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

The aim of the following study was to estimate the increase of the bone-inductive potency by DDM combined with BMP-2 in rat subcutaneous tissues.

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

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 BMPfree control.

#### **Bioassay in rats**

134 Biomaterials – Physics and Chemistry

The biopsy tissue showed that DDM granules were received to host and the biological width (4-6mm) was acquired. The DDM residues were partially observed during the implant placement. Bone biopsy revealed the DDM were remodeled by bone at 4 months. This patient was successfully restored with the dental implants after the DDM autograft (Fig. 6d). Though animal-derived atelocollagens have been generally used as medical materials, autogenous decalcified dentin is a highly insoluble collagenous matrix and a safe

Human DDM granules from vital teeth are collagenous matrics with osteoinductive potency, and the human dentin can be recycled as autogenous biomaterials for local bone

Case 1 and 2 were approved by the Ethical Committee in the Health Sciences University of Hokkaido. All subjects enrolled in this research have responded to an Informed Consent which has been approved by my Institutional Committee on Human Research and that this

a: 4 missing teeth and atrophied upper maxilla b: DDM autograft before suture c: just after operation d:

final view after prosthetic restoration using dental implantation Fig. 6. Case 2: Bone regeneration, 58 year-old woman

**Results and discussion** 

biomaterial. **Conclusion** 

engineering.

protocol has been found acceptable by them.

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 Sciences University of Hokkaido for Experiments on Animals.

#### **Histological findings and Morphometric analysis at 3 weeks**

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

#### **Conclusion**

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 bone-forming cells for bone engineering.

Human Dentin as Novel Biomaterial for Bone Regeneration 137

Biomaterials have had a major impact on the regenerative medicine and patient care for

We have been challenging to be able to develop bioabsorbable materials, harmonized with living body, especially bone remodelling, using an innovative supersonic and acid-etching technology (Akazawa et al. 2010). Implanted biomaterials first contact to body fluid and cells. Human cells never live in dry condition. Generally, organ and tissue have interconnected porous structure for dynamic flow of body fluid. Material walls inhibit the body fluid permeation and the cell invasion. Therefore, we focused on the permeability of body fluid into the bulk of materials and the biomimetic structure for the living and working cells (Murata et al., 2007). Body fluid can permeate into collagenous materials such as DDM and DBM. Novel DDM material contains native growth factors, and adsorbs several proteins derived from body fluid. In addition, DDM with RGD sequences supports

Most importantly, material scientists, engineers, and doctors must work together and cooperate as professionals for the development of functional materials and for the present

Akazawa, T., Murata, M., Sasaki, T., Tazaki, J., Kobayashi, M., Kanno, T., Matsushima, K.,

Akazawa, T., Murata, M., Sasaki, T., Tazaki, J., Kobayashi, M., Kanno, T., Matsushima, K., &

Akazawa, T., Murata, M., Hino, J., Nakamura, K., Tazaki, J., Kikuchi, M., & Arisue, M.

from cattle bone. *J Am Ceram Soc,* 88.,12., 3545-3548.

Itabashi, K., & Arisue, M. (2005). Bio-absorption and osteoinduction innovation of bone morphogenetic protein-supported functionally graded apatites originated

Arisue, M. (2006). Biodegradation and bioabsorption innovation of the functionally graded cattle-bone-originated apatite with blood compatibility. *J Biomed Mater Res,*

(2007). Materials design and application of demineralized dentin/apatite composite granules derived from human teeth. *Archives of Bioceramics Research,* 7., 25-28.

The volume of bone and marrow showing a dose-dependent increase.

The volume of DDM showing a dose-dependent decrease.

improving the quality of lives of human.

mesenchymal cell adhesion as anchorage matrix.

and future of all patients.

76A., 1., 44-51.

**6. References** 

Table 1. Morphometry of BMP-2 dose-dependent study.

**5. Material science for patients in the near future** 

bone 0 0 3.7 ± 1.41 7.4 ± 0.94 20.3 ± 4.64 cartilage 0 4.0 ± 0.81 2.3 ± 0.47 0 0 bone marrow 0 0 0 0 6.0 ± 1.63 DDM 57.0 ± 0.81 43.3 ± 3.39 41.0 ± 2.16 40.3 ± 1.69 37.0 ± 0.81 mesenchymal tissue 40.7 ± 0.94 49.0 ± 5.09 48.0 ± 3.85 46.0 ± 2.16 32.7 ± 5.73 connective tissue 2.3 ± 0.47 3.7 ± 1.24 5.0 ± 0.47 6.3 ± 0.47 4.0 ± 0.81 All tissue: 100 % , values: mean ± SD , N: 9, Explanted time: 3 weeks

Dose of BMP-2 ( μg ) 0 0.5 1 2 5

Induced bone (B) bridging between DDM (D) granules. Note: active osteoblast differentiation. Fig. 7. Photograph in BMP-2 (5.0μg)/DDM (70mg) at 3 weeks

Fibroblasts on surface of DDM granule with original dentinal tubes. Fig. 8. Photograph in DDM (70mg) alone at 3 weeks

Induced bone (B) bridging between DDM (D) granules. Note: active osteoblast differentiation.

Fig. 7. Photograph in BMP-2 (5.0μg)/DDM (70mg) at 3 weeks

Fibroblasts on surface of DDM granule with original dentinal tubes.

Fig. 8. Photograph in DDM (70mg) alone at 3 weeks


The volume of bone and marrow showing a dose-dependent increase. The volume of DDM showing a dose-dependent decrease.

Table 1. Morphometry of BMP-2 dose-dependent study.
