(J-377) 3D bioprinting neural scaffolds with neurogenic core-shell polydopamine- poly(lactic-co-glycolic acid) microparticles for improving neural stem cell growth and maturation

Introduction:: Constrained cellular expansion of implanted cells within neural tissue defects is a crucial obstacle to the clinical success of engineered neural grafts. This issue is thought to be a result of two major factors: (1) mechanical mismatch between the implanted cells, the graft material, and the tissue defect site, and (2) an insufficient availability of pro-neurogenic factors at the graft-to-defect interface to facilitate implanted cell survival and expansion. To address these critical issues, we 3D bioprinted neural scaffolds using a novel bioink formulation composed of a functionalized derivative of sodium alginate, here referred to as alginate methacrylate (AlgMA), polyethylene glycol diacrylate (PEGDA) and incorporated core-shell polylactic-co-glycolic acid (PLGA) - polydopamine (PDA) microparticles loaded with the neurogenic morphogen folic acid (FA). After our scaffolds were printed, they were seeded with neural stem cells (NSCs) and were cultured continuously over a 2-week period in vitro to assess the capacity of the microparticle-loaded AlgMA scaffolds to support NSC growth and maturation.

Materials and Methods:: The FA-loaded PLGA core of the microparticles was created by combining a 1:1 molar ratio of folic acid and PLGA in acetone and then extrusding the FA-PLGA prepolymer solution through an applied 8-16 kV potential difference. The assembled FA-PLGA microparticles were then coated with PDA by suspending the microparticles in a 5% (w/v) dopamine hydrochloride in PBS at pH = 10. PDA self-assembled in a solid layer onto the exterior of the electrosprayed FA-PLGA microparticles. The FA-PLGA loaded PDA solid:solid microparticles (FA-PLGA@PDA) were then harvested for further encapsulation within the 3D printed AlgMa scaffolds. Next, AlgMA was synthesized through the addition of methacrylate groups to the hydroxyl groups of sodium alginate. The bioink formulation used to fabricate the neural scaffolds was composed of 5% AlgMA, 5% (w/v) PEGDA, 25-100 µg • mL FA-PLGA@PDA microparticles, and 1% (w/v) I-2959 photoinitiator. The complete bioink was crosslinked into a complex 3D architecture using a movable stereolithography (SLA) laser mounted printhead. After printing, the scaffolds were washed, seeded with NSCs (5.0 x 10⁵ cells/scaffold), and continuously cultured for 7 days. The rate of proliferation of the NSCs and the cytological coverage of the scaffolds were determined using a cell counting kit (CCK-8) assay and live / Dead staining, respectively. After 3 days of culture, 10^-6 M all-trans­-retinoic acid (ATRA) was introduced to the culturing media to induce the neurogenic differentiation of the NSCs. The cytological morphology of developing neural cells and their degree of maturation were also assessed over the course of 7 days using F-actin staining, immunocytochemical staining approaches, respectively.