摘要
目的:前期实验应用过氧化氢-乙醚法已成功将天然骨制成脱蛋白骨支架材料,进一步观察脱蛋白骨支架材料与成骨细胞在体外的生物相容性,旨在为组织工程骨构建提供良好的支架材料。方法:实验于2003-01/2005-12在重庆医科大学实验动物中心进行。①酶消化-组织块联合培养法体外培养胎兔颅骨成骨细胞:取新西兰大白兔胎兔颅骨骨块,制成2mm×2mm骨块,Ⅰ型胶原酶室温下消化。再制成1mm×1mm骨块并孵育,待细胞爬满骨块间隙后传代,行细胞形态学观察、碱性磷酸酶染色、钙化结节染色等方法行鉴定。将第3代成骨细胞复苏培养,制成2×1010L-1的细胞悬液备用。②将成年兔干骺端松质骨用过氧化氢-乙醚法制成脱蛋白骨。将备用细胞悬液均匀浸滴在脱蛋白骨上,使细胞悬液渗入材料孔隙内,并行体外复合培养。通过酶消化法、倒置显微镜及扫描电镜了解脱蛋白骨的细胞相容性。结果:①酶消化-组织块联合培养法成功获得胎兔颅骨成骨细胞:倒置显微镜观察示原代细胞呈不规则多边形,传代细胞均为长梭状或长不规则多边型,碱性磷酸酶化学染色示大多数细胞染色呈黑色阳性,钙结节染色示大量黑色的矿化结节分布在细胞外基质。②成骨细胞与脱蛋白骨材料体外复合培养后第3日进入对数生长期;倒置相差显微镜可见复合培养后第7日成骨细胞数量有明显增加,部分细胞出现连接生长;扫描电镜证实复合培养后第7日可见连接成片生长的成骨细胞。结论:过氧化氢-乙醚法制备的脱蛋白骨具有良好的细胞相容性,可作为组织工程骨的支架材料。
AIM： Natural bone has been made into deproteinized bone （DPB） successfully by hydrogen dioxide and diethyl ether method. In this study, the cellular compatibility of DPB as bio-scaffold co-cultured with osteoblast in vitro was investigated, so as to provide good scaffold material for bone construction in tissue engineering.
METHODS： The experiment was conducted in the Experimental Animal Center of Chongqing University of Medical Sciences from January 2003 to December 2005.①Osteoblasts were collected from the rabbit fetus skull by collagenase digestion associated with tissue nubbles cultivation and cultured in vitro. The skull of New Zealand White fetus rabbit was cut into 2 mm ×2 mm bone blocks which were digested with collagenase I at room temperature. Then the bone blocks were cut into 1 mm ×1 mm smaller ones and incubated. After the cultured cells covered the block interspace, cells were subcultivated and identified by morphological characteristics, by alkaline phosphatase cytochemistry staining and calcium tubercle staining. The third generation cells were thawed and cultured, subsequently suspended at a concentration of 2× 10^10/L for implantation. ②The fresh metaphyseal cancellous bone of adult New Zealand white rabbits was processed into DPBs with hydrogen dioxide and diethyl ether. The DPB scaffolds were immersed by prepared cell suspension then co-cultured subsequently in vitro. The cellular compatibility of DPB was detected by enzyme digestion, inverted phase contrast microscopy and scanning electron microscopy.
RESULTS：①Osteoblasts were obtained successfully from the rabbit fetus skull by collagenase digestion associated with tissue nubbles cultivation. Primary cells presented irregular polygonal, while all subcultivated ones were long spindle-shaped or long irregular polygonal under inverted phase contrast microscope. Most cells were positive black in alkaline phosphatase cytochemistry staining test and considerable black calcified nudes distributed in extracellular matrix after calcium tubercle staining. ②The logarithmic growth phase of osteoblasts started at the 3^rd day after the co-culture in vitro. Under inverted phase contrast microscope, osteoblasts increased obviously accompanied with discovery of cellular connective development at the 7^th day after implantation. Under scanning electromicroscope, obvious connective and fused development form of osteoblasts was discovered at the 7^th day after implantation.
CONCLUSION： DPB prepared with hydrogen dioxide and diethyl ether processes satisfactory cellular compatibility, and it could serve as bio-scaffold of tissue engineered bone.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第35期6938-6941,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
