Healing Mechanism and Clinical Application of Autogenous Tooth Bone Graft Material

new arises [69]. for the autogenous graft, on cases. As for autograft, bone graft but the single use of each is not recommended in the method of augmenting bone tissue or horizontally [69,70]. For the vertical or horizontal ridge augmentation, AutoBTR may be a substitute method for autogenous bone graft and may be very useful in clinical practices when used in mixture with other graft materials in case of insufficient volume. Kim, et al. [71,72] reported the successful case of alveolar ridge augmentation using various autogenous tooth bone graft materials.


Introduction
Autogenous bone, allogenic bone, xenogenic bone, and alloplastic materials are bone graft materials that are presently used in dental clinics. According to bone healing mechanism, they can be categorized into materials that induce osteogenesis, osteoinduction, and osteoconduction. Among the many different types of bone graft materials, autogenous bone is the most ideal since it is capable of osteogenesis, osteoinduction, and osteoconduction. Its advantage is the rapid healing time without immune rejection. As its biggest shortcomings, however, the harvest amount is limited, bone resorption after graft is unavoidable, and second defect is generated in the donor area. Therefore, to overcome such shortcomings, allogenic bone and synthetic bone were developed and used in clinics, and efforts have been made to develop more ideal bone substitution materials [1]. Lately, researchers and clinicians have become interested in the use of human dentin from extracted teeth in the context of autogenous bone grafts [2,3]. Dentin has inorganic and organic components that are very similar to those of human bone. In dentin, the inorganic content is 70 ~ 75%, whereas the organic content is about 20%. In alveolar bone, the inorganic content is 65%, and the organic content is 25%. At least 90% of organic content of dentin is type I collagen, which plays an important role in bone formation and mineralization. Dentin also contains bone morphogenetic proteins (BMP), which promote the differentiation of mesenchymal stem cells into chondrocytes and consequently enhance bone formation. In addition, both alveolar bone and teeth are derived from neural crest cells [4][5][6]. Thus, studies have been done to use fresh tooth in the form of demineralized dentin matrix (DDM) as a biocompatible autogenous bone graft material in alveolar bone repair. Butler, et al [7] and Conover and Urist, et al [8] successfully extracted bone BMP Many researchers have observed that alveolar bone formation occurs around bone graft materials as a result of experiments on animals [43][44][45][46][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].
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. Recently, we conducted a study to demonstrate the osteoinductivity of AutoBT when fabricated 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. a b Cartilagenous structure

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 crystallization 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-ray 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. 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 bioceramic 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 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, hydroxyapatites 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 showed fibrosis, and there was no sign of bone formation in the control group. On the other 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 osteoconductive healing of autogenous tooth bone graft material was confirmed ( Figure 10, 11).

Clinical application of AutoBT
Kim, et al developed a novel bone grafting material using autogenous teeth (AutoBT) in 2008 and provided the basis for its clinical application. Having organic and inorganic mineral components, AutoBT is prepared from autogenous grafting material; thus eliminating the risk of immune reaction that may lead to rejection. AutoBT was used at the time of implant placement --simultaneously with guided bone regeneration --and excellent bone healing by osteoinduction and osteoconduction was confirmed [3]. In a total of 6 patients, guided bone regeneration was performed simultaneously at the time of implant placement, and tissue samples were then harvested at the time of the second surgery with the patient's consent. In the histomorphometric analysis of the samples collected from 6 patients during the 3 ~ 6 months' healing period, new bone formation was detected in 46 ~ 87% of the area of interest, and excellent bone remodeling was achieved (Table 3) (Figure 12). Clinically available AutoBT consists of powder, chips, and block ( Figure 13).

Tooth material
Newly formed bone Remodelling of Newly formed bone

Sinus bone graft
If there is any material whose resorption speed is not too high and whose bone healing process approximates that of autogenous bone graft, it may be useful in maxillary sinus bone grafting. Likewise, more excellent clinical achievement may be expected when these materials are used in mixture with other bone substitutes with slow resorption properties [61,62,63]. With evidence presented in the foregoing paragraphs, AutoBT ® developed by the author, et al was proven to exhibit bone healing ability through osteoinduction and osteoconduction, demonstrating a histological healing process similar to that of free bone grafting being resorbed over 3~6 months [3]. Accordingly, AutoBT ® is regarded as a possible substitute when autogenous bone is needed for sinus bone graft, and it may wield a useful effect on increasing the volume of bone graft materials and minimizing repneumatization ( Figure 14).  Figure 14. A case of sinus bone graft performed by the mixture of AutoBT, autogenous maxillary tuberosity bone and synthetic bone. a) Panoramic radiography of a 64-year-old man at the first examination. b) Radiography after placing implants simultaneously with the sinus bone graft on the right side. c) Panoramic radiography after 2 weeks of maxillary left 1 st molar extraction. The prosthodontic therapy for the upper right maxillary bone was completed, and the extracted tooth was replaced with bone graft materials. d) Intraoral photography before operation. e) View of mixture of AutoBT and maxillary tuberosity bone. f) Grafted in the mixture with a synthetic bone, OSTEON (GENOSS, Suwon, Korea). g) Panoramic radiography after sinus bone graft. h) Panoramic radiography taken in a private dental clinic after 3 months of bone grafting. Performing implant placement in a private dental clinic was decided due to the medical costs. i) Panoramic radiography one year after final prosthetic delivery. The bone materials grafted on the maxillary sinus are maintained stably.

Guided bone regeneration
Bone dehiscence or bone fenestration often develops after dental implant placement, and guided bone regeneration using bone graft materials has become a popular method. The most ideal material for guided bone regeneration is autogenous bone, but autogenous bone graft has limited sources and high risk of complications at the donor site and causes high resorption after bone graft. Therefore, alternative bone materials have been developed and used clinically, such as allogenic bone, xenogenic bone, and synthetic bone. Note, however, that they are often mixed with autogenous bone to maximize their advantages.
Autogenous teeth bone graft materials have very good osteoinductive and osteoconductive properties due to the organic and inorganic contents of the teeth, such as collagen, bone growth factors, and various forms of calcium phosphate. In our study, we achieved 46~74% new bone formation in 3~6 months compared with the results of Babbush [3,67]. Considering the histological healing of the sites where autogenous teeth bone graft materials were applied, bone graft materials were replaced with new bone following resorption, and new bone directly fused with the remaining autogenous teeth bone graft materials. A healing process associated with excellent osteoinduction and osteoconduction was observed in every sample, including abundant lamella bone; thus indicating that rapid bone reconduction was occurring [50,51,59,65,66]. Kim, et al [68] installed implants combined with guided bone regeneration using autogenous tooth bone graft material in 6 patients. In the 6 months' histological examination after operation, excellent osteoconductive bone healing was noted. A clinically favorable outcome was obtained ( Figure 19~21). Autogenous teeth bone graft materials have very good osteoinductive and osteoconductive properties due to the organic and inorganic contents of the teeth, such as collagen, bone growth factors, and various forms of calcium phosphate. In our study, we achieved 46~74% new bone formation in 3~6 months compared with the results of Babbush [3,67]. Considering the histological healing of the sites where autogenous teeth bone graft materials were applied, bone graft materials were replaced with new bone following resorption, and new bone directly fused with the remaining autogenous teeth bone graft materials. A healing process associated with excellent osteoinduction and osteoconduction was observed in every sample, including abundant lamella bone; thus indicating that rapid bone reconduction was occurring [50,51,59,65,66]. Kim, et al [68] installed implants combined with guided bone regeneration using autogenous tooth bone graft material in 6 patients. In the 6 months' histological examination after operation, excellent osteoconductive bone healing was noted. A clinically favorable outcome was obtained ( Figure 19~21).

Ridge augmentation (Figure 22)
Autogenous bone grafting produces the best results in case a large volume of bone increase is required, as in the reconstruction of a site with lots of bone defects or ridge augmentation. The autograft may be taken from the endochondral bone such as ilium, rib, tibia, etc., and from the intramembranous bone such as calvaria, facial bone, etc. Alveolar ridge augmentation is a method of augmenting the height or width of the alveolar ridge by implementing bone grafting on the upper part or lateral part of the ridge in particulate or block type in case bone volume is insufficient vertically or horizontally; vertical and horizontal augmentation may be done simultaneously, but it may also be carried out individually. Since it is a kind of onlay graft, bone absorption occurs considerably after grafting, and dehiscence on the upper soft tissue easily arises [69]. Meanwhile, as for the autogenous bone graft, there may be some complications on the donor site, and doing the grafting takes time. Likewise, there are several problems such as limit to the volume of collection. Consequently, patients and clinical doctors are inclined to avoid it in many cases. As substitutes for autograft, bone graft materials such as allograft, xenograft, synthetic bone, etc., were developed, but the single use of each is not recommended in the method of augmenting bone tissue vertically or horizontally [69,70]. For the vertical or horizontal ridge augmentation, AutoBTR may be a substitute method for autogenous bone graft and may be very useful in clinical practices when used in mixture with other graft materials in case of insufficient volume. Kim, et al. [71,72] reported the successful case of alveolar ridge augmentation using various autogenous tooth bone graft materials. Autogenous bone grafting produces the best results in case a large volume of bone increase is required, as in the reconstruction of a site with lots of bone defects or ridge augmentation. The autograft may be taken from the endochondral bone such as ilium, rib, tibia, etc., and from the intramembranous bone such as calvaria, facial bone, etc. Alveolar ridge augmentation is a method of augmenting the height or width of the alveolar ridge by implementing bone grafting on the upper part or lateral part of the ridge in particulate or block type in case bone volume is insufficient vertically or horizontally; vertical and horizontal augmentation may be done simultaneously, but it may also be carried out individually. Since it is a kind of onlay graft, bone resorption occurs considerably after grafting, and dehiscence on the upper soft tissue easily arises [69]. Meanwhile, as for the autogenous bone graft, there may be some complications on the donor site, and doing the grafting takes time. Likewise, there are several problems such as limit to the volume of collection. Consequently, patients and clinical doctors are inclined to avoid it in many cases. As substitutes for autograft, bone graft materials such as allograft, xenograft, synthetic bone, etc., were developed, but the single use of each is not recommended in the method of augmenting bone tissue vertically or horizontally [69,70]. For the vertical or horizontal ridge augmentation, AutoBT may be a substitute method for autogenous bone graft and may be very useful in clinical practices when used in mixture with other graft materials in case of insufficient volume. Kim, et al. [71,72] reported the successful case of alveolar ridge augmentation using various autogenous tooth bone graft materials. There was not much bone resorption when the state of titanium screws was examined. s): After removing the titanium screws, the implants were placed. t): Exposed #15 and16 areas. The titanium screws fixing the block is observed, and bone healing was very good. u): View of implants placed on the site. v): Panoramic radiography after the #12, 21, 15, and 16 implants were placed. w): View of the #45 and 46 implants exposed while doing the secondary surgery after 2 months. x): Panoramic radiography 6 months after the final prosthetic delivery.

Extraction socket preservation or reconstruction (Figure 23)
The resorption of the residual alveolar bone in the vicinity of extraction sockets reportedly occurs primarily during the initial period after tooth extraction; in cases wherein teeth are infected with periodontal diseases, it shows more severe resorption [73]. Severe resorption of the alveolar bone may cause aesthetic problems in the anterior teeth. In addition, normal, natural healing may be difficult since the soft tissues may fall down into the defective area if there is progressive periodontal disease or periapical inflammatory lesion, or in case of serious defects of the surrounding bone wall after tooth extraction. Therefore, the preservation or reconstruction of the extraction sockets should be considered positively in case of serious defects after tooth extraction [74]. Ridge preservation methods using various bone graft materials were introduced and reported to be effective in preventing vertical and horizontal ridge resorption [75][76][77]. Kim, et al [78] reported an actual case of extraction socket preservation and reconstruction using autogenous tooth bone powder and block. They reported good healing of extraction socket after 3~3.5 months, and they could successfully perform the placement of implants.

Conclusion
It is obvious that autogenous tooth bone graft materials(AutoBT) are safer than allogeneic and xenogeneic bon egraft materials; the fact that they are compared with the healing performance of free autogenous bone graft in histological view is clear evidence. AutoBT can be used safely