异种骨/重组人骨形态发生蛋白7/纤维蛋白胶复合材料的缓释性能及成骨活性
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摘要
背景:骨形态发生蛋白的稳定缓慢释放是其诱导成骨作用的前提,因此需要合适的载体使骨形态发生蛋白缓慢释放,然而迄今为止还没有一种医学界公认的骨形态发生蛋白理想载体。目的:评价异种骨复合材料中骨形态发生蛋白7的缓释情况及成骨活性。方法:以异种小牛松质骨为支架材料,采用真空负压吸附法将含重组人骨形态发生蛋白7的溶液吸附在支架材料上,再喷涂纤维蛋白胶涂层,制备异种骨/重组人骨形态发生蛋白7/纤维蛋白胶复合材料,作为实验组材料;将小牛松质骨、小牛松质骨/重组人骨形态发生蛋白7复合材料分别作为空白组、对照组材料。将3组材料分别在PBS中持续浸泡28 d,观察重组人骨形态发生蛋白7的缓释情况;将3种材料分别与第3代髓间充质干细胞共培养,检测培养1,3,7,14 d的细胞碱性磷酸酶活性、骨桥蛋白和骨钙素表达水平。结果与结论:(1)空白组无重组人骨形态发生蛋白7释放,实验组重组人骨形态发生蛋白7的缓释效果优于对照组;(2)实验组、对照组培养不同时间点的碱性磷酸酶活性、骨桥蛋白和骨钙素表达水平均高于空白组(P<0.05),实验组培养14 d的细胞碱性磷酸酶活性、骨桥蛋白和骨钙素表达水平均高于对照组(P<0.05);(3)结果表明:异种骨/重组人骨形态发生蛋白7/纤维蛋白胶复合材料具有良好的缓释性能及成骨活性。
BACKGROUND:Sustained release of bone morphogenetic proteins(BMPs) at stable speed is a prerequisite for osteogenic induction.Therefore,a suitable carrier for the sustained release of BMPs is required.However,there is no ideal carrier for BMPs as yet.OBJECTIVE:To explore the sustained release process of BMP-7 and osteogenic activity in the xenogeneic bone composite that is the compound of xenogeneic bone,recombinant human BMP-7(rh BMP-7) and fibrin sealant(FS).METHODS:With the xenogeneic cattle spongy bone as a scaffold material,the solution containing rh BMP-7 was adsorbed onto the scaffold by means of vacuum negative pressure absorption,and fibrin sealant(FS) was sprayed onto the surface of the scaffold.The xenogeneic bone composite made up of these three materials acted as a sustained material.All scaffold materials were randomly divided into three groups:blank group(the scaffold material was xenogeneic bone with no rh BMP-7 and FS),control group(the scaffold material was xenogeneic bone with rh BMP-7) and experimental group(the scaffold material was the composite material of xenogeneic bone,rh BMP-7 and FS).All samples were immerged into phosphate buffer solution for 28 continuous days,and the release levels of rh BMP-7 from the samples were measured by high performance liquid chromatography at various set time.All the scaffold materials were co-cultured with passage 3 bone marrow mesenchymal stem cells.Alkaline phosphatase activity,osteopontin and osteocalcin expression levels were detected at days1,3,7,and 14 after co-culture.RESULTS AND CONCLUSION:rh BMP-7 was not detected in the blank control group,and the sustained release of rh BMP-7 in the experimental group was better than that in the control group.The alkaline phosphatase activity,osteopontin and osteocalcin levels were ranked as follows:blank control group < control group < experimental group,and there was a significant difference between groups(P < 0.05).These findings indicate that the xenogeneic bone/rh BMP-7/FS composite material as a good sustained-release carrier of BMP-7 shows strong osteogenic activity.
BACKGROUND:Sustained release of bone morphogenetic proteins(BMPs) at stable speed is a prerequisite for osteogenic induction.Therefore,a suitable carrier for the sustained release of BMPs is required.However,there is no ideal carrier for BMPs as yet.OBJECTIVE:To explore the sustained release process of BMP-7 and osteogenic activity in the xenogeneic bone composite that is the compound of xenogeneic bone,recombinant human BMP-7(rh BMP-7) and fibrin sealant(FS).METHODS:With the xenogeneic cattle spongy bone as a scaffold material,the solution containing rh BMP-7 was adsorbed onto the scaffold by means of vacuum negative pressure absorption,and fibrin sealant(FS) was sprayed onto the surface of the scaffold.The xenogeneic bone composite made up of these three materials acted as a sustained material.All scaffold materials were randomly divided into three groups:blank group(the scaffold material was xenogeneic bone with no rh BMP-7 and FS),control group(the scaffold material was xenogeneic bone with rh BMP-7) and experimental group(the scaffold material was the composite material of xenogeneic bone,rh BMP-7 and FS).All samples were immerged into phosphate buffer solution for 28 continuous days,and the release levels of rh BMP-7 from the samples were measured by high performance liquid chromatography at various set time.All the scaffold materials were co-cultured with passage 3 bone marrow mesenchymal stem cells.Alkaline phosphatase activity,osteopontin and osteocalcin expression levels were detected at days1,3,7,and 14 after co-culture.RESULTS AND CONCLUSION:rh BMP-7 was not detected in the blank control group,and the sustained release of rh BMP-7 in the experimental group was better than that in the control group.The alkaline phosphatase activity,osteopontin and osteocalcin levels were ranked as follows:blank control group < control group < experimental group,and there was a significant difference between groups(P < 0.05).These findings indicate that the xenogeneic bone/rh BMP-7/FS composite material as a good sustained-release carrier of BMP-7 shows strong osteogenic activity.
引文
[1]应吕方,贾叙锋,程尹,等.硫酸钙人工骨复合培养骨髓间充质干细胞对骨诱导脊柱融合的影响[J].中国组织工程研究,2016,20(43):6396-6402.
[2]高维诺,侯晓薇,樊世锋,等.富血小板纤维蛋白复合异种骨修复兔牙槽骨缺损的实验研究[J].中国保健营养(中旬刊),2014,25(5):3354-3355.
[3]李冰婷,贾莹珍,刘志芳,等.富血小板纤维蛋白/异种冻干骨复合物修复骨缺损及骨整合的作用[J].中国组织工程研究,2014,8(52):8376-8381.
[4]蒋李青,方炳木,赵冬梅,等.富血小板血浆复合人工骨植骨治疗骨折不愈合合并骨缺损[J].中医正骨,2016,28(12):58-60.
[5]柴旭斌,周英杰,郑怀亮,等.纳米羟基磷灰石/聚酰胺66人工骨块在颈椎后路双开门椎管成形术中的应用[J].中医正骨,2016,28(8):32-34.
[6]郑园娜,吴刚,吴向阳,等.Rh BMP2/7和rh BMP2、rh BMP7诱导MC3T3-E1细胞成骨分化相关基因表达的比较研究[J].口腔生物医学,2010,1(1):10-15.
[7]Lu C,Xing Z,Yu YY,et al.Recombinant human bone morphogenetic protein-7 enhances fracture healing in an ischemic environment.J Orthop Res.2010;28(5):687-696.
[8]杨允,邓春梅,韩明,等.人骨形态发生蛋白7在中国仓鼠卵巢细胞中的表达研究[J].生物技术,2012,22(2):23-26.
[9]孙平.Rh BMP2/7异源二聚体促进种值体周围骨缺修复和诱导成骨细胞通路初步探讨[D].浙江大学,2011.
[10]董晓红,叶建明,刘其锋,等.BMP7对Cs A诱导的NRK-52E细胞EMT的影响[J].现代中西医结合杂志,2013,22(35):3893-3895.
[11]Valimaki VV,Aro HT.Molecular basis for action of bioactive glasses as bone graft substitute.Scand J Surg.2006;95(2):95-102.
[12]Keskin DS,Tezcancer A,Korkusuz P,et al.Collagen-chondroitin sulfate-based PLLA-SAIB-coated rh BMP-2 delivery system for bone repair.Biomaterials.2005;26(18):4023-4034.
[13]吴五洲,喻爱喜,孙艳玲,等.壳聚糖作为rh BMP-2体外缓释载体的实验研究[J].中华显微外科杂志,2006,29(3):214-216.
[14]朱慧勇,吴求亮,申屠建中,等.多孔聚乳酸-聚乙醇酸共聚物作为缓释重组人骨形态发生蛋白-2载体的实验研究[J].中华创伤杂志,2004,20(10):602-605.
[15]吴斌,郑启新,郭晓东,等.纳米羟基磷灰石和重组胶原及聚乳酸复合材料作为骨形态发生蛋白2活性多肽缓释载体的实验研究[J].中华关节外科杂志:电子版,2007,1(5):302-306.
[2]高维诺,侯晓薇,樊世锋,等.富血小板纤维蛋白复合异种骨修复兔牙槽骨缺损的实验研究[J].中国保健营养(中旬刊),2014,25(5):3354-3355.
[3]李冰婷,贾莹珍,刘志芳,等.富血小板纤维蛋白/异种冻干骨复合物修复骨缺损及骨整合的作用[J].中国组织工程研究,2014,8(52):8376-8381.
[4]蒋李青,方炳木,赵冬梅,等.富血小板血浆复合人工骨植骨治疗骨折不愈合合并骨缺损[J].中医正骨,2016,28(12):58-60.
[5]柴旭斌,周英杰,郑怀亮,等.纳米羟基磷灰石/聚酰胺66人工骨块在颈椎后路双开门椎管成形术中的应用[J].中医正骨,2016,28(8):32-34.
[6]郑园娜,吴刚,吴向阳,等.Rh BMP2/7和rh BMP2、rh BMP7诱导MC3T3-E1细胞成骨分化相关基因表达的比较研究[J].口腔生物医学,2010,1(1):10-15.
[7]Lu C,Xing Z,Yu YY,et al.Recombinant human bone morphogenetic protein-7 enhances fracture healing in an ischemic environment.J Orthop Res.2010;28(5):687-696.
[8]杨允,邓春梅,韩明,等.人骨形态发生蛋白7在中国仓鼠卵巢细胞中的表达研究[J].生物技术,2012,22(2):23-26.
[9]孙平.Rh BMP2/7异源二聚体促进种值体周围骨缺修复和诱导成骨细胞通路初步探讨[D].浙江大学,2011.
[10]董晓红,叶建明,刘其锋,等.BMP7对Cs A诱导的NRK-52E细胞EMT的影响[J].现代中西医结合杂志,2013,22(35):3893-3895.
[11]Valimaki VV,Aro HT.Molecular basis for action of bioactive glasses as bone graft substitute.Scand J Surg.2006;95(2):95-102.
[12]Keskin DS,Tezcancer A,Korkusuz P,et al.Collagen-chondroitin sulfate-based PLLA-SAIB-coated rh BMP-2 delivery system for bone repair.Biomaterials.2005;26(18):4023-4034.
[13]吴五洲,喻爱喜,孙艳玲,等.壳聚糖作为rh BMP-2体外缓释载体的实验研究[J].中华显微外科杂志,2006,29(3):214-216.
[14]朱慧勇,吴求亮,申屠建中,等.多孔聚乳酸-聚乙醇酸共聚物作为缓释重组人骨形态发生蛋白-2载体的实验研究[J].中华创伤杂志,2004,20(10):602-605.
[15]吴斌,郑启新,郭晓东,等.纳米羟基磷灰石和重组胶原及聚乳酸复合材料作为骨形态发生蛋白2活性多肽缓释载体的实验研究[J].中华关节外科杂志:电子版,2007,1(5):302-306.