The aim of this study is to estimate the increase of bone-inductive potency by human demineralized dentin matrix (DDM) with recombinant human bone morphogenetic protein-2 (BMP-2). Human teeth were crushed, completely demineralized in 0.6M HCl, and freeze-dried. The tooth-derived material is called DDM. The shape of DDM was a particle type and its size varied from 0.4 to 0.8 mm. The BMP-2 dose-dependent study in the rat subcutaneous tissues demonstrated that the volume of induced bone and marrow increased at a dose-dependent manner. The time-course study of bone induction by the BMP-2 (5.0 μg)/DDM (70 mg) was estimated histologically and biochemically. Histological findings showed that the BMP-2/DDM increased bone and marrow sequentially between the DDM particles. Calcium content in the BMP-2/DDM-induced tissue was compatible to the histological findings. ALP activity in the BMP-2/DDM showed a maximal value at 1 week and gradually decreased. The morphometric analysis demonstrated that the BMP-2/DDM showed 66.9%, 79.0% in the volume of bone and marrow, and 32.4%, 21.0% in that of DDM at 8, 32 weeks, respectively. We confirmed that BMP-2 significantly accelerated bone formation in the acid-insoluble human-dentin carriers. These results indicate that human DDM should be an effective carrier for delivering BMP-2 and superior scaffold for bone-forming cells.

Dentin matrix is well known as the most insoluble collagen in human body. We already confirmed the osteoinductive property by granules of human demineralized dentin matrix (DDM) histologically. Human DDM granules and human demineralized rootdentin (DRD) mass were prepared from adult vital teeth. The hard tissue induction by DDM alone or DRD alone was estimated at 4 weeks after implantation. The DDM alone induced bone and cartilage independently. In addition, the bioassay by recombinant human BMP-2 (5.0 µg)/DDM or BMP-2 (5.0 µg)/DRD was estimated in mouse subcutaneous or intramuscular tissues, respectively. Histological examination showed that the BMP-2/DDM induced bone and marrow, and the DDM granules were partially absorbed by new bone. The morphometric analysis demonstrated that the BMP-2/DDM showed 36.3% in the volume of bone and marrow, while the DDM alone showed 1.3% at 4 weeks. Moreover, the BMP-2/DRD also induced active bone formation on the root surface and in the pulp cavity. These results indicate that BMP-2 significantly accelerated bone formation in decalcified dentin implants. Human recycled DDM and DRD might be effective materials as osteoinductive collagenous carriers of BMP-2 for bone engineering.

The purpose of this study is to compare in vivo retention of BMP-2 and bone induction in HAp (porosity: 60-80%, pore size: 100-600 µm, sintering temperature: 800 • C, surface area: 1 m 2 /g) and β-TCP (porosity: 75%, pore size: 100-400 µm, sintering temperature: 1050 • C, surface area: 4 m 2 /g). We estimated the in vivo release profile of 125 I-labeled BMP-2 and bone induction of hard tissues histologically. The amount of BMP-2 remaining in the β-TCP at 1 day after implantation was 49.6%, while the amount was 34.0% in the HAp. Furthermore, the HAp and β-TCP containing 0.0, 0.05, 0.1, 0.3, 0.5, 1.0, 5.0 µg of BMP-2 were implanted into the back subcutis of 4-week old Wistar rats. At 3 weeks after implantation, the ceramics were explanted and evaluated histologically. The HAp/BMP-2 (5.0 µg) system showed 3.0% in the total volume of bone at 3 weeks, while only in the β-TCP/BMP-2 (5.0 µg) system showed 32.5%. These results indicate that the absorbable β-TCP block may be an effective bioceramic for bone induction to deliver BMP-2 to the site of action.

A novel automatic mill of teeth and bone has been developed for bone engineering. A human frozen-tooth and/or a human frozen-bone block were put into the Zirconium oxide (ZrO 2 ) ceramics vessel of the machine, and crushed for 1 min with 20 salineice blocks (1 © 1 © 1 cm 3 /block) at 12000 rpm of ZrO 2 blade. The crushed granules were demineralized completely in 2% HNO 3 solution for 20 min, and rinsed in cold saline. We named each biomaterial after the acid treatment and washing, demineralized dentin matrices (DDM), demineralized bone matrices (DBM). Five wisdom teeth (total wet volume: 10.0 g) were crushed, decalcified, and lyophilized. The distribution of freeze-dried DDM granules was fine granules (0.51.0 mm: 0.27 g), moderate (1.02.0 mm: 0.46 g), and large (2.05.0 mm: 0.64 g). The fine granules of human DDM or DBM were implanted into the subcutaneous tissue of 4 week-old nude mice, and their tissue-inductive properties were estimated at 4 weeks after implantation histologically. The explanted samples were demineralized, embedded in paraffin, and sectioned. The specimens were stained with hematoxylin and eosin. We confirmed that DDM induced bone and cartilage independently, and DBM induced cartilage, bone and marrow at 4 weeks in the back skin of nude mice. These results indicated that our material preparation system by the novel mill with a vessel and a blade of ZrO 2 under ice-cooling maintained the bone-inducing activity of human dentin and bone.

The purpose of this study was to determine whether bone augmentation could be obtained by the composite of recombinant human bone morphogenetic protein-2 (rhBMP-2) and bioabsorbable atelocollagen when the periosteum was resected, and to compare the efficacy of the rhBMP-2/collagen implant and the collagen alone implant. The onlay implant was inserted into the space between the elevated galea aponeurotica and the skull without the periosteum of 10-month-old rats. The rhBMP-2/collagen implant resulted in osteoblasts differentiation under the galea at 1 week and active bone formation without a prior formation of cartilage. At 4 weeks, the bony trabeculae were interconnected and connected directly with the compact bone of the skull. Histomorphometric analysis at 4 weeks demonstrated that the rhBMP-2/collagen implant showed 92.5% in the volume of bone tissue, whereas the collagen alone showed 0%. The implanted collagen was gradually replaced by bone tissue in the presence of rhBMP-2. Our present results indicate that rhBMP-2 stimulates undifferentiated mesenchymal cells in the galea overlying the implant to proliferate and differentiate directly into osteoblasts on the carrier collagen fibers. The collagen matrix was stably placed on the skull and suitable as a substitute for rhBMP-2. The rhBMP-2/collagen onlay implant might be clinically applicable for bone augmentation even under the condition without the periosteum.