Synergistic effect of chitosan-alginate composite hydrogel enriched with ascorbic acid and alpha-tocopherol under hypoxic conditions on the behavior of mesenchymal stem cells for wound healing
Tóm tắt
In regenerative medicine, especially skin tissue engineering, the focus is on enhancing the quality of wound healing. Also, several constructs with different regeneration potentials have been used for skin tissue engineering. In this study, the regenerative properties of chitosan-alginate composite hydrogels in skin wound healing under normoxic and hypoxic conditions were investigated in vitro. The ionic gelation method was used to prepare chitosan/alginate (CA) hydrogel containing CA microparticles and bioactive agents [ascorbic acid (AA) and α-tocopherol (TP)]. After preparing composite hydrogels loaded with AA and TP, the physicochemical properties such as porosity, pore size, swelling, weight loss, wettability, drug release, and functional groups were analyzed. Also, the hemo-biocompatibility of composite hydrogels was evaluated by a hemolysis test. Then, the rat bone marrow mesenchymal stem cells (rMSCs) were seeded onto the hydrogels after characterization by flow cytometry. The survival rate was analyzed using MTT assay test. The hydrogels were also investigated by DAPI and H&E staining to monitor cell proliferation and viability. To induce hypoxia, the cells were exposed to CoCl2. To evaluate the regenerative potential of rMSCs cultured on CA/AA/TP hydrogels under hypoxic conditions, the expression of the main genes involved in the healing of skin wounds, including HIF-1α, VEGF-A, and TGF-β1, was investigated by real-time PCR. The results demonstrated that the prepared composite hydrogels were highly porous, with interconnected pores that ranged in sizes from 20 to 188 μm. The evaluation of weight loss showed that the prepared hydrogels have the ability to biodegrade according to the goals of wound healing. The reduction percentage of CA/AA/TP mass in 21 days was reported as 21.09 ± 0.52%. Also, based on wettability and hemolysis tests of the CA/AA/TP, hydrophilicity (θ = 55.6° and 53.7°) and hemocompatibility with a hemolysis ratio of 1.36 ± 0.19 were evident for them. Besides, MTT assay, DAPI, and H&E staining also showed that the prepared hydrogels provide a suitable substrate for cell growth and proliferation. Finally, based on real-time PCR, increased expression levels of VEGF and TGF-β1 were observed in rMSCs in hypoxic conditions cultured on the prepared hydrogels. In conclusion, this study provides evidence that 3D CA/AA/TP composite hydrogels seeded by rMSCs in hypoxic conditions have great potential to improve wound healing.
Tài liệu tham khảo
Bagher Z, et al. Wound healing with alginate/chitosan hydrogel containing hesperidin in rat model. J Drug Deliv Sci Technol. 2020;55: 101379.
Ehterami A, et al. Chitosan/alginate hydrogels containing Alpha-tocopherol for wound healing in rat model. J Drug Deliv Sci Technol. 2019;51:204–13.
Hu Y, et al. Exosomes from human umbilical cord blood accelerate cutaneous wound healing through miR-21-3p-mediated promotion of angiogenesis and fibroblast function. Theranostics. 2018;8(1):169.
Stoica AE, et al. Scar-free healing: current concepts and future perspectives. Nanomaterials. 2020;10(11):2179.
Rahmani Del Bakhshayesh A, et al. Recent advances on biomedical applications of scaffolds in wound healing and dermal tissue engineering. Artif Cells Nanomed Biotechnol. 2018;46(4):691–705.
Rahmani Del Bakhshayesh A, et al. Recent advances in nano-scaffolds for tissue engineering applications: toward natural therapeutics. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2023. https://doi.org/10.1002/wnan.1882.
Rahmani Del Bakhshayesh A, et al. Fabrication of three-dimensional scaffolds based on nano-biomimetic collagen hybrid constructs for skin tissue engineering. ACS Omega. 2018;3(8):8605–11.
Del Bakhshayesh AR, et al. High efficiency biomimetic electrospun fibers for use in regenerative medicine and drug delivery: a review. Mater Chem Phys. 2022;279:125785.
Asadi N, et al. Nanocomposite electrospun scaffold based on polyurethane/polycaprolactone incorporating gold nanoparticles and soybean oil for tissue engineering applications. J Bion Eng. 2023. https://doi.org/10.1007/s42235-023-00345-x.
Akbarzadeh A, et al. Adipose tissue stem cells bioengineered in nano-biomimetic col scaffolds for skin tissue engineering. In: 2018 AIChE Annual Meeting. 2018. AIChE.
Nezhad-Mokhtari P, et al. Honey-loaded reinforced film based on bacterial nanocellulose/gelatin/guar gum as an effective antibacterial wound dressing. J Biomed Nanotechnol. 2022;18(8):2010–21.
Nezhadmokhtari P, et al. Development of a novel film based on bacterial nanocellulose reinforced gelatin/guar gum containing honey for wound healing applications. 2021.
Aeridou E, et al. Advanced functional hydrogel biomaterials based on dynamic B-O bonds and polysaccharide building blocks. Biomacromol. 2020;21(10):3984–96.
Izadpanah M, et al. Melatonin and endothelial cell-loaded alginate-fibrin hydrogel promoted angiogenesis in rat cryopreserved/thawed ovaries transplanted to the heterotopic sites. J Biol Eng. 2023;17(1):1–13.
Kurowiak J, et al. Analysis of the degradation process of alginate-based hydrogels in artificial urine for use as a bioresorbable material in the treatment of urethral injuries. Processes. 2020;8(3):304.
Naskar S, Sharma S, Kuotsu K. Chitosan-based nanoparticles: an overview of biomedical applications and its preparation. J Drug Deliv Sci Technol. 2019;49:66–81.
Cattelan G, et al. Alginate formulations: current developments in the race for hydrogel-based cardiac regeneration. Front Bioeng Biotechnol. 2020;8:414.
Abdullah M, Jamil RT, Attia FN, Vitamin C (Ascorbic Acid), in StatPearls. 2022, StatPearls Publishing Copyright © 2022, StatPearls Publishing LLC: Treasure Island (FL).
Kaźmierczak-Barańska J, et al. Two faces of vitamin C-antioxidative and pro-oxidative agent. Nutrients. 2020;12(5):1501.
Vivcharenko V, et al. Highly porous and superabsorbent biomaterial made of marine-derived polysaccharides and ascorbic acid as an optimal dressing for exuding wound management. Materials. 2021;14(5):1211.
Zhu Y, et al. Roxadustat promotes angiogenesis through HIF-1α/VEGF/VEGFR2 signaling and accelerates cutaneous wound healing in diabetic rats. Wound Repair Regener. 2019;27(4):324–34.
Shi Y, et al. Bone marrow mesenchymal stem cells facilitate diabetic wound healing through the restoration of epidermal cell autophagy via the HIF-1α/TGF-β1/SMAD pathway. Stem Cell Res Ther. 2022;13(1):314.
Kishimoto Y, et al. Ascorbic acid enhances the expression of type 1 and type 4 collagen and SVCT2 in cultured human skin fibroblasts. Biochem Biophys Res Commun. 2013;430(2):579–84.
Mahmoodzadeh A, et al. Biodegradable cellulose-based superabsorbent as potent hemostatic agent. Chem Eng J. 2021;418:129252.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402–8.
Madni A, et al. Fabrication and characterization of chitosan-vitamin C-lactic acid composite membrane for potential skin tissue engineering. Int J Polym Sci. 2019;2019:4362395.
Stoica A, et al. Fourier transform infrared (FTIR) spectroscopy for characterization of antimicrobial films containing chitosan. Anal Univ Ńii din Oradea Fascicula Ecotoxicologie, Zootehnie şi Tehnologii de Industrie Alimentară. 2010;2010:1234.
Drabczyk A, et al. Physicochemical investigations of chitosan-based hydrogels containing aloe vera designed for biomedical use. Materials. 2020;13(14):3073.
Peng J, Wang X, Lou T. Preparation of chitosan/gelatin composite foam with ternary solvents of dioxane/acetic acid/water and its water absorption capacity. Polym Bull. 2020;77(10):5227–44.
Kuczajowska-Zadrożna M, Filipkowska U, Jóźwiak T. Adsorption of Cu (II) and Cd (II) from aqueous solutions by chitosan immobilized in alginate beads. J Environ Chem Eng. 2020;8:103878.
Kuczajowska-Zadrożna M, Filipkowska U, Jóźwiak T. Adsorption of Cu (II) and Cd (II) from aqueous solutions by chitosan immobilized in alginate beads. J Environ Chem Eng. 2020;8(4):103878.
Daemi H, Barikani M. Synthesis and characterization of calcium alginate nanoparticles, sodium homopolymannuronate salt and its calcium nanoparticles. Sci Iran. 2012;19(6):2023–8.
Asadi S, Eris S, Azizian S. Alginate-based hydrogel beads as a biocompatible and efficient adsorbent for dye removal from aqueous solutions. ACS Omega. 2018;3:15140–8.
Sreeja V, Jayaprabha K, Joy P. Water-dispersible ascorbic-acid-coated magnetite nanoparticles for contrast enhancement in MRI. Appl Nanosci. 2014;5:435.
Raza ZA, et al. Synthesis of alpha-tocopherol encapsulated chitosan nano-assemblies and their impregnation on cellulosic fabric for potential antibacterial and antioxidant cosmetotextiles. Cellulose. 2020;27(3):1717–31.
Sreeja V, Jayaprabha K, Joy P. Water-dispersible ascorbic-acid-coated magnetite nanoparticles for contrast enhancement in MRI. Appl Nanosci. 2015;5:435–41.
Tian X, et al. Synthesis and evaluation of chitosan-vitamin C complex. Indian J Pharm Sci. 2009;71(4):371.
Lavrič G, et al. functional nanocellulose, alginate and chitosan nanocomposites designed as active film packaging materials. Polymers. 2021;13(15):2523.
Wang G, Wang X, Huang L. Feasibility of chitosan-alginate (Chi-Alg) hydrogel used as scaffold for neural tissue engineering: a pilot study in vitro. Biotechnol Biotechnol Equip. 2017;31(4):766–73.
Othman N, et al. Synthesis and optimization of chitosan nanoparticles loaded with l-ascorbic acid and thymoquinone. Nanomaterials. 2018;8(11):920.
Ndlovu SP, et al. Gelatin-based hybrid scaffolds: promising wound dressings. Polymers. 2021;13(17):2959.
Lutzweiler G, Ndreu Halili A, Engin Vrana N. The overview of porous, bioactive scaffolds as instructive biomaterials for tissue regeneration and their clinical translation. Pharmaceutics. 2020;12(7):602.
Archana D, et al. Chitosan-PVP-nano silver oxide wound dressing: In vitro and in vivo evaluation. Int J Biol Macromol. 2015;73:49–57.
Kamoun EA, Kenawy E-RS, Chen X. A review on polymeric hydrogel membranes for wound dressing applications: PVA-based hydrogel dressings. J Adv Res. 2017;8(3):217–33.
Taghipour YD, et al. The application of hydrogels based on natural polymers for tissue engineering. Curr Med Chem. 2020;27(16):2658–80.
Nokoorani YD, et al. Fabrication and characterization of scaffolds containing different amounts of allantoin for skin tissue engineering. Sci Rep. 2021;11(1):16164.
Chhabra P, et al. Optimization, characterization, and efficacy evaluation of 2% chitosan scaffold for tissue engineering and wound healing. J Pharm Bioallied Sci. 2016;8(4):300–8.
Cao J, et al. Double crosslinked HLC-CCS hydrogel tissue engineering scaffold for skin wound healing. Int J Biol Macromol. 2020;155:625–35.
Intini C, et al. 3D-printed chitosan-based scaffolds: an in vitro study of human skin cell growth and an in-vivo wound healing evaluation in experimental diabetes in rats. Carbohyd Polym. 2018;199:593–602.
Chen L, et al. Conditioned medium from hypoxic bone marrow-derived mesenchymal stem cells enhances wound healing in mice. PLoS ONE. 2014;9(4):e96161.
Eid BG, et al. Melittin and diclofenac synergistically promote wound healing in a pathway involving TGF-β1. Pharmacol Res. 2022;175:105993.
Pullar JM, Carr AC, Vissers MCM. The roles of vitamin c in skin health. Nutrients. 2017;9(8):866.
Ravetti S, et al. Ascorbic acid in skin. Health. 2019;6(4):58.
Bhatti FUR, et al. Cytoprotective role of vitamin E in porcine adipose-tissue-derived mesenchymal stem cells against hydrogen-peroxide-induced oxidative stress. Cell Tissue Res. 2018;374(1):111–20.
Ramirez H, Patel SB, Pastar I. The role of TGFβ signaling in wound epithelialization. Adv Wound Care. 2014;3(7):482–91.
Tai Y, et al. Myofibroblasts: function. Form Scope Mol Therap Skin Fib. 2021;11(8):1095.
Aseem SO, et al. Epigenomic evaluation of cholangiocyte transforming growth factor-β signaling identifies a selective role for histone 3 lysine 9 acetylation in biliary fibrosis. Gastroenterology. 2021;160(3):889-905.e10.
Hu HH, et al. New insights into TGF-β/Smad signaling in tissue fibrosis. Chem Biol Interact. 2018;292:76–83.
Mingyuan X, et al. Hypoxia-inducible factor-1α activates transforming growth factor-β1/Smad signaling and increases collagen deposition in dermal fibroblasts. Oncotarget. 2018;9(3):3188–97.
Mohammed BM, et al. Vitamin C promotes wound healing through novel pleiotropic mechanisms. Int Wound J. 2016;13(4):572–84.
Liu Y, et al. Reversal of TET-mediated 5-hmC loss in hypoxic fibroblasts by ascorbic acid. Lab Invest. 2019;99(8):1193–202.