Effect of Stem Cells from Human Exfoliated Deciduous Teeth Spent Medium (SHED-SM) on the Healing of Traumatic Buccal Mucosal Ulcers

Juni Handajani; Hendrik Setia Budi; Lisa R. Amir; Sri Angky Soekanto

doi:10.12688/f1000research.164339.1

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Brief Report

Effect of Stem Cells from Human Exfoliated Deciduous Teeth Spent Medium (SHED-SM) on the Healing of Traumatic Buccal Mucosal Ulcers

[version 1; peer review: 2 approved]

Juni Handajani ¹, Hendrik Setia Budi², Lisa R. Amir³, Sri Angky Soekanto³

PUBLISHED 21 May 2025

Author details Author details

¹ Department of Oral Biology, Faculty of Dentistry, Universitas Gadjah Mada, Yogyakarta, Indonesia
² Department of Oral Biology, Faculty of Dentistry, Airlangga University, Surabaya, Indonesia
³ Department of Oral Biology, Faculty of Dentistry, Indonesia University, Jakarta, Indonesia

Juni Handajani
Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Project Administration, Validation, Writing – Original Draft Preparation

Hendrik Setia Budi
Roles: Conceptualization, Funding Acquisition, Project Administration, Resources, Supervision, Writing – Review & Editing

Lisa R. Amir
Roles: Conceptualization, Investigation, Writing – Review & Editing

Sri Angky Soekanto
Roles: Conceptualization, Investigation, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

Introduction

Traumatic ulcers are among the most prevalent injuries with a reported incidence of up to 95%. This study aims to evaluate Stem Cells from Human Exfoliated Deciduous Teeth Spent Medium (SHED-SM) nanoemulgel on the cytotoxicity of NIH-3T3 and buccal mucosal ulcer wounds.

Materials and methods

The cytotoxicity test of SHED-SM was analyzed on NIH-3T3 fibroblast cells using MTT. Thirty male Wistar rats, 2-3 months, were used as experimental subjects. Traumatic ulcer models were created by excising the right buccal mucosa with a 3 mm punch biopsy. Subjects were divided into 2 groups @15 rats for the treatment (SHED-SM) and control. The gel was applied on the surface once a day for 4 days. Evaluation was conducted on days 1, 3, 5, 7, and 14. Hematoxylin Eosin (HE) and immunohistochemical were stained to evaluate a number of neutrophils and the expression of COX-2, VEGF, and Col-1.

Results

The ANOVA results showed significant differences in the cytotoxicity test, the number of neutrophils, and the expression of COX-2, Col-1, and VEGF. The number of neutrophils and the expression of COX-2, Col-1, and VEGF were higher in the treatment group compared to the control.

Conclusion

SHED-SM demonstrates potential to accelerate the healing of traumatic buccal mucosal ulcers.

Keywords

nanoemulgel, spent medium, Stem Cells from Human Exfoliated Deciduous Teeth (SHED), traumatic ulcers, wound healing.

Corresponding author: Juni Handajani

Competing interests: No competing interests were disclosed.

Grant information: This research received funding from Indonesian Collaborative Research Program (Program Riset Kolaborasi Indonesia) Grant No. 1917/UN1/DITLIT/PT.01.03/2024 between Universitas Airlangga, Universitas Gadjah Mada, and Universitas Indonesia.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2025 Handajani J et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Handajani J, Setia Budi H, R. Amir L and Angky Soekanto S. Effect of Stem Cells from Human Exfoliated Deciduous Teeth Spent Medium (SHED-SM) on the Healing of Traumatic Buccal Mucosal Ulcers [version 1; peer review: 2 approved]. F1000Research 2025, 14:510 (https://doi.org/10.12688/f1000research.164339.1) First published: 21 May 2025, 14:510 (https://doi.org/10.12688/f1000research.164339.1) Latest published: 21 May 2025, 14:510 (https://doi.org/10.12688/f1000research.164339.1)

Introduction

Ulcers are a type of injury of the oral mucosa caused by various forms of trauma, including thermal, mechanical, chemical, and electrical trauma.¹ The 2018 Riskesdas report stated that ulcers represent a significant dental and oral health issue in Indonesia, with a prevalence rate of 95%.² Clinically, ulcers are characterized by the loss of epithelium, often present with yellowish fibrin exudate at the center, erythematous edges, and a round shape. Their size varies depending on the duration, intensity, and cause.³ The natural healing process of ulcerative wounds typically takes 10-14 days if the causative factor is eliminated.⁴ Wound healing is a complex and long process involving multiple phases: hemostasis, inflammation, proliferation, and maturation.⁵ The hemostasis phase occurs immediately after the injury, and is characterized by vasoconstriction, and platelet activation that binds collagen to the extracellular matrix (ECM), and initiate the blood clotting process.⁶ Platelets then release chemical mediators, including transforming factor beta (TGF-β), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF), which stimulate the macrophage migration, neutrophil infiltration, and fibroblast proliferation.⁶

Regenerative therapies utilizing stem cells are being developed to accelerate wound healing. Stem cell products offer potential solutions to address deficiencies in components critical to the wound-healing process. The secretion products of stem cells, collectively referred to as the secretome, have garnered particular attention in this context Regenerative therapies using stem cells are currently being developed to accelerate wound healing. Stem cell products are expected to overcome the lack of components that can cause delays in the wound-healing process. The secretion products of stem cells, known as the secretome⁷ have drawn particular attention in this context. Stem cells can be derived from several parts of the body, including dental tissues, such as Dental Pulp Stem Cells (DPSCs), Stem Cells from Human Exfoliated Deciduous Teeth (SHED), Stem Cells from Apical Papilla (SCAP), Periodontal Ligament Stem Cells (PDLSCs), Dental Follicle Stem Cells (DFSCs), and Gingival Mesenchymal Stem Cells (GMSCs).⁸

Stem cells originating from deciduous tooth pulp tissue are referred to as Stem Cells from Human Exfoliated Deciduous Teeth (SHED). SHED is known as a potential source of regenerative therapy due to its ability to differentiate into various cells, such as fibroblasts, odontoblasts, adipocytes, nerves, and epithelial cells.⁷ Furthermore, SHED exhibits robust osteogenic and adipogenic differentiation capabilities and expresses several critical growth factors, including Fibroblast Growth Factor (FGF), Transforming Growth Factor (TGF), Connective Tissue Growth Factor (CTGF), Nerve Growth Factor (NGF), and Bone Morphogenetic Protein (BMP).^9,10 The elevated expression of these growth factors facilitates tissue regeneration by promoting cell migration, proliferation, and differentiation.¹¹ SHED demonstrates a higher proliferation rate than other types of stem cells, even in unfavorable conditions, such as high glucose levels and hypoxic conditions. High expression of several growth factors can help accelerate tissue regeneration by stimulating the process of cell migration, proliferation, and differentiation.¹¹

Despite its potential, cell-based therapy still has shortcomings, such as the risk of cell mutations that lead to tumorigenesis and rejection during transplantation.^12,13 To address these challenges, the use of stem cell secretion products, known as secretomes, is an alternative to overcome problems related to cell-based therapy.¹⁴ Secretomes are molecules and biological factors secreted by cells into the extracellular environment that play pivotal roles in homeostasis, inflammation, angiogenesis, apoptosis, proteolysis, and signaling.¹⁵ These products contain growth factors, cytokines, chemokines, extracellular matrix (ECM), and small molecules, including metabolites, microvesicles, and exosomes.

Secretomes can be obtained by isolating stem cells in growth media, resulting in a conditioned medium or spent medium. The use of spent medium as a culture medium offers several advantages since it is simple, practical, and affordable.¹⁶ The process for obtaining spent medium is relatively safe and minimally invasive, which allows the collection of cell secretion without using and taking the cells themselves.¹⁷ Based on the description above, developing a safe therapy method for buccal mucosal ulcer wounds using SHED-SM nanoemulgel is necessary.

Material and methods

This research was conducted as part of the Indonesian Collaborative Research Program (Riset Kolaborasi Indonesia) according to Contract No. 1917/UN1/DITLIT/PT.01.03/2024 between Universitas Airlangga, Universitas Gadjah Mada, and Universitas Indonesia. This research has obtained ethical approval from the Ethics Commission of the Faculty of Dentistry and Prof. Soedomo Dental Hospital (RSGM Prof. Soedomo), Universitas Gadjah Mada University as documented in Letter No. 153/UN1/Kep/FKG-RSGM/EC/2024 dated July 25, 2024 for animal research.

Isolation of SHED and SHED-SM

This study was followed previous research and adhered to ethical guidelines, ensuring that written informed consent was obtained from donors. The isolation of SHED and SHED Spent Medium (SHED-SM) was carried out at Universitas Airlangga. The ethics approval was granted by the Committee for Medical and Health Research Ethics at the Faculty of Dental Medicine (0530/HRECC.FODM/V/2024) Universitas Airlangga dated May 17, 2024 for human participant. Deciduous teeth were extracted under sterile conditions in a surgical setting from two 6-year-old patients. The selection criteria for the tooth samples included anterior teeth without caries, pulp exhibiting a red coloration, absence of gum infection, and extraction performed in an aseptic environment.¹⁸

Pulp tissues obtained from deciduous teeth were enzymatically digested using collagenase type I (4 mg/mL) and dispase (2 mg/mL) for one hour at 37°C before being plated onto a dish cell culture. The SHEDs were cultivated in alpha-minimum essential medium (α-MEM) Eagle, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cultures were cultivated in 35 mm dishes at 37°C in a humidified environment with 5% CO₂. The growth media were replaced every two to three days until the cells reached confluence.

Microscopic observation confirmed that the SHEDs were cultivated to guarantee a uniform population with a spindle-like shape, characteristic of mesenchymal stem cells (MSCs). Subcultures were performed across several passages. Passage 2 cells were used for surface marker characterization and differentiation analysis, while passage 4 cells were used for SHED-SM isolation.

To prepare SHED-SM, SHED cells obtained from the Indonesian Collaborative Research project at the fourth passage were cultured in complete growth medium after being inoculated into a T-75 flask at a density of 2 million cells. After reaching 70% to 80% confluency, the cells were rinsed twice with phosphate-buffered saline (PBS), and the medium was replaced with serum-free alpha-MEM (BM). The cells were incubated for days 1, 3, and 5 at 37°C in a humidified atmosphere containing 5% CO₂ to create SHED-SM (H1, H3, H5). This process generated 8–10 million SHEDs, equivalent to around 15 mL of SM. After collection, the SM was centrifuged at 1,200 rpm for 5 minutes, filtered through a 0.2-μm-pore-size filter and stored at -80°C.¹⁸

Cytotoxicity test of SHED-SM against NIH-3T3 fibroblast cells

Cytotoxicity was measured using the MTT assay using a microplate reader at wavelength of 490 nm (Thermo Scientific). NIH-3T3 fibroblast cells suspended in RPMI 1640 medium (100 μl, density 1.5x10^4 cells/well) were put into a 96-well plate and incubated for 24 hours in a 5% CO₂ incubator. Furthermore, SHED-SM was added to each well in three replications, followed by incubation at a 37°C in 5% CO₂ incubator for 24 hours. After incubation, the medium in each well was discarded and replaced with a mixture of of SHED-SM and medium at a ratio of 50:150 μl, for a total of 200 μl. Finally, 10 μl of 0.5% MTT in the culture medium was added during the final stage of incubation. The plate was then incubated again at 37°C for 2.5 hours. Living cells formed purple formazan due to reaction with MTT. The formazan was dissolved in an SDS solution and incubated at room temperature for 12 hours.

Treatment of experimental animals

Thirty Wistar rats aged 2-3 months, weighing 250-300 grams, were used as experimental subjects and adapted in individual cages for 3 days. The rats were then randomly divided into two groups of 15 each: the treatment group (SHED-SM nanoemulgel) and the control (Aloclair Plus) (Kalbe Farma). All experimental animals were treated at the Integrated Research and Testing Laboratory (LPPT) of Universitas Gadjah Mada.

The induction of traumatic ulcers began by anesthetizing the rats intramuscularly with ketamine at a dose of 0.1 ml/100 grams of body weight. A 3 mm diameter punch biopsy tool (Medax model Epitheasy) was then pressed and rotated on the buccal mucosa to create the ulcer. Therapy was performed once a day at 10:00 Western Indonesian Time for four consecutive days using a regular size 3.0 mm microbrush (Microbrush International) according to the treatment and control groups.

Decapitation was performed on days 1, 3, 5, 7, and 14, with three rats sacrificed per time point after being anesthetized intramuscularly with ketamine HCl (Ketamil) at the same dose (0.1 mL/100 grams of body weight). Ulcer tissue was excised from the buccal mucosa with a thickness of ± 2-3 mm. The tissue samples were fixed in 10% Buffered Natural Formalin (BNF) solution for 24 hours.

Histology preparation was carried out at the Integrated Research Laboratory (LRT) of the Faculty of Dentistry, Universitas Gadjah Mada. The ulcer tissue specimens were dehydrated using an automatic tissue processor (Tissue-Tek II Sakura Timing Disc, USA) for 20 hours. The specimens were then cleared with xylene and infiltrated with paraffin. The embedding process was carried out in a tissue embedding cassette (Refurbished Sakura TECTM 4 Tissue Embedding Center), and tissue sections 3 μm thick were cut using a rotary microtome (Accu-Cut® SRMTM Rotary Microtome).

Tissue staining

Tissue staining was performed using Hematoxylin Eosin (Harris Hematoxylin, Leica Biosystems, USA) and immunohistochemistry (Elabscience). Deparaffinization and rehydration procedures were conducted using graded xylol and alcohol solutions starting from 100%, 95%, 80% and 70% concentrations. The specimens were stained in hematoxylin solution for 5 minutes, then rinsing using running water for 3 minutes, and then stained with eosin solution for 1 minute, followed by another rinse with running water. Dehydration was performed with graded alcohol solutions at 70%, 80%, 96%, and 100% concentrations before being cleared using xylol and mounting.

Immunohistochemistry staining was performed using an IHC kit (Elabscience). Endogenous peroxidase activity was blocked by incubating the samples in 3% H₂O₂ at room temperature for 10 minutes, followed by washing with PBS. Normal Goat Blocking Buffer was applied to the specimens and incubated for 30 minutes at 37°C. The samples were then incubated overnight at 4°C with primary antibodies: anti-COX-2 (Rabbit Polyclonal antibody COX-2, NB100-689, Novusbio) 1:100, anti-Collagen 1 (Rabbit Polyclonal antibody Collagen 1, NB100-408, Novusbio) 1:100 and anti-VEGF (Rabbit Polyclonal antibody VEGF, sc-152, Santa Cruz Biotechnology) 1:50 as much as 50 μl of each antibody.

After washing with PBS, the samples were treated with Polyperoxidase-anti-Mouse/Rabbit IgG (E-IR-R217B) and incubated for 20 minutes at room temperature. Following another PBS wash, the samples were stained with the chromogen 3,3’-diaminobenzidine (DAB) in a 1:20 ratio with the substrate until a brown color appeared, indicating positive staining. The process concluded with dehydration using graded alcohol solutions, clearing with xylene, and mounting.

Observation of neutrophil infiltration and immunohistochemistry

Neutrophil infiltration was observed by counting the number of neutrophil cells in the connective tissue of the wound edge using a light microscope at 400× magnification. Each slide was analyzed in three fields of view, and the results were averaged to determine the neutrophils count per field.

COX-2, VEGF, and Collagen 1 (Col-1) protein expression was observed in the connective tissue at the ulcer edge. COX-2 expression was identified by brown staining in the cytoplasm of neutrophils, macrophages, lymphocytes, fibroblasts, and the extracellular matrix. The number of COX-2-positive cells in the ulcer edge epidermis was counted across three fields of view.

Col-1 expression was indicated by brown staining in fibroblasts and the extracellular matrix, while VEGF expression was marked by brown staining in endothelial cells, fibroblasts, and inflammatory cells within the ulcer area. Measurements of VEGF and Col-1 expression were conducted using ImageJ software (National Institutes of Health, version 1.54) based on specimen photographs. The data obtained were analyzed using parametric statistical tests, including ANOVA and LSD, to determine significant differences.

Results

The results of the cytotoxicity test of SHED Spent Medium (SHED-SM) nanoemulgel against NIH-3T3 fibroblast cells after MTT administration are presented in Figure 1. After MTT application, black living cells and fuzzy crystals were visible at the bottom of the well.

Figure 1. Image of NIH-3T3 fibroblast cells (A) and NIH-3T3 fibroblast cells after treatment using nanoemulgel spent medium SHED (SHED-SM) H1 (B), H3 (C) and H5 (D).

Cell viability was indicated by black living cells and fuzzy crystals at the bottom of the well after MTT application. The highest viability of NIH-3T3 fibroblast cells was shown after applying SHED-SM H1.

Figure 2 shows the percentage viability of NIH-3T3 fibroblast cells after treatment with SHED-SM nanoemulge. The results indicated that the highest cell viability was obtained in SHED H1 (SHED-SM H1) nanoemulgel at 65.95%, while the lowest was recorded for SHED-SM H5 at 37.01%. Statistical analysis using ANOVA and LSD tests revealed significant differences in cell viability among SHED-SM H1, H3, and H5 (p < 0.05). Based on these findings, SHED-SM H1 nanoemulgel was selected for application in experimental animal studies.

Figure 2. The highest percentage of NIH-3T3 fibroblast cell viability was shown after the application of nanoemulgel SHED-H1spent medium (SHED-SM H1) and significant differences in the comparison of each concentration (*) (p<0.05).

Figure 3 displays clinical observations of the buccal mucosa following the application of SHED-SM nanoemulgel. By the 3^rd day, the traumatic ulcers in both the treatment and control groups showed closure, and a reduction in wound diameter over each observation day. On the 5^th day, ulcers were still visible but had decreased in size. By the 7^th and 14^th days, no ulcers were clinically observable.

Figure 3. Clinical evaluation of traumatic ulcer after application of nanoemulgel spent medium SHED (SHED-SM) on the buccal mucosa of rats.

An ulcer with a diameter of Ø 3 mm was seen on day 1 (A) and the diameter of the ulcer decreased as the observation days 3^rd (B) and 5^th (C). The ulcer had closed and could not be observed clinically on days 7^th (D) and 14^th (E).

Figure 4 provides a histological evaluation of the healing process in traumatic ulcer wounds, comparing the treatment group (SHED-SM nanoemulgel) after application of spent medium SHED gel) and control (Aloclair Plus).

Figure 4. Evaluation of the wound healing process after application of nanoemulgel spent medium SHED (SHED-SM) (treatment) and control.

Inflammatory cell infiltration appeared on 1^st and 3^rd day. The epithelium closed on the 5^th day after treatment, while the epithelium was still open in the control group. On the 7^th days and 14^th, the epithelium has closed. Observation on the 7^th day of the treatment group showed thicker epithelium than the control group. Hematoxylin eosin staining at 40x magnification.

Figure 4 illustrates the ulcer healing process after applying treatment with SHED Spent Medium (SHED-SM) nanoemulgel compared to the control group treated with Aloclair Plus gel. On the 1^st day, inflammatory cell infiltration was seen until 3^rd day. On the 5^th day after treatment, epithelial closure was evident in the treatment group, while the control group demonstrated incomplete epithelial closure. On the 7^th and 14^th days, the ulcers were fully closed in both groups. On the 7^th day, the treatment group displayed thicker epithelial layers compared to the thinner epithelium in the control group.²⁶

Figures 5 and 6 present the results for neutrophil counts and COX-2 expression. The highest neutrophil count occurred on the 1^st day, while peak COX-2 expression was observed on the 3^rd day in both groups. Positive COX-2 expression was indicated by the presence of brown cytoplasmic staining in inflammatory cells within the ulcer area. Both neutrophil counts and COX-2 expression decreased progressively over the observation period.

Figure 5. Neutrophil infiltration (A) and COX-2 expression (B) after application of nanoemulgel spent medium SHED (SHED-SM) on the 3^rd day.

COX-2 expression was seen in the cytoplasm of cells. HE staining (A) and immunohistochemistry (B) at 400x magnification.

Figure 6. The number of neutrophils was highest on the 1^st day, while the positive expression of COX-2 on the 3^rd day decreased until the 14^th.

There was a significant difference in the number of neutrophils between the treatment and control group on the 1^st and 3^rd days and COX-2 expression on the 5^th day (*) (p<0.05).

The homogeneity and normality analyses of neutrophil count and COX-2 expression data confirmed normal and homogeneous distributions (p > 0.05). ANOVA results indicated significant differences (p < 0.05), with LSD analysis revealing significant differences in neutrophil counts between the treatment and control groups on the 1^st and 3^rd days, as well as in COX-2 expression on the 5^th day.

The brown staining observed in fibroblasts and the extracellular matrix, as shown in Figures 7 and 8, indicates collagen 1 (Col-1) expression. Homogeneity and normality tests for Col-1 expression data confirmed normal and homogeneous distributions (p > 0.05). ANOVA results revealed significant differences (p < 0.05), and LSD analysis identified significant differences (p < 0.05) in Col-1 expression between the treatment and control groups on the 3^rd and 7^th days.²⁶

Figure 7. Evaluation of Collagen-1 (Col-1) expression on the 3^rd day after treatment showed a brown color in the extracellular matrix area and fibroblasts in the ulcer area.

Magnification 40x (A) and 400x (B).

Figure 8. The highest Col-1 expression was shown on the 5^th day after treatment and control, and the expression decreased until the 14^th day.

There was a significant difference in Col-1 expression on 3^rd and 5^th days (*) (p<0.05).

Figures 9 and 10 present the evaluation of VEGF expression in response to SHED-SM nanoemulgel and the control. VEGF expression, marked by brown staining in endothelial cells, fibroblasts, and inflammatory cells at the ulcer margins, was analyzed.²⁵

Figure 9. Evaluation of VEGF expression on the 5^th day after application of nanoemulgel spent medium SHED (SHED-SM).

The brown color was seen in endothelial cells (Ο), fibroblasts, and inflammatory cells in the ulcer area. Magnification 100x (A) and 400x (B).

Figure 10. VEGF expression increased until the 5^th day in the control and treatment groups, then decreased until the 14^th day.

There was a significant difference in VEGF expression between the control and treatment groups on all days (*) (p<0.05).

Homogeneity and normality tests confirmed normal and homogeneous data distributions (p > 0.05). ANOVA results indicated significant differences (p < 0.05), with LSD analysis showing significant differences (p < 0.05) in VEGF expression between the treatment and control groups on all observation days.

This study demonstrates that SHED-SM nanoemulgel accelerates the healing of traumatic ulcers compared to the control. Clinically, this was evidenced by reduced ulcer size and epithelial closure by the 5^th day. Histological evaluation revealed that SHED-SM nanoemulgel treatment increased neutrophil counts on the 1^st day and COX-2 expression on the 3^rd day compared to the control. Additionally, Col-1 and VEGF expression levels were higher in the treatment group than in the control group, except on the 7^th and 14^th days, when VEGF expression was higher in the control group.

Discussion

The cytotoxicity test results revealed the highest cell viability ( Figures 1 and 2) obtained in SHED Spent Medium (SHED-SM) nanoemulgel H1, 65.95%. These results were used as the basis for application to experimental animals, specifically using SHED-SM nanoemulgel H1.

In the experimental animal treatment, clinical observations indicated that traumatic ulcers healed following both treatment and control applications. By the 5^th day, the ulcers were observed to have closed, and by the 7^th day, no ulcers were visible ( Figure 3). Histological observations using Hematoxylin Eosin staining corroborated these findings. In the treatment group, the epithelium appeared closed by the 5^th day, whereas in the control group, it remained open ( Figure 4).

Comparative analysis of neutrophil counts between the treatment and control groups showed the highest levels on the 1^st day. COX-2 expression peaked on the 3^rd day and decreased over subsequent observation days. COX-2 expression plays a critical role in the ulcer healing process, as it is known to increase rapidly within 12 hours of mucosal injury. The COX-2 enzyme is essential for producing prostaglandin E2 (PGE2), a key mediator of inflammation. The sharp increase in COX-2 expression on the 3^rd day indicates active debris and pathogen clearance in the ulcer area. By the 5^th day and in later observations, a decline in COX-2 expression was evident. This decline likely marks the transition to the proliferation phase of healing. Additionally, reduced COX-2 expression helps mitigate excessive inflammation and supports the formation of new tissue.¹⁹

The observed decrease in the number of neutrophil cell infiltration and COX-2 expression in the SHED-SM treatment group and the Aloclair Plus® control group was likely due to the anti-inflammatory effects of both components. The anti-inflammatory effects of SHED-SM may stem from its bioactive content, which could modulate the inflammatory response, including reducing the production of pro-inflammatory cytokines (TNF-α and IL-6).¹⁹ However, further research is still needed to prove this mechanism. Similarly, in the control group, the reduction in the number of neutrophils and COX-2 expression could be attributed to the anti-inflammatory effects of Aloclair Plus® ingredients, including Aloe Vera, Sodium Hyaluronate, Glycyrrhetinic Acid, and Polyvinylpyrrolidone (PVP). The four ingredients were likely to reduce inflammation, especially PVP, which can form a layer on the wound’s surface, thereby preventing further ulcer irritation.

The evaluation of Collagen-1 (Col-1) expression showed the highest expression on the 5^th day after treatment and control, followed by a gradual decline until the 14^th day. Collagen-1 was known to have an important role in the mucosal wound healing process. The increase in Col-1 expression was in line with the new tissue formation and remodeling process. Figure 8 showed a rise in Col-1 expression from the 1^st to 5^th days. This result was thought to be in the early stages of ulcer healing (inflammatory phase on 1^st and 3^rd days); col-1 played a role in strengthening the extracellular matrix and supporting the migration of inflammatory cells to the ulcer area. On the 5^th day, Col-1 expression continued to increased, likely marking the proliferation phase, during which fibroblasts migrate to the ulcer area and synthesize collagen, forming new granulation tissue. This day also marks the peak of Col-1 expression. Col-1 expression decreased in the remodeling phase, suggesting that the tissue had matured and become more organized.

Evaluation of VEGF expression revealed increased levels on the 3^rd and 5^th days, with higher expression in the treatment group compared to the control. Conversely, on the 7^th and 14^th days, VEGF expression in the control group was higher than in the treatment group. These results indicated that VEGF expression plays an important role in the proliferation phase by stimulating the growth of new blood vessels to replace ulcer damage. Strong VEGF expression was seen in endothelial cells, fibroblasts, and inflammatory cells. VEGF expression in endothelial cells contributing to cell proliferation and migration to form new capillaries.

The SHED-SM material can be applied topically to oral mucosal ulcer wounds. The advantages of the topical method may have high bioavailability and is more selective and efficient in drug delivery.²⁰ One factor affecting wound healing therapy was the particle size of the material used. Small particles were easier to absorb into the body. However, the disadvantage of small particles is that they cannot last long in wounds.²¹ The development of nanoemulsion-based formulations was known to increase the absorption of therapeutic materials into the skin. This particle shape can also carry hydrophilic and hydrophobic therapeutic materials. Gel preparations have a faster dissolution ability than cream or ointment preparations.²² However, gels also have limitations, such as potential irritation from additional gel-forming agents, a high susceptibility to microbial or fungal contamination, and drying due to solvent evaporation.²³ These challenges can be mitigated by converting the formulation into a nanoemulgel. Nanoemulgels combine the benefits of nanoemulsion and gel, offering improved therapeutic agent release characteristics and a stable gel matrix consistency. As a result, nanoemulgel is considered an effective dosage form for delivering therapeutic agents through the skin.^24,25

The results of this study indicate that the application of SHED-SM nanoemulgel can accelerate the healing of traumatic ulcers. This was characterized by a smaller ulcer diameter on the 5^th day, supported by the results of histological evaluation in the form of epithelial closure, the number of neutrophils on the 1^st day followed by COX-2 expression on the 3^rd day, expression of Col-1 and VEGF. All the parameters were consistently higher in the treatment group compared to the control. In conclusion, the application of SHED Spent Medium nanoemulgel could accelerate the healing of traumatic buccal mucosal ulcers, as demonstrated by clinical and histological outcomes, including epithelial closure and increased neutrophil count, as well as elevated expressions of COX-2, Col-1, and VEGF.

Data availability

Underlying data

Handajani, Juni (2025). Raw Data RKI-2024 https://figshare.com/articles/dataset/Raw_Data_RKI-2024/28816256.²⁶

https://doi.org/10.6084/m9.figshare.28816256.v1.²⁶

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Acknowledgment

We want to thank Shafira Zakia Rahma, Yesica Dea Cahyani, Fiorentina Desmonda Tabita, Aisyah Mardiani, and Dava Tiara Dewi for their assistance in this research. All names and affiliations have agreed to be included in this published article.

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13. Meissner TB, Schulze HS, Dale SM: Immune editing: overcoming immune barriers in stem cell transplantation. Curr. Stem Cell Rep. 2022 Dec; 8(4): 206–218. PubMed Abstract | Publisher Full Text | Free Full Text
14. Damayanti RH, Rusdiana T, Wathoni N: Mesenchymal stem cell secretome for dermatology application: a review. Clin. Cosmet. Investig. Dermatol. 2021 Oct 5; 14: 1401–1412. PubMed Abstract | Publisher Full Text | Free Full Text
15. Daneshmandi L, Shah S, Jafari T, et al.: Emergence of the stem cell secretome in regenerative engineering. Trends Biotechnol. 2020 Dec 1; 38(12): 1373–1384. PubMed Abstract | Publisher Full Text | Free Full Text
16. O’Neill EN, Ansel JC, Kwong GA, et al.: Spent media analysis suggests cultivated meat media will require species and cell type optimization. npj Science of Food. 2022 Sep 29; 6(1): 46. PubMed Abstract | Publisher Full Text | Free Full Text
17. Li P, Song Z, Yao Y, et al.: Preimplantation genetic screening with spent culture medium/blastocoel fluid for in vitro fertilization. Sci. Rep. 2018 Jun 18; 8(1): 9275. PubMed Abstract | Publisher Full Text | Free Full Text
18. Budi HS, Handajani J, Amir LR, et al.: Nanoemulgel Development of Stem Cells from Human Exfoliated Deciduous Teeth-Derived Conditioned Medium as a Novel Nanocarrier Growth Factors. Eur. J. Dent. 2025 Apr 23. PubMed Abstract | Publisher Full Text
19. Futagami A, Ishizaki M, Fukuda Y, et al.: Wound healing involves induction of cyclooxygenase-2 expression in rat skin. Lab. Investig. 2002 Nov; 82(11): 1503–1513. PubMed Abstract | Publisher Full Text
20. Jayaraman P, Nathan P, Vasanthan P, et al.: Stem cells conditioned medium: a new approach to skin wound healing management. Cell Biol. Int. 2013 Oct; 37(10): 1122–1128. PubMed Abstract | Publisher Full Text
21. Shelke MB, Godage RK, Mankar SD: Nanoemulgel as Recent Drug Delivery System: Updated Review. Asian Journal of Research in Pharmaceutical Sciences. 2024 Sep 19; 14(3): 327–330. Publisher Full Text
22. Mok ZH: The effect of particle size on drug bioavailability in various parts of the body. Pharmaceutical Science Advances. 2023 Nov 23; 2: 100010–100031. Publisher Full Text
23. Sharma MU, Arjariya S, Chouksey R, et al.: A review: formulation and evaluation of pharmaceutical gel. Journal of Pharmaceutical Negative Results. 2022 Oct 3; 1344–1362.
24. Ting TC, Rahim NFA, Zaudin NAC, et al.: Development and Characterization of Nanoemulgel Containing Piper betle Essential Oil as Active Ingredient. IOP Conference Series: Earth and Environmental Science. 2020; 596: 012032–012037. Publisher Full Text
25. Handajani J, Susilowati H, Cahyani YD, et al.: Effect of Ananas comosus nanoemulgel on traumatic ulcers in the inflammatory phase. J. Taibah Univ. Med. Sci. 2025 Mar 13; 20(2): 201–208. Publisher Full Text | PubMed Abstract | Free Full Text
26. Handajani J: Raw Data RKI-2024. Dataset. 2025. Publisher Full Text Reference Source

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¹ Department of Oral Biology, Faculty of Dentistry, Universitas Gadjah Mada, Yogyakarta, Indonesia
² Department of Oral Biology, Faculty of Dentistry, Airlangga University, Surabaya, Indonesia
³ Department of Oral Biology, Faculty of Dentistry, Indonesia University, Jakarta, Indonesia

Juni Handajani
Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Project Administration, Validation, Writing – Original Draft Preparation

Hendrik Setia Budi
Roles: Conceptualization, Funding Acquisition, Project Administration, Resources, Supervision, Writing – Review & Editing

Lisa R. Amir
Roles: Conceptualization, Investigation, Writing – Review & Editing

Sri Angky Soekanto
Roles: Conceptualization, Investigation, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

This research received funding from Indonesian Collaborative Research Program (Program Riset Kolaborasi Indonesia) Grant No. 1917/UN1/DITLIT/PT.01.03/2024 between Universitas Airlangga, Universitas Gadjah Mada, and Universitas Indonesia.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Handajani J, Setia Budi H, R. Amir L and Angky Soekanto S. Effect of Stem Cells from Human Exfoliated Deciduous Teeth Spent Medium (SHED-SM) on the Healing of Traumatic Buccal Mucosal Ulcers [version 1; peer review: 2 approved]. F1000Research 2025, 14:510 (https://doi.org/10.12688/f1000research.164339.1)

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Reviewer Report 10 Oct 2025

Zahreni Hamzah, University of Jember, Jember, Indonesia

Approved

https://doi.org/10.5256/f1000research.180835.r419329

Tittle. This title is already quite solid, clear, interesting, and relevant in the context of current research in the field of stem cells.

Abstract. The abstract you have written is quite good, clear, and concise.

... Continue reading

Tittle. This title is already quite solid, clear, interesting, and relevant in the context of current research in the field of stem cells.

Abstract. The abstract you have written is quite good, clear, and concise.

Introduction
1. Regenerative therapies using stem cells are currently being developed to accelerate wound healing (written twice, please remove one instance).
2. SHED-SM nanoemulgel (at the end of the introduction), please clarify the nanoemulgel in relation to secretome therapy.
3. Provide a brief explanation of the mechanism of action of secretomes in the wound healing process, with more specific details on wound healing.

Material and Methods
This section is described in great detail and methodologically, providing a clear description of the experimental design and protocols used, including cell isolation, cytotoxicity testing, animal experiments, and histological analysis. However, additional explanation is needed regarding the randomization of animal grouping.

Results
This study presents clear results, relevant images, accurate statistical analysis, and in-depth evaluation of the network, as well as measurement of relevant parameters, indicating that SHED-SM nanoemulgel can accelerate the healing of traumatic ulcers more effectively. This study has the potential to make a significant contribution to the development of regenerative therapies for wound healing. However, further discussion is needed on the role of VEGF and COX-2 in the wound healing process.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Oral Biology, Physiology, Physio-Geriatry Oromaxillofacial, Stress

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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Reviewer Report 08 Oct 2025

Dhona Afriza, Baiturrahmah University, Sumatera Barat, Indonesia

Approved

https://doi.org/10.5256/f1000research.180835.r415719

The study is relevant and well designed; however, some clarifications are needed. The statistical analysis is only described as “ANOVA,” without specifying the exact type used, post hoc procedures, or assumption tests. These details should be reported in the Methods ... Continue reading

The study is relevant and well designed; however, some clarifications are needed. The statistical analysis is only described as “ANOVA,” without specifying the exact type used, post hoc procedures, or assumption tests. These details should be reported in the Methods section, while the Results should present outcomes only. In addition, the MTT assay is presented as a cytotoxicity test, but the data reflect cell viability; given the focus on fibroblast survival, it would be clearer to describe this as a cell viability test. Clarifying these points will improve transparency and strengthen the validity of the conclusions.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Biology oral, molecular biology, molecular dynamics, oral cancer

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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VERSION 1 PUBLISHED 21 May 2025

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Version 1 21 May 25	read	read

Dhona Afriza, Baiturrahmah University, Sumatera Barat, Indonesia
Zahreni Hamzah, University of Jember, Jember, Indonesia

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Reviewer Report

1 Views

10 Oct 2025 | for Version 1

Zahreni Hamzah, University of Jember, Jember, Indonesia

1 Views Cite this report Responses(0)

Approved

Tittle. This title is already quite solid, clear, interesting, and relevant in the context of current research in the field of stem cells.

Abstract. The abstract you have written is quite good, clear, and concise.

Introduction
1. Regenerative therapies using stem cells are currently being developed to accelerate wound healing (written twice, please remove one instance).
2. SHED-SM nanoemulgel (at the end of the introduction), please clarify the nanoemulgel in relation to secretome therapy.
3. Provide a brief explanation of the mechanism of action of secretomes in the wound healing process, with more specific details on wound healing.

Material and Methods
This section is described in great detail and methodologically, providing a clear description of the experimental design and protocols used, including cell isolation, cytotoxicity testing, animal experiments, and histological analysis. However, additional explanation is needed regarding the randomization of animal grouping.

Results
This study presents clear results, relevant images, accurate statistical analysis, and in-depth evaluation of the network, as well as measurement of relevant parameters, indicating that SHED-SM nanoemulgel can accelerate the healing of traumatic ulcers more effectively. This study has the potential to make a significant contribution to the development of regenerative therapies for wound healing. However, further discussion is needed on the role of VEGF and COX-2 in the wound healing process.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Oral Biology, Physiology, Physio-Geriatry Oromaxillofacial, Stress

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Respond to this report

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Reviewer Report

2 Views

08 Oct 2025 | for Version 1

Dhona Afriza, Baiturrahmah University, Sumatera Barat, Indonesia

2 Views Cite this report Responses(0)

Approved

The study is relevant and well designed; however, some clarifications are needed. The statistical analysis is only described as “ANOVA,” without specifying the exact type used, post hoc procedures, or assumption tests. These details should be reported in the Methods section, while the Results should present outcomes only. In addition, the MTT assay is presented as a cytotoxicity test, but the data reflect cell viability; given the focus on fibroblast survival, it would be clearer to describe this as a cell viability test. Clarifying these points will improve transparency and strengthen the validity of the conclusions.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Biology oral, molecular biology, molecular dynamics, oral cancer

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Respond to this report

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[1] 1. Regezi JA, Sciubba J, Jordan RC: Oral pathology: clinical pathologic correlations. Elsevier Health Sciencesd. California: Elsevier; 7th Ed. 2017.

[2] 2. Ministry of Health Republic Indonesia (Kementerian Kesehatan RI). Riskesdas. Jakarta: Kemenkes; 2018.

[3] 3. Myers S, Curran A: General and oral pathology for dental hygiene practice. Philadelphia: FA Davis; 2014; 45–47.

[4] 4. Bilodeau EA, Lalla RV: Recurrent oral ulceration: Etiology, classification, management, and diagnostic algorithm. Periodontol. 2019 Jun; 80(1): 49–60. PubMed Abstract | Publisher Full Text

[5] 5. Gonzalez AC, Costa TF, Andrade ZD, et al.: Wound healing-A literature review. An. Bras. Dermatol. 2016; 91(5): 614–620. PubMed Abstract | Publisher Full Text | Free Full Text

[6] 6. Goldberg SR, Diegelmann RF: Basic science of wound healing. Switzerland: Springer International Publishing; 2017; 131–136.

[7] 7. Mohd Nor NH, Mansor NI, Mohd Kashim MI, et al.: From Teeth to Therapy: A Review of Therapeutic Potential within the Secretome of Stem Cells from Human Exfoliated Deciduous Teeth. Int. J. Mol. Sci. 2023 Jul 21; 24(14): 11763. PubMed Abstract | Publisher Full Text | Free Full Text

[8] 8. Lei T, Zhang X, Du H: Characteristics, classification, and application of stem cells derived from human teeth. Stem Cells Int. 2021; 2021: 1–11. PubMed Abstract | Publisher Full Text | Free Full Text

[9] 9. Al-Hadi MA: Combination of stem cell-derived secretome from human exfoliated deciduous teeth with Yemeni Sidr honey on cell viability and migration: an in vitro study. BDJ open. 2024 Mar 13; 10(1): 1–16. PubMed Abstract | Publisher Full Text

[10] 10. El Moshy S, Radwan IA, Rady D, et al.: Dental stem cell-derived secretome/conditioned medium: The future for regenerative therapeutic applications. Stem Cells Int. 2020; 2020: 1–29. PubMed Abstract | Publisher Full Text | Free Full Text

[11] 11. Ren X, Zhao M, Lash B, et al.: Growth factor engineering strategies for regenerative medicine applications. Front. Bioeng. Biotechnol. 2020 Jan 21; 7(469): 1–9. PubMed Abstract | Publisher Full Text | Free Full Text

[12] 12. Derks LL, van Boxtel R : Stem cell mutations, associated cancer risk, and consequences for regenerative medicine. Cell Stem Cell. 2023 Oct 12; 30(11): 1421–1433. PubMed Abstract | Publisher Full Text | Free Full Text

[13] 13. Meissner TB, Schulze HS, Dale SM: Immune editing: overcoming immune barriers in stem cell transplantation. Curr. Stem Cell Rep. 2022 Dec; 8(4): 206–218. PubMed Abstract | Publisher Full Text | Free Full Text

[14] 14. Damayanti RH, Rusdiana T, Wathoni N: Mesenchymal stem cell secretome for dermatology application: a review. Clin. Cosmet. Investig. Dermatol. 2021 Oct 5; 14: 1401–1412. PubMed Abstract | Publisher Full Text | Free Full Text

[15] 15. Daneshmandi L, Shah S, Jafari T, et al.: Emergence of the stem cell secretome in regenerative engineering. Trends Biotechnol. 2020 Dec 1; 38(12): 1373–1384. PubMed Abstract | Publisher Full Text | Free Full Text

[16] 16. O’Neill EN, Ansel JC, Kwong GA, et al.: Spent media analysis suggests cultivated meat media will require species and cell type optimization. npj Science of Food. 2022 Sep 29; 6(1): 46. PubMed Abstract | Publisher Full Text | Free Full Text

[17] 17. Li P, Song Z, Yao Y, et al.: Preimplantation genetic screening with spent culture medium/blastocoel fluid for in vitro fertilization. Sci. Rep. 2018 Jun 18; 8(1): 9275. PubMed Abstract | Publisher Full Text | Free Full Text

[18] 18. Budi HS, Handajani J, Amir LR, et al.: Nanoemulgel Development of Stem Cells from Human Exfoliated Deciduous Teeth-Derived Conditioned Medium as a Novel Nanocarrier Growth Factors. Eur. J. Dent. 2025 Apr 23. PubMed Abstract | Publisher Full Text

[19] 19. Futagami A, Ishizaki M, Fukuda Y, et al.: Wound healing involves induction of cyclooxygenase-2 expression in rat skin. Lab. Investig. 2002 Nov; 82(11): 1503–1513. PubMed Abstract | Publisher Full Text

[20] 20. Jayaraman P, Nathan P, Vasanthan P, et al.: Stem cells conditioned medium: a new approach to skin wound healing management. Cell Biol. Int. 2013 Oct; 37(10): 1122–1128. PubMed Abstract | Publisher Full Text

[21] 21. Shelke MB, Godage RK, Mankar SD: Nanoemulgel as Recent Drug Delivery System: Updated Review. Asian Journal of Research in Pharmaceutical Sciences. 2024 Sep 19; 14(3): 327–330. Publisher Full Text

[22] 22. Mok ZH: The effect of particle size on drug bioavailability in various parts of the body. Pharmaceutical Science Advances. 2023 Nov 23; 2: 100010–100031. Publisher Full Text

[23] 23. Sharma MU, Arjariya S, Chouksey R, et al.: A review: formulation and evaluation of pharmaceutical gel. Journal of Pharmaceutical Negative Results. 2022 Oct 3; 1344–1362.

[24] 24. Ting TC, Rahim NFA, Zaudin NAC, et al.: Development and Characterization of Nanoemulgel Containing Piper betle Essential Oil as Active Ingredient. IOP Conference Series: Earth and Environmental Science. 2020; 596: 012032–012037. Publisher Full Text

[25] 25. Handajani J, Susilowati H, Cahyani YD, et al.: Effect of Ananas comosus nanoemulgel on traumatic ulcers in the inflammatory phase. J. Taibah Univ. Med. Sci. 2025 Mar 13; 20(2): 201–208. Publisher Full Text | PubMed Abstract | Free Full Text

[26] 26. Handajani J: Raw Data RKI-2024. Dataset. 2025. Publisher Full Text Reference Source

Effect of Stem Cells from Human Exfoliated Deciduous Teeth Spent Medium (SHED-SM) on the Healing of Traumatic Buccal Mucosal Ulcers

Abstract

Introduction

Materials and methods

Results

Conclusion

Keywords

Introduction

Material and methods

Isolation of SHED and SHED-SM

Cytotoxicity test of SHED-SM against NIH-3T3 fibroblast cells

Treatment of experimental animals

Tissue staining

Observation of neutrophil infiltration and immunohistochemistry

Results

Figure 1. Image of NIH-3T3 fibroblast cells (A) and NIH-3T3 fibroblast cells after treatment using nanoemulgel spent medium SHED (SHED-SM) H1 (B), H3 (C) and H5 (D).

Figure 2. The highest percentage of NIH-3T3 fibroblast cell viability was shown after the application of nanoemulgel SHED-H1spent medium (SHED-SM H1) and significant differences in the comparison of each concentration (*) (p<0.05).

Figure 3. Clinical evaluation of traumatic ulcer after application of nanoemulgel spent medium SHED (SHED-SM) on the buccal mucosa of rats.

Figure 4. Evaluation of the wound healing process after application of nanoemulgel spent medium SHED (SHED-SM) (treatment) and control.

Figure 5. Neutrophil infiltration (A) and COX-2 expression (B) after application of nanoemulgel spent medium SHED (SHED-SM) on the 3rd day.

Figure 6. The number of neutrophils was highest on the 1st day, while the positive expression of COX-2 on the 3rd day decreased until the 14th.

Figure 7. Evaluation of Collagen-1 (Col-1) expression on the 3rd day after treatment showed a brown color in the extracellular matrix area and fibroblasts in the ulcer area.

Figure 8. The highest Col-1 expression was shown on the 5th day after treatment and control, and the expression decreased until the 14th day.

Figure 9. Evaluation of VEGF expression on the 5th day after application of nanoemulgel spent medium SHED (SHED-SM).

Figure 10. VEGF expression increased until the 5th day in the control and treatment groups, then decreased until the 14th day.

Discussion

Data availability

Underlying data

Acknowledgment

References

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Figure 5. Neutrophil infiltration (A) and COX-2 expression (B) after application of nanoemulgel spent medium SHED (SHED-SM) on the 3^rd day.

Figure 6. The number of neutrophils was highest on the 1^st day, while the positive expression of COX-2 on the 3^rd day decreased until the 14^th.

Figure 7. Evaluation of Collagen-1 (Col-1) expression on the 3^rd day after treatment showed a brown color in the extracellular matrix area and fibroblasts in the ulcer area.

Figure 8. The highest Col-1 expression was shown on the 5^th day after treatment and control, and the expression decreased until the 14^th day.

Figure 9. Evaluation of VEGF expression on the 5^th day after application of nanoemulgel spent medium SHED (SHED-SM).

Figure 10. VEGF expression increased until the 5^th day in the control and treatment groups, then decreased until the 14^th day.