载BMP2活性肽PLGA-NHAC仿生骨基质材料修复大鼠颅骨缺损的实验研究
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摘要
【目的】探讨自行研制的BMP2活性多肽与纳米羟基磷灰石胶原仿生骨材料修复大鼠颅骨缺损的效果,以及BMP2活性多肽与重组人骨形态发生蛋白2的诱导成骨活性的初步比较。
【方法】通过材料学生物自组装技术PLGA-NHAC复合材料,同时用固相合成法合成BMP2活性多肽P24。将P24和rhBMP2分别通过真空吸附到复合材料上。动物实验随机分为三组,制备5mm颅骨缺损模型,分别接受空白PLGA-NHAC材料(A组),3ug rhBMP2/PLGA-NHAC(B组), 3mg P24/PLGA-NHAC(C组)。于6、12周每组各处死5只大鼠,取颅骨行X线、CT三维扫描及组织学检查,并行放射计量学和灰度值测定。比较各组骨缺损区新骨形成、炎症及材料降解情况。
【结果】①12周X线骨缺损修复面积测定,A组与B组、C组均有明显差异(α<0.05),而B组、C组成骨量则近似(α>0.05)。灰度值的结果也存在同样的差异。②CT扫描:6周时A组未见成骨,B组、C组见部分高密度钙化影。12周时A组周边有部分新骨形成,B组、C组骨缺损已大部修复。③组织学检查:6周时A组材料少量降解,无炎症反应,B组、C组可见新生骨组织和大量成骨细胞。12周时A组缺损区纤维结缔组织密集,有少量新生骨在材料周边发现,B组、C组材料基本降解,有骨重建过程,但C组成骨量少于B组。
【结论】①P24多肽具备很强的诱导成骨活性和稳定性。②PLGA-NHAC是理想的生物支架和缓释载体。③P24/PLGA-NHAC系统是潜在的临床骨缺损修复基质材料。
Objective: To investigate the efficiency of bone repair in rat calvarial defects by Biomimetic hydroxyapatite-collagan complex carrier/BMP2-derived peptide P24 system, and the preliminary comparison of bioactivity between bmp2-derived peptide P24 and rhBMP2
Methods: Five-millimeter critical-size calvarial defects were created in 30 male Sprague–Dawley rats. The animals were divided into three groups of 10 animals each. The defects were treated with PLGA-NHAC (A group), rhBMP-2/PLGA-NHAC (B group), P24/PLGA-NHAC (C group). Defects were evaluated by X-ray, radiodensity, CT scan, histology, following a 6- and 12-week healing interval (5 animals/group/healing intervals).
Results:①Radiological examination: The percentages of the regenerated areas in A group were significantly lower than B group and C group at 12 weeks (α<0.05),but no significant difference between B group and C group(α>0.05).The comparison of the gray levels also shows the difference.②Histological examination: At 6 weeks post-surgery, Small amount of biomimetic material in group A degraded, free of inflammation; and B group and C group show a large number of new bone tissue and osteoblasts. At 12 weeks, the defect sites were filled with dense connective tissue and small particles of residual PLGA-NHAC, minimal new bone formation was observed in group A. The quantity of the newly formed bone was greater than that observed at 6 weeks in B and C group, and the bone showed more advanced stages of remodeling and consolidation.
Conclusion: P24 peptide is with strong osteoinductive activity and stability in vivo, and PLGA-NHAC is an ideal scaffold and a sustained release carrier. It is suggested that P24/PLGA-NHAC system may be a available matrix material for clinical bone defect.
【方法】通过材料学生物自组装技术PLGA-NHAC复合材料,同时用固相合成法合成BMP2活性多肽P24。将P24和rhBMP2分别通过真空吸附到复合材料上。动物实验随机分为三组,制备5mm颅骨缺损模型,分别接受空白PLGA-NHAC材料(A组),3ug rhBMP2/PLGA-NHAC(B组), 3mg P24/PLGA-NHAC(C组)。于6、12周每组各处死5只大鼠,取颅骨行X线、CT三维扫描及组织学检查,并行放射计量学和灰度值测定。比较各组骨缺损区新骨形成、炎症及材料降解情况。
【结果】①12周X线骨缺损修复面积测定,A组与B组、C组均有明显差异(α<0.05),而B组、C组成骨量则近似(α>0.05)。灰度值的结果也存在同样的差异。②CT扫描:6周时A组未见成骨,B组、C组见部分高密度钙化影。12周时A组周边有部分新骨形成,B组、C组骨缺损已大部修复。③组织学检查:6周时A组材料少量降解,无炎症反应,B组、C组可见新生骨组织和大量成骨细胞。12周时A组缺损区纤维结缔组织密集,有少量新生骨在材料周边发现,B组、C组材料基本降解,有骨重建过程,但C组成骨量少于B组。
【结论】①P24多肽具备很强的诱导成骨活性和稳定性。②PLGA-NHAC是理想的生物支架和缓释载体。③P24/PLGA-NHAC系统是潜在的临床骨缺损修复基质材料。
Objective: To investigate the efficiency of bone repair in rat calvarial defects by Biomimetic hydroxyapatite-collagan complex carrier/BMP2-derived peptide P24 system, and the preliminary comparison of bioactivity between bmp2-derived peptide P24 and rhBMP2
Methods: Five-millimeter critical-size calvarial defects were created in 30 male Sprague–Dawley rats. The animals were divided into three groups of 10 animals each. The defects were treated with PLGA-NHAC (A group), rhBMP-2/PLGA-NHAC (B group), P24/PLGA-NHAC (C group). Defects were evaluated by X-ray, radiodensity, CT scan, histology, following a 6- and 12-week healing interval (5 animals/group/healing intervals).
Results:①Radiological examination: The percentages of the regenerated areas in A group were significantly lower than B group and C group at 12 weeks (α<0.05),but no significant difference between B group and C group(α>0.05).The comparison of the gray levels also shows the difference.②Histological examination: At 6 weeks post-surgery, Small amount of biomimetic material in group A degraded, free of inflammation; and B group and C group show a large number of new bone tissue and osteoblasts. At 12 weeks, the defect sites were filled with dense connective tissue and small particles of residual PLGA-NHAC, minimal new bone formation was observed in group A. The quantity of the newly formed bone was greater than that observed at 6 weeks in B and C group, and the bone showed more advanced stages of remodeling and consolidation.
Conclusion: P24 peptide is with strong osteoinductive activity and stability in vivo, and PLGA-NHAC is an ideal scaffold and a sustained release carrier. It is suggested that P24/PLGA-NHAC system may be a available matrix material for clinical bone defect.
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] Vogelin E, Jones NF, Huang JI, et al. Healing of a critical-sized defect in the rat femur with use of a vascularized periosteal flap, a biodegradable matrix, and bone morphogenetic protein. J Bone Jt Surg Am, 2005,87(6):1323–1331.
[4] Miki T, Imai Y. Osteoinductive potential of freeze-dried, biodegradable, poly(glycolic acid-co-lactic acid) disks incorporated with bone morphogenetic protein in skull defects of rats. Int J Oral Maxillofac Surg, 1996, 25: 402–406.
[5] Cowan CM, Aalami OO, Shi YY, et al. Bone morphogenetic protein 2 and retinoic acid accelerate in vivo bone formation, osteoclast recruitment, and bone turnover. Tissue Eng,2005,11(3-4):645–658.
[6] Vogelin E, Jones NF, Huang JI, et al. Healing of a critical-sized defect in the rat femur with use of a vascularized periosteal flap, a biodegradable matrix, and bone morphogenetic protein. J Bone Jt Surg Am, 2005,87(6):1323–31.
[7] Takahashi Y, Yamamoto M, Tabata Y. Enhanced osteoinduction by controlled release of bone morphogenetic protein-2 from biodegradable sponge composed of gelatin and beta-tricalcium phosphate. Biomaterials, 2005,26(23):4856–65.
[8] Narumichi M, Naoto Sa, Jun T, Hiroshi O, Hiroshi H, Masashi N, et al. Repair of a proximal femoral bone defect in dogs using a porous surfaced prosthesis in combination with recombinant BMP-2 and a synthetic polymer carrier. Biomaterials, 2003,24(13):2153–9.
[9] Yamashita A, Takada T, Narita J, et al. Osteoblastic differentiation of monkey embryonic stem cells in vitro. Cloning Stem Cells, 2005,7(4):232–7.
[10] Meinel L, Hofmann S, Betz O, et al. Osteogenesis by human mesenchymal stem cells cultured on silk biomaterials: comparison of adenovirus mediated gene transfer andprotein delivery of BMP-2. Biomaterials ,2006,27(28):4993–5002..
[11] Zhao B, Katagiri T, Toyoda H, et al. Heparin potentiates the in vivo ectopic bone formation induced by bone morphogenetic protein-2. J Biol Chem ,2006,281(32): 23246–53.
[12] Alden TD ,Varady P , Kallemes DF ,et al . Bone morphogenetic protein gene therapy. Spine ,2002 ,27:S87～93
[13] Simon. Z, Deporter. D.A, Pilliar. R.M, et al. Heterotopic bone formation around sintered porous-surfaced Ti-6Al-4V implants coated with native bone morphogenetic proteins. Implant Dent, 2006, 15: 265–274.
[14] Seeherman, H, Wozney, J.M . Delivery of bone morphogenetic proteinsfor orthopedic tissue regeneration. Cytokine Growth Factor Rev, 2005, 16: 329–345.
[15] Zhao B, Katagiri T, Toyoda H, Takada T, Yanai T, Fukuda T, et al. Heparin potentiates the in vivo ectopic bone formation induced by bone morphogenetic protein-2. J Biol Chem,2006,281(32):23246–53.
[16] Kirsch T, Sebald W, Dreyer MK. Crystal structure of the BMP2-BRIA ectodomain complex. Nat struct Biol,2000, 7(6):492-296.
[17]段智霞,郑启新,郭晓东等. BMP2活性多肽体外定向诱导大鼠BMSC向成骨方向分化的实验研究.中国矫形外科杂志,2007,15(21):1647-1650.
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