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

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沿着胶原分子排列。在我们前期的研究中，将复合材料植入Beagle犬胫骨骨缺损中。组织学观察发现，复合材料表面沿着有成骨细胞，破骨细胞出现在复合材料表面形成的Howship骨陷窝样结构中。HAp/Col复合物具有很强的骨传导活性，能诱导骨重建单位的形成。为了防止移位，使用PLLA接骨板和钛螺钉固定植入物。结果表明，含rhBMP-2的植入物可缩短骨融合时间，该人工椎体系统可用于垂直应力型颈椎前路融合。为了提高HAp/Col复合材料对骨的置换速度， 关于我们 HAp/Col复合材料多孔体具有弹性、塑性和良好的透气性。在SD大鼠股骨远端钻3 mm直径的骨孔，植入复合材料多孔体，评价其骨传导活性。组织学观察显示，术后2周，新骨形成，TRAP阳性破骨细胞附着于退变的复合材料和新生骨上。骨组织中还可见ALP阳性细胞、成骨细胞排列。4周后，复合材料几乎全部被新骨取代。在pH 7.4、37℃的磷酸盐缓冲液（PBS）中沉淀HAp/Col纤维，经压缩脱水后形成厚度为0.5、0.9、1.5和3 mm的片状纤维。成骨细胞从小鼠颅骨收获，骨髓细胞从Fischer大鼠的股骨获得。在含15%胎牛血清的最低谷氨酰胺标准培养基中37℃培养。P1后用腺病毒载体将LacZ基因转入骨髓细胞，并以10^6个/ml的浓度在0.5或0.9 mm厚的HAp/Col片复合物上传代培养。制备6 mm长的骨缺损，用骨外固定器固定。将含有或不含有大鼠滑膜间充质干细胞的HAp/Col片植入大鼠骨缺损处。早在植入后2周就观察到愈伤组织形成。在股骨或胫骨外侧髁上钻孔（直径6 mm，深10 mm），植入0.5、0.9、1.5和3 mm厚的HAp/Col片（不同厚度和页码的组合）。在片材总数较少或片材厚度较薄的情况下，尽管片材的降解和新骨形成都发生得很快，但骨吸收速度也很快。当移植片厚度较厚时，复合材料的降解延迟，需要较长的时间才能替换成新形成的骨。总之，建议将1.0或1.5 mm厚的片材填充与骨缺损尺寸一样多，以平衡材料降解和新骨形成。少

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