Silk fibroin–gelatine haemostatic sponge loaded with thrombin for wound haemostasis and tissue regeneration

Abstract

Background: Wound haemostasis is an important part of clinical treatments, especially treatments for patients with avulsion injury, destructive injury and large-scale soft tissue injury. Therefore, developing fast and effective haemostatic materials is critical. This study aimed to design a novel and efficient silk fibroin–gelatine composite haemostatic sponge loaded with thrombin (SFG@TB) to assist in wound haemostasis. Methods: The SFG@TB composite haemostatic sponge was formed with gelatine, silk fibroin and thrombin through a freeze-drying technique. First, the material characteristics of SFG@TB were measured, including the elastic modulus, swelling rate and porosity. Second, in vitro cell coculture experiments, in vivo embedding experiments and haemolytic analyses were performed to evaluate the biocompatibility of SFG@TB. Then, coagulation experiments and femoral artery and liver bleeding models were used to evaluate the haemostatic performance of SFG@TB. Finally, the ability of SFG@TB to promote tissue healing was evaluated through experiments with Sprague–Dawley rat models of injury. Results: Compared with gelatine sponges, SFG@TB exhibited outstanding mechanical properties and water absorption properties. In addition, the excellent biosafety of the composite haemostatic sponge was confirmed by cell experiments, subcutaneous embedding experiments and haemolytic analysis. Based on the in vitro coagulation test results, SFG@TB exhibited greater adhesion of red blood cells and platelets and a shorter dynamic coagulation time. Compared to the use of silk fibroin–gelatine composite haemostatic sponges or gelatine sponges, the introduction of thrombin resulted in a shorter haemostasis time and a smaller bleeding volume, as revealed by in vivo coagulation tests. The experiments with Sprague–Dawley rat models of injury indicated that SFG@TB accelerated the wound healing process and reduced scar width, which was accompanied by thicker granulation tissue. Conclusions: Overall, the SFG@TB composite haemostatic sponge, which exhibits outstanding mechanical properties, good haemostatic performance and high biosafety, promoted wound haemostasis and tissue repair. Therefore, the SFG@TB composite haemostatic sponge could be a promising material for wound haemostasis.

Keywords

Composite haemostatic sponge / Gelatine / Silk fibroin / Thrombin / Wound haemostasis / Tissue regeneration / Biomaterial

References

[1] Guo B, Dong R, Liang Y, Li M. Haemostatic materials for wound healing applications. Nat Rev Chem. 2021; 5: 773-91.

[2] Montazerian H, Davoodi E, Baidya A, Baghdasarian S, Sarikhani E, Meyer CE, et al. Engineered Hemostatic biomaterials for sealing wounds. Chem Rev. 2022; 122: 12864-903.

[3] Yu P, Zhong W. Hemostatic materials in wound care. Burns Trauma. 2021; 9: tkab019.

[4] Pezeshki-Modaress M, Rajabi-Zeleti S, Zandi M, Mirzadeh H, Sodeifi N, Nekookar A, et al. Cell-loaded gelatin/chitosan scaffolds fabricated by salt-leaching/lyophilization for skin tissue engineering: In vitro and in vivo study. J Biomed Mater Res A. 2014; 102: 3908-17.

[5] Ji H, Wang X, Wang P, Gong Y, Wang Y, Liu C, et al. Lanthanide-based metal-organic frameworks solidified by gelatin-methacryloyl hydrogels for improving the accuracy of localization and excision of small pulmonary nodules. Journal of Nanobiotechnology. 2022; 20: 60.

[6] Zheng M, Wang X, Yue O, Hou M, Zhang H, Beyer S, et al. Skin-inspired gelatin-based flexible bio-electronic hydrogel for wound healing promotion and motion sensing. Biomaterials. 2021; 276: 121026.

[7] Han K, Bai Q, Wu W, Sun N, Cui N, Lu T. Gelatin-based adhesive hydrogel with self-healing, hemostasis, and electrical conductivity. Int J Biol Macromol. 2021; 183: 2142.

[8] Asadpour S, Kargozar S, Moradi L, Ai A, Nosrati H, Ai J. Natural biomacromolecule based composite scaffolds from silk fibroin, gelatin and chitosan toward tissue engineering applications. Int J Biol Macromol. 2020; 154: 1285.

[9] Xia S, Weng T, Jin R, Yang M, Yu M, Zhang W, et al. Curcumin-incorporated 3d bioprinting gelatin methacryloyl hydrogel reduces reactive oxygen species-induced adipose-derived stem cell apoptosis and improves implanting survival in diabetic wounds. Burns Trauma. 2022; 10: tkac001.

[10] Xie X, Li D, Chen Y, Shen Y, Yu F, Wang W, et al. Conjugate electrospun 3D Gelatin nanofiber sponge for rapid Hemostasis. Adv Healthc Mater. 2021; 10: e2100918.

[11] Chen K, Pan H, Yan Z, Li Y, Ji D, Yun K, et al. A novel alginate/gelatin sponge combined with curcumin-loaded electrospun fibers for postoperative rapid hemostasis and prevention of tumor recurrence. Int J Biol Macromol. 2021; 182: 1339.

[12] Ndlovu SP, Ngece K, Alven S, Aderibigbe BA. Gelatin-based hybrid scaffolds: promising wound dressings. Polymers (Basel). 2021; 13: 2959. https://doi.org/10.3390/polym13172959.

[13] Yung CW, Wu LQ, Tullman JA, Payne GF, Bentley WE, Barbari TA. Transglutaminase crosslinked gelatin as a tissue engineering scaffold. J Biomed Mater Res A. 2007; 83A: 1039-46.

[14] Koh LD, Cheng Y, Teng CP, Khin YW, Loh XJ, Tee SY, et al. Structures, mechanical properties and applications of silk fibroin materials. Prog Polym Sci. 2015; 46: 86-100.

[15] Sultan MT, Hong H, Lee OJ, Ajiteru O, Lee YJ, Lee JS, et al. Silk fibroin-based biomaterials for Hemostatic applications. Biomol Ther. 2022; 12: 660. https://doi.org/10.3390/biom12050660.

[16] Zhang X, Reagan MR, Kaplan DL. Electrospun silk biomaterial scaffolds for regenerative medicine. Adv Drug Deliv Rev. 2009; 61: 988-1006.

[17] Kundu B, Rajkhowa R, Kundu SC, Wang X. Silk fibroin biomaterials for tissue regenerations. Adv Drug Deliv Rev. 2013; 65: 457-70.

[18] Brown JE, Moreau JE, Berman AM, McSherry HJ, Coburn JM, Schmidt DF, et al. Shape memory silk protein sponges for minimally invasive tissue regeneration. Advanced Healthcare Materials. 2017; 6. https://doi.org/10.1002/adhm.201600762.

[19] Gomes JM, Silva SS, Fernandes EM, Lobo FCM, Martín-Pastor M, Taboada P, et al. Silk fibroin/cholinium gallate-based architectures as therapeutic tools. Acta Biomater. 2022; 147: 168-84.

[20] Li Z, Zheng A, Mao Z, Li F, Su T, Cao L, et al. Silk fibroin-gelatin photo-crosslinked 3D-bioprinted hydrogel with MOF-methylene blue nanoparticles for infected wound healing. Int J Bioprint. 2023; 9: 773.

[21] Shi W, Sun M, Hu X, Ren B, Cheng J, Li C, et al. Structurally and functionally optimized silk-fibroin-gelatin scaffold using 3D printing to repair cartilage injury in vitro and in vivo. Adv Mater. 2017; 29. https://doi.org/10.1002/adma.201701089.

[22] Di Cera E. Thrombin. Mol Asp Med. 2008; 29: 203-54.

[23] Ali-Mohamad N, Cau MF, Zenova V, Baylis JR, Beckett A, McFadden A, et al. Self-propelling thrombin powder enables hemostasis with no observable recurrent bleeding or thrombosis over 3 days in a porcine model of upper GI bleeding. Gastrointest Endosc. 2023; 98: 245-8.

[24] Cui D, Li M, Zhang P, Rao F, Huang W, Wang C, et al. Polydopamine-coated Polycaprolactone electrospun nanofiber membrane loaded with thrombin for wound Hemostasis. Polymers. 2023; 15: 3122. https://doi.org/10.3390/polym15143122.

[25] Ali-Mohamad N, Cau M, Baylis J, Zenova V, Semple H, Beckett A, et al. Severe upper gastrointestinal bleeding is halted by endoscopically delivered self-propelling thrombin powder: a porcine pilot study. Endoscopy International Open. 2021; 09: E693-8.

[26] Liu L, Hu E, Yu K, Xie R, Lu F, Lu B, et al. Recent advances in materials for hemostatic management. Biomaterials Science. 2021; 9: 7343-78.

[27] Hickman DA, Pawlowski CL, Sekhon UDS, Marks J, Gupta AS. Biomaterials and advanced Technologies for Hemostatic Management of bleeding. Adv Mater. 2018; 30. https://doi.org/10.1002/adma.201700859.

[28] Guo Y, Wang M, Liu Q, Liu G, Wang S, Li J. Recent advances in the medical applications of hemostatic materials. Theranostics. 2023; 13: 161-96.

[29] Nepal A, Tran HDN, Nguyen NT, Ta HT. Advances in haemostatic sponges: characteristics and the underlying mechanisms for rapid haemostasis. Bioactive Materials. 2023; 27: 231-56.

[30] Wang H, Liu Y, Cai K, Zhang B, Tang S, Zhang W, et al. Antibacterial polysaccharide-based hydrogel dressing containing plant essential oil for burn wound healing. Burns Trauma. 2021; 9: tkab041.

[31] Tamer TM, Alsehli MH, Omer AM, Afifi TH, Sabet MM, Mohy-Eldin MS, et al. Development of polyvinyl alcohol/kaolin sponges stimulated by marjoram as Hemostatic, antibacterial, and antioxidant dressings for wound healing promotion. Int J Mol Sci. 2021; 22: 13050. https://doi.org/10.3390/ijms222313050.

[32] Song Y, Li S, Chen H, Han X, Duns GJ, Dessie W, et al. Kaolin-loaded carboxymethyl chitosan/sodium alginate composite sponges for rapid hemostasis. Int J Biol Macromol. 2023; 233: 123532.

[33] Wei X, Ding S, Liu S, Yang K, Cai J, Li F, et al. Polysaccharides-modified chitosan as improved and rapid hemostasis foam sponges. Carbohydr Polym. 2021; 264: 118028.

[34] Al-Amer OM. The role of thrombin in haemostasis. Blood Coagul Fibrinolysis. 2022; 33: 145-8.

[35] Cardenas JC. Thrombin generation following severe trauma: mechanisms, modulators, and implications for Hemostasis and thrombosis. Shock. 2021; 56: 682-90.

[36] Crawley JT, Zanardelli S, Chion CK, Lane DA. The central role of thrombin in hemostasis. J Thromb Haemost. 2007; 5: 95-101.

[37] Dong R, Zhang H, Guo B. Emerging hemostatic materials for non-compressible hemorrhage control. Natl Sci Rev. 2022; 9: nwac162.

[38] Werner S, Grose R. Regulation of wound healing by growth factors and cytokines. Physiol Rev. 2003; 83: 835-70.

[39] Xu N, Yuan Y, Ding L, Li J, Jia J, Li Z, et al. Multifunctional chitosan/gelatin@tannic acid cryogels decorated with in situ reduced silver nanoparticles for wound healing. Burns Trauma. 2022; 10: tkac019. https://doi.org/10.1093/burnst/tkac019.

[40] Chouhan D, Mandal BB. Silk biomaterials in wound healing and skin regeneration therapeutics: from bench to bedside. Acta Biomater. 2020; 103: 24-51.

[41] Klotz BJ, Gawlitta D, Rosenberg AJWP, Malda J, Melchels FPW. Gelatin-Methacryloyl hydrogels: towards biofabrication-based tissue repair. Trends Biotechnol. 2016; 34: 394-407.

[42] Sun W, Gregory DA, Tomeh MA, Zhao X. Silk fibroin as a functional biomaterial for tissue engineering. Int J Mol Sci. 2021; 22: 1499. https://doi.org/10.3390/ijms22031499.

[43] Kurian AG, Singh RK, Patel KD, Lee JH, Kim HW. Multifunctional GelMA platforms with nanomaterials for advanced tissue therapeutics. Bioactive Materials. 2022; 8: 267-95.