Abstract
The overall goal for this study was to develop a novel cell-free, off-the-shelf, and easy-to-use regenerative treatment for spina bifida (SB). Every day in the United States, four children are born with SB. SB is a devastating congenital disorder with high healthcare costs—approximately 13 times greater than the cost for otherwise healthy children. The most severe form of SB is myelomeningocele (MMC), which results from the incomplete closure of the neural tube during fetal development, leaving the spinal cord exposed to mechanical and chemical trauma resulting in lifelong paralysis, cognitive disabilities due to hindbrain herniation, musculoskeletal deformities, and bowel and bladder dysfunction. We proposed to develop a bioengineered scaffold that will not only protect neurons of the spinal cord, but also regenerate the missing vertebral arch of the spine to achieve long-term durability of motor function. This study focused specifically on developing a novel collagen hydrogel loaded with extracellular vesicles (EVs) isolated from placenta-derived mesenchymal stromal/stem cells (PMSC-EVs). First, we optimized EV isolation and storage methods such that a large quantity of EVs would be available to characterize and modify for studying their release from hydrogel and their neuroprotective functions. The concentration, size, and zeta potential measurements obtained via nanoparticle tracking analysis (NTA) suggested that EV isolation using exosome detection via the ultrafast-isolation system (EXODUS) and preserving EVs in standard PBS at -80°C would provide the highest degree of predictability, counts, and concentrations for further experimentation. The presence of protein content was also confirmed in isolated EV suspension through microbial assays—a promising indication that our isolation techniques were successful and that these EVs might have neuroprotective properties through a protein-based payload. We also characterized classic EV surface proteins, tetraspanins CD63, CD81, and CD9, through super-resolution microscopy, further indicating successful EV isolation. Concentration, zeta potential, and tetraspanin staining results suggested that EVs from one of the PMSC donor lines, donor line 495, might be superior in these aspects compared to EVs from donors 892 and 488. After obtaining EVs, we characterized their release from type I collagen hydrogel and showed that EV release appears to increase rapidly after two days of incubation and decreases to a steady rate for at least 19 additional days. As an alternative method for characterizing EV release rate, we optimized protocols for quantifying fluorescence units of stained EVs rather than their particle number. Lastly, neuroprotection of EVs was demonstrated through a neuroprotection assay using the SH-SY5Y neuroblastoma cell line. Treatment of SH-SY5Y cells with EVs and collagen significantly increased neural circuitry lengths, branching points, and segmentation compared to PBS and collagen treatments alone, demonstrating that PBS is nontoxic and is an appropriate storage buffer for EVs. This same experiment also showed that collagen is nontoxic and likely safe to use in future in vivo studies. Once this innovative PMSC-EV sustained release system is established, we will apply the EV-loaded collagen hydrogel to in vivo animal models of MMC to further develop this cell-free, off-the-shelf, and easy-to-apply scaffold for a comprehensive fetal treatment of MMC.