SummaryAimsAstroglial‐fibrotic scar formation following central nervous system injury can help repair blood‐brain barrier and seal the lesion, whereas it also represents a strong barrier for axonal regeneration. Intensive preclinical efforts have been made to eliminate/reduce the inhibitory part and, in the meantime, preserve the beneficial role of astroglial‐fibrotic scar.MethodsIn this study, we established an in vitro system, in which coculture of astrocytes and meningeal fibroblasts was treated with exogenous transforming growth factor‐β1 (TGF‐β1) to form astroglial‐fibrotic scar‐like cell clusters, and thereby evaluated the efficacy of RNAi targeting ephrin‐B2 in preventing scar formation from the very beginning. We further tested the effect of RNAi‐based mitigation of astroglial‐fibrotic scar on spinal axon outgrowth on a custom‐made microfluidic platform.ResultsWe found that siRNA targeting ephrin‐B2 significantly reduced both the number and the diameter of cell clusters induced by TGF‐β1 and diminished the expression of aggrecan and versican in the coculture, and allowed for significantly longer extension of outgrowing spinal cord axons into astroglial‐fibrotic scar as assessed on the microfluidic platform.ConclusionsThese results suggest that astroglial‐fibrotic scar formation and particularly the expression of aggrecan and versican could be mitigated by ephrin‐B2 specific siRNA, thus improving the microenvironment for spinal axon regeneration.