2023, Vol. 27 ›› Issue (12): 1824-1831
Biomimetic microenvironment constructed from gelatin methacrylamide/platelet-rich plasma hydrogel promotes the function of insulinoma cell line MIN6 in mice
Zhu Biwen1, 2, 3, Wang Dongzhi1, 2, 3, Wu Di1, 2, 3, Gong Tiancheng2, 3, Pan Haopeng2, Lu Yuhua1, 2, 3, Guo Yibing2, 3, Wang Zhiwei1, 2, 3, Huang Yan1, 2, 3
1First Department of Hepatic and Splenic Surgery, 2Research Center of Clinical Medicine, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu Province, China; 3Medical School of Nantong University, Nantong 226001, Jiangsu Province, China
Abstract: BACKGROUND: In the field of pancreatic tissue engineering, constructing biomimetic microenvironment to promote the survival and functional exertion of insulin-secreting cells remains difficult point and hot problem.
OBJECTIVE: To construct a biomimetic microenvironment based on gelatin methacrylamide/platelet-rich plasma hydrogel to promote the survival and cellular functional of MIN6.
METHODS: 10%, 30% and 50% platelet-rich plasmas were mixed with 50 g/L final concentration of gelatin methacrylamide, gelation by the activation of Ca2+/thrombin and ultraviolet irradiation (abbreviated as G+10P, G+30P, G+50P). Simultaneously, pure gelatin methacrylamide hydrogel was prepared (denoted as G). The porosity, Young’s modulus, swelling properties and rheological behavior of hydrogels of the four groups were tested. MIN6 cells were seeded on the gel surface, and cell morphology and proliferation were detected, followed by qRT-PCR, immunofluorescence and release assays of insulin.
RESULTS AND CONCLUSION: (1) The porosity of the composite-component hydrogel was smaller than that of the single-component hydrogel, and the Young's modulus was higher than that of the single-component hydrogel. Furthermore, the porosity and Young's modulus of the composite hydrogel decreased with the increase of the platelet-rich plasma concentration. The swelling rate of the G+30P and G+50P groups was lower than that of the G and G+10P groups
(P < 0.05). The storage modulus and dissipation modulus of the composite hydrogels were higher than those of the single-component hydrogels (P < 0.05). (2) Under the light microscope, the cells on the surface of the monocomponent hydrogel were clumpy and scattered; the cells on the surface of the composite-component hydrogel were clumps, but grew faster and the cell clusters were tightly connected. Live-dead staining showed that the hydrogels in each group could promote cell survival; the number of dead cells in G+30P and G+50P groups was significantly less than that in G+10P and G groups. The CCK-8 assay displayed that the composite-component hydrogel had a stronger promoting effect on cell proliferation than the monocomponent hydrogel. Moreover, with the increase of platelet-rich plasma concentration, the promoting effect on proliferation was more obvious. (3) qRT-PCR demonstrated that compared with the monocomponent hydrogel, the composite-component hydrogel could significantly up-regulate the mRNA expression levels of islet-duodenum homeobox 1, insulin, and glucokinase; among them, the G+30P group was the most obvious. Immunofluorescence and insulin release assays exhibited that compared with monocomponent hydrogels, composite-component hydrogels could promote insulin protein expression and insulin release. (4) The results confirm that the gelatin methacrylamide/platelet-rich plasma hydrogel can be used to simulate the microenvironment of insulin-secreting cells and can significantly improve their survivals and functions.
Key words: gelatin methacrylamide, platelet-rich plasma, hydrogel, insulin, mouse insulinoma cells, biomimetic microenvironment, diabetes mellitus