Protective effects of platelet-rich plasma hydrogel on oxidative damage in L929 cells

doi:10.12307/2025.215

Abstract

Abstract: BACKGROUND: During healing process of chronic wounds, excessive production of reactive oxygen species can impair the function of L929 fibroblasts, thereby delaying wound repair. Therefore, protecting fibroblasts from oxidative stress is important to promote wound healing.
OBJECTIVE: To assess the protective effects of carboxymethyl chitosan-oxidized chondroitin sulfate/platelet-rich plasma (CMC-OCS/PRP) hydrogel on L929 cells under H2O2 stimulation.
METHODS: CMC-OCS/PRP hydrogels were prepared, and the micromorphology, degradation performance, scavenging ability of H2O2 and hydroxyl radical and biocompatibility of the hydrogels were characterized. L929 cells with good growth state were taken and cultured in five groups. The control group was cultured conventionally. H2O2 was added to the H2O2 group. Carboxymethyl chitosan-oxidized chondroitin sulfate hydrogel extract + H2O2 was added to the CMC-OCS group. Platelet-rich plasma gel extract + H2O2 was added to the PRP group. The CMC-OCS/PRP group was treated with carboxymethyl chitosan-oxidized chondroitin sulfate/platelet-rich plasma hydrogel extract + H2O2. Each group was treated with hydrogel extract for 6 hours, and then H2O2 for 24 hours. After culture, the levels of active oxygen and malondialdehyde, apoptosis and expression of collagen fiber I protein were detected. In the presence of H2O2, the above hydrogel extracts were directly or indirectly co-cultured with L929 fibroblasts for 36 hours, respectively. Migration ability of the cells was detected by scratch test and Transwell chamber test.
RESULTS AND CONCLUSION: (1) CMC-OCS/PRP hydrogels had uniform and interrelated porous structure and good degradation ability, could effectively remove H2O2 and hydroxyl radicals in vitro, and had good biocompatibility. (2) Compared with the control group, the apoptosis rate, reactive oxygen species, and malondialdehyde levels were increased (P < 0.05); the spread area of cells was decreased (P < 0.05), and the expression of collagen fiber I protein had no significant changes (P > 0.05) in the H2O2 group. Compared with the H2O2 group, reactive oxygen species level was decreased in the CMC-OCS group (P < 0.05), malondialdehyde level was decreased (P < 0.05), and cell spread area was increased (P <0.05) in the PRP group, CMC-OCS group, and CMC-OCS/PRP group; apoptosis rate was decreased in the CMC-OCS/PRP group (P < 0.05), and collagen fiber I protein expression was increased in the PRP group, CMC-OCS group, and CMC-OCS/PRP group (P < 0.05). (3) Compared with the control group, the number of cell migration was decreased (P < 0.05), and the migration area had no significant change (P > 0.05) in the H2O2 group. Compared with the H2O2 group, the number and area of cell migration were increased in the PRP group, CMC-OCS group, and CMC-OCS/PRP group (P < 0.05), and the increase was most significant in the CMC-OCS/PRP group. (4) Under oxidative stress, CMC-OCS/PRP hydrogel can improve the migration ability of fibroblasts, resist cell apoptosis, and preserve cell extension function.

Key words: platelet-rich plasma, carboxymethyl chitosan, oxidized chondroitin sulfate, hydrogel, L929 cell, antioxidation

CLC Number:

Wang Zilin, Mu Qiuju, Liu Hongjie, Shen Yuxue, Zhu Lili. Protective effects of platelet-rich plasma hydrogel on oxidative damage in L929 cells[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(4): 771-779.

Figures/Tables 7

References

[1] ZHANG X, WANG Z, JIANG H, et al. Self-powered enzyme-linked microneedle patch for scar-prevention healing of diabetic wounds. Sci Adv. 2023;9(28):h1415.
[2] DUNNILL C, PATTON T, BRENNAN J, et al. Reactive oxygen species (ROS) and wound healing: the functional role of ROS and emerging ROS-modulating technologies for augmentation of the healing process. Int Wound J. 2017;14(1):89-96.
[3] KIRK T, AHMED A, ROGNONI E. Fibroblast Memory in Development, Homeostasis and Disease. Cells. 2021;10(11):2840
[4] WOODLEY JP, LAMBERT DW, ASENCIO IO. Reduced Fibroblast Activation on Electrospun Polycaprolactone Scaffolds. Bioengineering (Basel). 2023;10(3):384.
[5] WOODLEY JP, LAMBERT DW, ASENCIO IO. Understanding Fibroblast Behavior in 3D Biomaterials. Tissue Eng Part B Rev. 2022;28(3):569-578.
[6] DAWLEE S, SUGANDHI A, BALAKRISHNAN B, et al. Oxidized chondroitin sulfate-cross-linked gelatin matrixes: a new class of hydrogels. Biomacromolecules. 2005;6(4):2040-2048.
[7] LI H, CHENG F, WEI X, et al. Injectable, self-healing, antibacterial, and hemostatic N,O-carboxymethyl chitosan/oxidized chondroitin sulfate composite hydrogel for wound dressing. Mater Sci Eng C Mater Biol Appl. 2021;118:111324.
[8] CHEN YR, ZHOU ZX, ZHANG JY, et al. Low-Molecular-Weight Heparin-Functionalized Chitosan-Chondroitin Sulfate Hydrogels for Controlled Release of TGF-β3 and in vitro Neocartilage Formation. Front Chem. 2019;7:745.
[9] MEDEIROS L, VASCONCELOS B, SILVA MB, et al. Chondroitin sulfate from fish waste exhibits strong intracellular antioxidant potential. Braz J Med Biol Res. 2021;54(10):e10730.
[10] ALINEJAD Y, ADOUNGOTCHODO A, HUI E, et al. An injectable chitosan/chondroitin sulfate hydrogel with tunable mechanical properties for cell therapy/tissue engineering. Int J Biol Macromol. 2018;113:132-141.
[11] FAN M, MA Y, TAN H, et al. Covalent and injectable chitosan-chondroitin sulfate hydrogels embedded with chitosan microspheres for drug delivery and tissue engineering. Mater Sci Eng C Mater Biol Appl. 2017; 71:67-74.
[12] LIU H, CHEN R, WANG P, et al. Electrospun polyvinyl alcohol-chitosan dressing stimulates infected diabetic wound healing with combined reactive oxygen species scavenging and antibacterial abilities. Carbohydr Polym. 2023;316:121050.
[13] SHAHRIARI-KHALAJI M, SATTAR M, CAO R, et al. Angiogenesis, hemocompatibility and bactericidal effect of bioactive natural polymer-based bilayer adhesive skin substitute for infected burned wound healing. Bioact Mater. 2023;29:177-195.
[14] RAMASAMY P, SUBHAPRADHA N, THINESH T, et al. Characterization of bioactive chitosan and sulfated chitosan from Doryteuthis singhalensis (Ortmann, 1891). Int J Biol Macromol. 2017;99:682-691.
[15] DEFAIL AJ, CHU CR, IZZO N, et al. Controlled release of bioactive TGF-beta 1 from microspheres embedded within biodegradable hydrogels. Biomaterials. 2006;27(8):1579-1585.
[16] CAI C, ZHU H, CHEN Y, et al. Platelet-Rich Plasma Composite Organohydrogel with Water-Locking and Anti-Freezing to Accelerate Wound Healing. Adv Healthc Mater. 2023;12(28):e2301477.
[17] WANG Y, ZHANG Y, LI T, et al. Adipose Mesenchymal Stem Cell Derived Exosomes Promote Keratinocytes and Fibroblasts Embedded in Collagen/Platelet-Rich Plasma Scaffold and Accelerate Wound Healing. Adv Mater. 2023;35(40):e2303642.
[18] CAKIN MC, OZDEMIR B, KAYA-DAGISTANLI F, et al. Evaluation of the in vivo wound healing potential of the lipid fraction from activated platelet-rich plasma. Platelets. 2020;31(4):513-520.
[19] ZHANG Y, WANG Z L, DENG ZP, et al. An extracellular matrix-inspired self-healing composite hydrogel for enhanced platelet-rich plasma-mediated chronic diabetic wound treatment. Carbohydr Polym. 2023; 315:120973.
[20] 梅和平,石孟琼,陈茂华,等.地连二心颗粒对H2O2致小鼠成纤维细胞损伤的保护作用研究[J].中药药理与临床,2017,33(1):139-144.
[21] 陈浩祥,杨舒展,陆智儿,等.白术多糖缓解环磷酰胺诱导的岭南黄鸡氧化应激和肝脏细胞凋亡[J]. 动物营养学报,2023,35(3): 1976-1984.
[22] DING Q, SUN T, SU W, et al. Bioinspired Multifunctional Black Phosphorus Hydrogel with Antibacterial and Antioxidant Properties: A Stepwise Countermeasure for Diabetic Skin Wound Healing. Adv Healthc Mater. 2022;11(12):e2102791.
[23] SHAHRIARI-KHALAJI M, SATTAR M, CAO R, et al. Angiogenesis, hemocompatibility and bactericidal effect of bioactive natural polymer-based bilayer adhesive skin substitute for infected burned wound healing. Bioact Mater. 2023;29:177-195.
[24] 胡晶晶.具有ROS清除作用的水凝胶包裹小檗碱@NMOF促进糖尿病足创面愈合[D].广州:南方医科大学,2022.
[25] 姜浩.ROS敏感材料载SDF-1α纳米粒趋化BMSCs归巢、促进创面血管化的研究[D].上海:第二军医大学,2016.
[26] HU S, YANG Z, ZHAI Q, et al. An All-in-One “4A Hydrogel”: through First-Aid Hemostatic, Antibacterial, Antioxidant, and Angiogenic to Promoting Infected Wound Healing. Small. 2023;19(27):e2207437.
[27] JIANG Y, ZHANG X, ZHANG W, et al. Infant Skin Friendly Adhesive Hydrogel Patch Activated at Body Temperature for Bioelectronics Securing and Diabetic Wound Healing. ACS Nano. 2022;16(6): 8662-8676.
[28] YANG R, HUANG J, ZHANG W, et al. Mechanoadaptive injectable hydrogel based on poly(γ-glutamic acid) and hyaluronic acid regulates fibroblast migration for wound healing. Carbohydr Polym. 2021;273:118607.
[29] YUAN F Z, WANG H F, GUAN J, et al. Fabrication of Injectable Chitosan-Chondroitin Sulfate Hydrogel Embedding Kartogenin-Loaded Microspheres as an Ultrasound-Triggered Drug Delivery System for Cartilage Tissue Engineering. Pharmaceutics. 2021;13(9):1487.
[30] KIM H, XUE X. Detection of Total Reactive Oxygen Species in Adherent Cells by 2’,7’-Dichlorodihydrofluorescein Diacetate Staining. J Vis Exp. 2020;(160):1048.
[31] MOHIDEEN K, SUDHAKAR U, BALAKRISHNAN T, et al. Malondialdehyde, an Oxidative Stress Marker in Oral Squamous Cell Carcinoma-A Systematic Review and Meta-Analysis. Curr Issues Mol Biol. 2021;43(2): 1019-1035.
[32] 王应辉,张娅,王自林,等.PRP通过NRF2/HO-1通路对LPS诱导的BV2小胶质细胞神经炎症的保护作用[J].中国输血杂志,2023, 36(1):19-25.
[33] TOGNOLONI A, BARTOLINI D, PEPE M, et al. Platelets Rich Plasma Increases Antioxidant Defenses of Tenocytes via Nrf2 Signal Pathway. Int J Mol Sci. 2023;24(17):13299.
[34] LIU F, LI HY, WANG Z, et al. Carboxymethyl chitosan reduces inflammation and promotes osteogenesis in a rabbit knee replacement model. BMC Musculoskelet Disord. 2020;21(1):775.
[35] EGEA J, GARCÍA AG, VERGES J, et al. Antioxidant, antiinflammatory and neuroprotective actions of chondroitin sulfate and proteoglycans. Osteoarthritis Cartilage. 2010;18 Suppl 1:S24-S27.
[36] 张琦,尚森,江轶南,等.0.1THz辐照对成纤维细胞迁移及细胞骨架的影响[J].真空电子技术,2022(4):31-35.
[37] WANG J, LIN J, CHEN L, et al. Endogenous Electric-Field-Coupled Electrospun Short Fiber via Collecting Wound Exudation. Adv Mater. 2022;34(9):e2108325.
[38] DUAN W, ZHAO J, LIU X, et al. Trapping and release of NIR-active dye in porous silicon as a theranostic strategy for ROS photothermal monitoring and chronic wound management. J Control Release. 2023; 359:428-440.
[39] 田馨如,唐乾利.脂多糖与成纤维细胞在创面修复中的作用机制研究进展[J].中国烧伤创疡杂志,2022,34(4):236-239.
[40] FUKUI M, LAI F, HIHARA M, et al. Activation of cell adhesion and migration is an early event of platelet-rich plasma (PRP)-dependent stimulation of human adipose-derived stem/stromal cells. Hum Cell. 2024;37(1):181-192.