Regulation Of Neural Stem Cells By Silk Nanofiber Hydrogels With Tunable Modulus

Objective:Reconstruction of damaged nerves remains a significant unmet challenge inclinical medicine. Neural stem cells play an important role in both neural developmentand neural regeneration. Transplantation of NSCs has been shown as a promisingapproach in various neurological disorders such as spinal cord injury, brain trauma,lzheimer’s disease and5arkinson disease. Controlling the differentiation,proliferation, migration and apoptosis of NSCs is the crucial factor to consider in stemcell transplantation. A multitude of biomaterials have been developed as substrates orscaffolds to regulate the behavior of NSCs by fabrication with control ofmicrostructure, surface charge, biomechanical properties, as well as composition, tosimulate the microenvironments of nerve tissues. Silks spun by silkworms and spidersrepresent some of the strongest and toughest biological materials. The ability to controlthe release of growth factors from silk, the control of silk materials morphology, andthe biocompatibility of silk make this protein a unique materials platform forcontrolling stem cell fate and tissue regeneration. However, whether the bioactive silknanofibers with distinct tunable mechanical properties have different regulation ofNSCs remains unclear. The goal of the present study was to generate bioactive silknanofibers with tunable mechanical properties by controlling the self-assembly of silkin aqueous solution, and to test the ability of these systems to regulate thedifferentiation of NSCs.Methods:Silk nanofiber hydrogels with distinct tunable mechanical properties wereprovided by5rof.6iang1v’s lab (8oochow university) as indicated: silk nanofiberhydrogel (SN-H， control), water annealed silk nanofiber hydrogel (WA-SN-H),50%methanol-annealed silk nanofiber hydroge (MA50-SN-H),80%methanol-annealed silknanofiber hydrogel (MA80-SN-H). NPCs were isolated from E14C57/BL6J mice telencephalon and cultured in presence of distinct silk nanofibers. BrdU incorporationanalysis was performed to examine the proliferation of NSCs. Differentiation andmigration anlysis was performed to exmian the differentiation and migration of NSCs.To examine apoptosis of NSCs, Caspase-3and DAPI was stained and the proportion ofCaspase-3+were analyzed.Results:1) All four different silk nanofiber hydrogels do not affect the proliferation ofNSCs.2) Compared to the untreated nanofibers, NSCs cultured on water annealed,50%methanol annealed and80%methanol annealed nanofibers displayed reduced numbersof Caspase3+cells.3) NSCs cultured on water annealed,50%methanol annealed and80%methanolannealed silk nanofiber hydrogels differentiated into more neurons compared to on theuntreated silk nanofiber hydrogels.4) More GFAP+cells were differentiated from the NSCs on the water annealedand50%methanol annealed hydrogels compared to on the untreated hydrogels. Incontrast, the NSCs cultured on80%methanol annealed hydrogels showed decreasednumbers of GFAP+cells compared to on the untreated hydrogels.5) Compared to untreated nanofiber hydrogels, the neurospheres on the waterannealed and80%methanol annealed nanofibers had significantly more migratingcells. In contrast, no significant differences in the numbers of migrating cells wereobserved from the neurospheres on untreated and50%methanol annealed nanofibers.Conclusion:Silk nanofiber hydrogels with distinct tunable mechanical properties significantlyaffect apoptois, proliferation, differentiation of migration of NSCs.Compared with untreated nanofiber hydrogels,1) Water annealed,50%methanolannealed and80%methanol annealed silk nanofiber hydrogels inibits apoptosis ofNSCs;2) Water annealed,50%methanol annealed and80%methanol annealed silknanofiber hydrogels promote neuronal differentiation of NSCs;3) Water annealed and50%methanol annealed hydrogels promotes, but80%methanol annealed hydrogelsinhibits astroglial differentiation of NSCs;4) Water annealed and80%methanolannealed nanofibers, but not50%methanol annealed nanofibers promote migration of NSCs.