INTRODUCTION

Scarring of the skin following burns and traumatic surgery forms a major load on healthcare. The children suffer a lot from long-term functional and psychological problems as a result of scars [14]. Stem cell therapy evolved in the last years to exosomes and microvesicles (MVs) treatment paradigm which exhibited emerging promising modalities [15]. Exosomes or MVs are small secretory organelles with single membranes and enormous amounts of proteins, lipids, nucleic acids, and carbohydrate conjugates. They are capable of extracellular matrix remodelling and signalling as well as molecules delivering to other cells with preferable usage over stem cells due to stability, easy storage, and dosage control, non-rejection by the immune system [1, 2]. Extracellular vesicles are the principal mediators of stem cells’ paracrine effects. They are plentiful with large numbers of miRNAs and act as transferral agents of miRNAs to recipient cells, where they can modulate the gene expression of recipient cells [22]. Platelet-rich plasma (PRP) has been widely used in post-burn disfiguring aesthetics with proven efficacy and tolerability. PRP is regarded as “tissue sealants” owing to their favourable effects on wound healing mediated by high levels of granular growth factors ready to be secreted [8]. Targeting the transforming growth factor beta (TGF-β) pathway is a still promising goal for wound scarring modulation in animals, although this matter is debated in human studies debated. In contrast, connective tissue growth factor (CTGF) pathway blocking agents for scar formation produced highly encouraging results in phase II clinical trials in human studies [14]. Fetal wounds possess the beverage of being scarless owing to large amounts of hyaluronic acid and an elevated ratio of collagen type III to type I [19]. The role of miRNA203 in the process of wound repair had been investigated. It was demonstrated that after 3 and 5 days from skin wound induction the GTPase Ran (a pro-proliferative factor) and Raph1 (a pro-migratory factor) are putative direct miR-203 targets whose expression could be essential wound for skin homeostasis and re-epithelialisation [18]. The present study investigated the possible role of MVs derived from mesenchymal stem cells and PRP in murine burn wound healing regarding inflammatory markers (interleukin [IL]-6, IL-10, tumour necrosis factor alpha [TNF-α]), scar regulating parameters (matrix metalloproteinases 3 [MMP-3], TGF-β, CTGF, hyaluronic acid, type I and III collagen), antioxidant marker (Nrf2), and involvement of miRNA203.

MATERIALS AND METHODS

Animals

This study was approved by CU-IACUC under the number CU-III-F-31-21 following all ethics of animal research studies. Forty adult Wistar rats weighing 170–220 g were housed in the animal house of the faculty of medicine, Cairo University, in isolated cages under standard temperature and light, fed ad libitum. Four groups of rats (n = 10) were utilized in this study; control, burn group, burn + PRP, burn + MVs.

Burn induction

After the removal of rats’ skin hair by epilating cream, the skin of the dorsum of rats was sterilised with alcohol, and the tip of a tuning fork was applied to the skin for 20 s after being put in boiling water for 15 s [9]. Anaesthesia with ketamine (Ketamax, India) and xylazine (Xylaject, Adwia, Egypt) was done to rats just before burn induction and on the 7th and 14th day to obtain proper images of the skin burn wounds. Ceftriaxone (100 mg/kg IM, Rocephin, Roche) and buprenorphine were injected into rats to obtain antibiosis and analgesia, respectively for the 1st 5 days [17]. Burn size was assessed by ImageJ software from the captured images on the 1st, 7th, 14th, and 21st days.

PRP preparation

Blood of the PRP animal group (III) was aspirated from a retro-orbital vein and underwent double centrifugation, the 1st at 160 G for 20 min, the 2nd at 400 G for 15 min [7] using Beckman centrifuge (USA). 0.5 mL of PRP was obtained and injected locally into each rat of this group.

Microvesicles preparation

Mesenchymal stem cells (MSC) were obtained from bones of rats similar to that described by Zaki et al. 2018 [23]. Cultured cells were counted and their viability was confirmed by trypan blue, whereas their proliferation was verified using the MTT (3-[4,5-dimethylthiazol-2-yl]-2.5-diphenyl tetrazolium bromide) cell proliferation kit (Thermo Fisher Scientific, USA) as manufacturer’s protocol. A flow cytometer (FACS Caliber, BD Bioscience USA) was used to delineate BM-MSCs cells which exhibited a negative reaction for CD34 (haematopoietic) and a strong positive reaction for CD90 and CD105 (MSC specific markers) (Fig. 1). MVs were obtained from supernatants of MSCs cultured overnight in RPMI deprived of fetal calf serum. The supernatants were centrifuged at 10,000 g for 20 min to remove their debris, then centrifuged at 100,000 g (Beckman Coulter Optima L-90K Ultracentrifuge) for 1 h at 4°C, washed in serum-free medium 199 containing N-2-hydroxyethyl piperazine-N’-2-ethane sulfonic acid (HEPES) 25 mM (Sigma) and subjected to repeated ultracentrifugation in the same conditions [4]. Obtained MVs were fixed with 2.5% glutaraldehyde for 2 h for demonstration by transmission electron microscopy (TEM) then ultra-centrifuged and suspended in 100 μL HSA. A 20 μL volume of MVs was loaded onto a formvar/carbon-coated grid, stained with 3% aqueous phosphotungstic acid for 1 min, and examined under TEM (Joel Jem 1400, Germany) [6]. TEM displayed their spheroid morphology and confirmed their size (Fig. 1). After preparation, they were injected locally into the burn site at a dosage of 800 μg (RNA concentration) MVs suspended in 1 mL phosphate buffered saline [12].

Figure 1. The flow cytometry of bone marrow derived-mesenchymal stem cells (MSCs) is showing negative (–ve) reaction to CD34 (A), whereas positive (+ve) reaction to CD90 (B) and CD105 (C); D. Electron microscope picture of MSCs-derived microvesicles (arrowheads).

Biochemical tests

Measurement of hyaluronic acid IL-10 and IL-6. Skin samples from different groups were subjected to measurement of hyaluronic acid IL-10 and IL-6 by ELISA technique with kits supplied from MyBioSource (USA).

Real-time reverse transcription polymerase chain reaction (PCR) for alpha smooth muscle actin (αSMA) and miRNA203. Extracting total RNA from tissue homogenates was performed by the RNeasy purification reagent (Qiagen). cDNA was generated by using high capacity cDNA Reverse Transcription Kit (Fermentas). Quantitative PCR amplification was done using SYBR Green I. The endogen for αSMA RNA was beta-actin, the forward primer was 5’-GACGTACAACTGGTATTGTG-3’ and reverse 5’-TCAGGATCTTCATGAGGTAG-3’. For miRNA203 detection, RNA extraction was performed according to the manufacturer’s protocol by acidic phenol/chloroform extraction (peqGOLD RNAPure; PEQLAB Biotechnologie), then RNA was subjected to DNase treatment (Ambion), reverse transcribed. The endogen for miRNA203 was SnU6RNA and the forward primmer for miRNA203 was 5’-CGGTAGTCTGATACTGTAA-3’ and the reverse primer was 5’-GTGCTCCGAAGGGGGT-3’. The αSMA RNA and miRNA203 expressions were analysed with the ABI Prism® 7500 detection system (Applied Biosystems) according to the 2-ΔΔCt method.

Western blot analysis. Skin tissue homogenates protein extracts were prepared via the BioRad system. A Bradford assay followed by loading on a polyacrylamide gel. Electrophoresed proteins on SDS-PAGE were transferred to a Hybond™ nylon membrane (GE Healthcare) and β-actin was applied as a housekeeping protein, then incubated in an antibody solution containing the anti-MMP-3 antibody (Abcam, USA). Detects a band of approximately 50 kDa and anti-β-actin antibody (Abcam, USA) separately, followed by incubation in HRP-conjugated 2ry antibody solution. Data analysis by Totallab analysis software (Ver.1.0.1) using a Gel documentation system (Geldoc-it, UVP, England). The same was done for the detection of TGF-β (Santa Cruz, USA) with the results were expressed using the image analysis software to read the band intensity by protein normalisation by β-actin on the ChemiDoc MP imager.

Histological and immunohistochemical examination [16]

Skin sections were prepared and stained with haematoxylin and eosin (H&E) as well as Masson’s trichrome. Deparaffinised sections underwent immunohistochemical study by antigen retrieval, H202 blocking, incubation with the following 1ry antibodies; collagen I and III (Abcam, USA, rabbit, polyclonal, dilution 1/100), TNF-α (Santa Cruz, USA, rabbit, polyclonal, dilution, 1/100), CTGF and Nrf2 (Invitrogen, USA, rabbit, polyclonal, dilution 1/100). The slides were then incubated for 30 min at room temperature with anti-rabbit IgG secondary antibodies (Envision + system HRP; Dako) to be visualised with diaminobenzidine commercial kits and finally counterstained with May’s haematoxylin. The slides were imaged with a Leica DFC camera attached to the microscope except that of TNF-α were imaged with a Leica ICC 50 microscope.

Histomorphometric measurements

The intensity of trichrome stain and that of immune stains (CTGF, TNF-α, collagen I and III, and Nrf2) were measured using ImageJ software (NIH, USA) and sent to statistical analysis.

Statistical analysis

All gathered data (biochemical, Western blotting (WB), PCR, and intensity of different stains) were analysed using GraphPad version 8, two-way ANOVA was used to analyse burn wound size followed by Tukey’s test. One-way ANOVA was performed on all other tests followed by a post-hoc Tukey’s test.

RESULTS

Microvesicles morphologically accelerate burn wound healing

Microvesicles significantly decreased burn wound size after 2 weeks from burn induction compared with burn and burn + PRP groups. After 3 weeks, the MVs group showed a significant decline in burn wound size as compared with the burn group (II) (Fig. 2).

Figure 2. A. Burn images at 1st day, 1st, 2nd, and 3rd weeks from burn induction showing accelerated burn wound healing, particularly in microvesicles (MVs) treated rats. B. The graph shows a significant decrease in burn wound size in platelet-rich plasma (PRP) and MVs groups compared to the burn group after 2 weeks from burn induction, whereas only a significant decrease in MVs compared to the burn group after 3 weeks; number of animals = 10 in each group; *p < 0.05, ***p < 0.0001.

Microvesicles and PRP modulate ELISA levels of IL-10, IL-6, and hyaluronic acid in burn wounds

Burn had significantly elevated IL-6 and hyaluronic acid, whereas decreased IL-10 in skin tissue compared with the control. MVs and PRP significantly alleviated levels of IL-6 and hyaluronic acid, whereas MVs significantly upregulated IL-10 in burn tissue compared with the burn group (II) with significantly favourable results in the MVs group as compared with PRP (Fig. 3).

Figure 3. Bar charts of ELISA levels of hyaluronic acid, interleukin 6 (Il-6) and interleukin 10 (IL-10) (A), polymerase chain reaction (PCR) levels of alpha smooth muscle actin (αSMA) and miRNA203 (B), and intensity of different histological and immune stains (Trichrome, CTGF, TNF-α, collagen I and III, and Nrf2) (C); n = 10 for each group; *when p < 0.05, **when p < 0.01, ***when p < 0.001, ****when p < 0.0001 (error bar = standard error of the mean).

Microvesicles and PRP attenuate PCR levels of α-SMA and miRNA203 burn wound

Microvesicles and PRP significantly attenuate PCR levels of α-SMA and miRNA203 in burn tissue as compared with the burn group (II) with significantly better results in the MVs group as compared with PRP (Fig. 3).

Microvesicles and PRP mitigate WB levels of TGF-β and MMP-3 in burn wounds

Microvesicles and PRP significantly attenuate WB levels of TGF-β and MMP-3 in burn tissue as compared with the burn group (II) (Fig. 4).

Figure 4. A. Western blotting (WB) analysis of matrix metalloproteinases 3 (MMP-3) and transforming growth factor beta (TGF-β) in skin tissues among different groups with β-actin as a housekeeping protein. Both proteins are overexpressed in the burn group, whereas exhibited a decreased expression in platelet-rich plasma (PRP) and microvesicles (MVs) treated groups. B. The graph shows the WB results among different groups; n = 10 for each group; *when p < 0.05; **when p < 0.01; ***when p < 0.001; ****when p < 0.0001 (error bar = standard error of the mean).

Histological results

Via H&E staining (Fig. 5), the skin of the control animals showed normal epidermal and dermal layers, whereas the skin of burned animals displayed complete epithelial necrosis with the formation of scab, with still signs of inflammation and haemorrhage, and without any signs of epithelisation. The skin of PRP-treated animals showed marked epithelisation and granulation tissues formation with still collagenous matrix, still the presence of blood capillaries, and marked proliferation of collagen fibrous tissues. The skin of MVs treated animals exhibited complete epithelial layer formation, decreased number of inflammatory cells, and marked noticeable remodelling of collagen fibrous tissues. Via Masson’s Trichrome staining (Fig. 6), the skin of the control animals exhibited normal collagen bundles within the dermal layer, whereas the skin of burned animals showed a marked reduction of fibrous connective tissues. The skin of PRP-treated animals showed marked proliferation of collagen fibrous tissues, whereas the skin of burned animals treated with MVs displayed marked remodelling and maturation of collagen fibrous tissues. Histomorphometric data revealed a dramatic reduction in the intensity of Trichrome stains in the burn group (II), whereas a significant elevation was recorded in both treated groups particularly MVs treated one (Fig. 3).

Figure 5. A, A*. Skin of control animal showing normal epidermal and dermal layers (arrowheads indicate normal stratified squamous epithelium with keratin covering); B, B*. Skin of burned animal showing complete epithelial necrosis with formation of scab (black arrowhead), with still signs of inflammation and haemorrhage (white arrowhead), and without any signs of epithelization; C, C*. Platelet-rich plasma treated group shows marked epithelisation (arrow) and granulation tissues formation with still collagenous matrix (white arrowhead), still the presence of blood capillaries (black arrowhead), and marked proliferation of collagen fibrous tissues (white arrowhead); D, D*. With microvesicles treated group shows complete epithelial layer formation (arrow), decreased number of inflammatory cells (black arrowhead), and marked noticeable remodelling of collagen fibrous tissues (white arrowhead), haematoxylin and eosin stain, ×100, bar = 100 µm, ×200, bar = 50 µm.

Figure 6. A. The skin of the control animal shows normal collagen bundles within the dermal layer (arrowheads); B. Skin of burned animal showing marked reduction of fibrous connective tissues (arrowheads); C. Platelet-rich plasma treated group shows marked proliferation of collagen fibrous tissues (arrowheads); D. Microvesicles treated group shows marked remodelling and maturation of collagen fibrous tissues (arrowheads), Masson’s Trichrome stain, ×200, bar = 50 µm.

Immunohistochemical results

Microvesicles and PRP downregulate CTGF immune expression in burn wounds. Burn increased the CTGF immune expression in group II, whereas in MVs and PRP treated groups, CTGF was markedly reduced (Fig. 7). Morphometrically, MVs, and PRP significantly downregulated CTGF immuno-staining intensity in burn tissue as compared with the burn group (II) with significantly more downregulation in the MVs group as compared with PRP (Fig. 3).

Figure 7. Connective tissue growth factor (CTGF) immuno-expression in different groups; A. Skin of the control animal shows a mild expression of CTGF within the epidermal epithelium (arrowheads), whereas burn group (II) (B) shows marked expression; C. Platelet-rich plasma treated group displays a declined expression of CTGF within the epidermal covering; D. Burn group treated with microvesicles shows a marked decrease in the expression; CTGF ×400, bar = 20 µm.

Microvesicles ameliorate the inflammatory cytokine TNF-α immune expression. Burn increased the TNF-α immune expression in group II, whereas PRP partially reduced it. in MVs, it was markedly reduced (Fig. 8). Histomorphometrically, MVs significantly ameliorated TNF-α immuno-staining intensity in burn wounds compared with the burn group (II) (Fig. 3).

Figure 8. Tumour necrosis factor alpha (TNF-α) immuno-expression in different groups; A. Skin of the control animal shows minimal expres­sion within the dermis, whereas the burn group II (B) is showing increased expression; C. Platelet-rich plasma treated group shows a declined dermal TNF-α immune expression compared to the burn group; D. Microvesicles treated group exhibits normal expression; TNF-α ×200, bar = 100 µm.

Microvesicles modulate collagen I and III depositions in burn wounds. MVs and PRP (particularly the MVs) decreased collagen I and III depositions in burn wounds (Fig. 9). Via histomorphometric analysis, a significant decline in the immuno-staining intensity of collagen I and III was recorded in the MVs and PRP groups compared with the burn group with significantly favourable results in MVs than the PRP group (Fig. 3).

Figure 9. Collagen I and III among different groups; A. The skin of the control animal displays a mild expression of collagen I within the dermal fibrous tissues (arrowheads), whereas burn group II (B) displays a marked expression; C. Platelet-rich plasma (PRP) treated group displays a decrease in the expression of collagen I within the dermal fibrous tissues (arrowheads); D. Microvesicles (MVs) treated group displays a marked decrease in its expression; A*. The skin of the control animal shows a mild expression of collagen III within the dermal fibrous tissues (arrowheads). In contrast the burn group (B*) displays a marked collagen III expression; C*. PRP treated group shows a decrease in the expression of collagen III within the dermal fibrous tissues (arrowheads); D*. MVs treated group displays a marked decrease in collagen III expression (collagen I and III immunohistochemistry, bar = 20 µm).

Microvesicles and PRP enhance the antioxidant Nrf2 immune expression in burn tissue. MVs and PRP increased the Nrf2 in the burn, particularly in the MVs group (Fig. 10). Via histomorphometric analysis, Nrf2 immune expression was dramatically enhanced especially in the MVs group (Fig. 3).

Figure 10. A. The skin of the control animal displays a mild expression of Nrf2 within the dermal fibrous tissues (arrowheads), whereas burned animal group II (B) displays a marked expression; C. Platelet-rich plasma treated group displays a decrease in the expression of Nrf2; D. Microvesicles-treated group displays a marked decrease in expression; Nrf2 ×200, bar = 20 µm.

DISCUSSION

Microvesicles and PRP considerably accelerate burn wound healing and improved the parameters involved in little scar formation in the present work. Previously, Xiao et al. (2016) [21] reported that human umbilical cord mesenchymal stem cells (hcMSC)-derived exosomes downregulated inflammatory reaction in severely burned rat model via reducing the inflammatory cytokine TNF-α levels and increasing IL-10 levels. Recently, adipose-derived MSC exosomes proved to regulate collagen remodelling to counteract scar hyperplasia which changes the aesthetic appearance and impairs organ function [1]. MVs significantly decreased the burn wound size after 3 weeks from burn induction compared with the burn group (II) and PRP group (III). Despite limited available data regarding the impact of MVs on burn wound size, it was reported that at any time point from 1st to 4th week from burn induction, adipose-derived stem cells-significantly decreased burn wound size with more hair growth when compared to the control group [5], supporting the results of the present work. PRP failed to change the burn wound size significantly in this work, conversely, PRP was proved to attenuate burn wound size 14 days from burn induction in diabetic rats [8]. This might be attributed to the larger burn wound size and induction of diabetes in the latter study. MVs dramatically attenuated IL-6 and immunohistochemical expression of TNF-α, whereas increased IL-10 in burn tissue elucidating the anti-inflammatory and immune-modulatory mechanism of these MVs. In accordance, ADSCs-EXOs were reported to mitigate in-vitro interferon-α secretion, with the resultant deactivation of T cells proposing a presumable immunosuppressive role [3]. In addition, ADSCs-EXOs were evidenced to comprise immunoregulatory proteins such as TNF-α, macrophage colony-stimulating factor, and retinol binding protein-4 [10]. Briefly, An et al. 2021 [1], recently concluded that exosomes upregulate early inflammation, thereby accelerating wound healing. Interestingly, MVs considerably modulated some scar regulating parameters (hyaluronic acid, αSMA, TGF-β, MMP-3, and immunohistochemical CTGF) in burn tissue associated with modulation of the collagen I and III immune expression in the present work elucidating a presumable action of these MVs towards a scarless burn wound healing. Supporting these findings, ADSCs-EXOs upregulated the MMP-3 expression in skin dermal fibroblasts enhancing the remodelling of extracellular matrix, and ameliorating scaring [20]. ADSCs-EXOs also might regulate fibroblast differentiation and gene expression, enhancing the reconstruction of the extracellular matrix, thus preventing scar proliferation [1]. Fetal wounds heal without scar because of higher amounts of hyaluronic acid, higher extracellular matrix production by fibroblast, and the absence of myofibroblast [15, 14]. Although the ratio of type III to type I collagen is higher in the fetal wound than in the adult, collagen III is the main type of collagen in pathologic scar hyperplasia [14]. MVs decreased the immune expression of collagen I and III in burn wounds in this work, more studies on the effect of MVs on collagen deposition and their association with scar proliferation still deserve further work. MVs and PRP had attenuated TGF-β in burn wounds in this work, TGF-β action is not restricted to scar formation after injury but shares in the development of fibrosis in the lung, liver, and kidneys that follows chronic inflammation [11]. TGF-β blocking agents were clinically tried for scar prevention with disappointing results, whereas clinical trials with CTGF blocking drugs are encouraging [11]. Accordingly, MVs might provide hope for scarless burn wounds as they considerably modulated CTGF expression in this work. Down-regulation of mi­RNA203 in MVs and PRP groups in this work elucidate the involvement of miRNA203 in the process of burn wound repair. It was demonstrated that after 3 and 5 days from skin wound induction the GTPase Ran (a pro-proliferative factor) and Raph1 (a pro-migratory factor) are putative direct miR-203 targets whose expression could be essential for skin wound homeostasis and re-epithelialisation [18]. Limited available knowledge about miRNA203 after 3 weeks from burn induction and future works are still warranted. MVs and PRP dramatically enhanced the antioxidant Nrf2 immune expression in this work elucidating the mechanistic beyond their role in burn wound healing. Agreeing with this finding, ADSCs-EXOs overexpressing the antioxidant receptors (Nrf2), had noticeably accelerated the healing of diabetic foot ulcers [13]. From the previous findings, we could conclude that MVs and PRP may improve burn wound healing via controlling scar regulation parameters, antioxidant role, and miRNA203 recruitment.

CONCLUSIONS

Microvesicles derived from mesenchymal stem cells and PRP may improve burn wound healing via regulating scar formation and antioxidant mechanism.

Conflict of interest: None declared