Acute Implantation of Aligned Hydrogel Tubes Supports Delayed Spinal Progenitor Implantation

Abstract

An important role of neural stem cell transplantation is repopulating neural and glial cells that actively promote repair following spinal cord injury (SCI). However, stem cell survival after transplantation is severely hampered by the inflammatory environment that arises after SCI. Biomaterials have a demonstrated history of managing post-SCI inflammation and can serve as a vehicle for stem cell delivery. In this study, we utilize macroporous polyethylene glycol (PEG) tubes, which were previously found to modulate the post-SCI microenvironment, to serve as a viable, soft substrate for injecting mouse embryonic day 14 (E14) spinal progenitors 2 weeks after tube implantation into a mouse SCI model. At 2 weeks after transplantation (4 weeks after injury), 4.3% of transplanted E14 spinal progenitors survived when transplanted directly into tubes compared to 0.7% when transplanted into the injury alone. Surviving E14 spinal progenitors exhibited a commitment to the neuronal lineage at 4 weeks post-injury, as assessed by both early and late phenotypic markers. Mice receiving tubes with E14 spinal progenitor transplantations had on average 21 ± 4 axons/mm 2 regenerated compared to 8 ± 1 axons/mm 2 for the injury only control, which corresponded with a significant increase in remyelination compared to the injury only control, while all conditions exhibited improved forelimb control 4 weeks after injury compared to the injury only. Collectively, we have demonstrated the feasibility of using PEG tubes to modify the implantation site and improve survival of transplanted E14 spinal progenitors.

IL‐10 lentivirus‐laden hydrogel tubes increase spinal progenitor survival and neuronal differentiation after spinal cord injury

“…Once implanted, the tubes create a more pro-regenerative environment, serving as a privileged injection site for E14 spinal progenitors 2 weeks after injury. Our previous work showed that transplanted cell survival is enhanced when injected into the tubes, compared with direct transplantation into the injury, demonstrating the feasibility of using this as a cell-biomaterial system for SCI (Ciciriello et al, 2020). While survival did improve with the integrated tubes, the stem cell-influenced results of that study were minimized as functional and histological assessments occurred only 2 weeks after cell transplantation (4 weeks after injury).…”

“…In this study, we expand the passive microenvironment modulation of our macroporous PEG tubes to actively modify the transplantation microenvironment through lentiviral-mediated delivery of IL-10. Previously, we demonstrated the short-term feasibility of temporally flexible transplantation of enhanced green fluorescent protein (EGFP + ) embryonic Day 14 (E14) spinal progenitors into integrated PEG tubes (Ciciriello et al, 2020). Expanding on this prior work, in our current study we will implant IL-10 encoding lentivirus-laden tubes into a cervical (C5) lateral hemisection, and 2 weeks later inject EGFP + E14 spinal progenitors directly into the tubes at the lesion epicenter.…”

“…Bromodeoxyuridine (BrdU) was used to identify proliferating cells in the first 7 days post-SCI, as described previously (Ciciriello et al, 2020). Intraperitoneal injections of 50 mg/kg BrdU (Roche) were pulsed every day for 7 days, allowing 7 additional days for unbound BrdU to wash out before exogenous spinal progenitors were implanted, as done by others (Ciciriello et al, 2020;Z. Yang et al, 2015).…”

“…Isolated spinal cords were flash frozen and then cryosectioned transversely (4 or 12-week tissue) in 12 μm sections. Immunohistochemistry analysis was performed using nine transverse sections evenly spaced across the rostral, middle, and caudal regions of the tubes and average across each animal allowing for comparisons between animals while considering regional differences (Ciciriello et al, 2020;Dumont et al, 2019). Samples were fixed, permeabilized (0.5% triton-X for 10 min) and/or prepared with BrdU staining (described above) as necessary, and incubated overnight at 4°C with primary antibodies.…”

“…Macroporous tubes, molded from PEG microspheres, function similar to rigid, polymeric scaffolds, but better match native tissue mechanical properties with the potential to conform to individual injury anatomy, making them advantageous. Biomaterial strategies for SCI can additionally support stem cell transplantation through ex vivo culture on a rigid scaffold (Dumont et al, 2018;Li et al, 2016;Mothe et al, 2013), suspension in hydrogel injections (Assunção-Silva et al, 2015;Cai et al, 2015;Marquardt & Heilshorn, 2016), or direct injection into an integrated implant (Ciciriello et al, 2020).…”