Grant-in-Aid for Scientific Research (B)
|Allocation Type||Single-year Grants |
|Research Institution||Tokyo Medical and Dental University |
ITOH Soichiro Tokyo Medical and Dental University, Division of Molecular Tissue Engineering, Human Gene and Sciences Center, Associate Professor, 疾患遺伝子実験センター, 助教授 (10242190)
SHINOMIYA Kenichi Tokyo Medical and Dental University, Department of orthopedic surgery, associate professor, 大学院・医歯学総合研究科, 教授 (20111594)
TAKAKUDA Kazuo Tokyo Medical and Dental University, Institute of Biomaterials and Bioengineering, professor, 生体材料工学研究所, 教授 (70108223)
TANAKA Junzo National Institute for Materials Science, Biomaterials Center, director-general, 生体材料研究センター・センター長 (10343831)
KIKUCHI Masanori National Institute for Materials Science, Biomaterials Center, senior researcher, 生体材料研究センター・主任研究員 (00354267)
ISHII Tsnehiro Kyocera Co., division of BIOCERAM【○!R】, Tissue engineering, senior researcher, バイオセラム事業部, 組織工学統括研究者
麻生 義則 国立大学法人東京医科歯科大学, 疾患遺伝子実験センター, 助手 (50345279)
|Project Period (FY)
2001 – 2004
Completed (Fiscal Year 2004)
|Budget Amount *help
¥13,800,000 (Direct Cost: ¥13,800,000)
Fiscal Year 2004: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2003: ¥4,300,000 (Direct Cost: ¥4,300,000)
Fiscal Year 2002: ¥4,300,000 (Direct Cost: ¥4,300,000)
Fiscal Year 2001: ¥4,000,000 (Direct Cost: ¥4,000,000)
|Keywords||Hydroxyapatite / type I collagen (HAp / Col) composite / anterior fusion of spine / Porous body / sheet / osteoblast / osteoclast / mesenchymal stem cell / scaffold / コラーゲンナノ複合体 / 骨髄細胞 / 骨芽細胞 / 骨形成能 / コラーゲン(HAp / Col) / 頚椎前方固定システム / 骨代替材料 / コラーゲン / 骨伝導能 / 骨髄間葉系細胞 / HAp / Col複合体 / 人工椎体 / rhBMP-2|
Hydroxyapatite/type I collagen (HAp/Col) composite in which the hydroxyapatite nanocrystals were aligned along the collagen molecules, was prepared. In our previous study the composite materials were implanted into bone defects of tibia in beagle dogs. Histological determination revealed that osteoblasts were found along its surface, and osteoclasts appeared in a Howship's lacunae-like structure formed on the composite. It is suggested that HAp/Col has a very high osteoconductive activity and is able to induce bone remodeling units.
HAp/Col implants were used in anterior fusion between C3 and C4 vertebrae in beagles. To prevent displacement, the implants were fixed with a PLLA plate and titanium screws. It is suggested that the implants containing rhBMP-2 may shorten the time of bone fusion, and that this artificial vertebra system may be available for anterior fusion of the cervical spine suffering from vertical stress. To enhance the replacement speed of the HAp/Col composite to bone,
porous body and sheet have been developed.
Porous body of HAp/Col composite has elasticity, plasticity and good permeability. Furthermore, cultured cells can easily penetrate into the porous body when they are subcultured onto it. Bone hole of 3mm diameter was perforated into the distal end of femur of SD rats, and the porous body of composite was implanted to evaluate its osteoconductive activity. Histological findings revealed that new bone is formed into the central part of composite as early as after 2 weeks of operation, and TRAP-positive cells, osteoclasts, attached on both the degenerated composite and newly formed bone. And ALP-positive cells, osteoblasts were also observed arranged on the bone. Almost whole body of the composite was replaced to newly formed bone after 4 weeks. These findings suggest that porous body of HAp/Col is absorbed rapidly by osteoclasts activity after implantation, and replaced to bone newly formed by osteoblasts.
HAp/Col fibers were precipitated in phosphate buffered saline (PBS) at pH of 7.4 and temperature of 37℃, and dehydrated by compression to form sheets of 0.5, 0.9, 1.5 and 3mm thickness. Osteoblasts were harvested from mouse calvaria, and bone marrow cells were obtained from the femur of Fischer rats. They were cultured in a standard medium of Eagle-minimum containing 15% fetal bovine serum at 37℃. LacZ gene was transferred into the marrow cells by Adenovirus vector after P1, and subcultured onto HAp/Col sheet composites of 0.5 or 0.9 mm thickness at a concentration of 10^6 cells/ml. Both type of cells attached alive on the sheet, suggesting that it is suitable for cultured cell scaffold.
Bone defect of 6 mm length was prepared and fixed with an extraskeletal fixator. HAp/Col sheet with or without mesenchymal stem cells derived from synovium of rat was implanted into the bone defect of rats. Callus formation was observed as early as 2 weeks after implantation. And enhancement of callus formation was confirmed with implantation of a sheet onto which mesenchymal stem cells were subcultured.
Bone hole (6 mm diameter, 10 mm depth) was drilled into the lateral femoral or tibial condyle, and HAp/Col sheet of 0.5, 0.9, 1.5 and 3mm thickness were implanted with combination of various thickness and pagination. In the cases that the total number of the sheet was less or the thickness of sheets were thin, although both degradation of the sheets and new bone formation occurred rapidly, bone absorption speed was also fast. When thickness of each grafted sheet was thick, however, degradation of the composites delayed and it took long time for replacement to newly formed bone. In conclusion, it is recommend that the sheet of 1.0 or 1.5 mm thickness is packed as many as bone defect size for bringing balance of material degradation and newly bone formation. Less