Scaffolds based on chitosan with phenolic acids for tissue engineering – in vivo assessment

Scaffolds based on chitosan with phenolic acids for tissue engineering – in vivo assessment | 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 Article Scaffolds based on chitosan with phenolic acids for tissue engineering – in vivo assessment Beata Kaczmarek-Szczepańska, Izabela Polkowska, Katarzyna Paździor-Czapula, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1788379/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 Chitosan-based scaffolds modified by gallic acid, ferulic acid, and tannic acid were fabricated. The aim of the experiment was to study the compatibility of scaffolds by in vivo method. For such purpose materials were implanted into rabbit in the middle of the latissimus dorsi muscle length. Scaffold based on unmodified chitosan was implanted by the same method as a control. The results showed that all studied materials were safe and nontoxic. However, chitosan scaffolds modified by gallic acid and tannic acid were resorbed faster and as a result tissues were organized faster than those modified by ferulic acid or unmodified. Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Phenolic acids are natural compounds proposed as effective cross-linkers for biopolymers [ 1 ]. They are able to form strong hydrogen bonds with functional groups present in the polymeric chain such as hydroxyl and amine [ 2 ]. As a result, they improve the properties of the biopolymer-based materials, mainly the mechanical parameters. Also, the stability is improved as biopolymer-based materials without cross-linkers have low resistance to the enzymes and may easily dissolve in aqueous conditions [ 3 ]. Chitosan-based materials cross-linked by different phenolic acids have been already studied [ 4 – 6 ]. Tannic acid classified into a polyphenolic group presents unique antiviral and antibacterial properties [ 7 ]. It has many hydroxyl groups which easily interact with functional groups of chitosan. Thereby, tannic acid was considered as chitosan cross-linker. The results showed that the mechanical properties and surface free energy were improved, and also films showed antibacterial activity against Staphylococcus aureus [ 8 ]. In vitro studies were also carried out where the normal and cancer cells (MNT-1, SK-MEL-28, Saos-2, HaCaT and BMSC) were seeded on the film composed of chitosan and tannic acid. The results showed that films with the lowest tannic acid (CTS/TA 80/20) content inhibit the cell growth of MNT-1 cells; however, the lowest inhibition was observed for BMSC [ 9 ]. It was also studied that a safe method for the sterilization of films enriched by phenolic acids may be using UVC light (254 nm). Moreover, it was noticed that the addition of ferulic acid to chitosan causes effective antimicrobial activity [ 10 ]. It was also reported that chitosan-based films cross-linked by tannic acid degrade in soil and compost. After 14 days of biodegradation, the chemical structure of the materials was changed, resulting from adhesion of the microorganisms. Based on the obtained results, it was assumed that the proposed films were interesting for medical application due to the their nontoxicity and antibacterial activity [ 11 ]. The last stage for the primary studies of chitosan-based materials modified by selected phenolic acids includes in vivo tests. In this study a pre-clinical in vivo animal test was performed in a tracheal defected rabbit model for 4 weeks to confirm epithelium and cartilage regeneration. The aim of the study was to examine the biological properties of chitosan/phenolic acid-based scaffolds by the histological assessment of tissue with implanted materials to assess the applicability of scaffolds as potentially suitable for cartilage tissue regeneration. The study is reported in accordance with ARRIVE guidelines. Results In the control sample (Fig. 1 ), within the subcutaneous adipose tissue fragments of fibrous material (fragments of scaffold) were observed. These fragments were surrounded by moderate infiltration of lymphocytes, macrophages, moderately numerous multinucleated giant cells (of foreign body type) with proliferation of the connective tissue. Figure 2 shows the tissue after implantation of CTS_GA sample, no implant was observed. In the skin focal proliferation of cell-poor, collagen-rich connective tissue was observed, with subsequent atrophy of the adnexa (cutaneous fibrosis). It suggests that the scaffold was resorbed and replaced by connective tissue. Tissues undergo organization relating to the healing and repair of the tissue. In the CTS_FA sample (Fig. 3 ), the partial (minimal) resorption of the implant was observed. Large implant was located within the subcutaneous tissue, surrounded by infiltration of macrophages, single multinucleated giant cells and neutrophils, variably numerous lymphocytes, with peripheral proliferation of the connective tissue. In the CTS_TA sample (Fig. 4 ), no implant was observed. There was a slight cutaneous fibrosis with the atrophy of adnexa, but indiscernible grossly. Discussion Hypodermis is composed of adipose and collagen. In deep skin defects it takes a long period of time for re-epithelialization to be complete. Also, on the skin skars may be formed as a base. Wound healing is a constant process which can be define by four phases: hemostasis, inflammation, proliferation and remodeling/maturation [ 12 ]. Interactions of biomaterial with surrounding environment after implantation should be studied to consider its biocompatibility. It have been reported that chitosan-based materials result in the formation of a foreign body granuloma. In the control sample with implanted chitosan scaffold the regeneration of skin was observed. However, small fragments of chitosan scaffold was also still visible, surrounded by inflammatory cells and fibrous tissue. No formation of blood vessels was noticed. Formed tissue was compared with the skin after material implantation. For CTS_GA sample the collagen-rich connective tissue was observed. However, the subsequent atrophy of the adnexa (cutaneous fibrosis) was noticed. The cutaneous fibrosis is the accumulation of extracellular matrix (ECM) components in the dermis, leading to compromised function and altered architecture of the dermis. The formation of fibrosis occurs as natural process in the scar formation. During fibrosis two major processes are carried out such as synthesis and degradation of ECM which normally are in equilibrium. It is important to shift processes in the synthesis side during skin regeneration [ 13 ]. CTS_GA samples did not show such trend. Considering CTS_FA the material was still observed in the implantation place. It was not resorbed completely. It was located within the subcutaneous tissue, surrounded by infiltration of macrophages, single multinucleated giant cells and neutrophils, variably numerous lymphocytes, with peripheral proliferation of the connective tissue. It suggests that collagen was formed even if the scaffold was not degrade [ 14 ]. For the comparison, CTS_TA scaffold was not observed. It was resorbed without any immunological reaction. Slight cutaneous fibrosis was observed, but indiscernible grossly. Methods Samples preparation Chitosan (CTS; low molecular weight; Sigma-Aldrich, shrimp sourced) was used for the studies. Gallic acid (GA), ferulic acid (FA), and tannic acid (TA) were purchased from Sigma-Aldrich company. Chitosan, tannic acid, gallic acid, and ferulic acid were dissolved in 0.1M acetic acid at 1% concentration separately. Chitosan solution was mixed with 10 (w/w%) addition of phenolic acid solution for 1h. The mixtures were then placed in the 24-well sterile cell culture plates (2.5 mL/well), frozen for 24 h in -18oC, and lyophilized (ALPHA 1–2 LDplus, CHRIST,−20°C, 100 Pa, 48 h). As a result, the three-dimensional dry samples (scaffolds) were obtained. In Vivo Experiment The in vivo experiment was carried out on a group of male New Zealand rabbits weighing 2.8–3.2 kg. The rabbits were purchased at the Experimental Medicine Center of the Medical University of Silesia in Katowice, fak. No.KCM / FPS / 0041/06/20. Before the procedure, the animals' health was checked. The animals were under constant veterinary supervision and were given a vaccine: Castomix by Pharmagal Bio against Myxomatosis (MXT) and rabbit haemorrhagic disease (RHDV). All research protocols were approved by the Local Ethics Committee of the University of Life Sciences in Lublin No. 104/2017, and the experiment was conducted in accordance with the provisions on animal protection. The animals were placed in the animal facility of the Experimental Medicine Center of the Medical University of Lublin. During this time, their natural habits were monitored and the temperature of each animal was measured daily. The general condition of the rabbits was very good, with no clinical signs of disease. The daily measured body temperature was within the reference range. For 7 days after the herd was introduced to the Vivarium, their body temperature was measured, the food intake and the behavior of the animals were observed. After a week's adaptation, the rabbits were prepared for surgery. After weighing, each individual was premedicated. On the day of surgery, each animal in the group was sedated by intramuscular injection (Domitor-Orion Corporation, Fin-land) of medetomidine (0.5 mg / kg) and Butomidor, Richter Pharma, Austria) butorphanol (0.2 mg / kg) depending on their weight. Then, after about 15 minutes, a mask was put on in order to administer inhalation anesthesia (isofluorane). The period of anesthesia of each individual lasted about 30 minutes. After the rabbit was immobilized, the skin was shaved, disinfected with alcohol and iodine. The material for implantation was prepared according to the recommendations. The skin incision was made parallel, in the intercostal area, in the middle of the latissimus dorsi muscle length, 3 cm above the dorsal line. Subcutaneous tissue and fascia were dissected in the same line and the prepared material was placed (cylindrical shape height 1 cm, diameter 1,5 cm). Two materials were implanted into the one organism (one on the left one on the right side). Experimental samples were chitosan modified by gallic acid (CTS_GA), ferulic acid (CTS_FA) and tannic acid (CTS_TA). Control was prepared by implantation of chitosan scaffold without addition of phenolic acid (CTS). The implantation site was closed with a mattress suture using Dexon 3 − 0. After the operation, all the rabbits could move freely in the cages without additional dressings in the area operated on. In order to minimize the risk of infection and reduce postoperative discomfort, an antibiotic and an anti-inflammatory drug (gentamicin 5 mg / kg and meloxicam 0.4 mg / kg) were administered for 5 days after the procedure. In the postoperative period, a mild swelling was observed around the skin suture in most rabbits. After two weeks, all the operated animals were in good general condition. Three months after surgery, all rabbits were sacrificed. First, animals were anesthetized intramuscularly and sedated by intramuscular injection of medetomidine (0.5 mg / kg) and butorphanol (0.2 mg / kg) depending on their weight. The rabbits were then sacrificed by barbiturate injection. Tissue fragments with a margin (3cm x 3cm x 3cm) were taken from the implantation site along with the implanted material and placed in a buffered paraformaldehyde solution at pH 7.4. All the samples were placed in appropriate transporters and accurately described according to the implanted material. All the animal surgical procedures were carried and approved by the Local Ethical Committee for Experiments on Animals in Lublin (Agreement no. 104/2017). Histological Assessment Tissue samples were immediately fixed in 10% buffered formalin, processed routinely for histopathology using paraffin method, cut at 5µm and stained with Mayer’s haematoxylin and eosin. Samples were evaluated in a blind fashion by an experienced pathologist (IOD). Microphotographs were prepared using Olympus BX43 microscope (Tokyo, Japan), equipped with Olympus SC 180 camera (Hamburg, Germany) and cellSens software (Olympus). Conclusions Chitosan scaffolds modified by gallic acid, tannic acid, and ferulic acid were successfully implanted into the subcutaneous tissue of rabbits. The histological images allowed to compare the tissue regeneration processes occurring after scaffold implantation. It may be assumed that chitosan scaffolds modified by gallic acid and tannic acid were resorbed faster and tissues are organized faster than those modified by ferulic acid. All tested materials are considered biocompatible and safe. Declarations Data availability The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. Funding This work was supported by financial support of Nicolaus Copernicus University in Torun grant number 282/2021 IDUB SD (B.K.-S.). References Kaczmarek B. & Mazur O. Collagen-based materials modified by phenolic acids—a review. Materials 13(16), 3641, DOI: https://10.3390/ma13163641 (2020). Hutchins K.M. Functional materials based on molecules with hydrogen-bonding ability: applications to drug co-crystals and polymer complexes. R. Soc. Open Sci. 5(6), 180564, DOI: https://doi.org/10.1098/rsos.180564 (2018). Vieira M.G.A., da Silva M.A., dos Santos L.O. & Beppu M.M. Natural-based plasticizers and biopolymer films: A review. Eur. Polym. J. 47(3), 254-263, DOI: https://doi.org/10.1016/j.eurpolymj.2010.12.011 (2011). Riccucci G., Ferraris S., Reggio C., Bosso A., Orlygsson G., Ng C.H. & Spriano S. Polyphenols from grape pomace: functionalization of chitosan-coated hydroxyapatite for modulated swelling and release of polyphenols. Langmuir 37(51), 14793-14804, DOI: https://doi.org/10.1021/acs.langmuir.1c01930 (2021). Huber D., Grzelak A., Baumann M., Borth N., Schleining G., Nyanhongo G.S. & Guebitz G.M. Anti-inflammatory and anti-oxidant properties of laccase-synthesized phenolic-O-carboxymethyl chitosan hydrogels. New Biotechnol. 40B, 236-244, DOI: http:// 10.1016/j.nbt.2017.09.004 (2018). Liu J., Yong H., Yao X., Hu H., Yun D. & Xiao L. Recent advances in phenolic–protein conjugates: synthesis, characterization, biological activities and potential applications. RSC Adv. 9, 35825-35840, DOI: https://doi.org/10.1039/C9RA07808H (2019). Kaczmarek B. Tannic acid with antiviral and antibacterial activity as a promising component of biomaterials-a minireview. Materials 13(14), 3224, DOI: http://10.3390/ma13143224. (2020). Kaczmarek B., Wekwejt M., Nadolna K., Owczarek A., Mazur O. & Pałubicka A. The mechanical properties and bactericidal degradation effectiveness of tannic acid-based thin films for wound care. J. Mech. Beh. Biomed. Mater. 110, 103916, DOI: https://doi.org/10.1016/j.jmbbm.2020.103916 (2020). Kaczmarek B., Miłek O., Nadolna K., Owczarek A., Kleszczyński K. & Osyczka A.M. Normal and cancer cells response on the thin films based on chitosan and tannic acid. Toxicol. Vitro 62, 104688, DOI: http://10.1016/j.tiv.2019.104688 (2020). Kaczmarek-Szczepanska B., Wekwejt M., Mazur O., Zasada L., Palubicka A. & Olewnik-Kruszkowska E. The physicochemical and antibacterial properties of chitosan‐ based materials modified with phenolic acids irradiated by UVC light. Int. J. Mol. Sci. 22, 6472, DOI: https://doi.org/10.3390/ijms22126472 (2021). Kaczmarek-Szczepanska B., Michalska-Sionkowska M., Mazur O., Swiatczak J. & Swiontek Brzezinska M. The role of microorganisms in biodegradation of chitosan/tannic acid materials. Int. J. Biol. Macromol. 104, 584-592, DOI: https://doi.org/10.1016/j.ijbiomac.2021.06.133 (2021). Norouzi M. & Boroujeni S.M, Omidvarkordshouli N, Soleimani M. Advances in skin regeneration: application of electrospun scaffolds. Adv. Healthcare Mater. 4(8), 1114-1133, DOI: http:// 10.1002/adhm.201500001 (2015). Do N.N. & Eming S.A. Skin fibrosis: Models and mechanisms. Curr. Res. Transl. Med. 64(4), 185-193, DOI: http:// 10.1016/j.retram.2016.06.003 (2016). Kaczmarek B., Sionkowska A., Otrocka-Domagala I. & Polkowska I. In vivo studies of novel scaffolds with tannic acid addition. Polym. Deg. Stab. 158, 26-30, DOI: https://doi.org/10.1016/j.polymdegradstab.2018.10.018 (2018). Additional Declarations No competing interests reported. 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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-1788379","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":119877507,"identity":"9dbf94aa-9c48-4926-bd97-51db1cb845be","order_by":0,"name":"Beata Kaczmarek-Szczepańska","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYBACAwiVAOMnyDEwsAHpArxaGBuQtRhDtBiQoCWxgZAWc/bm5w9+MKTJ8/efTvxcUZGWvuHasQTmAjxaLHuOGTb2MOQYzjhwdrPkmTM5uRtupx1gnoHPYTcSDBt4GCoYNzD2bpBsbKsAaklvYObBp+X+84+Nfxgq7Dcw827+2fivIt2AoJYbPIbNPAw5iRvYeLdJNjbkJBiAHIZPi2VPTuFsGYO05BlneLdZNhxLM5x5Oy3hMD6/mLMf3/DxTUWybX//2c03G2qS5flupxk+LqjArQXqPDT+YUIaMAEz6VpGwSgYBaNgGAMA8xpU9Sxca3UAAAAASUVORK5CYII=","orcid":"","institution":"Nicolaus Copernicus University in Torun","correspondingAuthor":true,"prefix":"","firstName":"Beata","middleName":"","lastName":"Kaczmarek-Szczepańska","suffix":""},{"id":119877509,"identity":"4d5c9d66-81b3-4164-ab3a-16d86e69ecb6","order_by":1,"name":"Izabela Polkowska","email":"","orcid":"","institution":"University of Life Sciences in Lublin","correspondingAuthor":false,"prefix":"","firstName":"Izabela","middleName":"","lastName":"Polkowska","suffix":""},{"id":119877511,"identity":"6a0543b0-f7fa-4598-ac2f-80b7556a8a7e","order_by":2,"name":"Katarzyna Paździor-Czapula","email":"","orcid":"","institution":"University of Warmia and Mazury","correspondingAuthor":false,"prefix":"","firstName":"Katarzyna","middleName":"","lastName":"Paździor-Czapula","suffix":""},{"id":119877514,"identity":"1c1b79d6-90c4-44ae-ac03-ea5b3beccaf9","order_by":3,"name":"Beata Nowicka","email":"","orcid":"","institution":"University of Life Sciences in Lublin","correspondingAuthor":false,"prefix":"","firstName":"Beata","middleName":"","lastName":"Nowicka","suffix":""},{"id":119877516,"identity":"796e0d7c-e3ab-438e-81e3-0dd1d481bbd3","order_by":4,"name":"Iwona Otrocka-Domagała","email":"","orcid":"","institution":"University of Warmia and Mazury","correspondingAuthor":false,"prefix":"","firstName":"Iwona","middleName":"","lastName":"Otrocka-Domagała","suffix":""}],"badges":[],"createdAt":"2022-06-23 13:14:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1788379/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1788379/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":23779465,"identity":"7d491f35-83f4-427f-bc5b-5f722b55d05e","added_by":"auto","created_at":"2022-07-12 17:45:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1079120,"visible":true,"origin":"","legend":"\u003cp\u003eThe controls ample – tissue with implanted chitosan scaffold. Small fragments of resorbed chitosan scaffold are visible (asterisks), surrounded by inflammatory cells.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-1788379/v1/3987afb551cf47995f0a8893.png"},{"id":23779050,"identity":"7d35f88c-e3ea-417a-a815-bf8a9b69df89","added_by":"auto","created_at":"2022-07-12 17:40:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1524107,"visible":true,"origin":"","legend":"\u003cp\u003eThe tissue after implantation of CTS_GA sample with subsequent proliferation of collagen-rich connective tissue (asterisk).\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-1788379/v1/10d74842c446712ce68f7ac1.png"},{"id":23779048,"identity":"e1106839-1a75-4a38-9b47-3aa70c930589","added_by":"auto","created_at":"2022-07-12 17:40:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1616127,"visible":true,"origin":"","legend":"\u003cp\u003eTissue with implanted CTS_FA scaffold (asterisks).\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-1788379/v1/978f126be40d1189239628d9.png"},{"id":23779047,"identity":"9f05ab57-be5d-4aed-8aac-6d05051abe25","added_by":"auto","created_at":"2022-07-12 17:40:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1399003,"visible":true,"origin":"","legend":"\u003cp\u003eThe tissue after implantation of CTS_TA sample with subsequent cutaneous fibrosis (asterisk).\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-1788379/v1/890b3165b3ee910437983c53.png"},{"id":24397174,"identity":"a44d256d-b85a-4003-a434-4755c73d9e4f","added_by":"auto","created_at":"2022-07-27 11:59:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5067613,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1788379/v1/04f7edfb-7325-46c2-baca-ce3a717a9683.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Scaffolds based on chitosan with phenolic acids for tissue engineering – in vivo assessment","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePhenolic acids are natural compounds proposed as effective cross-linkers for biopolymers [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. They are able to form strong hydrogen bonds with functional groups present in the polymeric chain such as hydroxyl and amine [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. As a result, they improve the properties of the biopolymer-based materials, mainly the mechanical parameters. Also, the stability is improved as biopolymer-based materials without cross-linkers have low resistance to the enzymes and may easily dissolve in aqueous conditions [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eChitosan-based materials cross-linked by different phenolic acids have been already studied [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Tannic acid classified into a polyphenolic group presents unique antiviral and antibacterial properties [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. It has many hydroxyl groups which easily interact with functional groups of chitosan. Thereby, tannic acid was considered as chitosan cross-linker. The results showed that the mechanical properties and surface free energy were improved, and also films showed antibacterial activity against Staphylococcus aureus [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In vitro studies were also carried out where the normal and cancer cells (MNT-1, SK-MEL-28, Saos-2, HaCaT and BMSC) were seeded on the film composed of chitosan and tannic acid. The results showed that films with the lowest tannic acid (CTS/TA 80/20) content inhibit the cell growth of MNT-1 cells; however, the lowest inhibition was observed for BMSC [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. It was also studied that a safe method for the sterilization of films enriched by phenolic acids may be using UVC light (254 nm). Moreover, it was noticed that the addition of ferulic acid to chitosan causes effective antimicrobial activity [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. It was also reported that chitosan-based films cross-linked by tannic acid degrade in soil and compost. After 14 days of biodegradation, the chemical structure of the materials was changed, resulting from adhesion of the microorganisms. Based on the obtained results, it was assumed that the proposed films were interesting for medical application due to the their nontoxicity and antibacterial activity [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The last stage for the primary studies of chitosan-based materials modified by selected phenolic acids includes in vivo tests.\u003c/p\u003e \u003cp\u003eIn this study a pre-clinical in vivo animal test was performed in a tracheal defected rabbit model for 4 weeks to confirm epithelium and cartilage regeneration. The aim of the study was to examine the biological properties of chitosan/phenolic acid-based scaffolds by the histological assessment of tissue with implanted materials to assess the applicability of scaffolds as potentially suitable for cartilage tissue regeneration. The study is reported in accordance with ARRIVE guidelines.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIn the control sample (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), within the subcutaneous adipose tissue fragments of fibrous material (fragments of scaffold) were observed. These fragments were surrounded by moderate infiltration of lymphocytes, macrophages, moderately numerous multinucleated giant cells (of foreign body type) with proliferation of the connective tissue.\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e shows the tissue after implantation of CTS_GA sample, no implant was observed. In the skin focal proliferation of cell-poor, collagen-rich connective tissue was observed, with subsequent atrophy of the adnexa (cutaneous fibrosis). It suggests that the scaffold was resorbed and replaced by connective tissue. Tissues undergo organization relating to the healing and repair of the tissue.\u003c/p\u003e\n\u003cp\u003eIn the CTS_FA sample (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e), the partial (minimal) resorption of the implant was observed. Large implant was located within the subcutaneous tissue, surrounded by infiltration of macrophages, single multinucleated giant cells and neutrophils, variably numerous lymphocytes, with peripheral proliferation of the connective tissue.\u003c/p\u003e\n\u003cp\u003eIn the CTS_TA sample (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e), no implant was observed. There was a slight cutaneous fibrosis with the atrophy of adnexa, but indiscernible grossly.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHypodermis is composed of adipose and collagen. In deep skin defects it takes a long period of time for re-epithelialization to be complete. Also, on the skin skars may be formed as a base. Wound healing is a constant process which can be define by four phases: hemostasis, inflammation, proliferation and remodeling/maturation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Interactions of biomaterial with surrounding environment after implantation should be studied to consider its biocompatibility. It have been reported that chitosan-based materials result in the formation of a foreign body granuloma.\u003c/p\u003e \u003cp\u003eIn the control sample with implanted chitosan scaffold the regeneration of skin was observed. However, small fragments of chitosan scaffold was also still visible, surrounded by inflammatory cells and fibrous tissue. No formation of blood vessels was noticed. Formed tissue was compared with the skin after material implantation. For CTS_GA sample the collagen-rich connective tissue was observed. However, the subsequent atrophy of the adnexa (cutaneous fibrosis) was noticed. The cutaneous fibrosis is the accumulation of extracellular matrix (ECM) components in the dermis, leading to compromised function and altered architecture of the dermis. The formation of fibrosis occurs as natural process in the scar formation. During fibrosis two major processes are carried out such as synthesis and degradation of ECM which normally are in equilibrium. It is important to shift processes in the synthesis side during skin regeneration [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. CTS_GA samples did not show such trend. Considering CTS_FA the material was still observed in the implantation place. It was not resorbed completely. It was located within the subcutaneous tissue, surrounded by infiltration of macrophages, single multinucleated giant cells and neutrophils, variably numerous lymphocytes, with peripheral proliferation of the connective tissue. It suggests that collagen was formed even if the scaffold was not degrade [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. For the comparison, CTS_TA scaffold was not observed. It was resorbed without any immunological reaction. Slight cutaneous fibrosis was observed, but indiscernible grossly.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSamples preparation\u003c/h2\u003e \u003cp\u003eChitosan (CTS; low molecular weight; Sigma-Aldrich, shrimp sourced) was used for the studies. Gallic acid (GA), ferulic acid (FA), and tannic acid (TA) were purchased from Sigma-Aldrich company. Chitosan, tannic acid, gallic acid, and ferulic acid were dissolved in 0.1M acetic acid at 1% concentration separately. Chitosan solution was mixed with 10 (w/w%) addition of phenolic acid solution for 1h. The mixtures were then placed in the 24-well sterile cell culture plates (2.5 mL/well), frozen for 24 h in -18oC, and lyophilized (ALPHA 1\u0026ndash;2 LDplus, CHRIST,\u0026minus;20\u0026deg;C, 100 Pa, 48 h). As a result, the three-dimensional dry samples (scaffolds) were obtained.\u003c/p\u003e \u003c/div\u003e\n\u003ch2\u003eIn Vivo Experiment\u003c/h2\u003e\n\u003cp\u003eThe in vivo experiment was carried out on a group of male New Zealand rabbits weighing 2.8\u0026ndash;3.2 kg. The rabbits were purchased at the Experimental Medicine Center of the Medical University of Silesia in Katowice, fak. No.KCM / FPS / 0041/06/20. Before the procedure, the animals' health was checked. The animals were under constant veterinary supervision and were given a vaccine: Castomix by Pharmagal Bio against Myxomatosis (MXT) and rabbit haemorrhagic disease (RHDV). All research protocols were approved by the Local Ethics Committee of the University of Life Sciences in Lublin No. 104/2017, and the experiment was conducted in accordance with the provisions on animal protection. The animals were placed in the animal facility of the Experimental Medicine Center of the Medical University of Lublin. During this time, their natural habits were monitored and the temperature of each animal was measured daily. The general condition of the rabbits was very good, with no clinical signs of disease. The daily measured body temperature was within the reference range.\u003c/p\u003e \u003cp\u003eFor 7 days after the herd was introduced to the Vivarium, their body temperature was measured, the food intake and the behavior of the animals were observed. After a week's adaptation, the rabbits were prepared for surgery. After weighing, each individual was premedicated. On the day of surgery, each animal in the group was sedated by intramuscular injection (Domitor-Orion Corporation, Fin-land) of medetomidine (0.5 mg / kg) and Butomidor, Richter Pharma, Austria) butorphanol (0.2 mg / kg) depending on their weight. Then, after about 15 minutes, a mask was put on in order to administer inhalation anesthesia (isofluorane).\u003c/p\u003e \u003cp\u003eThe period of anesthesia of each individual lasted about 30 minutes. After the rabbit was immobilized, the skin was shaved, disinfected with alcohol and iodine. The material for implantation was prepared according to the recommendations.\u003c/p\u003e \u003cp\u003eThe skin incision was made parallel, in the intercostal area, in the middle of the latissimus dorsi muscle length, 3 cm above the dorsal line. Subcutaneous tissue and fascia were dissected in the same line and the prepared material was placed (cylindrical shape height 1 cm, diameter 1,5 cm). Two materials were implanted into the one organism (one on the left one on the right side). Experimental samples were chitosan modified by gallic acid (CTS_GA), ferulic acid (CTS_FA) and tannic acid (CTS_TA). Control was prepared by implantation of chitosan scaffold without addition of phenolic acid (CTS). The implantation site was closed with a mattress suture using Dexon 3\u0026thinsp;\u0026minus;\u0026thinsp;0.\u003c/p\u003e \u003cp\u003eAfter the operation, all the rabbits could move freely in the cages without additional dressings in the area operated on. In order to minimize the risk of infection and reduce postoperative discomfort, an antibiotic and an anti-inflammatory drug (gentamicin 5 mg / kg and meloxicam 0.4 mg / kg) were administered for 5 days after the procedure.\u003c/p\u003e \u003cp\u003eIn the postoperative period, a mild swelling was observed around the skin suture in most rabbits. After two weeks, all the operated animals were in good general condition. Three months after surgery, all rabbits were sacrificed. First, animals were anesthetized intramuscularly and sedated by intramuscular injection of medetomidine (0.5 mg / kg) and butorphanol (0.2 mg / kg) depending on their weight.\u003c/p\u003e \u003cp\u003eThe rabbits were then sacrificed by barbiturate injection. Tissue fragments with a margin (3cm x 3cm x 3cm) were taken from the implantation site along with the implanted material and placed in a buffered paraformaldehyde solution at pH 7.4. All the samples were placed in appropriate transporters and accurately described according to the implanted material.\u003c/p\u003e \u003cp\u003e All the animal surgical procedures were carried and approved by the Local Ethical Committee for Experiments on Animals in Lublin (Agreement no. 104/2017).\u003c/p\u003e\n\u003ch2\u003eHistological Assessment\u003c/h2\u003e\n\u003cp\u003eTissue samples were immediately fixed in 10% buffered formalin, processed routinely for histopathology using paraffin method, cut at 5\u0026micro;m and stained with Mayer\u0026rsquo;s haematoxylin and eosin. Samples were evaluated in a blind fashion by an experienced pathologist (IOD). Microphotographs were prepared using Olympus BX43 microscope (Tokyo, Japan), equipped with Olympus SC 180 camera (Hamburg, Germany) and cellSens software (Olympus).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eChitosan scaffolds modified by gallic acid, tannic acid, and ferulic acid were successfully implanted into the subcutaneous tissue of rabbits. The histological images allowed to compare the tissue regeneration processes occurring after scaffold implantation. It may be assumed that chitosan scaffolds modified by gallic acid and tannic acid were resorbed faster and tissues are organized faster than those modified by ferulic acid. All tested materials are considered biocompatible and safe.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by financial support of Nicolaus Copernicus University in Torun grant number 282/2021 IDUB SD (B.K.-S.).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKaczmarek B. \u0026amp; Mazur O. Collagen-based materials modified by phenolic acids\u0026mdash;a review. Materials 13(16), 3641, DOI: https://10.3390/ma13163641 (2020).\u003c/li\u003e\n\u003cli\u003eHutchins K.M. Functional materials based on molecules with hydrogen-bonding ability: applications to drug co-crystals and polymer complexes. R. Soc. Open Sci. 5(6), 180564, DOI: https://doi.org/10.1098/rsos.180564 (2018).\u003c/li\u003e\n\u003cli\u003eVieira M.G.A., da Silva M.A., dos Santos L.O. \u0026amp; Beppu M.M. Natural-based plasticizers and biopolymer films: A review. Eur. Polym. J. 47(3), 254-263, DOI: https://doi.org/10.1016/j.eurpolymj.2010.12.011 (2011).\u003c/li\u003e\n\u003cli\u003eRiccucci G., Ferraris S., Reggio C., Bosso A., Orlygsson G., Ng C.H. \u0026amp; Spriano S. Polyphenols from grape pomace: functionalization of chitosan-coated hydroxyapatite for modulated swelling and release of polyphenols. Langmuir 37(51), 14793-14804, DOI: https://doi.org/10.1021/acs.langmuir.1c01930 (2021).\u003c/li\u003e\n\u003cli\u003eHuber D., Grzelak A., Baumann M., Borth N., Schleining G., Nyanhongo G.S. \u0026amp; Guebitz G.M. Anti-inflammatory and anti-oxidant properties of laccase-synthesized phenolic-O-carboxymethyl chitosan hydrogels. New Biotechnol. 40B, 236-244, DOI: http:// 10.1016/j.nbt.2017.09.004 (2018).\u003c/li\u003e\n\u003cli\u003eLiu J., Yong H., Yao X., Hu H., Yun D. \u0026amp; Xiao L. Recent advances in phenolic\u0026ndash;protein conjugates: synthesis, characterization, biological activities and potential applications. RSC Adv. 9, 35825-35840, DOI: https://doi.org/10.1039/C9RA07808H (2019).\u003c/li\u003e\n\u003cli\u003eKaczmarek B. Tannic acid with antiviral and antibacterial activity as a promising component of biomaterials-a minireview. Materials 13(14), 3224, DOI: http://10.3390/ma13143224. (2020).\u003c/li\u003e\n\u003cli\u003eKaczmarek B., Wekwejt M., Nadolna K., Owczarek A., Mazur O. \u0026amp; Pałubicka A. The mechanical properties and bactericidal degradation effectiveness of tannic acid-based thin films for wound care. J. Mech. Beh. Biomed. 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Stab. 158, 26-30, DOI: https://doi.org/10.1016/j.polymdegradstab.2018.10.018 (2018).\u003c/li\u003e\n\n\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":"","lastPublishedDoi":"10.21203/rs.3.rs-1788379/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1788379/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChitosan-based scaffolds modified by gallic acid, ferulic acid, and tannic acid were fabricated. The aim of the experiment was to study the compatibility of scaffolds by in vivo method. For such purpose materials were implanted into rabbit in the middle of the latissimus dorsi muscle length. Scaffold based on unmodified chitosan was implanted by the same method as a control. The results showed that all studied materials were safe and nontoxic. However, chitosan scaffolds modified by gallic acid and tannic acid were resorbed faster and as a result tissues were organized faster than those modified by ferulic acid or unmodified.\u003c/p\u003e","manuscriptTitle":"Scaffolds based on chitosan with phenolic acids for tissue engineering – in vivo assessment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-07-12 17:40:28","doi":"10.21203/rs.3.rs-1788379/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":"48a0276b-541a-43f4-930f-5abbeeaae6fc","owner":[],"postedDate":"July 12th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-07-27T11:59:15+00:00","versionOfRecord":[],"versionCreatedAt":"2022-07-12 17:40:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1788379","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1788379","identity":"rs-1788379","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}