壳聚糖／硫酸葡聚糖／重组人骨形态发生蛋白-2微球诱导异位成骨的实验研究

Ectopic osteogenesis induced by microspheres of chiston/dextran sulfate/recombinant human bone morphogenetic protein-2

【摘要】目的探讨壳聚糖／硫酸葡聚糖／重组人骨形态发生蛋白-2（CS／DS／rhBMP-2）异位诱导成骨能力。方法将48只SD大鼠随机分为4组，制作大鼠左大腿股四头肌肌袋模型，分别将明胶海绵、CS／DS微球、rhBMP-2和CS／DS／rhBMP．2微球适当压缩后分别植入股四头肌肌袋肌间隙中，并于4、8、12、16周后取材行苏木精-伊红（HE）染色、碱性磷酸酶（ALP）活性与钙含量检测；植入后第16周行X线片检查。结果①组织学切片：植入后第4周，4组均无明显骨组织形成；植入后第8周，CS／DS／rhBMP-2微球组可见少量骨细胞、骨髓，骨小梁转化为成熟的骨质，骨小梁间有骨髓腔和骨髓样物质形成；rhBMP-2组可见少量骨小梁及骨细胞，骨细胞周围见少量成骨细胞及软骨细胞，但骨组织不及CS／DS／rhBMP-2微球组成熟。植入后第12、16周，rhBMP-2组和CS／DS／rhBMP-2微球组均有成熟的骨组织形成，骨小梁中可见骨陷窝，骨陷窝中有骨细胞，骨细胞周围有大量成骨细胞。但CS／DS／rhBMP-2微球组骨组织较rhBMP-2组成熟，骨陷窝及骨小梁量均多于rhBMP．2组；明胶海绵组及CS／DS微球组无明显骨组织形成。②ALP活性及钙含量检测：除第4周外，第8、12、16周CS／DS／rhBMP-2组纳米微球的ALP活性及钙含量均高于rhBMP-2组，差异有统计学意义（P〈0．05）。（3）X线检查：植入后第16周，CS／DS／rhBMP．2微球组及rhBMP-2组植入部位均有X线不透性物质，密度较均匀，CS／DS／rhBMP．2微球组阻射密度与周围股骨接近，rhBMP-2组阻射密度较周围股骨稍低，CS／DS／rhBMP-2微球组高密度区面积明显较rhBMP-2组大，明胶海绵组及CS／DS微球组无高密度组织形成。结论CS／DS／rhBMP-2复合微球异位成骨能力较单独rhBPM-2强，可能有助于CS／DS／rhBMP-2复合微球在骨组织工程领域的应用。

Objective To explore the capacity of microspheres of chiston/dextran sulfate/recom- binant human bone morphogenetic protein-2 （CS/DS/rhBMP-2） to induce ectopic osteogenesis. Methods Quadriceps muscle bags were created at the left legs in 48 SD rats which were randomly divided into 4 groups. Gelatin sponge, CS/DS, rhBMP-2 and CS/DS/rhBMP-2 microspheres were implanted into the muscle bags of the 4 groups respectively. At 4, 8, 12 and 16 weeks after implantation, samples were har- vested for examinations of histology by hematoxylin-eosin （HE） staining, alkaline phosphatase （ALP） activity and calcium content. An X-ray check was done at the 16th weekend. Results At 4 weeks, no obvious bone tissue formed in the 4 groups. At 8 weeks, in CS/DS/rhBMP-2 group, a small number of bone cells were observed, bone marrow formed, trabecular bone turned into mature sclerotin, and bone marrow and bone-like materials formed between the trabecular bones. In rhBMP-2 group, some trabecular bone and bone cells were seen, there were a small number of osteoblasts and chondrocytes around the bone cells, but the bone tissues were less mature than in CS/DS/rhBMP-2 group. At 12 and 16 weeks in CS/DS/rhBMP-2 and rhBMP-2 groups, mature bone tissues were seen, and on bone pits in trabecular bone there were bone cells around which a large number of osteoblasts appeared. The bone tissues in CS/DS/rhBMP-2 group were more mature than in rhBMP-2 group, and there were more bone lacunas and bone trabeculae in CS/DS/rhBMP-2 group. No obvious bone tissues formed in gelatin sponge or CS/DS groups. At 8, 12 and 16 weeks, the ALP activity and calcium content in CS/DS/rhBMP-2 group were significantly higher than in rhBMP-2 group （P 〈 0. 05）. X-ray check at 16 weeks revealed radio-opaque substance with uniform density at the implantation sites in both CS/DS/rhBMP-2 and rhBMP-2 groups. The radiation density of CS/DS/rhBMP-2 microspheres was close to that of nearby femur while the radiation density of rhBMP-2 microspheres was lower than that of nearby femur. The high density radiation area in CS/DS/rhBMP-2 group was larger than in rhBMP-2 group. No high density tissues formed in gelatin sponge or CS/DS group. Conclusion Since CS/DS/rhBMP-2 microspheres are more capable of inducing ectopic bone formation than rhBPM-2 ones, they may have a promising prospect of application in bone tissue engineering.