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Development of the artificial bone using a novel biomaterial HAp/Col composite and PLLA

使用新型生物材料 HAp/Col 复合材料和 PLLA 开发人造骨

基本信息

批准号：
13557126
负责人：
ITOH Soichiro
金额：
$ 8.83万
依托单位：
Tokyo Medical and Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2004
项目状态：
已结题

项目摘要

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, … More 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

制备了羟基磷灰石/ I型胶原（HAp/Col）复合材料，其中羟基磷灰石纳米晶体沿胶原分子排列。本研究将复合材料植入比格犬胫骨骨缺损。组织学检测显示其表面有成骨细胞，破骨细胞出现在复合材料表面形成的Howship氏腔隙样结构中。提示HAp/Col具有非常高的骨传导活性，能够诱导骨重塑单位。HAp/Col植入物用于小猎犬C3和C4椎体的前路融合。为了防止移位，种植体用PLLA钢板和钛螺钉固定。提示含有rhBMP-2的植入物可缩短骨融合时间，该人工椎体系统可用于颈椎垂直应力前路融合。为了提高HAp/Col复合材料对骨的替代速度，开发了多孔体和多孔板。HAp/Col复合材料多孔体具有弹性、塑性和良好的渗透性。此外，培养的细胞在其上传代培养时，可以很容易地渗透到多孔体中。在SD大鼠股骨远端穿孔直径3mm的骨孔，植入复合材料多孔体，评价其导骨活性。组织学结果显示，早在手术2周后，新骨形成复合材料的中心部分，并且trap阳性细胞，破骨细胞，附着在退化的复合材料和新形成的骨上。骨上还可见到alp阳性细胞、成骨细胞。4周后，几乎整个复合材料被替换成新形成的骨。这些结果表明，HAp/Col的多孔体在植入后被破骨细胞活性迅速吸收，并被成骨细胞新形成的骨所取代。将HAp/Col纤维在pH为7.4、温度为37℃的磷酸盐缓冲盐水（PBS）中沉淀，压缩脱水成0.5、0.9、1.5和3mm厚度的薄片。从小鼠颅骨中获取成骨细胞，从Fischer大鼠股骨中获取骨髓细胞。在含有15%胎牛血清的Eagle-minimum标准培养基中，37℃培养。P1后用腺病毒载体将LacZ基因转移到骨髓细胞中，以10^6个细胞/ml的浓度传代到0.5或0.9 mm厚度的HAp/Col复合片上。两种类型的细胞都在薄片上存活，表明它适合于培养细胞支架。制备长度为6mm的骨缺损，用骨外固定架固定。将大鼠滑膜间充质干细胞的HAp/Col片植入大鼠骨缺损。早在植入后2周就观察到愈伤组织的形成。移植间充质干细胞后，证实了愈伤组织形成的增强。在股骨外侧髁或胫骨外侧髁上钻孔（直径6 mm，深度10 mm），分别植入0.5、0.9、1.5、3mm厚度的HAp/Col片，并采用不同厚度、不同排列组合。在骨片总数较少或骨片厚度较薄的情况下，虽然骨片的降解和新骨的形成都发生得很快，但骨吸收速度也很快。然而，当每个移植片的厚度较厚时，复合材料的降解延迟，并且需要较长的时间来替换新形成的骨。综上所述，为了平衡材料降解和新骨形成，建议将1.0或1.5 mm厚度的骨片填入骨缺损尺寸。少