聚磷酸钙纤维增强磷酸钙骨水泥复合骨髓间充质干细胞构建人工骨
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摘要
背景:磷酸钙骨水泥作为生物医用材料的研究热点现广泛应用于临床中,但由于磷酸钙骨水泥支架材料抗压抗折强度低,在应用中受到限制,而且磷酸钙骨水泥不具有骨诱导活性,这也是作为人工骨的一大缺憾。因此,对于磷酸钙骨水泥材料的增强成为这一领域的研究热点。目的:探讨聚磷酸钙纤维增强磷酸钙骨水泥复合骨髓间充质干细胞构建人工骨的可行性。方法:构建合理配比的磷酸钙骨水泥与聚磷酸钙纤维复合支架材料,按聚磷酸钙纤维分别占总质量的10%,20%,30%,40%,50%,60%分为6组,分别进行压缩试验、三点弯曲试验及扭转试验,记录各组复合支架材料的最大抗压负荷及抗弯负荷,从而确定达到最佳强度时的聚磷酸钙纤维/磷酸钙骨水泥比例。采用密度梯度离心法分离培养骨髓间充质干细胞并进行成骨诱导,接种于最适比例的聚磷酸钙纤维/磷酸钙复合支架材料上,扫描电镜观察复合支架材料上骨髓间充质干细胞的生长情况。结果与结论:(1)聚磷酸钙纤维占支架材料总质量的20%时,复合支架材料的硬度及韧性达到最佳;(2)扫描电镜观察复合支架上骨髓间充质干细胞生长良好,约在第7天时,骨髓间充质干细胞完全填充于复合支架材料的空隙中;(3)结果表明,聚磷酸钙纤维在支架材料中起到了"钢筋"的作用,增强了支架材料的强度及韧性;骨髓间充质干细胞的加入使支架材料的成骨方式从单一的骨传导方式成骨变为同时具有骨传导性、骨诱导性、骨生成性3种方式成骨,增强了材料的生物相容性。
BACKGROUND: Calcium phosphate cement is a research focus of biomedical materials and has been widely used in clinical practice. However, the low compressive strength and bending strength of calcium phosphate cement limit its applications, and as the artificial bone, the lack of osteoinductive activity is also a major defect. Therefore, the enhancement of calcium phosphate cement has become a hot spot in this field. OBJECTIVE: To explore the feasibility of constructing artificial bone by bone marrow mesenchymal stem cells combined with calcium phosphate fiber/bone cement. METHODS: Calcium phosphate cement/calcium polyphosphate fiber composite scaffold materials at a reasonable proportion were prepared, in which calcium polyphosphate fibers accounted for 10%, 20%, 30%, 40%, 50% and 60% of the total mass respectively. Then, compression test, three-point bending test and torsion test were performed. The maximum compressive load and bending load were recorded, so as to determine the proportion of calcium phosphate/calcium polyphosphate fibers under the best strength. Bone marrow mesenchymal stem cells were isolated and cultured by density gradient centrifugation and induced by osteogenesis. The cultured cells were inoculated onto the calcium polyphosphate fiber/calcium phosphate cement composite scaffold at the most suitable proportion. The growth of bone marrow mesenchymal stem cells on the composite scaffold was observed by scanning electron microscope. RESULTS AND CONCLUSION: The hardness and toughness of the composite scaffold material reached the best when the calcium phosphate fibers accounted for 20% of the total mass of the scaffold. Bone marrow mesenchymal stem cells grew well on the composite scaffold under the scanning electron microscope. Approximately 7 days after culture, the bone marrow mesenchymal stem cells were completely filled in the space of the composite scaffold. To conclude, the calcium polyphosphate fiber plays the role of "steel bar" in the composite scaffold, enhancing the strength and toughness of the scaffold material. The addition of bone marrow mesenchymal stem cells changes the osteogenesis from a single bone conduction mode to an osteogenic mode with bone conductivity, bone induction and bone generation, which enhances the biocompatibility of the scaffold material.
BACKGROUND: Calcium phosphate cement is a research focus of biomedical materials and has been widely used in clinical practice. However, the low compressive strength and bending strength of calcium phosphate cement limit its applications, and as the artificial bone, the lack of osteoinductive activity is also a major defect. Therefore, the enhancement of calcium phosphate cement has become a hot spot in this field. OBJECTIVE: To explore the feasibility of constructing artificial bone by bone marrow mesenchymal stem cells combined with calcium phosphate fiber/bone cement. METHODS: Calcium phosphate cement/calcium polyphosphate fiber composite scaffold materials at a reasonable proportion were prepared, in which calcium polyphosphate fibers accounted for 10%, 20%, 30%, 40%, 50% and 60% of the total mass respectively. Then, compression test, three-point bending test and torsion test were performed. The maximum compressive load and bending load were recorded, so as to determine the proportion of calcium phosphate/calcium polyphosphate fibers under the best strength. Bone marrow mesenchymal stem cells were isolated and cultured by density gradient centrifugation and induced by osteogenesis. The cultured cells were inoculated onto the calcium polyphosphate fiber/calcium phosphate cement composite scaffold at the most suitable proportion. The growth of bone marrow mesenchymal stem cells on the composite scaffold was observed by scanning electron microscope. RESULTS AND CONCLUSION: The hardness and toughness of the composite scaffold material reached the best when the calcium phosphate fibers accounted for 20% of the total mass of the scaffold. Bone marrow mesenchymal stem cells grew well on the composite scaffold under the scanning electron microscope. Approximately 7 days after culture, the bone marrow mesenchymal stem cells were completely filled in the space of the composite scaffold. To conclude, the calcium polyphosphate fiber plays the role of "steel bar" in the composite scaffold, enhancing the strength and toughness of the scaffold material. The addition of bone marrow mesenchymal stem cells changes the osteogenesis from a single bone conduction mode to an osteogenic mode with bone conductivity, bone induction and bone generation, which enhances the biocompatibility of the scaffold material.
引文
[1]Wang P, Zhao L, Chen W, et al. Stem Cells and Calcium Phosphate Cement Scaffolds for Bone Regeneration. J Dent Res. 2014;93(7):618-625.
[2]Brown WE, Chow LC. A new calcium setting cement. J Dent Res.1983;63:762-765.
[3]Janicki P, Boeuf S, Steck E, et al. Prediction of in vivo bone forming potency of bone marrow-derived human mesenchymal stem cells. Eur Cell Mater. 2011;21:488-507.
[4]Kagami H, Agata H, Tojo A. Bone marrow stromal cells(bone marrow-derived multipotent mesenchymal stromal cells)for bone tissue engineering:basic science to clinical translation. Int J Biochem Cell Biol. 2011;43(3):286-289.
[5]Sommeling CE, Heyneman A, Hoeksema H, et al. The use of platelet-rich plasma in plastic surgery:a systematic review. J Plast Reconstr Aesthet Surg. 2013;66(3):301-311.
[6]Namazi H. Enhancing bone healing during distraction osteogenesis with platelet-rich plasma:a novel molecular mechanism. Injury.2012;43(7):1225-1226.
[7]Latalski M, Elbatrawy YA, Thabet AM, et al. Enhancing bone healing during distraction osteogenesis with platelet-rich plasma. Injury.2011;42(8):821-824.
[8]Kanthan SR, Kavitha G, Addi S, et al. Platelet-rich plasma(PRP)enhances bone healing in non-united critical-sized defects:a preliminary study involving rabbit models. Injury. 2011;42(8):782-789.
[9]St-Pierre JP, Gan L, Wang J, et al. The incorporation of a zone of calcified cartilage improves the interfacial shear strength between in vitro-formed cartilage and the underlying substrate. Acta Biomater.2012;8(4):1603-1615.
[10]St-Pierre JP, Pilliar RM, Grynpas MD, et al. Calcification of cartilage formed in vitro on calcium polyphosphate bone substitutes is regulated by inorganic polyphosphate. Acta Biomater. 2010;6(8):3302-3309.
[11]Waldman SD, Grynpas MD, Pilliar RM, et al. Characterization of cartilagenous tissue formed on calcium polyphosphate substrates in vitro. J Biomed Mater Res. 2002;62(3):323-330.
[12]Jones JR, Ahir S, Hench LL. Large-scale production of 3D bioactive glass macroporous. Sol-Gel Sci Technol. 2004;29:179-188.
[13]周磊,闫景龙,胡春杰.聚磷酸钙纤维/磷酸钙骨水泥复合材料修复骨缺损的实验研究[J].中国脊柱脊髓杂志,2006,16(11):851-855.
[14]周磊,闫景龙,胡春杰.聚磷酸钙纤维/磷酸钙骨水泥/微小颗粒骨修复骨缺损的实验研究[J].中国骨与关节损伤杂志, 2007,22(12):1002-1005.
[15]周磊,闫景龙,胡春杰.聚磷酸钙纤维/磷酸钙骨水泥/微小颗粒骨复合人工骨的体外降解[J].中国组织工程研究与临床康复, 2007,11(1):33-36.
[16]Wei J, Li YB. Tissue engineering scaffold material of nano-apatite crystals and polyamide composite. Eur Polymer J. 2004;40(3):509-515.
[17]Granero-Molto F, Weis JA, Longobardi L, et al. Role of mesenchymal stem cells in regenerative medicine:application to bone and cartilage repair. Expert Opin Biol Ther. 2008;8(3):255-268.
[18]Kang SW, Lee JS, Park MS, et al. Enhancement of in vivo bone regeneration efficacy of human mesenchymal stem cells. J Microbiol Biotechnol. 2008;18(5):975-982.
[19]Matsushima A, Kotobuki N, Tadokoro M, et al. In vivo osteogenic capability of human mesenchymal cells cultured on hydroxyapatite and on beta-tricalcium phosphate. Artif Organs. 2009;33(6):474-481.
[20]黄粹业.骨缺损的临床修复与研究进展[J].右江民族医学院学报, 2002,24(3):427-429.
[21]Larsen KH, Frederiksen CM, Burns JS, et al. Identifying a molecular phenotype for bone marrow stromal cells with in vivo bone-forming capacity. J Bone Miner Res. 2010;25(4):796-808.
[22]Shahdadfar A, Fr?nsdal K, Haug T, et al. In vitro expansion of human mesenchymal stem cells:choice of serum is a determinant of cell proliferation, differentiation, gene expression, and transcriptome stability. Stem Cells. 2005;23(9):1357-1366.
[23]张铭杰,张庆文,何伟,等.成人骨髓间充质干细胞体外培养、鉴定与成骨诱导[J].中国组织工程研究,2013,17(45):7947-7953.
[24]Chen L, Tredget EE, Wu PY, et al. Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing. PLoS One. 2008;3(4):e1886.
[25]Arinzeh TL, Peter SJ, Archambault MP, et al. Allogeneic mesenchymal stem cells regenerate bone in a critical-sized canine segmental defect.J Bone Joint Surg Am. 2003;85-A(10):1927-1935.
[26]Mendes SC, Tibbe JM, Veenhof M, et al. Relation between in vitro and in vivo osteogenic potential of cultured human bone marrow stromal cells. J Mater Sci Mater Med. 2004;15(10):1123-1128.
[27]Stolzing A, Jones E, McGonagle D, et al. Age-related changes in human bone marrow-derived mesenchymal stem cells:consequences for cell therapies. Mech Ageing Dev. 2008;129(3):163-173.
[28]周常艳,周庆焕,边竞,等.骨髓间充质干细胞与磷酸钙骨水泥复合物修复兔关节软骨缺损[J].中国组织工程研究, 2015,19(8):1195-1199.
[29]Winet H, Hollinger JO, Stevanovic M. Incorporation of polylactide-polyglycolide in a cortical defect:neoangiogenesis and blood supply in a bone chamber. J Orthop Res. 1995;13(5):679-689.
[30]Thomas KA. Vascular endothelial growth factor, a potent and selective angiogenic agent. J Biol Chem. 1996;271(2):603-606.
[31]杨显声,阎景龙,麻松.自体颗粒骨磷酸钙骨水泥复合细胞移植的实验研究[J].中国骨质疏松杂志,2005,11(2):225-229.
[2]Brown WE, Chow LC. A new calcium setting cement. J Dent Res.1983;63:762-765.
[3]Janicki P, Boeuf S, Steck E, et al. Prediction of in vivo bone forming potency of bone marrow-derived human mesenchymal stem cells. Eur Cell Mater. 2011;21:488-507.
[4]Kagami H, Agata H, Tojo A. Bone marrow stromal cells(bone marrow-derived multipotent mesenchymal stromal cells)for bone tissue engineering:basic science to clinical translation. Int J Biochem Cell Biol. 2011;43(3):286-289.
[5]Sommeling CE, Heyneman A, Hoeksema H, et al. The use of platelet-rich plasma in plastic surgery:a systematic review. J Plast Reconstr Aesthet Surg. 2013;66(3):301-311.
[6]Namazi H. Enhancing bone healing during distraction osteogenesis with platelet-rich plasma:a novel molecular mechanism. Injury.2012;43(7):1225-1226.
[7]Latalski M, Elbatrawy YA, Thabet AM, et al. Enhancing bone healing during distraction osteogenesis with platelet-rich plasma. Injury.2011;42(8):821-824.
[8]Kanthan SR, Kavitha G, Addi S, et al. Platelet-rich plasma(PRP)enhances bone healing in non-united critical-sized defects:a preliminary study involving rabbit models. Injury. 2011;42(8):782-789.
[9]St-Pierre JP, Gan L, Wang J, et al. The incorporation of a zone of calcified cartilage improves the interfacial shear strength between in vitro-formed cartilage and the underlying substrate. Acta Biomater.2012;8(4):1603-1615.
[10]St-Pierre JP, Pilliar RM, Grynpas MD, et al. Calcification of cartilage formed in vitro on calcium polyphosphate bone substitutes is regulated by inorganic polyphosphate. Acta Biomater. 2010;6(8):3302-3309.
[11]Waldman SD, Grynpas MD, Pilliar RM, et al. Characterization of cartilagenous tissue formed on calcium polyphosphate substrates in vitro. J Biomed Mater Res. 2002;62(3):323-330.
[12]Jones JR, Ahir S, Hench LL. Large-scale production of 3D bioactive glass macroporous. Sol-Gel Sci Technol. 2004;29:179-188.
[13]周磊,闫景龙,胡春杰.聚磷酸钙纤维/磷酸钙骨水泥复合材料修复骨缺损的实验研究[J].中国脊柱脊髓杂志,2006,16(11):851-855.
[14]周磊,闫景龙,胡春杰.聚磷酸钙纤维/磷酸钙骨水泥/微小颗粒骨修复骨缺损的实验研究[J].中国骨与关节损伤杂志, 2007,22(12):1002-1005.
[15]周磊,闫景龙,胡春杰.聚磷酸钙纤维/磷酸钙骨水泥/微小颗粒骨复合人工骨的体外降解[J].中国组织工程研究与临床康复, 2007,11(1):33-36.
[16]Wei J, Li YB. Tissue engineering scaffold material of nano-apatite crystals and polyamide composite. Eur Polymer J. 2004;40(3):509-515.
[17]Granero-Molto F, Weis JA, Longobardi L, et al. Role of mesenchymal stem cells in regenerative medicine:application to bone and cartilage repair. Expert Opin Biol Ther. 2008;8(3):255-268.
[18]Kang SW, Lee JS, Park MS, et al. Enhancement of in vivo bone regeneration efficacy of human mesenchymal stem cells. J Microbiol Biotechnol. 2008;18(5):975-982.
[19]Matsushima A, Kotobuki N, Tadokoro M, et al. In vivo osteogenic capability of human mesenchymal cells cultured on hydroxyapatite and on beta-tricalcium phosphate. Artif Organs. 2009;33(6):474-481.
[20]黄粹业.骨缺损的临床修复与研究进展[J].右江民族医学院学报, 2002,24(3):427-429.
[21]Larsen KH, Frederiksen CM, Burns JS, et al. Identifying a molecular phenotype for bone marrow stromal cells with in vivo bone-forming capacity. J Bone Miner Res. 2010;25(4):796-808.
[22]Shahdadfar A, Fr?nsdal K, Haug T, et al. In vitro expansion of human mesenchymal stem cells:choice of serum is a determinant of cell proliferation, differentiation, gene expression, and transcriptome stability. Stem Cells. 2005;23(9):1357-1366.
[23]张铭杰,张庆文,何伟,等.成人骨髓间充质干细胞体外培养、鉴定与成骨诱导[J].中国组织工程研究,2013,17(45):7947-7953.
[24]Chen L, Tredget EE, Wu PY, et al. Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing. PLoS One. 2008;3(4):e1886.
[25]Arinzeh TL, Peter SJ, Archambault MP, et al. Allogeneic mesenchymal stem cells regenerate bone in a critical-sized canine segmental defect.J Bone Joint Surg Am. 2003;85-A(10):1927-1935.
[26]Mendes SC, Tibbe JM, Veenhof M, et al. Relation between in vitro and in vivo osteogenic potential of cultured human bone marrow stromal cells. J Mater Sci Mater Med. 2004;15(10):1123-1128.
[27]Stolzing A, Jones E, McGonagle D, et al. Age-related changes in human bone marrow-derived mesenchymal stem cells:consequences for cell therapies. Mech Ageing Dev. 2008;129(3):163-173.
[28]周常艳,周庆焕,边竞,等.骨髓间充质干细胞与磷酸钙骨水泥复合物修复兔关节软骨缺损[J].中国组织工程研究, 2015,19(8):1195-1199.
[29]Winet H, Hollinger JO, Stevanovic M. Incorporation of polylactide-polyglycolide in a cortical defect:neoangiogenesis and blood supply in a bone chamber. J Orthop Res. 1995;13(5):679-689.
[30]Thomas KA. Vascular endothelial growth factor, a potent and selective angiogenic agent. J Biol Chem. 1996;271(2):603-606.
[31]杨显声,阎景龙,麻松.自体颗粒骨磷酸钙骨水泥复合细胞移植的实验研究[J].中国骨质疏松杂志,2005,11(2):225-229.