Islet biomimetic microenvironment constructed by chitosan oligosaccharide protects islets from hypoxia-induced damage by reducing intracellular reactive oxygen species_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WANG Dongzhi ^1,2 , GUO Yibing ² , HUANG Yan ¹ , ZHU Biwen ^1,2 , PAN Haopeng ³ ,  WANG Zhiwei ¹

1. Department of General Surgery, Affiliated Hospital of Nantong University, Nantong Jiangsu, 226001, P. R. China;
2. Research Center of Clinical Medicine, Affiliated Hospital of Nantong University, Nantong Jiangsu, 226001, P. R. China;
3. Key Laboratory of Neuroregeneration, Nantong University, Nantong Jiangsu, 226001, P. R. China;

Corresponding author：

WANG Zhiwei, Email: wzw3639@163.com

Keywords：

Islets; chitosan oligosaccharide; reactive oxygen species; biomimetic microenvironment

DOI：

10.7507/1002-1892.202201063

Video：

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Objective Gelatin methacryloyl (GelMA)/hyaluronic acid methacryloyl (HAMA)/chitosan oligosaccharide (COS) hydrogel was used to construct islet biomimetic microenvironment, and to explore the improvement effect of GelMA/HAMA/COS on islet activity and function under hypoxia. Methods Islets cultured on the tissue culture plate was set as the control group, on the GelMA/HAMA/COS hydrogel with COS concentrations of 0, 1, 5, 10, and 20 mg/mL respectively as the experimental groups. Scanning electron microscopy was used to observe the microscopic morphology, rheometer test to evaluate the gel-forming properties, contact angle to detect the hydrophilicity, and the biocompatibility was evaluated by the scaffold extract to L929 cells [using cell counting kit 8 (CCK-8) assay]. The islets were extracted from the pancreas of 8-week-old Sprague Dawley rats and the islet purity and function were identified by dithizone staining and glucose-stimulated insulin secretion (GSIS) assays, respectively. Islets were cultured under hypoxia (1%O₂) for 24, 48, and 72 hours, respectively. Calcein-acetyl methyl/propidium iodide (Calcein-AM/PI) staining was used to evaluate the effect of hypoxia on islet viability. Islets were cultured in GelMA/HAMA/COS hydrogels with different COS concentrations for 48 hours, and the reactive oxygen species kits were used to evaluate the antagonism of COS against islet reactive oxygen species production under normoxia (20%O₂) and hypoxia (1%O₂) conditions. Calcein-AM/PI staining was used to evaluate the effect of COS on islet activity under hypoxia (1%O₂) conditions. Islets were cultured in tissue culture plates (group A), GelMA/HAMA hydrogels (group B), and GelMA/HAMA/COS hydrogels (group C) for 48 hours, respectively. Immunofluorescence and GSIS assays were used to evaluate the effect of COS on islet activity under hypoxia (1%O₂) conditions, respectively. Results GelMA/HAMA/COS hydrogel had a porous structure, the rheometer test showed that it had good gel-forming properties, and the contact angle test showed good hydrophilicity. CCK-8 assay showed that the hydrogel in each group had good biocompatibility. The isolated rat islets were almost round, with high islet purity and insulin secretion ability. Islets were treated with hypoxia for 24, 48, and 72 hours, Calcein-AM/PI staining showed that the number of dead cells gradually increased with time, which were significantly higher than those in the non-hypoxia-treated group (P<0.001). Reactive oxygen staining showed that GelMA/HAMA/COS hydrogels with different COS concentrations could antagonize the production of reactive oxygen under normal oxygen and hypoxia conditions, and this ability was positively correlated with COS concentration. Calcein-AM/PI staining indicated that GelMA/HAMA/COS hydrogels with different COS concentrations could improve islet viability under hypoxia conditions, and cell viability was positively correlated with COS concentration. Immunofluorescence staining showed that GelMA/HAMA/COS hydrogel could promote the expression of islet function-related genes under hypoxia conditions. GSIS assay results showed that the insulin secretion of islets in hypoxia condition of group C was significantly higher than that of groups B and C (P<0.05). Conclusion GelMA/HAMA/COS hydrogel has good biocompatibility, promotes islet survival and function by inhibiting reactive oxygen species, and is an ideal carrier for building islet biomimetic microenvironment for islet culture and transplantation.

Citation： WANG Dongzhi, GUO Yibing, HUANG Yan, ZHU Biwen, PAN Haopeng, WANG Zhiwei. Islet biomimetic microenvironment constructed by chitosan oligosaccharide protects islets from hypoxia-induced damage by reducing intracellular reactive oxygen species. Chinese Journal of Reparative and Reconstructive Surgery, 2022, 36(5): 633-642. doi: 10.7507/1002-1892.202201063 Copy

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2.	Xu Y, Tan M, Ma X, et al. Human mesenchymal stem cells-derived conditioned medium inhibits hypoxia-induced death of neonatal porcine islets by inducing autophagy. Xenotransplantation, 2020, 27(1): e12556. doi: 10.1111/xen.12556.
3.	Tan Y, Nie W, Chen C, et al. Mesenchymal stem cells alleviate hypoxia-induced oxidative stress and enhance the pro-survival pathways in porcine islets. Exp Biol Med (Maywood), 2019, 244(9): 781-788.
4.	Wang G, Wang Z, Sun N, et al. Reactive oxygen species-responsive silk sericin microcapsules used for antioxidative stress damage. Microsc Res Tech, 2021, 84(4): 618-626.
5.	Keshtkar S, Kaviani M, Jabbarpour Z, et al. Significant reduction of apoptosis induced via hypoxia and oxidative stress in isolated human islet by resveratrol. Nutr Metab Cardiovasc Dis, 2020, 30(7): 1216-1226.
6.	Coronel MM, Liang JP, Li Y, et al. Oxygen generating biomaterial improves the function and efficacy of beta cells within a macroencapsulation device. Biomaterials, 2019, 210: 1-11.
7.	Lee EM, Jung JI, Alam Z, et al. Effect of an oxygen-generating scaffold on the viability and insulin secretion function of porcine neonatal pancreatic cell clusters. Xenotransplantation, 2018, 25(2): e12378. doi: 10.1111/xen.12378.
8.	Liang JP, Accolla RP, Soundirarajan M, et al. Engineering a macroporous oxygen-generating scaffold for enhancing islet cell transplantation within an extrahepatic site. Acta Biomater, 2021, 130: 268-280.
9.	Razavi M, Primavera R, Kevadiya BD, et al. A collagen based cryogel bioscaffold that generates oxygen for islet transplantation. Adv Funct Mater, 2020, 30(15): 1902463. doi: 10.1002/adfm.201902463.
10.	Wang DW, Li SJ, Tan XY, et al. Engineering of stepwise-targeting chitosan oligosaccharide conjugate for the treatment of acute kidney injury. Carbohydr Polym, 2021, 256: 117556. doi: 10.1016/j.carbpol.2020.117556.
11.	Zhang X, Liang S, Gao X, et al. Protective effect of chitosan oligosaccharide against hydrogen peroxide-mediated oxidative damage and cell apoptosis via activating Nrf2/ARE signaling pathway. Neurotox Res, 2021, 39(6): 1708-1720.
12.	Velasco-Rodriguez B, Diaz-Vidal T, Rosales-Rivera LC, et al. Hybrid methacrylated gelatin and hyaluronic acid hydrogel scaffolds. Preparation and systematic characterization for prospective tissue engineering applications. Int J Mol Sci, 2021, 22(13): 6758. doi: 10.3390/ijms22136758.
13.	Wang S, Guan S, Zhu Z, et al. Hyaluronic acid doped-poly (3, 4-ethylenedioxythiophene)/chitosan/gelatin (PEDOT-HA/Cs/Gel) porous conductive scaffold for nerve regeneration. Mater Sci Eng C Mater Biol Appl, 2017, 71: 308-316.
14.	Mouser VHM, Levato R, Mensinga A, et al. Bio-ink development for three-dimensional bioprinting of hetero-cellular cartilage constructs. Connect Tissue Res, 2020, 61(2): 137-151.
15.	Guan G, Qizhuang Lv None, Liu S, et al. 3D-bioprinted peptide coupling patches for wound healing. Mater Today Bio, 2021, 13: 100188. doi: 10.1016/j.mtbio.2021.100188.
16.	Posniak S, Chung JHY, Liu X, et al. Bioprinting of chondrocyte stem cell co-cultures for auricular cartilage regeneration. ACS Omega, 2022, 7(7): 5908-5920.
17.	Fan Y, Yue Z, Lucarelli E, et al. Hybrid printing using cellulose nanocrystals reinforced GelMA/HAMA hydrogels for improved structural integration. Adv Healthc Mater, 2020, 9(24): e2001410. doi: 10.1002/adhm.202001410.
18.	Colli ML, Szymczak F, Eizirik DL. Molecular footprints of the immune assault on pancreatic beta cells in type 1 diabetes. Front Endocrinol (Lausanne), 2020, 11: 568446. doi: 10.3389/fendo.2020.568446.
19.	Yoshihara E, O’Connor C, Gasser E, et al. Immune-evasive human islet-like organoids ameliorate diabetes. Nature, 2020, 586(7830): 606-611.
20.	Nishime K, Miyagi-Shiohira C, Kuwae K, et al. Preservation of pancreas in the University of Wisconsin solution supplemented with AP39 reduces reactive oxygen species production and improves islet graft function. Am J Transplant, 2021, 21(8): 2698-2708.
21.	Sayadi LR, Alexander M, Sorensen AM, et al. Micro/nanobubbles: Improving pancreatic islet cell survival for transplantation. Ann Plast Surg, 2019, 83(5): 583-588.
22.	Lau J, Henriksnäs J, Svensson J, et al. Oxygenation of islets and its role in transplantation. Curr Opin Organ Transplant, 2009, 14(6): 688-693.
23.	Zhang J, Sun X, Chen Y, et al. Preparation of 2, 6-diurea-chitosan oligosaccharide derivatives for efficient antifungal and antioxidant activities. Carbohydr Polym, 2020, 234: 115903. doi: 10.1016/j.carbpol.2020.115903.
24.	Zhang J, Wang W, Mao XZ, et al. Chitopentaose protects HaCaT cells against H₂O₂-induced oxidative damage through modulating MAPKs and Nrf2/ARE signaling pathways. Journal of Functional Foods, 2020, 72: 10408. doi: 10.1016/j.jff.2020.104086.
25.	Chang Q, Cai H, Wei L, et al. Chitosan oligosaccharides alleviate acute heat stress-induced oxidative damage by activating ERK1/2-mediated HO-1 and GSH-Px gene expression in breast muscle of broilers. Poult Sci, 2022, 101(1): 101515. doi: 10.1016/j.psj.2021.101515.
26.	Zhang Y, Ahmad KA, Khan FU, et al. Chitosan oligosaccharides prevent doxorubicin-induced oxidative stress and cardiac apoptosis through activating p38 and JNK MAPK mediated Nrf2/ARE pathway. Chem Biol Interact, 2019, 305: 54-65.
27.	Wang G, An Y, Zhang X, et al. Chondrocyte spheroids laden in GelMA/HAMA hybrid hydrogel for tissue-engineered cartilage with enhanced proliferation, better phenotype maintenance, and natural morphological structure. Gels, 2021, 7(4): 247. doi: 10.3390/gels7040247.
28.	Duchi S, Onofrillo C, O’Connell C, et al. Bioprinting stem cells in hydrogel for in situ surgical application: A case for articular cartilage. Methods Mol Biol, 2020, 2140: 145-157.
29.	Lam T, Dehne T, Krüger JP, et al. Photopolymerizable gelatin and hyaluronic acid for stereolithographic 3D bioprinting of tissue-engineered cartilage. J Biomed Mater Res B Appl Biomater, 2019, 107(8): 2649-2657.

1. Wu S, Wang L, Fang Y, et al. Advances in Encapsulation and Delivery Strategies for Islet Transplantation. Adv Healthc Mater, 2021, 10(20): e2100965. doi: 10.1002/adhm.202100965.
2. Xu Y, Tan M, Ma X, et al. Human mesenchymal stem cells-derived conditioned medium inhibits hypoxia-induced death of neonatal porcine islets by inducing autophagy. Xenotransplantation, 2020, 27(1): e12556. doi: 10.1111/xen.12556.
3. Tan Y, Nie W, Chen C, et al. Mesenchymal stem cells alleviate hypoxia-induced oxidative stress and enhance the pro-survival pathways in porcine islets. Exp Biol Med (Maywood), 2019, 244(9): 781-788.
4. Wang G, Wang Z, Sun N, et al. Reactive oxygen species-responsive silk sericin microcapsules used for antioxidative stress damage. Microsc Res Tech, 2021, 84(4): 618-626.
5. Keshtkar S, Kaviani M, Jabbarpour Z, et al. Significant reduction of apoptosis induced via hypoxia and oxidative stress in isolated human islet by resveratrol. Nutr Metab Cardiovasc Dis, 2020, 30(7): 1216-1226.
6. Coronel MM, Liang JP, Li Y, et al. Oxygen generating biomaterial improves the function and efficacy of beta cells within a macroencapsulation device. Biomaterials, 2019, 210: 1-11.
7. Lee EM, Jung JI, Alam Z, et al. Effect of an oxygen-generating scaffold on the viability and insulin secretion function of porcine neonatal pancreatic cell clusters. Xenotransplantation, 2018, 25(2): e12378. doi: 10.1111/xen.12378.
8. Liang JP, Accolla RP, Soundirarajan M, et al. Engineering a macroporous oxygen-generating scaffold for enhancing islet cell transplantation within an extrahepatic site. Acta Biomater, 2021, 130: 268-280.
9. Razavi M, Primavera R, Kevadiya BD, et al. A collagen based cryogel bioscaffold that generates oxygen for islet transplantation. Adv Funct Mater, 2020, 30(15): 1902463. doi: 10.1002/adfm.201902463.
10. Wang DW, Li SJ, Tan XY, et al. Engineering of stepwise-targeting chitosan oligosaccharide conjugate for the treatment of acute kidney injury. Carbohydr Polym, 2021, 256: 117556. doi: 10.1016/j.carbpol.2020.117556.
11. Zhang X, Liang S, Gao X, et al. Protective effect of chitosan oligosaccharide against hydrogen peroxide-mediated oxidative damage and cell apoptosis via activating Nrf2/ARE signaling pathway. Neurotox Res, 2021, 39(6): 1708-1720.
12. Velasco-Rodriguez B, Diaz-Vidal T, Rosales-Rivera LC, et al. Hybrid methacrylated gelatin and hyaluronic acid hydrogel scaffolds. Preparation and systematic characterization for prospective tissue engineering applications. Int J Mol Sci, 2021, 22(13): 6758. doi: 10.3390/ijms22136758.
13. Wang S, Guan S, Zhu Z, et al. Hyaluronic acid doped-poly (3, 4-ethylenedioxythiophene)/chitosan/gelatin (PEDOT-HA/Cs/Gel) porous conductive scaffold for nerve regeneration. Mater Sci Eng C Mater Biol Appl, 2017, 71: 308-316.
14. Mouser VHM, Levato R, Mensinga A, et al. Bio-ink development for three-dimensional bioprinting of hetero-cellular cartilage constructs. Connect Tissue Res, 2020, 61(2): 137-151.
15. Guan G, Qizhuang Lv None, Liu S, et al. 3D-bioprinted peptide coupling patches for wound healing. Mater Today Bio, 2021, 13: 100188. doi: 10.1016/j.mtbio.2021.100188.
16. Posniak S, Chung JHY, Liu X, et al. Bioprinting of chondrocyte stem cell co-cultures for auricular cartilage regeneration. ACS Omega, 2022, 7(7): 5908-5920.
17. Fan Y, Yue Z, Lucarelli E, et al. Hybrid printing using cellulose nanocrystals reinforced GelMA/HAMA hydrogels for improved structural integration. Adv Healthc Mater, 2020, 9(24): e2001410. doi: 10.1002/adhm.202001410.
18. Colli ML, Szymczak F, Eizirik DL. Molecular footprints of the immune assault on pancreatic beta cells in type 1 diabetes. Front Endocrinol (Lausanne), 2020, 11: 568446. doi: 10.3389/fendo.2020.568446.
19. Yoshihara E, O’Connor C, Gasser E, et al. Immune-evasive human islet-like organoids ameliorate diabetes. Nature, 2020, 586(7830): 606-611.
20. Nishime K, Miyagi-Shiohira C, Kuwae K, et al. Preservation of pancreas in the University of Wisconsin solution supplemented with AP39 reduces reactive oxygen species production and improves islet graft function. Am J Transplant, 2021, 21(8): 2698-2708.
21. Sayadi LR, Alexander M, Sorensen AM, et al. Micro/nanobubbles: Improving pancreatic islet cell survival for transplantation. Ann Plast Surg, 2019, 83(5): 583-588.
22. Lau J, Henriksnäs J, Svensson J, et al. Oxygenation of islets and its role in transplantation. Curr Opin Organ Transplant, 2009, 14(6): 688-693.
23. Zhang J, Sun X, Chen Y, et al. Preparation of 2, 6-diurea-chitosan oligosaccharide derivatives for efficient antifungal and antioxidant activities. Carbohydr Polym, 2020, 234: 115903. doi: 10.1016/j.carbpol.2020.115903.
24. Zhang J, Wang W, Mao XZ, et al. Chitopentaose protects HaCaT cells against H₂O₂-induced oxidative damage through modulating MAPKs and Nrf2/ARE signaling pathways. Journal of Functional Foods, 2020, 72: 10408. doi: 10.1016/j.jff.2020.104086.
25. Chang Q, Cai H, Wei L, et al. Chitosan oligosaccharides alleviate acute heat stress-induced oxidative damage by activating ERK1/2-mediated HO-1 and GSH-Px gene expression in breast muscle of broilers. Poult Sci, 2022, 101(1): 101515. doi: 10.1016/j.psj.2021.101515.
26. Zhang Y, Ahmad KA, Khan FU, et al. Chitosan oligosaccharides prevent doxorubicin-induced oxidative stress and cardiac apoptosis through activating p38 and JNK MAPK mediated Nrf2/ARE pathway. Chem Biol Interact, 2019, 305: 54-65.
27. Wang G, An Y, Zhang X, et al. Chondrocyte spheroids laden in GelMA/HAMA hybrid hydrogel for tissue-engineered cartilage with enhanced proliferation, better phenotype maintenance, and natural morphological structure. Gels, 2021, 7(4): 247. doi: 10.3390/gels7040247.
28. Duchi S, Onofrillo C, O’Connell C, et al. Bioprinting stem cells in hydrogel for in situ surgical application: A case for articular cartilage. Methods Mol Biol, 2020, 2140: 145-157.
29. Lam T, Dehne T, Krüger JP, et al. Photopolymerizable gelatin and hyaluronic acid for stereolithographic 3D bioprinting of tissue-engineered cartilage. J Biomed Mater Res B Appl Biomater, 2019, 107(8): 2649-2657.

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Islet biomimetic microenvironment constructed by chitosan oligosaccharide protects islets from hypoxia-induced damage by reducing intracellular reactive oxygen species

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