Introduction: Many forms of injectable hydrogels are explored as a way to deliver stem cells for regenerative purposes. One of the bottleneck is attrition of injected cells prior to engraftment. For complete engraftment of injected stem cells, hydrogels that support cell survival and stability during the regenerative process are essential. In order to improve cell survival, we have developed chitosan-gelatin based hydrogels containing nutrients and with mechanical properties matching many of the soft tissues. We reported on the improvements in cell survival using human fibroblasts[1]. We hypothesized that increasing the available oxygen will is essential to further improve the cell survival. To test the hypothesis, we incorporated calcium peroxide which releases oxygen slowly to the hydrogel preparation. Also, we incorporated laminin to use this configuration for cardiac tissue regeneration, and tested survival of human adipocyte stem cells. We have performed different mechanical tests, such as cyclical tension tests, that mimic the cardiac tissue environment. Also, cell viability was evaluated.
Methods: Gelatin, chitosan, powder of growth media that supports adipocyte stem cells, transglutaminase, laminin, calcium peroxide, doxycycline encapsulated in PLGA nanoparticles, and β-glycerophosphate were used to create hydrogels. Sterile solutions were prepared, samples were subjected to compressive properties, stress-relaxation properties, and cyclical testing (at 1Hz) using the INSTRON 5542 at 37°C in hydrated conditions. Fresh porcine pericardium was used as the control to compare the mechanical properties. Oxygen release profiles were recorded by placing oxygen microelectrode and Chart™ 5 for Windows via the Powerlab/4SP System. To investigate the viability of the hydrogels in a 7 day culture, hydrogels were seeded with 500,000 human adipocyte stem cells (from Life Technologies) and incubated at 37°C with 5% CO2. Cell viability was assessed using CFDA SE staining[2], and distribution via histology.
Results and Discussion: Presence of growth medium, transglutaminase, calcium peroxide, laminin, and PLGA nanoparticles did not hinder the gelation characteristics. Further, addition of low amount of transglutaminase improved mechanical stability after cell seeding. The optimum types of chitosan and gelatin and the concentration of growth medium were determined. Increasing gelatin content significantly improved the compressive properties due to post cross-linking with TG. Performed stress relaxation experiments showed that hydrogels behave identical to porcine myocardium as strain hardening materials. We also performed cyclical tests to mimic the cardiac cycle at a frequency of 72 beats per minute for 30 min using 10% and 30% strains. Except for the first cycle, the rest of the cycles were identical. Oxygen release profile correlated to the amount of the Calcium peroxide. For gels with calcium peroxide and laminin we see that at the end of a 7-day experiment, 70% of viable cells were retrieved from the hdyrogel. Histology analyses showed uniform distribution of cells.
Conclusions: Chitosan-gelatin hydrogels that release oxygen is prepared with mechanical properties mimicking cardiac tissue. Addition of laminin does not alter the gelation characteristics. Viability of adipocyte stem cells improved[1].
Christian Tormos is supported by Ronald E. McNair graduate fellowship program; We like to thank Edward Jouallian Endowment
References:
[1] Tormos CM, Abraham C, Madihally SV. Improving the Stability of Chitosan-Gelatin Based Hydrogels for Cell Delivery Using Transglutaminase and Controlled Release of Doxycycline. Drug Delivery and Translational Research. DOI:10.1007/s13346-015-0258-7, 2015
[2] Iyer P, Walker KJ, Madihally SV. Increased Matrix Synthesis by Fibroblasts with Decreased Proliferation on Synthetic Porous Structures. Biotechnology/Bioengineering. 109(5): 1314-1325. 2012.