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. 2019 Nov 21;16(6):675–684. doi: 10.1007/s13770-019-00227-6

Chemical Regeneration of Wound Defects: Relevance to the Canine Palatal Mucosa and Cell Cycle Up-Regulation in Human Gingival Fibroblasts

Kyungho Lee 1, Heithem Ben Amara 2, Sang Cheon Lee 3, Richard Leesungbok 4, Min Ah Chung 1, Ki-Tae Koo 2,✉, Suk Won Lee 4,✉
PMCID: PMC6879698  PMID: 31824829

Abstract

Background:

Trichloroacetic acid (TCA) is an agent widely applied in dermatology for skin regeneration. To test whether TCA can offer an advantage for the regeneration of oral soft tissue defects, the cellular events following TCA application were explored in vitro and its influence on the oral soft tissue wound healing was evaluated in a canine palate model.

Methods:

The cytotoxicity and growth factor gene expression in human gingival fibroblasts were tested in vitro following the application of TCA at four concentrations (0.005%, 0.05%, 0.5% and 1%) with different time intervals (0, 3, 9 and 21 h). One concentration of TCA was selected to screen the genes differentially expressed using DNA microarray and the associated pathways were explored. TCA was injected in open wound defects of the palatal mucosa from beagle dogs (n = 3) to monitor their healing and regeneration up to day 16-post-administration.

Results:

While the 0.5–1% concentration induced the cytoxicity, a significantly higher expression of growth factor genes was observed after 3 and 9 h following the 0.5% TCA application in comparison to other groups. DNA microarray analysis in 0.5% TCA group showed 417 genes with a significant 1.5-fold differential expression, involving pathways of cell cycle, FoxO signaling, p53 signaling, ubiquitin mediated proteolysis and cAMP signaling. In vivo results showed a faster reepithelialization of TCA-treated wounds as compared to spontaneous healing.

Conclusion:

TCA promoted the healing and regeneration of oral soft tissue wound defects by up-regulating the cell cycle progression, cell growth, and cell viability, particularly at a concentration of 0.5%.

Electronic supplementary material

The online version of this article (10.1007/s13770-019-00227-6) contains supplementary material, which is available to authorized users.

Keywords: Chemical regeneration, Wound defect, Oral mucosa, Canine palate, Trichloroacetic acid, Cell cycle

Introduction

All tissues are endowed with the capacity to repair themselves. However, they differ greatly in the degree of injury they can withstand. Although oral mucosa and cutaneous systems are able to restore their functions even after major injury, wound healing does not always result in a restitutio ad integrum and may end up with a scar tissue. This is not only true for the classical skin injury, but also relevant for the healing following periodontal surgery [1]. While it is generally reported that wound healing in the oral cavity is faster than healing in skin [2], it appears that under conditions where a considerable amount of new connective tissue synthesis is required, oral healing is slower then dermal repair [3]. In the setting of oral excisional injuries, a factor of healing delay might be the high degree of inflammatory stimulation secondary to the never-ending insult of potentially harmful substances and pathogenic organisms at the oral environment [4].

Chemical regeneration of skin scars (CROSS) is an ablative modality through which the partial removal of epidermis by chemical agents triggers the growth of new skin to replace scarred or aged tissue. Experience has shown that chemical peels such as trichloroacetic acid (TCA) may have a therapeutic benefit when injury, disease or ageing cause cosmetic changes in the skin [5]. The application of TCA adjusted at various concentrations induces tissue coagulation due to acid frosting and prompts the reconstruction of the injured skin [6]. Because TCA application is a simple treatment, it is widely applied in clinical settings, and several indications have yet been explored including acne scars, skin rejuvenation, burn scars, striae distensae, androgenetic alopecia, melasma and acne vulgaris [7]. This popularity of TCA-based approaches is supported by the evidence accumulated from the in vitro and in vivo trials investigating its effects on cutaneous wound healing [8]. Promotion of collagen formation [9, 10], increased angiogenesis [11] and activation of growth factors synthesis [12, 13] are among the cellular events that characterize the healing of injuries treated with TCA.

In contrast, the application of TCA to hasten the healing of oral wounds has been restricted. A reason behind the paucity of the data on TCA-based oral treatments is the high acidity of the chemical peels which might hamper the oral structures. Nonetheless, reports on the beneficial use of TCA as a remedy for oral mucosal lesions [14] exemplifies the potential of this chemical agent purposed as an oral mucosal therapy. Data are missing in terms of (1) the biochemical events downstream of the TCA’s interaction with the oral soft tissue and (2) the biological potential of TCA on oral mucosa wound defects in pre-clinical settings.

The aim of this study was to test whether TCA adjusted at different concentration contribute or not to the promotion of cell viability, and to verify if the application of TCA in an animal model of oral soft tissue accelerates the regeneration of wound defects.

Materials and methods

Cell culture

Human gingival fibroblasts (HGFs) were purchased from ATCC (ATCC® PCS-201-018™, Manassas, VA, USA). The cells were floated on Dulbecco’s minimum essential medium (DMEM, Invitrogen Corporation, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS, Sigma-Aldrich Co., St Louis, MO, USA) and antibiotics. Suspended fibroblasts were transferred and incubated in a humidified incubator at 95% air, 5% CO2, and 37 °C. This study was carried out using cultured 2–3 cycle human gingival fibroblasts.

Cytotoxicity assessment

TCA with various concentrations was applied HGFs under different time conditions to evaluate their cytotoxicity (Supplementary Table 1). Concentrations of 0%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5% and 1% TCA were applied to the cultured cells. After 21 h, the cell viability was assessed using phenol red-MTT assay (CellQuanti-MTT™ Cell Viability Assay Kits; Bioassay Systems, Hayward, CA, USA). The pH was 8.53 ± 0.04 for 0.005% TCA solution, 8.36 ± 0.02 for 0.05% TCA solution, 6.48 ± 0.34 for 0.5% TCA solution and was 3.76 ± 0.12 for 1% TCA solution. Because a TCA concentration superior to 1% was very acidic, the maximum value was set to 1%. The appropriate application time was determined by evaluating the cytotoxicity using MTT assay immediately, 3, 9, and 21 h after the application of 0.005%, 0.05%, 0.5%, and 1% TCA solutions in HGF.

RT PCR

RT PCR was performed to screen the expression of 9 key genes related to wound healing (EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FGF-1, fibroblast growth factor-1; FGF-2, fibroblast growth factor-2; KGF, keratinocyte growth factor; PDGF-β, platelet derived growth factor-β; TGF-α, transforming growth factor-α; TGF-β1, transforming growth factor-β1; VEGF, vascular endothelial growth factor).

For this purpose, total RNA was extracted using TRIZOL lysis solution (TRIZOL® Reagent, GIBCO BRL, Carlsbad, CA, USA). Probe labeling was performed using cyanine dye and concentration was processed. To identify changes in expression level, the oligonucleotide primer sequences of the growth factors were confirmed (Supplementary Table 2). The synthesized cDNA samples were heated at 94 °C for 5 min and amplified at 94 °C for 1 min, 59 °C for 1 min, and 72 °C for 1.5 min for 35 cycles. PCR products were extracted with ethidium bromide-containing Tris–acetate–EDTA buffer (Sigma-Aldrich Co., St Louis, MO, USA), which were then extracted on 2% agarose gel and separated under ultraviolet light. β-actin was identified as a loading control to confirm the significance of the experiment.

DNA microarray and KEGG pathway analysis

In order to determine the upregulated and downregulated genes secondary to the application of TCA, DNA microarray and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses were performed 9 h after HGF were exposed to a solution of 0.5% TCA. Total RNA was first extracted with TRIZOL lysis solution (TRIZOL® Reagent, GIBCO BRL, Carlsbad, CA, USA). Probe labeling was performed using cyanine dye and concentration was proceeded. Affymetrix gene chip (Affymetrix GeneChip®, Affymetrix, Santa Clara, CA, USA) was used and images were extracted with a genechip scanner (Affymetrix GeneChip® Scanner 3000 7G, Affymetrix, Santa Clara, CA, USA). Data were extracted using a dedicated software (Affymetrix GeneChip® Command Console®, Affymetrix, Santa Clara, CA, USA) and standardized (Expression Console 1.4, Affymetrix, Santa Clara, CA, USA). Genes showing significant differences in expression by more than 1.5-fold were identified through comparison of control group and experimental group with Basic Differentially Expressed Gene (DEG) finding. KEGG pathway analysis was used to map the expression of the DNA chip, and the change in gene expression level was determined at the pathway level according to experimental conditions.

Real-time PCR

After 0.5% TCA concentration was applied to human gingival fibroblasts, quantitative real-time PCR was performed to confirm differences in expression levels of genes. The aim was to confirm the significance of up-regulation or down-regulation of a total of twenty-six genes identified in DNA microarray. The gene numbers and predesigned primer sets for the selected genes were used (Supplementary Table 3). Human gingival fibroblasts were seeded in 24-well tissue culture plates of the control (CON) and the 0.5% TCA treatment group (TCA) at a density of 1 × 104 cells/well and cultured for 9 h at 37 °C in 5% CO2. The total RNA extraction and quantitative real-time PCR were performed as previously described [15]. Relative expression levels were analyzed by normalizing the experimental values to those of the internal control, GAPDH. The relative expression levels obtained on the TCA group are presented as fold-change relative to the levels obtained on the CON group.

In vivo experiment

In order to pre-clinically verify the biologic potential of TCA on the healing of oral mucosa wounds, 1-year old, 10 kg-weighing beagle dogs (N = 3) were employed following approval by the Institutional Animal Care and Use Committee, Cronex, Hwasung, Korea (CRONEX-IACUC 201712004). All surgical and follow-up interventions were performed under general anesthesia induced by the injection of tiletamine/zolazepam (0.1 mg/kg, ZOLETIL® 100, VIRBAC FRANCE, Carros, France), xylazine (2.3 mg/kg, Rompun Inj., BAYER KOREA, Seoul, Korea), and atropine sulfate (0.05 mg/kg, Atropine sulfate Inj. 10 mg, JE IL PHARMACEUTICAL, Daegu, Korea). Further analgesia was given for 3 days following surgical procedures (Zipan Inj. 100 mg, ILSUNG Phamaceuticals, Seoul, Korea).

Surgical procedures: a previously described defect model of the masticatory mucosa of the palate was adapted for the purpose for the present study [16, 17]. In each animal, standardized cylindrical wounds with a 6 mm-diameter were created by harvesting full thickness palatal mucosa using a biopsy punch (Acuderm inc., Fort Lauderdale, FL, USA) inserted until contact with the palatal bone was reached. Three defects were symmetrically allocated in each hemi-palate to obtain a total of six wound defects per animal. Treatment of oral wounds and follow-up: at day 1-post-surgery, the following treatment modalities were randomly applied to the dogs. Spontaneous healing: No TCA administered. (control group; SH; n = 4 defects). TCA application: four concentrations were tested (0.005%, 0.05%, 0.5% and 1%; n = 2 defects for each concentration). A single administration of a 1 mL-volume of TCA solutions was performed using disposable syringes individually for each defect (Kovax-Syringe® 1 mL, Korea Vaccine Co. LTD, Ansan, Gyeonggi, Korea). Immediately after surgery (day 0) and at post-surgery days 1, 3, 11, and 17, clinical photographs were taken at an angle of 90° to the upper jaw and a distance of 80 cm using identical resolution, exposure and enlargement [18]. Wound healing monitoring: photographical recordings of the wounds were imported into a software program (ImageJ, NIH, Bethesda, MD, USA). The surface covered by an unkeratinized tissue was digitally marked on the screen to calculate the relative Wound Area expressed in mm2 and the percentage of Wound Reepithelialization [18].

Statistical analysis

Cytotoxicity tests were performed independently three times; RT-PCR, DNA microarray, and real-time PCR was performed independently five times; three beagles were used for animal experiment; and the mean and standard deviation were calculated. One-way ANOVA was used for the cytotoxicity tests for TCA concentration, and two-way ANOVA test with Tukey’s multiple comparison test was for post hoc analysis were applied to evaluate the interaction between incubation periods and TCA concentrations. For the real-time PCR results, student’s t test was performed to compare the mean values of the control and experimental groups. SPSS 18.0 software program (SPSS Inc., Chicago, IL, USA) was used for all statistical analyses with p < 0.05 set for significance.

Results

Cytotoxicity assessment

Twenty-one hours following the application of 0%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5% and 1% TCA on HGF, increasing cytotoxicity was observed when the concentration of TCA solution raised (Fig. 1A). Whilst almost all cells were killed with 1% and 0.5% TCA concentrations, the cytotoxicity did not exceed 50% for concentration of 0.1% or lower. Statistical difference was depicted by all groups except between 0.005, 0.01 and 0.05% and between 0 and 0.001% (p < 0.001).

Fig. 1.

Fig. 1

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A Cytotoxicity (%) test results of trichloroacetic acid (TCA)-treated human gingival fibroblasts (HGFs) using the MTT assay after 21 h incubation at various concentrations of TCA. Note that, under identical letters, the groups show no significant differences. As a result, significant differences were noted between a (0 and 0.001%), b (0.005, 0.01 and 0.05%), c (0.1%), d (0.5%) and e (1%). One-way ANOVA (p < 0.05). B Cytotoxicity (%) test results of TCA-treated HGFs using the MTT assay after 0, 3, 9, and 21 h incubation at various concentrations of TCA. Significant differences were noted between the TCA concentration (%) groups of 0.005, 0.05, 0.5, and 1. Two-way ANOVA (p < 0.05)

When the cytotoxicity was sequentially assessed at 0, 3, 9, and 21 h after the application of 0.005%, 0.05%, 0.5%, and 1% TCA solutions, at least 80.1% (± 1.1) of the HGF were killed after 9 h for a TCA concentration of 0.5% or higher. A longer time of TCA application resulted in a higher cytotoxicity. The interaction between the TCA concentration and the application time was statistically confirmed by the Tukey’s multiple comparison tests (Fig. 1B and Supplementary Table 4).

RT-PCR

In comparison with 0%, 0.005%, 0.05% TCA concentration, the gene expression of EGF, EGFR, FGF-1, FGF-2, KGF, PDGF-β, TGF-α, TGF-β1 and VEGF was higher with the 0.5% TCA group (Fig. 2), leading to the selection of 0.5% concentration of TCA as the main experimental group for following experiments in this study. This upregulation was more evident at 3 and 9 h following the application of 0.5% TCA group than the gene expression observed at 0 and 21 h. Besides the weak gene expression featured by the 0% TCA group, an up-regulation of PDGF-β, TGF-α, TGF-β1, and VEGF genes was observed with the 0.005% TCA group, albeit not significant. In 0.05% TCA group, PDGF-β, TGF-α, TGF-β1, VEGF, EGF and EGFR gene expression remained low.

Fig. 2.

Fig. 2

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RT-PCR results of the expression of various growth factors in trichloroacetic acid (TCA)-treated human gingival fibroblasts (HGFs) after 0, 3, 9, and 21 h incubation at 0, 0.005, 0.05, and 0.5% concentrations of TCA. Note that the 0.05% TCA group after 3- and 9 h incubation induces up-regulation of all the genes tested

DNA microarray and KEGG pathway

Of the 53617 genes analyzed, 417 genes were identified with a significant differential expression up to 1.5-fold (p < 0.05). These genes involved seven significantly enriched pathways related to cell proliferation, growth and regeneration as demonstrated by the KEGG pathway. The expression levels of genes related to cell cycles necessary for cell growth (BUB1, CCNA2, CCNB1, CCNB2, CDC20, CDC25C, CDK1, GADD45G, MAD2L1, PLK1, PTTG1, TTK, DBF4) increased in the HGF treated with 0.5% TCA solution whereas SMAD4 gene was down-regulated (Supplementary Fig. 1). The upregulation of genes related to the FoxO signaling pathway involved CCNB1, CCNB2, PLK1, PLK4 and GADD45G whereas, the expression levels of SMAD4 gene was decreased in the 0.5% TCA group (Supplementary Fig. 2). Six genes related to p53 pathway were upregulated by 0.5% TCA (CCNB1, CCNB2, CDK1, GADD45G, GTSE1, and RRM2) (Supplementary Fig. 3), while the upregulated genes of CDC20, UBE2C and UBE2S belonged to the ubiquitin mediated proteolysis pathway (Supplementary Fig. 4). In protein processing in endoplasmic reticulum pathway, HSPH1, HSPA4L, and DNAJB1 were up-regulated (Supplementary Fig. 5). In addition to the MAPK signaling pathway which featured two upregulated genes FOS and GADD45G (Supplementary Fig. 6), FOS and GNAI3 genes, belonging to the cAMP pathway, exhibited an increase of their expression levels (Supplementary Fig. 7).

Real-time PCR

The real-time PCR indicated that BUB1, CCNA2, CCNB1, CCNB2, CDC20, CDC25C, CDK1, DBF4, DNAJB1, FOS, GADD45G, GNAI3, GTSE1, HMMR, HSPA4L, HSPH1, LMNB1, MAD2L1, PLK1, PLK4, PTTG1, RRM2, SMAD4, TTK, UBE2C and UBE2S mRNA levels were significantly increased. The down-regulation of GNAI1 and SMAD4 mRNA levels did not reach statistical significance (Fig. 3A, B). Also, mRNA levels of selected genes representing major cell cycle progression other than the genes verified by the significant up-regulation in the DNA microarray analysis in this study such as CCNA1, CCND1, CCND2, CCNE1, CCNE2, CDK2, CDK4, CDK6, CDC25A, CDC7, E2F5, ERK1, and ERK2 were significantly increased (Fig. 3C).

Fig. 3.

Fig. 3

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A, B Relative fold change mRNA expression of various genes responsible for cell cycle progression, FoxO signaling, p53 signaling, ubiquitin mediated proteolysis and cAMP signaling in human gingival fibroblasts (HGFs) after 9 h incubation with 0.5% TCA treatment. Note that the selected genes in C represent major cell cycle progression genes other than the ones verified by the significant up-regulation in the DNA microarray analysis in this study. The results are expressed as a ratio to the mRNA levels of the reference gene, GAPDH, followed by a standardization of the Ct (threshold cycle) expressions on the control surface as 1. One-way ANOVA (n = 5). ***: Significant difference (p < 0.001). **: Significant difference (p < 0.01). *: Significant difference (p < 0.05)

In vivo assessment of wound defect regeneration

Immediately following the application of TCA, a white to greyish frosting reaction of wound tissues was observed. The tissue frosting was observed with the concentration 1% and 0.5% while wounds treated with 0.05% TCA and 0.005% TCA displayed an attenuated reaction. Photographic recordings at post-surgery days 3, 11 and 17 demonstrated a constant decrease of the wound areas over time in all groups (Table 1). From post-surgery days 3 to 17, the control group SH displayed the largest mean wound area, while the smallest was observed in group 0.5% TCA very close to concentrations 0.005%, 0.05% and 1%. This difference between the control group SH and TCA-treated defects was clear particularly at post-surgery day 11, translating in a faster reepithelialization (Fig. 4). The 0.005-, 0.05-, 0.5-, and 1% TCA group all showed statistically significant amount of reepithelialization and regeneration of the oral soft tissue wound defects in canine palate compared to the control group SH at post-surgery day 11 (Fig. 5).

Table 1.

The results of the wound area measurement for the control spontaneous healing group (SH) and the experimental groups (0.005%, 0.05%, 0.5% and 1% of TCA). Mean, median and standard deviation (SD) are given for each parameter

D0 D1 D3 D11 D17
Group Mean SD Mean SD Mean SD Mean SD Mean SD
SH 25.12 1.02 23.8 0.72 21.93 0.78 10.15 0.49 0.69 0.98
0.005% 26.29 1.7 22.24 0.27 20.8 0.02 7.18 0.23 0.39 0.79
0.05% 27.89 1.97 22.416 0.42 21.1 0.57 7.31 0.29 0 0
0.5% 25.97 1.68 24.03 1.33 19.75 1.54 6.26 0.9 0 0
1% 27.24 2.07 21.975 1.28 21.18 0.18 6.48 1.16 0.33 0.46
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Results of volume area (mm2) comparing the situation D0: immediately after the surgery. D1: 1 day post-surgery. D3: 3 days post-surgery. D11: 11 days post-surgery. D17: 17 days post-surgery

Fig. 4.

Fig. 4

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A, B Surgical procedure, TCA administration and follow-up. Full-thickness punch biopsies were harvested creating 6 defects on the hard palate mucosa of each dog. C At day-1 post-surgery, TCA solutions were injected in the wound of the masticatory mucosa of the palate using 1 mL-disposable syringes. Immediately after TCA injection, a frosting reaction was observed in the healing tissue of the wound. D Wounds receiving 0.5% displayed an even frosting response while this reaction was less evident in sites treated with 0.005% TCA. E Immediately after surgery (day 0, D0) and at days 1, 3, 11 and 17 post-surgery (D1, D3, D11 and D17), clinical photographs of the wounds were recorded to calculate the remaining wound area. F It was observed that the wound closure and tissue regeneration was faster in the 0.5% TCA group compared to the control group with spontaneous healing (SH). G From the software analysis of the photographs, the epithelialized area (yellow) ratio to the remaining wounds were calculated and the TCA-treated groups all showed superior epithelialization at day-11 post-surgery compared to the control, SH. Scale bar: 10 mm

Fig. 5.

Fig. 5

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Results from the software analysis of the photographs analyzing the epithelialized area ratio to the remaining wounds at day-11 post-surgery. The TCA-treated groups of 0.005%, 0.05%, 0.5%, and 1% all showed superior epithelialization at day-11 post-surgery compared to the control, SH. One-way ANOVA (n = 3). *: Significant difference (p < 0.05)

Discussion

Understanding the biological mechanisms that underly the process of wound healing is a litmus test for candidate therapies aimed at promoting the repair of skin or oral mucosa deformities. The present study provides insights on the cellular events triggered by TCA on human gingival fibroblasts in an attempt to answer the hypothesis questioning the biological potential of this chemical agent on oral soft tissues defect regeneration. First, cytotoxicity experiments were conducted with the objective to screen the appropriate concentration and duration of TCA application. Because concentrations of 1% or above were previously linked with a high rate of cell apoptosis [12], 1% TCA concentration was set as a limit. The apoptosis of 100% of the HGF 3 h following the incubation with the 1% TCA is therefore an expected finding in the present study and may be correlated to its low pH (3.76) [11]. This early phase cytotoxicity was less evident for 0.5% TCA, while concentrations below 0.05% appeared to provoke limited HGF death even after 21 h of incubation.

In clinical dermatology, the full necrosis of the superficial layers of the skin secondary to TCA application is followed by the reconstruction of the epidermis and papillary dermis through a process of wound healing [12]. In order to verify whether TCA is endowed with the ability to activate oral mucosa healing, the gene expression of HGF incubated with 0%, 0.005%, 0.05%, and 0.5% TCA concentrations was investigated for several durations varying between 0 and 21 h. Specifically, the expression levels of growth factors such as EGF, FGF1, FGF2, PDGF-β, KGF, TGF-β1 and VEGF were targeted due to their pivotal role in the wound healing process. For instance, PDGF-β has been demonstrated to induce the proliferation of vascular endothelial cells, vascular smooth muscle cells, fibroblasts and glial cells [19], while VEGF plays a role in stimulating angiogenesis [20]. Another example is the contribution of KGF, EGF, FGF1 and FGF221 to cells proliferation as well as TGF-β1 which promotes the production of extracellular matrix such as collagen [21]. A major finding in the present study was the upregulation of the above-mentioned growth factors by 0.5% TCA particularly after 9 h of incubation. In line with similar records from skin settings [12], this result claims the potential of 0.5% TCA in promoting the early phase of oral mucosa healing.

Consistent with this assumption were the KEGG-identified pathways, which included cell regulation, proliferation, and differentiation among others. For example, several genes involved in the promotion of cell activity through cell cycle progression, mitosis regulation, and DNA amplification and cleavage [22] were significantly up-regulated. These included BUB1, cyclin A2, cyclin B1, cyclin B2, CDK1, Cdc20, CDC25C, Dbf4, GADD45, Mad2, Plk1, PTTG1, and PTTG4. From the results of the DNA microarray analysis, we verified up-regulation of major cell cycle progression genes such as cyclin A1, cyclin D1, cyclin D2, cyclin E1, cyclin E2, Cdc25A, CDK2, CDK4, CDK6, Cdc25A, Cdc7, E2F5, ERK1 and ERK2 to a relatively lower levels compared to those of the determined genes above. These major cell cycle genes were also determined to be significantly up-regulated as verified by the real time PCR in this study, suggesting, as far as we know, for the first time that TCA administration to human fibroblasts under specific conditions up-regulates the major cell cycle gene expression.

Elevated mRNA expression of GADD45G, GTSE1, MAD2L1, PTTG1, TKK and DBF4 in HGF was further validated by real time PCR. GADD45G is released in cells upon stress reaction, leading to DNA repair or apoptosis [23]. The up-regulation of GADD45G is believed to be in association with the cytotoxic response of TCA. In a similar fashion, the higher expression of GTSE1, which induces apoptotic cell death after DNA damage, appears to be linked to the cytotoxic reaction to TCA [24]. On the other hand, the differential expression of MAD2L1, PTTG1, TKK and DBF4 supports the potential of TCA to fasten the wound healing. MAD2L1 is a component of the mitotic spindle, which inhibits mitosis progression until all chromosomes are aligned in the middle of mitosis. The deficiency of this protein in cancer patients highlights its role in the normal cell division [25]. The upregulation of PTTG1, a cecilin protein necessary to chromosome division [26]. Purport the increase of mitosis triggered by TCA. The elevated expression levels of TTK, which regulates the chromosomal arrangement in mitogens [27], and DBF4, which interact with cell cycle-related genes [28], may be associated with the increased cell activity.

Of the differentially expressed genes identified in the present study, several belong to pathways linked with cell recovery and regeneration. One example is FoxO signaling pathway which regulates cell cycle and cell metabolism [29]. Highly upregulated genes include also the p53 pathway, a major actor of cell cycle, cell death, and DNA repair [30]. In ubiquitin mediated proteolysis pathway, which plays a role in the regeneration process [31], genes such as UBE3C and UBE2S were up-regulated. MAPK and cAMP are other examples of activated pathways which sustain the assumption that HGF triggered by 0.5% TCA was not restricted to features of cell toxicity. Overall, the increased activity of cell regulation, proliferation and differentiation strengthen the belief that TCA may fasten the wound healing of oral mucosa.

In order to verify this hypothesis, TCA was tested in an animal model of critical size oral soft tissue wound defect. Because the morphological features of oral epithelium and molecular patterns in connective tissue of the healing oral palate from dog are the most similar to humans as compared to other species including rodents and pigs [32, 33], the canine palatal mucosa was selected as a wounding substrate. Considering the excisional nature of the wounds, healing requires the closure of the wound site by formation of new connective tissue and coverage by epithelium, events that might be hampered not only by the direct insults from the oral commensal flora but also by the full-thickness of the excisions [3]. Thus, TCA was administrated to test whether it improves or not the wound repair. Due to the acidic nature of the solutions, a frosting reaction was instantly detected in the excisions following the application of the solutions, although substantially varying between the different TCA concentrations tested. Histologically, frosting translates in a coagulation of the proteins and cells that constitute the tissues exposed to the acidic solution. Thus, the intensity of frosting correlates directly with the penetration’s depth of the chemical peel [34]. Although the concentration used in the present experiment may appear low as compared to the usual dosages applied for dermatological indications (starting from 10%), the frosting response is explained by the absence of the epithelial barrier.

Then, wound defects were monitored during the first and second week following the excisional wounding, time-points at which the healing masticatory mucosa displayed typical signs of erythema/edema and wound closure respectively. Hence, as illustrated by the photographical recordings, injuries treated with various TCA solutions showed a tendency toward a faster recovery of inflammation and a hastened reepithelialization as compared to the control group SH. In particular, the 0.5% TCA-group wounds exhibited the smallest mean wound area at post-surgery day 11 and were fully reepithelialized at post-surgery day 17. The latter result confirms the in vitro outcomes of this study asserting the biological potential of TCA for the purpose of oral mucosa healing and regeneration acceleration. In contrast, an unexpected finding was the smaller wound area observed with a concentration of TCA as small as 0.005% in comparison to the control SH wounds. An explanation could be the nociceptive stimulation by the acidic solution triggering the licking by the animals’ tongue, a factor of healing promotion [35]. Given the limited number of animals, precaution is needed when interpreting the outcomes of the present in vivo study which was designed as a pilot. Further experiments employing a larger number of animals and harnessing histological and molecular verifications are mandatory before any robust conclusion could be drawn on the effect of TCA in preclinical settings.

The results of the present study suggest the following conclusions. A chemical agent of 0.5% TCA induces growth factor expression and gene expression changes in human gingival fibroblasts involving pathways of cell cycle progression. To our knowledge, an attempt, such as ours, to promote the oral mucosal healing and regeneration by applying the TCA has not been reported. The clinical features of oral soft tissue wound defects treated with TCA in this study suggest a faster regeneration as compared to injuries healing spontaneously.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 21 kb) (21.9KB, docx)
Supplementary material 2 (PPTX 316 kb) (316.8KB, pptx)

Acknowledgement

This research was supported by a Grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant Number: HI16C1838).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

The animal experiment protocol was approved by the Institutional Animal Care and Use Committee, Cronex, Hwasung-si, Gyeonggi-do, Republic of Korea (CRONEX-IACUC 201712004).

Footnotes

Publisher's Note

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Contributor Information

Ki-Tae Koo, Phone: 82-2-2072-2005, Email: periokoo@snu.ac.kr.

Suk Won Lee, Phone: 82-2-440-7519, Email: ysprosth@hanmail.net.

References

  • 1.Sculean A, Gruber R, Bosshardt DD. Soft tissue wound healing around teeth and dental implants. J Clin Periodontol. 2014;41:S6–S22. doi: 10.1111/jcpe.12206. [DOI] [PubMed] [Google Scholar]
  • 2.Wong JW, Gallant-Behm C, Wieb C, Mak K, Hart DA, Larjava H, et al. Wound healing in oral mucosa results in reduced scar formation as compared with skin: evidence from the red Duroc pig model and humans. Wound Repair Regen. 2009;17:717–729. doi: 10.1111/j.1524-475X.2009.00531.x. [DOI] [PubMed] [Google Scholar]
  • 3.Nooh N, Graves DT. Healing is delayed in oral compared to dermal excisional wounds. J Periodontol. 2003;74:242–246. doi: 10.1902/jop.2003.74.2.242. [DOI] [PubMed] [Google Scholar]
  • 4.Graves DT, Nooh N, Gillen T, Davey M, Pate S, Cottrell D, et al. IL-1 plays a critical role in oral, but not dermal, wound healing. J Immunol. 2001;167:5316–5320. doi: 10.4049/jimmunol.167.9.5316. [DOI] [PubMed] [Google Scholar]
  • 5.Lee JB, Chung WG, Kwahck H, Lee KH. Focal treatment of acne scars with trichloroacetic acid: chemical reconstruction of skin scars method. Dermatol Surg. 2002;28:1017–1021. doi: 10.1046/j.1524-4725.2002.02095.x. [DOI] [PubMed] [Google Scholar]
  • 6.Roberts HL. The chloracetic acids: a biochemical study. Br J Dermatol. 1926;38:375–391. doi: 10.1111/j.1365-2133.1926.tb09235.x. [DOI] [Google Scholar]
  • 7.Brodland DG, Cullimore KC, Roenigk RK, Gibson LE. Depths of chemexfoliation induced by various concentrations and application techniques of trichloroacetic acid in a porcine model. J Dermatol Surg Oncol. 1989;15:967–971. doi: 10.1111/j.1524-4725.1989.tb03183.x. [DOI] [PubMed] [Google Scholar]
  • 8.Cho SB, Park CO, Chung WG, Lee KH, Lee JB, Chung KY. Histometric and histochemical analysis of the effect of trichloroacetic acid concentration in the chemical reconstruction of skin scars method. Dermatol Surg. 2006;32:1231–1236. doi: 10.1111/j.1524-4725.2006.32281.x. [DOI] [PubMed] [Google Scholar]
  • 9.Humphreys TR, Werth V, Dzubow L, Kligman A. Treatment of photodamaged skin with trichloroacetic acid and topical tretinoin. J Am Acad Dermatol. 1996;34:638–644. doi: 10.1016/S0190-9622(96)80065-X. [DOI] [PubMed] [Google Scholar]
  • 10.El-Domyati M, Attia SK, Saleh FY, Ahmad HM, Uitto JJ. Trichloroacetic acid peeling versus dermabrasion: a histometric, immunohistochemical, and ultrastructural comparison. Dermatol Surg. 2004;30:179–188. doi: 10.1111/j.1524-4725.2004.30061.x. [DOI] [PubMed] [Google Scholar]
  • 11.Rakic L, Lapière CM, Nusgens BV. Comparative caustic and biological activity of trichloroacetic and glycolic acids on keratinocytes and fibroblasts in vitro. Skin Pharmacol Appl Skin Physiol. 2000;13:52–59. doi: 10.1159/000029908. [DOI] [PubMed] [Google Scholar]
  • 12.Yonei N, Kanazawa N, Ohtani T, Furukawa F, Yamamoto Y. Induction of PDGF-B in TCA-treated epidermal keratinocytes. Arch Dermatol Res. 2007;299:433–440. doi: 10.1007/s00403-007-0781-6. [DOI] [PubMed] [Google Scholar]
  • 13.Kimura A, Kanazawa N, Li HJ, Yonei N, Yamamoto Y, Furukawa F. Influence of chemical peeling on the skin stress response system. Exp Dermatol. 2012;21:S8–S10. doi: 10.1111/j.1600-0625.2012.01495.x. [DOI] [PubMed] [Google Scholar]
  • 14.Mahmoudi H, Balighi K, Tavakolpour S, Daneshpazhooh M, Chams-Davatchi C. Trichloroacetic acid as a treatment for persistent oral mucosal lesions in pemphigus vulgaris. J Am Acad Dermatol. 2019;80:e51–e52. doi: 10.1016/j.jaad.2018.08.039. [DOI] [PubMed] [Google Scholar]
  • 15.Lee MH, Kang JH, Lee SW. The significance differential expression of genes and proteins in human primary cells caused by microgrooved biomaterial substrata. Biomaterials. 2012;33:3216–3234. doi: 10.1016/j.biomaterials.2012.01.034. [DOI] [PubMed] [Google Scholar]
  • 16.Thoma DS, Sancho-Puchades M, Ettlin DA, Hämmerle CH, Jung RE. Impact of a collagen matrix on early healing, aesthetics and patient morbidity in oral mucosal wounds–a randomized study in humans. J Clin Periodontol. 2012;39:157–165. doi: 10.1111/j.1600-051X.2011.01823.x. [DOI] [PubMed] [Google Scholar]
  • 17.Ayvazyan A, Morimoto N, Kanda N, Takemoto S, Kawai K, Sakamoto Y, et al. Collagen-gelatin scaffold impregnated with bFGF accelerates palatal wound healing of palatal mucosa in dogs. J Surg Res. 2011;171:e247–e257. doi: 10.1016/j.jss.2011.06.059. [DOI] [PubMed] [Google Scholar]
  • 18.Rennert R, Golinko M, Kaplan D, Flattau A, Brem H. Standardization of wound photography using the wound electronic medical record. Adv Skin Wound Care. 2009;22:32–38. doi: 10.1097/01.ASW.0000343718.30567.cb. [DOI] [PubMed] [Google Scholar]
  • 19.Itoh T, Suzuki M, Mitsui Y. Keratinocyte growth factor as a mitogen for primary culture of rat hepatocytes. Biochem Biophys Res Commun. 1993;192:1011–1015. doi: 10.1006/bbrc.1993.1517. [DOI] [PubMed] [Google Scholar]
  • 20.Tarkowski E, Issa R, Sjögren M, Wallin A, Blennow K, Tarkowski A, et al. Increased intrathecal levels of the angiogenic factors VEGF and TGF-β in Alzheimer’s disease and vascular dementia. Neurobiol Aging. 2002;23:237–243. doi: 10.1016/S0197-4580(01)00285-8. [DOI] [PubMed] [Google Scholar]
  • 21.Aaronson SA. Growth factors and cancer. Science. 1991;254:1146–1153. doi: 10.1126/science.1659742. [DOI] [PubMed] [Google Scholar]
  • 22.Murray AW. Recycling the cell cycle: cyclins revisited. Cell. 2004;116:221–234. doi: 10.1016/S0092-8674(03)01080-8. [DOI] [PubMed] [Google Scholar]
  • 23.Vairapandi M, Balliet AG, Hoffman B, Liebermann DA. GADD45b and GADD45g are cdc2/cyclinB1 kinase inhibitors with a role in S and G2/M cell cycle checkpoints induced by genotoxic stress. J Cell Physiol. 2002;192:327–338. doi: 10.1002/jcp.10140. [DOI] [PubMed] [Google Scholar]
  • 24.Monte M, Benetti R, Buscemi G, Sandy P, Del Sal G, Schneider C. The cell cycle-regulated protein human GTSE-1 controls DNA damage-induced apoptosis by affecting p53 function. J Biol Chem. 2003;278:30356–30364. doi: 10.1074/jbc.M302902200. [DOI] [PubMed] [Google Scholar]
  • 25.Percy MJ, Myrie KA, Neeley CK, Azim JN, Ethier SP, Petty EM. Expression and mutational analyses of the human MAD2L1 gene in breast cancer cells. Genes Chromosomes Cancer. 2000;29:356–362. doi: 10.1002/1098-2264(2000)9999:9999<::AID-GCC1044>3.0.CO;2-N. [DOI] [PubMed] [Google Scholar]
  • 26.Ying H, Furuya F, Zhao L, Araki O, West BL, Hanover JA, et al. Aberrant accumulation of PTTG1 induced by a mutated thyroid hormone β receptor inhibits mitotic progression. J Clin Invest. 2006;116:2972–2984. doi: 10.1172/JCI28598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Mills GB, Schmandt R, McGill M, Amendola A, Hill M, Jacobs K, et al. Expression of TTK, a novel human protein kinase, is associated with cell proliferation. J Biol Chem. 1992;267:16000–16006. [PubMed] [Google Scholar]
  • 28.Sheu YJ, Stillman B. Cdc7-Dbf4 phosphorylates MCM proteins via a docking site-mediated mechanism to promote S phase progression. Mol Cell. 2006;24:101–113. doi: 10.1016/j.molcel.2006.07.033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Ouyang W, Li MO. Foxo: in command of T lymphocyte homeostasis and tolerance. Trends Immunol. 2011;32:26–33. doi: 10.1016/j.it.2010.10.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Morikawa T, Hino R, Uozaki H, Maeda D, Ushiku T, Shinozaki A, et al. Expression of ribonucleotide reductase M2 subunit in gastric cancer and effects of RRM2 inhibition in vitro. Hum Pathol. 2010;41:1742–1748. doi: 10.1016/j.humpath.2010.06.001. [DOI] [PubMed] [Google Scholar]
  • 31.Chu BW, Kovary KM, Guillaume J, Chen LC, Teruel MN, Wandless TJ. The E3 ubiquitin ligase UBE3C enhances proteasome processivity by ubiquitinating partially proteolyzed substrates. J Biol Chem. 2013;288:34575–34587. doi: 10.1074/jbc.M113.499350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Sa G, Xiong X, Wu T, Yang J, He S, Zhao Y. Histological features of oral epithelium in seven animal species: as a reference for selecting animal models. Eur J Pharm Sci. 2016;81:10–17. doi: 10.1016/j.ejps.2015.09.019. [DOI] [PubMed] [Google Scholar]
  • 33.Sa G, Xiong X, Ren J, Zhao Y, He S. Expression of fibrosis-related molecules in the oral mucosa of six animal species: a reference for selecting animal models. Eur J Pharm Sci. 2017;96:472–478. doi: 10.1016/j.ejps.2016.10.018. [DOI] [PubMed] [Google Scholar]
  • 34.Collins PS. Trichloroacetic acid peels revisited. J Dermatol Surg Oncol. 1989;15:933–940. doi: 10.1111/j.1524-4725.1989.tb03180.x. [DOI] [PubMed] [Google Scholar]
  • 35.Brand HS, Ligtenberg AJ, Veerman EC. Saliva and wound healing. Monogr Oral Sci. 2014;24:52–60. doi: 10.1159/000358784. [DOI] [PubMed] [Google Scholar]

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