printed bionic scaffolds and engineered ADSC nanovesicles: Towards an optimized regenerative microenvironment. Biomaterials, 308, 122566. [DOI] [PubMed] [Google Scholar]
  20. Jiang, Z. , Liu, G. , & Li, J. (2020). Recent progress on the isolation and detection methods of exosomes. Chemistry ‐ An Asian Journal, 15, 3973–3982. [DOI] [PubMed] [Google Scholar]
  21. Jing, S. , Zhou, H. , Zou, C. , Chen, D. P. C. , Ye, Q. , Ai, Y. , & He, Y. (2022). Application of telomere biology and telomerase in mesenchymal stem cells. Nano TransMed, 1, e9130007. [Google Scholar]
  22. Kalluri, R. , & LeBleu, V. S. (2020). The biology, function, and biomedical applications of exosomes. Science, 367(6478), eaau6977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kim, S. N. , Lee, C. J. , Nam, J. , Choi, B. , Chung, E. , & Song, S. U. (2021). The effects of human bone marrow‐derived mesenchymal stem cell conditioned media produced with fetal bovine serum or human platelet lysate on skin rejuvenation characteristics. International Journal of Stem Cells, 14, 94–102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Li, S. , Luo, L. , He, Y. , Li, R. , Xiang, Y. , Xing, Z. , Li, Y. , Albashari, A. A. , Liao, X. , Zhang, K. , Gao, L. , & Ye, Q. (2021). Dental pulp stem cell‐derived exosomes alleviate cerebral ischaemia‐reperfusion injury through suppressing inflammatory response. Cell Proliferation, 54, e13093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Li, T. , Lu, H. , Zhou, L. , Jia, M. , Zhang, L. , Wu, H. , & Shan, L. (2022). Growth factors‐based platelet lysate rejuvenates skin against ageing through NF‐κB signalling pathway: In vitro and in vivo mechanistic and clinical studies. Cell Proliferation, 55, e13212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Li, X. , Liu, L. , Yang, J. , Yu, Y. , Chai, J. , Wang, L. , Ma, L. , & Yin, H. (2016). Exosome derived from human umbilical cord mesenchymal stem cell mediates MiR‐181c attenuating burn‐induced excessive inflammation. EBioMedicine, 8, 72–82. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lim, T. , Tang, Q. , Zhu, Z. , Wei, X. , & Zhang, C. (2020). Sustained release of human platelet lysate growth factors by thermosensitive hydroxybutyl chitosan hydrogel promotes skin wound healing in rats. Journal of Biomedical Materials Research. Part A, 108, 2111–2122. [DOI] [PubMed] [Google Scholar]
  28. Mahmood, R. , Mehmood, A. , Choudhery, M S. , Awan, S. J. , Khan, S N. , & Riazuddin, S. (2019). Human neonatal stem cell‐derived skin substitute improves healing of severe burn wounds in a rat model. Cell Biology International, 43, 147–157. [DOI] [PubMed] [Google Scholar]
  29. Pu, C. M. , Chen, Y. C. , Chen, Y. C. , Lee, T. L. , Peng, Y. S. , Chen, S. H. , Yen, Y. H. , Chien, C. L. , Hsieh, J. H. , & Chen, Y. L. (2019). Interleukin‐6 from adipose‐derived stem cells promotes tissue repair by the increase of cell proliferation and hair follicles in ischemia/reperfusion‐treated skin flaps. Mediators of Inflammation, 2019, 2343867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shafiee, A. , Cavalcanti, A S. , Saidy, N T. , Schneidereit, D. , Friedrich, O. , Ravichandran, A. , De‐Juan‐Pardo, E M. , & Hutmacher, D W. (2021). Convergence of 3D printed biomimetic wound dressings and adult stem cell therapy. Biomaterials, 268, 120558. [DOI] [PubMed] [Google Scholar]
  31. Shi, A. , Li, J. , Qiu, X. , Sabbah, M. , Boroumand, S. , Huang, T. C. , Zhao, C. , Terzic, A. , Behfar, A. , & Moran, S. L. (2021). TGF‐β loaded exosome enhances ischemic wound healing in vitro and in vivo. Theranostics, 11, 6616–6631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Song, Y. , You, Y. , Xu, X. , Lu, J. , Huang, X. , Zhang, J. , Zhu, L. , Hu, J. , Wu, X. , Xu, X. , Tan, W. , & Du, Y. (2023). Adipose‐derived mesenchymal stem cell‐derived exosomes biopotentiated extracellular matrix hydrogels accelerate diabetic wound healing and skin regeneration. Advanced Science (Weinh), 10, e2304023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sovkova, V. , Vocetkova, K. , Rampichova, M. , Mickova, A. , Buzgo, M. , Lukasova, V. , Dankova, J. , Filova, E. , Necas, A. , & Amler, E. (2018). Platelet lysate as a serum replacement for skin cell culture on biomimetic PCL nanofibers. Platelets, 29, 395–405. [DOI] [PubMed] [Google Scholar]
  34. Sun, D. , Mou, S. , Chen, L. , Yang, J. , Wang, R. , Zhong, A. , Wang, W. , Tong, J. , Wang, Z. , & Sun, J. (2022). High yield engineered nanovesicles from ADSC with enriched miR‐21‐5p promote angiogenesis in adipose tissue regeneration. Biomaterials Research, 26, 83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Surowiecka, A. , Chrapusta, A. , Klimeczek‐Chrapusta, M. , Korzeniowski, T. , Drukala, J. , & Struzyna, J. (2022). Mesenchymal stem cells in burn wound management. International Journal of Molecular Sciences, 23, 15339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tian, M. , Zhou, L. , Fan, C. , Wang, L. , Lin, X. , Wen, Y. , Su, L. , & Dong, H. (2023). Bimetal‐organic framework/GOx‐based hydrogel dressings with antibacterial and inflammatory modulation for wound healing. Acta Biomaterialia, 158, 252–265. [DOI] [PubMed] [Google Scholar]
  37. Wang, J. , Li, M. , Jin, L. , Guo, P. , Zhang, Z. , Zhanghuang, C. , Tan, X. , Mi, T. , Liu, J. , Wu, X. , Wei, G. , & He, D. (2022). Exosome mimetics derived from bone marrow mesenchymal stem cells deliver doxorubicin to osteosarcoma in vitro and in vivo. Drug Delivery, 29, 3291–3303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wang, L. , Wei, X. , He, X. , Xiao, S. , Shi, Q. , Chen, P. , Lee, J. , Guo, X. , Liu, H. , & Fan, Y. (2024). Osteoinductive dental pulp stem cell‐derived extracellular vesicle‐loaded multifunctional hydrogel for bone regeneration. ACS Nano, 18, 8777–8797. [DOI] [PubMed] [Google Scholar]
  39. Wang, W. , Lu, K. J. , Yu, C. H. , Huang, Q. L. , & Du, Y. Z. (2019). Nano‐drug delivery systems in wound treatment and skin regeneration. Journal of Nanobiotechnology, 17, 82. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wang, Y. , Cao, Z. , Wei, Q. , Ma, K. , Hu, W. , Huang, Q. , Su, J. , Li, H. , Zhang, C. , & Fu, X. (2022a). VH298‐loaded extracellular vesicles released from gelatin methacryloyl hydrogel facilitate diabetic wound healing by HIF‐1alpha‐mediated enhancement of angiogenesis. Acta Biomaterialia, 147, 342–355. [DOI] [PubMed] [Google Scholar]
  41. Wang, Y. , Cao, Z. , Wei, Q. , Ma, K. , Hu, W. , Huang, Q. , Su, J. , Li, H. , Zhang, C. , & Fu, X. (2022b). VH298‐loaded extracellular vesicles released from gelatin methacryloyl hydrogel facilitate diabetic wound healing by HIF‐1α‐mediated enhancement of angiogenesis. Acta Biomaterialia, 147, 342–355. [DOI] [PubMed] [Google Scholar]
  42. Wang, Y. , He, G. , Guo, Y. , Tang, H. , Shi, Y. , Bian, X. , Zhu, M. , Kang, X. , Zhou, M. , Lyu, J. , Yang, M. , Mu, M. , Lai, F. , Lu, K. , Chen, W. , Zhou, B. , Zhang, J. , & Tang, K. (2019). Exosomes from tendon stem cells promote injury tendon healing through balancing synthesis and degradation of the tendon extracellular matrix. Journal of Cellular and Molecular Medicine, 23, 5475–5485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Welsh, J. A. , Goberdhan, D. C. I. , O'Driscoll, L. , Buzas, E. I. , Blenkiron, C. , Bussolati, B. , Cai, H. , Di Vizio, D. , Driedonks, T. A. P. , Erdbrugger, U. , Falcon‐Perez, J. M. , Fu, Q. L. , Hill, A. F. , Lenassi, M. , Lim, S. K. , Mahoney, M. G. , Mohanty, S. , Moller, A. , Nieuwland, R. , … Witwer, K. W. (2024). Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches. Journal of Extracellular Vesicles, 13, e12404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wen, Y. , Fu, Q. , Soliwoda, A. , Zhang, S. , Zheng, M. , Mao, W. , & Wan, Y. (2022). Cell‐derived nanovesicles prepared by membrane extrusion are good substitutes for natural extracellular vesicles. Extracell Vesicle, 1, 100004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yerneni, S. S. , Yalcintas, E. P. , Smith, J. D. , Averick, S. , Campbell, P. G. , & Ozdoganlar, O. B. (2022). Skin‐targeted delivery of extracellular vesicle‐encapsulated curcumin using dissolvable microneedle arrays. Acta Biomaterialia, 149, 198–212. [DOI] [PubMed] [Google Scholar]
  46. Zhang, Y. , Bi, J. , Huang, J. , Tang, Y. , Du, S. , & Li, P. (2020). Exosome: A review of its classification, isolation techniques, storage, diagnostic and targeted therapy applications. International Journal of Nanomedicine, 15, 6917–6934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zheng, Y. , Xu, P. , Pan, C. , Wang, Y. , Liu, Z. , Chen, Y. , Chen, C. , Fu, S. , Xue, K. , Zhou, Q. , & Liu, K. (2022). Production and biological effects of extracellular vesicles from adipose‐derived stem cells were markedly increased by low‐intensity ultrasound stimulation for promoting diabetic wound healing. Stem Cell Reviews and Reports, 19, 784–806. [DOI] [PubMed] [Google Scholar]
  48. Zhou, Z. , Wang, R. , Wang, J. , Hao, Y. , Xie, Q. , Wang, L. , & Wang, X. (2022). Melatonin pretreatment on exosomes: Heterogeneity, therapeutic effects, and usage. Frontiers in Immunology, 13, 933736. [DOI] [PMC free article] [PubMed] [Google Scholar]

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