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Acellular peripheral allograft scaffolds can be fabricated using chemical extraction techniques, but methods for producing acellular scaffold derived from spinal cord tissue are not currently available.The present study demonstrated that chemical extraction using Triton X-100 and sodium deoxycholate could be used to completely remove the cells, axons and neural sheaths in spinal cord extracellular matrix-derived scaffolds. The matrix fibers were longitudinally arranged in a wave-like formation, and were connected by fiber junctions. Lattice-shaped fiber cages appeared and developed into bone trabecula-like changes. The natural structure of matrix fibers in the scaffolds was maintained; this helps to guide the differentiation and migration of implanted stem cells. Decellularized spinal cord extracellular matrix-derived scaffolds can provide an ideal substance for fabricating tissue-engineered spinal cord.