Gelatin collagen composite hydrogel and inducible factor regulate differentiation of rat bone marrow mesenchymal stem cells into hepatocyte-like cells

doi:10.12307/2022.249

Abstract

Abstract: BACKGROUND: In vitro artificial liver system has become a very effective and practical treatment for liver failure, but there are still some problems such as limited source of cells and limited function of cells.
OBJECTIVE: To optimize and screen gelatin collagen composite hydrogel that meet the needs of cell growth, the hydrogel was used as the basal cell growth material to explore the scheme of inducing hepatic differentiation of bone marrow mesenchymal stem cells.
METHODS: Gelatin collagen composite hydrogel with gelatin concentration of 50, 100, 150, 200 g/L was prepared by mixing gelatin solution and collagen solution at different volume ratios (1:1, 2:1, 4:1). The volume ratio of the solution and the gelatin concentration were selected for subsequent experiments by melting point, microstructure, water absorption and porosity. Rat bone marrow mesenchymal stem cells seeded in composite hydrogel were used as experimental group; rat bone marrow mesenchymal stem cells cultured alone were used as controls. Cell proliferation was detected by CCK-8 assay. The two-step induction regimen was used to induce the hepatic differentiation of two groups of rat bone marrow mesenchymal stem cells. The levels of albumin production and urea secretion in the supernatant were tested at 3, 7, 14, and 21 days after induced culture.
RESULTS AND CONCLUSION: (1) After testing the melting point, microscopic morphology, water absorption and porosity of the experimental groups with different ratios, the composite gel was most suitable for cell culture when the 150 g/L gelatin solution was mixed with collagen solution at 4:1 volume ratio. (2) CCK-8 assay showed that the proliferation activity of the experimental group was lower than that of the control group at 1, 3 and 5 days, and the difference between the two groups gradually decreased, and there was no difference at 10 days. (3) Experiment of hepatic differentiation showed that with the extension of induction time, the amount of albumin secretion in the two groups increased gradually, and the amount of albumin in the experimental group was higher than that in the control group at 14 and 21 days (P < 0.05). The amount of urea secretion in the two groups increased significantly within 3-7 days of induction, and stabilized after 7 days. The amount of urea secretion in the experimental group was slightly higher than that in the control group at 14 and 21 days, but the difference was not significant (P > 0.05). (4) The results showed that gelatin collagen composite hydrogel could induce rat bone marrow mesenchymal stem cells to differentiate into hepatocyte-like cells.

Key words: composite hydrogel, artificial liver, bone marrow mesenchymal stem cells, hepatocytes, induced differentiation, gelatin, collagen, EDC/NHS

CLC Number:

Yuan Yihang, Xu Menghan, Niu Xufeng. Gelatin collagen composite hydrogel and inducible factor regulate differentiation of rat bone marrow mesenchymal stem cells into hepatocyte-like cells[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(16): 2510-2515.

Figures/Tables 7

References

[1] DU T, NIU X, HOU S, et al. Apatite minerals derived from collagen phosphorylation modification induce the hierarchical intrafibrillar mineralization of collagen fibers. J Biomed Mater Res A. 2019;107(11): 2403-2413.
[2] NIU X, FAN R, TIAN F, et al. Calcium concentration dependent collagen mineralization. Mater Sci Eng C Mater Biol Appl. 2017;73:137-143.
[3] NIU X, FAN R, GUO X, et al. Shear-mediated orientational mineralization of bone apatite on collagen fibrils. J Mater Chem B. 2017;5(46):9141-9147.
[4] DU T, NIU X, LI Z, et al. Crosslinking induces high mineralization of apatite minerals on collagen fibers. Int J Biol Macromol. 2018;113: 450-457.
[5] DE BARTOLO L, SALERNO S, MORELLI S, et al. Long-term maintenance of human hepatocytes in oxygen-permeable membrane bioreactor. Biomaterials. 2006;27(27):4794-4803.
[6] HOFFMAN AS. Hydrogels for biomedical applications. Adv Drug Deliv Rev. 2012;64:18-23.
[7] 张林,孙磊,徐梦浛,等.可吸收胶原膜的体内免疫反应评价[J].北京航空航天大学学报,2018,44(4):879-886.
[8] 孙磊,袁源,牛睿,等.辐照和EO灭菌对SIS材料免疫原性的对比研究[J].北京航空航天大学学报,2020,46(12):2245-2252.
[9] JOSHI S, SINGH V. Gelatin–rosin gum complex nanoparticles: preparation, characterization and colon targeted delivery of 5-fluorouracil. Chem Pap. 2020;74(12):4241-4252.
[10] LIU J, WANG Q, SUN Y, et al. A core-shell structured collagen hydrogel microsphere with removable superparamagnetic alginate coating for cell coculture and rapid separation. Materials Letters. 2019;249:49-52.
[11] ZHAO X, WU H, GUO B, et al. Antibacterial anti-oxidant electroactive injectable hydrogel as self-healing wound dressing with hemostasis and adhesiveness for cutaneous wound healing. Biomaterials. 2017;122: 34-47.
[12] YOUNG S, WONG M, TABATA Y, et al. Gelatin as a delivery vehicle for the controlled release of bioactive molecules. J Control Release. 2005; 109(1-3):256-274.
[13] WANG J, TABATA Y, BI D, et al. Evaluation of gastric mucoadhesive properties of aminated gelatin microspheres. J Control Release. 2001; 73(2-3):223-231.
[14] KIM EH, KIM JW, HAN GD, et al. Biocompatible, drug-loaded anti-adhesion barrier using visible-light curable furfuryl gelatin derivative. Int J Biol Macromol. 2018;120:915-920.
[15] BUIE T, MCCUNE J, COSGRIFF-HERNANDEZ E. Gelatin Matrices for Growth Factor Sequestration. Trends Biotechnol. 2020;38(5):546-557.
[16] CHOWDHURY DK, MITRA AK. Kinetics of in vitro release of a model nucleoside deoxyuridine from crosslinked insoluble collagen and collagen–gelatin microspheres. Int J Pharm.1999;193(1):113-122.
[17] JIANG J, LIU A, CHEN C, et al. An efficient two-step preparation of photocrosslinked gelatin microspheres as cell carriers to support MC3T3-E1 cells osteogenic performance. Colloids and Surf B Biointerfaces. 2020;188:110798.
[18] PARK C, VO CLN, KANG T, et al. New method and characterization of self-assembled gelatin–oleic nanoparticles using a desolvation method via carbodiimide/N-hydroxysuccinimide (EDC/NHS) reaction. Eur J Pharm Biopharm. 2015;89:365-373.
[19] WISSINK MJB, BEERNINK R, PIEPER JS, et al. Immobilization of heparin to EDC/NHS-crosslinked collagen. Characterization and in vitro evaluation. Biomaterials. 2001;22(2):151-163.
[20] BHATIA SN, UNDERHILL GH, ZARET S, et al. Cell and tissue engineering for liver disease. Sci Transl Med. 2014;6(245):245sr2-245sr2.
[21] ZHANG J, ZHAO X, LIANG L, et al. A decade of progress in liver regenerative medicine. Biomaterials. 2018;157:161-176.
[22] HUCH M, DORRELL C, BOJ SF, et al. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature. 2013; 494(7436):247-250.
[23] LORVELLEC M, PELLEGATA AF, MAESTRI A, et al. An In Vitro Whole-Organ Liver Engineering for Testing of Genetic Therapies. iScience. 2020;23(12):101808.
[24] WU G, WU D, LO J, et al. A bioartificial liver support system integrated with a DLM/GelMA-based bioengineered whole liver for prevention of hepatic encephalopathy via enhanced ammonia reduction. Biomater Sci. 2020;8(10):2814-2824.
[25] MONA S , MOHAMMAD B , RASOUL G , et al. Human mesenchymal stem cells-conditioned medium improves diabetic wound healing mainly through modulating fibroblast behaviors. Arch Dermatol Res. 2020;312(5):325-336.
[26] GEMA VALLÉS, FÁTIMA BENSIAMAR, MAESTRO-PARAMIO L , et al. Influence of inflammatory conditions provided by macrophages on osteogenic ability of mesenchymal stem cells. Stem Cell Res Ther. 2020;11(1):1-15.
[27] YAO S, HE F, CAO Z, et al. Mesenchymal Stem Cell-Laden Hydrogel Microfibers for Promoting Nerve Fiber Regeneration in Long-Distance Spinal Cord Transection Injury. ACS Biomater Sci Eng. 2020;6(2):1165-1175.
[28] JAYARAMAYYA K, MAHALAXMI I, SUBRAMANIAM MD, et al. Immunomodulatory effect of mesenchymal stem cells and mesenchymal stem-cell-derived exosomes for COVID-19 treatment. BMB Rep. 2020;53(8):400-412.
[29] YAN L, SHA L, FENG Y, et al. Bone Marrow Mesenchymal Stem Cell treatment for end stage liver disease: A case report. Asian J of Surg. 2020;43(2):454-455.
[30] EISSA M, ELARABANY N, HYDER A. In vitro efficacy of liver microenvironment in bone marrow mesenchymal stem cell differentiation. In Vitro Cell Dev Biol Anim. 2020;56(4):341-348.
[31] MATTA A, DYM M ZK, HODA GERAMI BS, et al. Study comparing NTG-101, a growth factor-based therapy with mesenchymal stem cell (MSC) based treatment of degenerative disc disease (DDD) in pre-clinical rodent model. Spine J. 2020;20(9):S105-S106.
[32] NITTA S, KUSAKARI Y, YAMADA Y, et al. Conversion of mesenchymal stem cells into a canine hepatocyte-like cells by Foxa1 and Hnf4a. Regen Ther. 2020;14:165-176.
[33] CHRIST B, BRÜCKNER S, WINKLER S. The Therapeutic Promise of Mesenchymal Stem Cells for Liver Restoration. Trends Mol Med. 2015; 21(11):673-686.
[34] DUNCAN AW, DORRELL C, GROMPE M. Stem Cells and Liver Regeneration. Gastroenterology. 2009;137(2):466-481.
[35] SHI D, XIN J, LU Y, et al. Transcriptome Profiling Reveals Distinct Phenotype of Human Bone Marrow Mesenchymal Stem Cell-derived Hepatocyte-like cells. Int J Med Sci. 2020;17(2):263-273.
[36] LIN N, LIN J, BO L, et al. Differentiation of bone marrow-derived mesenchymal stem cells into hepatocyte-like cells in an alginate scaffold. Cell Prolif. 2010;43(5):427-434.
[37] LI J, TAO R, WU W, et al. 3D PLGA Scaffolds Improve Differentiation and Function of Bone Marrow Mesenchymal Stem Cell–Derived Hepatocytes. Stem Cells and Dev. 2010;19(9):1427-1436.
[38] JOSSÉ R, ANINAT C, GLAISE D, et al. Long-Term Functional Stability of Human HepaRG Hepatocytes and Use for Chronic Toxicity and Genotoxicity Studies. Drug Metab Dispos. 2008;36(6):1111-1118.
[39] SANCHO-BRU P, ROELANDT P, NARAIN N, et al. Directed differentiation of murine-induced pluripotent stem cells to functional hepatocyte-like cells. J Hepatol. 2011;54(1):98-107.
[40] TOSTÕES RM, LEITE SB, MIRANDA JP, et al. Perfusion of 3D encapsulated hepatocytes-A synergistic effect enhancing long-term functionality in bioreactors. Biotechnology and Bioengineering, Biotechnol Bioeng. 2011;108(1):41-49.
[41] UKAIRO O, KANCHAGAR C, MOORE A, et al. Long-Term Stability of Primary Rat Hepatocytes in Micropatterned Cocultures. J Biochem Mol Toxicol. 2013;27(3):204-212.
[42] SCHWARTZ RE, REYES M, KOODIE L, et al. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest. 2002;109(10):1291-1302.
[43] JANG M, NEUZIL P, VOLK T, et al. On-chip three-dimensional cell culture in phaseguides improves hepatocyte functionsin vitro. Biomicrofluidics. 2015;9(3):034113.
[44] ANIRUDHAN TS, MOHAN AM. Novel pH sensitive dual drug loaded-gelatin methacrylate/methacrylic acid hydrogel for the controlled release of antibiotics. Int J Biol Macromol. 2018;110:167-178.