Angiogenic potential of endothelial and tumor cells seeded on gelatin–based hydrogels in response to electrical stimulations

Abstract

Angiogenesis is one of the key processes during development, wound healing and tumor formation. Prerequisite for its existence is the presence of endogenous electrical fields (EFs) generated by active ion transport across polarized epithelia and endothelia, and appearance of the transcellular potentials. During angiogenesis cellular factor as endothelial growth factor (VEGF), synthesis of adhesive proteins and membrane metalloproteinases (MMPs) govern the angiogenic response to different external stimuli as biomaterials interactions and/or exogenous EF. Gelatin-based hydrogels with elasticities comparable to human tissues have shown to influence cell behavior as well as cell attachment, protein synthesis, VEGF and MMP’s production after the application of EF. Gelatin-based matrices with 3 (G10_LNCO3), 5 (G10_LNCO5), and 8 (G10_LNCO8) fold excess of isocyanate groups per mol of amine groups present in gelatin were used. Human umbilical endothelial cells (HUVEC) (Lonza Basel, Switzerland) and highly invasive breast cancer MDA-MB-231 cells (ATCC®HTB-26^TM) were used. For an estimation of the amount of VEGF released from cells a commercially available VEGF ELISA (Thermo Fisher Scientific, Germany) kit was used. Fibronectin (FN) enzyme immunoassay (EIA) was used to analyze the secreted amount of FN by cells seeded on the materials. Secreted MMPs were analyzed by zymography. Gelatin-based hydrogels attracted HUVEC adhesion and diminished the adhesion of MDA-MB-231 cells. The applied direct current (DC) EF induced an almost 5–fold increase in VEGF production by HUVEC seeded on gelatin-based hydrogels, while in contrast, the applied EF decreased the production of VEGF by cancer cells. FN synthesis was elevated in HUVEC cells seeded on gelatin-based materials in comparison to FN synthesis by cancer cells. HUVEC seeded on gelatin hydrogels showed an expression mainly of MMP-2. The application of EF increased the production of MMP-2 in HUVEC seeded on gelatin materials. In contrast, for MDA-MB-231 the production of MMPs on gelatin materials was lower compared to control materials. With the application of EF the levels of MMP-9 decreased but MMP-2 expression raised significantly for gelatin materials. Overall, the results showed that studied gelatin materials suppressed attachment of cancerous cells, as well as suppressed their angiogenic potential revealed by decreased VEGF and MMP production. Thus, this study approved gelatin-based hydrogels with proper elasticity characteristics and different degradation behavior as useful matrices for use in vascular tissue regeneration or in restriction of tumor growth after tumor resection.