Peptides

Synthetic peptide SVVYGLR upregulates cell motility and facilitates oral mucosal wound healing
Susumu Tanaka a,*, Takuji Yasuda a, Yoshinosuke Hamada b, c, d, Naomasa Kawaguchi e, f, g, Yohei Fujishita a, Seiji Mori b, h, Yuhki Yokoyama b, Hirofumi Yamamoto b, Mikihiko Kogo a

a The 1st Department of Oral and Maxillofacial Surgery, Graduate School of Dentistry, Osaka University, Osaka, Japan
b Department of Molecular Pathology, Division of Health Sciences, Graduate School of Medicine, Osaka University, Osaka, Japan
c Department of Health Economics and Management, Graduate School of Medicine, Osaka University, Osaka, Japan
d Department of Pediatric Dentistry, Osaka Dental University, Osaka, Japan
e Department of Cardiovascular Pathology, Division of Health Sciences, Graduate School of Medicine, Osaka University, Osaka, Japan
f Departments of Drug Discovery Cardiovascular Regeneration, Graduate School of Medicine, Osaka University, Osaka, Japan
g Graduate School of Health Sciences, Morinomiya University of Medical Sciences, Osaka, Japan
h Department of Medical Technology, Faculty of Health Sciences, Morinomiya University of Medical Sciences, Osaka, Japan

Keywords: SVVYGLR , Osteopontin
Oral wound healing Cell motility
TGF-β1
Myofibroblast

A B S T R A C T

Osteopontin-derived SVVYGLR (SV) 7-amino-acid sequence is a multifunctional and synthetic SV peptide implicated in angiogenesis, production of collagen III, and fibroblast differentiation into myofibroblasts. This study investigated the effect of the SV peptide on mucosal wound healing activity. Normal human-derived gingival fibroblasts (NHGF) and human oral mucosa keratinocytes (HOMK) were used for in vitro experiments. In addition, an oral punch wound was prepared at the buccal mucosa in male rats aged 11 weeks, and we evaluated the effect of local injection of SV peptide on wound healing. The synthetic SV peptide showed no influence on the proliferation and adhesion properties of NHGF and HOMK, but it enhanced the cell motility and
migration activities. TGF-β1 receptor inhibitor, SB431542 or SB505124, substantially suppressed the SV peptide- induced migration activity, suggesting an involvement of TGF-β1 receptor activation. Furthermore, SV peptide accelerated the healing process of an in vivo oral wound model, compared with control groups. Further immu- nohistological staining of wound tissue revealed that an increase in capillary growth and the greater number of fibroblasts and myofibroblasts that migrated into the wound area might contribute to the facilitation of the healing process produced by the SV peptide. The SV peptide has beneficial effects on oral wound healing through enhancement of the earlier phase consisting of angiogenesis and remodeling with granulation tissue. The syn- thetic SV peptide can be a useful treatment option, particularly for intractable mucosal wounds caused by trauma or surgery for progressive lesions such as oral cancer.

1. Introduction

In general, rapid wound healing can reduce the risk of infection and prevent adverse effects on growth and development. Although wounds
of the oral mucosa tend to exhibit accelerated healing and minimal scarring compared with dermal wounds [1], soft tissue repair from injury also depends on the wound size, location, and oral environment such as saliva secretion related to immunity [2].

Abbreviations: SV, SVVYGLR; PBS, phosphate-buffered saline; NHGF, normal human-derived gingival fibroblasts; HOMK, human oral mucosa keratinocytes; TGF- β1, transforming growth factor beta 1; OPN, osteopontin; ECM, extracellular matrix; VEGF, vascular endothelial growth factor; Col III, Collagen type III; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; rSV, scrambled SV random; Fmoc, fluorenylmethyloxycarbonyl; vWF, von Willebrand factor; HSP-47,heat shock protein-47; αSMA, alpha smooth muscle actin; SD, standard deviation.

* Corresponding author at: The 1st Department of Oral and Maxillofacial Surgery, Graduate School of Dentistry, Osaka University, 1-8 Yamada-oka, Suita, Osaka, 565-0871, Japan.
E-mail addresses: [email protected] (S. Tanaka), [email protected] (T. Yasuda), [email protected] (Y. Hamada), kawaguch@ sahs.med.osaka-u.ac.jp (N. Kawaguchi), [email protected] (Y. Fujishita), [email protected] (S. Mori), [email protected] (Y. Yokoyama), [email protected] (H. Yamamoto), [email protected] (M. Kogo).

https://doi.org/10.1016/j.peptides.2020.170405

Received 8 July 2020; Received in revised form 6 September 2020; Accepted 7 September 2020
Available online 10 September 2020
0196-9781/© 2020 Elsevier Inc. All rights reserved. demonstrated that wound contraction and granulation tissue formation are delayed in sialoadenetomized animals [3], and open mucosal wounds with denuded bone due to the operation for primary cleft palate repair have often been associated with disadvantageous effects on maxillary growth due to scar tissue formation [4]. Also, large-sized mucosal defects caused by trauma or surgery for progressive diseases such as oral cancer could undergo dysregulations or delays in the healing process, which often results in a rough texture with discomfort or oral functional impairment such as difficulty in eating, swallowing, and articulation, primarily due to the development of hypertrophic scarring.

One of the most frequently used treatment options for superficial or deep mucosal defects is dermal graft or dermal fat grafting. Transposed skin can maintain its morphological characteristics and compromise rapid wound healing, but it does not allow natural wound closure, and intraoral keloids in the transposed skin often cause discomfort and dysfunction of the oral cavity [5]. As an alternative option, various types of artificial skin substitutes, including the polyglycolic acid sheet attached with fibrin glue derived from human blood have been used in the treatment of defects of the oral mucosa. However, these substitutes basically serve as scaffolding, require more time for sufficient vascular ingrowth, and have a lower rate of uptake, particularly when applied to mobile intraoral soft tissue or sites of possible infection and hematoma formation, which might result in scar contraction [6]. In vitro-engi- neered mucosal cell sheets [7], or an ex vivo-produced oral mucosa equivalent [8], have also demonstrated potential applicability for rapid oral wound healing with less fibrosis. Nevertheless, these treatment options have certain limitations, such as the need for a certain period of time and dedicated facilities for preparation of a transplant sheet, the risk of onset of unknown side effects due to transplantation of cultured cells, and the issue of medical costs.

Although not clinically applied, other studies have demonstrated the usefulness of collagen-gelatin scaffold retaining basic fibroblast growth factor thymosin β4 and leptin in accelerating oral wound repair through the enhancement of angiogenesis [9], which is critically involved in the
early phase of the wound healing process. Some peptide agents such as tetrapeptide, AcSDKP, and substance P, one of the neuropeptides, have also been reported to be effective agents for the induction of therapeutic angiogenesis [10]. Osteopontin (OPN), which is present in various tissues as a compo- nent of the extracellular matrix (ECM), has been implicated in the regulation of several pathological and physiological processes including tissue repair [11] and has been reported to exhibit angiogenic activity [12]. Furthermore, the 7-amino-acid sequence Ser-Val-Val-Tyr-Gly-Leu-Arg (SVVYGLR) exposed at the thrombin-cleaved N-terminal OPN has strong angiogenic activity through the enhancement of adhesion and migration of endothelial cells as well as VEGF [13,14]. It is considered that the expression of OPN and N-terminal OPN is increased in response to inflammation upon injury [15], and the peptide synthesized from these amino acid residues (SV peptide) has previously been reported to promote the differentiation of cardiac fibroblasts into myofibroblasts, synthesize Col III via TGF-β/Smad signaling [16,17], and facilitate the regeneration of injured
skeletal muscle with scarless healing [18].

Consistent with the abovementioned findings, this short peptide resulted in a significant acceleration of the dermal wound healing pro- cess through the enhancement of cell motility and migration activity in both dermal keratinocytes and fibroblasts [19]. Immunohistochemical staining also demonstrated that the SV peptide promoted beneficial ef- fects on wound healing by stimulating angiogenesis and myofibroblastic differentiation of dermal fibroblasts [19].Oral mucosal wound healing consists of the same basic processes as those of dermal wound healing and cell migration, including oral ker- atinocytes and fibroblasts in the early phase and wound contraction promoted by myofibroblasts differentiated from resident or migratory
oral keratinocytes or fibroblasts differ in terms of phenotype [20,21] and exhibit different intrinsic characteristics in response to inflammatory stimuli or growth factors such as TGF-β [20], which is likely to be involved in the accelerated and favorable repair of the oral mucosa.
Therefore, we hypothesized that the SV peptide has some effects on the oral wound healing activity as well. The present study aimed to investigate the effect of SV peptide on the intrinsic properties of oral keratinocytes and fibroblasts. We further explored the SV peptide- induced effect on the actual mucosal wound healing and the subse- quent responses associated with angiogenesis or fibroblast differentia- tion into myofibroblasts.

2. Materials and methods
2.1. Primary culture of human-derived fibroblasts and epithelial keratinocytes
Normal human gingival fibroblasts (NHGF) (Lifeline Cell Technol- ogy, CA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal bovine serum (FBS) (Equitech-Bio, Inc, Kerrville, TX, USA) and 1% penicillin/streptomycin solution (Sigma- Aldrich, MO, USA) at 37 ◦C in 5 % CO2. Human oral mucosa keratino- cytes (HOMK) (Cell Research Corporation, Singapore) were cultured in EpiLife medium containing growth supplement (Gibco, Thermo Fisher Scientific, MA, USA) and incubated under 5 % CO2 at 37 ◦C.

2.2. Proliferation assay
SV peptide and scrambled SV random (GYRVLSV; rSV) peptide were synthesized using Fmoc (fluorenylmethyloxycarbonyl) through a high- efficacy solid-phase method [13,14]. The effects of SV peptide on the proliferation of NHGF or HOMK were assessed by the WST-1 assay. Either NHGF (3.0 104 cells) or HOMK (4.0 104 cells) were added into each well of 96-well plates and cultured in a medium containing SV peptide (20 ng/mL), rSV peptide (20 ng/mL), or phosphate-buffered saline (PBS). The WST-1 solution (Takara Bio, Shiga, Japan) was added to each well after an incubation period of 0, 24, 48, or 72 h at 37 ◦C in the presence of 5% CO2. The absorbance at an optical wavelength of 450 nm was determined, with a reference wavelength of 630 nm, using a microplate reader.

2.3. Cell adhesion assay
Fibronectin (10 μg/mL)-precoated 96-well microplates were used for the adhesion assay. Briefly, 2.0 105 cells of NHGF or DMEM were
plated into each well and incubated in a serum-free medium containing SV peptide (20 ng/mL), rSV peptide (20 ng/mL), or PBS in a 5 % CO2 incubator at 37 ◦C for 2 h. After removing the nonadherent cells by
washing twice with PBS, the attached cells were stained with 0.04 % crystal violet for 10 min at room temperature. The treated cells were
lysed in 20 μL DMSO, and the absorbance was measured at an optical
wavelength of 550 nm, with a reference wavelength of 630 nm, using a microplate reader.

2.4. Migration assay
The Boyden chamber principle was applied for the cell migration assay. A total of 1.0 × 105 cells of NHGF or HOMK seeded in the upper chamber were allowed to migrate through a fibronectin (10 μg/mL)-coated polycarbonate membrane with a pore size of 8 μm (Chemo-
taxicell; Kurabou, Osaka, Japan), and serum-free medium was added in the lower chamber along with SV peptide (20 ng/mL), rSV peptide (20 ng/mL), or PBSas a chemoattractant. The chamber was incubated at 37 ◦C for 12 h in 5% CO2, and then, the cells from the lower chamberm were fixed with 10 % neutral buffered formalin solution (Wako, Osaka, mfibroblasts in the later phase [20]. As previously described, dermal andm Japan), stained with hematoxylin, and counted under an optical microscope. To examine the effect of TGF-β receptor activation on the induction of migration activity, TGF-β1 (5 ng/mL; Pepro Tech Inc, NJ, USA) was applied as a chemoattractant, and a TGF-β1 receptor-specific inhibitor, SB431542 (10 μM; Selleck Chemicals, TX, USA) or SB505124 (10 μM; Selleck Chemicals, TX, USA), was added with the chemoattractant.

2.5. Wound healing assay

A standard scratch wound healing assay was adopted to evaluate the motility of NHGF or HOMK. The cells were seeded in φ 16.2-mm Petri dish plates (Iwaki, Tokyo, Japan) and cultured to confluence as a monolayer. After a cell-free gap was created in the center of the monolayer by scratching using a 2-mm disposable micropipette tip, the well was replenished with fresh medium containing SV peptide (20 ng/ mL), rSV peptide (20 ng/mL), or PBS. Images of the scratched cell monolayers were taken at regular time points of 0, 6, 12, and 24 h for the NHGF sample and at 0, 12, 24, and 36 h for the HOMK sample under a light microscope with 200 magnification for monitoring cell migra- tion. For quantitative analyses, each image was captured and used to measure the rate of reduction in the gap relative to the total scratched area to estimate the rate of cell motility using the ImageJ software (National Institutes of Health, MD, USA).

2.6. Animal model

Male Sprague–Dawley rats aged 11 weeks were housed under the following conditions: 12/12 h light–dark cycle, a controlled room tem- perature of 20 ◦C – 22 ◦C, and a humidity level of 40 %–50 %. The rats were anesthetized by intraperitoneal injection of a mixture of mid- azolam (2 mg/kg), medetomidine (0.375 mg/kg), and butorphanol (2.5 mg/kg) and then fixed in the supine position. The depth of anes- thesia was monitored during surgery and adjusted appropriately. All experiments involving procedures and experimental animals were con- ducted in accordance with the guidelines for the proper conduct of an- imal experiments established by the ARRIVE guidelines, Animals (Scientific Procedures) Act 1986, Directive 2010/63/EU for animal ex- periments, Science Council of Japan 2006 (The Guide), and approved by the Department of Animal Care and Use Committee, Osaka University Graduate School ofDentistry (Animal Care and Use Committee approval number: Doha -30—013-0). An oral punch wound was prepared at the left buccal mucosa approximately 7–8 mm behind the angle of mouth. A5-mm dermal biopsy punch was used for this purpose, and a full- thickness mucosal wound was created in each animal. After the prepa- ration of the wound, SV peptide (20 ng/mL, n 5), rSV peptide (20 ng/ mL, n 5), or PBS (n 5) was dividedly injected into four sites of wound periphery as a total amount of 1 mL solution for each siteFrom the day after the creation of the wound, the wound area was photographed for 15 days at a magnification of 22.2 using a digital camera (TG-5; OLYMPUS, Tokyo, Japan). The remaining area of the wound, which did not achieve macroscopic re-epithelialization, was evaluated, and the rate of wound closure was calculated using ImageJ software (National Institutes of Health, Bethesda, MA, USA).

2.7. Immunohistochemical analysis
Immunohistochemical analysis was performed, as previously described [17,19]. After 1 or 3 days of wound preparation and treatment with SV peptide (n 18), rSV peptide (n 18), or PBS (n 18), the mucosal tissue involving the wound region was resected under anes- thesia as mentioned earlier. The specimens were fixed with 10 % formalin and embedded in paraffin. Immunohistochemical staining was performed using antibodies against von Willebrand factor (vWF, Abcam Ltd, Cambridge, UK) for assessing angiogenic capillary formation, heat shock protein (HSP)-47 as a marker for fibroblasts (Santa Cruz, TX, USA), and anti-alpha smooth muscle actin (αSMA, Abcam Ltd,Cambridge, UK) for myofibroblasts. The sections were incubated with biotinylated secondary antibody and peroxidase-conjugated streptavi- din (Vector Laboratories, CA, USA) and then visualized by 3,3-diamino- benzidine solution (Sigma-Aldrich, MO, USA). Image acquisition and quantitative analysis for each sample were subsequently performed using the NIS Elements system (Nikon, Tokyo, Japan) [17].

2.8. Statistical analyses

Statistical analyses were performed using SPSS version 24.0 (SPSS
Inc, Chicago, IL, USA). Continuous data were represented as mean ± standard deviation (SD). For comparisons among the three groups, one-way ANOVA or repeated-measures ANOVA with Tukey’s HSD post-hoc test were used, respectively. P < 0.05 was considered to be statistically significant, unless otherwise stated.

3. Results
3.1. Effects of SV peptide on proliferation and adhesion of NHGF and HOMK
Treatment with SV peptide showed no proliferative or anti- proliferative effects on NHGF and HOMK, and there was no significant difference in the absorbance measured at 450 nm for 72 h of incubation under all the treatment conditions examined in this study (Fig. 1A and B). In the quantification of cell adhesion to fibronectin, SV peptide also did not cause any significant change in the absorbance value at 550 nm in either cultured NHGF or HOMK compared with those obtained from rSV, PBS, and BSA groups, which were used as negative controls (Fig. 1C and D).

3.2. Effects of SV peptide on cell motility, cell migration activity of NHGF and HOMK, and involvement of TGF-β1 receptor activation

In the wound healing assay, the healing process and the cell motility of both NHGF and HOMK demonstrated accelerated wound closure in the monolayer with a significant increase in the rate of cell motility 24–36 h after scratching compared with that observed in rSV and PBStreatment controls (Fig. 2A-D).The migration assay revealed that SV peptide significantly increased the migration activities of NHGF (Fig. 2E) and HOMK (Fig. 2F) compared with the rSV and PBS groups. A previous study demonstrated the possible involvement of TGF-β1 receptor activation and subsequent
phosphorylation, including Smad2 and Smad3, in SV peptide-induced migration activity of cardiac fibroblasts [8]. Therefore, we investi- gated the effect of TGF-β1 and its inhibitor on the migration activity of NHGF and HOMK. The addition of TGF-β1 as a chemoattractant signif- icantly increased the migration activities of both cell populations compared with those of the control groups. In contrast, in the presence of either SB431542 or SB505124, the SV peptide-induced migration activities of both cell populations were substantially suppressed to the same level as that in the control groups (Fig. 2E and F).

3.3. Effects of SV peptide on mucosal wound healing in animal model

In all the groups examined in this study, we detected a tendency to contract the punch wound prepared at the left buccal mucosa over time. SV peptide accelerated the wound healing activity (Fig. 3A), and the rate
of wound contraction calculated based on the remaining wound area was significantly increased, particularly in the earlier period (days 1–7) compared with that in the control groups (Fig. 3B).

3.4. Influence of SV peptide on histological changes in mucosal wound healing
Immunohistological staining performed on day 1 revealed a greater Effects of SV peptide on the prolifera- tion and adhesion of NHGF and HOMK.
A and B: Cell proliferation assessed by the WST assay for NHGF (A) (n = 4) or DMEM (B) (n = 7) under the following three different conditions: treated with PBS, rSV peptide, and SV peptide.C and D: Assay of cell adhesion to fibronectin for NHGF (C) and DMEM (D) treated with three different conditions (n = 4). Data are presente as mean ± SD.number of vWF-positive new blood vessels in SV group than that in the control groups treated with rSV peptide or PBS (Fig. 4A and B). Furthermore, there was a significant increase in the number of positive
cells for HSP-47 and αSMA in the tissues treated with SV peptide on day 3 compared with those in the control groups (Fig. 4C–F).

4. Discussion

In this study, we observed that the cell migration ability and cell motility of both NHGF and HOMK were increased by SV peptide, which is consistent with the findings of a previous study on dermal keratino- cytes and fibroblasts [19]. Since the migration of fibroblasts and kera- tinocytes is believed to be crucially involved in wound healing accompanied by adhesion to the ECM and cytoplasmic elongation and contraction, our results suggest that wound healing is promoted by the rapid movement of these cells to the damaged area.

Previous studies have demonstrated the ability of SV peptide to bind to TGF-β2 receptor with a high affinity and that treatment with SV peptide resulted in the phosphorylation of the TGF-β1 receptor, Smad2,and Smad3 in cardiac fibroblasts or dermal fibroblasts [16,17]. In this study, the TGF-β1
receptor inhibitor significantly suppressed the SV peptide-induced migration activities in oral mucosa-derived keratino-cytes or gingival fibroblasts, suggesting an involvement of the TGF-β/Smad signaling pathway in the SV peptide-induced cell migra- tion, as previously reported in dermal fibroblasts [17]. Conversely, both fibroblasts and keratinocytes showed no significant difference in cell proliferation and cell adhesion ability among the SV, PBS, and RSV groups. SV peptide or thrombin-cleaved N-osteopontin also did not change the proliferative activities of cardiac fibroblasts [17].

Our immunohistological staining experiment conducted using cheek mucosal wound animal models showed that the number of vWF-positive capillaries on the first day after wound preparation was significantly higher in the SV group than in the control groups. During the early phase of wound repair manifesting inflammatory symptoms, new capillaries invade the wound clot, and a functional vascular network is important to supply nutrients and oxygen to the wound area, whereas a delayed or defective process of angiogenesis has been implicated in healing im- pairments, as previously reported [22]. Wound angiogenesis is believed to be regulated by interactions among endothelial cells, angiogenic cy-tokines, and ECM environments [23]. Vascular endothelial growth fac- tor (VEGF), TGF-β1, and FGF are well-known potent proangiogenic agents that exert angiogenic activity [24], and SV peptide also has the potential of angiogenesis comparable to that of VEGF [14]. Our results suggest that SV peptide can facilitate the wound healing process, in part, by stimulating angiogenic activity as observed in the dermal wound healing process [19].

In addition, in the histological specimens treated with SV peptide, a large number of Hsp47- and αSMA-positive cells were also observed. Fibroblasts are activated in the granulation tissue and differentiate into myofibroblasts, which express SMA filaments and contribute to contraction and wound strength [25]. A previous study demonstrated that SV peptide facilitated the differentiation of cardiac fibroblasts into active myofibroblasts [17]. Consistent with these results, our study showed that SV peptide could promote increased expression of fibro- blasts and myofibroblasts in the process of oral wound repair, as pre- viously demonstrated in dermal wound healing [19].

Upon dermal or oral wounding, resident and migrated fibroblasts also play a crucial role in the deposition and remodeling of the provi- sional ECM. Expression of collagen and other components of the ECM is essential for maintaining tissue integrity and organ architecture and has a vital role in the migration of epithelium during wound healing and tissue repair processes [26]. Herein, secreted TGF-β functions to stim-
ulate the production of ECM by fibroblasts and restore tissue architecture. OPN is an ECM protein with multifunctional domains and has been implicated in the regulation of several physiological and pathological processes, including tissue repair [15]. A previous study demonstrated that acute OPN expression promotes inflammation following a single severe muscle injury and that its deficiency significantly delayed the inflammatory responses and muscle regeneration [27]. In addition, the increased expression of thrombin by inflammation upon injury cleavage full length of OPN and SVVYGLR amino acids sequence would be Effects of SV peptide on the cell motility and the cell migration activity of NHGF and HOMK and involvement of TGF-β1 receptor activation. Photomicrographs of scratch wound healing assay using NHGF (A) and HOMK (B) captured at 0–24 and 0–36 h, respectively. The rate of wound closure representing cell motility of NHGF (C) and HOMK (D) was significantly enhanced by SV peptide (n = 4) compared with control (rSV peptide, n = 4, PBS, n = 4) groups. Migration activity was assessed by Boyden chamber assay using independent cultured NHGF (E) and HOMK (F) under different treatment conditions (n = 6). TGF-β1 receptor- specific inhibitor, SB431542 or SB505124 suppressed SV peptide-induced migration activities. Data are presented as mean ± SD. *P < 0.05, **P < 0.01 vs control groups (PBS and rSV), †P < 0.05, ††P < 0.01 vs SV.

Effects of SV peptide on mucosal wound healing in vivo. A: Photographs taken for healing progression of punch wound prepared at the buccal mucosa from day 0 to day 15. B: The rate of wound closure on days 1, 3, 5, and 7 showed signifi-cant differences between the treatment condi- tion of SV peptide and control groups (n = 5). Data are presented as mean ± SD. *P < 0.05 vs control groups.exposed on N-terminal fragment, which gains multifunctional effects including angiogenesis and increased adhesive properties binding to integrins [15,28], comparable with those revealed in SV peptide [17]. In the rat oral wound model, the application of synthetic SV peptide in addition to resident thrombin-cleaved N-terminal OPN caused by inflammation upon injury was believed to increase the local expression level of SVVYGLR motif. As a result of subsequent reactions, including the enhancement of cell motility and migration activities in oral kera- tinocytes and fibroblasts, mucosal wound healing was believed to be accelerated. The present study demonstrated that the local application of SV peptide stimulated TGF-β1 receptor activation and increased the
migration of myofibroblasts into the wound area. Although an increase or persistence of these reactions in the later phase of wound healing causes fibrosis or hypertrophic scars [4,25,29], the SV peptide, a short-length peptide composed of seven amino acids, could rapidly be
degenerated and might lose its potency to activate TGF-β1 receptor and induce normal apoptosis of myofibroblasts in the later period of wound healing, thus contributing to less scar formation.In support of this idea, our recent study conducted using a volumetric muscle loss model demonstrated that SV peptide facilitated skeletal muscle regeneration with scarless healing [18]. Although further investigation is necessary, this functional peptide can be a useful treat- ment option, especially for intractable mucosal wounds caused by trauma or surgery for progressive lesions such as oral cancer.

5. Conclusions
Within the limitations of this study, it can be concluded that Synthetic SV peptide promoted an increase in the cell motility of oral fibroblasts and keratinocytes. TGF-β receptor activation was poten- tially involved in the SV peptide-induced migration activities of these
cell populations.In vivo experiment revealed that SV peptide accelerated the healing process of oral mucosal wounds by facilitating angiogenesis and increasing the production of fibroblasts and myofibroblasts.

Funding
This research was supported by a Grant-in-Aid for Scientific Research
(C) (19K10265 to S.T.) from the Japan Society for the Promotion of Science.
CRediT authorship contribution statement
Susumu Tanaka: Conceptualization, Methodology, Formal analysis, Writing - original draft, Writing - review & editing, Funding acquisition. Takuji Yasuda: Investigation, Visualization. Yoshinosuke Hamada: Conceptualization, Methodology, Formal analysis, Writing - review & editing. Naomasa Kawaguchi: Conceptualization, Methodology, Formal analysis. Yohei Fujishita: Investigation, Visualization. Seiji Mori: Investigation, Visualization, Formal analysis. Yuhki Yokoyama: Investigation, Visualization. Hirofumi Yamamoto: Supervision. Miki- hiko Kogo: Supervision.

Declaration of Competing Interest
The authors report no declarations of interest.
Histological evaluation of vWF, Hsp-47 and αSMA expression in mucosal wound healing.
A: Immunohistological staining with anti-vWF antibody in mucosal wound sections prepared on day 1 after treatment at different conditions (n = 6). Scale bars represent 100 μm. B: Capillary density was significantly increased by treatment with SV peptide. C: Immunostaining with antibodies against heat shock protein-47 on day 3. Scale bars represent 50 μm. D: Quantitation of the number of fibroblasts (n = 6). E: Immunostaining with anti-α-smooth muscle actin on day 3. Scale bars represent 50 μm. F: Quantitation of the number of myofibroblasts (n = 6). Data are presented as mean ± SD. *P < 0.05, **P < 0.01.

Acknowledgements
None.

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