BMP-2活性肽/纳米晶胶原基骨修复材料复合移植修复大鼠颅骨缺损的实验研究
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摘要
【目的】探讨自行研发合成的BMP-2活性多肽与纳米晶胶原基骨修复材料复合移植促进大鼠颅骨缺损修复的有效性与实用性,并对比分析其与已商业化的rhBMP2生物学效应。
【方法】采用固相合成法合成BMP-2活性多肽P24,并与rhBMP-2分别通过冷冻干燥法真空吸附到nHAC多孔支架材料上。动物实验分为四组。A组:5mg P24/ nHAC复合材料;B组:3μg rhBMP2/ nHAC复合材料:C组:空白nHAC材料;D组:单纯缺损。取19只大白鼠随机分配到四组中,建立直径为5mm的大鼠颅骨缺损模型,分别将材料植入颅骨缺损处,术后6周、12周分别处死各组动物,分别进行X线、CT三维扫描及组织学检查,并进行骨缺损修复面积测定,比较各组材料的成骨作用。
【结果】①6周X线, A、B组骨缺损可见小片高密度影;C组骨缺损边缘有轻微高密度影;D组缺损无改变。12周X线, A、B组骨缺损有大片高密度影,B组基本修复缺损;C组骨缺损高密度影有所增强,但未能修复;D组缺损边缘可见极少量高密度影。②CT三维重建:6周时A、B组可见部分高密度钙化影,断层面少许骨桥连接;C组边缘少量成骨,断层面未见骨桥生成。12周时A组骨缺损已大部修复;B组骨缺损已完全修复,断层面可见缺损由骨桥完全连接;C组周边有部分新骨形成,断层面骨桥未连接缺损;D组缺损未见明显成骨。骨缺损处高密度影测定,A、B组与C、D组相比均有极显著差异(p<0.05),而A、B组成骨量则近似(p>0.05)。③组织学检查:6周时A、B组可见少量新生骨组织和活跃的成骨细胞;C组材料少许降解,无明显炎症反应。12周时A、B组材料基本降解,有骨重建过程,但A组成骨量少于B组;C组缺损区有大量纤维结缔组织,有少量新生骨在材料周边发现。
【结论】①P24/ nHAC复合材料促进骨缺损修复能力明显强于空白的nHAC,略低于3ug剂量的rhBMP2/ nHAC复合材料。②nHAC是一种理想的生物支架和缓释材料。③P24/ nHAC复合材料是一种理想的具有稳定骨诱导活性的新型骨缺损修复材料。
Objective: To investigate the osteogenetic capacity of bone repair in rat cranial bone defects by nHAC loading with a BMP-2-derived peptide P24 biomimetic scaffold materials.
Methods:19 male Sprague–Dawley rats were divided into four groups. Five-millimeter critical-size cranial bone defects were created in each one. The defects were treated with P24/nHAC scaffold (A group), rhBMP-2/ nHAC scaffold (B group), nHAC scaffold (C group)and only bone defect(D group). Up to the 6th and 12th weeks, the rats were sacrificed in batch respectively. Defects were evaluated by X-ray、three-dimensional reconctruction of computer tomography、histology and the percentage of bone formation areas.
Results:①Radiological examination indicated that there were some flaky radiodense areas in A and B gruoups of each rat, but the density of the radiodense areas of C group were obviously lower than the density of A and B groups at the 6th week.At the 12 week,the radiodense areas of C group were denser than the 6th week,but defects not repaired. The defects nearly healed at the 12th week in the A and B groups. A trifle of radiodense areas were discovered in the margin of defects in the D group.②Three-dimensional reconstruction of computed tomography indicated that there were fewer radiodense areas and not saw bone bridge in the C group, but the density of the radiodense areas of A and B groups were obviously denser than the density of C group and bone brige was discovered at the 6th week. The defects completely healed in B group and nearly reparied in A group that was treated with BMP-2- derived peptide loaded nHAC at the 12th week ,while not saw defects were connected by bone bridge in C group and D group. The percentages of the regenerated areas in C and D group were significantly lower than A group and B group at 6 and 12 weeks (p<0.05),but no significant difference between A group and B group(p>0.05).③Histological examination: At 6 weeks post-surgery, Small amount of composite in group C degraded, few of inflammation; and A group and B group showed some new bone tissue and osteoblasts. At 12 weeks, in the A group and B group, the bony-union between new bone and host bone was observed. Meanwhile, the composite was almost completely degraded. In the C group, there were still slight amounts of new bone, the scaffolds were only partly degraded and the residual materials were surrounded by areas of new bone formation.
Conclusion:①The osteogenic capability of BMP-2- derived peptide loaded nHAC is obviously superior to nHAC alone.②nHAC is an ideal scaffold and a sustained release carrier.③It is suggested that P24/ n nHAC biomimetic scaffold material can be a ideal and steady repairing material for bone defects.
【方法】采用固相合成法合成BMP-2活性多肽P24,并与rhBMP-2分别通过冷冻干燥法真空吸附到nHAC多孔支架材料上。动物实验分为四组。A组:5mg P24/ nHAC复合材料;B组:3μg rhBMP2/ nHAC复合材料:C组:空白nHAC材料;D组:单纯缺损。取19只大白鼠随机分配到四组中,建立直径为5mm的大鼠颅骨缺损模型,分别将材料植入颅骨缺损处,术后6周、12周分别处死各组动物,分别进行X线、CT三维扫描及组织学检查,并进行骨缺损修复面积测定,比较各组材料的成骨作用。
【结果】①6周X线, A、B组骨缺损可见小片高密度影;C组骨缺损边缘有轻微高密度影;D组缺损无改变。12周X线, A、B组骨缺损有大片高密度影,B组基本修复缺损;C组骨缺损高密度影有所增强,但未能修复;D组缺损边缘可见极少量高密度影。②CT三维重建:6周时A、B组可见部分高密度钙化影,断层面少许骨桥连接;C组边缘少量成骨,断层面未见骨桥生成。12周时A组骨缺损已大部修复;B组骨缺损已完全修复,断层面可见缺损由骨桥完全连接;C组周边有部分新骨形成,断层面骨桥未连接缺损;D组缺损未见明显成骨。骨缺损处高密度影测定,A、B组与C、D组相比均有极显著差异(p<0.05),而A、B组成骨量则近似(p>0.05)。③组织学检查:6周时A、B组可见少量新生骨组织和活跃的成骨细胞;C组材料少许降解,无明显炎症反应。12周时A、B组材料基本降解,有骨重建过程,但A组成骨量少于B组;C组缺损区有大量纤维结缔组织,有少量新生骨在材料周边发现。
【结论】①P24/ nHAC复合材料促进骨缺损修复能力明显强于空白的nHAC,略低于3ug剂量的rhBMP2/ nHAC复合材料。②nHAC是一种理想的生物支架和缓释材料。③P24/ nHAC复合材料是一种理想的具有稳定骨诱导活性的新型骨缺损修复材料。
Objective: To investigate the osteogenetic capacity of bone repair in rat cranial bone defects by nHAC loading with a BMP-2-derived peptide P24 biomimetic scaffold materials.
Methods:19 male Sprague–Dawley rats were divided into four groups. Five-millimeter critical-size cranial bone defects were created in each one. The defects were treated with P24/nHAC scaffold (A group), rhBMP-2/ nHAC scaffold (B group), nHAC scaffold (C group)and only bone defect(D group). Up to the 6th and 12th weeks, the rats were sacrificed in batch respectively. Defects were evaluated by X-ray、three-dimensional reconctruction of computer tomography、histology and the percentage of bone formation areas.
Results:①Radiological examination indicated that there were some flaky radiodense areas in A and B gruoups of each rat, but the density of the radiodense areas of C group were obviously lower than the density of A and B groups at the 6th week.At the 12 week,the radiodense areas of C group were denser than the 6th week,but defects not repaired. The defects nearly healed at the 12th week in the A and B groups. A trifle of radiodense areas were discovered in the margin of defects in the D group.②Three-dimensional reconstruction of computed tomography indicated that there were fewer radiodense areas and not saw bone bridge in the C group, but the density of the radiodense areas of A and B groups were obviously denser than the density of C group and bone brige was discovered at the 6th week. The defects completely healed in B group and nearly reparied in A group that was treated with BMP-2- derived peptide loaded nHAC at the 12th week ,while not saw defects were connected by bone bridge in C group and D group. The percentages of the regenerated areas in C and D group were significantly lower than A group and B group at 6 and 12 weeks (p<0.05),but no significant difference between A group and B group(p>0.05).③Histological examination: At 6 weeks post-surgery, Small amount of composite in group C degraded, few of inflammation; and A group and B group showed some new bone tissue and osteoblasts. At 12 weeks, in the A group and B group, the bony-union between new bone and host bone was observed. Meanwhile, the composite was almost completely degraded. In the C group, there were still slight amounts of new bone, the scaffolds were only partly degraded and the residual materials were surrounded by areas of new bone formation.
Conclusion:①The osteogenic capability of BMP-2- derived peptide loaded nHAC is obviously superior to nHAC alone.②nHAC is an ideal scaffold and a sustained release carrier.③It is suggested that P24/ n nHAC biomimetic scaffold material can be a ideal and steady repairing material for bone defects.
引文
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2. DeLuca L., Raszewski R., Tresser N, et al. The fate of preserved autogenous bone graft. Plast. Reconstructr. Surg,1997,99:1324–1328.
3. El-Ghannam A.Bone reconstruction: from bioceramics to tissue engineering. Expert Rev Med Devices. 2005 Jan;2(1):87-101.
4. Khan Y, Yaszemski MJ, Mikos AG,et al. Tissue engineering of bone: material and matrix considerations. J Bone Joint Surg Am. 2008 Feb;90 Suppl 1:36-42.
5.Clemens Scheufler, Walter Sebald, Martin Huèlsmeyer.Crystal Structure of Human Bone Morphogenetic Protein-2 at 2.7 AêResolution. J. Mol. Biol, 1999;287:103-115.
6.David M,Patrical A,James M,et.al. Sequential actions of BMP receptors control neural precursor cell production and fate.Genes Dev,2001,15:2094-2110.
7.Chun-Ya E. Han , Youping Wang, Longchuan Yu , et.al. Small molecules with potent osteogenic-inducing activity in osteoblast cells.Bioorganic & Medicinal Chemistry Letters , 2009, 19 :1442–1445.
8.Milan Milovancev, Peter Muir, Paula Manley, et al. Clinical Application of Recombinant Human Bone Morphogenetic Protein-2 in 4 Dogs. Journal of The American College of Veterinary Surgeons, 2007,36:132-140.
9.Seeherman H, Bouxsein M, Kim H, et al: rhBMP-2/calcium phosphate matrix accelerates osteotomy-site healing in a non-human primate model at multiple treatment times and concentrations. J Bone Joint Surg Am ,2006,88:144–160.
10.Zhao B, Katagiri T, Toyoda H, Takada T, Yanai T, Fukuda T, et al. Heparinpotentiates the in vivo ectopic bone formation induced by bone morphogenetic protein-2. J Biol Chem,2006,281(32):23246–53.
11.Kirsch T, Sebald W, Dreyer MK. Crystal structure of the BMP2-BRIA ectodomain complex. Nat struct Biol,2000, 7(6):492-296.
12.Dionys Weber, Alexander Kotzsch, Joachim Nickel,et,al. A silent H-bond can be mutationally activated for high-affinity interaction of BMP-2 and activin type IIB receptor. BMC Structural Biology 2007, 7:6 1472-6807-7-6
13. Bin Wu, Qixin Zheng, Xiaodong Guo, et al. Preparation of scaffold based on mineralized recombinant human-like collagen laoding with synthetic BMP-2-derived peptide and its ectopic osteogenesis in vivo. Biomed Mater. 2008 Dec;3(4):44111.
14.Duan Z, Zheng Q, Guo X, et al. Experimental research on ectopic osteogenesis of BMP-2-derived peptide P24 combined with PLGA copolymers. J Huazhong Univ Sci Technolog Med Sci. 2007 Apr;27(2):179-82.
15.Riminucci M, Bianco P. Building bone tissue: matrices and scaffolds in physiology and biotechnology. Braz J Med Biol Res, 2003, 36(8): 1027-1036.
16.廖素三,崔福斋,张伟.组织工程中胶原基纳米骨复合材料的研制.中国医学科学院学报,2003,25(1):36-38
17.Urist MR. Bone formation by autoinduction. Science,1965;150:893–899.
18.Wozney JM, Rosen V, Celeste AJ, et al. Novel regulators of bone formation: Molecular clones and activities. Science, 1988,242:1528–1534.
19.Tsumaki, Noriyuk6; Yoshikawa, Hideki. The role of bone morphogenetic proteins in endochondral bone formation. Cytokine and Growth Factor Reviews, 2005, 16(3): 279-285.
20. Wozney J,Seeherman H. Protein-based tissue engineering in bone andcartilage repair. Curr Opin Biotechnol,2004,15(5):392-398.
21. Boden SD. The ABCs of BMPs. Orthop Nurs,2005,24(1):49-54.
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