Using extracellular vesicles derived from human umbilical cord mesenchymal stem cells for a topical coating promotes oral mucositis healing in rats

Introduction

Oral mucositis (OM) is one of the most common complications in cancer therapy (1). Existing literature shows that OM-related treatment interruptions seriously affect the quality of life and therapeutic outcome of cancer patients (2,3). Although the multinational association of supportive care in cancer and international society of oral oncology (MASCC/ISOO) has updated the guidelines of prevention and treatment of OM induced by cancer therapy, the control methods of OM have not made great progress in the past 10 years (4,5). Therefore, more effective drugs and methods for treating OM are urgently required for clinical application.

The pathogenesis of OM secondary to anticancer therapy is very complex. The fundamental pathobiology of OM accepted currently is divided into five overlapping stages (6,7). Inflammatory reaction is an important pathobiology of OM development, especially nuclear factor kappa (NF-κB) and pro-inflammatory cytokines (8). The activation of NF-κB can up-regulate many pro-inflammatory cytokines, including interleukin-6 (IL-6), IL-1β, tumor necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2) (3,9). These pro-inflammatory cytokines also regulate NF-κB by positive feedback resulting in further damage and inflammation in oral mucosa (10,11).

Extracellular vesicles (EVs) are lipid bilayers released naturally by cells and are unable to replicate themselves (12). They are enriched with proteins, micro RNA (miRNA), messenger RNA (mRNA), non-coding RNA (ncRNA), DNA, cytokines, and chemokines (13). According to the size and biogenesis, EVs can be divided into three subtypes (12): exosomes (Exos), which are 50–150 nm and produced by exocytosis; microvesicles (MVs), which are 100–1,000 nm in diameter and produced through outward budding from the plasma membrane; and apoptotic bodies, which are 500–5,000 nm in diameter and are produced by blebbing of the plasma membrane of the apoptotic cells. EVs can transmit information derived from their mother cells to the target cells and play important roles in immune-regulation, tissue repair, and angiogenesis (14). Recently, many studies have confirmed that EVs administered by tail vein injection or local subcutaneous injection can promote cutaneous wound healing in rodents by inhibiting the pro-inflammatory cytokines (15) and angiogenesis (16), and polarizing the macrophages from M1 (promoting inflammatory response) to M2 (inhibiting inflammatory response) (17).

Therefore, we hypothesized that EVs derived from human umbilical cord mesenchymal stem cells (hUC-MSC-EVs) have a positive role in the promotion of OM repair. To validate our hypothesis, we compared the healing rate of OM coated topically with hUC-MSC-EVs or without hUC-MSC-EVs and investigated the possible mechanisms. Our results showed for the first time that coating the OM with hUC-MSC-EVs topically can promote the healing of OM. We present the following article in accordance with the ARRIVE reporting checklist (available at https://atm.amegroups.com/article/view/10.21037/atm-22-767/rc).

Methods

Cell culture and medium collection

HUC-MSCs were provided by the Institute of Radiology, Academy of Military Medical Sciences. HUC-MSCs were cultured with serum free medium (Clin-Biotechnology Co., Ltd, China) for MSCs in a 37 ℃, 5% CO₂ incubator. The frequency of passage was about 1 week. The conditioned medium was collected every 3–4 days and stored at −80 ℃.

Isolation and identification of hUC-MSC-EVs

The conditioned medium of hUC-MSCs was centrifuged at 2,000 ×g for 30 minutes at 4 ℃ to remove cell debris, and then the supernatants of the medium were ultracentrifuged (Optima XPN-100, Beckman Coulter, USA) at 100,000 ×g for 2 hours at 4 ℃. Discarding the supernatants, the sediment was re-suspended in phosphate buffer saline (PBS) and ultracentrifuged at 100,000 g for 2 hours again. The concentrated hUC-MSC-EVs were re-suspended in PBS (18).

Electron microscopy analysis morphology: HUC-MSC-EVs were negatively stained with 1% uranyl acetate, and morphologies were observed using transmission electron microscopy (TEM, HT7800, Hitachi, Japan).

BCA protein assay analysis concentration: The concentration of hUC-MSC-EVs was determined using the BCA Protein Assay Kit (CWBIO, China) according to the manufacturer’s instructions.

Bead-based flow cytometric analysis surface markers: HUC-MSC-EVs were coupled with aldehyde/sulfate latex beads (4 µm, Invitrogen, USA) according to the previous protocol (19). The bead-coupled EVs were re-suspended in MES buffer and incubated respectively with anti-FITC-CD9 (eBioscience, USA), anti-PECY7-CD63 (eBioscience, USA), and anti-PE-CD81 (eBioscience, USA). After being washed 2 times in MES buffer, the bead-coupled EVs were resuspended in 250 µL of PBS by a flow cytometry (FACSCalibur, BD Biosciences, USA). The fluorescence was analyzed with FlowJo7.6 software.

Animal model of OM and coating topically with hUC-MSC-EVs

Wistar rats (male, 6–8 weeks, SPF Beijing Biotechnology Co., Ltd) were anesthetized with 10% chloral hydrate through intraperitoneal injection (300 mg/kg). All rats were treated with a filter paper (5 mm × 5 mm) soaked glacial acetic acid on the inner mucosa of the lower lip for 30 seconds (20) (Figure 1A). Their body weights were measured on alternative days. The glacial acetic acid induced OM model (oral ulcer) was visually evaluated 24 hours after acetic acid treatment. Experiments were performed under a project license (No. IACUC-4th Hos Hebmu-2020009) granted by the Laboratory Animal Ethical Committee Fourth Hospital of Hebei Medical University, in compliance with Chinese guidelines for the care and use of animals.

Figure 1 The position of rat’s OM induced by glacial acetic acid (A) and the position of rats when treated with rhaFGF or hUC-MSCs-EVs (B). OM, oral mucositis; rhaFGF, recombinant human acidic fibroblast growth factor; hUC-MSCs-EVs, extracellular vesicles derived from human umbilical cord mesenchymal stem cells.

The dose of hUC-MSC-EVs was based on previous literatures (16,21,22) and the results of our pre-experiment. Rats of OM were randomized into five groups (n=5 for each group). The groups and their treatments were as follows. Blank control group: without treatment. Positive control group: coated topically with lyophilized recombinant human acidic fibroblast growth factor for external use (rhaFGF, 25,000 U, 100 U/cm², Shanghai Tenry Pharmaceutical Co., Ltd). Experimental groups: coated topically with 0.25, 0.75, or 1.5 µg/µL hUC-MSC-EVs, respectively.

After anesthesia, rats were kept in prone position with their head down (Figure 1B). This position ensured that the surface of the oral ulcer was completely covered by rhaFGF or hUC-MSC-EVs. Twenty µL of rhaFGF or hUC-MSC-EVs was used to cover the oral ulcer for 5 minutes, and then this operation was repeated once. Rats with OM in the positive control group and experimental groups were treated with rhaFGF or hUC-MSC-EVs once a day. The rats were euthanized by an intraperitoneal injection of 10% chloral hydrate (900 mg/kg) on days 5 and 8 after modeling. All the experiments were repeated three times.

Macroscopic and histologic evaluation

The degree of OM was assessed according to the macroscopic scoring standard (23,24) (Table 1). Oral mucosal tissues were fixed in 10% neutral formalin and paraffin-embedded. The samples were cut at 4 µm thick. Then, they were stained using hematoxylin and eosin (HE). The degree of OM healing was assessed according to the histologic scoring standard by light microscopy (Olympus, Japan) (25) (Table 2).

Immunohistochemistry for NF-κB, IL-6, TNF-α and IL-1β

The immunohistochemistry technique was used for the levels of NF-κB, IL-6, TNF-α, and IL-1β expression according to the methodology described previously (23). Paraffin sections were deparaffinized and underwent antigen repairing. Then, sections were incubated with the primary antibody (1:50) at room temperature for 1-2 hours. Subsequently, sections were incubated with the secondary antibody. Then, they were visualized by incubating them with 3, 3'-diaminobenzidine etrahydrochloride (DAB) (Servicebio, China). The primary antibodies were the following: IL-1β polyclonal antibody (Bioworld Technology, co. Ltd., China; Cat.no. BS6067; rabbit), anti-IL-6 antibody (Boster, China; Cat. no. BA4339; rabbit IgG), TNF-α antibody (Affinity Biosciences, China; Cat. no. AF7014; rabbit polyclonal), and anti-NF-κBp65 antibody (Boster, China; Cat. no. BA0610; rabbit IgG).

The sections were evaluated with a high-power objective lens (25×) using optical microscopy (E100 LED, Nikon Corporation, Japan). The immunostaining intensity of pro-inflammation was evaluated by two examiners under double-blinded conditions. Possible scores were as follows. Score 1: 0% of positive cells. Score 2: <10% of positive cells or isolated cells. Score 3: 11–50% of positive cells. Score 4: >50% of positive cells (25).

Statistical analysis

SPSS 20.0 (SPSS Inc., USA) statistical software was used for statistical analysis. Mean and standard deviation were used in the description of quantitative data with a normal distribution. Median was used in the description of non-normally distributed data. Normally distributed data were compared using analysis of variance (ANOVA) among multiple groups, followed by the least significant difference (LSD) test. Kruskal-Wallis test was applied to compare non-normally distributed data. P<0.05 indicated a statistically significant difference. GraphPad Prism 9.0 (GraphPad Software, USA) was used for plotting.

Results

Identification of hUC-MSCs

HUC-MSCs was identified by morphology and its surface antigen markers (26). HUC-MSCs under an inverted microscope showed relatively uniform spindle shape and growth parallel or vortex adherent. The expression of surface antigen markers in each generation of hUC-MSCs was detected by flow cytometry. More than 95% of hUC-MSCs population expresses CD73, CD90 and CD105. Less than 2% of hUC-MSCs population expresses CD34, CD45 and CD14.

Characteristics of hUC-MSC-EVs

HUC-MSC-EVs extracted by ultracentrifugation were observed using TEM after negative staining with 1% uranyl acetate. The EVs had typical characteristics as shown by TEM (×30.0 K), which was a heterogeneous spherical membrane structure with a diameter distribution of 50–150 nm (Figure 2A). The expression level of surface markers CD9, CD63, and CD81 of hUC-MSC-EVs was identified by flow cytometry (Figure 2B). The results indicated that hUC-MSC-EVs expressed the specific surface markers CD9, CD63, and CD81, but that CD63 was expressed at a lower level.

Figure 2 Characterization of hUC-MSC-EVs. hUC-MSC-EVs were isolated by differential centrifugation. (A) The main morphological characteristics of hUC-MSC-EVs by transmission electron microscopy. Surveyor’s rod =1 µm/500 nm. (B) The surface markers of hUC-MSC-EVs are detected using flow cytometry. hUC-MSC-EVs, extracellular vesicles derived from human umbilical cord mesenchymal stem cells.

HUC-MSC-EVs promote OM healing

The median healing time of OM in the blank control, rhaFGF, 25 µg/µL EVs, 0.75 µg/µL EVs, and 1.50 µg/µL EVs groups was 14, 11, 10, 7, and 11 days, respectively. A statistically significant difference was observed among these 5 groups starting from day 5 (*P<0.05). The 0.75 µg/µL EVs group had a significant difference compared with other 4 groups starting from day 7 (*P<0.05). These results showed that the most significant effect in promoting healing was at the concentration of 0.75 µg/µL (Figure 3A,3B).

Figure 3 Macroscopic images representatively of rat oral mucosa with OM induced by glacial acetic acid and the healing degree of OM after treated with rhaFGF or hUC-MSC-EVs (n=5). (A) Blank control group: rats with OM without treatment. rhaFGF group: rats with OM treated with rhaFGF once a day. 0.75 µg/µL EVs group: rats with OM treated with 0.75 µg/µL hUC-MSC-EVs once a day. (B) Macroscopic scores of OM in the blank control, rhaFGF, 0.25 µg/µL EVs, 0.75 µg/µL EVs and 1.5 µg/µL EVs groups. A statistically significant difference was observed among these 5 groups, starting from day 5 (*P<0.05). The 0.75 µg/µL EVs group had a significant difference comparing with other 4 groups, starting from day 7 (*P<0.05 n=5). OM, oral mucositis; rhaFGF, recombinant human acidic fibroblast growth factor; hUC-MSCs-EVs, extracellular vesicles derived from human umbilical cord mesenchymal stem cells.

After HE staining, the sections were evaluated using optical microscopy and divided into 4 grades (Table 2). Histopathological scores in the 0.75 µg/µL EVs group were lower vs. the blank control group on day 5 and 8. On day 5, the blank control group had large number of inflammatory cells infiltrations, interstitial edema, and red blood cell extravasation in oral mucosa, and the healing grade as assessed by light microscope was 4. On day 8, there were fewer inflammatory cells infiltrations, interstitial edema, and red blood cell extravasation in the mucosa, and the grade was 3. For the 0.75 µg/µL EVs group, on day 5, a small number of inflammatory cells infiltrated in the oral mucosa of rats, without obvious interstitial edema and erythrocyte extravasation, and the healing grade was 2. On day 8, stratified epithelium cells appeared, covering the whole ulcer surface. There was no obvious inflammatory cell infiltration in the deep part of the ulcer, and no small blood vessels were congested and dilated. The healing grade was 1 (Figure 4A,4B).

Figure 4 Histopathological images representatively of rat oral mucosa with OM induced by glacial acetic acid and the healing degree of OM after treated with 0.75 µg/µL hUC-MSC-EVs. (A) Histopathological images representatively of OM on day 5 and 8 (stained using hematoxylin and eosin, 20×). Blank control group: On day 5, a large number of inflammatory cells infiltration, interstitial edema and red blood cell extravasation (Grade 4). On day 8, a less number of inflammatory cells infiltration, interstitial edema, and red blood cell extravasation (Grade 3). 0.75 µg/µL EVs group: On day 5, a small amount of inflammatory cells infiltrated in the oral mucosa of rats, without obvious interstitial edema and erythrocyte extravasation (Grade 2). On day 8, stratified epithelium cells appeared. There was no obvious inflammatory cell infiltration and no small blood vessels were obviously congested and dilated (Grade 1). (B) Histopathological scores from rats with OM in the blank control group and 0.75 µg/µL EVs group. *P<0.05, **P<0.01. (C) The body weights of rats in the blank control group and 0.75 µg/µL EVs group. The body weight started to recover since day 8 in the blank control group and day 4 in 0.75 µg/µL EVs group (blank control group *P<0.003; EVs group *P<0.026) (n=5). OM, oral mucositis; hUC-MSCs-EVs, extracellular vesicles derived from human umbilical cord mesenchymal stem cells.

HUC-MSC-EVs relieve dietary pain

According to the pre-experiment, the healing speed of the 0.75 µg/µL EVs group was the fastest compared with other groups. We recorded the body weight of rats in the 0.75 µg/µL EVs group and the blank control group. Their body weights all decreased on day 2. The body weight started to recover from day 8 in the blank control group and day 4 in the 0.75 µg/µL EVs group (the means ± the standard deviation for the blank control group: *P<0.003; and for the 0.75 µg/µL EVs group: *P<0.026) (Figure 4C). The results suggested that hUC-MSCs-EVs can promote the healing of OM and relieve dietary pain.

HUC-MSC-EVs down-regulated the expression levels of NF-κB, IL-6 and TNF-α

The 0.75 µg/µL EVs group showed lower immunostaining intensity of NF-κB than the blank control group on the day 5 and 8 after modeling, and immunostaining scores of the 0.75 µg/µL EVs group were 2 and 1 on day 5 and 8, respectively (P<0.05; Figure 5). To further verify the underlying mechanism of promoting OM healing, we investigated the levels of IL-6, TNF-α, and IL-1β expression, which are regulated through the NF-κB signaling pathway. The 0.75 µg/µL EVs group showed lower immunostaining intensity of IL-6 and TNF-α compared with the blank control group on days 5 and 8 (P<0.05). However, there was no significant difference between the blank control group and the 0.75 µg/µL EVs group for the intensity of IL-1β. The immunostaining intensity of IL-1β in these 2 groups gradually diminished from day 5 (Figure 6).

Figure 5 Immunohischemistry representative photomicrographs and scores for NF-κB in the blank control group and 0.75 µg/µL EVs group (25×). On day 5 and 8, the staining intensity of NF-κB in the 0.75 µg/µL EVs group were weaker comparing with blank control group (*P<0.05 n=5). EVs, extracellular vesicles.

Figure 6 Immunohischemistry representative photomicrographs and scores for IL-6, TNF-α and IL-1β in the blank control group and 0.75 µg/µL EVs group (25×). On day 5 and 8, the staining intensity of IL-6 and TNF-α in the 0.75 µg/µL EVs group were weaker comparing with blank control group (*P<0.05 n=5). There is no statistical difference between blank control group and 0.75 µg/µL EVs group on day 5 and 8 in IL-1β. EVs, extracellular vesicles.

Discussion

Up to now, this is the first report of an experiment that topically coated the OM of rats once a day with hUC-MSC-EVs. Our results confirmed that coating the OM with hUC-MSC-EVs topically can promote healing of OM in rats.

We measured the protein concentration of hUC-MSC-EVs using the BCA method and selected three concentrations (0.25, 0.75, and 1.5 µg/µL) as experimental groups according to the pre-experimental results. From day 7 of treatment, the healing degree of OM in the 0.75 µg/µL EVs group was better than other 4 groups. This suggested that hUC-MSC-EVs did not have a dose-dependent relationship with the efficacy. However, Yang et al. (27) used EVs derived from bone marrow mesenchymal stem cells (BMSC-EVs) to protect against colitis in a rat model. A total of 50, 100, and 200 µg of EVs were injected by tail vein in separate groups. Their results showed that the 200 µg EVs group had the best efficacy compared with the other 2 groups. The study concluded that BMSC-EVs can accelerate the healing of skin wounds in a dose-dependent manner. However, the mother cell sources, animal models, and administration modes of EVs differed, therefore the relationship between the concentration and the efficacy of hUC-MSC-EVs still requires further studied.

OM is a common complication of radiotherapy or chemotherapy (28). Its pathological process can be divided into five overlapping stages: initial injury, information formation, signal amplification, ulcer formation, and healing (9). In studies of the potential mechanisms for OM induced by radiotherapy or chemotherapy, the NF-κB signal transduction pathway and pro-inflammatory cytokines, such as TNF-α, IL-6 and IL-1β, have been reported to contribute to the pathogenesis of OM (9,29-31). Therefore, inhibition of NF-κB and its downstream pro-inflammatory cytokines has become the main biological target for treating OM. Yang et al. (27) found that BMSC-EVs could treat an inflammatory bowel disease model in rats by down-regulating the expression levels of pro-inflammatory factors TNF-α and IL-1β and inhibiting the NF-κB signal transduction pathway. Wu et al. (32) used BMSC-EVs enriched with miR-146a to down-regulate NF-κB and significantly inhibit the expression levels of IL-6 and IL-1β in rat colitis. Ti et al. (33) found that MSC-Exos can promote the transformation of macrophages into M2 type (anti-inflammatory). Considering the similarity in the mechanisms of OM induced by cancer therapy and trauma (34,35), we used a rat model of glacial acetic acid-induced OM to study the efficacy of hUC-MSC-EVs in the treatment of OM that was secondary to cancer therapy. Our results are consistent with the previous studies above. In our study, the immunohistochemistry results showed that the expression level of NF-κBp65 in OM induced by glacial acetic acid was down-regulated significantly after it was topically coated with hUC-MSC-EVs. NF-κB plays important roles in mediating the transcription of pro-inflammatory genes (11), therefore we further examined the expression levels of TNF-α, IL-1β, and IL-6. We also observed that the levels of TNF-α and IL-6 expression were markedly decreased in the mucosal tissue after hUC-MSC-EVs treatment. These results demonstrated that hUC-MSC-EVs can promote healing of OM induced by glacial acetic acid, presumably by inhibiting the NF-κB signaling pathways. Therefore, we speculate that hUC-MSC-EVs can promote healing of OM related to radio-chemotherapy by inhibiting the NF-κB signaling pathways.

IL-1β is an important inflammatory mediator and is produced by M1 macrophages (36). Our results showed that the expression level of IL-1β decreased gradually from day 5 both in the blank control group and the 0.75 µg/µL EVs group. However, the two groups had no significant difference. We speculate that a topical coating of hUC-MSC-EVs may not inhibit IL-1β expression, or the inhibition may occur earlier than day 5 of treatment.

HUC-MSC-EVs are enriched with various biological active substances (13) and act at the same time through multiple mechanisms on anti-inflammatory, re-epithelization and angiogenesis (16,17,37). In the therapeutic process by hUC-MSC-EVs, a variety of molecular and cellular events are closely coordinated, and many kinds of cells are highly coordinated and interact to repair damaged tissues (38). Therefore, hUC-MSC-EVs have unique advantages to treat OM induced by cancer therapy. However, there are still several challenges need to be resolved, including yield improvement, standardized production, transportation and storage (39). Further clinical trials of MSC-EVs are needed to demonstrate their role in promoting wound healing (40).

Conclusions

Our study demonstrated for the first time that coating hUC-MSC-EVs topically (once a day) can promote the healing of OM in rats. The possible mechanism involves suppressing the activation of the NF-κB signaling pathway. Therefore, hUC-MSC-EVs may become a potential approach in clinical applications for treating OM induced by radiotherapy or chemotherapy.

Acknowledgments

The authors would like to thank Hongyu Liu from the first hospital of Hebei Medical University, for his help with animal experiments, and Dezhi Kong and Wei Zhang from Hebei Medical University, for the device support.

Funding: The study was supported by the Beijing Natural Science Foundation (No. 7202185).

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://atm.amegroups.com/article/view/10.21037/atm-22-767/rc

Data Sharing Statement: Available at https://atm.amegroups.com/article/view/10.21037/atm-22-767/dss

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-22-767/coif). The authors have no conflicts of interest to declare.

Ethical Statement:The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Experiments were performed under a project license (No. IACUC-4th Hos Hebmu-2020009) granted by the Laboratory Animal Ethical Committee Fourth Hospital of Hebei Medical University, in compliance with Chinese guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: C. Mullens)