Multiple Mesenchymal Stem Cell Derived Extracellular Vesicle Administration Accelerates Healing and Ameliorates Tissue Scarring Compared with Single Application in Full Thickness Cutaneous Wounds

doi:10.21203/rs.3.rs-5026947/v1

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Multiple Mesenchymal Stem Cell Derived Extracellular Vesicle Administration Accelerates Healing and Ameliorates Tissue Scarring Compared with Single Application in Full Thickness Cutaneous Wounds

https://doi.org/10.21203/rs.3.rs-5026947/v1

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Background

Impaired wound healing is still challenging for patients and health care providers. Healing is a complex multiphasic process that is mediated by paracrine signaling. In recent years, human umbilical cord mesenchymal stem cell (hUC-MSC) derived extracellular vesicles (EVs) have shown promising effects on healing acceleration by modifying intercellular interactions. However, they will be promptly washed out after local injection and are almost depleted from the injured site in five days. Therefore, single-dose administration may fail to affect all phases of the healing process. In this study, we evaluated the advantages of multi-administration over its single injection following full-thickness cutaneous wound induction in rats.

Methods

EVs were isolated from human umbilical cord mesenchymal stem cells and characterized. These particles were administered locally in the EV-treated wounds. The level of inflammatory (TNF-α and TGF-β) and angiogenesis (CD31) factors were evaluated through the study to compare multiple dose EV administered, single dose EV administered, and control wounds with each other.

Results

In vivo results demonstrate that triple EV administration significantly attenuates inflammation and improves angiogenesis and collagen deposition in the injured area (p < 0.05).

Conclusion

This study suggests that multiple injections of EVs promotes wound closure and decreases scar formation mainly by maintaining adequate concentration in the wounded area.

extracellular vesicles

wound healing

angiogenesis

neovascularization

mesenchymal stem cells

stem cells

Impaired wound healing is still challenging, despite the progress made in the medical fields. The global cost for wound care in recent years was billions of dollars for both under-healed (chronic) and over-healed (scarred) wounds [1].

The wound healing process consists of four overlapping phases; homeostasis, inflammation, proliferation, and remodeling. Different cells and cytokines play a part in each stage [2]. Smooth muscle cells and platelets are the principal mediators of homeostasis, which starts right after the injury. Smooth muscle cells prevent blood loss owing to the vasoconstriction reflex. Also, platelet activation promotes blood coagulation and releases cytokines, including Tissue Growth Factor-β (TGF-β), Platelet-Derived Growth Factor (PDGF), and Vascular Endothelial Growth Factor (VEGF) [3]. These cytokines attract neutrophils and monocytes to the injured area, the pivotal cells in the inflammatory phase [4, 5]. Neutrophils immigrate to the wounded site after 24-36h, and phagocyte tissue debris, foreign particles, and invasive bacteria to sterilize the wound. In the late stage of the inflammatory phase, macrophages predominate in the wound and activate fibroblasts and keratinocytes by secreting mediators such as Heparin Binding Epidermal Growth Factor (HB-EGF), TGF-β, and VEGF. The proliferation phase starts around three days post-injury, and fibroblasts and myofibroblasts play a crucial role in this stage. Extracellular matrix (ECM) formation, granulation formation, and neovascularization occur during this phase. The remodeling phase starts around 14 days post-injury and may last several years. During this phase, scar tissue develops, and a balance occurs between ECM degradation and formation [6]. Bidirectional intercellular interactions mainly mediate the transition between these phases [7].

Extracellular vesicles (EVs), are endosomal particles ranging from 30 nm to 10 µm in size and are secreted by diverse types of cells [8]. They contain a multitude of signaling components, such as growth factors, cytokines, and nucleic acids, allowing them to regulate paracrine communications [8]. However, their cargo varies from each other depending on their progenitor cells [9].

Previous studies have shown that umbilical cord mesenchymal stem cell (UC-MSC) derived EVs improve the wound healing process, primarily through the presence of healing prompting cytokines, including TGF-β, PDGF, and (VEGF) in their cargo, and UC-MSC-derived EVs enhance healing by modifying the cellular milieu in the wounded area [10, 11]. Additionally, the application of MSC-derived EV is superior to direct stem cell therapy because these particles lack the risk of possible consequences such as immunological rejection and improper cell differentiation [12]. However, they have a relatively short half-life at the injured site [13–15].

For instance, EV concentration significantly decreases after two days, and the wound site is almost depleted in five days [13, 16]. Therefore, a single administration might fail to influence all healing phases. Repeated local injection of EVs can boost tissue repair and maintain an adequate concentration of healing-promoting factors in the injured area. At this time, the advantages of repeated EV administration in different healing phases over a single-dose injection remain unclear. In this study, we verified the favorable benefits of multiple administrations compared to single-dose therapy by evaluating inflammatory factors, angiogenesis, and collagen deposition for several days throughout the healing process.

2.1. Isolation of human umbilical cord mesenchymal stem cells and extracellular vesicles.

Human umbilical cord mesenchymal stem cells (hUC-MSC) were isolated and characterized with the same method discussed in our previous study [17]. After 2 passages, the cells were cultured for 48 hours in serum-free DMEM. In order to EV purification, the supernatant was centrifuged at 300g for 10 minutes, 2000g for 10 minutes, and 10,000g for 30 minutes to remove cells, dead cells and cell debris, respectively. The precipitates were centrifuged at 100,000g for 70 minutes to remove contaminating proteins and filtered through a 0.22m membrane to remove larger vesicles. EV pellets were diluted with 200 µl phosphate buffered saline (PBS) and stored at -80 ºC.

2.2. Extracellular Vesicle characterization

The expression of including CD9, CD63, and CD81 was assessed by western blotting to confirm the accuracy of EV isolation [17]. Briefly, Radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor was added to the cellular extract for EV lysis. After that, SDS buffer was used to denaturize the vesicles at 95 ºC. A total of 30 µg protein per lane was loaded to the SDS-PAGE, and electrophoresis was performed. Subsequently, the protein was transferred to a nitrocellulose (NC) page and blocked with 3% BSA for 2 hours. Then, samples were incubated with primary antibodies against CD63, CD9, and CD81 at 4 ºC for 8 hours; and were probed with corresponding HRP-conjugated (secondary) antibodies at room temperature for 2 hours. The Proteins were imaged using a chemiluminescence kit (Roche Molecular Biochemicals, NJ, USA).

Transmission Electron Microscopy (TEM) and Dynamic light scattering (DLS) techniques were performed to evaluate the morphological characteristics of the extracted particles, including their size and spherical shape.

2.3. Animals and Ethics

Eighteen male Wistar rats, aged 8–9 weeks, were purchased from Pasture Institute. Individual animals were housed in cages with free access to food pellets and tap water. They were subjected to a 12-12hour cycle of light and dark. All procedures were performed in agreement with NIH guidelines for the care and use of laboratory animals and the protocol provided by the Research Ethics Committees of Laboratory Animals-Tehran University of Medical Sciences (IR.TUMS.AEC.1400.048).

2.4. Excisional wound model and treatment

All rats were anesthetized with an intraperitoneal injection of ketamine (Alfasan, Woerden, Netherlands; 87mg/kg) and xylazine (Alfasan, Woerden, Netherlands; 13mg/kg)[18]. Before the operation, rats’ hair was shaved on the upper dorsal skin, and the skin was disinfected using povidone-iodine. Four circular full-cutaneous wounds (10 mm in diameter) were made on the dorsum of each animal using an external template and surgical scissors, with a 1.5 cm distance between every two wounds. The right-sided wounds were assigned as Group A (Single) wounds in one group that received a single injection of 200µg/µl EV immediately after wound induction and a similar injection of PBS on days 2nd and 7th of the study to eliminate the effect of the injection. In the other group, right-sided wounds were assigned to Group B (Multiple) wounds that received three doses of 200 µg/µl EV injection on days 0, 2nd, and 7th post-wounding. All left-sided wounds on each rat were treated with an equal amount of PBS on the same days. A 25G syringe was used for subcutaneous injection. 160µl of EV solution or PBS was injected subcutaneously in four different spots around each wound (40µl per spot), and the last 40µl of the solution was topically applied on the wound bed. In each group, animals were randomly assigned into three subgroups and euthanized using the CO₂ chamber on days 4th ,7th, and 14th post-operation. Tissue samples were harvested from the wounded sites with a 1-2mm edge from the adjacent skin. Half of the specimens were kept in 10% formalin solution; the other half were stabilized in Trizol (Sigma Aldrich, USA) and kept at -80 ºC temperature for further quantitative reverse transcription polymerase chain reaction (qRT-PCR) investigations.

2.5. Macroscopic evaluation of the healing rate:

Digital photos of wounds were taken on days 0, 4th, 7th, and 14th of the study to monitor wound closure rates. The wounded area was calculated using ImageJ software (ver 1.8.0_172).

2.6. Histopathological analysis

Samples kept in 10% formalin solution were subsequently embedded in paraffin and sectioned into 5µm cross-sections. Then, these slices were stained with hematoxylin and eosin (H&E) and Masson’s trichrome to assess the healing process, collagen deposition, and orientation in the wounded areas. Primary antibodies against TNF-α (1:100; ab220210, Abcam, Cambridge, UK) and TGF-β (1:100; ab229856, Abcam, Cambridge, UK), and secondary antibodies; Horseradish peroxidase (HRP)-Conjugated Rabbit Anti-Mouse antibodies (ab6728, Abcam, Cambridge, UK) and HRP-Conjugated Goat Anti-Rabbit (ab6721, Abcam, Cambridge, UK) to evaluate pro-inflammatory and anti-inflammatory response through the study.

For assessing vascularization, specimens from the 14th day of the study were incubated with primary antibody against CD31 (1:100; ab182981, Abcam, Cambridge, UK) and secondary HRP-Conjugated Goat Anti-Rabbit (ab6721, Abcam, Cambridge, UK).

A single pathologist blinded to the study evaluated samples using a light microscope (Olympus-CX43, MA, USA). The quantity of inflammatory markers in stained sections was determined using the ImageJ software (ver 1.8.0_172), and angiogenesis was evaluated by counting the number of vessels per high-power field.

2.7. Total RNA extraction and Quantitative RT-PCR

According to the manufacturer’s guidance, a Trizol reagent (Sigma-Aldrich, USA) was used to extract the total RNA of samples taken on days 4th, 7th, and 14th of the study. Then extracted RNA was reversed-transcribed into complementary DNA (cDNA) using (M-MULV Reverse Transcriptase, fermentas) according to the manufacturer’s guidance. Real time RT-PCR performed with following program; denaturation at 95°C for 30s, annealing at 60°C for 30s, and extension at 72°C for 30s, in 40 cycles. Primers used in this investigation are shown in Table 1

2.8. Statistics

Graphpad Prism was used to conduct all statistical analyses (version 9.3.1; Graphpad Software, La Jolla, CA). All values are represented as mean and Standard error of the Mean (SEM). The Two-Way analysis of variance (ANOVA) was used to compare wound closure rates and inflammatory markers (TGF-β and TNF-α) expression levels within and between the groups. The one-Way ANOVA test was performed to compare the number of vessels between the groups. P-values less than 0.05 are considered to be statistically significant.

3.1. Extracellular vesicle characterization

EVs collected from UC-MSCs were characterized by western blotting, TEM, and DLS techniques. The corresponding bands in the results demonstrate the expression of CD9, CD63, and CD81 (Fig. 1A). TEM results show the spherical shape of the particles and their 100–500 nm (Fig. 1, B). DLS results also confirm EV diameter ranging from 100–500 nm (mean = 238.9nm, SD = 52.7nm).

3.2. Multiple local administrations of EVs enhance cutaneous wound closure in rats

Wound closure after 14 days was significantly enhanced in group B that received three doses of subcutaneous injection (mean = 96.42 ± 1.36%) compared to group A, treated with a single dose (mean = 84.30 ± 2.24%) and PBS treated control wounds (mean = 64.08 ± 1.46%). Also, multiple EV administration attenuated scar formation in Group B wounds compared to the other experimental groups (Fig. 2A, B).

3.3. Repeated sub-cutaneous UC-MSC derived extracellular vesicles enhanced collagen deposition and re-epithelialization

For microscopic evaluation of wound healing in experimental groups, tissue samples of the 4th, 7th, and 14th days post-wounding were stained with H&E and Masson’s trichrome techniques (Fig. 3A, B).

H&E results demonstrate enhanced re-epithelialization in Group B subjects compared to Group A and control wounds. Also, repeated local injection of UC-MSC-derived EVs promoted hair follicle maturation in Group B in the late stages of wound healing (Fig. 3A).

Collagen maturity and orientation are determining parameters in the wound healing process. Wounds treated with three doses of UC-MSC EVs developed more mature and oriented collagen fibers than the other groups, leading to less scar formation (Fig. 3B).

3.4. Repeated UC-MSC EV treatment improves angiogenesis and revascularization

Angiogenesis is a critical step in the wound healing process. Immunohistochemical staining against CD31 represents higher density and maturity of newly formed vessels in the late stages of the healing process (on the 14th day of study) in group B wounds compared to two other groups. (Fig. 4A, B)

3.5. Multiple local administrations of UC-MSC extracellular vesicles ameliorate inflammation

Repeated injection of UC-MSC-derived EVs facilitates the inflammatory phase transition through wound healing process. We measured TNF-α and TGF-β mRNA and protein levels to evaluate the inflammatory responses of all three experimental groups, as shown in Figs. 5 and 6.

The mRNA expression of TNF-α was significantly lower in group B wounds than in group A wounds on days 4th and 14th. It was also lower in group B compared with the PBS-treated wounds during the healing process on days 4th, 7th, and 14th. On the 4th day post-wounding, which is representative of the inflammatory phase, the mean TNF-α level in group B wounds was 0.63 of group A (p-value < 0.05) and 0.31 of control (p-value < 0.05) wounds (Fig. 5A).

In addition, the results of IHC staining against TNF-α were consistent with its mRNA expression levels. These results demonstrated suppressed inflammation in group B wounds compared to the group A and control groups (Fig. 5B, C). On the 4th day of the study, TNF-α in group A and control wounds were expressed higher than in group B wounds (p-value < 0 .01 and p-value < .0001, respectively). Furthermore, TNF-α was least expressed in group B wounds compared with other study groups on days 7th and 14th post-surgery, which indicates attenuated inflammation in group B wounds (Fig. 5B, C).

TGF-β mRNA and protein expression were significantly higher in EV-treated groups (groups A and B) compared with the control subjects on the 4th day (P < 0.001) without any significant difference between groups A and B. On the 7th day of the study, group B wounds showed the highest expression of TGF-β mRNA compared with other study groups (P < 0.05). However, this trend was reversed on the 14th day of the study, and TGF-β was least expressed in group B wounds and significantly lower than TGF-β level in the control subject (P < 0.05). Though the TGF-β level in group B subjects was lower than in group A wounds, the difference between the EV-treated groups was slightly insignificant (P ~ 0.06) (Fig. 6A).

As shown in IHC results, samples of group B wounds exhibited the highest levels of TGF-β compared with the other study groups in the first week of the experiment. TGF-β level in group B was significantly higher than in group A on 7th day of the study (p-value < 0.05) and PBS-treated controls on days 4th and 7th of the study (p-value < 0.001). However, similar to PCR analysis, subjects in group B expressed lower levels of TGF-β compared to group A and control wounds at the late stages of the healing process (p-value < 0.001 and p-value < 0.0001, respectively) (Fig. 6B, C).

In this study, we showed the advantages of multiple-dose EV administration over single-dose therapy. Repeated EV injection regulated inflammatory responses, enhanced angiogenesis, and improved collagen deposition, which promoted wound closure.

Mesenchymal Stem Cell (MSC) therapy enhances the healing process by improving angiogenesis, cellular immigration, differentiation, and tissue regeneration [19–21]. Among different Mesenchymal Stem Cells (MSC) sources, UC-MSC has superiorities to adult-derived MSCs. Compared with Bone Marrow and Adipose tissue-derived MSC, UC-MSC has lower immunogenicity, greater capacity for cellular expansion, and higher differentiation potential [22, 23]. Apart from these benefits, direct stem cell therapy has limitations, including undesirable differentiation, possible tumorigenicity, and graft versus host immune reactions [24, 25]. The therapeutic effects of MSC therapy are in favor of the extracellular vesicles released by these cells [26]. EVs modify the cellular milieu by altering paracrine signaling and promoting the healing process without the risk of deleterious side effects of direct Stem Cell therapy [27].

Previous studies have evinced that UC-MSC-derived EVs improve wound closure after subcutaneous injection owing to their cargo [28–30]. The structural characteristics of EVs, including their spherical shape and diameter, facilitate their uptake in the target cells by the caveolae system while scaping the phagolysosome degradation [31]. However, due to their short half-life, EV concentration significantly decreases in two days and will be almost abolished five days post-injection [13, 16]. Hence, single-dose therapy may fail to affect all phases of the healing process. Previous studies evince that the transition between the inflammation and proliferation phases is a key determinant of the healing process, and prolonged inflammation results in compromised healing [32, 33]. Considering the EVs’ short half-life and their modulatory effects on inflammation, the inflammatory phase, which starts 36h after injury, is suggested as the best time for single-dose local therapy.

To investigate the inflammatory responses in different groups, we evaluated TNF-α and TGF-β levels in different phases of our study. TNF-α is a potent pro-inflammatory cytokine that inhibits fibroblast differentiation into myofibroblast cells and hinders α-SMA¹ expression in these cells [34]. Therefore, high levels of TNF-α deteriorate the healing process [35]. Based on the current body of evidence, suppressed TNF-α accelerates wound closure and attenuates inflammation by reducing leukocyte infiltration [36, 37]. Also, TNF-α has a positive feedback regulatory effect on the inflammatory pathway NF-ĸB. Hence, higher levels of TNF-α exacerbate inflammation [38]. In our study, repeated local injection of hUC-MSC-derived EV alleviated inflammation and developed tissue regeneration by attenuating TNF-α expression (Fig. 5).

TGF-β, a well-known growth factor, acts like a double-edged sword in wound healing. At the early stages of the healing process, TGF-β contributes to the inflammation phase by recruiting macrophages and monocytes to the injured site. Surprisingly when the wounded area becomes sterilized, TGF-β halts the production of reactive oxygen species (ROS) by macrophages [4, 39]. It also promotes granulation formation, ECM formation, fibronectin expression, and angiogenesis in the wounded area [4, 40]. However, high levels of TGF-β expression in the late stages of the wound healing process result in excessive collagen type 1 and 3 expression and will cause scar tissue formation [41, 42]. In our study, repeated EV injection caused higher expression of TGF-β levels in the wounded area till the 7th day post-surgery. However, TGF-β levels fell and were least expressed in Group B wounds in the last days of our study, which resulted in less scar formation in this group (Fig. 6).

Additionally, another key determinant in the wound healing process is sufficient angiogenesis and neovascularization. A Proper blood supply is needed to provide nutrition and oxygen to different cells involved in the healing process [43]. Wei et al. demonstrated that UC-MSC derived EVs enhance wound healing and promote angiogenesis through its MiR-17-5p component, downregulating PTEN and activating AKT/HIF-1α/VEGF pathway to enhance angiogenesis [30]. In our study, Group B (multiple) wounds had a higher density and maturity of blood vessels than Group A (single) and control wounds. This is mainly caused by adequate EV concentration in the injured area maintained by multiple injections.

Also, there have been clinical trials investigating the safety and efficacy of EV injection in wound treatment. Johnson et al. declared the safety of platelet-derived extracellular vesicle (pEV) application in wound treatment in humans. Their study consists of two parts, including in vitro studies and a double-blind safety clinical trial. The in vitro investigations demonstrate that pEV application enhances wound healing through activating ERK and Akt pathways. In the clinical part of their study, 11 healthy individuals with cutaneous wounds were enrolled and received a single dose of either pEV or placebo. This study declared safety of pEV application but could not show its therapeutic advantages probably due to the small sample size and enrollment of healthy individuals, and further studies are warranted to evaluate the therapeutic outcomes and optimum dose of pEV application [44]. In another study, Kwon et al. applied adipose tissue stem cell (ASC)-derived exosomes for acne scar treatment in a double-blind trial. In this study patients received three doses of topical exosome for treatment after CO₂ laser therapy and the results demonstrate that exosomes significantly ameliorated the side effects including erythema and improved healing in patients. However, all patients had the same ethnic background, and the number of studying populations was limited. Therefore, the results might not be attributable to all populations. The therapeutic effects and underlying mechanisms are still unclear and require more investigation [45].

Also, many other studies in recent years aimed to overcome their rapid clearance by recruiting biomaterials, including hydrogels and scaffolds, which release vesicles sustainably [46, 47]. These methods’ advantages over each other must be further studied in different disorders. Maintenance and storage of some of these materials require specific conditions (e.g., sterilized conditions and a specific range of temperature), which might not be easily affordable [48]. Thus, multiple local injections can be a more cost-effective and feasible route for EV administration in wound treatment, especially in clinical settings. Moreover, recent studies have provided evidence that EVs therapy improves the healing process in a dose-dependent manner [49, 50]. Although these introduced biomaterials release particles in a sustained manner, they may fail to provide their optimum dose in the course of healing. However, the precise ideal concentration can be achieved through subcutaneous injection in the wounded area, which indicates its potential priority in wound treatment.

Taking all into consideration, our study shows that repeated local administration of UC-MSC EVs accelerates the wound healing process by addressing the short half-life challenge. However, the underlying mechanisms need more investigation. Also, the optimum dose for multiple-EV therapy and its comparison with the optimum dose of single-EV administration can be further studied.

This study suggests that multiple local UC-MSC-derived EV administrations promote cutaneous wound healing by attenuating inflammation and improving neovascularization. These beneficial effects are potentially due to enhanced EV half-life in the wounded area and adequate EV concentration during the healing process.

hUC-MSC

Human umbilical cord mesenchymal stem cell

UC-MSC

umbilical cord mesenchymal stem cell

Extracellular Vesicles

TNF-α

Tumor necrosis factor alpha

TGF-β

Transforming growth factor-β

PDGF

Platelet-Derived Growth Factor

VEGF

Vascular Endothelial Growth Factor

HB-EGF

Heparin Binding Epidermal Growth Factor

ECM

Extracellular matrix

RIPA

Radioimmunoprecipitation assay

Nitrocellulose

TEM

Transmission Electron Microscopy

DLS

Dynamic light scattering

qRT-PCR

Quantitative reverse transcription polymerase chain reaction

H&E

Hematoxylin and eosin

HRP

Horseradish peroxidase

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

SEM

Standard error of the mean

ANOVA

Analysis of variance

Declaration of ethics:

All procedures were performed in agreement with National Institute of Health (NIH) guidelines for the care and use of laboratory animals and the protocol provided by the Research Ethics Committees of Laboratory Animals-Tehran University of Medical Sciences (IR.TUMS.AEC.1400.048, 28 February 2022).

Consent for publication:

Not applicable

Availability of data and material:

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Conflict of interests:

The authors declare that they have no competing interests.

Funding:

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Authors’ contribution:

Azadeh Tarafdari, Behnam Behboudi, and Alireza Kazemeini participated in the design and methodology. Hasti Tashak Golroudbari, Nadia Rajablou, and Armaghan Banikarimiplayed major roles in data acquisition and scientific writing. Asieh Heirani-Tabasi and Hojjatollah Nazari supplied extracellular vesicles and chemical reagents (including Trizol, Ketamine, and Xylazine) and revised the written manuscript. Parisa Arabmohammadiand Zahra Ebrahim Soltanianalyzed and interpreted the data. Seyed Mohsen Ahmadi Tafticonceptualized and supervised the project. All authors have read and approved the manuscript for submission to the journal.

Acknowledgement:

The authors warmly thank Ahmadreza Pourrashidi for contributing to designing the graphical abstract.

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α-smooth muscle actin that contributes to granulation formation and enhanced wound closure.

No competing interests reported.

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Graphical Abstract This study demonstrates that multiple-time administration of MSC-derived EV therapy accelerates wound healing by alleviating inflammation and scar formation and enhancing the wound closure rate and angiogenesis.

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Multiple Mesenchymal Stem Cell Derived Extracellular Vesicle Administration Accelerates Healing and Ameliorates Tissue Scarring Compared with Single Application in Full Thickness Cutaneous Wounds

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Abstract

Background

Methods

Results

Conclusion

Figures

1. Introduction

2. Materials and Methods

2.1. Isolation of human umbilical cord mesenchymal stem cells and extracellular vesicles.

2.2. Extracellular Vesicle characterization

2.3. Animals and Ethics

2.4. Excisional wound model and treatment

2.5. Macroscopic evaluation of the healing rate:

2.6. Histopathological analysis

2.7. Total RNA extraction and Quantitative RT-PCR

2.8. Statistics

3. Results

3.1. Extracellular vesicle characterization

3.2. Multiple local administrations of EVs enhance cutaneous wound closure in rats

3.3. Repeated sub-cutaneous UC-MSC derived extracellular vesicles enhanced collagen deposition and re-epithelialization

3.4. Repeated UC-MSC EV treatment improves angiogenesis and revascularization

3.5. Multiple local administrations of UC-MSC extracellular vesicles ameliorate inflammation

4. Discussion

5. Conclusion

Abbreviations

Declarations

References

Footnotes

Additional Declarations

Supplementary Files

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