Human Mesenchymal stromal cells (hMSC) are appealing candidates for regenerative medicine applications. However, upon implantation, they encounter an ischemic microenvironment depleted of oxygen and nutrients responsible for their massive death post-transplantation, a major roadblock to successful clinical therapies. To date, various approaches have been proposed to address this issue, albeit with limited clinical success. We hereby propose a paradigm shift for enhancing hMSC survival by designing, developing, and testing an enzyme-controlled, nutritive hydrogel with an inbuilt glucose delivery system for the first time. This novel hydrogel is composed of fibrin, wheat starch (a glucose polymer), and amyloglucosidase (AMG), which hydrolyze glucose from starch.
In vitro: Glucose concentration at the core of hydrogels was determined using a custom-made glucose electrode biosensor. hMSC survival was assessed by cytometry after releasing cells from cell-loaded hydrogels exposed at 0.1% oxygen for up to day 14. The chemotactic potential of hMSCs towards hMSC and Human Umbilical Vein Endothelial Cells (HUVEC) was assessed by collecting conditioned Media (CM) from these hMSC-loaded hydrogels and evaluating migration in Boyden chambers. Moreover, chemotactic and angiogenic cytokines in CM were quantified using Luminex®.
In vivo: fluorescent-labelled AMG leakage from cell-free fibrin hydrogels was monitored using Xenogen live imaging until day 14 after ectopic implantation in nude mice. Luciferase-labelled hMSC survival within both fibrin/starch/AMG and fibrin hydrogels was assessed in an ectopic nude mice model by bioluminescence imaging until day 14. New blood vessel formation in the hydrogel vicinity was determined by µCT scanner, using a radiopaque agent Microfil® perfused within blood vessels at day 7 and 14.
In vitro fibrin/starch/AMG hydrogels released glucose at physiological concentrations and exhibited a 95 fold increase in hMSC survival compared to fibrin hydrogels after 14 days. CM collected from hMSC loaded fibrin/starch/AMG hydrogels showed (i) a 9- and a 4-fold increase in chemotactic potential towards hMSCs and HUVECs and (ii) a statistically significant rise in most but not all chemotactic and angiogenic cytokines compared to hMSC loaded fibrin hydrogels. In vivo glucose concentration within cell-free fibrin/starch/AMG hydrogels was within physiological ranges at days 7 and 14. Fluorescence monitoring revealed that AMG had completely disappeared within 7 days. hMSCs viability (measured by bioluminescent signal intensity compared to day 1) was 76.4% within fibrin/starch/AMG hydrogels and 22.1% within fibrin hydrogels at day 7. The hMSCs viability decreased drastically between days 7 and 14, corroborating the AMG time course. Last but not least, the formation of new blood vessels in the hydrogel vicinity exhibited a 4-fold increase when using fibrin/starch/AMG hydrogels compared to fibrin hydrogels at day 21.
We hereby establish the proof of concept that a fibrin/starch/AMG hydrogel provides glucose to hMSCs and maintain their viability and angioinductive potential in vitro and in vivo. AMG sustained delivery is required to extend the survival time of the transplanted hMSCs."