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 secretome ⁷ 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.

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.

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 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 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 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 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.

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

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

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.

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.