Bilayer cellulose-coated hyaluronic acid-based scaffold for accelerating oral wound healing

Bilayer cellulose-coated hyaluronic acid-based scaffold for accelerating oral wound healing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Bilayer cellulose-coated hyaluronic acid-based scaffold for accelerating oral wound healing Yun Sun Jung, Ju Ri Ye, Myoung-Han Lee, Dong-Keun Kweon, Yong Kwon Chae, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4263630/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Sep, 2024 Read the published version in Cellulose → Version 1 posted 4 You are reading this latest preprint version Abstract Objectives To assess the effects of hyaluronic acid (HA)-based scaffolds on oral wound healing. Materials and Methods A film-type 3% HA scaffold with bilayer cellulose coating was prepared and compared with one without coating. For cytocompatibility, human gingival fibroblasts were exposed to both scaffolds. Cell viability, flow cytometry, and scratch wound assays were performed. Additionally, in vivo and` ex vivo wound-healing assays were also conducted. Results Cytocompatibility tests showed no cytotoxicity for either HA scaffold. The scratch wound assay revealed a significant reduction in the open wound area in both HA scaffolds compared with the control ( p < 0.05). However, no differences were observed in the open wound areas between the scaffolds with and without cellulose coating. Regarding in vivo wound healing analysis, the healing rates on day 3 were significantly higher in the HA scaffolds than in the control ( p < 0.05), and there were no differences between the scaffolds. The HA scaffolds with the coating showed lower CD68 and higher vimentin expression than the control ( p < 0.05). In contrast, the HA scaffolds without the coating did not. The ex vivo wound healing analysis showed significantly higher re-epithelialization rates in both scaffolds compared to the control ( p < 0.05). Conclusions Within the limits of this study, the HA scaffold with coating showed enhanced wound-healing efficacy, indicating its potential for oral wound-healing applications. cellulose drug vehicle hyaluronic acid wound healing hyaluronic acid Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Oral wound healing is a significant topic in the field of dentistry as many dental procedures involve surgical wounds in the oral cavity (Toma et al., 2021 ). Oral wounds generally heals quickly and effectively. However, constant oral environmental changes, such as food consumption, may impact the healing process. In some instances, the normal healing progression may even be disrupted. Exudates containing high levels of tissue-destroying proteinases and inflammatory responses induced by foreign bodies may also disrupt the normal progression (Boateng et al., 2008 ). For free gingival grafts, concerns regarding donor site healing remain unresolved (Agarwal et al., 2015 ). These grafts generally extend into the connective tissue and/or bone in the palate following a full-thickness flap elevation, thus, healing at the donor site occurs via secondary intention. This results in delayed healing by 2–6 weeks, prolonging patient discomfort and pain. Hence, novel products that facilitate oral wound healing are warranted. Recently, hyaluronic acid (HA) has attracted significant attention. A randomized controlled trial showed that the topical delivery of an HA-containing gel improved soft tissue and bone healing, and reduced post-operative symptoms after third molar extractions (Bonatto et al., 2024 ). Another study, published in the same year, found that a sodium bicarbonate/HA hybrid dressing material improved epidermal regeneration, collagen synthesis, and angiogenesis in an infected wound skin model (Li et al., 2024 ). Similarly, Cho et al. (2021) found that HA films provided a faster healing rate in rat perianal wounds by increasing re-epithelialization and fibroblast proliferation. Moreover, this finding is supported by recent in vivo studies, which suggested that HA films accelerate the healing of palatal and tongue biopsy wounds (Lee et al., 2024 , Lee et al., 2022 ). Collectively, the available evidence supports the potential use of HA for oral wound healing. Cellulose is a polysaccharide found abundantly in plant cell walls that can be as a drug vehicle (Ciolacu et al., 2020 ). Its molecular structure comprises tightly connected chains formed by strong hydrogen bonds, rendering it insoluble in water. Its molecular structure renders cellulose a useful coating material in biomedical applications. For example, a previous study reported that hydroxyethyl cellulose loaded with 0.04% tungsten oxide showed the highest efficacy in wound closure and inhibited abnormal immune responses by regulating pro-inflammatory cytokine levels (El Fawal et al., 2018 ). Several studies have investigated the synergistic effects of HA and cellulose. A previous rat study showed that the topical delivery of HA-carboxymethylcellulose (CMC) solution reduced peripheral nerve scar formation and adhesion, as well as promoted nerve regeneration at the repair site, after peripheral nerve repair (Park et al., 2011 ). In another study, cellulose synthesized with HA increased fibroblast proliferation, suggesting its potential application as a wound-dressing material (Wang et al., 2020 ). Similarly, HA/cellulose scaffolds upregulated RUNX2 , alkaline phosphatase, and BMP-2 expression in human osteoblast-like cells and increased new bone formation in rabbit calvarial defects (Daugela et al., 2018 ). We recently developed a new type of HA-based scaffold with bilayer cellulose coating for oral wound healing. The aim of this study was to evaluate the effects of HA scaffolds with and without cellulose coating on oral wound healing. The null hypothesis is that the HA scaffold with added cellulose has no greater potential for oral wound healing than the HA scaffold and control. 2. Materials And Methods 2.1. Scaffold fabrication Briefly, a film-type product of 3% low molecular weight (0.5 MDa) HA was prepared according to a previously described method (Lee et al., 2024 ). A 3% HA solution with the same molecular weight was prepared from sodium hyaluronates and cast onto a base film using an automated coating machine (COAD 411, Ocean Sci., Uiwang, South Korea) to achieve a thickness of 1300 µm. The HA scaffold was then dried under 60% humidity at 40°C. The HA scaffolds were converted into HA-C scaffolds by CMC coating using the double-layer casting method (Wu et al., 2017 ). Considering the uniformity and reproducibility of the HA-C scaffolds, the casting ratio between HA and CMC was set to 8:2. 2.2. Characterization of hyaluronic acid-based scaffolds 2.2.1. Structural characterization The Fourier transform infrared (FT-IR) spectra of the HA and HA-C scaffolds were obtained using an FT-IR spectrometer system (Nicolet5700; Thermo Fisher Scientific, Inc. Waltham, MA, USA). The samples were scanned in the range of 4000–400 cm − 1 at room temperature. The chemical composition was assessed to confirm the consistency of the sodium hyaluronate. 2.2.2. Mechanical measurements The mechanical properties of HA and HA-C scaffolds were evaluated at room temperature using a universal testing machine (UTM Series IX version 8.32.00; Instron, MA, US). The cylindrical samples of HA and HA-C scaffolds were prepared and exposed to controlled tensile-relaxation cycles with a 12.5 mm/min of tensile rate and various stretching strains (2018). Stress–strain curves were plotted. 2.3. In vitro wound healing analysis 2.3.1. Cell culture Human gingival fibroblasts (HGF; PCS-201-018) were purchased from ATCC® Cell Lines. Cells were cultured in Dulbecco's Modified Eagle's medium (DMEM; GibcoBRL, Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Giboco BRL, Life Technologies, Grand Island, NY, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin (Pen Strep; Giboco BRL, Life Technologies, Grand Island, NY, USA). The cells were cultivated to the 4th generation, under 5% CO 2 at 37°C. 2.3.2. Cell viability test Cell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan) assays were used to assess cell viability. Each well was loaded with a specific number of cells (5 × 10 3 cells/well) on scaffold surfaces. Subsequently, the cells were incubated for 0 h, 12 h, and 24 h. Following the respective incubation periods, the CCK-8 reagent was applied. The optical density was measured at 450 nm using a plate microreader (AMR-100; Allsheng, Hangzhou, Zhejiang, China). 2.3.3. Flow cytometry Flow cytometry analysis was used to assess cytotoxicity and apoptotic cell levels. Annexin V staining was used for quantitative analysis. Using Fluorescence-activated cell sorting (FACS), cells treated with Annexin V staining and reagents were quantitatively assessed to determine the ratio of dead, apoptotic, and live cells. In each well, HGF cells were loaded onto the scaffold surface at a density of 5 × 10 3 cells/well. After incubation for 0 h, 12 h, and 24 h, the medium was removed, and the cells were pelleted. Subsequently, the cells were stained with Annexin V-FITC and propidium iodide in a 1:1 ratio, and mixed with 500 µL of binding buffer. Flow cytometry data were acquired using a flow cytometer (BD FACSVerseTM, BD Biosciences, NJ, USA) and analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA). Additionally, flow cytometry (FACS) was used to count the number of live, apoptotic, and dead cells. 2.3.4. In vitro wound healing assays Wound healing assays were performed to evaluate the efficacy of the HA and HA-C scaffolds. Each sample was exposed to the culture medium for 24 h to collect the scaffold extracts. HGF cells were cultured in Culture-Insert 2 Well in µ-Dish 35 mm (SPLScarTM Block, SPL Life Sciences, Gyeonggido, Korea) to form a cell monolayer. After 24 h of cell seeding, the silicone insert was removed to induce wounds of 0.5 ± 0.05 mm. The cells were then exposed to the scaffold extracts. The scaffold extracts were prepared using the recommended extraction ratios outlined in the ISO 10993-12 guidelines (2021). Cell strainers (SPL 40 µm Cell Strainer; SPL Life Sciences, Gyeonggido, Korea) were used to filter the extraction solutions. Subsequently, the cells were incubated for 48 h. Digital images were captured at 0 h, 24 h, and 48 h using a JuLI microscope (NanoEnTek, Seoul, Korea) and the wound area was calculated. The open wound area (%) was calculated as follows: Open wound area (%) = (Evaluation time / Average distance of wounds at 0 h) × 100 2.4. In vivo wound healing analysis 2.4.1. Study design The study design received approval from the ethics committee of the institution where this study was conducted (KHMC-IACUC-2023-008) and complied with the National Institutes of Health guidelines for the care and use of laboratory animals (National Research Council Committee for the Update of the Guide for the & Use of Laboratory, 2011). G*Power (v. 3.1.9.7., Heinrich Heine University, Düsseldorf, Germany) was used to calculate sample size, with a significance level of 0.05, 90% statistical power, and an effect size of 0.97 (Lee et al., 2024 ). A total of 36 male Sprague-Dawley rats were used in this study. The rats were randomly allocated to three groups: control ( n = 12), HA scaffolds ( n = 12), and HA-C scaffolds ( n = 12). Each group was subsequently divided into two groups: one for the 3-day sacrifice ( n = 6 for each group) and another for the 7-day sacrifice ( n = 6 for each group). 2.4.2. Surgical protocol Animals were intraperitoneally anesthetized with 30 mg/kg Zoletil 50 (Virbac Lab). After disinfecting the oral cavity with a 2% chlorhexidine solution, 2% lidocaine with a 1:80,000 epinephrine solution was administered. A disposable biopsy punch with 5-mm diameter for a full-thickness flap was used to make a palatal wound. After hemostasis was achieved, the materials were applied and gauze was placed over the wound for 2 min. (Lee et al., 2024 ). The animals were sacrificed at 3 or 7 days after surgery. 2.4.3. Histological wound healing analysis The extracted specimens were sectioned to an appropriate thickness of 2–3 mm after being fixed in neutral buffered formalin (10%). Specimens were processed using the Myr Spin Tissue Processor STP120 automat for 13 h. Using a microtome (RM2235; Leica), specimens were sliced into 3-µm section in the middle of the wound along the sagittal plane and sections were stained with Masson's trichrome. Histological wound healing was evaluated by two examiners by measuring the healing rate between the inner re-epithelial margins using Motic DSAssistant 4K. 2.4.4. Immunohistochemical (IHC) analysis Specimens from the palate were processed in neutral-buffered formalin and paraffin and sectioned at a thickness of 3 mm. IHC staining was performed using CD68 specific antibody (1:200 dilution; Ab125212; Abcam) and vimentin specific antibody (1:500 dilution; Ab137321; Abcam). CD68 staining was performed on the third day, whereas vimentin staining was performed on the specimens sacrificed on the seventh day. Regions of interest (ROI) were set at both wound margins. The EnVision + System-HRP-labeled polymer anti-rabbit was applied for 20 min, followed by color development using a labeled DAB kit. A slide scanner was used for sample scanning, after counterstaining with Mayer’s hematoxylin. And two trained examiners assessed stained protein areas of scanned slides with ImageJ software. 2.5. Ex vivo wound healing analysis Neoderm®-OD (TEGO Science, Seoul, Korea), a 3 dimensionally-reconstructed human oral mucosa model, was utilized for ex vivo wound healing analysis. The rats were divided into three groups similar to those used in the rat model ( n = 6 for each group). A round-shape-wound with 4mm diameter was created in each model using a biopsy punch. The models were incubated for 5 days at 37°C in a humidified environment with 10% CO 2 . The maintenance medium was replaced every 2 days. After incubation, the model was processed with 4% paraformaldehyde and paraffin, and sectioned to make slides. And hematoxylin and eosin was used to stain the slides. With use of Image Pro software (Media Cybernetics, Inc., Rockville, MD, USA), re-epithelialization was evaluated. Re-epithelialization (%) was calculated as (Re-epithelialization length / Total wound length) × 100. 2.6. Statistical analysis Statistical analysis of acquired data were conducted with IBM SPSS Statistics, version 20 (SPSS Inc., Chicago, IL, USA). One-way ANOVA and Tukey’s honest significant difference post hoc tests were performed after the normality test. The threshold for p -values was set to 0.05. To assess the interobserver reliability of both IHC and histological analyses, intraclass coefficient values (ICC) were computed between the two examiners. The ICC values indicated high reliability, with scores of 0.991 and 0.890 for histological and IHC analyses, respectively. 3. Results 3.1. Characterization of hyaluronic acid-based scaffolds 3.1.1. FT-IR analysis HA exhibited strong and broad peaks at 3390 cm − 1 that originated from the -OH and -NH groups. Adding CMC to the HA scaffold leads to a corresponding decrease in the peaks attributed to HA characteristics (Fig. 1 ). Particularly, a significant increase in the peaks was noted around the 3390 cm − 1 region, indicating distinct HA properties. However, no distinct peaks were observed. 3.1.2. Mechanical properties In the experiments conducted to assess the mechanical properties, including tensile strength and elongation, adding the CMC scaffold layer resulted in increased brittleness, thus reducing tensile strength and elongation (Fig. 2 ). The addition of CMC (20%) to the HA scaffold reduced the tensile strength and elongation rate. * p < 0.05 vs. HA scaffold. 3.2. In vitro evaluation 3.2.1. Cytocompatibility To assess the cytocompatibility of the HA-C scaffold, the CCK-8 test and flow cytometry were performed on HGF cells. The CCK-8 assay showed that the viability of HGF cells on the HA-C scaffold was similar to that of the control at 24 h (Fig. 3 ). The flow cytometry assay also confirmed that apoptotic or dead cells among the materials did not differ significantly, thus, confirming the cytocompatibility of the HA-C scaffold (Fig. 4 ). 3.2.2. In vitro wound healing assay In vitro wound healing assay showed a significant reduction in the open wound area in the HA-C scaffold group compared with that in the control group ( p < 0.05). In contrast, the open wound areas between the HA and HA-C scaffold groups did not differ significantly (Fig. 5 ). 3.3. In vivo evaluation On day 3, healing rates were higher in the HA (21.17 ± 10.65%) and HA-C scaffold groups (22.08 ± 6.50%), compared to the control group (10.98 ± 8.33%) ( p < 0.05). Conversely, the healing rates among the 7-day groups did not differ significantly (Fig. 6 ). Furthermore, the HA-C scaffold group demonstrated a significantly lower CD68 expression and a greater vimentin expression than the control group ( p < 0.05). However, marker expression in the HA scaffold group did not significantly differ from the control group (Fig. 7 ). 3.4. Ex vivo evaluation Figure 8 shows the results of the ex vivo evaluation. The re-epithelialization rates in the HA scaffold group (94.77 ± 11.73%) and HA-C group (96.15 ± 7.97%) were significantly higher than the controls (14.55 ± 5.95%). However, there were no significant differences between the HA and HA-C scaffold groups. 4. Discussion This study investigated the effects of HA-C scaffolds on oral wound healing, both in vitro and in vivo . The results of our study confirmed that the wound-healing effects of the HA and HA-C scaffolds did not differ significantly. However, wound-healing marker expression in the HA-C scaffold differed from that in the control, whereas the HA scaffold did not. Thus, the null hypothesis was partially rejected. Based on the CCK-8 assay and flow cytometry, the HA-C scaffold did not cause detrimental effects on HGF cell proliferation, indicating its cytocompatibility. This finding suggests that the scaffold has the potential to support cellular activity and tissue regeneration. Taken together, HA-C scaffolds are suitable for biomedical applications. Maintaining high cytocompatibility is crucial for wound dressings (Wu et al., 2016 ). Previous studies proposed an acceptable range (approximately > 70%~75% viability) for biomedical applications, including wound dressings (Khorasani et al., 2019 , Archana et al., 2013 ). The in vitro healing assays confirmed that the open wound area waw significantly reduced when both scaffolds were used, compared with the control. Similarly, the HA-C scaffolds significantly promoted wound healing during the initial phase in both in vivo and ex vivo analyses. Previous studies have shown that palatal wound healing progressively slows during the first 7 days after injury (Kozlovsky et al., 2007 , Weinberg et al., 2020 ). Considering these findings, our results suggest that HA-C scaffolds can accelerate palatal wound healing during the initial phase. Furthermore, our findings, particularly from the initial phase (0–3 days) are consistent with those of a previous study (Lee et al., 2024 ). In this study, at day 7, wound healing did not differ significantly between the HA-C scaffold and control groups. In contrast, a difference was demonstrated in a previous study (Lee et al., 2024 ). These discrepancies may be attributed to the different cutting planes of the specimens used in the two studies. According to the zipper theory, most wound healing dynamics in the initial stage of mucoperiosteal wounds occur in the sagittal plane, with minimal changes observed in the coronal plane (Weinberg et al., 2020 ). Taken together, these results support that HA-C scaffolds offer the most beneficial effects during the initial phase of oral wound healing. As concerns regarding water resistance in cellulose-based materials have been well documented because of the abundance of hydroxyl groups in cellulose (Abdelmouleh et al., 2004 ), in the future, studies to explore the exact interaction between HA and cellulose, as well as the stability of the materials, are warranted To assess the effects of HA-C scaffolds on wound healing markers, CD68 and vimentin expression were evaluated. CD68 serves as an indicator of macrophages and inflammatory responses (Lee et al., 2024 ). We observed a significant decrease in CD68 expression in the HA-C scaffold group, indicating that HA-C scaffolds may hinder the migration of macrophages and neutrophils. Macrophages, particularly M1-like macrophages, are critical in the inflammatory-proliferation phase transition (Kim et al., 2019 ). M1-like macrophages are active during the early inflammatory phase and promote inflammatory responses (Zhou et al., 2022 ). Our findings suggest that the HA-C scaffolds can mitigate inflammatory responses by inhibiting the formation of M1-like macrophages. This validates the potential of HA-C scaffolds in improving wound healing by modulating immune responses. Vimentin is a marker of fibroblasts. HA-C scaffolds significantly upregulated vimentin expression compared to the HA scaffold 7 days after injury. During the proliferation phase of wound healing, fibroblasts play a crucial role in initial wound contraction, leading to wound closure (Tomasek et al., 2002 ). According to recent findings, a significant increase in fibroblast density was observed during palatal wound healing in the first 1–2 weeks after injury (Weinberg et al., 2020 ). Considering these findings, the HA-C scaffolds can also contribute to wound healing by improving fibroblast recruitment and proliferation. Thus, it is plausible that cellulose has a positive effect on fibroblast colonization of the scaffold. Available evidence suggests that the structure of cellulose contributes to the attachment and overgrowth of fibroblasts on cellulose-coated scaffolds to a greater extent than uncoated surfaces (Dydak et al., 2022 ). This study has several limitations. First, we only observed the wound healing process up to 7 days post-operatively. The fibroblast-driven wound healing process may continue until the wound closes. Further studies with longer evaluation periods are warranted. Second, we did not directly assess HA release. To better understand the action of HA-C scaffolds, the residual amount of HA in the wound area should be assessed. Finally, various combinations of HA and cellulose were not evaluated. Previous studies on HA-C composites have shown superior functionality, such as anti-adhesive barriers and vehicles for controlled drug release (Huang et al., 2016 ). To explore the full potential of HA-C scaffolds on wound-healing and tissue-engineering applications, further studies on the formulation and optimization of HA/CMC scaffolds, are warranted. Conclusions In conclusion, HA-C scaffolds are cytocompatible with oral wound healing. Importantly, HA-C scaffolds accelerated the wound healing process by suppressing inflammatory responses and encouraging fibroblast recruitment and proliferation at wound sites. These results support their potential use in oral wound-healing applications. Declarations Conflict of interest statement The authors report no direct or indirect conflicts of interest, such as associations with commercial entities, financial associations involving family members, or other non-financial associations. Data availability statement Datasets related to this article are available upon request to the corresponding author. Ethical approval statement This study was reviewed and approved by the Institutional Animal Care and Use Committee of Kyung Hee University Medical Center, Seoul, South Korea (KHMC-IACUC-2023-008). Author contributions Yun Sun Jung: Data curation, formal analysis, and writing-original draft. Ju Ri Ye: Data curation, formal analysis, and visualization. Myoung-Han Lee: Data curation and resources. Dong-Keun Kewon: Data curation and resources. Yong Kwon Chae: Writing-review and editing. Hyo-Seol Lee: Writing-review and editing. Sung Chul Choi: Writing-review and editing. Ok Hyung Nam: Conceptualization, data curation, validation, formal analysis, funding acquisition, writing-original draft, writing-review, and editing. Funding statement This study was supported by the Technology Development Program, funded by the Ministry of SMEs and Startups (MSS, No. S3290500). References Abdelmouleh M, Boufi S, Belgacem M, Duarte A, Salah AB, Gandini A (2004) Modification of cellulosic fibres with functionalised silanes: development of surface properties. Int J Adhes Adhes 24:43-54 Agarwal C, Tarun Kumar AB, Mehta DS (2015) Comparative evaluation of free gingival graft and AlloDerm® in enhancing the width of attached gingival: A clinical study. Contemp Clin Dent 6:483-488 American Society for Testing and Materials (2018) ASTM D882: Standard Test Method for Tensile Properties of Thin Plastic Sheeting. American Society for Testing and Materials Archana D, Dutta J, Dutta PK (2013) Evaluation of chitosan nano dressing for wound healing: characterization, in vitro and in vivo studies. Int J Biol Macromol 57:193-203 Boateng JS, Matthews KH, Stevens HN, Eccleston GM (2008) Wound healing dressings and drug delivery systems: a review. J Pharm Sci 97:2892-2923 Bonatto MDS, Feltran GDS, Barbosa TP, Pereira DA, Santos SS, Mendes PGJ, RS EP, Bezerra FJB, Zambuzzi WF, de Oliveira G (2024) Green tea and hyaluronic acid gel enhance fibroblast activation and improves the gingival healing post-third molar extraction. Sci Rep 14:7124 Ciolacu DE, Nicu R, Ciolacu F (2020) Cellulose-Based Hydrogels as Sustained Drug-Delivery Systems. Materials (Basel) 13 Daugela P, Pranskunas M, Juodzbalys G, Liesiene J, Baniukaitiene O, Afonso A, Sousa Gomes P (2018) Novel cellulose/hydroxyapatite scaffolds for bone tissue regeneration: In vitro and in vivo study. J Tissue Eng Regen Med 12:1195-1208 Dydak K, Junka A, Nowacki G, Paleczny J, Szymczyk-Ziółkowska P, Górzyńska A, Aniołek O, Bartoszewicz M (2022) In Vitro Cytotoxicity, Colonisation by Fibroblasts and Antimicrobial Properties of Surgical Meshes Coated with Bacterial Cellulose. Int J Mol Sci 23 El Fawal GF, Abu-Serie MM, Hassan MA, Elnouby MS (2018) Hydroxyethyl cellulose hydrogel for wound dressing: Fabrication, characterization and in vitro evaluation. Int J Biol Macromol 111:649-659 Huang YC, Huang KY, Yang BY, Ko CH, Huang HM (2016) Fabrication of Novel Hydrogel with Berberine-Enriched Carboxymethylcellulose and Hyaluronic Acid as an Anti-Inflammatory Barrier Membrane. Biomed Res Int 2016:3640182 International Organization for Standardization (2021) ISO 10993-12:2021: Biological evaluation of medical devices Part 12: Sample preparation and reference materials. ISO Khorasani MT, Joorabloo A, Adeli H, Mansoori-Moghadam Z, Moghaddam A (2019) Design and optimization of process parameters of polyvinyl (alcohol)/chitosan/nano zinc oxide hydrogels as wound healing materials. Carbohydr Polym 207:542-554 Kim H, Wang SY, Kwak G, Yang Y, Kwon IC, Kim SH (2019) Exosome-Guided Phenotypic Switch of M1 to M2 Macrophages for Cutaneous Wound Healing. Adv Sci 6:1900513 Kozlovsky A, Artzi Z, Hirshberg A, Israeli-Tobias C, Reich L (2007) Effect of local antimicrobial agents on excisional palatal wound healing: a clinical and histomorphometric study in rats. J clin periodontol 34:164-171 Lee JH, Lee KE, Kang SW, Park SH, Chae YK, Lee MH, Kweon DK, Choi SC, Nam OH (2024) Effect of orodispersible hyaluronic acid film on palatal mucosa wound healing. Oral Dis 30:518-527 Lee JH, Lee KE, Nam OH, Chae YK, Lee MH, Kweon DK, Kim MS, Lee HS, Choi SC (2022) Orodispersible hyaluronic acid film delivery for oral wound healing in rats. J Dent Sci 17:1595-1603 Li Y, Li H, Yu Z, Liu J, Lin Y, Xu J, Zhang C, Chen Q, Han X, Peng Q (2024) Drug-free and multifunctional sodium bicarbonate/hyaluronic acid hybrid dressing for synergistic healing of infected wounds. Int J Biol Macromol 259:129254 National Research Council (2011) Guide for the Care and Use of Laboratory Animals: Eighth Edition. Washington, DC: The National Academies Press. https://doi.org/10.17226/12910 Park JS, Lee JH, Han CS, Chung DW, Kim GY (2011) Effect of hyaluronic acid-carboxymethylcellulose solution on perineural scar formation after sciatic nerve repair in rats. Clin Orthop Surg 3:315-24 Toma AI, Fuller JM, Willett NJ, Goudy SL (2021) Oral wound healing models and emerging regenerative therapies. Transl Res 236:17-34 Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, Brown RA (2002) Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol 3:349-363 Wang X, Tang J, Huang J, Hui M (2020) Production and characterization of bacterial cellulose membranes with hyaluronic acid and silk sericin. Colloids Surf B Biointerfaces 195:111273 Weinberg E, Vered M, Atzil S, Chaushu G, Chaushu L (2020) The dynamics of closure following excisional mid-palatal mucoperiosteal wound in a rat model. Clin Oral Investig 24:4385-4393 Wu Y, Li X, Shi X, Zhan Y, Tu H, Du Y, Deng H, Jiang L (2017) Production of thick uniform-coating films containing rectorite on nanofibers through the use of an automated coating machine. Colloids Surf B Biointerfaces 149:271-279 Wu Y, Wang L, Zhao X, Hou S, Guo B, Ma PX (2016) Self-healing supramolecular bioelastomers with shape memory property as a multifunctional platform for biomedical applications via modular assembly. Biomater 104:18-31 Zhou Y, Zhang X-L, Lu S-T, Zhang N-Y, Zhang H-J, Zhang J, Zhang J (2022) Human adipose-derived mesenchymal stem cells-derived exosomes encapsulated in pluronic F127 hydrogel promote wound healing and regeneration. Stem Cell Res Ther 13:407 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 30 Sep, 2024 Read the published version in Cellulose → Version 1 posted Editorial decision: Revision requested 15 Apr, 2024 Submission checks completed at journal 15 Apr, 2024 Editor assigned by journal 15 Apr, 2024 First submitted to journal 14 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4263630","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":291594603,"identity":"51da4d9d-3ca0-4154-b53a-ecabce4c25a7","order_by":0,"name":"Yun Sun Jung","email":"","orcid":"","institution":"Department of Dentistry, Graduate School, Kyung Hee University","correspondingAuthor":false,"prefix":"","firstName":"Yun","middleName":"Sun","lastName":"Jung","suffix":""},{"id":291594604,"identity":"57b4f278-2b88-4183-ab18-b233afc66a62","order_by":1,"name":"Ju Ri Ye","email":"","orcid":"","institution":"Department of Dentistry, Graduate School, Kyung Hee University","correspondingAuthor":false,"prefix":"","firstName":"Ju","middleName":"Ri","lastName":"Ye","suffix":""},{"id":291594609,"identity":"59ded6ac-2e18-4321-ba96-d1ebb619186d","order_by":2,"name":"Myoung-Han Lee","email":"","orcid":"","institution":"Jinwoo Bio Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Myoung-Han","middleName":"","lastName":"Lee","suffix":""},{"id":291594610,"identity":"428537a3-d8e3-4c88-af22-de52e1c9b879","order_by":3,"name":"Dong-Keun Kweon","email":"","orcid":"","institution":"Jinwoo Bio Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Dong-Keun","middleName":"","lastName":"Kweon","suffix":""},{"id":291594611,"identity":"26d1f179-53f4-4688-a872-d9b061c4b80b","order_by":4,"name":"Yong Kwon Chae","email":"","orcid":"","institution":"Department of Pediatric Dentistry, Kyung Hee University, College of Dentistry, Kyung Hee University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"Kwon","lastName":"Chae","suffix":""},{"id":291594612,"identity":"e28395b3-a8c8-4cdf-9895-50986a32569a","order_by":5,"name":"Hyo-Seol Lee","email":"","orcid":"","institution":"Department of Pediatric Dentistry, Kyung Hee University, College of Dentistry, Kyung Hee University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Hyo-Seol","middleName":"","lastName":"Lee","suffix":""},{"id":291594613,"identity":"96cf0d58-c0a6-4cb2-adfa-22ec38e870bb","order_by":6,"name":"Sung Chul Choi","email":"","orcid":"","institution":"Department of Pediatric Dentistry, Kyung Hee University, College of Dentistry, Kyung Hee University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Sung","middleName":"Chul","lastName":"Choi","suffix":""},{"id":291594614,"identity":"5e6c93d0-406a-4538-a5ad-5aa7ceea1c2a","order_by":7,"name":"Ok Hyung Nam","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIiWNgGAWjYHACA8bGBhDNfABISMiQooUtAaSFhxQtPAZgkqB6c/bmjQ9n7jicuJ1/zedXN2oseBjYDx/dgE+LZc+xYsONZw4n7pzxdpt1zjGgw3jS0m7gddWNHDPJh22HEzfcOLvNOIcNqEWCxwy/lvtvYFrOPDPO+UeMlhs8ZpIbQVrO9zA/zm0jQotlT1qx4cy2dOMNN9jMmHP7JHjYCPnFnP3wxoe9bdayG84ffvw551udHD/74WP4HQahmoGRmMAmAWKy4VOOpKWOgYH/APMHQqpHwSgYBaNgZAIA5dRSD0IdlJ4AAAAASUVORK5CYII=","orcid":"","institution":"Department of Pediatric Dentistry, Kyung Hee University, College of Dentistry, Kyung Hee University Medical Center","correspondingAuthor":true,"prefix":"","firstName":"Ok","middleName":"Hyung","lastName":"Nam","suffix":""}],"badges":[],"createdAt":"2024-04-14 04:59:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4263630/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4263630/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10570-024-06198-9","type":"published","date":"2024-09-30T15:57:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54807466,"identity":"80491703-10f0-4e0a-b0d6-748d2ea316dc","added_by":"auto","created_at":"2024-04-17 05:00:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":409283,"visible":true,"origin":"","legend":"\u003cp\u003eFourier transform infrared (FT-IR) spectra of HA scaffolds\u003cstrong\u003e.\u003c/strong\u003e HA exhibited strong and broad peaks at 3390 cm\u003csup\u003e-1\u003c/sup\u003e that originated from the -OH and -NH groups. Adding CMC to the HA scaffold leads to a corresponding decrease in the peaks attributed to HA characteristics.\u003c/p\u003e\n\u003cp\u003eHA, Hyaluronic acid; CMC, carboxymethylcellulose.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4263630/v1/ea41e441aec0594222c62fbc.png"},{"id":54807468,"identity":"ac7c1ae6-1bf0-4ab0-9e59-a2fe0a8ac146","added_by":"auto","created_at":"2024-04-17 05:00:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":168188,"visible":true,"origin":"","legend":"\u003cp\u003eMechanical properties of HA and HA-C scaffolds. (a) Tensile stress–strain curves of the HA scaffold and HA–C scaffold. (b) The corresponding stress and strain.\u003c/p\u003e\n\u003cp\u003eThe addition of CMC (20%) to the HA scaffold reduced the tensile strength and elongation rate. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. HA scaffold.\u003c/p\u003e\n\u003cp\u003eHA, Hyaluronic acid; HA-C, Hyaluronic acid with carboxymethylcellulose; CMC, carboxymethylcellulose.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4263630/v1/2f764f426c1bea7e22eec517.png"},{"id":54807467,"identity":"b9ab7179-8f8e-410a-b1eb-1c32d6a9395d","added_by":"auto","created_at":"2024-04-17 05:00:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":161858,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability test\u003cstrong\u003e.\u003c/strong\u003e Mean optical density of human gingival fibroblast cell treated by control, HA, and HA-C groups for 24 h. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. control, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. HA scaffold.\u003c/p\u003e\n\u003cp\u003eOD, Optical density; HA, Hyaluronic acid; HA-C, Hyaluronic acid with carboxymethylcellulose.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4263630/v1/a08dcf39b4105e2fd5847969.png"},{"id":54807470,"identity":"09eafc22-f48a-48d4-80fb-9ecda2ead38e","added_by":"auto","created_at":"2024-04-17 05:00:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":677519,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometry. (a) Representative dot charts of Annexin-V/7-AAD bivariate flow cytometry. Live cells are presented in Q4, dead cells in Q2, and apoptotic cells in Q3. (b) Bar graph of %cell comparison. The scaffolds did not affect the HGF cell viability. DMEM served as the control.\u003c/p\u003e\n\u003cp\u003eHGF, Human gingival fibroblast; DMEM, Dulbecco's Modified Eagle's medium.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4263630/v1/3df9a1fa8c4cad7b7f4b1db0.png"},{"id":54807473,"identity":"5daf0adc-8d1d-4a5d-ada2-5d4c8d465bb3","added_by":"auto","created_at":"2024-04-17 05:00:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3244102,"visible":true,"origin":"","legend":"\u003cp\u003eCell scratch wound assay.\u003cstrong\u003e \u003c/strong\u003e(a) Representative microscopic images. Scale bar = 100 µm, (b) Results from the open wound area (%). Oral wound healing is significantly accelerated in both HA and HA-C scaffolds compared to the control group (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. control)\u003c/p\u003e\n\u003cp\u003eHA, Hyaluronic acid; HA-C, Hyaluronic acid with carboxymethylcellulose.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4263630/v1/178fb06dd0da682440b682a4.png"},{"id":54807472,"identity":"d7a7ed77-b2a8-4895-9215-ded1b3b2901a","added_by":"auto","created_at":"2024-04-17 05:00:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1260476,"visible":true,"origin":"","legend":"\u003cp\u003eHA, Hyaluronic acid; HA-C, Hyaluronic acid with carboxymethylcellulose.\u003c/p\u003e\n\u003cp\u003eMasson's trichrome stain. (a) Histological sections from the control, HA, and HA-C groups. (b) Histological healing rates (%). At day 3, the HA and HA-C groups showed significantly higher healing rates compared to the control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). At day 7, similar healing rates among the groups are noted. Scale bar = 200 μm, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. control\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4263630/v1/13496d6d657a3ed8c0d86ba2.png"},{"id":54807471,"identity":"38d72ae8-cc40-4280-bce2-a692fafe4542","added_by":"auto","created_at":"2024-04-17 05:00:10","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3245638,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemistry (IHC) analysis.\u003cstrong\u003e \u003c/strong\u003e(a)\u003cstrong\u003e \u003c/strong\u003eIHC stained samples from the control, HA, and HA-C groups. (b) IHC-positive area (%) of CD68. (c) IHC-positive area (%) of vimentin. CD68 expression is significantly lower in the HA-C group, while vimentin expression is significantly higher in the HA-C group. Scale bar = 30 μm, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. control\u003c/p\u003e\n\u003cp\u003eHA, Hyaluronic acid; HA-C, Hyaluronic acid with carboxymethylcellulose.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-4263630/v1/097390280df5d9995cdacaf5.png"},{"id":54807474,"identity":"c70c47e0-29aa-4dcd-8a7c-79742770f4fd","added_by":"auto","created_at":"2024-04-17 05:00:10","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":531413,"visible":true,"origin":"","legend":"\u003cp\u003eHuman oral mucosa equivalent \u003cem\u003eex vivo\u003c/em\u003e model.\u003cstrong\u003e \u003c/strong\u003e(a) Hematoxylin \u0026amp; eosin (H \u0026amp; E) stained samples from the control, HA, and HA-C groups. (b) Re-epithelialization (%). The HA and HA-C groups showing significant re-epithelialization compared to the controls (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Scale bar = 200 μm, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. control\u003c/p\u003e\n\u003cp\u003eHA, Hyaluronic acid; HA-C, Hyaluronic acid with carboxymethylcellulose.\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-4263630/v1/dab14af4ed4a2c1493d9210d.png"},{"id":66097079,"identity":"d419853d-a358-4788-9e46-f562ee343464","added_by":"auto","created_at":"2024-10-07 16:13:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11975914,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4263630/v1/5881d81f-85f2-4f97-8cd3-c0b65bcb15a5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bilayer cellulose-coated hyaluronic acid-based scaffold for accelerating oral wound healing","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eOral wound healing is a significant topic in the field of dentistry as many dental procedures involve surgical wounds in the oral cavity (Toma et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Oral wounds generally heals quickly and effectively. However, constant oral environmental changes, such as food consumption, may impact the healing process. In some instances, the normal healing progression may even be disrupted. Exudates containing high levels of tissue-destroying proteinases and inflammatory responses induced by foreign bodies may also disrupt the normal progression (Boateng et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). For free gingival grafts, concerns regarding donor site healing remain unresolved (Agarwal et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). These grafts generally extend into the connective tissue and/or bone in the palate following a full-thickness flap elevation, thus, healing at the donor site occurs via secondary intention. This results in delayed healing by 2\u0026ndash;6 weeks, prolonging patient discomfort and pain. Hence, novel products that facilitate oral wound healing are warranted.\u003c/p\u003e \u003cp\u003eRecently, hyaluronic acid (HA) has attracted significant attention. A randomized controlled trial showed that the topical delivery of an HA-containing gel improved soft tissue and bone healing, and reduced post-operative symptoms after third molar extractions (Bonatto et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Another study, published in the same year, found that a sodium bicarbonate/HA hybrid dressing material improved epidermal regeneration, collagen synthesis, and angiogenesis in an infected wound skin model (Li et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Similarly, Cho et al. (2021) found that HA films provided a faster healing rate in rat perianal wounds by increasing re-epithelialization and fibroblast proliferation. Moreover, this finding is supported by recent \u003cem\u003ein vivo\u003c/em\u003e studies, which suggested that HA films accelerate the healing of palatal and tongue biopsy wounds (Lee et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Lee et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Collectively, the available evidence supports the potential use of HA for oral wound healing.\u003c/p\u003e \u003cp\u003eCellulose is a polysaccharide found abundantly in plant cell walls that can be as a drug vehicle (Ciolacu et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Its molecular structure comprises tightly connected chains formed by strong hydrogen bonds, rendering it insoluble in water. Its molecular structure renders cellulose a useful coating material in biomedical applications. For example, a previous study reported that hydroxyethyl cellulose loaded with 0.04% tungsten oxide showed the highest efficacy in wound closure and inhibited abnormal immune responses by regulating pro-inflammatory cytokine levels (El Fawal et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral studies have investigated the synergistic effects of HA and cellulose. A previous rat study showed that the topical delivery of HA-carboxymethylcellulose (CMC) solution reduced peripheral nerve scar formation and adhesion, as well as promoted nerve regeneration at the repair site, after peripheral nerve repair (Park et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In another study, cellulose synthesized with HA increased fibroblast proliferation, suggesting its potential application as a wound-dressing material (Wang et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Similarly, HA/cellulose scaffolds upregulated \u003cem\u003eRUNX2\u003c/em\u003e, alkaline phosphatase, and \u003cem\u003eBMP-2\u003c/em\u003e expression in human osteoblast-like cells and increased new bone formation in rabbit calvarial defects (Daugela et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). We recently developed a new type of HA-based scaffold with bilayer cellulose coating for oral wound healing.\u003c/p\u003e \u003cp\u003eThe aim of this study was to evaluate the effects of HA scaffolds with and without cellulose coating on oral wound healing. The null hypothesis is that the HA scaffold with added cellulose has no greater potential for oral wound healing than the HA scaffold and control.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Scaffold fabrication\u003c/h2\u003e \u003cp\u003eBriefly, a film-type product of 3% low molecular weight (0.5 MDa) HA was prepared according to a previously described method (Lee et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). A 3% HA solution with the same molecular weight was prepared from sodium hyaluronates and cast onto a base film using an automated coating machine (COAD 411, Ocean Sci., Uiwang, South Korea) to achieve a thickness of 1300 \u0026micro;m. The HA scaffold was then dried under 60% humidity at 40\u0026deg;C. The HA scaffolds were converted into HA-C scaffolds by CMC coating using the double-layer casting method (Wu et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Considering the uniformity and reproducibility of the HA-C scaffolds, the casting ratio between HA and CMC was set to 8:2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Characterization of hyaluronic acid-based scaffolds\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1. Structural characterization\u003c/h2\u003e \u003cp\u003eThe Fourier transform infrared (FT-IR) spectra of the HA and HA-C scaffolds were obtained using an FT-IR spectrometer system (Nicolet5700; Thermo Fisher Scientific, Inc. Waltham, MA, USA). The samples were scanned in the range of 4000\u0026ndash;400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at room temperature. The chemical composition was assessed to confirm the consistency of the sodium hyaluronate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2. Mechanical measurements\u003c/h2\u003e \u003cp\u003eThe mechanical properties of HA and HA-C scaffolds were evaluated at room temperature using a universal testing machine (UTM Series IX version 8.32.00; Instron, MA, US). The cylindrical samples of HA and HA-C scaffolds were prepared and exposed to controlled tensile-relaxation cycles with a 12.5 mm/min of tensile rate and various stretching strains (2018). Stress\u0026ndash;strain curves were plotted.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.3. \u003cem\u003eIn vitro\u003c/em\u003e wound healing analysis\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. Cell culture\u003c/h2\u003e \u003cp\u003eHuman gingival fibroblasts (HGF; PCS-201-018) were purchased from ATCC\u0026reg; Cell Lines. Cells were cultured in Dulbecco's Modified Eagle's medium (DMEM; GibcoBRL, Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Giboco BRL, Life Technologies, Grand Island, NY, USA), 100 U/mL penicillin, and 100 \u0026micro;g/mL streptomycin (Pen Strep; Giboco BRL, Life Technologies, Grand Island, NY, USA). The cells were cultivated to the 4th generation, under 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2. Cell viability test\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan) assays were used to assess cell viability. Each well was loaded with a specific number of cells (5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well) on scaffold surfaces. Subsequently, the cells were incubated for 0 h, 12 h, and 24 h. Following the respective incubation periods, the CCK-8 reagent was applied. The optical density was measured at 450 nm using a plate microreader (AMR-100; Allsheng, Hangzhou, Zhejiang, China).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3. Flow cytometry\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFlow cytometry analysis was used to assess cytotoxicity and apoptotic cell levels. Annexin V staining was used for quantitative analysis. Using Fluorescence-activated cell sorting (FACS), cells treated with Annexin V staining and reagents were quantitatively assessed to determine the ratio of dead, apoptotic, and live cells. In each well, HGF cells were loaded onto the scaffold surface at a density of 5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well. After incubation for 0 h, 12 h, and 24 h, the medium was removed, and the cells were pelleted. Subsequently, the cells were stained with Annexin V-FITC and propidium iodide in a 1:1 ratio, and mixed with 500 \u0026micro;L of binding buffer. Flow cytometry data were acquired using a flow cytometer (BD FACSVerseTM, BD Biosciences, NJ, USA) and analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA). Additionally, flow cytometry (FACS) was used to count the number of live, apoptotic, and dead cells.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.3.4. \u003cem\u003eIn vitro\u003c/em\u003e wound healing assays\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWound healing assays were performed to evaluate the efficacy of the HA and HA-C scaffolds. Each sample was exposed to the culture medium for 24 h to collect the scaffold extracts. HGF cells were cultured in Culture-Insert 2 Well in \u0026micro;-Dish 35 mm (SPLScarTM Block, SPL Life Sciences, Gyeonggido, Korea) to form a cell monolayer. After 24 h of cell seeding, the silicone insert was removed to induce wounds of 0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 mm. The cells were then exposed to the scaffold extracts. The scaffold extracts were prepared using the recommended extraction ratios outlined in the ISO 10993-12 guidelines (2021). Cell strainers (SPL 40 \u0026micro;m Cell Strainer; SPL Life Sciences, Gyeonggido, Korea) were used to filter the extraction solutions. Subsequently, the cells were incubated for 48 h. Digital images were captured at 0 h, 24 h, and 48 h using a JuLI microscope (NanoEnTek, Seoul, Korea) and the wound area was calculated. The open wound area (%) was calculated as follows:\u003c/p\u003e \u003cp\u003eOpen wound area (%) = (Evaluation time / Average distance of wounds at 0 h) \u0026times; 100\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.4. \u003cem\u003eIn vivo\u003c/em\u003e wound healing analysis\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. Study design\u003c/h2\u003e \u003cp\u003eThe study design received approval from the ethics committee of the institution where this study was conducted (KHMC-IACUC-2023-008) and complied with the National Institutes of Health guidelines for the care and use of laboratory animals (National Research Council Committee for the Update of the Guide for the \u0026amp; Use of Laboratory, 2011). G*Power (v. 3.1.9.7., Heinrich Heine University, D\u0026uuml;sseldorf, Germany) was used to calculate sample size, with a significance level of 0.05, 90% statistical power, and an effect size of 0.97 (Lee et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). A total of 36 male Sprague-Dawley rats were used in this study. The rats were randomly allocated to three groups: control (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12), HA scaffolds (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12), and HA-C scaffolds (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12). Each group was subsequently divided into two groups: one for the 3-day sacrifice (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6 for each group) and another for the 7-day sacrifice (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6 for each group).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2. Surgical protocol\u003c/h2\u003e \u003cp\u003eAnimals were intraperitoneally anesthetized with 30 mg/kg Zoletil 50 (Virbac Lab). After disinfecting the oral cavity with a 2% chlorhexidine solution, 2% lidocaine with a 1:80,000 epinephrine solution was administered. A disposable biopsy punch with 5-mm diameter for a full-thickness flap was used to make a palatal wound. After hemostasis was achieved, the materials were applied and gauze was placed over the wound for 2 min. (Lee et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The animals were sacrificed at 3 or 7 days after surgery.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3. Histological wound healing analysis\u003c/h2\u003e \u003cp\u003eThe extracted specimens were sectioned to an appropriate thickness of 2\u0026ndash;3 mm after being fixed in neutral buffered formalin (10%). Specimens were processed using the Myr Spin Tissue Processor STP120 automat for 13 h. Using a microtome (RM2235; Leica), specimens were sliced into 3-\u0026micro;m section in the middle of the wound along the sagittal plane and sections were stained with Masson's trichrome. Histological wound healing was evaluated by two examiners by measuring the healing rate between the inner re-epithelial margins using Motic DSAssistant 4K.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.4.4. Immunohistochemical (IHC) analysis\u003c/h2\u003e \u003cp\u003eSpecimens from the palate were processed in neutral-buffered formalin and paraffin and sectioned at a thickness of 3 mm. IHC staining was performed using CD68 specific antibody (1:200 dilution; Ab125212; Abcam) and vimentin specific antibody (1:500 dilution; Ab137321; Abcam). CD68 staining was performed on the third day, whereas vimentin staining was performed on the specimens sacrificed on the seventh day. Regions of interest (ROI) were set at both wound margins. The EnVision\u0026thinsp;+\u0026thinsp;System-HRP-labeled polymer anti-rabbit was applied for 20 min, followed by color development using a labeled DAB kit. A slide scanner was used for sample scanning, after counterstaining with Mayer\u0026rsquo;s hematoxylin. And two trained examiners assessed stained protein areas of scanned slides with ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.5. \u003cem\u003eEx vivo\u003c/em\u003e wound healing analysis\u003c/h2\u003e \u003cp\u003eNeoderm\u0026reg;-OD (TEGO Science, Seoul, Korea), a 3 dimensionally-reconstructed human oral mucosa model, was utilized for \u003cem\u003eex vivo\u003c/em\u003e wound healing analysis. The rats were divided into three groups similar to those used in the rat model (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6 for each group). A round-shape-wound with 4mm diameter was created in each model using a biopsy punch. The models were incubated for 5 days at 37\u0026deg;C in a humidified environment with 10% CO\u003csub\u003e2\u003c/sub\u003e. The maintenance medium was replaced every 2 days. After incubation, the model was processed with 4% paraformaldehyde and paraffin, and sectioned to make slides. And hematoxylin and eosin was used to stain the slides. With use of Image Pro software (Media Cybernetics, Inc., Rockville, MD, USA), re-epithelialization was evaluated. Re-epithelialization (%) was calculated as (Re-epithelialization length / Total wound length) \u0026times; 100.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis of acquired data were conducted with IBM SPSS Statistics, version 20 (SPSS Inc., Chicago, IL, USA). One-way ANOVA and Tukey\u0026rsquo;s honest significant difference post hoc tests were performed after the normality test. The threshold for \u003cem\u003ep\u003c/em\u003e-values was set to 0.05. To assess the interobserver reliability of both IHC and histological analyses, intraclass coefficient values (ICC) were computed between the two examiners. The ICC values indicated high reliability, with scores of 0.991 and 0.890 for histological and IHC analyses, respectively.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. Characterization of hyaluronic acid-based scaffolds\u003c/h2\u003e\n \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.1. FT-IR analysis\u003c/h2\u003e\n \u003cp\u003eHA exhibited strong and broad peaks at 3390 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e that originated from the -OH and -NH groups. Adding CMC to the HA scaffold leads to a corresponding decrease in the peaks attributed to HA characteristics (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Particularly, a significant increase in the peaks was noted around the 3390 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e region, indicating distinct HA properties. However, no distinct peaks were observed.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.2. Mechanical properties\u003c/h2\u003e\n \u003cp\u003eIn the experiments conducted to assess the mechanical properties, including tensile strength and elongation, adding the CMC scaffold layer resulted in increased brittleness, thus reducing tensile strength and elongation (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe addition of CMC (20%) to the HA scaffold reduced the tensile strength and elongation rate. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. HA scaffold.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. \u003cem\u003eIn vitro\u003c/em\u003e evaluation\u003c/h2\u003e\n \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.1. Cytocompatibility\u003c/h2\u003e\n \u003cp\u003eTo assess the cytocompatibility of the HA-C scaffold, the CCK-8 test and flow cytometry were performed on HGF cells. The CCK-8 assay showed that the viability of HGF cells on the HA-C scaffold was similar to that of the control at 24 h (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The flow cytometry assay also confirmed that apoptotic or dead cells among the materials did not differ significantly, thus, confirming the cytocompatibility of the HA-C scaffold (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.2. \u003cem\u003eIn vitro\u003c/em\u003e wound healing assay\u003c/h2\u003e\n \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e wound healing assay showed a significant reduction in the open wound area in the HA-C scaffold group compared with that in the control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, the open wound areas between the HA and HA-C scaffold groups did not differ significantly (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. \u003cem\u003eIn vivo\u003c/em\u003e evaluation\u003c/h2\u003e\n \u003cp\u003eOn day 3, healing rates were higher in the HA (21.17\u0026thinsp;\u0026plusmn;\u0026thinsp;10.65%) and HA-C scaffold groups (22.08\u0026thinsp;\u0026plusmn;\u0026thinsp;6.50%), compared to the control group (10.98\u0026thinsp;\u0026plusmn;\u0026thinsp;8.33%) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Conversely, the healing rates among the 7-day groups did not differ significantly (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eFurthermore, the HA-C scaffold group demonstrated a significantly lower CD68 expression and a greater vimentin expression than the control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, marker expression in the HA scaffold group did not significantly differ from the control group (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4. \u003cem\u003eEx vivo\u003c/em\u003e evaluation\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e shows the results of the \u003cem\u003eex vivo\u003c/em\u003e evaluation. The re-epithelialization rates in the HA scaffold group (94.77\u0026thinsp;\u0026plusmn;\u0026thinsp;11.73%) and HA-C group (96.15\u0026thinsp;\u0026plusmn;\u0026thinsp;7.97%) were significantly higher than the controls (14.55\u0026thinsp;\u0026plusmn;\u0026thinsp;5.95%). However, there were no significant differences between the HA and HA-C scaffold groups.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study investigated the effects of HA-C scaffolds on oral wound healing, both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. The results of our study confirmed that the wound-healing effects of the HA and HA-C scaffolds did not differ significantly. However, wound-healing marker expression in the HA-C scaffold differed from that in the control, whereas the HA scaffold did not. Thus, the null hypothesis was partially rejected.\u003c/p\u003e \u003cp\u003eBased on the CCK-8 assay and flow cytometry, the HA-C scaffold did not cause detrimental effects on HGF cell proliferation, indicating its cytocompatibility. This finding suggests that the scaffold has the potential to support cellular activity and tissue regeneration. Taken together, HA-C scaffolds are suitable for biomedical applications. Maintaining high cytocompatibility is crucial for wound dressings (Wu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Previous studies proposed an acceptable range (approximately\u0026thinsp;\u0026gt;\u0026thinsp;70%~75% viability) for biomedical applications, including wound dressings (Khorasani et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Archana et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e healing assays confirmed that the open wound area waw significantly reduced when both scaffolds were used, compared with the control. Similarly, the HA-C scaffolds significantly promoted wound healing during the initial phase in both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003eex vivo\u003c/em\u003e analyses. Previous studies have shown that palatal wound healing progressively slows during the first 7 days after injury (Kozlovsky et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, Weinberg et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Considering these findings, our results suggest that HA-C scaffolds can accelerate palatal wound healing during the initial phase. Furthermore, our findings, particularly from the initial phase (0\u0026ndash;3 days) are consistent with those of a previous study (Lee et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, at day 7, wound healing did not differ significantly between the HA-C scaffold and control groups. In contrast, a difference was demonstrated in a previous study (Lee et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These discrepancies may be attributed to the different cutting planes of the specimens used in the two studies. According to the zipper theory, most wound healing dynamics in the initial stage of mucoperiosteal wounds occur in the sagittal plane, with minimal changes observed in the coronal plane (Weinberg et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Taken together, these results support that HA-C scaffolds offer the most beneficial effects during the initial phase of oral wound healing. As concerns regarding water resistance in cellulose-based materials have been well documented because of the abundance of hydroxyl groups in cellulose (Abdelmouleh et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), in the future, studies to explore the exact interaction between HA and cellulose, as well as the stability of the materials, are warranted\u003c/p\u003e \u003cp\u003eTo assess the effects of HA-C scaffolds on wound healing markers, CD68 and vimentin expression were evaluated. CD68 serves as an indicator of macrophages and inflammatory responses (Lee et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). We observed a significant decrease in CD68 expression in the HA-C scaffold group, indicating that HA-C scaffolds may hinder the migration of macrophages and neutrophils. Macrophages, particularly M1-like macrophages, are critical in the inflammatory-proliferation phase transition (Kim et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). M1-like macrophages are active during the early inflammatory phase and promote inflammatory responses (Zhou et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Our findings suggest that the HA-C scaffolds can mitigate inflammatory responses by inhibiting the formation of M1-like macrophages. This validates the potential of HA-C scaffolds in improving wound healing by modulating immune responses.\u003c/p\u003e \u003cp\u003eVimentin is a marker of fibroblasts. HA-C scaffolds significantly upregulated vimentin expression compared to the HA scaffold 7 days after injury. During the proliferation phase of wound healing, fibroblasts play a crucial role in initial wound contraction, leading to wound closure (Tomasek et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). According to recent findings, a significant increase in fibroblast density was observed during palatal wound healing in the first 1\u0026ndash;2 weeks after injury (Weinberg et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Considering these findings, the HA-C scaffolds can also contribute to wound healing by improving fibroblast recruitment and proliferation. Thus, it is plausible that cellulose has a positive effect on fibroblast colonization of the scaffold. Available evidence suggests that the structure of cellulose contributes to the attachment and overgrowth of fibroblasts on cellulose-coated scaffolds to a greater extent than uncoated surfaces (Dydak et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study has several limitations. First, we only observed the wound healing process up to 7 days post-operatively. The fibroblast-driven wound healing process may continue until the wound closes. Further studies with longer evaluation periods are warranted. Second, we did not directly assess HA release. To better understand the action of HA-C scaffolds, the residual amount of HA in the wound area should be assessed. Finally, various combinations of HA and cellulose were not evaluated. Previous studies on HA-C composites have shown superior functionality, such as anti-adhesive barriers and vehicles for controlled drug release (Huang et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). To explore the full potential of HA-C scaffolds on wound-healing and tissue-engineering applications, further studies on the formulation and optimization of HA/CMC scaffolds, are warranted.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConclusions\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn conclusion, HA-C scaffolds are cytocompatible with oral wound healing. Importantly, HA-C scaffolds accelerated the wound healing process by suppressing inflammatory responses and encouraging fibroblast recruitment and proliferation at wound sites. These results support their potential use in oral wound-healing applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no direct or indirect conflicts of interest, such as associations with commercial entities, financial associations involving family members, or other non-financial associations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDatasets related to this article are available upon request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was reviewed and approved by the Institutional Animal Care and Use Committee of Kyung Hee University Medical Center, Seoul, South Korea (KHMC-IACUC-2023-008).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYun Sun Jung:\u003c/strong\u003e Data curation, formal analysis, and writing-original draft. \u003cstrong\u003eJu Ri Ye:\u003c/strong\u003e Data curation, formal analysis, and visualization. \u003cstrong\u003eMyoung-Han Lee:\u003c/strong\u003e Data curation and resources. \u003cstrong\u003eDong-Keun Kewon:\u003c/strong\u003e Data curation and resources. \u003cstrong\u003eYong Kwon Chae:\u003c/strong\u003e Writing-review and editing. \u003cstrong\u003eHyo-Seol Lee:\u003c/strong\u003e Writing-review and editing. \u003cstrong\u003eSung Chul Choi:\u003c/strong\u003e Writing-review and editing. \u003cstrong\u003eOk Hyung Nam:\u003c/strong\u003e Conceptualization, data curation, validation, formal analysis, funding acquisition, writing-original draft, writing-review, and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Technology Development Program, funded by the Ministry of SMEs and Startups (MSS, No. S3290500).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdelmouleh M, Boufi S, Belgacem M, Duarte A, Salah AB, Gandini A (2004) Modification of cellulosic fibres with functionalised silanes: development of surface properties. Int J Adhes Adhes 24:43-54\u003c/li\u003e\n\u003cli\u003eAgarwal C, Tarun Kumar AB, Mehta DS (2015) Comparative evaluation of free gingival graft and AlloDerm\u0026reg; in enhancing the width of attached gingival: A clinical study. Contemp Clin Dent 6:483-488\u003c/li\u003e\n\u003cli\u003eAmerican Society for Testing and Materials (2018) ASTM D882: Standard Test Method for Tensile Properties of Thin Plastic Sheeting. American Society for Testing and Materials\u003c/li\u003e\n\u003cli\u003eArchana D, Dutta J, Dutta PK (2013) Evaluation of chitosan nano dressing for wound healing: characterization, in vitro and in vivo studies. Int J Biol Macromol 57:193-203\u003c/li\u003e\n\u003cli\u003eBoateng JS, Matthews KH, Stevens HN, Eccleston GM (2008) Wound healing dressings and drug delivery systems: a review. J Pharm Sci 97:2892-2923\u003c/li\u003e\n\u003cli\u003eBonatto MDS, Feltran GDS, Barbosa TP, Pereira DA, Santos SS, Mendes PGJ, RS EP, Bezerra FJB, Zambuzzi WF, de Oliveira G (2024) Green tea and hyaluronic acid gel enhance fibroblast activation and improves the gingival healing post-third molar extraction. Sci Rep 14:7124\u003c/li\u003e\n\u003cli\u003eCiolacu DE, Nicu R, Ciolacu F (2020) Cellulose-Based Hydrogels as Sustained Drug-Delivery Systems. Materials (Basel) 13\u003c/li\u003e\n\u003cli\u003eDaugela P, Pranskunas M, Juodzbalys G, Liesiene J, Baniukaitiene O, Afonso A, Sousa Gomes P (2018) Novel cellulose/hydroxyapatite scaffolds for bone tissue regeneration: In vitro and in vivo study. J Tissue Eng Regen Med 12:1195-1208\u003c/li\u003e\n\u003cli\u003eDydak K, Junka A, Nowacki G, Paleczny J, Szymczyk-Zi\u0026oacute;łkowska P, G\u0026oacute;rzyńska A, Aniołek O, Bartoszewicz M (2022) In Vitro Cytotoxicity, Colonisation by Fibroblasts and Antimicrobial Properties of Surgical Meshes Coated with Bacterial Cellulose. Int J Mol Sci 23\u003c/li\u003e\n\u003cli\u003eEl Fawal GF, Abu-Serie MM, Hassan MA, Elnouby MS (2018) Hydroxyethyl cellulose hydrogel for wound dressing: Fabrication, characterization and in vitro evaluation. Int J Biol Macromol 111:649-659\u003c/li\u003e\n\u003cli\u003eHuang YC, Huang KY, Yang BY, Ko CH, Huang HM (2016) Fabrication of Novel Hydrogel with Berberine-Enriched Carboxymethylcellulose and Hyaluronic Acid as an Anti-Inflammatory Barrier Membrane. Biomed Res Int 2016:3640182\u003c/li\u003e\n\u003cli\u003eInternational Organization for Standardization (2021) ISO 10993-12:2021: Biological evaluation of medical devices Part 12: Sample preparation and reference materials. ISO\u003c/li\u003e\n\u003cli\u003eKhorasani MT, Joorabloo A, Adeli H, Mansoori-Moghadam Z, Moghaddam A (2019) Design and optimization of process parameters of polyvinyl (alcohol)/chitosan/nano zinc oxide hydrogels as wound healing materials. Carbohydr Polym 207:542-554\u003c/li\u003e\n\u003cli\u003eKim H, Wang SY, Kwak G, Yang Y, Kwon IC, Kim SH (2019) Exosome-Guided Phenotypic Switch of M1 to M2 Macrophages for Cutaneous Wound Healing. Adv Sci 6:1900513\u003c/li\u003e\n\u003cli\u003eKozlovsky A, Artzi Z, Hirshberg A, Israeli-Tobias C, Reich L (2007) Effect of local antimicrobial agents on excisional palatal wound healing: a clinical and histomorphometric study in rats. J clin periodontol 34:164-171\u003c/li\u003e\n\u003cli\u003eLee JH, Lee KE, Kang SW, Park SH, Chae YK, Lee MH, Kweon DK, Choi SC, Nam OH (2024) Effect of orodispersible hyaluronic acid film on palatal mucosa wound healing. Oral Dis 30:518-527\u003c/li\u003e\n\u003cli\u003eLee JH, Lee KE, Nam OH, Chae YK, Lee MH, Kweon DK, Kim MS, Lee HS, Choi SC (2022) Orodispersible hyaluronic acid film delivery for oral wound healing in rats. J Dent Sci 17:1595-1603\u003c/li\u003e\n\u003cli\u003eLi Y, Li H, Yu Z, Liu J, Lin Y, Xu J, Zhang C, Chen Q, Han X, Peng Q (2024) Drug-free and multifunctional sodium bicarbonate/hyaluronic acid hybrid dressing for synergistic healing of infected wounds. Int J Biol Macromol 259:129254\u003c/li\u003e\n\u003cli\u003eNational Research Council (2011) Guide for the Care and Use of Laboratory Animals: Eighth Edition. Washington, DC: The National Academies Press. https://doi.org/10.17226/12910\u003c/li\u003e\n\u003cli\u003ePark JS, Lee JH, Han CS, Chung DW, Kim GY (2011) Effect of hyaluronic acid-carboxymethylcellulose solution on perineural scar formation after sciatic nerve repair in rats. Clin Orthop Surg 3:315-24\u003c/li\u003e\n\u003cli\u003eToma AI, Fuller JM, Willett NJ, Goudy SL (2021) Oral wound healing models and emerging regenerative therapies. Transl Res 236:17-34\u003c/li\u003e\n\u003cli\u003eTomasek JJ, Gabbiani G, Hinz B, Chaponnier C, Brown RA (2002) Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol 3:349-363\u003c/li\u003e\n\u003cli\u003eWang X, Tang J, Huang J, Hui M (2020) Production and characterization of bacterial cellulose membranes with hyaluronic acid and silk sericin. Colloids Surf B Biointerfaces 195:111273\u003c/li\u003e\n\u003cli\u003eWeinberg E, Vered M, Atzil S, Chaushu G, Chaushu L (2020) The dynamics of closure following excisional mid-palatal mucoperiosteal wound in a rat model. Clin Oral Investig 24:4385-4393\u003c/li\u003e\n\u003cli\u003eWu Y, Li X, Shi X, Zhan Y, Tu H, Du Y, Deng H, Jiang L (2017) Production of thick uniform-coating films containing rectorite on nanofibers through the use of an automated coating machine. Colloids Surf B Biointerfaces 149:271-279\u003c/li\u003e\n\u003cli\u003eWu Y, Wang L, Zhao X, Hou S, Guo B, Ma PX (2016) Self-healing supramolecular bioelastomers with shape memory property as a multifunctional platform for biomedical applications via modular assembly. Biomater 104:18-31\u003c/li\u003e\n\u003cli\u003eZhou Y, Zhang X-L, Lu S-T, Zhang N-Y, Zhang H-J, Zhang J, Zhang J (2022) Human adipose-derived mesenchymal stem cells-derived exosomes encapsulated in pluronic F127 hydrogel promote wound healing and regeneration. Stem Cell Res Ther 13:407\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellulose","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cels","sideBox":"Learn more about [Cellulose](https://www.springer.com/journal/10570)","snPcode":"10570","submissionUrl":"https://submission.nature.com/new-submission/10570/3","title":"Cellulose","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"cellulose, drug vehicle, hyaluronic acid, wound healing, hyaluronic acid","lastPublishedDoi":"10.21203/rs.3.rs-4263630/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4263630/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eTo assess the effects of hyaluronic acid (HA)-based scaffolds on oral wound healing.\u003c/p\u003e\u003ch2\u003eMaterials and Methods\u003c/h2\u003e \u003cp\u003eA film-type 3% HA scaffold with bilayer cellulose coating was prepared and compared with one without coating. For cytocompatibility, human gingival fibroblasts were exposed to both scaffolds. Cell viability, flow cytometry, and scratch wound assays were performed. Additionally, \u003cem\u003ein vivo\u003c/em\u003e and` \u003cem\u003eex vivo\u003c/em\u003e wound-healing assays were also conducted.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCytocompatibility tests showed no cytotoxicity for either HA scaffold. The scratch wound assay revealed a significant reduction in the open wound area in both HA scaffolds compared with the control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, no differences were observed in the open wound areas between the scaffolds with and without cellulose coating. Regarding \u003cem\u003ein vivo\u003c/em\u003e wound healing analysis, the healing rates on day 3 were significantly higher in the HA scaffolds than in the control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and there were no differences between the scaffolds. The HA scaffolds with the coating showed lower CD68 and higher vimentin expression than the control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, the HA scaffolds without the coating did not. The \u003cem\u003eex vivo\u003c/em\u003e wound healing analysis showed significantly higher re-epithelialization rates in both scaffolds compared to the control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eWithin the limits of this study, the HA scaffold with coating showed enhanced wound-healing efficacy, indicating its potential for oral wound-healing applications.\u003c/p\u003e","manuscriptTitle":"Bilayer cellulose-coated hyaluronic acid-based scaffold for accelerating oral wound healing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-17 05:00:04","doi":"10.21203/rs.3.rs-4263630/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-04-15T22:51:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-15T07:47:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-15T07:47:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellulose","date":"2024-04-14T04:56:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellulose","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cels","sideBox":"Learn more about [Cellulose](https://www.springer.com/journal/10570)","snPcode":"10570","submissionUrl":"https://submission.nature.com/new-submission/10570/3","title":"Cellulose","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6aaa782d-906a-4997-ae86-96c504a78c54","owner":[],"postedDate":"April 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-07T16:06:43+00:00","versionOfRecord":{"articleIdentity":"rs-4263630","link":"https://doi.org/10.1007/s10570-024-06198-9","journal":{"identity":"cellulose","isVorOnly":false,"title":"Cellulose"},"publishedOn":"2024-09-30 15:57:16","publishedOnDateReadable":"September 30th, 2024"},"versionCreatedAt":"2024-04-17 05:00:04","video":"","vorDoi":"10.1007/s10570-024-06198-9","vorDoiUrl":"https://doi.org/10.1007/s10570-024-06198-9","workflowStages":[]},"version":"v1","identity":"rs-4263630","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4263630","identity":"rs-4263630","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}