两种壳聚糖载rhBMP-2纳米微球对SD大鼠体内异位成骨的影响
余翔,夏远军,郑晓辉,章莹,陈泽鹏,夏虹,尹庆水
中国临床解剖学杂志 ›› 2016, Vol. 34 ›› Issue (4) : 412-418.
两种壳聚糖载rhBMP-2纳米微球对SD大鼠体内异位成骨的影响
Ectopic osteogenesis evaluation of two kind of chitosan rhBMP-2 loaded microspheres in SD rats
目的 通过SD大鼠异位成骨实验来探究重组人骨形态发生蛋白-2/壳聚糖/硫酸葡聚糖(rhBMP-2/CS/DS) 复合微球和重组人骨形态发生蛋白-2/壳聚糖 (rhBMP-2/CS) 微球对SD大鼠体内异位成骨的影响。 方法 随机将36只SD大鼠平均分为三组（n=12），分别为A组 (rhBMP-2), B组(rhBMP-2/CS), C组(rhBMP-2/CS/DS)。制备股四头肌肌袋模型后，分别将三种材料植入股四头肌肌袋肌间隙中。分别在4，8和12周时大体观察植入区组织硬度，每组处死4只大鼠后取出异位骨块，并切取异位骨化的组织行micro-CT扫描及 Mimics软件三维重建；检测各组织块骨体积分数（bone volume fraction,BVF）、骨小梁厚度（trabecular thichness,Tb.Th）、骨密度（bone mineral density,BMD）；并行组织学观察和ALP活性、钙含量检测。 结果 4周时，A、B、C三组植入区周围组织质地均稍硬，三者并无明显区别；8周和12周时，三组植入区硬度明显增加，且C组比A、B组质地更硬。4周时，HE染色可见三组有少量骨组织形成，但不明显；B、C两组BVF、Tb.Th、BMD，碱性磷酸酶（ALP）活性、钙含量均高于A组；B、C两组以上指标差异无统计学意义。8、12周时，HE染色可见到三组骨组织逐渐增多，并逐渐成熟，且B、C两组可见到比A组更成熟的骨组织，C组骨组织比B组更成熟；B、C两组BVF、Tb.Th、BMD，ALP活性、钙含量均高于A组，C组以上指标均高于B组。 结论 rhBMP-2/CS/DS纳米缓释微球的成骨效果明显强于rhBMP-2/CS纳米微球和单独rhBMP-2，其可能在骨组织工程领域有较好的运用前景。
Objective To explore the ectopic osteogenesis introducing ability of recombinant human bone morphogenetic protein-2/chitosan/dextran sulfate composite microspheres and recombinant human bone morphogenetic protein-2/chitosan composite microspheres on SD rats. Method 36 SD rats were randomly divided into three groups, in which the quadriceps muscle bag model of the rats were established; the bags were implanted respectively with rhBMP-2(group A), CS/DS microspheres (group B), and rhBMP-2/CS/DS microspheres (group C). Hardness of the implanted regions tissuesat the 4th,8th,12th weekend were observed, and four rats were sacrificed in each group, from which the ectopic bone tissues were harvested. The bone formation situation of plantation areas were generally observed, and the heterotopic ossification organization tissues were scanned by micro-CT, and were three-dimensionally reconstructed by using the Mimics software；parameters such astissue bone volume fraction (BVF), trabecular thickness (Tb.Th), bone mineral density (BMD) were detected; Some heterotopic ossification organization tissues were taken for histological observation and analysis of ALP activity and calcium content. Results At the 4th weekend, tissues around implanted regions of each group had slightly harder texture, and there was no significant difference among the three groups; at the 8th and 12th weekend, hardness of tissues around implanted regions of each group increased significantly, and the group C had harder tissues formed than group A and B. At the 4th weekend, HE staining showed that the three groups had a small amount of bone tissue formation, but not obviously; the BVF, Tb.Th, BMD, ALP activity and calcium levels of B, C groups were significantly higher than group A; there were no statistically significance on the above indicators of group B and C. At the 8th and 12th weekend, gradually-increased and marture bone tissues could be seen in the three groups under HE staining. However,there were more mature ectopic bone tissues in group B and C than group A; And the bone tissues of group C were more mature than group B. Meanwhile, the BVF, Tb.Th, BMD, ALP activity and calcium levels of group B and C were significantly higher than group A, and the above-mentioned indicators of group C were higher than that of group B. Conclusion rhBMP-2/CS/DS microspheres have stronger ability of inducing ectopic bone than rhBMP-2/CS microspheres and single rhBMP-2, and it mayhave good prospects for applicationin bone tissue engineering.
重组人骨形态发生蛋白-2 / 壳聚糖 / 硫酸葡聚糖 / 异位成骨 / Micro-CT / 三维重建
Bone morphogenetic proteins-2 / Chitosan / Dextran sulfate / Ectopic osteogenesis / Micro-CT / Three-dimensional reconstruction
[1] Urist MR. Bone formation by autoinduction[J].Science,1965,150(698):893-899.
[2] Martínez A, Arana P, Fernandez A, et al. Synthesis and characterisation of alginate/chitosan nanoparticles as tamoxifen controlled delivery systems[J].J Microencapsul, 2013,30(4):398-408.
[3] 杨强, 彭江, 卢世璧, 等. 软骨细胞外基质与壳聚糖复合制备组织工程软骨支架及其性能研究[J].中华骨科杂志, 2011, 31(8):904-910.
[4] Wink JD, Gerety PA, Sherif RD, et al. Sustained delivery of rhBMP-2 by means of Poly(Lactic-co-Glycolic Acid) microspheres: cranial bone regeneration without heterotopic ossification or craniosynostosis[J]. Plast Reconstr Surg, 2014, 134(1): 51-59.
[5] 王玮, 尹庆水, 张余. 重组人骨形态发生蛋白-2壳聚糖纳米微球的制备及检测[J].中国骨与关节损伤杂志, 2011，26(7):598-600.
[6] 夏远军, 章莹, 李丽华, 等. 重组人骨形态发生蛋白-2/壳聚糖/硫酸葡聚糖复合微球对SD大鼠骨髓基质干细胞诱导及分化作用实验研究[J].中华创伤骨科志, 2014, 16(5):421-426.
[7] 夏远军,余翔,章莹,等.BMP-2/壳聚糖/硫酸葡聚糖复合微球诱导异位成骨的Micro-CT评价[J].中国修复重建外科杂志, 2016, 30(3):286-291.
[8] Jun SH, Lee EJ, Jang TS, et al. Bone morphogenic protein-2 (BMP-2) loaded hybrid coating on porous hydroxyapatite scaffolds for bone tissue engineering[J].J Mater Sci Mater Med，2013, 24(3):773-782.
[9] Kanczler JM, Ginty PJ, Barry JA, et al. The effect of mesenchymal populations and vascular endothelial growth factor delivered from biodegradable polymer scaffolds on bone formation[J]. Biomaterials, 2008, 29(12): 1892-1900.
[10]Huang D, Zuo Y, Zou Q, et al. Reinforced nanohydroxyapatite/polyamide66 scaffolds by chitosan coating for bone tissue engineering[J]. J Biomed Mater Res B Appl Biomater, 2012，100(1):51-57.
[11]Palazzo B, Gallo A, Casillo A, et al. Fabrication, characterization and cell cultures on a novel composite chitosan-nano-hydroxyapatite scaffold[J].Int J Immunopathol Pharmacol, 2011, 24(1 Suppl 2):73-78.
[12]Patel ZS, Ya mamoto M, Ueda H, et al. Biodegradable gelatin microparticles as deliveiy systems for the controlled release of bone morphogenetic Protein-2[J]. Acta Bioniater, 2008, 4(5):l 126-1138.
[13]Guliyeva U, Oner F, Ozsoy S, et al. Chitosan microparticles containing plasmid DNA as potential oral gene delivery system[J].Eur J Pharm Biopharm, 2006, 62(1):17-25.
[14] Shah RK, Moncayo VM, Smitson RD, et al. Recombinant human bone morphogenetic protein 2-induced heterotopic ossification of the retroperitoneum, psoas muscle, pelvis and abdominal wall following lumbar spinal fusion[J]. Skeletal Radiol, 2010, 39(5):501-504.
[15] Inoda H, Yanmmoto G, Hattori T. rhBMP2 induced ectopic bone for grafting critical size defects: a preliminary histological evaluation in rat calvariae[J]. Int J Oral Maxillofac Surg, 2007, 36(1): 39-44.
[16]Iwata H, Sakano S, Itoh T, et al. Demineralized bone matrix and native bone morphgenetic protein in orthpadic surgery[J] .Clin Orthop, 2002, 395:99-109.
[17]Nakagawa T, Tagawa T. UItrastructural study of direct bone formation induced by Uitrastructural study of direct bone formation induced by BMPs-collagen complex implanted into an ecropic site[J].Oral Dis, 2000, 6(3):172-179.
[18]Vehof JW, Takita H, Kuboki Y, et al. Histological characterization of the carly stages of bone morphpgenetic protein-induced osteogencsis[J].J Biomed Mater Res, 2002, 61(3):440-449.
[19] Sullad AG，Manjeshwar LS，Aminabhavi TM. Novel Semi-interpenetrating microspheres of dextran-grafted-acrylamide and poly(Vinyl Alcohol) for controlled release of abacavir sulfate[J]. Indu strial & Engineering Chemistry Research, 2011, 50(21): 11778-11784.
[20] Gribova V, Crouzier T, Picart C. A material’s point of view on recent developments of polymeric biomaterials: control of mechanical and biochemical properties[J]. J Mater Chem, 2011, 21(38), 14354-14364.
[21]Lee KY, Mooney DJ. Alginate: properties and biomedical applications[J]. Prog Polym Sci, 2012, 37(1):106-126.
[22] Wang M, Abbah SA, Hu T，et al. Minimizing the severity of rhBMP-2-induced inflammation and heterotopic ossification with a polyelectrolyte carrier incorporating heparin on microbead templates[J]. Spine, 2013, 38(17):1452-1458.
[23]Hudalla GA, Murphy WL. Biomaterials that regulate growth factor activity via bioinspired interactions[J]. Adv Funct Mater, 2011, 21(10):1754-1768 .
国家自然科学基金青年基金资助项目（51303031）；广东省自然科学基金资助项目（S2012010009743）；广东省应用基础研究计划项目（2013J4100120）
京ICP备08002354号-3/
| 〈 |
|
〉 |
