**2. Bone induction of human dentin**

In 1967, bone-inducing property in rabbit dentin was confirmed in the intramuscular pockets (Yeoman &Urist, 1967; Bang & Urist, 1967), after the discovery of bone induction by rabbit demineralized bone matrix (DBM) in 1965 (Urist, 1965). The rabbit studies reported that completely demineralized dentin matrix (DDM) induced bone at 4 weeks, while nondemineralized dentin (so-called, calcified dentin) induced bone at 8-12 weeks after implantation (Yeoman & Urist, 1967). In our study, human DDM including small patches of cementum derived from wisdom teeth, and human DBM derived from adult femur induced bone and cartilage independently in the subcutaneous tissues at 4 weeks (Murata et al., 2010a). The delayed inductive properties of the calcified dentin and bone may be related to the inhibition of BMP-release by the apatite crystals. Highly calcified tissues such as cortical bone and dentin are not earlier in osteoinduction and bone fornmation than spongy bone, decalcified bone (DBM), and decalcified dentin (DDM) (Huggins et al., 1970).

In this chapter, human dentin will be introduced as novel biomaterial and also as carrier matrix of the recombinant human bone morphogenetic protein-2 (BMP-2) delivery for bone

engineering.

a: whole appearance of molar.

Fig. 1. Human wisdom tooth

c: crushed tooth granules.

b: divided tooth (E; enamel, D; dentin, P; pulp).

**2. Bone induction of human dentin** 

d: SEM photograph of calcified dentin after crushing and washing. Note; dentinal tubes

decalcified bone (DBM), and decalcified dentin (DDM) (Huggins et al., 1970).

In 1967, bone-inducing property in rabbit dentin was confirmed in the intramuscular pockets (Yeoman &Urist, 1967; Bang & Urist, 1967), after the discovery of bone induction by rabbit demineralized bone matrix (DBM) in 1965 (Urist, 1965). The rabbit studies reported that completely demineralized dentin matrix (DDM) induced bone at 4 weeks, while nondemineralized dentin (so-called, calcified dentin) induced bone at 8-12 weeks after implantation (Yeoman & Urist, 1967). In our study, human DDM including small patches of cementum derived from wisdom teeth, and human DBM derived from adult femur induced bone and cartilage independently in the subcutaneous tissues at 4 weeks (Murata et al., 2010a). The delayed inductive properties of the calcified dentin and bone may be related to the inhibition of BMP-release by the apatite crystals. Highly calcified tissues such as cortical bone and dentin are not earlier in osteoinduction and bone fornmation than spongy bone, Dentin and bone are mineralized tissues and almost similar in chemical components. Both DDM and DBM are composed of predominantly type I collagen (95%) and the remaining as non-collagenous proteins including small amount of growth factors (Finkelman et al., 1990). In other words, DDM and DBM can be defined as acid-insoluble collagen binding bone morphogenetic proteins (BMPs), which are member of transforming growth factor-beta (TGF-β) super-family. BMPs were discovered from bone matrix (Urist, 1965; Sampath & Reddi., 1983), and had bone-inducing property in non-skeletal site (Murata et al., 1998). Animal dentin-derived BMPs were extracted with 4M guanidine HCl, and partially purified from rat, rabbit, and bovine (Butler et al., 1977; Urist & Mizutani, 1982; Kawai & Urist, 1989; Bessho et al, 1990). In addition, the concentration of TGF-β, Insulin growth factor-I (IGF-I) and Insulin growth factor-II (IGF-II) were detected in human dentin (DDM). Briefly, the three growth factors were measured in the following concentration (ng/μg 4M guanidine hydrochloride-EDTA protein): TGF-β (0.017), IGF-I (0.06) and IGF-II (0.52). All 3 growth factors were present in concentrations lower than that in human bone (Finkelman et al., 1990). Recently, both mature and immature types of BMP-2 were detected in human dentin and dental pulps (Ito et al., 2008).

a: SEM of DDM (granule size: 0.5mm), Note: smooth surface and no crack.

b: bone induction by DDM at 4 weeks.

c: SEM of DBM (granule size: 0.5mm), Note: micro-cracks and spaces of blood vessels.

d: bone induction by DBM at 4 weeks.

Fig. 2. Dematerialized dentin matrix (DDM) and dematerialized bone matrix (DBM)

Human Dentin as Novel Biomaterial for Bone Regeneration 131

into the augmented bone under local anesthesia (Fig. 4c). At the same time, bone biopsy was

a: intraoral initial view (before operation), Note: 3 missing teeth and atrophied maxilla.

Fig. 3. Case 1: DDM autograft for sinus lifting, 48 year-old man

b: oval shaped window

c: autogenous DDM derived from 2 molars

d: view just after DDM autograft

carried out for the tissue observation (Fig. 4d).

Even after the demineralization of dentin, active types of BMPs bind collagen-rich matrices, similar to bone (Urist et al., 1973). The decalcified dentin (DDM) was known to be more active bone-inducing matrix than the calcified dentin (Yeoman & Urist, 1967), and roll type of decalcified dentin membrane revealed better activity of bone induction (Inoue et al., 1986).

Very interestingly, the demineralized treatment for bone and dentin increased their osteoinductivity and decreased their antigenesity (Reddi, 1974). These facts are scientifically very important for the processing procedures of hard tissue-derived graft materials (Kim et al., 2010; Murata et al, 2010a).

The acid-insoluble dentin matrix (DDM) after demineralization is an organic, absorbable material with original dentin structures. Human DDM, prepared from vital teeth-origin, were implanted into the subcutaneous tissue in 4 week-old nude mice, deficient in immunogenic reactions. The DDM induced bone and cartilage independently at 4 weeks after the subcutaneous implantation, similar to human DBM (Murata et al., 2010b). The independent differentiation of bone and cartilage was compatible to our previous study using ceramic and collagen combined with BMPs (Murata et al., 1998). The acid-insoluble collagen, DBM and DDM, possess the ability to coagulate platelet-free heparinized, citrated, and oxalated blood plasmas (Huggins & Reddi., 1973). Clotting constituents become denatured in contact with the insoluble coagulant proteins. The coagulation action of blood plasma by DBM and DDM should become advantageous for surgical operations. Collagenous materials has been commercially available as medical uses for more 30 years.
