Angiogenic Biomaterials for Stem Cell Transplantation

Introduction: Traumatic spinal cord injury (SCI) stems from compression, dislocation, or penetration to the spine. SCIs cause chronic inflammation at the site and damage to nerve tracts that relay signals up and down the body, disrupting sensory, motor, and autonomic functions. A possible therapeutic for SCI patients is the use of stem cell transplantations for immunomodulation, regeneration, and pain reduction. Stem cells differentiated into chromaffin cells or GABAergic neurons have been used to mitigate pain associated with SCI (Jergova et al., 2016; Chen et al., 2017), but their localization after implantation has remained a challenge. Due to the inflammatory nature of SCI, the main concern with the success of cell therapy is poor survival post-transplantation and maintaining cells within the transplant site. By designing injectable polyethylene glycol (PEG) hydrogel microspheres, we can reduce inflammation while localizing cells at the site of injury (Ciciriello et al., 2020). Another crucial component in spinal cord regeneration is the need for vascularization to promote functional recovery and create a supportive microenvironment. The vascular network plays a vital role in facilitating nutrient and oxygen supply for increased cell proliferation, modulating the immune response, and providing structural support for axonal regeneration. Using the addition of an angiogenic moiety, we have developed a biomaterial that supports pain alleviating cell populations while also promoting angiogenesis.

Materials and

Methods: PEG microspheres were fabricated using an 8-arm PEG maleimide and a YKND crosslinker by a water/oil emulsion method (Dumont, 2019). Vascular Endothelial Growth Factor human 165 (VEGF165) was integrated into the biomaterial in two distinct methods. VEGF165 was directly incorporated into the polymer solution during microsphere fabrication. Alternatively, polylactic-co-glycolic acid (PLGA)-PEG-heparin nanoparticles were fabricated, incubated with VEGF165, and incorporated during microsphere crosslinking. PEG, VEGF-PEG, and VEGF-sequestering nanoparticle-loaded PEG microspheres were characterized using rheological and diameter measurements to compare their mechanical and physical properties. Microspheres were degraded in trypsin or saline over 10 days. Both the supernatant and degraded gel were collected to assess VEGF loading efficiency and release kinetics. In vitro live/dead viability assays were performed on human induced pluripotent stem cell (hiPSC) derived chromaffin cells and GABAergic neural progenitor cells seeded on PEG, VEGF-PEG, and VEGF-sequestering nanoparticle-loaded microspheres. The phenotype, metabolism, and functional output of the stem cell populations within each biomaterial will also be tested in vitro, along with endothelial cell co-cultures to assess angiogenesis. All conditions were compared using a two-way ANOVA test to determine significance.

Results, Conclusions, and Discussions: No statistically significant differences (p>0.05) in diameter (Figure 1A) or mechanical properties were reported between the PEG, VEGF-PEG, and VEGF-sequestering nanoparticle-loaded microspheres. These results indicate that the addition of the growth factor did not significantly alter the measured properties of the microspheres. An average of 2.159 ng of the added 20 ng of VEGF was loaded into the microspheres. After altering the fabrication method to include an incubation with heparin nanoparticles, we still saw a similar loading of only approximately 2.201 ng in the gel. The release kinetics showed us that approximately 50% of the loaded VEGF concentration was released by day 7 (Figure 1B). A slow release of the growth factor was also displayed. The lack of a burst release of VEGF is crucial because it reduces the chance of tumorigenesis occurring. Our next goal is to continue optimizing the VEGF microsphere fabrication process to reach a loading efficiency of at least 50%. We are currently investigating alternative steps in the fabrication process in which the VEGF-bound nanoparticles can be integrated to prevent the growth factor from being released before loading into the microspheres. The in vitro live/dead viability assays performed on chromaffin stem cells and GABA cells seeded on PEG microspheres showed approximately 90% live cells (Figure 1C), suggesting that the biomaterial maintains viability over different stem cell populations. The testing of phenotype and function of the different stem cell populations within the VEGF-PEG microspheres is currently underway. Additionally, we are investigating the formation of new vessels in vitro using endothelial cells. This study shows the potential of injectable PEG hydrogel microspheres combined with VEGF for stem cell transplantations to promote both vascularization at the site and pain alleviation after SCI. By engineering an effective hydrogel matrix, we are increasing the survival and localization of stem cells injected into the inflammatory injury site. With the addition of an angiogenic moiety that does not affect the properties of the biomaterial, we create an even more desirable environment for wound repair and for the differentiation and function of these pain alleviating stem cell populations.

Acknowledgements (Optional): I would like to thank the NeuSTEM Lab of the University of Miami and the Miami Project to Cure Paralysis for their support, mentorship, guidance, and for providing materials/funding for this research. I would also like to thank the Nielsen Foundation’s SCIRT grant #890307 awarded to J. Sagen for supporting this work.