**2. Osteoinduction of AutoBT**

Many researchers have examined tooth dentin as a potential carrier for human proteins and as grafting material because its biological composition is very similar to that of alveolar bone [9, 24-28]. Both tooth and alveolar bone are derived from neural crest cells and are made up of the same Type I collagen. Furthermore, dentin contains BMPs, which induce bone formation and noncollagenous proteins such as osteocalcin, osteonectin, and dentin phosphoprotein [29, 30]. Since its investigation by Urist in 1965, BMP has been widely studied and used in clinical applications [31]. As a result, Yeoman and Urist, et al (1967) and Bang and Urist, et al (1967) showed the osteoinductivity of rabbit DDM by BMP [32, 33]. Bessho, et al extracted BMP from bone matrix, dentin matrix, and wound tissue after extracting teeth from rabbits. Each BMP was confirmed to have induced the formation of new bone when xenogenic implantation was performed [9]. Bessho, et al extracted human dentin matrix containing 4mol/L guanidine HC1 and refined it into liquid chromatography and found out based on SDS-PAGE and IEF that purified BMP is homogenous, inducing the formation of new bone within 3 weeks of implan‐ tation in muscle pouches in Wistar rats. Dentin matrix-derived BMP is not exactly same as bone matrix-derived BMP, but they are very similar. In other words, two types of BMP exhibit the same action in the body [34]. The organic component accounts for about 20% of dentin weight and mostly consists of type I collagen. Moreover, it was proven to have BMP promoting cartilage and bone formation, and differentiating undifferentiated mesenchymal stem cells into chondrocytes and osteogenic cells [30, 35-37]. Noncollagenous proteins of dentin such as osteocalcin, osteonectin, phosphoprotein, and sialoprotein are known to be involved in bone calcification [38,39].

Patterns of matrix protein in teeth must have osteoinductive potential even though it does not perfectly match the protein in alveolar bone. Moreover, the apatite in teeth has long been known to play the role of protecting proteins [40]. According to Boden, et al, LIM mineraliza‐ tion protein 1 (LMP-1) is an essential positive regulator of osteoblast differentiation and maturation and bone formation [41]. Wang, et al found that LIM-1 was expressed primarily in predentin, odontoblasts, and endothelial cells of the blood vessels of teeth [42].

Many researchers have observed that alveolar bone formation occurs around bone graft materials as a result of experiments on animals [43-47]. Chung registered the patent for the technology of extracting proteins from teeth in 2002 and 2004; this carries an important, serving as evidence that teeth contain bone morphogenic protein [48,49]. Ike and Urist suggested that root dentin prepared from extracted teeth may be recycled for use as carrier of rhBMP-2 because it induces new bone formation in the periodontium [10]. Murata, et al reported that demineralized dentin matrix (DDM) does not inhibit BMP-2 activity but shows better release profile of BMP-2. Human recycled DDM is an unique, absorbable matrix with osteoinductivity, and DDM should be an effective graft material as a carrier of BMP-2 and a scaffold for boneforming cells for bone engineering [2].

from rabbit DDM, and Bessho, et al [9] secured new bone formation *in situ* by BMP from human DDM. Furthermore, Ike and Urist [10] used dentin root matrix as a carrier of recombinant human bone morphogenetic protein (rhBMP). Starting in 1993, we developed bone graft materials using human teeth with which we conducted experimental studies [11-22]. In 2008, we developed an autogenous tooth bone graft material (AutoBT; Korea Tooth Bank Co., Seoul, Korea) from extracted teeth prepared as powder and grafted it to the donor patient himself. The mineral components of autogenous tooth bone graft materials have 4 stages (types) of calcium phosphate (HA, TCP, OCP, and ACP). Under scanning electron microscopic exami‐ nation, HA crystalline structures and collagen fibers around the dentinal tubules were detected. Short-term clinical studies reported that, even when wounds became dehiscent, the bone graft materials were not infected, and good secondary healing was achieved [3,23].

Many researchers have examined tooth dentin as a potential carrier for human proteins and as grafting material because its biological composition is very similar to that of alveolar bone [9, 24-28]. Both tooth and alveolar bone are derived from neural crest cells and are made up of the same Type I collagen. Furthermore, dentin contains BMPs, which induce bone formation and noncollagenous proteins such as osteocalcin, osteonectin, and dentin phosphoprotein [29, 30]. Since its investigation by Urist in 1965, BMP has been widely studied and used in clinical applications [31]. As a result, Yeoman and Urist, et al (1967) and Bang and Urist, et al (1967) showed the osteoinductivity of rabbit DDM by BMP [32, 33]. Bessho, et al extracted BMP from bone matrix, dentin matrix, and wound tissue after extracting teeth from rabbits. Each BMP was confirmed to have induced the formation of new bone when xenogenic implantation was performed [9]. Bessho, et al extracted human dentin matrix containing 4mol/L guanidine HC1 and refined it into liquid chromatography and found out based on SDS-PAGE and IEF that purified BMP is homogenous, inducing the formation of new bone within 3 weeks of implan‐ tation in muscle pouches in Wistar rats. Dentin matrix-derived BMP is not exactly same as bone matrix-derived BMP, but they are very similar. In other words, two types of BMP exhibit the same action in the body [34]. The organic component accounts for about 20% of dentin weight and mostly consists of type I collagen. Moreover, it was proven to have BMP promoting cartilage and bone formation, and differentiating undifferentiated mesenchymal stem cells into chondrocytes and osteogenic cells [30, 35-37]. Noncollagenous proteins of dentin such as osteocalcin, osteonectin, phosphoprotein, and sialoprotein are known to be involved in bone

Patterns of matrix protein in teeth must have osteoinductive potential even though it does not perfectly match the protein in alveolar bone. Moreover, the apatite in teeth has long been known to play the role of protecting proteins [40]. According to Boden, et al, LIM mineraliza‐ tion protein 1 (LMP-1) is an essential positive regulator of osteoblast differentiation and maturation and bone formation [41]. Wang, et al found that LIM-1 was expressed primarily in

predentin, odontoblasts, and endothelial cells of the blood vessels of teeth [42].

**2. Osteoinduction of AutoBT**

406 Advances in Biomaterials Science and Biomedical Applications

calcification [38,39].

Lee [50] performed quantitative analysis of proliferation and differentiation of the MG-63 cell line on the bone grafting material using human tooth. This study demonstrated that the cellular adhesion and proliferation activity of the MG-63 cell on partially demineralized dentin matrix (PDDM) were comparable to control with enhanced osteogenic differentiation (Figure 1). Kim & Choi [51] reported a case on tooth autotransplantation with autogenous tooth bone graft. The extracted right mandibular third molar of a 37-year-old man was transplanted into the first molar area, and a bone graft procedure using autogenous tooth-bone graft material was performed for the space between the root and the alveolar socket. Reattachment was achieved (Figure 2). Therefore, the autogenous tooth bone graft material is considered reasonable for bone inducement and healing in the autotransplantation of teeth.

**Figure 1.** MG-63 cells adhered to PDDM, and they were spread out. This means excellent biocompatibility between cells and PDDM. (Lee H.J. Quantitative Analysis of Proliferation and Differentiation of MG-63 Cell Line on the Bone Grafting Material Using Human Tooth. PhD Thesis. School of Dentistry, Seoul National University, 2011.)

**Figure 2.** Periapical radiograph 2 years after autotransplantation.

Recently, we conducted a study to demonstrate the osteoinductivity of AutoBT when fabri‐ cated from bio-recycled dysfunctional teeth after patented processing. A total of 46 extracted dysfunctional teeth samples were collected from actual patients. *In vivo* study was done on 15 athymic mice by inserting AutoBT in dorsal subcutaneous muscular tissues. Samples were then biopsied in 2, 5, and 8 weeks. For additional analyses, Bradford assay, SDS-PAGE, and western blotting were performed *in vitro*. Histologic analyses *in vivo* showed new active bone formation as early as 2 weeks later (Figure 3,4,5). The Bradford assay indicated the existence of noncollagenous proteins in AutoBT. Nonetheless, rhBMP-2 was not extractable from AutoBT according to electrophoresis and immunoblotting analyses (Figure 6). In conclusion, this study provided an evidence of osteoinductivity of AutoBT th rough noncollagenous proteins.

**Figure 3.** Histologic analyses of 2-week biopsy sample. a) The new cell lining and attachment to AutoBT powder and b) Newly deposited osteoid formations were observed. (H&E staining, X 200).

**Figure 2.** Periapical radiograph 2 years after autotransplantation.

408 Advances in Biomaterials Science and Biomedical Applications

a b

b) Newly deposited osteoid formations were observed. (H&E staining, X 200).

proteins.

Recently, we conducted a study to demonstrate the osteoinductivity of AutoBT when fabri‐ cated from bio-recycled dysfunctional teeth after patented processing. A total of 46 extracted dysfunctional teeth samples were collected from actual patients. *In vivo* study was done on 15 athymic mice by inserting AutoBT in dorsal subcutaneous muscular tissues. Samples were then biopsied in 2, 5, and 8 weeks. For additional analyses, Bradford assay, SDS-PAGE, and western blotting were performed *in vitro*. Histologic analyses *in vivo* showed new active bone formation as early as 2 weeks later (Figure 3,4,5). The Bradford assay indicated the existence of noncollagenous proteins in AutoBT. Nonetheless, rhBMP-2 was not extractable from AutoBT according to electrophoresis and immunoblotting analyses (Figure 6). In conclusion, this study provided an evidence of osteoinductivity of AutoBT th rough noncollagenous

**Figure 3.** Histologic analyses of 2-week biopsy sample. a) The new cell lining and attachment to AutoBT powder and

**Figure 4.** Cartilages were formed at the periphery of AutoBT in 5-week biopsy sample (H&E staining X 200).

**Figure 5.** a) Endochondral ossification and b) lamellar bone formation were identified 8 weeks after the insertion of AutoBT powder in the intramuscular pouch of athymic mice (H&E, staining, X 200).

**Figure 6.** SDS-PAGE of purified fractions from AutoBT powder fabricated from a dried tooth in 25 ºC (A) and from wisdom tooth in fresh state (B).

#### **3. Osteoconduction of AutoBT**

The analytic results showed that AutoBT consisted of low-crystalline hydroxyapatite (HA) and possibly other calcium phosphate minerals (ß-tricalcium phosphate (ß-TCP), ACP, and OCP), similar to the minerals of human bone tissues. Note, however, that the level of HA crystalli‐ zation and the amount of HA differed greatly depending on the area of the tooth. The XRD pattern was much stronger in the crown portion with enamel than in the root portion (Figure 7). Likewise, the dental crown portion consisted of high-crystalline calcium phosphate minerals (mainly HA) with higher Ca/P ratio, whereas the root portion was mainly made up of low-crystalline calcium phosphates with relatively low Ca/P ratio [3, 23]. Kim, et al [52] performed the study to evaluate the surface structures and physicochemical characteristics of a novel autogenous tooth bone graft material currently in clinical use. The material's surface structure was compared with a variety of other bone graft materials via scanning electron microscope (SEM). The crystalline structure of the autogenous tooth bone graft material from the crown (AutoBT crown) and root (AutoBT root), xenograft (BioOss), alloplastic material (MBCP), allograft (ICB), and autogenous mandibular cortical bone were compared using xray diffraction (XRD) analysis. The solubility of each material was measured with the Ca/P dissolution test. The result of the SEM analysis showed that the pattern associated with AutoBT was similar to that from autogenous cortical bone (Figure 8). In the XRD analysis, AutoBT root and allograft showed a low crystalline structure similar to that of autogenous cortical bone (Figure 9). In the CaP dissolution test, the amount of calcium and phosphorus dissolution in AutoBT was significant from the beginning, displaying a pattern similar to that of autogenous cortical bone (Tables 1, 2). In conclusion, autogenous tooth bone graft materials can be considered to have physicochemical characteristics similar to those of autogenous bone.


m/z: mass-to-charge ratio

**Table 1.** Ca (m/z; 42.959) ion dissolution (Kim Y.K., et al. Autogenous teeth used for bone grafting: a comparison to traditional grafting materials. Oral Surg. Oral Med. Oral Pathol. Oral Radiol., 2013, in press)


m/z: mass-to-charge ratio

**Figure 6.** SDS-PAGE of purified fractions from AutoBT powder fabricated from a dried tooth in 25 ºC (A) and from

The analytic results showed that AutoBT consisted of low-crystalline hydroxyapatite (HA) and possibly other calcium phosphate minerals (ß-tricalcium phosphate (ß-TCP), ACP, and OCP), similar to the minerals of human bone tissues. Note, however, that the level of HA crystalli‐ zation and the amount of HA differed greatly depending on the area of the tooth. The XRD pattern was much stronger in the crown portion with enamel than in the root portion (Figure 7). Likewise, the dental crown portion consisted of high-crystalline calcium phosphate minerals (mainly HA) with higher Ca/P ratio, whereas the root portion was mainly made up of low-crystalline calcium phosphates with relatively low Ca/P ratio [3, 23]. Kim, et al [52] performed the study to evaluate the surface structures and physicochemical characteristics of a novel autogenous tooth bone graft material currently in clinical use. The material's surface structure was compared with a variety of other bone graft materials via scanning electron microscope (SEM). The crystalline structure of the autogenous tooth bone graft material from the crown (AutoBT crown) and root (AutoBT root), xenograft (BioOss), alloplastic material (MBCP), allograft (ICB), and autogenous mandibular cortical bone were compared using x-

wisdom tooth in fresh state (B).

**3. Osteoconduction of AutoBT**

410 Advances in Biomaterials Science and Biomedical Applications

**Table 2.** P (m/z; 30.994) ion dissolution (Kim Y.K., et al. Autogenous teeth used for bone grafting: a comparison to traditional grafting materials. Oral Surg. Oral Med. Oral Pathol. Oral Radiol., 2013, in press)

In an in vitro dissolution test, AutoBT showed excellent biodegradability, whereas apatite reprecipitation was actively visible immediately after transplantation. We conjecture that this material plays an effective role in inducing bone regrowth [52]. Priya, et al [53] reported that the extensive dissolution of calcium phosphate composites, which release calcium and phosphorus ions, induces the re-precipitation of the apatite onto the surfaces. According to them, the combination of dissolution and re-precipitation was the mechanism behind apatite formation. Apatite layer formation was expected to encourage the osseointegration of biocer‐ amic composites.

Both the organic and inorganic compositions differ between the crown and root of autogenous tooth bone graft materials. Thus, when the material is grafted, crown and root show different healing mechanisms. Apatites present in bone tissues form a ceramic/high-molecular weight

**Figure 7.** X-ray diffraction patterns of human tooth. (#1: root portion, #2: crown portion, 3: whole tooth) (Kim Y.K., et al. Development of a novel bone grafting material using autogenous teeth. Oral Surg. Oral Med. Oral Pathol. Oral Ra‐ diol. Endod., 2010.)

nanocomplex pattern [54]. In particular, apatites present in human bone tissues have low crystallinity and crystal size that are several tens of nanometers. On the other hand, hydroxya‐ patites prepared via the sintering process at high temperatures have high crystallinity. Grain growth occurs during the sintering process, resulting in sizes that are at least ten times larger than those apatites present in bone tissues [55]. The biodegradation of large particles with high crystallinity is almost impossible. Their osteoconduction capacity is very low, and osteoclasts cannot degrade them. Low-crystalline carbonic apatites show the best osteoconduction effects [56,57].

Nampo, et al introduced alveolar bone repair using extracted teeth for the graft material. DSP is a dentin-specific noncollagenous protein involved in the calcification of dentin. Based on immunohistochemical staining with anti-DSP antibody, the positive reaction was localized to the dentin of the extracted tooth fragments; thus suggesting that dentin has high affinity for and marked osteoconductive effect on the jaw bone [58].

Kim, et al reported bone healing capacity of demineralized dentin matrix materials in a minipig cranium defect [59]. A defect was induced in the cranium of mini-pigs, and those without defect were used as control. In the experimental group, teeth extracted from the mini-pig were manufactured into autogenous tooth bone graft material and grafted to the defect. The minipigs were sacrificed at 4, 8, and 12 weeks to evaluate histologically the bone healing ability and observe the osteonectin gene expression pattern with RT-PCR. At 4 weeks, the inside of the bur hole showedfibrosis, andthere was no sign of bone formation in the control group.On the other Healing Mechanism and Clinical Application of Autogenous Tooth Bone Graft Material http://dx.doi.org/10.5772/53200 413

nanocomplex pattern [54]. In particular, apatites present in human bone tissues have low crystallinity and crystal size that are several tens of nanometers. On the other hand, hydroxya‐ patites prepared via the sintering process at high temperatures have high crystallinity. Grain growth occurs during the sintering process, resulting in sizes that are at least ten times larger than those apatites present in bone tissues [55]. The biodegradation of large particles with high crystallinity is almost impossible. Their osteoconduction capacity is very low, and osteoclasts cannot degrade them. Low-crystalline carbonic apatites show the best osteoconduction effects

**Figure 7.** X-ray diffraction patterns of human tooth. (#1: root portion, #2: crown portion, 3: whole tooth) (Kim Y.K., et al. Development of a novel bone grafting material using autogenous teeth. Oral Surg. Oral Med. Oral Pathol. Oral Ra‐

Nampo, et al introduced alveolar bone repair using extracted teeth for the graft material. DSP is a dentin-specific noncollagenous protein involved in the calcification of dentin. Based on immunohistochemical staining with anti-DSP antibody, the positive reaction was localized to the dentin of the extracted tooth fragments; thus suggesting that dentin has high affinity for

Kim, et al reported bone healing capacity of demineralized dentin matrix materials in a minipig cranium defect [59]. A defect was induced in the cranium of mini-pigs, and those without defect were used as control. In the experimental group, teeth extracted from the mini-pig were manufactured into autogenous tooth bone graft material and grafted to the defect. The minipigs were sacrificed at 4, 8, and 12 weeks to evaluate histologically the bone healing ability and observe the osteonectin gene expression pattern with RT-PCR. At 4 weeks, the inside of the bur hole showedfibrosis, andthere was no sign of bone formation in the control group.On the other

and marked osteoconductive effect on the jaw bone [58].

[56,57].

diol. Endod., 2010.)

412 Advances in Biomaterials Science and Biomedical Applications

**Figure 8.** SEM views of the different types of bone graft materials. (Kim Y.K., et al. Autogenous teeth used for bone grafting: a comparison to traditional grafting materials. Oral Surg. Oral Med. Oral Pathol. Oral Radiol., 2013, in press) a): AutoBT crown (x500), b): AutoBT crown (x5,000), c): AutoBT root (x500), d): AutoBT root (x5,000), e): Autogenous cortical bone (x500), f): Autogenous cortical bone (x5,000), g): ICB (x500), h): ICB ( x5,000), i): BioOss (x500), j): BioOss (x5,000), k): MBCP (x500), l): MBCP (x5,000)

hand, bone formation surrounding the tooth powder granule was observed at 4 weeks in the experimental group wherein the bur hole was filled with tooth powder. There was practically no osteonectin expression in the control group, whereas active osteonectin expression was observed from 4 to 12 weeks in the experimental group. In this study, excellent osteoconduc‐ tive healing of autogenous tooth bone graft material was confirmed (Figure 10, 11).

**Figure 9.** Results of the X-ray diffraction analysis. AutoBT D+: AutoBT root, AutoBT E+: AutoBT crown, Cancellous bone: ICB. (Kim Y.K., et al. Autogenous teeth used for bone grafting: a comparison to traditional grafting materials. Oral Surg. Oral Med. Oral Pathol. Oral Radiol., 2013, in press)

**Figure 10.** Experimental group of 8 weeks. New bone is actively formed around the tooth powder granules. Asterisks and arrows indicate graft tooth granule materials and new bone formation around the tooth granules, respectively. Hematoloxylin and eosin staining (×100). (Kim J.Y., et al. Bone healing capacity of demineralized dentin matrix materi‐ als in a mini-pig cranium defect. J. Korean Dent. Sci., 2012.)

Healing Mechanism and Clinical Application of Autogenous Tooth Bone Graft Material http://dx.doi.org/10.5772/53200 415

**Figure 11.** Experimental group of 12 weeks. Extensive new bone formation was noted around the bone powder gran‐ ules in the bur hole. Asterisks indicate tooth powder materials. Hematoloxylin and eosin staining (×100). (Kim J.Y., et al. Bone healing capacity of demineralized dentin matrix materials in a mini-pig cranium defect. J. Korean Dent. Sci., 2012.)
