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Dentin is a suitable scaffold for tooth tissue engineering and can provide the appropriate combinations and concentrations of bioactive proteins to accommodate odontogenic cells. Freeze drying is a possible strategy to treat dentin because it ensures long-term stability of biopharmaceutical products and can increase the stability of dentin for improved distribution and storage. We hypothesized that freeze-dried dentin matrix (FDDM) constitutes a novel bioinstructive scaffold for tooth tissue engineering.This study used a modified technique to prepare an FDDM that preserved the mechanical and biological properties of dentin. The compression resistance and microhardness of FDDM were determined. Similarly, various biological characteristics, including cell morphology, cell proliferation, collagen secretion, alkaline phosphatase activity, and gene and protein expression, were investigated. To assess the inductive capacity of FDDM in vivo, FDDM and human dental pulp stem cell (DPSC) sheets were subcutaneously implanted in the dorsal pockets of nude mice. At 8 weeks postimplantation, the transplants were removed and histologically studied.The results show that FDDM has mechanical and biological characteristics similar to those of dentin (P > .05). DPSCs cultured on FDDM and dentin showed superior attachment, growth, viability, and collagen secretion capacity but decreased mineral capability compared with DPSCs cultured with alpha minimum essential medium or hydroxyapatite (P < .05). The histologic results show that FDDM, which is similar to dentin, supported dentin/pulplike tissue regeneration in vivo as shown by the related expression levels of dentin markers, such as dentin sialoprotein and alkaline phosphatase.These results suggest that FDDM constitutes a novel superior scaffold for tooth tissue engineering.