# Interleukin-10-Modified Adipose-Derived Mesenchymal Stem Cells Prevent Hypertrophic Scar Formation via Regulating the Biological Characteristics of Fibroblasts and Inflammation

Authors: Fang Xie; Li Teng; Jianjian Lu; Jiajie Xu; Chao Zhang; Liya Yang; Xiaoyang Ma; Minghao Zhao
Journal: Mediators of Inflammation
 (2022)

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## Abstract

Hypertrophic scar causes serious functional and cosmetic problem, but no treatment method is known to achieve a satisfactory therapeutic effect. However, mesenchymal stem cells show a possible cure prospect. Here, we investigated the effect of interleukin-10-modified adipose-derived mesenchymal stem cells (IL-10-ADMSC) on the formation of hypertrophic scar. In vitro, IL-10-ADMSC could highly express IL-10 and exhibited stronger inhibition of hypertrophic scar fibroblasts (HSFs) proliferation, migration, and extracellular matrix synthesis (the expression of collagen I, collagen III, FN, andα-SMA protein) than ADMSC. In vivo, we found that IL-10-ADMSC speeded up wound healing time and reduced scar area and scar outstanding height. Same as in vitro, IL-10-ADMSC also exhibited stronger inhibition of extracellular matrix synthesis (the expression of collagen I, collagen III protein) in wound than ADMSC. In addition, we also found that IL-10-ADMSC is also a stronger inhibitory effect on inflammation in wound than ADMSC, and IL-10-ADMSC inhibited TGF-β/Smads and NF-κB pathway. In conclusion, IL-10-ADMSC demonstrated the ability to prevent hypertrophic scar formation. And its possible molecular mechanism might be related to IL-10-ADMSC inhibiting the proliferation and migration of the synthesis of extracellular matrix of HSFs, and IL-10-ADMSC inhibited the inflammation during the wound healing.


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## Body

## 1. Introduction

Scars are the appearance and histopathological changes of normal skin tissue that appear after wound healing. Scars is an inevitable product after wound healing, but it will cause various complications after growing beyond a certain limit, such as itching, pain, ulceration, and even serious dysfunction or disfigurement [1, 2]. Hypertrophic scars, a kind of scar with red and protrusion in the original position of wound healing, are characterized by excessive fibrosis and extra cellular matrix (ECM) deposition [3, 4]. Although the exact mechanism of hypertrophic scar formation has not yet been elucidated, the abnormal migration of fibroblasts, excessive accumulation of extracellular matrix, and excessive inflammation are thought to be related to the formation of hypertrophic scars [5, 6], especially excessive inflammation [7, 8]. Therefore, these known mechanisms that were related to the formation of hypertrophic scars are the current targets for the development of prevention and treatment of hypertrophic scars.Recently, mesenchymal stem cells have entered the public eye due to their potential to treat and prevent hypertrophic scar formation. The results of many clinical trials and preclinical trials have shown that many mesenchymal stem cells had the ability to promote scarless healing and inhibit tissue fibrosis in skin wounds, such as bone marrow mesenchymal stem cells (BMSCs) [9], umbilical cord mesenchymal stem cells [10], chorionic lining mesenchymal stem cells [11], and adipose-derived mesenchymal stem cells (ADMSC) [12]. In the present study, our research topic is ADMSC, not only because it is an adult stem cell with self-renewal ability and multidirectional differentiation potential but also because ADMSC has a wide range of sources and simple preparation [13, 14]. Importantly, genetically modified ADMSC has been found to be used for the treatment of some diseases, such as the repair of bone defects [15], anterior cruciate ligament reconstruction [16], and improved erectile dysfunction in diabetic rats [17]. These studies showed that genetically modified ADMSC was feasible for disease treatment.Interleukin 10 (IL-10) is a multicell-derived, multifunctional cytokine that regulates cell growth and differentiation, participates in inflammatory and immune responses, and is a recognized inflammation and immunosuppressive factor [18, 19]. And it has been reported that inflammation and immune responses play important roles in the healing of scar wounds, and inhibition of excessive inflammation during wound healing may help prevent the formation of hypertrophic scars [20, 21]. Moreover, IL-10-modified bone marrow mesenchymal stem cells could attenuate the severity of acute graft-versus-host disease after experimental allogeneic stem cell transplantation [22], and IL-10-modified human amniotic mesenchymal stem cells promoted wound healing by exerting multiple synergistic effects [23]. However, there is no study which reports the effect of IL-10-modified ADMSCs on preventing hypertrophic scarring. In this study, we create a new treatment method to prevent hypertrophic scar formation, namely, local injection of IL-10-modified ADMSC (IL-10-ADMSC) in the wound. And our results suggested that IL-10-ADMSC prevented hypertrophic scar formation, and its preventive effect is better than ADMSC.


## 2. Material and Methods

### 2.1. Isolation, Culture, and Identification of ADMSC

All animal experiments are in accordance with the Animal Ethics Committee of Plastic Surgery Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (Ethics approval number 2021-23) and meet the NIH guidelines for the care and use of laboratory animals [24]. The New Zealand white female rabbit (12 weeks old, 2800-3000 g) was anesthetized by intramuscular administration of 5% pelltobarbitalum natricum (25 mg/kg) and Sumianxin (0.1 mL/kg), followed by euthanasia by ear vein air embolization. We firstly separated the adipose tissue of the rabbit’s groin under aseptic conditions, cleaned it, and cut it into a paste, added 0.1% type I collagen (17018029, Invitrogen, USA) and incubated for 45 minutes at 37°C, and then, collected cells by centrifugation. At last, cells were cultured in DMEM medium (21885108, Gibco, USA) at 5% CO2 and 37°C. We used flow cytometry and immunofluorescence to detect CD11b, CD34, CD29, and CD90 to assess the purity of ADMSC.


### 2.2. Establishment of IL-10-Modified ADMSC

We inserted the synthetic rabbit IL-10 gene (C_13684.1, NCBI, USA) into the pDc316 plasmid (VT1805, Youbao Biotec, China) and then transferred the recombinant plasmid into HEK293 cells to prepare the virus. Finally, the prepared virus was used for infection ADMSC cells to prepare IL-10-modified ADMSC, namely, IL-10-ADMSC.


### 2.3. Cell Proliferation Assay

In an independent cell culture system, we seeded3.0×104 cells into 24-well culture dishes. In the coculture system of ADMSC/IL-10-ADMSC and HSFs, we seeded 3.0×104 ADMSC or IL-10-ADMSC in the upper chamber of the Transwell chamber (140620, Thermo Fisher, USA) and seeded 3.0×104 HSFs in the lower chamber of the Transwell chamber. After the cells were cultured for different times, we removed the cell culture medium and added 100 μL/well cell culture medium and 10 μL/well CCK-8 regent. After 1 hour, we determined the OD450 value of each well and calculated the relative cell viability as stated in the manufacturing instructions.


### 2.4. Cell Migration Assay

HSFs were cultured alone, or added 1μg/L IL-10 in the medium, or cocultured with ADMSC/IL-10-ADMSC for 3 days. And then, a Transwell chamber (140652, Thermo Fisher, USA) was used to assess the ability of migration in different HSFs. In brief, 3.0×103 HSFs inoculated into the upper chamber with the culture medium. A medium containing 20% FBS (Gibco, Sacramento, USA) was added into lower chamber for 24 h at 37°C. At last, we removed the medium and washed cells for 3 times with PBS. Next, we added methanol to fix cells and dried after fixed for 30 minutes. After being stained with crystal violet for 20 minutes, we counted the migration cells.


### 2.5. Real-Time Quantitative PCR

Real-time quantitative PCR (RT-qPCR) was performed to assess the influence of IL-10-ADMSC on gene expression in wound tissues [25]. In brief, we used a total RNA extraction kit (DP431, Tian Gen, China) to extract total RNA from tissues and prepared cDNA using a reverse transcription kit (A3500, Promega, USA). As stated in manufacturing instructions of a GoTaq qPCR mix kit (A6006A, Promega, USA), we prepared 200 μL RT-qPCR reaction system and detected the gene expression using a RT-qPCR instrument (CFX384, BIO-RAD, USA). The relative expression of gene was calculated by 2−ΔΔCt method. β-Actin was loaded as control for mRNA, and U6 was loaded as control for miRNA. Primers was showed as follow: MCP-1-F:5′-GCATCAACCTGACCCCTCAA-3′, MCP-1-F:5′-ATCACACACGCATCTGAGCA-3′; MIP-1β-F:5′-TGCTCGCTTCTCCGAACAAT-3′, MIP-1β-R:5′-CCCTTCCATGCGGTTAGGTT-3′; IL-1β-F:5′-CGCATGTTCCTGGGGAGATT-3′, IL-1β-R:5′-ATCTTTTGGGGTCCGTCAACT-3′; IL-6-F:5′-CCTGAACCTTCCAAAGATGGC-3′, IL-6-R:5′-TTCACCAGGCAAGTCTCCTCA-3′.


### 2.6. Western Blot

The total protein is extracted from tissues and cells using a Tissue Protein Extraction Reagent (78510, Thermo Fisher, USA) and RIPA lysis buffer (89900, Thermo Fisher, USA), respectively. And a BCA kit (23227, Thermo Fisher, USA) is used to determine the protein concentration. Next, 40μg total protein is analyzed using a 10% SDS-PAGE. After transfer, PVDF membranes (LC2002, Thermo Fisher, USA) is first blocked with 5% skimmed milk powder and then is probed with primary antibodies against collagen-I (ab34710), collagen-III (ab184993), FN (ab2413,), α-SMA(ab5694), TGF-β1 (ab215715), Smad2 (ab40855), p-Smad2 (ab188334), Smad7 (ab216428), p-IκBα (ab133462), IκBα (ab32518), p-p65 (ab86299), and p65 (ab16502) overnight at 4°C. The PVDF membrane was washed 3 times with PBS-Tween 20 buffer solution, and the secondary antibody was added to incubate for 1 hour at room temperature. Protein bands were visualized with ECL solution, followed by densitometry analysis using Image J 3.0 (IBM, USA), and β-actin was loading as control. And all antibodies used for Western blot detection were purchased from Abcam.


### 2.7. Immunofluorescence

For cell immunofluorescence, 1 X 105 ADMSC cells were seeded into Lab-Tek chambered (Thermo Scientific, USA) to culture for 24 hours at 37°C with 5% CO2. Next day, we removed the cell culture medium and fixed cell with 4% paraformaldehyde for 10 minutes at room temperature. And then, cells were blocked with 5% BSA for one hour at room temperature. For tissue immunofluorescence, we first prepared frozen tissue sections: the tissue was first incubated in a 4% paraformaldehyde solution for 6-8 hours, and then, the tissue was transferred to a 20% sucrose solution until the tissue sinks to the bottom, and we prepared frozen tissue sections of 8-10 microns. After fixing and blocking, cells were incubated with a primary antibody against CD11b, CD34, CD29, and CD90 overnight at 4°C, and then, cells were incubated with a secondary antibody. Similarly, tissue sections were incubated with a primary antibody against CD45 (13917, Cell Signaling Technology) and CD3 (85061, Cell Signaling Technology) overnight at 4°C, and then, cells were incubated with a secondary antibody: goat anti-rabbit IgG H&L (Alexa Fluor® 594) (ab150080, ABCAM). Next, cells/tissue sections should counterstained the nucleus with 5μg/mL DAPI for 5 minutes at room temperature. At last, all samples were analyzed by a Leica TCS SP5 microscope (Leica microsystem) with the LAS AF Lite 4.0 image browser software.


### 2.8. Rabbit Ear Hypertrophic Scar Model and Treatment

According to the previous description [25], we prepared a rabbit ear hypertrophic scar model using the New Zealand white rabbit. In brief, after being anesthetized, we used a 4 mm dermal biopsy punch (15110-40, PELCO, USA) to build 4 wounds on the ventral side of each ear (we defined this time as day 0). The wounds were randomly divided into 3 groups, 5 rabbits in each group: control group, ADMSC group, and IL-10-ADMSC group. After the wound is established, 100 μL of phosphate buffer saline (PBS) containing nothing or 0.5×106 ADMSC or 0.5×106 IL-10-ADMSC were intradermally injected around each wound in control group, ADMSC group, or IL-10-ADMSC group, respectively.


### 2.9. Immunochemistry

We first prepared frozen tissue sections: the tissue was first incubated in a 4% paraformaldehyde solution for 6-8 hours, and then, the tissue was transferred to a 20% sucrose solution until the tissue sinks to the bottom. At last, we prepared frozen tissue sections of 8-10 microns. Next, we blocked the tissue sections with 5% BSA for 1 hour at room temperature, and then, tissue sections were incubated with the primary antibody against collagen-I (ab34710, Abcam, UK) and collagen-III (ab184993, Abcam, UK) overnight at 4°C. After the incubation of primary antibody, tissue sections were incubated with HRP-labeled secondary antibody for 1 hour at room temperature. At last, all samples were analyzed by a Leica TCS SP5 microscope (Leica microsystem) with the LAS AF Lite 4.0 image browser software.


### 2.10. Statistical Analysis

GraphPad Prism 8 (GraphPad Software, USA) was used to analyze the data and drawn figures in this study. The Kolmogorov-Smirnov test was used to check whether quantitative data conformed to a normal distribution, data that conformed to a normal distribution were presented as (mean±standarddeviation), and the different between two groups was analyzed by Student’s t test, and one-way ANOVA was used to compare the difference between multiples groups. P value less than 0.05 indicated significantly different.


## 2.1. Isolation, Culture, and Identification of ADMSC

All animal experiments are in accordance with the Animal Ethics Committee of Plastic Surgery Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (Ethics approval number 2021-23) and meet the NIH guidelines for the care and use of laboratory animals [24]. The New Zealand white female rabbit (12 weeks old, 2800-3000 g) was anesthetized by intramuscular administration of 5% pelltobarbitalum natricum (25 mg/kg) and Sumianxin (0.1 mL/kg), followed by euthanasia by ear vein air embolization. We firstly separated the adipose tissue of the rabbit’s groin under aseptic conditions, cleaned it, and cut it into a paste, added 0.1% type I collagen (17018029, Invitrogen, USA) and incubated for 45 minutes at 37°C, and then, collected cells by centrifugation. At last, cells were cultured in DMEM medium (21885108, Gibco, USA) at 5% CO2 and 37°C. We used flow cytometry and immunofluorescence to detect CD11b, CD34, CD29, and CD90 to assess the purity of ADMSC.


## 2.2. Establishment of IL-10-Modified ADMSC

We inserted the synthetic rabbit IL-10 gene (C_13684.1, NCBI, USA) into the pDc316 plasmid (VT1805, Youbao Biotec, China) and then transferred the recombinant plasmid into HEK293 cells to prepare the virus. Finally, the prepared virus was used for infection ADMSC cells to prepare IL-10-modified ADMSC, namely, IL-10-ADMSC.


## 2.3. Cell Proliferation Assay

In an independent cell culture system, we seeded3.0×104 cells into 24-well culture dishes. In the coculture system of ADMSC/IL-10-ADMSC and HSFs, we seeded 3.0×104 ADMSC or IL-10-ADMSC in the upper chamber of the Transwell chamber (140620, Thermo Fisher, USA) and seeded 3.0×104 HSFs in the lower chamber of the Transwell chamber. After the cells were cultured for different times, we removed the cell culture medium and added 100 μL/well cell culture medium and 10 μL/well CCK-8 regent. After 1 hour, we determined the OD450 value of each well and calculated the relative cell viability as stated in the manufacturing instructions.


## 2.4. Cell Migration Assay

HSFs were cultured alone, or added 1μg/L IL-10 in the medium, or cocultured with ADMSC/IL-10-ADMSC for 3 days. And then, a Transwell chamber (140652, Thermo Fisher, USA) was used to assess the ability of migration in different HSFs. In brief, 3.0×103 HSFs inoculated into the upper chamber with the culture medium. A medium containing 20% FBS (Gibco, Sacramento, USA) was added into lower chamber for 24 h at 37°C. At last, we removed the medium and washed cells for 3 times with PBS. Next, we added methanol to fix cells and dried after fixed for 30 minutes. After being stained with crystal violet for 20 minutes, we counted the migration cells.


## 2.5. Real-Time Quantitative PCR

Real-time quantitative PCR (RT-qPCR) was performed to assess the influence of IL-10-ADMSC on gene expression in wound tissues [25]. In brief, we used a total RNA extraction kit (DP431, Tian Gen, China) to extract total RNA from tissues and prepared cDNA using a reverse transcription kit (A3500, Promega, USA). As stated in manufacturing instructions of a GoTaq qPCR mix kit (A6006A, Promega, USA), we prepared 200 μL RT-qPCR reaction system and detected the gene expression using a RT-qPCR instrument (CFX384, BIO-RAD, USA). The relative expression of gene was calculated by 2−ΔΔCt method. β-Actin was loaded as control for mRNA, and U6 was loaded as control for miRNA. Primers was showed as follow: MCP-1-F:5′-GCATCAACCTGACCCCTCAA-3′, MCP-1-F:5′-ATCACACACGCATCTGAGCA-3′; MIP-1β-F:5′-TGCTCGCTTCTCCGAACAAT-3′, MIP-1β-R:5′-CCCTTCCATGCGGTTAGGTT-3′; IL-1β-F:5′-CGCATGTTCCTGGGGAGATT-3′, IL-1β-R:5′-ATCTTTTGGGGTCCGTCAACT-3′; IL-6-F:5′-CCTGAACCTTCCAAAGATGGC-3′, IL-6-R:5′-TTCACCAGGCAAGTCTCCTCA-3′.


## 2.6. Western Blot

The total protein is extracted from tissues and cells using a Tissue Protein Extraction Reagent (78510, Thermo Fisher, USA) and RIPA lysis buffer (89900, Thermo Fisher, USA), respectively. And a BCA kit (23227, Thermo Fisher, USA) is used to determine the protein concentration. Next, 40μg total protein is analyzed using a 10% SDS-PAGE. After transfer, PVDF membranes (LC2002, Thermo Fisher, USA) is first blocked with 5% skimmed milk powder and then is probed with primary antibodies against collagen-I (ab34710), collagen-III (ab184993), FN (ab2413,), α-SMA(ab5694), TGF-β1 (ab215715), Smad2 (ab40855), p-Smad2 (ab188334), Smad7 (ab216428), p-IκBα (ab133462), IκBα (ab32518), p-p65 (ab86299), and p65 (ab16502) overnight at 4°C. The PVDF membrane was washed 3 times with PBS-Tween 20 buffer solution, and the secondary antibody was added to incubate for 1 hour at room temperature. Protein bands were visualized with ECL solution, followed by densitometry analysis using Image J 3.0 (IBM, USA), and β-actin was loading as control. And all antibodies used for Western blot detection were purchased from Abcam.


## 2.7. Immunofluorescence

For cell immunofluorescence, 1 X 105 ADMSC cells were seeded into Lab-Tek chambered (Thermo Scientific, USA) to culture for 24 hours at 37°C with 5% CO2. Next day, we removed the cell culture medium and fixed cell with 4% paraformaldehyde for 10 minutes at room temperature. And then, cells were blocked with 5% BSA for one hour at room temperature. For tissue immunofluorescence, we first prepared frozen tissue sections: the tissue was first incubated in a 4% paraformaldehyde solution for 6-8 hours, and then, the tissue was transferred to a 20% sucrose solution until the tissue sinks to the bottom, and we prepared frozen tissue sections of 8-10 microns. After fixing and blocking, cells were incubated with a primary antibody against CD11b, CD34, CD29, and CD90 overnight at 4°C, and then, cells were incubated with a secondary antibody. Similarly, tissue sections were incubated with a primary antibody against CD45 (13917, Cell Signaling Technology) and CD3 (85061, Cell Signaling Technology) overnight at 4°C, and then, cells were incubated with a secondary antibody: goat anti-rabbit IgG H&L (Alexa Fluor® 594) (ab150080, ABCAM). Next, cells/tissue sections should counterstained the nucleus with 5μg/mL DAPI for 5 minutes at room temperature. At last, all samples were analyzed by a Leica TCS SP5 microscope (Leica microsystem) with the LAS AF Lite 4.0 image browser software.


## 2.8. Rabbit Ear Hypertrophic Scar Model and Treatment

According to the previous description [25], we prepared a rabbit ear hypertrophic scar model using the New Zealand white rabbit. In brief, after being anesthetized, we used a 4 mm dermal biopsy punch (15110-40, PELCO, USA) to build 4 wounds on the ventral side of each ear (we defined this time as day 0). The wounds were randomly divided into 3 groups, 5 rabbits in each group: control group, ADMSC group, and IL-10-ADMSC group. After the wound is established, 100 μL of phosphate buffer saline (PBS) containing nothing or 0.5×106 ADMSC or 0.5×106 IL-10-ADMSC were intradermally injected around each wound in control group, ADMSC group, or IL-10-ADMSC group, respectively.


## 2.9. Immunochemistry

We first prepared frozen tissue sections: the tissue was first incubated in a 4% paraformaldehyde solution for 6-8 hours, and then, the tissue was transferred to a 20% sucrose solution until the tissue sinks to the bottom. At last, we prepared frozen tissue sections of 8-10 microns. Next, we blocked the tissue sections with 5% BSA for 1 hour at room temperature, and then, tissue sections were incubated with the primary antibody against collagen-I (ab34710, Abcam, UK) and collagen-III (ab184993, Abcam, UK) overnight at 4°C. After the incubation of primary antibody, tissue sections were incubated with HRP-labeled secondary antibody for 1 hour at room temperature. At last, all samples were analyzed by a Leica TCS SP5 microscope (Leica microsystem) with the LAS AF Lite 4.0 image browser software.


## 2.10. Statistical Analysis

GraphPad Prism 8 (GraphPad Software, USA) was used to analyze the data and drawn figures in this study. The Kolmogorov-Smirnov test was used to check whether quantitative data conformed to a normal distribution, data that conformed to a normal distribution were presented as (mean±standarddeviation), and the different between two groups was analyzed by Student’s t test, and one-way ANOVA was used to compare the difference between multiples groups. P value less than 0.05 indicated significantly different.


## 3. Results

### 3.1. IL-10-Modified ADMSC Inhibited the Proliferation of HSFs In Vitro

We isolated and cultured rabbit ADMSC and observed under a microscope: ADMSC mainly showed a long spindle shape (Figure1(a)). Then, we use the CCK8 kit to determine the third (P3) and tenth (P10) generation growth curves (Figure 1(b)). Next, we used flow cytometry to detect cell surface markers to evaluate the purity of ADMSC, such as CD11b, CD34, CD29, and CD90, and found that (Figures 1(c) and 1(d)) CD11b and CD34 were negative (<1%) and CD29 and CD90 were positive (>98%). At the same time, we also detected the cell surface marker using cellular immunofluorescence (Figure 1(e)): CD11b and CD34 were also mostly negative, and CD29 and CD90 were also mostly positive.Figure 1
Identification of rabbit adipose-derived mesenchymal stem cells (ADMSC). (a) Representative images of the tenth (right, scale bar: 25μm) generation rabbit ADMSC under an optical microscope; (b) draw the growth curve of ADMSC by CCK8 assay kit. P3: third generation ADMSC; P10: tenth generation ADMSC; (c) flow cytometry was used to analyze the cell surface markers of ADMSC and the statistical comparison of the data; (d) the representative flow cytometry picture; (e) cellular immunofluorescence was used to analyze the cell surface markers of ADMSC. Scale bar: 50 μm; data was expressed as (SD±mean), and each analysis was repeated at least 3 times independently.
(a)(b)(c)(d)(e)To investigate the effect of IL-10 modification on ADMSC, we first tested the effect of IL-10 modification on the expression of IL-10 in ADMSC and found that the expression of IL-10 mRNA in IL-10-modified ADMSC (IL-10-ADMSC) was significantly higher than that in wild type ADMSC (Figure2(a)). At the same time, we also found that the concentration of IL-10 protein in the cell culture medium of IL-10-ADMSC was significantly higher than that in the cell culture medium of ADMSC (Figure 2(b)). Additionally, we compared the proliferation ability of ADMSC and IL-10-ADMSC and found that the proliferation of IL-10-ADMSC was reduced, and it decreased significantly from the 6th day which was compared with ADMSC (Figure 2(c)). Importantly, we use a Transwell chamber to establish a coculture system of ADMSC/IL-10-ADMSC and hypertrophic scar fibroblasts (HSFs) to assess the effect of IL-10 modification on the proliferation of HSFs. Interestingly, we found that ADMSC could significantly decrease the proliferation of HSFs, and the proliferation of HSFs was lowest in a coculture system of IL-10-ADMSC and HSFs (Figure 2(d)), which suggested IL-10-modified ADMSC inhibited the proliferation of HSFs in vitro.Figure 2
IL-10-modified ADMSC highly expresses IL-10 and inhibits the proliferation of HSFs. (a) 72 hours after lentivirus infection, we detected the expression of IL-10 mRNA in ADMSC; (b) 72 hours after lentivirus infection, we detected the expression of IL-10 protein in the medium of ADMSC; (c) IL-10 modification reduces the proliferation of ADMSC using CCK8 assay kit; (d) IL-10-modified ADMSC (IL-10-ADMSC) reduced the proliferation of hypertrophic scar fibroblasts (HSFs) in a cocultivation system of ADMSC and HSFs. Data was expressed as (SD±mean), and each analysis was repeated at least 3 times independently; P value was calculated by Student’s t test or one-way ANOVA; ∗ was P<0.05, ∗∗ was P<0.01, and ∗∗∗ was P<0.001 vs. ADMSC group; ### was P<0.001 vs. control group.
(a)(b)(c)(d)


### 3.2. IL-10-Modified ADMSC Inhibited the Migration of HSFs In Vitro

To investigate the effect of IL-10 modification on the migration of HSFs in a coculture system of ADMSC/IL-10-ADMSC and HSFs, as shown in Figure3, the migration of HSFs in a coculture system of ADMSC and HSFs was significantly lower than the migration of HSFs in alone culture system. Similarly, when we added 1 μg/L IL-10 recombinant protein to the HSFs alone culture system, the migration of HSFs was significantly inhibited, which showed that IL-10 inhibited the migration of HSFs in vitro. In the above finding, we found that the concentration of IL-10 protein in the cell culture medium of IL-10-ADMSC was significantly higher than that in the cell culture medium of ADMSC, so we assumed that IL-10-ADMSC would also inhibit the migration of HSFs, and its effect might be better than ADMSC and IL-10. Fortunately, the results of the cell migration test showed that the migration of HSFs was lowest in a coculture system of IL-10-ADMSC and HSFs.Figure 3
IL-10-modified ADMSC inhibited the migration of HSFs. HSFs were cultured alone, or added 1μg/L IL-10 in the medium, or cocultured with ADMSC/IL-10-ADMSC for 3 days, and then, Transwell test was used to evaluate the migration ability of HSFs in a cocultivation system of ADMSC and HSFs, and representative migration maps were displayed (a) and statistically compared the number of migrated cells in each group (b). Scale bar: 25 μm. Data was expressed as (SD±mean), and each analysis was repeated at least 3 times independently; P value was calculated by one-way ANOVA; ∗∗∗ was P<0.001 vs. ADMSC group; ### was P<0.001 vs. control group.
(a)(b)


### 3.3. IL-10-Modified ADMSC Inhibited the Synthesis of Extracellular Matrix in HSFs

The excessive synthesis and secretion of extracellular matrix during skin wound healing is one of the main causes of scar formation [26]. Therefore, the inhibition of synthesis and secretion of extracellular matrix could help to inhibit the scar formation. To investigate the effect of IL-10 modification on the synthesis of extracellular matrix in HSFs in a coculture system of ADMSC/IL-10-ADMSC and HSFs. We cultured HSFs alone, or added 1 μg/L IL-10 in the medium, or cocultured with ADMSC/IL-10-ADMSC for 3 days, and then, we harvested the HSFs to detect the expression of collagen-I, collagen-III, α-SMA, and FN protein using Western blot (Figure 4(a)) and found that the expression of collagen I, collagen III, FN, and α-SMA protein in HSFs in a coculture system of ADMSC and HSFs was significantly lower than these in HSFs alone culture system. Similarly, when we added 1 μg/L IL-10 recombinant protein to the HSFs alone culture system, the expression of collagen I (Figure 4(b)), collagen III (Figure 4(c)), FN (Figure 4(d)), and α-SMA (Figure 4(e)) protein in HSFs was significantly decreased, which showed that IL-10 inhibited the expression of collagen I, collagen III, FN, and α-SMA protein in HSFs in vitro. In the above finding, we found that the concentration of IL-10 protein in the cell culture medium of IL-10-ADMSC was significantly higher than that in the cell culture medium of ADMSC, so we assumed that IL-10-ADMSC would also decrease the expression of collagen I, collagen III, FN, and α-SMA protein in HSFs, and its effect might be better than ADMSC and IL-10. Fortunately, the results of the Western blot showed that the expression of collagen I, collagen III, FN, and α-SMA protein in HSFs were lowest in a coculture system of IL-10-ADMSC and HSFs. In addition, we also found that the concentration of 4-hydroxyproline in the culture medium was lowest in a coculture system of IL-10-ADMSC and HSFs (Figure 4(f)).Figure 4
IL-10-modified ADMSC inhibited the synthesis of extracellular matrix in HSFs. HSFs were cultured alone, or added 1μg/L IL-10 in the medium, or cocultured with ADMSC/IL-10-ADMSC for 3 days. (a–e) We harvested the HSFs to detect the expression of collagen-I, collagen-III, α-SMA, and FN protein; (f) we collected the cell culture medium to determine the concentration of 4-hydroxyproline. Data was expressed as (SD±mean), and each analysis was repeated at least 3 times independently; P value was calculated by one-way ANOVA; ∗∗∗ was P<0.001 vs. ADMSC group; ### was P<0.001 vs. control group.
(a)(b)(c)(d)(e)(f)


### 3.4. IL-10-Modified ADMSC Prevented Hypertrophic Scar Formation in a Rabbit Ear Model

According to the results in vitro, we suspected that IL-10-ADMSC would inhibit scar formation in vivo, and its effect might be better than ADMSC. To verify this hypothesis, we first established a rabbit ear wound model and then explored suitable treatment conditions for IL-10-ADMSC. Due to the IL-10 recombinant plasmid carries the GFP label, we can observe the green fluorescence to evaluate the amount of IL-10-ADMSC in the wound. The results of fluorescence analysis showed that the green fluorescence gradually decreased with the extension of the local injection time of IL-10-ADMSC (Figure5(a)). After 3 days of local injection in the wound, the number of IL-10-ADMSC in the wound was only (33.9±5.2) % of 0.5 days (Figure 5(b)). At the same time, we also analyzed the dynamic changes of IL-10 protein in the wound within 9 days of local injection of IL-10-ADMSC using Western blot (Figure 5(c)); the results showed that the expression of IL-10 protein in wound without the injection of IL-10-ADMSC was very low; after local injection of IL-10-ADMSC in the wound, IL-10 underwent a rapidly upregulation and was characterized by the peaking stage reached at the first 1 day postinjection. Nevertheless, the production of IL-10 in wound exhibited a gradual decline until day 7 and still maintained at a high level on the 3rd and 5th day (Figure 5(d)).Figure 5
IL-10-modified ADMSC underwent extensive apoptosis and survived for a short time after injection. (a) In the wound, the locally injected IL-10-ADMSC gradually reduced which was evaluated by observing green fluorescence, and representative tissue fluorescence image was displayed; (b) the GFP-labeled IL-10-ADMSC in the wound after different time of local injection of IL-10-ADMSC were statistically compared; (c–d) after different time of local injection of IL-10-ADMSC, Western blot was used to detect the expression of IL-10 in the wound. Representative protein bands were displayed and statistically compared protein band gray value. Scale bar: 10μm. Data was expressed as (SD±mean), and each analysis was repeated at least 3 times independently; P value was calculated by Student’s t test; ns was P>0.05, ∗ was P<0.05, ∗∗ was P<0.01, and ∗∗∗ was P<0.001 vs. ADMSC group.
(a)(b)(c)(d)To sum up, we formulated IL-10-ADMSC to treat wounds as a local injection of IL-10-ADMSC every 3 days until the wound was healed. At the start of the third treatment, we observed the lowest skin pathological damage at the wound in the IL-10-ADMSC group (FigureS1). We observed the healing of wounds every day and recorded the healing time of each group of wounds, the healing time of wound in control group (without any treat) was (33.9±5.2) days, and the healing time of wound in ADMSC group (with local injection of ADMSC) was (9.7±5.2) days, while the healing time of wound in IL-10-ADMSC group (with local injection of ADMSC) was only (7.2±1.2) days (Figure 6(a)). 28th day after treatment, we analyzed the scar area and the scar outstanding height and found that both the scar area (Figure 6(b)) and the scar outstanding height (Figure 6(c)) in IL-10-ADMSC group were the smallest. Note that the scar area and the scar outstanding height in ADMSC group was also significantly smaller than that in control group, which indicated that ADMSC prevented hypertrophic scar formation in a rabbit ear model, and its inhibitory effect was better after IL-10 modification.Figure 6
IL-10-modified ADMSC prevented hypertrophic scar formation. (a) The time of wound healing time in different group in rabbit ear hypertrophic scar model. (b) After 28 days of treatment in different ways, the comparison of scar area in different group; (c) after 28 days of treatment in different ways, the scar outstanding height of hypertrophic scar were statistically compared.n=10; data was expressed as (SD±mean); P value was calculated by one-way ANOVA; ∗∗ was P<0.01 and ∗∗∗ was P<0.001 vs. ADMSC group; ### was P<0.001 vs. control group.
(a)(b)(c)


### 3.5. IL-10-Modified ADMSC Reduced the Extracellular Matrix and Inflammation in Wound

Similarly, we also investigated the effect of IL-10-ADMSC on the extracellular matrix in wound. After 7 days of treatment in different ways, we detected the expression of collagen I/III protein in wound using immunochemistry (Figure7(a)) and immunoblotting (Figures 7(b) and 7(c)), the results showed that the expression of collagen I/III protein in wound in ADMSC group and IL-10-ADMSC group were all significantly lower than that in control group. Importantly, the expression of collagen I/III protein in wound in IL-10-ADMSC group was significantly lower than that in ADMSC group, which indicated that ADMSC prevented extracellular matrix in wound of rabbit ear model, and its inhibitory effect was better after IL-10 modification.Figure 7
IL-10-modified ADMSC inhibited the expression of collagen I and III in wound. (a) After 7 days of treatment in different ways, we detected the expression of collagen I/III protein in wound using immunochemistry; (b, c) After 7 days of treatment in different ways, we detected the expression of collagen I/III protein in wound using immunoblotting.n=6; scale bar: 50 μm. Data was expressed as (SD±mean); P value was calculated by one-way ANOVA; ∗ was P<0.05 and ∗∗∗ was P<0.001 vs. ADMSC group; ### was P<0.001 vs. control group.
(a)(b)(c)Furthermore, previous studies have shown that IL-10 is a cytokine with multidirectional regulation, such as immunosuppression and inflammation suppression [27, 28]. And it has been reported that inflammation and immune responses play important roles in the healing of scar wounds, and inhibition of excessive inflammation during wound healing may help prevent the formation of hypertrophic scars [20, 21]. Therefore, we hypothesized that IL-10-ADMSC could also exert the effect of inhibiting inflammation as IL-10. To test this hypothesis, we detected the expression of inflammatory cytokines in the wound and found that the expression of MIP (Figure 8(a)), MIP-1β (Figure 8(b)), IL-1β (Figure 8(c)), and IL-6 (Figure 8(d)) in wound in ADMSC group and IL-10-ADMSC group were all significantly lower than that in control group. Importantly, the expression of MIP, MIP-1β, IL-1β, and IL-6 in wound in IL-10-ADMSC group was significantly lower than that in ADMSC group, which indicated that ADMSC prevented inflammation in wound of rabbit ear model, and its inhibitory effect was better after IL-10 modification. Moreover, immune cell infiltration, such as T cells, white blood cells, and macrophages, is the initial stage of inflammation, and we found that the CD45 or CD3 positive cells in wound in ADMSC group and IL-10-ADMSC group were all significantly lower than that in control group, and IL-10-ADMSC was the most (Figures 8(e) and 8(f)). All in all, these results indicated that IL-10-ADMSC inhibited the inflammation in wound.Figure 8
IL-10-modified ADMSC regulated wound inflammation. (a–d) After 3 days of treatment in different ways, we detected the expression of MCP-1, MIP-1β, IL-1β, and IL-6mRNA expression in wound by RT-qPCR. (e, f) After 3 days of treatment in different ways, we detected CD45 and CD3 positive cell in the wound using immunofluorescence (e, f). n=6; scale bar: 10 μm. Data was expressed as (SD±mean); P value was calculated by one-way ANOVA; ∗∗∗ was P<0.001 vs. ADMSC group; ns was P>0.05, ## was P<0.001, and ### was P<0.001 vs. control group.
(a)(b)(c)(d)(e)(f)


### 3.6. IL-10-Modified ADMSC Inhibited TGF-β/Smads and NF-κB Pathway in Wound

To explore molecular mechanisms, we detected the expression of some signaling pathways, such as TGF-β/Smads pathway which was closely associated with cell proliferation and migration, and extracellular matrix synthesis in fibroblasts [29, 30], and NF-κB pathway which was closely related to inflammation [31, 32]. And the results showed that (Figure 9) the expressions of TGF-β1, p-Smad 2/Smad 2, Smad 7, p-IκBα/IκBα, and p-p65/p65 in wound in ADMSC group and IL-10-ADMSC group were all significantly lower than that in control group. Importantly, the expression of MIP, MIP-1β, IL-1β, and IL-6 in wound in IL-10-ADMSC group was significantly lower than that in ADMSC group.Figure 9
IL-10-modified ADMSC regulated TGF-β/Smads and NF-κB pathway in wound. After 3 days of treatment in different ways, we detected the expression of key proteins in TGF-β/Smads and NF-κB pathway in wound using immunoblotting. n=6; data was expressed as (SD±mean); P value was calculated by one-way ANOVA; ∗∗∗ was P<0.001 vs. ADMSC group; ### was P<0.001 vs. control group.
(a)(b)(c)(d)


## 3.1. IL-10-Modified ADMSC Inhibited the Proliferation of HSFs In Vitro

We isolated and cultured rabbit ADMSC and observed under a microscope: ADMSC mainly showed a long spindle shape (Figure1(a)). Then, we use the CCK8 kit to determine the third (P3) and tenth (P10) generation growth curves (Figure 1(b)). Next, we used flow cytometry to detect cell surface markers to evaluate the purity of ADMSC, such as CD11b, CD34, CD29, and CD90, and found that (Figures 1(c) and 1(d)) CD11b and CD34 were negative (<1%) and CD29 and CD90 were positive (>98%). At the same time, we also detected the cell surface marker using cellular immunofluorescence (Figure 1(e)): CD11b and CD34 were also mostly negative, and CD29 and CD90 were also mostly positive.Figure 1
Identification of rabbit adipose-derived mesenchymal stem cells (ADMSC). (a) Representative images of the tenth (right, scale bar: 25μm) generation rabbit ADMSC under an optical microscope; (b) draw the growth curve of ADMSC by CCK8 assay kit. P3: third generation ADMSC; P10: tenth generation ADMSC; (c) flow cytometry was used to analyze the cell surface markers of ADMSC and the statistical comparison of the data; (d) the representative flow cytometry picture; (e) cellular immunofluorescence was used to analyze the cell surface markers of ADMSC. Scale bar: 50 μm; data was expressed as (SD±mean), and each analysis was repeated at least 3 times independently.
(a)(b)(c)(d)(e)To investigate the effect of IL-10 modification on ADMSC, we first tested the effect of IL-10 modification on the expression of IL-10 in ADMSC and found that the expression of IL-10 mRNA in IL-10-modified ADMSC (IL-10-ADMSC) was significantly higher than that in wild type ADMSC (Figure2(a)). At the same time, we also found that the concentration of IL-10 protein in the cell culture medium of IL-10-ADMSC was significantly higher than that in the cell culture medium of ADMSC (Figure 2(b)). Additionally, we compared the proliferation ability of ADMSC and IL-10-ADMSC and found that the proliferation of IL-10-ADMSC was reduced, and it decreased significantly from the 6th day which was compared with ADMSC (Figure 2(c)). Importantly, we use a Transwell chamber to establish a coculture system of ADMSC/IL-10-ADMSC and hypertrophic scar fibroblasts (HSFs) to assess the effect of IL-10 modification on the proliferation of HSFs. Interestingly, we found that ADMSC could significantly decrease the proliferation of HSFs, and the proliferation of HSFs was lowest in a coculture system of IL-10-ADMSC and HSFs (Figure 2(d)), which suggested IL-10-modified ADMSC inhibited the proliferation of HSFs in vitro.Figure 2
IL-10-modified ADMSC highly expresses IL-10 and inhibits the proliferation of HSFs. (a) 72 hours after lentivirus infection, we detected the expression of IL-10 mRNA in ADMSC; (b) 72 hours after lentivirus infection, we detected the expression of IL-10 protein in the medium of ADMSC; (c) IL-10 modification reduces the proliferation of ADMSC using CCK8 assay kit; (d) IL-10-modified ADMSC (IL-10-ADMSC) reduced the proliferation of hypertrophic scar fibroblasts (HSFs) in a cocultivation system of ADMSC and HSFs. Data was expressed as (SD±mean), and each analysis was repeated at least 3 times independently; P value was calculated by Student’s t test or one-way ANOVA; ∗ was P<0.05, ∗∗ was P<0.01, and ∗∗∗ was P<0.001 vs. ADMSC group; ### was P<0.001 vs. control group.
(a)(b)(c)(d)


## 3.2. IL-10-Modified ADMSC Inhibited the Migration of HSFs In Vitro

To investigate the effect of IL-10 modification on the migration of HSFs in a coculture system of ADMSC/IL-10-ADMSC and HSFs, as shown in Figure3, the migration of HSFs in a coculture system of ADMSC and HSFs was significantly lower than the migration of HSFs in alone culture system. Similarly, when we added 1 μg/L IL-10 recombinant protein to the HSFs alone culture system, the migration of HSFs was significantly inhibited, which showed that IL-10 inhibited the migration of HSFs in vitro. In the above finding, we found that the concentration of IL-10 protein in the cell culture medium of IL-10-ADMSC was significantly higher than that in the cell culture medium of ADMSC, so we assumed that IL-10-ADMSC would also inhibit the migration of HSFs, and its effect might be better than ADMSC and IL-10. Fortunately, the results of the cell migration test showed that the migration of HSFs was lowest in a coculture system of IL-10-ADMSC and HSFs.Figure 3
IL-10-modified ADMSC inhibited the migration of HSFs. HSFs were cultured alone, or added 1μg/L IL-10 in the medium, or cocultured with ADMSC/IL-10-ADMSC for 3 days, and then, Transwell test was used to evaluate the migration ability of HSFs in a cocultivation system of ADMSC and HSFs, and representative migration maps were displayed (a) and statistically compared the number of migrated cells in each group (b). Scale bar: 25 μm. Data was expressed as (SD±mean), and each analysis was repeated at least 3 times independently; P value was calculated by one-way ANOVA; ∗∗∗ was P<0.001 vs. ADMSC group; ### was P<0.001 vs. control group.
(a)(b)


## 3.3. IL-10-Modified ADMSC Inhibited the Synthesis of Extracellular Matrix in HSFs

The excessive synthesis and secretion of extracellular matrix during skin wound healing is one of the main causes of scar formation [26]. Therefore, the inhibition of synthesis and secretion of extracellular matrix could help to inhibit the scar formation. To investigate the effect of IL-10 modification on the synthesis of extracellular matrix in HSFs in a coculture system of ADMSC/IL-10-ADMSC and HSFs. We cultured HSFs alone, or added 1 μg/L IL-10 in the medium, or cocultured with ADMSC/IL-10-ADMSC for 3 days, and then, we harvested the HSFs to detect the expression of collagen-I, collagen-III, α-SMA, and FN protein using Western blot (Figure 4(a)) and found that the expression of collagen I, collagen III, FN, and α-SMA protein in HSFs in a coculture system of ADMSC and HSFs was significantly lower than these in HSFs alone culture system. Similarly, when we added 1 μg/L IL-10 recombinant protein to the HSFs alone culture system, the expression of collagen I (Figure 4(b)), collagen III (Figure 4(c)), FN (Figure 4(d)), and α-SMA (Figure 4(e)) protein in HSFs was significantly decreased, which showed that IL-10 inhibited the expression of collagen I, collagen III, FN, and α-SMA protein in HSFs in vitro. In the above finding, we found that the concentration of IL-10 protein in the cell culture medium of IL-10-ADMSC was significantly higher than that in the cell culture medium of ADMSC, so we assumed that IL-10-ADMSC would also decrease the expression of collagen I, collagen III, FN, and α-SMA protein in HSFs, and its effect might be better than ADMSC and IL-10. Fortunately, the results of the Western blot showed that the expression of collagen I, collagen III, FN, and α-SMA protein in HSFs were lowest in a coculture system of IL-10-ADMSC and HSFs. In addition, we also found that the concentration of 4-hydroxyproline in the culture medium was lowest in a coculture system of IL-10-ADMSC and HSFs (Figure 4(f)).Figure 4
IL-10-modified ADMSC inhibited the synthesis of extracellular matrix in HSFs. HSFs were cultured alone, or added 1μg/L IL-10 in the medium, or cocultured with ADMSC/IL-10-ADMSC for 3 days. (a–e) We harvested the HSFs to detect the expression of collagen-I, collagen-III, α-SMA, and FN protein; (f) we collected the cell culture medium to determine the concentration of 4-hydroxyproline. Data was expressed as (SD±mean), and each analysis was repeated at least 3 times independently; P value was calculated by one-way ANOVA; ∗∗∗ was P<0.001 vs. ADMSC group; ### was P<0.001 vs. control group.
(a)(b)(c)(d)(e)(f)


## 3.4. IL-10-Modified ADMSC Prevented Hypertrophic Scar Formation in a Rabbit Ear Model

According to the results in vitro, we suspected that IL-10-ADMSC would inhibit scar formation in vivo, and its effect might be better than ADMSC. To verify this hypothesis, we first established a rabbit ear wound model and then explored suitable treatment conditions for IL-10-ADMSC. Due to the IL-10 recombinant plasmid carries the GFP label, we can observe the green fluorescence to evaluate the amount of IL-10-ADMSC in the wound. The results of fluorescence analysis showed that the green fluorescence gradually decreased with the extension of the local injection time of IL-10-ADMSC (Figure5(a)). After 3 days of local injection in the wound, the number of IL-10-ADMSC in the wound was only (33.9±5.2) % of 0.5 days (Figure 5(b)). At the same time, we also analyzed the dynamic changes of IL-10 protein in the wound within 9 days of local injection of IL-10-ADMSC using Western blot (Figure 5(c)); the results showed that the expression of IL-10 protein in wound without the injection of IL-10-ADMSC was very low; after local injection of IL-10-ADMSC in the wound, IL-10 underwent a rapidly upregulation and was characterized by the peaking stage reached at the first 1 day postinjection. Nevertheless, the production of IL-10 in wound exhibited a gradual decline until day 7 and still maintained at a high level on the 3rd and 5th day (Figure 5(d)).Figure 5
IL-10-modified ADMSC underwent extensive apoptosis and survived for a short time after injection. (a) In the wound, the locally injected IL-10-ADMSC gradually reduced which was evaluated by observing green fluorescence, and representative tissue fluorescence image was displayed; (b) the GFP-labeled IL-10-ADMSC in the wound after different time of local injection of IL-10-ADMSC were statistically compared; (c–d) after different time of local injection of IL-10-ADMSC, Western blot was used to detect the expression of IL-10 in the wound. Representative protein bands were displayed and statistically compared protein band gray value. Scale bar: 10μm. Data was expressed as (SD±mean), and each analysis was repeated at least 3 times independently; P value was calculated by Student’s t test; ns was P>0.05, ∗ was P<0.05, ∗∗ was P<0.01, and ∗∗∗ was P<0.001 vs. ADMSC group.
(a)(b)(c)(d)To sum up, we formulated IL-10-ADMSC to treat wounds as a local injection of IL-10-ADMSC every 3 days until the wound was healed. At the start of the third treatment, we observed the lowest skin pathological damage at the wound in the IL-10-ADMSC group (FigureS1). We observed the healing of wounds every day and recorded the healing time of each group of wounds, the healing time of wound in control group (without any treat) was (33.9±5.2) days, and the healing time of wound in ADMSC group (with local injection of ADMSC) was (9.7±5.2) days, while the healing time of wound in IL-10-ADMSC group (with local injection of ADMSC) was only (7.2±1.2) days (Figure 6(a)). 28th day after treatment, we analyzed the scar area and the scar outstanding height and found that both the scar area (Figure 6(b)) and the scar outstanding height (Figure 6(c)) in IL-10-ADMSC group were the smallest. Note that the scar area and the scar outstanding height in ADMSC group was also significantly smaller than that in control group, which indicated that ADMSC prevented hypertrophic scar formation in a rabbit ear model, and its inhibitory effect was better after IL-10 modification.Figure 6
IL-10-modified ADMSC prevented hypertrophic scar formation. (a) The time of wound healing time in different group in rabbit ear hypertrophic scar model. (b) After 28 days of treatment in different ways, the comparison of scar area in different group; (c) after 28 days of treatment in different ways, the scar outstanding height of hypertrophic scar were statistically compared.n=10; data was expressed as (SD±mean); P value was calculated by one-way ANOVA; ∗∗ was P<0.01 and ∗∗∗ was P<0.001 vs. ADMSC group; ### was P<0.001 vs. control group.
(a)(b)(c)


## 3.5. IL-10-Modified ADMSC Reduced the Extracellular Matrix and Inflammation in Wound

Similarly, we also investigated the effect of IL-10-ADMSC on the extracellular matrix in wound. After 7 days of treatment in different ways, we detected the expression of collagen I/III protein in wound using immunochemistry (Figure7(a)) and immunoblotting (Figures 7(b) and 7(c)), the results showed that the expression of collagen I/III protein in wound in ADMSC group and IL-10-ADMSC group were all significantly lower than that in control group. Importantly, the expression of collagen I/III protein in wound in IL-10-ADMSC group was significantly lower than that in ADMSC group, which indicated that ADMSC prevented extracellular matrix in wound of rabbit ear model, and its inhibitory effect was better after IL-10 modification.Figure 7
IL-10-modified ADMSC inhibited the expression of collagen I and III in wound. (a) After 7 days of treatment in different ways, we detected the expression of collagen I/III protein in wound using immunochemistry; (b, c) After 7 days of treatment in different ways, we detected the expression of collagen I/III protein in wound using immunoblotting.n=6; scale bar: 50 μm. Data was expressed as (SD±mean); P value was calculated by one-way ANOVA; ∗ was P<0.05 and ∗∗∗ was P<0.001 vs. ADMSC group; ### was P<0.001 vs. control group.
(a)(b)(c)Furthermore, previous studies have shown that IL-10 is a cytokine with multidirectional regulation, such as immunosuppression and inflammation suppression [27, 28]. And it has been reported that inflammation and immune responses play important roles in the healing of scar wounds, and inhibition of excessive inflammation during wound healing may help prevent the formation of hypertrophic scars [20, 21]. Therefore, we hypothesized that IL-10-ADMSC could also exert the effect of inhibiting inflammation as IL-10. To test this hypothesis, we detected the expression of inflammatory cytokines in the wound and found that the expression of MIP (Figure 8(a)), MIP-1β (Figure 8(b)), IL-1β (Figure 8(c)), and IL-6 (Figure 8(d)) in wound in ADMSC group and IL-10-ADMSC group were all significantly lower than that in control group. Importantly, the expression of MIP, MIP-1β, IL-1β, and IL-6 in wound in IL-10-ADMSC group was significantly lower than that in ADMSC group, which indicated that ADMSC prevented inflammation in wound of rabbit ear model, and its inhibitory effect was better after IL-10 modification. Moreover, immune cell infiltration, such as T cells, white blood cells, and macrophages, is the initial stage of inflammation, and we found that the CD45 or CD3 positive cells in wound in ADMSC group and IL-10-ADMSC group were all significantly lower than that in control group, and IL-10-ADMSC was the most (Figures 8(e) and 8(f)). All in all, these results indicated that IL-10-ADMSC inhibited the inflammation in wound.Figure 8
IL-10-modified ADMSC regulated wound inflammation. (a–d) After 3 days of treatment in different ways, we detected the expression of MCP-1, MIP-1β, IL-1β, and IL-6mRNA expression in wound by RT-qPCR. (e, f) After 3 days of treatment in different ways, we detected CD45 and CD3 positive cell in the wound using immunofluorescence (e, f). n=6; scale bar: 10 μm. Data was expressed as (SD±mean); P value was calculated by one-way ANOVA; ∗∗∗ was P<0.001 vs. ADMSC group; ns was P>0.05, ## was P<0.001, and ### was P<0.001 vs. control group.
(a)(b)(c)(d)(e)(f)


## 3.6. IL-10-Modified ADMSC Inhibited TGF-β/Smads and NF-κB Pathway in Wound

To explore molecular mechanisms, we detected the expression of some signaling pathways, such as TGF-β/Smads pathway which was closely associated with cell proliferation and migration, and extracellular matrix synthesis in fibroblasts [29, 30], and NF-κB pathway which was closely related to inflammation [31, 32]. And the results showed that (Figure 9) the expressions of TGF-β1, p-Smad 2/Smad 2, Smad 7, p-IκBα/IκBα, and p-p65/p65 in wound in ADMSC group and IL-10-ADMSC group were all significantly lower than that in control group. Importantly, the expression of MIP, MIP-1β, IL-1β, and IL-6 in wound in IL-10-ADMSC group was significantly lower than that in ADMSC group.Figure 9
IL-10-modified ADMSC regulated TGF-β/Smads and NF-κB pathway in wound. After 3 days of treatment in different ways, we detected the expression of key proteins in TGF-β/Smads and NF-κB pathway in wound using immunoblotting. n=6; data was expressed as (SD±mean); P value was calculated by one-way ANOVA; ∗∗∗ was P<0.001 vs. ADMSC group; ### was P<0.001 vs. control group.
(a)(b)(c)(d)


## 4. Discussion

Hypertrophic scar is a pathological tissue structure that is higher than the surrounding skin, red in color, and hard in texture, after the integrity of the external skin of the human body is destroyed, and accompanied with uncomfortable symptoms such as contractures, itching, and pain, not only affects the appearance but also causes great pain to the patient [3, 4]. Excessive proliferation of fibroblasts on the wound surface and excessive deposition of extracellular matrix are the main causes of hypertrophic scar formation and are also the main characteristics of hypertrophic scars [5, 6]. In this study, we found that ADMSC could inhibit the proliferation, migration, and synthesis of extracellular matrix of HSFs in vitro and inhibit the synthesis of extracellular matrix in wound in vivo. Importantly, the inhibitory effect of IL-10-ADMSC is better than ADMSC. By comparison, we found that IL-10-ADMSC could produce and secrete more IL-10 than ADMSC; this might be the reason why IL-10-ADMSC has better inhibitory effect than ADMSC.IL-10 was first found to be synthesized and secreted by murine CD4+ Th2 cells and had the function of inhibiting the synthesis of IFN-γ by Th1-T cells [33]. As the study progressed, IL-10 has been identified as a candidate scar improving therapy based on preclinical studies, due to the fibrosis and inflammation regulation function of IL-10 is thought to be related to scar formation [34]. IL-10 was found to regulate the biological characteristics of human skin fibroblasts, such as IL-10 suppressed proliferation and remodeling of extracellular matrix of cultured human skin fibroblasts [35, 36]. And its molecular mechanism might be related to IL-10 regulating the expression of transforming growth factor-β and monocyte chemoattractant protein-1 [37], and IL-10 also regulated the conduction of the downstream signaling pathway (STAT3-AKT-mTOR pathway) through its interaction with its receptor, thereby regulating the biological characteristics of hypertrophic scar fibroblasts [38]. In addition, CD206, a molecule was closely related to human dermal fiber cell proliferation, migration, apoptosis, cell cycle, and extracellular matrix synthesis, was found to be a downstream gene of IL-10 and be regulated by IL-10 [39]. Taking together, these previous studies indicated IL-10 was potential drug to prevent hypertrophic scar formation by regulating the proliferation and migration of fibroblasts and the synthesis of extracellular matrix. For our research, IL-10-ADMSC could also play the regulatory role of IL-10 on fibroblasts by synthesizing and secreting IL-10.Inflammation is very important for scar formation. After the integrity of tissue is destroyed, the body will undergo a series of complex pathophysiological processes to promote the restoration of tissue integrity. This process mainly includes the inflammatory reaction period, the granulation hyperplasia period, and the matrix remodeling period, and various types of cells, cytokines, and extracellular matrix components are involved in regulating various stages of wound repair [40]. Hypertrophic scar formation is closely related to excessive wound repair and is the result of the combined effects of enhanced local inflammation, abnormal cytokine secretion, abnormal fibroblast proliferation and apoptosis, and excessive deposition of extracellular matrix [41], and inflammation plays a prime role [42]. In the process of scar formation, inflammatory factors first promote the activation of immune cells, induce immune cells to migrate to the wound, and synthesize and secrete cytokines, and cytokines further aggravate the inflammatory response at the wound. In this way, the interaction between inflammation and immune response promotes the development of scars formation [42]. Therefore, inhibiting the excessive inflammation at the wound can help inhibit the formation of scars.In this study, we found that IL-10-ADMSC speeded up wound healing time and reduced scar area and scar outstanding height. Further, we also found that IL-10-ADMSC/ADMSC decreased the expression of proinflammatory factors and the infiltration of inflammatory cells in wound, and IL-10-ADMSC has better therapeutic effect. This indicated IL-10-ADMSC prevented the formation of hypertrophic scar and might be related to IL-10-ADMSC which played a combined role of ADMSC and IL-10. The inhibition of inflammation by ADMSC has been widely confirmed [43, 44]. Similarly, IL-10 has also been found to have inflammation suppression and immunosuppressive effects [33]. IL-10 has the ability to prevent the proliferation of antigen-specific T cells, inhibit the ability of the antigen-presenting cells to present, and inhibit the synthesis and expression of inflammatory cytokines and inflammatory mediators [33]. In addition, Kieran et al. [45] found IL-10 could promote wound healing and reduce scar formation by inhibiting inflammatory reaction by comparing the skin soft tissue defect of IL-10 knockout mice and wild type mice. And King et al. [46] found that IL-10 could prevent scar formation by inhibiting the synthesis of IL-6, IL-8, and TGF-β. The inhibition of inflammation is also the main reason why IL-10 is used to prevent scar formation, but its short half-life and poor targeting have led to the poor effectiveness of IL-10 in preventing and treating scar formation [45, 46]. According to our experimental results in this study, local injection of IL-10-ADMSC could maintain high levels of IL-10 for 3-5 days in wound, which solved the problem of short half-life of IL-10 in vivo, and the inhibitory effect of IL-10-ADMSC on wound inflammation and the prevention of hypertrophic scar formation have also proved that this is feasible.To explore molecular mechanisms, we detected the expression of some signaling pathways, such as TGF-β/Smads pathway which was closely associated with cell proliferation and migration, and to extracellular matrix synthesis in fibroblasts [29, 30], and NF-κB pathway which was closely related to inflammation [31, 32]. Fortunately, we found that IL-10-ADMSC inhibited TGF-β/Smads and NF-κB pathway in wound.


## 5. Conclusion

All in all, our data showed that IL-10-ADMSC demonstrated the ability to prevent hypertrophic scar formation via inhibiting the proliferation and migration of the synthesis of extracellular matrix of HSFs and by inhibiting the inflammation during the wound healing. However, due to the limitation of experimental conditions, more pathological detection of fibrosis has not been carried out. Nonetheless, our study also suggests that IL-10-modified ADMSCs have the potential to prevent scarring.



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*Source: 6368311-2022-06-21.xml*