Demineralized dentin matrix (DDM) granules with excellent biocompatibility were easily prepared using unnecessary human teeth by a new cooling-pulverizing and demineralizing technique. Extracted human teeth were pulverized together with saline ice at 12,000 rpm-rotation number of a ZrO2 blade for 30 s in a ZrO2 vessel. The pulverized granules exhibited the particle size distribution of 0.5-2 mm that was efficient for regeneration of alveolar bone. The (Ca/P) ratios of the granules were 1.60-1.66, which were close to the stoichiometric value of 1.67 for standard hydroxyapatite (HAp). Small amounts of Na+ and Mg2+ ions present at less than 1% were detected. The pulverized granules were dissolved with stirring under 500 rpm for 10-60 min in 2.0%-HNO3 solutions to obtain partial or complete DDM granules. As the dissolution time increased, crystallinity of HAp phase lowered and asperity on surfaces of the granules became outstanding due to elution of mineral components. At the dissolution of 60 min, the pulverizing granules were completely demineralized and the weight decreased to about one-fifth. To improve surface activity of the DDM granules without denaturation of bone growth factors, the DDM granules were soaked at 309.5 K and pH 7.40 in a simulated body fluid (SBF). HAp microcrystals were gradually precipitated on surfaces of the DDM granules with increasing the soaking time. Different morphology of the precipitates was observed, depending on the demineralization situation of the pulverized granules. For the DDM with low dissolution efficiency of 42%, porous bone-like apatites at 24 h after the soaking and fiber-oriented aggregates at 144 h were recognized. The bioactive DDM granules were implanted into the subcutaneous tissues of the back region of rats. At 4 weeks after the implantation, bio-absorption by comparatively small amounts of multi-giant cells was recognized around the surface layers of DDM granules. (c) 2012 Elsevier B.V. All rights reserved.