多孔磷酸钙骨水泥/纤维蛋白胶性状及对成骨细胞生物学行为的影响
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摘要
背景:在多孔材料磷酸钙骨水泥粉末中复合纤维蛋白胶是否能有效改善磷酸钙骨水泥的生物力学性能,同时加速其在体内的降解代谢,促进体内骨再生,目前还未被证实。目的:构建不同配比的多孔磷酸钙骨水泥/纤维蛋白胶复合材料,探索其诱导成骨的生物学特性。方法:将磷酸钙骨水泥与纤维蛋白胶分别以1∶1、3∶1、5∶1(mL/g)的配比复合,以单纯的磷酸钙骨水泥为对照,检测各组材料的初凝和终凝时间、压缩强度与弹性模量,扫描电镜观察材料表面结构。将第3代成骨细胞分别接种于4组材料表面,以单纯培养细胞为空白对照,观察不同时间点细胞黏附、增殖及碱性磷酸酶活性。结果与结论:1∶1、3∶1复合材料组初凝时间和终凝时间长于对照组(P<0.05),5∶1组复合材料组初凝时间和终凝时间短于对照组(P<0.05)。磷酸钙骨水泥/纤维蛋白胶复合材料表面较单纯的磷酸钙骨水泥致密而光滑,孔径随着纤维蛋白胶浓度的增加而降低。3∶1、5∶1复合材料组压缩强度高于对照组(P<0.05),1∶1、3∶1、5∶1复合材料组弹性模量高于对照组(P<0.05)。4组材料上的细胞黏附、增殖及碱性磷酸酶活性差异无显著性意义,但均高于空白对照组(P<0.05)。表明纤维蛋白胶的加入克服了磷酸钙骨水泥脆性较高的缺点,同时也保留了其促进成骨的作用。
BACKGROUND: Fibrin glue introduced into calcium phosphate cement has not been confirmed whether this way could overcome the compressive limits and the low degradation of calcium phosphate cement and to modify the biological properties of calcium phosphate cement. OBJECTIVE: To explore the mechanical and biological properties of calcium phosphate cement/fibrin glue at different powder/liquid ratio for bone regeneration in vitro. METHODS: Calcium phosphate cement and fibrin glue were mixed at ratios of 1:1, 3:1, 5:1(mL/g), and the pure calcium phosphate cement served as controls. Setting time, scanning electron microscope and the biomechanical test were used to analyze the composite scaffold structure, physical performance and the mechanical properties. Passage 3 osteoblasts were respectively inoculated on the material surface of the four groups, and pure cellsserved as blank controls. Cell adhesion, proliferation and alkaline phosphatase activity were observed. RESULTS AND CONCLUSION: The initial and final setting time of calcium phosphate cement/fibrin glue at 1:1 and 3:1(mL/g) was higher than that in the control group(P < 0.05), while the initial and final setting time of calcium phosphate cement/fibrin glue at 5:1(mL/g) was lower than that of the control group(P < 0.05). Scanning electron microscope showed smoother and denser surface of composite scaffolds compared with the pure calcium phosphate cement. The aperture of the composite scaffolds was decreased with the increasing concentration of fibrin glue. The compressive strength of composite scaffolds at 3:1 and 5:1 was higher than that of the control group(P < 0.05), while the modulus of the composite scaffolds at 1:1, 3:1, 5:1 was higher than that of the control group(P < 0.05). Cell adhesion, proliferation and alkaline phosphatase activity showed no difference among the three composite scaffold and control groups, but all higher than the blank control group(P < 0.05). These findings indicate that fibrin glue introduced into calcium phosphate cement can overcome the low-strength limits of calcium phosphate cement, and maintain the good biological properties of calcium phosphate cement for bone regeneration.
BACKGROUND: Fibrin glue introduced into calcium phosphate cement has not been confirmed whether this way could overcome the compressive limits and the low degradation of calcium phosphate cement and to modify the biological properties of calcium phosphate cement. OBJECTIVE: To explore the mechanical and biological properties of calcium phosphate cement/fibrin glue at different powder/liquid ratio for bone regeneration in vitro. METHODS: Calcium phosphate cement and fibrin glue were mixed at ratios of 1:1, 3:1, 5:1(mL/g), and the pure calcium phosphate cement served as controls. Setting time, scanning electron microscope and the biomechanical test were used to analyze the composite scaffold structure, physical performance and the mechanical properties. Passage 3 osteoblasts were respectively inoculated on the material surface of the four groups, and pure cellsserved as blank controls. Cell adhesion, proliferation and alkaline phosphatase activity were observed. RESULTS AND CONCLUSION: The initial and final setting time of calcium phosphate cement/fibrin glue at 1:1 and 3:1(mL/g) was higher than that in the control group(P < 0.05), while the initial and final setting time of calcium phosphate cement/fibrin glue at 5:1(mL/g) was lower than that of the control group(P < 0.05). Scanning electron microscope showed smoother and denser surface of composite scaffolds compared with the pure calcium phosphate cement. The aperture of the composite scaffolds was decreased with the increasing concentration of fibrin glue. The compressive strength of composite scaffolds at 3:1 and 5:1 was higher than that of the control group(P < 0.05), while the modulus of the composite scaffolds at 1:1, 3:1, 5:1 was higher than that of the control group(P < 0.05). Cell adhesion, proliferation and alkaline phosphatase activity showed no difference among the three composite scaffold and control groups, but all higher than the blank control group(P < 0.05). These findings indicate that fibrin glue introduced into calcium phosphate cement can overcome the low-strength limits of calcium phosphate cement, and maintain the good biological properties of calcium phosphate cement for bone regeneration.
引文
[1]Miyazaki M,Tsumura H,Wang JC,et al.An update on bone substitutes for spinal fusion.Eur Spine J.2009;18(6):783-799.
[2]Bueno EM,Glowacki J.Cell-free and cell-based approaches for bone regeneration.Nat Rev Rheumatol.2009;5(12):685-697.
[3]Kimelman N,Pelled G,Helm GA,et al.Review:Gene-and cell-based therapeutics for bone regeneration and repair.Tissue Eng.2007;13(6):1135-1150.
[4]Awad HA,Zhang X,Reynolds DG,et al.Recent advances in gene delivery for structural bone allografts.Tissue Eng.2007;13(8):1973-1985.
[5]LeGeros RZ.Properties of osteoconductive biomaterials:calcium phosphates.Clin Orthop.2002;1:81-98.
[6]Zhang Z,Mao J,Feng X,et al.In vitro cytotoxicity of polyphosphoester as a novel injectable alveolar replacement material.J Huazhong Univ Sci Technolog Med Sci.2008;28(5):604-607.
[7]Hesaraki S,Zamanian A,Moztarzadeh F.The influence of the acidic component of the gas-foaming porogen used in preparing an injectable porous calcium phosphate cement on its properties:acetic acid versus citric acid.J Biomed Mater Res B Appl Biomater.2008;86(1):208-216.
[8]Le Guehennec L,Goyenvalle E,Aguado E,et al.Influence of calcium chloride and aprotinin in the in vivo biological performance of a composite combing biphasic calcium phosphate granules and fibrin sealant.J Mater Sci Mater Med.2007;18(8):1489-1495.
[9]Le Nihouannen D,Saffarzadeh A,Gauthier O,et al.Bone tissue formation in sheep muscles induced by a biphasic calcium phosphate ceramic and fibrin glue composite.J Mater Sci Mater Med.2008;19(2):667-675.
[10]Baroth S,Bourges X,Goyenvalle E,et al.Injectable biphasic calcium phosphate bioceramic:the hydros concept.Biomed Mater Eng.2009;19(1):71-76.
[11]Matsushima A,Kotobuki N,Tadokoro M,et al.In vivo osteogenic capability of human mesenchymal cell cultured on hydroxyapatite and on beta-tricalcium phosphate.Artif Organs.2009;33(6):471-481.
[12]Schneider OD,Weber F,Brunner TJ,et al.In vivo and in vitro evaluation of flexible,cottonwool-like nanocomposites as bone substitute material for complex defects.Acta Biomater.2009;5(5):1775-1784.
[13]Tian M,Chen F,Song W,et al.In vivo study of porous strontiumdoped calcium polyphosphate scaffolds for bone substitute applications.J Mater Sci Mater Med.2009;20(7):1505-1512.
[14]Huang Y,Jin X,Zhang X,et al.In vitro and in vivo evaluation of akermanite bioceramics for bone regeneration.Biomaterials.2009;30(28):5041-5048.
[15]Martinowitz U,Saltz R.Fibrin sealant.Curr Opin Hematol.1996;3(5):395-402.
[16]Brunk B.Fibrin glue.Dtsch Med Wochenschr.1989;114:2028-2029.
[17]Cederholm-Williams SA.Fibrin glue.BMJ.2002;308:1570.
[18]Schlag G,Redl H.Fibrin sealant in orthopedic surgery.Clin Orthop.1988;227:269-285.
[19]Radosevich M,Goubran HI,Burnouf T.Fibrin sealant:scientific rationale,produc-tion method,properties and current clinical use.Vox Sang.1997;72(3):133-143.
[20]Reiss RF,Oz MC.Autologous fibrin glue:production and clinical use.Transfus Med Rev.1996;10(2):85-92.
[21]Park YJ,Ku Y,Chung CP,et al.Controlled release of platelet-derived growth factor from porous poly(L-lactide)membranes for guided tissue regeneration.J Controlled Release.1998;51(2):201-211.
[22]Brown W,Chow L.A new calcium phosphate setting cement.J Dent Res.1983;62:672.
[23]Perez RA,Del Valle S,Altankov G,et al.Porous hydroxyapatite and gelatin/hydroxyapatite microspheres obtained by calcium phosphate cement emulsion.J Biomed Mater Res B Appl Biomater.2011;97(1):156-166.
[24]Moreau JL,Weir MD,Xu HHK.Self‐setting collagen‐calcium phosphate bone cement:Mechanical and cellular properties.J Biomed Mater Res A.2009;9(2):605-613.
[25]Wang X,Ma J,Wang Y,et al.Structural characterization of phosphorylated chitosan and their applications as effective additives of calcium phosphate cements.Biomaterials.2001;22:2247-2255.
[26]Kim NH,Yang KH,Lee HM.Effect of porcine bone morphogenetic protein on healing of bone defect in the rabbit radius.Yonsei Med J.1992;33(1):54-63.
[27]陈克明,李旭升,刘兴炎.骨形态发生蛋白复合纤维蛋白载体修复骨缺损的实验研究[J].中华骨科杂志,1998,18(2):69-70.
[28]Bosch P,Lintner F,Arbes H,et al.Experimental investigations of the effect of the fibrin adhesive on the Kiel heterologous bone graft.Arch Orthop Trauma Surg.1980;96(3):177-185.
[29]Minamide A,Kawakami M,Hashizume H.Evaluation of carriers of bone morphogenetic protein for spinal fusion.Spine.2001;26:933-939.
[30]Coulombe J,Faure H,Robin B,et al.In vitro effects of strontium ranelate on extracellular calcium-sensing receptor.Biochem Biophys Res Commun.2004;323:1184-1190.
[31]Sahni A,Francis CW.Vascular endothelial growth factor binds to fibrinogen and fibrin and stimulates endothelial cell proliferation.Blood.2000;96:3772-3778.
[32]Sahni A,Odrljin T,Francis CW.Binding of basic fibroblast growth factor to fibrinogen and fibrin.J Biol Chem.1998;273:7554-7559.
[33]Catelas I,Dwyer JF,Helgerson S.Controlled release of bioactive transforming growth factor beta-1 from fibrin gels in vitro.Tissue Eng Part C Methods.2008;14(2):119-128.
[34]Campbell PG,Durham SK,Hayes JD,et al.Insulin-like growth factor-binding protein-3 binds fibrinogen and fibrin.J Biol Chem.1999;274:30215-30221.
[35]Greco F,de Palma L,Specchia N,et al.Experimental investigation into reparative osteogenesis with fibrin adhesive.Arch Orthop Trauma Surg.1988;107(2):99-104.
[36]Oberg S,Kahnberg K.Combined use of hydroxy-apatite and Tisseel in experim-ental bone defects in the rabbit.Swedish Dent J.1993;17:147.
[37]Le Nihouannen D,Guehennec LL,Rouillon T,et al.Micro-architecture of calcium phosphate granules and fibrin glue composites for bone tissue engineering.Biomaterials.2006;27:2716-2722.
[38]Miyazaki M,Tsumura H,Wang JC,et al.An update on bone substitutes for spinal fusion.Eur Spine J.2009;18(6):783-799.
[39]Bueno EM,Glowacki J.Cell-free and cell-based approaches for bone regeneration.Nat Rev Rheumatol.2009;5(12):685-697.
[40]Le Guehennec L,Goyenvalle E,Aguado E,et al.Influence of calcium chloride and aprotinin in the in vivo biological performance of a composite combing biphasic calcium phosphate granules and fibrin sealant.J Mater Sci Mater Med.2007;18(8):1489-1495.
[41]Jegoux F,Goyenvalle E,Bagot D'arc M,et al.In vivo biological performance of composites combining micro-macroporous biphasic calcium phosphate granules and fibrin sealant.Arch Orthop Trauma Surg.2005;125(3):153-159.
[42]Le Guehennec L,Goyenvalle E,Aguado E,et al.MBCP biphasic calcium phosphate granules and tissucol fibrin sealant in rabbit femoral defects:the effect of fibrin on bone ingrowth.J Mater Sci Mater Med.2005;16(1):29-35.
[43]Yu H,VandeVord PJ,Mao L,et al.Improved tissue-engineered bone regeneration by endothelial cell mediated vascularization.Biomaterials.2009;30(4):508-517.
[44]Oliveira JM,Rodrigues MT,Silva SS,et al.Novel hydroxyapatite/chitosan bilayered scaffold for osteochondral tissue-engineering applications:Scaffold design and its performance when seeded with goat bone marrow stromal cells.Biomaterials.2006;27(36):6123-6137.
[45]Liu G,Sun J,Li Y,et al.Evaluation of partially demineralized osteoporotic cancellous bone matrix combined with human bone marrow stromal cells for tissue engineering:an in vitro and in vivo study.Calcif Tissue Int.2008;83(3):176-185.
[46]Mastrogiacomo M,Papadimitropoulos A,Cedola A,et al.Engineering of bone using bone marrow stromal cells and a silicon-stabilized tricalcium phosphate bioceramic:evidence for a coupling between bone formation and scaffold resorption.Biomaterials.2007;28(7):1376-1384.
[2]Bueno EM,Glowacki J.Cell-free and cell-based approaches for bone regeneration.Nat Rev Rheumatol.2009;5(12):685-697.
[3]Kimelman N,Pelled G,Helm GA,et al.Review:Gene-and cell-based therapeutics for bone regeneration and repair.Tissue Eng.2007;13(6):1135-1150.
[4]Awad HA,Zhang X,Reynolds DG,et al.Recent advances in gene delivery for structural bone allografts.Tissue Eng.2007;13(8):1973-1985.
[5]LeGeros RZ.Properties of osteoconductive biomaterials:calcium phosphates.Clin Orthop.2002;1:81-98.
[6]Zhang Z,Mao J,Feng X,et al.In vitro cytotoxicity of polyphosphoester as a novel injectable alveolar replacement material.J Huazhong Univ Sci Technolog Med Sci.2008;28(5):604-607.
[7]Hesaraki S,Zamanian A,Moztarzadeh F.The influence of the acidic component of the gas-foaming porogen used in preparing an injectable porous calcium phosphate cement on its properties:acetic acid versus citric acid.J Biomed Mater Res B Appl Biomater.2008;86(1):208-216.
[8]Le Guehennec L,Goyenvalle E,Aguado E,et al.Influence of calcium chloride and aprotinin in the in vivo biological performance of a composite combing biphasic calcium phosphate granules and fibrin sealant.J Mater Sci Mater Med.2007;18(8):1489-1495.
[9]Le Nihouannen D,Saffarzadeh A,Gauthier O,et al.Bone tissue formation in sheep muscles induced by a biphasic calcium phosphate ceramic and fibrin glue composite.J Mater Sci Mater Med.2008;19(2):667-675.
[10]Baroth S,Bourges X,Goyenvalle E,et al.Injectable biphasic calcium phosphate bioceramic:the hydros concept.Biomed Mater Eng.2009;19(1):71-76.
[11]Matsushima A,Kotobuki N,Tadokoro M,et al.In vivo osteogenic capability of human mesenchymal cell cultured on hydroxyapatite and on beta-tricalcium phosphate.Artif Organs.2009;33(6):471-481.
[12]Schneider OD,Weber F,Brunner TJ,et al.In vivo and in vitro evaluation of flexible,cottonwool-like nanocomposites as bone substitute material for complex defects.Acta Biomater.2009;5(5):1775-1784.
[13]Tian M,Chen F,Song W,et al.In vivo study of porous strontiumdoped calcium polyphosphate scaffolds for bone substitute applications.J Mater Sci Mater Med.2009;20(7):1505-1512.
[14]Huang Y,Jin X,Zhang X,et al.In vitro and in vivo evaluation of akermanite bioceramics for bone regeneration.Biomaterials.2009;30(28):5041-5048.
[15]Martinowitz U,Saltz R.Fibrin sealant.Curr Opin Hematol.1996;3(5):395-402.
[16]Brunk B.Fibrin glue.Dtsch Med Wochenschr.1989;114:2028-2029.
[17]Cederholm-Williams SA.Fibrin glue.BMJ.2002;308:1570.
[18]Schlag G,Redl H.Fibrin sealant in orthopedic surgery.Clin Orthop.1988;227:269-285.
[19]Radosevich M,Goubran HI,Burnouf T.Fibrin sealant:scientific rationale,produc-tion method,properties and current clinical use.Vox Sang.1997;72(3):133-143.
[20]Reiss RF,Oz MC.Autologous fibrin glue:production and clinical use.Transfus Med Rev.1996;10(2):85-92.
[21]Park YJ,Ku Y,Chung CP,et al.Controlled release of platelet-derived growth factor from porous poly(L-lactide)membranes for guided tissue regeneration.J Controlled Release.1998;51(2):201-211.
[22]Brown W,Chow L.A new calcium phosphate setting cement.J Dent Res.1983;62:672.
[23]Perez RA,Del Valle S,Altankov G,et al.Porous hydroxyapatite and gelatin/hydroxyapatite microspheres obtained by calcium phosphate cement emulsion.J Biomed Mater Res B Appl Biomater.2011;97(1):156-166.
[24]Moreau JL,Weir MD,Xu HHK.Self‐setting collagen‐calcium phosphate bone cement:Mechanical and cellular properties.J Biomed Mater Res A.2009;9(2):605-613.
[25]Wang X,Ma J,Wang Y,et al.Structural characterization of phosphorylated chitosan and their applications as effective additives of calcium phosphate cements.Biomaterials.2001;22:2247-2255.
[26]Kim NH,Yang KH,Lee HM.Effect of porcine bone morphogenetic protein on healing of bone defect in the rabbit radius.Yonsei Med J.1992;33(1):54-63.
[27]陈克明,李旭升,刘兴炎.骨形态发生蛋白复合纤维蛋白载体修复骨缺损的实验研究[J].中华骨科杂志,1998,18(2):69-70.
[28]Bosch P,Lintner F,Arbes H,et al.Experimental investigations of the effect of the fibrin adhesive on the Kiel heterologous bone graft.Arch Orthop Trauma Surg.1980;96(3):177-185.
[29]Minamide A,Kawakami M,Hashizume H.Evaluation of carriers of bone morphogenetic protein for spinal fusion.Spine.2001;26:933-939.
[30]Coulombe J,Faure H,Robin B,et al.In vitro effects of strontium ranelate on extracellular calcium-sensing receptor.Biochem Biophys Res Commun.2004;323:1184-1190.
[31]Sahni A,Francis CW.Vascular endothelial growth factor binds to fibrinogen and fibrin and stimulates endothelial cell proliferation.Blood.2000;96:3772-3778.
[32]Sahni A,Odrljin T,Francis CW.Binding of basic fibroblast growth factor to fibrinogen and fibrin.J Biol Chem.1998;273:7554-7559.
[33]Catelas I,Dwyer JF,Helgerson S.Controlled release of bioactive transforming growth factor beta-1 from fibrin gels in vitro.Tissue Eng Part C Methods.2008;14(2):119-128.
[34]Campbell PG,Durham SK,Hayes JD,et al.Insulin-like growth factor-binding protein-3 binds fibrinogen and fibrin.J Biol Chem.1999;274:30215-30221.
[35]Greco F,de Palma L,Specchia N,et al.Experimental investigation into reparative osteogenesis with fibrin adhesive.Arch Orthop Trauma Surg.1988;107(2):99-104.
[36]Oberg S,Kahnberg K.Combined use of hydroxy-apatite and Tisseel in experim-ental bone defects in the rabbit.Swedish Dent J.1993;17:147.
[37]Le Nihouannen D,Guehennec LL,Rouillon T,et al.Micro-architecture of calcium phosphate granules and fibrin glue composites for bone tissue engineering.Biomaterials.2006;27:2716-2722.
[38]Miyazaki M,Tsumura H,Wang JC,et al.An update on bone substitutes for spinal fusion.Eur Spine J.2009;18(6):783-799.
[39]Bueno EM,Glowacki J.Cell-free and cell-based approaches for bone regeneration.Nat Rev Rheumatol.2009;5(12):685-697.
[40]Le Guehennec L,Goyenvalle E,Aguado E,et al.Influence of calcium chloride and aprotinin in the in vivo biological performance of a composite combing biphasic calcium phosphate granules and fibrin sealant.J Mater Sci Mater Med.2007;18(8):1489-1495.
[41]Jegoux F,Goyenvalle E,Bagot D'arc M,et al.In vivo biological performance of composites combining micro-macroporous biphasic calcium phosphate granules and fibrin sealant.Arch Orthop Trauma Surg.2005;125(3):153-159.
[42]Le Guehennec L,Goyenvalle E,Aguado E,et al.MBCP biphasic calcium phosphate granules and tissucol fibrin sealant in rabbit femoral defects:the effect of fibrin on bone ingrowth.J Mater Sci Mater Med.2005;16(1):29-35.
[43]Yu H,VandeVord PJ,Mao L,et al.Improved tissue-engineered bone regeneration by endothelial cell mediated vascularization.Biomaterials.2009;30(4):508-517.
[44]Oliveira JM,Rodrigues MT,Silva SS,et al.Novel hydroxyapatite/chitosan bilayered scaffold for osteochondral tissue-engineering applications:Scaffold design and its performance when seeded with goat bone marrow stromal cells.Biomaterials.2006;27(36):6123-6137.
[45]Liu G,Sun J,Li Y,et al.Evaluation of partially demineralized osteoporotic cancellous bone matrix combined with human bone marrow stromal cells for tissue engineering:an in vitro and in vivo study.Calcif Tissue Int.2008;83(3):176-185.
[46]Mastrogiacomo M,Papadimitropoulos A,Cedola A,et al.Engineering of bone using bone marrow stromal cells and a silicon-stabilized tricalcium phosphate bioceramic:evidence for a coupling between bone formation and scaffold resorption.Biomaterials.2007;28(7):1376-1384.