Regeneration of the skin wound by two different crosslinkers: In vitro and in vivo studies

Document Type : Original Article

Authors

1 Student Research Committee, School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran

2 Department of Tissue Engineering and Biomaterials, School of Advanced Medical Sciences and Technologies, Hamadan University of Medical Sciences, Hamadan, Iran

3 Tissue Engineering and stem cells research center, Shahroud University of Medical Sciences, Shahroud, Iran

4 Department of Ophthalmology and Visual Sciences, University of Illinois, Chicago, IL 60612, USA

5 Department of Tissue Engineering, School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran

10.22038/ijbms.2024.80137.17361

Abstract

Objective(s): For designing a suitable hydrogel, two crosslinked Alginate/ Carboxymethyl cellulose (Alg/CMC) hydrogel, using calcium chloride (Ca2+) and glutaraldehyde (GA) as crosslinking agents were synthesized and compared.
Materials and Methods: All samples were characterized by Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM), Blood compatibility (BC), Blood clotting index (BCI), weight loss (WL), water absorption (WA), pH, and Electrochemical Impedance Spectroscopy (EIS). Cell viability and cell migration were investigated using the MTT assay and the wound scratch test, respectively. Besides, the wound healing potential of prepared hydrogels was evaluated on the rat models with full-thickness skin excision. To further investigation, TGF β1, IGF-I, COL1, ACT-A (alfa-SMA), and GAPDH expression levels were also reported by RT-PCR. 
Results: Water absorption and weight loss properties were compared between different crosslinker agents, and the most nontoxic crosslinker concentration was determined. We have shown that GA (20 µl/ml) and Ca2+ (50 or 75 mM) enhanced the physical stability of Alg-CMC hydrogel, and they are nontoxic and suitable crosslinkers for wound dressing applications. Although in vivo assessments indicated that the GA (20 µl/ml) had a cytotoxic effect on tissue repair, Ca2+ (75 mM) boosted the wound healing process. Further, RT-PCR results revealed that TGF β1, IGF-I, COL1, ACT-A (alfa-SMA), and GAPDH expression levels were increased in GA (20 µl/ml). Moreover, this trend is the opposite in the Ca2+ (75 mM) treatment groups.
Conclusion: This research shows that Ca2+ (75 mM) boosts tissue regeneration and wound healing process.

Keywords

Main Subjects

References

1. El-Ela FIA, Farghali AA, Mahmoud RK, Mohamed NA, Moaty SAA. New approach in ulcer prevention and wound healing treatment using doxycycline and amoxicillin/ldh nanocomposites. Sci Rep 2019; 9:6418-6432.
2. Gaharwar AK, Singh I, Khademhosseini A. Engineered biomaterials for in situ tissue regeneration. Nat Rev Mater 2020; 5:686-705.
3. Jeong K-H, Park D, Lee Y-C. Polymer-based hydrogel scaffolds for skin tissue engineering applications: A mini-review. J Polym Res 2017; 24:112-121.
4. Bagher Z, Ehterami A, Safdel MH, Khastar H, Semiari H, Asefnejad A, et al. Wound healing with alginate/chitosan hydrogel containing hesperidin in rat model. J Drug Deliv Technol 2020; 55:101379.
5. Wu S, Wang T-W, Du Y, Yao B, Duan S, Yan Y, et al. Tough, anti-freezing and conductive ionic hydrogels. NPG Asia Mater 2022; 14:65-72.
6. Nazarnezhad S, Salehi M, Samadian H, Ehtermi A, Kasaiyan N, Khastar H, et al. In vitro and in vivo evaluation of porous alginate hydrogel containing retinoic acid for skin wound healing applications. J Bioact Compat Pol 2022; 37:332-342.
7. Cascone S, Lamberti G. Hydrogel-based commercial products for biomedical applications: A review. Int J Pharm 2020; 573:118803.
8. Bahrami N, Farzin A, Bayat F, Goodarzi A, Salehi M, Karimi R, et al. Optimization of 3D alginate scaffold properties with interconnected porosity using freeze-drying method for cartilage tissue engineering application.  Arch Neurosci 2019; 6:e85122.
9. Abasalizadeh F, Moghaddam SV, Alizadeh E, akbari E, Kashani E, Fazljou SMB, et al. Alginate-based hydrogels as drug delivery vehicles in cancer treatment and their applications in wound dressing and 3D bioprinting. J Biol Eng 2020; 14:8-29.
10. Ehterami A, Salehi M, Farzamfar S, Samadian H, Vaez A, Ghorbani S, et al. Chitosan/alginate hydrogels containing alpha-tocopherol for wound healing in rat model. J Drug Deliv Sci Technol 2019; 51:204-213.
11. Chen Y, Tang Y, Wang Q, Lei L, Zhao J, Zhang Y, et al. Carboxymethylcellulose‐induced changes in rheological properties and microstructure of wheat gluten proteins under different pH conditions. J Food Sci 2021; 86:677-686.
12. Barbucci R, Magnani A, Consumi M. Swelling behavior of carboxymethylcellulose hydrogels in relation to cross-linking, pH, and charge density. Macromol 2000; 33:7475-7480.
13. Liu S, Zhang H, Ahlfeld T, Kilian D, Liu Y, Gelinsky M, et al. Evaluation of different crosslinking methods in altering the properties of extrusion-printed chitosan-based multi-material hydrogel composites. Bio-Design and Manufacturing 2023; 6:150-173.
14. Pourjavadi A, Barzegar S, Mahdavinia GR. MBA-crosslinked Na-Alg/CMC as a smart full-polysaccharide superabsorbent hydrogels. carbohydr polym 2006; 66:386-395.
15. Hu Y, Hu S, Zhang S, Dong S, Hu J, Kang L, et al. A double-layer hydrogel based on alginate-carboxymethyl cellulose and synthetic polymer as sustained drug delivery system. Sci Rep 2021; 11:1-14.
16. Hu W, Wang Z, Xiao Y, Zhang S, Wang J. Advances in crosslinking strategies of biomedical hydrogels. Biomater Sci 2019; 7:843-855.
17. Mironi-Harpaz I, Wang DY, Venkatraman S, Seliktar D. Photopolymerization of cell-encapsulating hydrogels: Crosslinking efficiency versus cytotoxicity. Acta Biomater 2012; 8:1838-1848.
18. Hennink WE, van Nostrum CF. Novel crosslinking methods to design hydrogels. Adv Drug Deliv Rev 2012; 64:223-236.
19. He X, Zeng L, Cheng X, Yang C, Chen J, Chen H, et al. Shape memory composite hydrogel based on sodium alginate dual crosslinked network with carboxymethyl cellulose. Eur Polym J 2021; 156:110592.
20. Sritweesinsub W, Charuchinda S, editors. Alginate/carboxymethyl cellulose hydrogel films in relation to crosslinking with glutaraldehyde and copper sulfate. EDP Sci 2015; 30:1-4.
21. Dey RK, Ray AR. Synthesis, characterization, and blood compatibility of polyamidoamines copolymers. Biomater 2003; 24:2985-2993.
22. Mahheidari N, Kamalabadi-Farahani M, Nourani MR, Atashi A, Alizadeh M, Aldaghi N, et al. Biological study of skin wound treated with alginate/carboxymethyl cellulose/chorion membrane, diopside nanoparticles, and botox A. NPJ Regen Med 2024; 9:9-28.
23. Felice F, Zambito Y, Belardinelli E, Fabiano A, Santoni T, Di Stefano R. Effect of different chitosan derivatives on in vitro scratch wound assay: A comparative study. Int J Biol Macromol 2015; 76:236-241.
24. Buhus G, Popa M, Desbrieres J. Hydrogels based on carboxymethylcellulose and gelatin for inclusion and release of chloramphenicol. J Bioact Compat 2009; 24:525-545.
25. Liu Y, Yang Y, Wu F. Effects of L-arginine immobilization on the anticoagulant activity and hemolytic property of polyethylene terephthalate films. Appl Surf Sci 2010; 256:3977-3981.
26. Xue H, Wang D, Jin M, Gao H, Wang X, Xia L, et al. Hydrogel electrodes with conductive and substrate-adhesive layers for noninvasive long-term EEG acquisition. Microsyst Nanoeng 2023; 9:79-92.
27. Badawi NM, Batoo KM, Subramaniam R, Kasi R, Hussain S, Imran A, et al. Highly conductive and reusable cellulose hydrogels for supercapacitor applications. Micromachines 2023; 14:1461-1479.
28. Wrobel TM, Kiełbus M, Kaczor AA, Kryštof V, Karczmarzyk Z, Wysocki W, et al. Discovery of nitroaryl urea derivatives with antiproliferative properties. J Enzyme Inhib Med Chem 2016; 31:608-618.
29. Krizanova O, Penesova A, Sokol J, Hokynkova A, Samadian A, Babula P. Signaling pathways in cutaneous wound healing. Front Physiol 2022; 13:2438-2448..
30. Lei X-X, Zou C-Y, Hu J-J, Jiang Y-L, Zhang X-Z, Zhao L-M, et al. Click-crosslinked in-situ hydrogel improves the therapeutic effect in wound infections through antibacterial, anti-oxidant and anti-inflammatory activities. J Chem Eng 2023; 461:142092.
31. Yu T, Yang W, Zhuang W, Tian Y, Kong Q, Chen X, et al. A bioprosthetic heart valve cross-linked by a non-glutaraldehyde reagent with improved biocompatibility, endothelialization, anti-coagulation and anti-calcification properties. J Mater Chem B 2021; 9:4031-4038.
32. Shah SA, Sohail M, Khan SA, Kousar M. Improved drug delivery and accelerated diabetic wound healing by chondroitin sulfate grafted alginate-based thermoreversible hydrogels. Mater Sci Eng C Mater Biol Appl 2021; 126:112169.
33. Yang S, Leong K-F, Du Z, Chua C-K. The design of scaffolds for use in tissue engineering. part I. traditional factors. Tissue Eng 2001; 7:679-689.
34. Mansur HS, Sadahira CM, Souza AN, Mansur AAP. FTIR spectroscopy characterization of poly (vinyl alcohol) hydrogel with different hydrolysis degree and chemically crosslinked with glutaraldehyde. Mater Sci Eng: C 2008; 28:539-548.
35. Ramadhani S, Helmiyati H, editors. Alginate/CMC/ZnO nanocomposite for photocatalytic degradation of congo red dye. 2020: AIP Publishing.
36. Saarai A, Kasparkova V, Sedlacek T, Sáha P.  On the development and characterisation of crosslinked sodium alginate/gelatine hydrogels. J Mech Behav Biomed Mater 2013; 18:152-166.
37. Hua F, Qian M. Synthesis of self-crosslinking sodium polyacrylate hydrogel and water-absorbing mechanism. J Mater Sci 2001; 36:731-738.
38. Salehi M, Bagher Z, Kamrava SK, Ehterami A, Alizadeh R, Farhadi M, et al. Alginate/chitosan hydrogel containing olfactory ectomesenchymal stem cells for sciatic nerve tissue engineering. J Cell Physiol 2019; 234:15357-15368.
39. Ali MK, Tayyab S. Differential resistance to calcium-induced bilirubin-dependent hemolysis in mammalian erythrocytes. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 1999; 122:109-113.
40. Kumar A, Sah DK, Khanna K, Rai Y, Yadav AK, Ansari MS, et al. A Calcium and zinc composite alginate hydrogel for pre-hospital hemostasis and wound care. Carbohydr Polym 2023; 299:120186.
41. Baghaie S, Khorasani MT, Zarrabi A, Moshtaghian J. Wound healing properties of PVA/starch/chitosan hydrogel membranes with nano zinc oxide as antibacterial wound dressing material. J Biomater Sci Polym Ed 2017; 28:2220-2241.
42. Paddle-Ledinek JE, Nasa Z, Cleland HJ. Effect of different wound dressings on cell viability and proliferation. Plast Reconstr Surg 2006; 117:110S-118S.
43. Boynton A, Whitfield J, Isaacs R, Morton H. Control of 3T3 cell proliferation by calcium. In vitro 1974; 10:12-17.
44. Sun HW, Feigal R, Messer H. Cytotoxicity of glutaraldehyde and formaldehyde in relation to time of exposure and concentration. Pediatr Dent 1990; 12:303-307.
45. Ishfaq B, Khan IU, Khalid SH, Asghar S. Design and evaluation of sodium alginate-based hydrogel dressings containing betula utilis extract for cutaneous wound healing. Front Bioeng Biotechnol 2023; 11:1042077.
46. Rodrigues IR, de Camargo Forte MM, Azambuja DS, Castagno KR. Synthesis and characterization of hybrid polymeric networks (HPN) based on polyvinyl alcohol/chitosan. React Funct Pol 2007; 67:708-715.
47. Pal K, Paulson AT, Rousseau D. 14 - Biopolymers In Controlled-Release Delivery Systems. In: Ebnesajjad S, Editor. Handbook of Biopolymers and Biodegradable Plastics. Boston: William Andrew Publishing; 2013. p. 329-363.
48. Choudhury NA, Sampath S, Shukla AK. Gelatin hydrogel electrolytes and their application to electrochemical supercapacitors. Journal of The Electrochemical Society 2007; 155:A74.
49. Danaee I, Jafarian M, Forouzandeh F, Gobal F, Mahjani MG. Impedance spectroscopy analysis of glucose electro-oxidation on Ni-modified glassy carbon electrode. Electrochimacta 2008; 53:6602-6609.
50. Al-Zubaidi R-Smith N, Kasper M, Kumar P, Nilsson D, Mårlid B, Kienberger F. Advanced electrochemical impedance spectroscopy of industrial Ni-Cd batteries. Batteries 2022; 8:1-14.
51. Yu R, Zhang H, Guo B. Conductive biomaterials as bioactive wound dressing for wound healing and skin tissue engineering. Nanomicro Lett 2021; 14:1-46.
52. Ishfaq B, Khan IU, Khalid SH, Asghar S. Design and evaluation of sodium alginate-based hydrogel dressings containing Betula utilis extract for cutaneous wound healing. Front Bioeng Biotechnol 2023; 11:1042077.
53. Harriger MD, Supp AP, Warden GD, Boyce ST. Glutaraldehyde crosslinking of collagen substrates inhibits degradation in skin substitutes grafted to athymic mice. J Biomed Mater Res 1997; 35:137-145.
54. Margineanu DG, Van Driessche W. Dose dependence of glutaraldehyde-induced changes in the electrical properties of the amphibian skin. Arch Int Physiol Biochim Biophys 1991; 99:83-88.
55. Subramaniam T, Fauzi MB, Lokanathan Y, Law JX. The role of calcium in wound healing. Int J Mol Sci 2021; 22:1-14.
56. Hesketh M, Sahin KB, West ZE, Murray RZ. Macrophage phenotypes regulate scar formation and chronic wound healing. Int J Mol Sci 2017; 18:1545-1554.
57. Kahnberg K-E, Thilander H. Healing of experimental excisional wounds in the rat palate: (I) histological study of the interphase in wound healing after sharp dissection. Int J Oral Surg 1982; 11:44-51.
58. Liliac IM, Popescu EL, Văduva IA, Pirici D, Mogoşanu GD, Streba CT, et al. Nanoparticle-functionalized dressings for the treatment of third-degree skin burns - histopathological and immunohistochemical study. Rom J Morphol Embryol 2021; 62:159-168.
59. Zhang Q, Liu L-N, Yong Q, Deng J-C, Cao W-G. Intralesional injection of adipose-derived stem cells reduces hypertrophic scarring in A rabbit ear model. Stem Cell Res Ther 2015; 6:1-11.
60. Beare AHM, O’Kane S, Ferguson MWJ, Krane SM. Severely impaired wound healing in the collagenase-resistant mouse. J Invest Dermatol 2003; 120:153-163.
61. Emmerson E, Campbell L, Davies FCJ, Ross NL, Ashcroft GS, Krust A, et al. Insulin-like growth factor-1 promotes wound healing in estrogen-deprived mice: New insights into cutaneous IGF-1R/ERα cross talk. J Invest Dermatol 2012; 132:2838-2848.
62. Garoufalia Z, Papadopetraki A, Karatza E, Vardakostas D, Philippou A, Kouraklis G, et al. Insulin-like growth factor-I and wound healing, a potential answer to non-healing wounds: A systematic review of the literature and future perspectives. Biomed Rep 2021; 15:66-70.
63. Wei G, Xu Q, Liu L, Zhang H, Tan X, Zhang C, et al. LY2109761 reduces TGF-β1-induced collagen production and contraction in hypertrophic scar fibroblasts. Arch Dermatol Res 2018; 310:615-623.
64. Wang Y, Moges H, Bharucha Y, Symes A. Smad3 null mice display more rapid wound closure and reduced scar formation after A stab wound to the cerebral cortex. Exp Neurol 2007; 203:168-184.
Iranian Journal of Basic Medical Sciences
Volume 28, Issue 2
February 2025
Pages 194-208

Files

Share

How to cite

Statistics