**3. Clinical study of human dentin**

### **3.1 Case 1: Bone augmentation, 48 year-old man**

First clinical study was reported at 81st IADR conference, Sweden in 2003 that DDM autograft had succeeded for bone augmentation (Murata et al., 2003).

The aim of this pioneering study is to observe new bone formation in the tissues obtained from the dental implant-placed region after the DDM graft for sinus lifting.

#### **Patient**

A 48-year-old male presented with missing teeth (#24-#26, #45-#47). Clinical examinations revealed an atrophied upper jaw in the region (Fig. 3,4). His medical history was unremarkable.

#### **Surgical procedure 1**

Four teeth (#17,#18,#25,#28) were extracted and 2 molars (#17,#18) were stocked at -80℃ for DDM. His right occlusion was restored using dental implants as the first clinical step (Fig. 4b).

#### **Preparations of DDM**

The autogenous DDM were obtained from non-functional vital teeth (#17, #18) (Fig. 4a). The molars were crushed by hand-made under the cooling with liquid nitrogen. The crushed tooth granules were decalcified completely in 0.6N HCl solution. The DDM granules including cementum were extensively rinsed in cold distilled water, and then freeze-dried (Murata et al., 2010a).

#### **Surgical procedure 2**

Sinus lifting procedure was done using autogenous dry DDM for bone augmentation (Fig. 3). At 5 months after the operation, 3 fixtures (FLIALIT-2®, FRIADENT) were implanted

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

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.

First clinical study was reported at 81st IADR conference, Sweden in 2003 that DDM

The aim of this pioneering study is to observe new bone formation in the tissues obtained

A 48-year-old male presented with missing teeth (#24-#26, #45-#47). Clinical examinations revealed an atrophied upper jaw in the region (Fig. 3,4). His medical history was

Four teeth (#17,#18,#25,#28) were extracted and 2 molars (#17,#18) were stocked at -80℃ for DDM. His right occlusion was restored using dental implants as the first clinical step

The autogenous DDM were obtained from non-functional vital teeth (#17, #18) (Fig. 4a). The molars were crushed by hand-made under the cooling with liquid nitrogen. The crushed tooth granules were decalcified completely in 0.6N HCl solution. The DDM granules including cementum were extensively rinsed in cold distilled water, and then freeze-dried

Sinus lifting procedure was done using autogenous dry DDM for bone augmentation (Fig. 3). At 5 months after the operation, 3 fixtures (FLIALIT-2®, FRIADENT) were implanted

al., 2010; Murata et al, 2010a).

**3. Clinical study of human dentin** 

**Patient** 

(Fig. 4b).

unremarkable.

**Surgical procedure 1** 

**Preparations of DDM** 

(Murata et al., 2010a). **Surgical procedure 2** 

**3.1 Case 1: Bone augmentation, 48 year-old man** 

autograft had succeeded for bone augmentation (Murata et al., 2003).

from the dental implant-placed region after the DDM graft for sinus lifting.

into the augmented bone under local anesthesia (Fig. 4c). At the same time, bone biopsy was carried out for the tissue observation (Fig. 4d).

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

c: autogenous DDM derived from 2 molars d: view just after DDM autograft

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

Human Dentin as Novel Biomaterial for Bone Regeneration 133

which have gained the approval of Food and Drug Administration (FDA) for human use. The ZrO2 ceramics were fabricated by sintering at 1400℃ for 2 h after the slip casting of the mixture of ZrO2 powder and distilled water (Fig. 5a). As the results of characteristics analyses of ZrO2 objects, the contraction rate, the relative density, and the bending strength were 21%, 99%, and 400MPa, respectively. The automatic mill could crush a tooth and/or a cortical bone block (1x1x1cm3) under the condition of cooling using saline ice blocks (1cm3) (Fig. 5b). The crushed tooth granules were decalcified completely in 0.026N HNO3 solution for 20 min. The DDM granules including cementum were extensively rinsed in cold distilled

a: ZrO2 vessel and blade, b: tooth with ice blocks, c: stainless cover, d: mill, e: DDM granules before

Splitting osteotomy and cortical perforations were performed in the atrophied jaw and the autogenous DDM were transplanted to the treated bone in 2006 (Fig. 6a,b,c). At 4 months after the operation, 3 same fixtures (Synchro-steppted screw type: diameter; 3.4mm, length; 11mm, FLIALIT-2® , FRIADENT) were implanted into the augmented bone under local anesthesia (Fig. 6b). At the same time, bone biopsy was carried out for the tissue

Fig. 5. Preparation of DDM using automatic tooth mill (DENTMILL®, Tokyo Iken)

clinical use.

observation.

**Surgical procedure** 

water (Fig. 5e), (Murata et al., 2009; Murata et al., 2010a).

a: initial view, b: 4 months after DDM graft, c: dental implant placement, d: mature bone with marrow

Fig. 4. X-ray photography and bone biopsy

#### **Results and discussion**

The biopsy tissue showed that mature bone was interconnected with the remained DDM granules (Fig. 4d). We found that DDM facilitated its adaption of the grafted site and was slowly absorbed as new bone began to form.

#### **Conclusion**

This patient was successfully restored with the dental implants after the DDM autograft. These results demonstrated that autogenous dentin could be recycled as an innovative biomaterial.

#### **3.2 Case 2: Bone regeneration, 58 year-old woman**

#### **Patient**

A 58-year-old female presented with missing teeth (#12-#22). A clinical examination revealed an atrophied upper jaw in the section. Her medical history was unremarkable.

#### **Preparations of DDM**

The autogenous DDM were obtained from a non-functional vital tooth (#17). The second molar was crushed with saline ice by our newly developed tooth- mill (DENTMILL®, Tokyo Iken Co., Ltd) at 12000rpm for 30 sec (Fig. 5). Briefly, vessel and blade were made in ZrO2 ,

a: initial view, b: 4 months after DDM graft, c: dental implant placement, d: mature bone with marrow

The biopsy tissue showed that mature bone was interconnected with the remained DDM granules (Fig. 4d). We found that DDM facilitated its adaption of the grafted site and was

This patient was successfully restored with the dental implants after the DDM autograft. These results demonstrated that autogenous dentin could be recycled as an innovative

A 58-year-old female presented with missing teeth (#12-#22). A clinical examination revealed an atrophied upper jaw in the section. Her medical history was unremarkable.

The autogenous DDM were obtained from a non-functional vital tooth (#17). The second molar was crushed with saline ice by our newly developed tooth- mill (DENTMILL®, Tokyo Iken Co., Ltd) at 12000rpm for 30 sec (Fig. 5). Briefly, vessel and blade were made in ZrO2 ,

Fig. 4. X-ray photography and bone biopsy

slowly absorbed as new bone began to form.

**3.2 Case 2: Bone regeneration, 58 year-old woman** 

**Results and discussion** 

**Preparations of DDM** 

**Conclusion** 

biomaterial.

**Patient** 

which have gained the approval of Food and Drug Administration (FDA) for human use. The ZrO2 ceramics were fabricated by sintering at 1400℃ for 2 h after the slip casting of the mixture of ZrO2 powder and distilled water (Fig. 5a). As the results of characteristics analyses of ZrO2 objects, the contraction rate, the relative density, and the bending strength were 21%, 99%, and 400MPa, respectively. The automatic mill could crush a tooth and/or a cortical bone block (1x1x1cm3) under the condition of cooling using saline ice blocks (1cm3) (Fig. 5b). The crushed tooth granules were decalcified completely in 0.026N HNO3 solution for 20 min. The DDM granules including cementum were extensively rinsed in cold distilled water (Fig. 5e), (Murata et al., 2009; Murata et al., 2010a).

a: ZrO2 vessel and blade, b: tooth with ice blocks, c: stainless cover, d: mill, e: DDM granules before clinical use.

Fig. 5. Preparation of DDM using automatic tooth mill (DENTMILL®, Tokyo Iken)

### **Surgical procedure**

Splitting osteotomy and cortical perforations were performed in the atrophied jaw and the autogenous DDM were transplanted to the treated bone in 2006 (Fig. 6a,b,c). At 4 months after the operation, 3 same fixtures (Synchro-steppted screw type: diameter; 3.4mm, length; 11mm, FLIALIT-2® , FRIADENT) were implanted into the augmented bone under local anesthesia (Fig. 6b). At the same time, bone biopsy was carried out for the tissue observation.

Human Dentin as Novel Biomaterial for Bone Regeneration 135

BMP-2, 4, and 7 are strong accelerating factors of bone induction. Currently, BMP-2 and BMP-7 have been shown in clinical studies to be beneficial in the therapy of a variety of bone-related conditions including delayed union and non-union. BMP-2 (Medtronic Co.Ltd.) and BMP-7 (Stryker Biotech Co.Ltd.) have received Food and Drug Administration (FDA) approval for human clinical uses (fractures of long bones, inter-vertebral disk regeneration), by delivery in purified collagen matrix or ceramics. Moreover, the BMP-2 product has been approved for certain dental applications. BMP-7 has also found use in the treatment of chronic kidney disease. In 2002, Curis licensed BMP-7 to Ortho Biotech

The aim of the following study was to estimate the increase of the bone-inductive potency

One hundred micro-liter of recombinant human BMP-2 solution (0.0, 0.5, 1.0, 2.0, 5.0μg of BMP-2) was mixed with 70 mg of human DDM in a sterilized syringe. The composite was called as the BMP-2/DDM. The DDM alone with 100μl of PBS was also prepared as a BMP-

Wistar rats (male, 4 week-old) were subjected to intraperitoneal anesthesia and incisions were added to the back skin under the sterile conditions. Each animal received three BMPcontaining composites (BMP-2/DDM) and one BMP-free control (DDM alone). The implanted materials were removed at 3 weeks after implantation, and prepared for histomorphological examinations. All procedures were followed the Guidelines in Health

In the BMP-2 (5.0μg)/DDM (70mg) group, bone with hematopoietic bone marrow developed extensively at 3 weeks. Chondrocytes were found only in the BMP-2 (0.5, 1.0μg)/DDM groups (Table 1). The BMP-2 (2.0, 5.0μg)/DDM groups accelarated bone induction predominantly (Fig. 7). In the DDM alone group, mesenchymal tissue was seen between DDM particles, and hard tissue induction was not observed at 3 weeks (Fig. 8). Morphometric analysis demonstrated that the volume of the induced bone and marrow increased at BMP-2 dose-dependent manner, while the DDM decreased at the dosedependent (Table 1). Briefly, the volume of the bone and marrow in BMP-2 (1.0μg)/DDM and BMP-2 (5.0μg)/DDM showed 3.7% and 26.3%, respectively. BMP-2 (0.5μg)/DDM

BMP-2 strongly accelerated bone formation in the DDM carrier system. DDM never inhibited BMP-2 activity and revealed better release profile of BMP-2. These results indicate that human recycled DDM are unique, absorbable matrix with osteoinductivity and the DDM should be an effective graft material as a carrier of BMP-2 delivering and a scaffold for

**4. Dentin scaffold for recombinant human BMP-2** 

**4.2 Acceleration of bone induction by BMP2 in human DDM scaffold** 

by DDM combined with BMP-2 in rat subcutaneous tissues.

Sciences University of Hokkaido for Experiments on Animals. **Histological findings and Morphometric analysis at 3 weeks** 

showed 0.0% and 4.0% in the volume of bone and cartilage, respectively.

**4.1 Recombinant human BMP products**

Products, a subsidiary of Johnson & Johnson.

**Composition of BMP-2 solution and DDM** 

free control. **Bioassay in rats** 

**Conclusion** 

bone-forming cells for bone engineering.
