## 3 DDM Preparation  
To prepare DDM, clinically extracted teeth were collected, and all the soft tissues and enamel were removed. Dentin was grinded into particles, cut into slices or blocks, or perforated to get macropores. The resulting dentin particles or blocks were demineralized. Hydrochloric acid (HCl) was most commonly used in demineralization. The demineralizing time ranged from 30 min to 24 h [38; 39; 40]. HNO\({}_{3}\) was also used in demineralization [17; 41; 42]. Dentin matrix was demineralized by 0.34 N HNO\({}_{3}\) for 30 mins, and the HNO\({}_{3}\)-DDM was grafted into critical-sized defect in the iliac crest of sheep. New bone formation was observed around DDM, and more bone volume was detected after 4 months than that at 2 months [41]. In another study, dentin matrix was treated with 2% HNO\({}_{3}\) for 10 min, then transplanted into the tooth extraction pocket, and bone augmentation was observed [42]. Ethylene diamine tetra acetic acid (EDTA) was also used in demineralization [16; 32; 43; 44]. The EDTA-demineralized dentin matrix is named treated dentin matrix (TDM) in some reports [32; 44]. Gradient EDTA (17%, 5%) was employed to demineralize dentin matrix, and dentin tubules were exposed [43]. The EDTA concentration and demineralization time were also varied in different reports [16; 32; 43; 44]. Besides the demineralized agent, the size of the particles is also related to the effect of demineralization. A previous study divided dentin particles into three groups: 200, 500, and 1000 \(\upmu\)m [17]. To get 70% demineralization of the dentin matrix (partially demineralized dentin matrix, PDDM), the 200, 500, 1000 \(\upmu\)m, respectively, took 5, 10, and 20 min of treatments [17]. The terms DDM and PDDM are interchangeable in almost all the reports, because DDM is always partially demineralized. The bone regeneration effect of dentin matrix was evaluated via implantation into the rat calvaria bone defects. The results showed that the completely demineralized dentin matrix (CDDM) was largely resorbed, the UDM was remained, and both the CDDM and UDM induced little bone formation (Figure 2A). In sharp contrast, PDDM induced much more new bone than other groups (Figure 2A). In addition, the newly-formed bone (n-Bone%) was quantitatively evaluated (Figure 2B). At 4 weeks, there was a significant difference of n-Bone % between the PDDM and the other groups in each particle size. At 8 weeks, there was a significant difference of the n-Bone % between the PDDM and the other groups in the 200 and 500 \(\upmu\)m particle size groups [17]. Few osteoblasts attached to the surface of UDM, while many osteoblasts attached and spread on the surface of DDM. In addition, UDM had limited growth factor exposure, while PDDM released several bioactive proteins. The demineralization opened the dentin tubules which served as channels for the release of these bioactive proteins when implanted in bone defects. Although CDDM also had opened tubules, the bioactive proteins may have already been released from dentin matrix during the process of demineralization, and therefore were lost before implantation. It was possible that PDDM contained more growth factors than CDDM. Furthermore, the n-Bone% of the 1000 \(\upmu\)m group showed more bone regeneration than the smaller sizes; this was true in all groups of UDD, PDDM and CDDM (Figure 3B). Smaller particles had more resorbability, and they may have been resorbed in vivo before the initiation of new bone formation. Regarding the optimal particle size of DDM, there was no agreement in the literature, which varied in defect sites and graft materials, warranting further and more systematic investigations [17; 42; 45]. Besides DDM particles, DDM blocks were also investigated [14; 18; 23; 41; 46]. In one study, DDM blocks with macropores were implanted into critical-sized iliac defects and new bone formation around the DDM had good union with the native bone [41].