Effect of Subcutaneous Tissue Transglutaminase on Growth Factors and Type I Collagen in Surgical Incision Wound Model

Effect of Subcutaneous Tissue Transglutaminase on Growth Factors and Type I Collagen in Surgical Incision Wound Model | 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 Effect of Subcutaneous Tissue Transglutaminase on Growth Factors and Type I Collagen in Surgical Incision Wound Model Orcun ISLER OZGUN, Suna CEBESOY, Emine Nazlı HAYIRLI, Oya EVIRGEN, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9103237/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study aims to determine the effects of subcutaneous Tissue Transglutaminase (TG2) application in surgical incision models in healthy male Wistar Albino rats, including macroscopic observations and its impact on selected growth factors and Type I collagen in vivo. A total of 24 healthy male Wistar Albino rats (200–250 g) were randomly assigned to three groups (n = 8 each): healthy tissue group, control surgical incision group, and surgical incision group treated with TG2. A standardized 4 cm dorsal incision was made on the groups and sutured. The treatment group received subcutaneous TG2 (10 IU/kg) on days 0 and 15 after suturing, and the incision sites were photographed to assess macroscopic healing. On day 15, all rats were euthanized under general anesthesia. Then, wound tissues were collected for microscopic examination. Levels of selected growth factors and Type I collagen were measured by the ELISA method, and the data were analyzed statistically using one-way ANOVA test. The wounds in the TG2-treated group healed faster and showed improved tissue organization compared to the untreated incision group, as macroscopically and histopathologically. The levels of selected growth factors and Type I collagen were increased in the TG2-treated group compared with both the control incision and healthy tissue groups, as indicated by ELISA results. The findings of this study suggest that TG2, alone or in combination with other growth factors, holds promise for future medical and veterinary clinical applications to accelerate wound repair and improve tissue regeneration outcomes. Growth factors Tissue transglutaminase (TG2) Type I Collagen Wound healing Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION The wound healing process, comprising three phases – inflammation, cell proliferation, and remodeling – consists of sequential, interconnected, complex biochemical and cellular events such as cell migration, cross-linking, and collagen synthesis (Mathew-Steiner, Roy, & Sen, 2021 ). Tissue transglutaminase (TG2) assumes extracellular roles in matrix stabilization, which is significant in tissue repair, wound healing, angiogenesis, bone repair, inflammation, and platelet aggregation. It, which has been proven to be effective in the wound healing process, has also been shown in many studies to be actively involved in many stages of wound healing, including cell adhesion, cell proliferation, and migration of fibroblasts to the wound site. TG2's ability to remodel the Extracellular Matrix (ECM) is associated with its cross-linking activity (Aydin et al., 2016 ; Verderio, Johnson, & Griffin, 2004 ; Yiu, Holman, Kaidonis, Graham, & Iismaa, 2023 ). Experimental studies demonstrate that growth factors play distinct roles at different phases of wound healing (e.g., tissue division, differentiation, proliferation, and organization). In this context, researchers have implemented numerous applications using growth factors, both externally and internally, for the treatment of different kinds of wounds (da Silva et al., 2024 ; Partoazar, Kianvash, & Goudarzi, 2022 ). A few hours after the injury, growth factors such as Fibroblast Growth Factor (FGF), Epidermal Growth Factor (EGF), and Vascular Endothelial Growth Factor (VEGF), continuously secreted from neutrophils and macrophages following a significant amount of phagocytosis, stimulate the repopulation of the wound and the reconstruction of new tissue (Table 1 ) (Singer & Clark, 1999 ; Yiu et al., 2023 ). Table 1 Growth factors in wound healing (Singer & Clark, 1999 ; Yiu et al., 2023 ) Growth factor Producer cells Biological activity EGF Macrophages Keratinocytes Stimulating collagenase activity, Keratinocyte proliferation and migration, FGF Macrophages Endothelial cells Fibroblasts Keratinocyte Smooth muscle cells Keratinocyte proliferation and migration, Angiogenesis, Endothelial cell activation VEGF Macrophages Keratinocytes Fibroblasts Angiogenesis Collagen is a structural protein of connective tissue that is widely found in the body, such as in skin, bone, and cartilage (Singh, Rai, & Agrawal, 2023 ). The best indicator of wound healing from a surgical, morphological, and chemical perspective is the achievement of wound tension resistance at the level of healthy tissue. Wound tension resistance depends on collagen maturation. The collagen maturation begins on day 15 after the wound and continues for one year (Mathew-Steiner et al., 2021 ). TG2 cell-surface interactions regulate signaling by growth factor receptors. These receptors support and maintain growth factor signaling. Therefore, TG2 cell surface interactions are effective in maintaining important events such as keratinocyte migration, fibroblast activation, and angiogenesis that occur during wound healing processes (Belkin, 2011 ; Telci & Griffin, 2006 ). This study aims to determine the effects of subcutaneous TG2 administration in surgical incision models in healthy male Wistar Albino rats, through both macroscopic and microscopic observations, as well as its impact on specific growth factors and Type I collagen in vivo. MATERIALS and METHODS Ethical Statement and Experimental Animals Before the experimental studies, confirmation was obtained from the Animal Experiments Ethics Committee of the Animal Research Center, Ministry of Health, Xxxxxx Xxxxxxx Xxxxxxxx Xxxxxxx Training and Research Hospital, on June 6, 2012, with protocol number 2012/8. In this study, 24 randomly selected healthy male Wistar-Albino rats weighing 200–250 grams, provided from the Xxxxxxx Xxxxxxxx Xxxxxxx Training and Research Hospital's Experimental Animal Production and Research Laboratory in Xxxxxx, were used. They were divided into three groups of eight (healthy tissue, surgical incision, and surgical incision + TG2 treatment), housed under a 12-hour light-dark cycle with ad libitum access to food and water. Creation of Wounds The dorsal area was shaved of each rat before surgery, and povidone-iodine was used for antisepsis as part of the preoperative preparation. Sixteen rats were anesthetized intraperitoneally with Ketamine HCL at 60 mg/kg and Xylazine at 5 mg/kg. After that, a 4 cm long full incision was made in the dorsal region of the rats with a scalpel, and then the incision edges were sutured together using simple interrupted sutures with 3.0 silk surgical suture and a sharp needle. No application was made to the rats in the healthy tissue group (n = 8) until the day of euthanasia. Preparation Process of TG2 In the study group, TG2 was applied subcutaneously at a dose of 10 IU/kg, optimized in cell culture medium, to the sutured area every other day, until the day of euthanasia. Macroscopic Examination of Wounds by Photographing During the experiment, the wound-healing process and infection status of rats in the sutured groups were monitored daily; photographs of the rats were taken on days 3, 5, 7, 10, 12, and 15 (the euthanasia day) after application. Tissue Sampling On day 15, all the rats were anesthetized with Ketamine HCl (60 mg/kg) and Xylazine (5 mg/kg) by intraperitoneal injection, and then their dorsal areas were shaved widely. Under sterile conditions, a 4 cm 2 square area of skin involving the suture line was excised. A portion of the extracted tissues was stored at -80 degrees for ELISA measurements, while the remaining portion was transferred to capped tubes containing 4% paraformaldehyde for histological examination. Preparation of the Skin Material for ELISA Skin materials stored at -80°C were homogenized in radioimmunoprecipitation assay (RIPA) buffer containing 2 µL/mL of protease inhibitor cocktail, using a homogenizer (Powergen ® homogenizer 125, Fisher Scientific) according to the procedures in the ELISA kits. The samples were then centrifuged at 5000 x g for 5 minutes, and the supernatant was collected. The concentrations of growth factors (FGF, EGF, VEGF) and Type I Collagen in the supernatants were determined using the ELISA kits (e-BioScience ® , Inc.) according to the manufacturer's instructions. Samples with determined concentrations in ELISA kits were read at 450 nm using a spectrophotometer (Bio-Tek Instruments Inc., MWGt Lambda Scan 200, USA). Histopathological Examination The histopathological examination was carried out at the Xxxxxxxxxx xx Xxxxxxxxx xxx Xxxxxxxxxx, Xxxxxx Xxxxxxxxxx Xxxxxxx xx Xxxxxxxx. On day 15 of the procedure, tissue samples taken from rats were fixed in 4% paraformaldehyde and processed according to the routine histological follow-up protocol. The tissues were embedded in paraffin for long-term storage. They were stored in a refrigerator at + 4°C. The sectioning process was carried out using a sliding microtome (Leica ® , SM2000R), yielding longitudinal sections 3 µm thick. Histopathological examination was performed by light microscopy. In the histological tissue examinations, organization of collagen type 1 fibers in the connective tissue, inflammatory cell infiltration, and increased vascularization were evaluated. The tissue samples were stained with Hematoxylin-Eosin (H3136, Sigma-Aldrich; E4009, Sigma-Aldrich) to examine basic parameters of wound healing and photographed with a microscope camera (Leica ® ICC50 HD). Statistical Analyse The difference between ELISA results obtained from all experimental groups was determined using one-way ANOVA test in the SPSS (IBM SPSS Statistics 2023) program, and significance controls were evaluated at the 0.01 level. RESULTS Macroscopic Examination The macroscopic examination was conducted by photographing some physical changes in the skin after the formation of the wound areas. In the group that received subcutaneous TG2 in the wound area, the wound showed faster, more robust healing morphologically. It was also determined that hair growth in the wound area increased more rapidly in this group (Figs. 1 and 2). Microscopic Evaluation of Tissue Samples Microscopic examination revealed migration of hair follicles and sebaceous glands into the granulation tissue only in the TG2-treated group (Fig. 3). On day 15 of wound formation, evaluations of the granulation tissue in the damaged dermis of the TG2-treated group, conducted via random scans across 5 areas at 40x magnification, revealed an average of 1–5 new blood vessel formations (Fig. 4). This finding was consistent with the maturation and remodeling phase of the healing process. The study presented here concludes that subcutaneous TG2 application increases new blood vessel formation during wound healing. The formation of new collagen, another indication of healing, was also observed in the TG2 group, appearing as fine collagen fibers in the wound area (Fig. 4). Evaluation of Growth Factors and Type I Collagen The mean concentration values of growth factors (FGF, EGF, VEGF) obtained by spectrophotometer measurement after the ELISA method were compared between all experimental groups, and the evaluation results of the mean for each growth factor are shown in Graphic 1. According to the data in Graphic 1, the mean FGF concentration ranged from 0.13 in the control group to 0.17 in the TG2-treated group. Regarding the mean EGF concentration, the highest value was 0.18 in the TG2-treated group, whereas the lowest was 0.10 in the control group. Lastly, the mean VEGF concentration was 0.41 in the TG2-treated group and 0.31 in the control group. Another parameter used to evaluate wound healing is the measurement of type I collagen concentration using a spectrophotometer after the ELISA method; the mean type I collagen concentrations for all experimental groups are presented in Graphic 2. According to the data in Graphic 2, the mean concentration of type 1 collagen in the experimental groups ranged from 0.09 to 0.13, with the highest value observed in the surgical incision group treated with TG2 and the lowest in the control surgical incision group. The mean data in the graphics show that subcutaneous TG2 application increases FGF, EGF, VEGF, and Type I collagen levels compared with healthy tissue and the control group during the wound-healing process. Statistical Analyse ELISA results were statistically evaluated, and a significant difference was observed among all groups (p < 0.01). DISCUSSION Numerous studies have been conducted to accelerate wound healing and contribute directly or indirectly to healing in diabetic models, where the healing process is significantly prolonged, and in healthy individuals (Armstrong, Tan, Boulton, & Bus, 2023 ; Baktir, Yeni, Üniversitesi, Fakültesi, & Dalı, 2019 ). The studies about skin wounds conducted in rats revealed that TG2 activity increases during healing, and knockout mice lacking TG2 have impaired wound repair (Eckert et al., 2014 ; C. S. Lee & Park, 2017 ; Sarang et al., 2009 ). Overall, TG2 appears to be essential for effective tissue regeneration. In this study, when overall results on growth factors effective in wound healing were evaluated, it was determined that subcutaneous TG2 administration increased growth factor levels during the wound-healing process. In the macroscopic evaluation, no bleeding or irritation was observed in the TG2 incision group, and the wound area was almost completely closed by day 15. In addition, hair growth in the wound area was better than in the control group (Figs. 1 and 2). This indicated that the healing in the TG2-treated group was faster and smoother. These findings are consistent with results from in vitro cell studies, indicating that TG2 plays a supportive role during wound healing (Kurt-Celep, Nihan Kilinc, Griffin, & Telci, 2022; Yiu et al., 2023 ). Experimental and clinical wound studies have also proven that growth factors have positive effects in the wound healing process (Yamakawa & Hayashida, 2019 ). FGF has been reported to induce angiogenesis and accelerate neovascularization by increasing endothelial cell proliferation and motility during the wound-healing process (Koike, Yozaki, Utani, & Murota, 2020 ). In the present study, when comparing the mean FGF concentrations obtained from ELISA measurements in the incision group treated with TG2 with those in the surgical incision and healthy tissue groups, it was observed that TG2 increased FGF levels during the wound-healing process (Graphic 1). Furthermore, a statistically significant difference was observed between the groups (p < 0.01). Studies have shown that TG2 supports wound closure by promoting angiogenesis and coordinating cell-matrix signaling (Badarau et al., 2015 ; Yadav & Kim, 2024 ). Considering the stimulating effect of FGF on angiogenesis as well, it appears that FGF and TG2 have a synergistic impact on the wound healing process. The results obtained in this study also support this statement. Increasing EGF secretion after injury accelerates wound healing by supporting epidermal differentiation and maintaining the skin's internal balance, while also protecting the injured tissue from the external environment by organizing keratinocytes (Barrientos, Brem, Stojadinovic, & Tomic-Canic, 2014 ). It has also been demonstrated that TG2 is induced by various stimuli, including EGF, ultraviolet (UV) light, oxidative stress, and viral infection (Lai, Lin, & Greenberg, 2016 ). In this study, ELISA measurements in the TG2-treated group showed that the mean EGF concentration was higher than that in the control and healthy tissue groups, indicating that TG2 increased EGF levels during the wound-healing process (Graphic 1). This has also been supported by macroscopic images (Figs. 1 and 2). These findings are attributed to the increase in EGF levels, as observed in other studies, which enhances wound healing rates and keratinocyte proliferation (Barrientos et al., 2014 ). Additionally, a statistically significant difference was observed between the groups (p < 0.01). A study showed that angiogenesis during wound healing began earlier in the EGF- and FGF-treated groups than in the control group. They particularly showed that the healing in the EGF group was faster and of better quality than in the FGF group. They suggested that this situation was due to early fibroblast proliferation, collagen release, and vascularization (Xing et al., 2013 ). In this study, it was hypothesized that the increase in EGF and FGF levels induced by TG2 contributed to increased vascularization and collagen secretion (Graphic 1). It has been revealed that TG2 supports neovascularization and angiogenesis in the wound healing process by increasing VEGF release (S. H. Lee et al., 2020 ). In this study, when the mean VEGF concentration levels obtained from ELISA measurements of the TG2-treated incision group were compared with the mean VEGF levels obtained from the control surgical incision and healthy tissue groups, it was shown that TG2 increased the level of VEGF during the wound healing process (Graphic 1). The data obtained were consistent with the literature. At the same time, a statistically significant difference was observed between the groups (p < 0.01). Studies have shown that TG2 has a direct effect on the cross-linking of type I collagen and is added to collagen I during fibril binding. The cross-linking activity of TG2 also increases the rate of collagen fibrillogenesis (Collighan & Griffin, 2009 ). In this study, it was observed that the mean Type I collagen concentration obtained from ELISA measurements in the incision group treated with TG2 increased Type I collagen levels during wound healing compared with the other experimental groups. This finding is consistent with studies proving the positive effect of type I collagen on wound healing (Mathew-Steiner et al., 2021 ). Furthermore, due to the collagen cross-linking activity of TG2, it is thought that subcutaneous administration of TG2 increases the rate of collagen fibrillogenesis (Graphic 2). However, a statistically significant difference was observed between the groups. (p < 0.01). Within the first 24 hours after injury, type I collagen is primarily found in the deeper layers of granulation tissue. Between days 6 and 13 post-injury, it is also seen in the granulation tissue in the upper layers (Singh et al., 2023 ). In the present study, microscopic examination of tissues obtained on the 15th day of the wound-healing process revealed Type I collagen fibers forming in the upper layers of the granulation tissue, as observed in the surgical incision group treated with TG2, consistent with the literature (Fig. 4). These findings of increased type I collagen levels and improved tissue formation in the TG2 surgical incision group are consistent with the literature data in the present study (Velez & Howard, 2012 ), which is associated with the maturation and organization of collagen fibers during the wound healing process. This suggests that TG2 application promotes faster, more effective deposition of mature collagen during wound healing in rats. In the study, the hair follicles and sebaceous glands observed in the surgical incision group treated with TG2 during microscopic examination (Fig. 3) are considered a highly positive finding for wound healing and granulation tissue maturation, as stated in the review by Hakkinen et al. (Häkkinen, Larjava, & Koivisto, 2011 ). Moreover, the results of the presented study indicate that subcutaneous application of TG2 also increases new blood vessel formation in the wound healing process (Fig. 4), which is consistent with the study by Haroon et al (Haroon, Hettasch, Lai, Dewhirst, & Greenberg, 1999 ). Both the examination of these findings and the analysis of macroscopic evaluation data suggest that subcutaneous administration of TG2 accelerates wound healing by increasing growth factors. Alongside these positive findings, under pathological conditions, TG2 can exert effects that lead to fibrosis at different phases of wound healing. In the inflammation phase, TG2 gene expression, induced by certain inflammatory cytokines, attracts inflammatory cells and T cells to the damaged tissues, thereby exaggerating responses in the wound-healing process (Mohan, Pinto, & Issekutz, 2003 ). TG2 can also act as a receptor to attract T cells to tissues, thereby further increasing injury responses (Le, Gohr, & Rosenthal, 2001 ). It is also possible that tissue injury leads to excessive transforming growth factor beta (TGFb) production, resulting in increased TG2 production and intense ECM cross-linking, thereby creating a microenvironment that promotes fibrosis (Lai, Lin, Wu, & Wu, 2017 ). The statistical results for growth factors and collagen parameters were significant in the study. However, to increase the reliability of the study results, it is recommended that further studies be conducted with a larger sample size and a longer experimental duration. As also stated in other studies in the article, the findings are consistent with the idea that growth factors positively affect the wound-healing process. Investigating the structure and function of TG2 offers a deeper understanding of treatment methods for fibrosis and impaired wound healing. When the data from this study are evaluated as a whole, subcutaneous TG2 application has positive effects on growth factors during the wound-healing process. It has also been suggested that TG2 may prevent undesirable conditions, such as scar formation, hypertrophic scars, or hair loss, in the wound area by promoting angiogenesis. Given TG2's role in wound healing and cell motility, it can also be considered a beneficial active agent in the treatment of chronic wounds. Due to these properties, the clinical use of TG2 can be further evaluated through studies on wound healing. However, the excessive accumulation of TG2 during wound healing, driven by inflammation, suggests that TG2 should be used clinically in limited amounts, as it can increase tissue scarring and lead to fibrosis. Although studies on the treatment of various human diseases with TG2 continue, its clinical applications are limited due to its numerous biological functions. For this reason, drug development processes targeting specific functions of TG2 are being pursued for therapeutic use. Moreover, while the current study provides important insights into TG2’s enhancement of wound healing and its effects on growth factor promotion during this process, several limitations must be acknowledged. Although this study is useful for controlled in vivo experiments, the very small sample size and the use of a rat model do not fully reflect human cellular responses. Additionally, despite macroscopic, histological, and immunohistochemical evaluations, biomechanical tests that would directly demonstrate functional improvement at the tissue level (e.g., tensile strength or elasticity measurements) were not included in the study. Furthermore, the study’s limitation to 15 days does not cover long-term outcomes such as potential recurrences and delayed complications. Future studies should incorporate larger sample sizes, longitudinal follow-up, and mechanical testing to better understand the durability and strength of wound areas treated with TG2. Declarations Conflict of interest There is no financial conflict of interest with any institution, organization, or individual regarding our article titled " Effect of Subcutaneous Tissue Transglutaminase on Growth Factors and Type I Collagen in Surgical Incision Wound Model" and there is no conflict of interest among the authors. Author Contribution The idea and design of this study belong to O IO, SC., and NY.; the collection and analysis of data belong to OIO, ENH, and NY; the interpretation of the data, preparation of the article draft, critical revision, review of the research results, and approval of the final version of the article belong to all authors. Acknowledgement This study, produced from a doctoral thesis, was supported by the Industrial Thesis (SAN-TEZ, Number: 0443-STZ.2013-2) Project in Turkey. Therefore, we would like to thank the Turkish Ministry of Science, Industry, and Technology for their financial support. Before the experiment, an application was submitted to the Animal Research Center of the Ankara Dışkapı Yıldırım Beyazıt Training and Research Hospital of the Ministry of Health, and the Animal Experiment Ethics Committee approved the appropriateness of the procedures to be performed on the animals throughout the study.Moreover, we thank High Food Engineer Seyma AGIRAL, Asst. 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Supplementary Files 5.jpg Graphic 1: EGF, FGF, and VEGF levels in all experimental groups (n=24, mean±SD) 6.jpg Graphic 2: Type I Collagen levels in all experimental groups (n=24, mean±SD) Cite Share Download PDF Status: Posted Version 1 posted 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. 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OZGUN","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYDACZgbGAwwMNgbIYgY41MK1MAC1pBmAWQgtCfh1AbUcJkELPzv7gwMf95w35u8/f/DTjYo7iQ3szdskGH/cw6lFspnH4OCMZ7fNJG4kM0vnnHmW2MBzrEyCIaEYpxaDwzwMh3kO3LZhuMHMIJ3bdjixQSLHDKgFt8sMDrM/OPznwDkb+fOHmX+Dtci/IaQFhA4cMDM4kMwGtYUHvxawX3oOJBsb3kg2s845c9i4jSet2CIhDbcWfv7jDx/8OGBnOO/8wce3cyoOy/azH95444MNbi2YgA1EkKJhFIyCUTAKRgEmAAD9WFYlcgDDMgAAAABJRU5ErkJggg==","orcid":"","institution":"Kastamonu University","correspondingAuthor":true,"prefix":"","firstName":"Orcun","middleName":"ISLER","lastName":"OZGUN","suffix":""},{"id":606459538,"identity":"e50f8fc2-c55f-4952-bc54-5157f3f60bec","order_by":1,"name":"Suna CEBESOY","email":"","orcid":"","institution":"Ankara University","correspondingAuthor":false,"prefix":"","firstName":"Suna","middleName":"","lastName":"CEBESOY","suffix":""},{"id":606459539,"identity":"fa738632-1b32-4cd9-921b-83b12ec7cb36","order_by":2,"name":"Emine Nazlı HAYIRLI","email":"","orcid":"","institution":"Hitit University","correspondingAuthor":false,"prefix":"","firstName":"Emine","middleName":"Nazlı","lastName":"HAYIRLI","suffix":""},{"id":606459540,"identity":"079dfa5a-ccda-4471-b6e5-4e6f8dc8ae04","order_by":3,"name":"Oya EVIRGEN","email":"","orcid":"","institution":"Ankara University","correspondingAuthor":false,"prefix":"","firstName":"Oya","middleName":"","lastName":"EVIRGEN","suffix":""},{"id":606459541,"identity":"53130e3e-1be4-4620-80f9-a4d9313996a2","order_by":4,"name":"Nuray YAZIHAN","email":"","orcid":"","institution":"Ankara University","correspondingAuthor":false,"prefix":"","firstName":"Nuray","middleName":"","lastName":"YAZIHAN","suffix":""}],"badges":[],"createdAt":"2026-03-12 09:54:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9103237/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9103237/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104876346,"identity":"d8d2c195-38d6-4015-b3e1-74dd31c4de50","added_by":"auto","created_at":"2026-03-18 08:42:18","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":79301,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9103237/v1/1f1cf2e3389916f9e509931a.jpg"},{"id":104876664,"identity":"202b2ab7-e99e-4513-a7ac-62c2a42cc9dd","added_by":"auto","created_at":"2026-03-18 08:43:18","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":85904,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9103237/v1/302c25faf745728837d0cec3.jpg"},{"id":104876560,"identity":"8fe6fee4-74f0-476e-9510-996d8c96aab5","added_by":"auto","created_at":"2026-03-18 08:42:53","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":163095,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9103237/v1/312604b1a7e15c20e5920432.jpg"},{"id":104876379,"identity":"d1caf57b-7bed-4fe4-b132-fa9fb12ecdf0","added_by":"auto","created_at":"2026-03-18 08:42:22","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":151744,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9103237/v1/1a3a2af3578ffdfad51b81b8.jpg"},{"id":108806024,"identity":"b4a0804d-9537-4451-8e17-c956dffdbded","added_by":"auto","created_at":"2026-05-08 15:27:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":685288,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9103237/v1/c862ace3-4ebe-4012-b99e-16b08a766f40.pdf"},{"id":104876288,"identity":"787d12d7-d72c-4c85-b1f7-55f732522604","added_by":"auto","created_at":"2026-03-18 08:42:12","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":42142,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphic 1:\u003c/strong\u003e EGF, FGF, and VEGF levels in all experimental groups (n=24, mean±SD)\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9103237/v1/c724ec6da20663f58a211446.jpg"},{"id":104876438,"identity":"087632de-18a2-48ae-b188-e26e8cf6a6e7","added_by":"auto","created_at":"2026-03-18 08:42:29","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":40139,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphic 2:\u003c/strong\u003e Type I Collagen levels in all experimental groups (n=24, mean±SD)\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9103237/v1/781bd2056810fa7a27a00846.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eEffect of Subcutaneous Tissue Transglutaminase on Growth Factors and Type I Collagen in Surgical Incision Wound Model\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe wound healing process, comprising three phases \u0026ndash; inflammation, cell proliferation, and remodeling \u0026ndash; consists of sequential, interconnected, complex biochemical and cellular events such as cell migration, cross-linking, and collagen synthesis (Mathew-Steiner, Roy, \u0026amp; Sen, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTissue transglutaminase (TG2) assumes extracellular roles in matrix stabilization, which is significant in tissue repair, wound healing, angiogenesis, bone repair, inflammation, and platelet aggregation. It, which has been proven to be effective in the wound healing process, has also been shown in many studies to be actively involved in many stages of wound healing, including cell adhesion, cell proliferation, and migration of fibroblasts to the wound site. TG2's ability to remodel the Extracellular Matrix (ECM) is associated with its cross-linking activity (Aydin et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Verderio, Johnson, \u0026amp; Griffin, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Yiu, Holman, Kaidonis, Graham, \u0026amp; Iismaa, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eExperimental studies demonstrate that growth factors play distinct roles at different phases of wound healing (e.g., tissue division, differentiation, proliferation, and organization). In this context, researchers have implemented numerous applications using growth factors, both externally and internally, for the treatment of different kinds of wounds (da Silva et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Partoazar, Kianvash, \u0026amp; Goudarzi, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA few hours after the injury, growth factors such as Fibroblast Growth Factor (FGF), Epidermal Growth Factor (EGF), and Vascular Endothelial Growth Factor (VEGF), continuously secreted from neutrophils and macrophages following a significant amount of phagocytosis, stimulate the repopulation of the wound and the reconstruction of new tissue (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (Singer \u0026amp; Clark, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Yiu et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGrowth factors in wound healing (Singer \u0026amp; Clark, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Yiu et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGrowth factor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProducer cells\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBiological activity\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEGF\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMacrophages\u003c/p\u003e \u003cp\u003eKeratinocytes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStimulating collagenase activity, Keratinocyte proliferation and migration,\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFGF\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMacrophages\u003c/p\u003e \u003cp\u003eEndothelial cells\u003c/p\u003e \u003cp\u003eFibroblasts\u003c/p\u003e \u003cp\u003eKeratinocyte\u003c/p\u003e \u003cp\u003eSmooth muscle cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKeratinocyte proliferation and migration, Angiogenesis, Endothelial cell activation\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVEGF\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMacrophages\u003c/p\u003e \u003cp\u003eKeratinocytes\u003c/p\u003e \u003cp\u003eFibroblasts\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAngiogenesis\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eCollagen is a structural protein of connective tissue that is widely found in the body, such as in skin, bone, and cartilage (Singh, Rai, \u0026amp; Agrawal, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The best indicator of wound healing from a surgical, morphological, and chemical perspective is the achievement of wound tension resistance at the level of healthy tissue. Wound tension resistance depends on collagen maturation. The collagen maturation begins on day 15 after the wound and continues for one year (Mathew-Steiner et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTG2 cell-surface interactions regulate signaling by growth factor receptors. These receptors support and maintain growth factor signaling. Therefore, TG2 cell surface interactions are effective in maintaining important events such as keratinocyte migration, fibroblast activation, and angiogenesis that occur during wound healing processes (Belkin, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Telci \u0026amp; Griffin, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study aims to determine the effects of subcutaneous TG2 administration in surgical incision models in healthy male Wistar Albino rats, through both macroscopic and microscopic observations, as well as its impact on specific growth factors and Type I collagen in vivo.\u003c/p\u003e"},{"header":"MATERIALS and METHODS","content":"\u003cp\u003e \u003cb\u003eEthical Statement and Experimental Animals\u003c/b\u003e \u003c/p\u003e \u003cp\u003e Before the experimental studies, confirmation was obtained from the Animal Experiments Ethics Committee of the Animal Research Center, Ministry of Health, Xxxxxx Xxxxxxx Xxxxxxxx Xxxxxxx Training and Research Hospital, on June 6, 2012, with protocol number 2012/8. In this study, 24 randomly selected healthy male Wistar-Albino rats weighing 200\u0026ndash;250 grams, provided from the Xxxxxxx Xxxxxxxx Xxxxxxx Training and Research Hospital's Experimental Animal Production and Research Laboratory in Xxxxxx, were used. They were divided into three groups of eight (healthy tissue, surgical incision, and surgical incision\u0026thinsp;+\u0026thinsp;TG2 treatment), housed under a 12-hour light-dark cycle with ad libitum access to food and water.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCreation of Wounds\u003c/h2\u003e \u003cp\u003eThe dorsal area was shaved of each rat before surgery, and povidone-iodine was used for antisepsis as part of the preoperative preparation. Sixteen rats were anesthetized intraperitoneally with Ketamine HCL at 60 mg/kg and Xylazine at 5 mg/kg. After that, a 4 cm long full incision was made in the dorsal region of the rats with a scalpel, and then the incision edges were sutured together using simple interrupted sutures with 3.0 silk surgical suture and a sharp needle. No application was made to the rats in the healthy tissue group (n\u0026thinsp;=\u0026thinsp;8) until the day of euthanasia.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation Process of TG2\u003c/h3\u003e\n\u003cp\u003eIn the study group, TG2 was applied subcutaneously at a dose of 10 IU/kg, optimized in cell culture medium, to the sutured area every other day, until the day of euthanasia.\u003c/p\u003e\n\u003ch3\u003eMacroscopic Examination of Wounds by Photographing\u003c/h3\u003e\n\u003cp\u003eDuring the experiment, the wound-healing process and infection status of rats in the sutured groups were monitored daily; photographs of the rats were taken on days 3, 5, 7, 10, 12, and 15 (the euthanasia day) after application.\u003c/p\u003e\n\u003ch3\u003eTissue Sampling\u003c/h3\u003e\n\u003cp\u003eOn day 15, all the rats were anesthetized with Ketamine HCl (60 mg/kg) and Xylazine (5 mg/kg) by intraperitoneal injection, and then their dorsal areas were shaved widely. Under sterile conditions, a 4 cm\u003csup\u003e2\u003c/sup\u003e square area of skin involving the suture line was excised. A portion of the extracted tissues was stored at -80 degrees for ELISA measurements, while the remaining portion was transferred to capped tubes containing 4% paraformaldehyde for histological examination.\u003c/p\u003e\n\u003ch3\u003ePreparation of the Skin Material for ELISA\u003c/h3\u003e\n\u003cp\u003eSkin materials stored at -80\u0026deg;C were homogenized in radioimmunoprecipitation assay (RIPA) buffer containing 2 \u0026micro;L/mL of protease inhibitor cocktail, using a homogenizer (Powergen\u003csup\u003e\u0026reg;\u003c/sup\u003e homogenizer 125, Fisher Scientific) according to the procedures in the ELISA kits. The samples were then centrifuged at 5000 x g for 5 minutes, and the supernatant was collected.\u003c/p\u003e \u003cp\u003eThe concentrations of growth factors (FGF, EGF, VEGF) and Type I Collagen in the supernatants were determined using the ELISA kits (e-BioScience\u003csup\u003e\u0026reg;\u003c/sup\u003e, Inc.) according to the manufacturer's instructions. Samples with determined concentrations in ELISA kits were read at 450 nm using a spectrophotometer (Bio-Tek Instruments Inc., MWGt Lambda Scan 200, USA).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological Examination\u003c/h2\u003e \u003cp\u003eThe histopathological examination was carried out at the Xxxxxxxxxx xx Xxxxxxxxx xxx Xxxxxxxxxx, Xxxxxx Xxxxxxxxxx Xxxxxxx xx Xxxxxxxx. On day 15 of the procedure, tissue samples taken from rats were fixed in 4% paraformaldehyde and processed according to the routine histological follow-up protocol.\u003c/p\u003e \u003cp\u003eThe tissues were embedded in paraffin for long-term storage. They were stored in a refrigerator at +\u0026thinsp;4\u0026deg;C. The sectioning process was carried out using a sliding microtome (Leica\u003csup\u003e\u0026reg;\u003c/sup\u003e, SM2000R), yielding longitudinal sections 3 \u0026micro;m thick.\u003c/p\u003e \u003cp\u003eHistopathological examination was performed by light microscopy. In the histological tissue examinations, organization of collagen type 1 fibers in the connective tissue, inflammatory cell infiltration, and increased vascularization were evaluated.\u003c/p\u003e \u003cp\u003eThe tissue samples were stained with Hematoxylin-Eosin (H3136, Sigma-Aldrich; E4009, Sigma-Aldrich) to examine basic parameters of wound healing and photographed with a microscope camera (Leica\u003csup\u003e\u0026reg;\u003c/sup\u003e ICC50 HD).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStatistical Analyse\u003c/h3\u003e\n\u003cp\u003eThe difference between ELISA results obtained from all experimental groups was determined using one-way ANOVA test in the SPSS (IBM SPSS Statistics 2023) program, and significance controls were evaluated at the 0.01 level.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMacroscopic Examination\u003c/h2\u003e \u003cp\u003eThe macroscopic examination was conducted by photographing some physical changes in the skin after the formation of the wound areas. In the group that received subcutaneous TG2 in the wound area, the wound showed faster, more robust healing morphologically. It was also determined that hair growth in the wound area increased more rapidly in this group (Figs.\u0026nbsp;1 and 2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMicroscopic Evaluation of Tissue Samples\u003c/h2\u003e \u003cp\u003eMicroscopic examination revealed migration of hair follicles and sebaceous glands into the granulation tissue only in the TG2-treated group (Fig.\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003cdiv description=\"\" class=\"Drawing\" id=\"1816147957\" name=\"Resim 6\"\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eOn day 15 of wound formation, evaluations of the granulation tissue in the damaged dermis of the TG2-treated group, conducted via random scans across 5 areas at 40x magnification, revealed an average of 1\u0026ndash;5 new blood vessel formations (Fig.\u0026nbsp;4). This finding was consistent with the maturation and remodeling phase of the healing process. The study presented here concludes that subcutaneous TG2 application increases new blood vessel formation during wound healing.\u003c/p\u003e \u003cp\u003eThe formation of new collagen, another indication of healing, was also observed in the TG2 group, appearing as fine collagen fibers in the wound area (Fig.\u0026nbsp;4).\u003c/p\u003e \u003cp\u003e \u003cdiv description=\"\" class=\"Drawing\" id=\"1357610808\" name=\"Resim 8\"\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of Growth Factors and Type I Collagen\u003c/h2\u003e \u003cp\u003eThe mean concentration values of growth factors (FGF, EGF, VEGF) obtained by spectrophotometer measurement after the ELISA method were compared between all experimental groups, and the evaluation results of the mean for each growth factor are shown in Graphic 1. According to the data in Graphic 1, the mean FGF concentration ranged from 0.13 in the control group to 0.17 in the TG2-treated group. Regarding the mean EGF concentration, the highest value was 0.18 in the TG2-treated group, whereas the lowest was 0.10 in the control group. Lastly, the mean VEGF concentration was 0.41 in the TG2-treated group and 0.31 in the control group.\u003c/p\u003e\u003cp\u003eAnother parameter used to evaluate wound healing is the measurement of type I collagen concentration using a spectrophotometer after the ELISA method; the mean type I collagen concentrations for all experimental groups are presented in Graphic 2. According to the data in Graphic 2, the mean concentration of type 1 collagen in the experimental groups ranged from 0.09 to 0.13, with the highest value observed in the surgical incision group treated with TG2 and the lowest in the control surgical incision group.\u003c/p\u003e\u003cp\u003eThe mean data in the graphics show that subcutaneous TG2 application increases FGF, EGF, VEGF, and Type I collagen levels compared with healthy tissue and the control group during the wound-healing process.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analyse\u003c/h2\u003e \u003cp\u003eELISA results were statistically evaluated, and a significant difference was observed among all groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eNumerous studies have been conducted to accelerate wound healing and contribute directly or indirectly to healing in diabetic models, where the healing process is significantly prolonged, and in healthy individuals (Armstrong, Tan, Boulton, \u0026amp; Bus, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Baktir, Yeni, \u0026Uuml;niversitesi, Fak\u0026uuml;ltesi, \u0026amp; Dalı, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe studies about skin wounds conducted in rats revealed that TG2 activity increases during healing, and knockout mice lacking TG2 have impaired wound repair (Eckert et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; C. S. Lee \u0026amp; Park, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sarang et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Overall, TG2 appears to be essential for effective tissue regeneration.\u003c/p\u003e \u003cp\u003eIn this study, when overall results on growth factors effective in wound healing were evaluated, it was determined that subcutaneous TG2 administration increased growth factor levels during the wound-healing process.\u003c/p\u003e \u003cp\u003eIn the macroscopic evaluation, no bleeding or irritation was observed in the TG2 incision group, and the wound area was almost completely closed by day 15. In addition, hair growth in the wound area was better than in the control group (Figs.\u0026nbsp;1 and 2). This indicated that the healing in the TG2-treated group was faster and smoother. These findings are consistent with results from in vitro cell studies, indicating that TG2 plays a supportive role during wound healing (Kurt-Celep, Nihan Kilinc, Griffin, \u0026amp; Telci, 2022; Yiu et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eExperimental and clinical wound studies have also proven that growth factors have positive effects in the wound healing process (Yamakawa \u0026amp; Hayashida, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFGF has been reported to induce angiogenesis and accelerate neovascularization by increasing endothelial cell proliferation and motility during the wound-healing process (Koike, Yozaki, Utani, \u0026amp; Murota, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the present study, when comparing the mean FGF concentrations obtained from ELISA measurements in the incision group treated with TG2 with those in the surgical incision and healthy tissue groups, it was observed that TG2 increased FGF levels during the wound-healing process (Graphic 1). Furthermore, a statistically significant difference was observed between the groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Studies have shown that TG2 supports wound closure by promoting angiogenesis and coordinating cell-matrix signaling (Badarau et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Yadav \u0026amp; Kim, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Considering the stimulating effect of FGF on angiogenesis as well, it appears that FGF and TG2 have a synergistic impact on the wound healing process. The results obtained in this study also support this statement.\u003c/p\u003e \u003cp\u003eIncreasing EGF secretion after injury accelerates wound healing by supporting epidermal differentiation and maintaining the skin's internal balance, while also protecting the injured tissue from the external environment by organizing keratinocytes (Barrientos, Brem, Stojadinovic, \u0026amp; Tomic-Canic, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). It has also been demonstrated that TG2 is induced by various stimuli, including EGF, ultraviolet (UV) light, oxidative stress, and viral infection (Lai, Lin, \u0026amp; Greenberg, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In this study, ELISA measurements in the TG2-treated group showed that the mean EGF concentration was higher than that in the control and healthy tissue groups, indicating that TG2 increased EGF levels during the wound-healing process (Graphic 1). This has also been supported by macroscopic images (Figs.\u0026nbsp;1 and 2).\u003c/p\u003e \u003cp\u003eThese findings are attributed to the increase in EGF levels, as observed in other studies, which enhances wound healing rates and keratinocyte proliferation (Barrientos et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Additionally, a statistically significant difference was observed between the groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003eA study showed that angiogenesis during wound healing began earlier in the EGF- and FGF-treated groups than in the control group. They particularly showed that the healing in the EGF group was faster and of better quality than in the FGF group. They suggested that this situation was due to early fibroblast proliferation, collagen release, and vascularization (Xing et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In this study, it was hypothesized that the increase in EGF and FGF levels induced by TG2 contributed to increased vascularization and collagen secretion (Graphic 1).\u003c/p\u003e \u003cp\u003eIt has been revealed that TG2 supports neovascularization and angiogenesis in the wound healing process by increasing VEGF release (S. H. Lee et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this study, when the mean VEGF concentration levels obtained from ELISA measurements of the TG2-treated incision group were compared with the mean VEGF levels obtained from the control surgical incision and healthy tissue groups, it was shown that TG2 increased the level of VEGF during the wound healing process (Graphic 1). The data obtained were consistent with the literature. At the same time, a statistically significant difference was observed between the groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003eStudies have shown that TG2 has a direct effect on the cross-linking of type I collagen and is added to collagen I during fibril binding. The cross-linking activity of TG2 also increases the rate of collagen fibrillogenesis (Collighan \u0026amp; Griffin, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In this study, it was observed that the mean Type I collagen concentration obtained from ELISA measurements in the incision group treated with TG2 increased Type I collagen levels during wound healing compared with the other experimental groups. This finding is consistent with studies proving the positive effect of type I collagen on wound healing (Mathew-Steiner et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Furthermore, due to the collagen cross-linking activity of TG2, it is thought that subcutaneous administration of TG2 increases the rate of collagen fibrillogenesis (Graphic 2). However, a statistically significant difference was observed between the groups. (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003eWithin the first 24 hours after injury, type I collagen is primarily found in the deeper layers of granulation tissue. Between days 6 and 13 post-injury, it is also seen in the granulation tissue in the upper layers (Singh et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In the present study, microscopic examination of tissues obtained on the 15th day of the wound-healing process revealed Type I collagen fibers forming in the upper layers of the granulation tissue, as observed in the surgical incision group treated with TG2, consistent with the literature (Fig.\u0026nbsp;4).\u003c/p\u003e \u003cp\u003eThese findings of increased type I collagen levels and improved tissue formation in the TG2 surgical incision group are consistent with the literature data in the present study (Velez \u0026amp; Howard, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), which is associated with the maturation and organization of collagen fibers during the wound healing process. This suggests that TG2 application promotes faster, more effective deposition of mature collagen during wound healing in rats.\u003c/p\u003e \u003cp\u003eIn the study, the hair follicles and sebaceous glands observed in the surgical incision group treated with TG2 during microscopic examination (Fig.\u0026nbsp;3) are considered a highly positive finding for wound healing and granulation tissue maturation, as stated in the review by Hakkinen et al. (H\u0026auml;kkinen, Larjava, \u0026amp; Koivisto, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Moreover, the results of the presented study indicate that subcutaneous application of TG2 also increases new blood vessel formation in the wound healing process (Fig.\u0026nbsp;4), which is consistent with the study by Haroon et al (Haroon, Hettasch, Lai, Dewhirst, \u0026amp; Greenberg, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1999\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBoth the examination of these findings and the analysis of macroscopic evaluation data suggest that subcutaneous administration of TG2 accelerates wound healing by increasing growth factors.\u003c/p\u003e \u003cp\u003eAlongside these positive findings, under pathological conditions, TG2 can exert effects that lead to fibrosis at different phases of wound healing. In the inflammation phase, TG2 gene expression, induced by certain inflammatory cytokines, attracts inflammatory cells and T cells to the damaged tissues, thereby exaggerating responses in the wound-healing process (Mohan, Pinto, \u0026amp; Issekutz, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). TG2 can also act as a receptor to attract T cells to tissues, thereby further increasing injury responses (Le, Gohr, \u0026amp; Rosenthal, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). It is also possible that tissue injury leads to excessive transforming growth factor beta (TGFb) production, resulting in increased TG2 production and intense ECM cross-linking, thereby creating a microenvironment that promotes fibrosis (Lai, Lin, Wu, \u0026amp; Wu, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe statistical results for growth factors and collagen parameters were significant in the study. However, to increase the reliability of the study results, it is recommended that further studies be conducted with a larger sample size and a longer experimental duration.\u003c/p\u003e \u003cp\u003eAs also stated in other studies in the article, the findings are consistent with the idea that growth factors positively affect the wound-healing process. Investigating the structure and function of TG2 offers a deeper understanding of treatment methods for fibrosis and impaired wound healing. When the data from this study are evaluated as a whole, subcutaneous TG2 application has positive effects on growth factors during the wound-healing process.\u003c/p\u003e \u003cp\u003eIt has also been suggested that TG2 may prevent undesirable conditions, such as scar formation, hypertrophic scars, or hair loss, in the wound area by promoting angiogenesis. Given TG2's role in wound healing and cell motility, it can also be considered a beneficial active agent in the treatment of chronic wounds. Due to these properties, the clinical use of TG2 can be further evaluated through studies on wound healing.\u003c/p\u003e \u003cp\u003eHowever, the excessive accumulation of TG2 during wound healing, driven by inflammation, suggests that TG2 should be used clinically in limited amounts, as it can increase tissue scarring and lead to fibrosis. Although studies on the treatment of various human diseases with TG2 continue, its clinical applications are limited due to its numerous biological functions. For this reason, drug development processes targeting specific functions of TG2 are being pursued for therapeutic use.\u003c/p\u003e \u003cp\u003eMoreover, while the current study provides important insights into TG2\u0026rsquo;s enhancement of wound healing and its effects on growth factor promotion during this process, several limitations must be acknowledged. Although this study is useful for controlled in vivo experiments, the very small sample size and the use of a rat model do not fully reflect human cellular responses. Additionally, despite macroscopic, histological, and immunohistochemical evaluations, biomechanical tests that would directly demonstrate functional improvement at the tissue level (e.g., tensile strength or elasticity measurements) were not included in the study. Furthermore, the study\u0026rsquo;s limitation to 15 days does not cover long-term outcomes such as potential recurrences and delayed complications. Future studies should incorporate larger sample sizes, longitudinal follow-up, and mechanical testing to better understand the durability and strength of wound areas treated with TG2.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThere is no financial conflict of interest with any institution, organization, or individual regarding our article titled \" Effect of Subcutaneous Tissue Transglutaminase on Growth Factors and Type I Collagen in Surgical Incision Wound Model\" and there is no conflict of interest among the authors.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eThe idea and design of this study belong to O IO, SC., and NY.; the collection and analysis of data belong to OIO, ENH, and NY; the interpretation of the data, preparation of the article draft, critical revision, review of the research results, and approval of the final version of the article belong to all authors.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis study, produced from a doctoral thesis, was supported by the Industrial Thesis (SAN-TEZ, Number: 0443-STZ.2013-2) Project in Turkey. Therefore, we would like to thank the Turkish Ministry of Science, Industry, and Technology for their financial support. Before the experiment, an application was submitted to the Animal Research Center of the Ankara Dışkapı Yıldırım Beyazıt Training and Research Hospital of the Ministry of Health, and the Animal Experiment Ethics Committee approved the appropriateness of the procedures to be performed on the animals throughout the study.Moreover, we thank High Food Engineer Seyma AGIRAL, Asst. Prof. Dr. Didem MIMIROGLU, Faculty Member at Sivas Cumhuriyet University, for their invaluable contributions to our work.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eYes. I used or generated research data in this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArmstrong DG, Tan TW, Boulton AJM, Bus SA (2023) Diabetic Foot Ulcers: A Review. 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Int J Mol Sci 24(14):11475. ttps://doi.org/10.3390/IJMS241411475/S1\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Growth factors, Tissue transglutaminase (TG2), Type I Collagen, Wound healing","lastPublishedDoi":"10.21203/rs.3.rs-9103237/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9103237/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study aims to determine the effects of subcutaneous Tissue Transglutaminase (TG2) application in surgical incision models in healthy male Wistar Albino rats, including macroscopic observations and its impact on selected growth factors and Type I collagen in vivo.\u003c/p\u003e \u003cp\u003eA total of 24 healthy male Wistar Albino rats (200\u0026ndash;250 g) were randomly assigned to three groups (n\u0026thinsp;=\u0026thinsp;8 each): healthy tissue group, control surgical incision group, and surgical incision group treated with TG2. A standardized 4 cm dorsal incision was made on the groups and sutured. The treatment group received subcutaneous TG2 (10 IU/kg) on days 0 and 15 after suturing, and the incision sites were photographed to assess macroscopic healing. On day 15, all rats were euthanized under general anesthesia. Then, wound tissues were collected for microscopic examination. Levels of selected growth factors and Type I collagen were measured by the ELISA method, and the data were analyzed statistically using one-way ANOVA test.\u003c/p\u003e \u003cp\u003eThe wounds in the TG2-treated group healed faster and showed improved tissue organization compared to the untreated incision group, as macroscopically and histopathologically. The levels of selected growth factors and Type I collagen were increased in the TG2-treated group compared with both the control incision and healthy tissue groups, as indicated by ELISA results.\u003c/p\u003e \u003cp\u003eThe findings of this study suggest that TG2, alone or in combination with other growth factors, holds promise for future medical and veterinary clinical applications to accelerate wound repair and improve tissue regeneration outcomes.\u003c/p\u003e","manuscriptTitle":"Effect of Subcutaneous Tissue Transglutaminase on Growth Factors and Type I Collagen in Surgical Incision Wound Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-18 08:39:11","doi":"10.21203/rs.3.rs-9103237/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"acf0565d-7d37-4dd5-9263-a5546aeb344b","owner":[],"postedDate":"March 18th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Withdrawn","date":"2026-05-07T07:36:10+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-07T07:42:49+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-18 08:39:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9103237","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9103237","identity":"rs-9103237","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}