仿生大孔骨支架的制备及生物相容性评估
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摘要
背景:骨组织工程的发展,为解决骨缺损修复提供了新手段,而其中的支架载体选材和结构构建是目前的热点问题。目的:采用石蜡微球沥滤技术制备具有仿生大孔结构的丝素蛋白/纳米羟基磷灰石支架,分析其细胞相容性。方法:以丝素蛋白和纳米羟基磷灰石为原料,采用石蜡微球沥滤技术制成具有仿生大孔结构的丝素蛋白/纳米羟基磷灰石支架,利用体式显微镜和扫描电镜观察支架结构,测量支架的弹性模量。将第3代兔脂肪间充质干细胞接种于丝素蛋白/纳米羟基磷灰石支架上,培养48 h后,LIVE/DEAD染色观察细胞活性;培养1周,苏木精-伊红染色观察细胞黏附情况;培养3 d,扫描电镜观察支架-细胞复合物;培养1,3,5,7 d,CCK-8法检测细胞增殖,以单纯细胞培养为对照。结果与结论:①体式显微镜下可见丝素蛋白/纳米羟基磷灰石支架呈乳白色,扫描电镜显示无论在支架横截面还是纵切面上,均可见均匀排列的大孔结构,连通性好,支架孔径为(362.23±26.52)μm,弹性模量为(54.93±5.44) kPa;②扫描电镜显示,脂肪间充质干细胞能够很好地在支架孔壁及连通孔隙内黏附和伸展,分泌大量细胞外基质,填充在丝素蛋白/纳米羟基磷灰石支架孔隙内;③苏木精-伊红染色显示,脂肪间充质干细胞均匀黏附在丝素蛋白/纳米羟基磷灰石支架孔隙内壁上,并且大量增殖;④LIVE/DEAD染色显示脂肪间充质干细胞活性良好;⑤CCK-8检测结果显示,脂肪间充质干细胞在丝素蛋白/纳米羟基磷灰石支架可良好地生长与增殖;⑥结果表明,丝素蛋白/纳米羟基磷灰石支架具有良好的孔径与细胞相容性。
BACKGROUND: Development of bone tissue engineering provides a new method to solve bone defect repair. Scaffold material and structure construction are issues of concern. OBJECTIVE: To fabricate a silk fibroin and hydroxyapatite scaffold with biomimetic interconnected macropores structure using the paraffin-sphere leaching technology and to evaluate its possibility of cytocompatibility. METHODS: The scaffold with biomimetic interconnected macropores structure was made by the paraffin-sphere leaching technology. The structure of scaffold was observed by the stereomicroscope and scanning electron microscope. The pore size and elasticity modulus were calculated. Passage 3 rabbit adipose-derived mesenchymal stem cells were seeded into the scaffold. The cell viability was detected by live/dead staining at 48 hours after culture. The cell adhesion was observed by hematoxylin-eosin staining at 1 week of culture. The scaffold-cell composite was observed under scanning electron microscope at 3 days of culture. The cell proliferation was detected by the cell counting-kit 8 assay at 1, 3, 5 and 7 days of culture. Those cells cultured alone were considered as control group. RESULTS AND CONCLUSION:(1) Stereomicroscope showed the ivory silk fibroin/hydroxyapatite scaffold. Scanning electron microscope revealed pore structures in cross-section and longitudinal-section with good connectivity. The scaffold pore size was(362.23±26.52) μm and the elasticity modulus was(54.93±5.44) kPa.(2) Scanning electron microscope showed that adipose-derived mesenchymal stem cells adhered and stretched well in the pore wall and connected pore, secreted abundant extracellular matrix, and filled in the pores of silk fibroin/hydroxyapatite scaffold.(3) Hematoxylin-eosin staining results found that adipose-derived mesenchymal stem cells evenly adhered onto the inner wall of silk fibroin/hydroxyapatite scaffold, and proliferated well.(4) Live/dead staining revealed a good viability of adipose-derived mesenchymal stem cells.(5) Cell counting-kit 8 assay results showed the good proliferation of adipose-derived mesenchymal stem cells on the scaffold.(6) To conclude, the silk fibroin/hydroxyapatite scaffold possesses good pore size and cytocompatibility.
BACKGROUND: Development of bone tissue engineering provides a new method to solve bone defect repair. Scaffold material and structure construction are issues of concern. OBJECTIVE: To fabricate a silk fibroin and hydroxyapatite scaffold with biomimetic interconnected macropores structure using the paraffin-sphere leaching technology and to evaluate its possibility of cytocompatibility. METHODS: The scaffold with biomimetic interconnected macropores structure was made by the paraffin-sphere leaching technology. The structure of scaffold was observed by the stereomicroscope and scanning electron microscope. The pore size and elasticity modulus were calculated. Passage 3 rabbit adipose-derived mesenchymal stem cells were seeded into the scaffold. The cell viability was detected by live/dead staining at 48 hours after culture. The cell adhesion was observed by hematoxylin-eosin staining at 1 week of culture. The scaffold-cell composite was observed under scanning electron microscope at 3 days of culture. The cell proliferation was detected by the cell counting-kit 8 assay at 1, 3, 5 and 7 days of culture. Those cells cultured alone were considered as control group. RESULTS AND CONCLUSION:(1) Stereomicroscope showed the ivory silk fibroin/hydroxyapatite scaffold. Scanning electron microscope revealed pore structures in cross-section and longitudinal-section with good connectivity. The scaffold pore size was(362.23±26.52) μm and the elasticity modulus was(54.93±5.44) kPa.(2) Scanning electron microscope showed that adipose-derived mesenchymal stem cells adhered and stretched well in the pore wall and connected pore, secreted abundant extracellular matrix, and filled in the pores of silk fibroin/hydroxyapatite scaffold.(3) Hematoxylin-eosin staining results found that adipose-derived mesenchymal stem cells evenly adhered onto the inner wall of silk fibroin/hydroxyapatite scaffold, and proliferated well.(4) Live/dead staining revealed a good viability of adipose-derived mesenchymal stem cells.(5) Cell counting-kit 8 assay results showed the good proliferation of adipose-derived mesenchymal stem cells on the scaffold.(6) To conclude, the silk fibroin/hydroxyapatite scaffold possesses good pore size and cytocompatibility.
引文
[1]Kim HD,Amirthalingam S,Kim SL,et al.Biomimetic Materials and Fabrication Approaches for Bone Tissue Engineering.Adv Healthc Mater.2017;6(23).doi:10.1002/adhm.201700612.Epub 2017 Nov24.
[2]张屹,陈建常,史振满.组织工程支架在修复骨缺损中的研究进展[J].中国矫形外科杂志,2013,21(16):1617-1619.
[3]张淦,程迅生.骨组织工程的研究进展[J].安徽医学,2016,37(8):1057-1061.
[4]Su P,Tian Y,Yang C,et al.Mesenchymal Stem Cell Migration during Bone Formation and Bone Diseases Therapy.Int J Mol Sci.2018;19(8):E2343.
[5]Noori A,Ashrafi SJ,Vaez-Ghaemi R,et al.A review of fibrin and fibrin composites for bone tissue engineering.Int J Nanomedicine.2017;12(12):4937-4961.
[6]Kim HJ,Kim UJ,Kim HS,et al.Bone tissue engineering with premineralized silk scaffolds.Bone.2008;42(6):1226-1234.
[7]徐桂文,乌日开西·艾依提,滕勇.复合型组织工程化人工骨支架:应用进展与未来方向[J].中国组织工程研究,2018,22(14):2245-2250.
[8]葛振,邹刚,刘毅,等.组织工程支架材料:特征及在组织工程中的应用[J].中国组织工程研究,2018,22(26):4222-4228.
[9]陈海霞,谢志刚.骨组织工程支架材料:脱矿骨基质[J].中国组织工程研究,2014,18(3):426-431.
[10]Zheng Z,Ning M,Li D.Silicification of silk fibroin and their application in bone tissue engineering.Sheng Wu Yi Xue Gong Cheng Xue Za Zhi.2018;35(4):643-646.
[11]高欣欣,许燕,周建平,等.丝素蛋白-聚乙烯醇复合凝胶对骨组织工程支架性能的影响[J].燕山大学学报,2017,41(2):127-132.
[12]Mandal BB,Park SH,Gil ES,et al.Multilayered silk scaffolds for meniscus tissue engineering.Biomaterials.2011;32(2):639-651.
[13]陆史俊,左保齐,刘洪臣.丝素蛋白生物支架材料在骨组织工程中的应用进展[J].中国修复重建外科杂志,2014,28(10):1307-1310.
[14]Meinel L,Karageorgiou V,Hofmann S,et al.Engineering bone-like tissue in vitro using human bone marrow stem cells and silk scaffolds.J Biomed Mater Res A.2004;71(1):25-34.
[15]余文,孟昊业,孙逊,等.3D打印生物陶瓷在骨组织工程中的研究现状[J].中国矫形外科杂志,2018,26(14):1306-1310.
[16]赵呈智,张悠,付睿捷,等.骨组织工程中纳米羟基磷灰石复合材料现状、评价及展望[J].全科口腔医学电子杂志,2018,5(17):4-5.
[17]Bhumiratana S,Grayson WL,Castaneda A,et al.Nucleation and growth of mineralized bone matrix on silk-hydroxyapatite composite scaffolds.Biomaterials.2011;32(11):2812-2820.
[18]赵呈智,张悠,付睿捷,等.骨组织工程中纳米羟基磷灰石复合材料现状、评价及展望[J].全科口腔医学电子杂志,2018,5(17):4-5.
[19]Jo YY,Kim S,Kwon K,et al.Silk Fibroin-Alginate-Hydroxyapatite Composite Particles in Bone Tissue Engineering Applications In Vivo.Int J Mol Sci.2017;18(4):E858.
[20]Zhou J,Xu C,Wu G,et al.In vitro generation of osteochondral differentiation of human marrow mesenchymal stem cells in novel collagen-hydroxyapatite layered scaffolds.Acta Biomater.2011;7(11):3999-4006.
[21]Lee CM,Yang SW,Jung SC,et al.Oxygen Plasma Treatment on3D-Printed Chitosan/Gelatin/Hydroxyapatite Scaffolds for Bone Tissue Engineering.J Nanosci Nanotechnol.2017;17(4):2747-2750.
[22]Zeng C,Yang Q,Zhu M,et al.Silk fibroin porous scaffolds for nucleus pulposus tissue engineering.Mater Sci Eng C Mater Biol Appl.2014;37:232-240.
[23]杨强,丁晓明,徐宝山,等.取向性丝素蛋白仿生软骨支架的制备及其生物相容性评估[J].天津医药,2015,43(7):709-712,834.
[24]Bunnell BA,Flaat M,Gagliardi C,et al.Adipose-derived stem cells:isolation,expansion and differentiation.Methods.2008;45(2):115-120.
[25]Liew LJ,Ong HT,Dilley RJ.Isolation and Culture of Adipose-Derived Stromal Cells from Subcutaneous Fat.Methods Mol Biol.2017;1627:193-203.
[26]Shafieian R,Matin MM,Rahpeyma A,et al.Effects of Human Adipose-derived Stem Cells and Platelet-Rich Plasma on Healing Response of Canine Alveolar Surgical Bone Defects.Arch Bone JSurg.2017;5(6):406-418.
[27]Angele P,Kujat R,Nerlich M,et al.Engineering of osteochondral tissue with bone marrow mesenchymal progenitor cells in a derivatized hyaluronan-gelatin composite sponge.Tissue Eng.1999;5(6):545-554.
[28]Moriguchi Y,Mojica-Santiago J,Grunert P,et al.Total disc replacement using tissue-engineered intervertebral discs in the canine cervical spine.PLoS One.2017;12(10):e0185716.
[29]Mohan N,Dormer NH,Caldwell KL,et al.The Tissue-Engineered Tendon-Bone Interface:In Vitro and In Vivo Synergistic Effects of Adipose-Derived Stem Cells,Platelet-Rich Plasma,and Extracellular Matrix Hydrogel.Plast Reconstr Surg.2017;140(6):1169-1184.
[30]Wang Y,Wang K,Li X,et al.3D fabrication and characterization of phosphoric acid scaffold with a HA/β-TCP weight ratio of 60:40 for bone tissue engineering applications.PLoS One 2017;12(4):e0174870.
[31]Campbell GM,Ominsky MS,Boyd SK.Bone quality is partially recovered after the discontinuation of RANKL administration in rats by increased bone mass on existing trabeculae:an in vivo micro-CTstudy.Osteoporos Int.2011;22(3):931-942.
[2]张屹,陈建常,史振满.组织工程支架在修复骨缺损中的研究进展[J].中国矫形外科杂志,2013,21(16):1617-1619.
[3]张淦,程迅生.骨组织工程的研究进展[J].安徽医学,2016,37(8):1057-1061.
[4]Su P,Tian Y,Yang C,et al.Mesenchymal Stem Cell Migration during Bone Formation and Bone Diseases Therapy.Int J Mol Sci.2018;19(8):E2343.
[5]Noori A,Ashrafi SJ,Vaez-Ghaemi R,et al.A review of fibrin and fibrin composites for bone tissue engineering.Int J Nanomedicine.2017;12(12):4937-4961.
[6]Kim HJ,Kim UJ,Kim HS,et al.Bone tissue engineering with premineralized silk scaffolds.Bone.2008;42(6):1226-1234.
[7]徐桂文,乌日开西·艾依提,滕勇.复合型组织工程化人工骨支架:应用进展与未来方向[J].中国组织工程研究,2018,22(14):2245-2250.
[8]葛振,邹刚,刘毅,等.组织工程支架材料:特征及在组织工程中的应用[J].中国组织工程研究,2018,22(26):4222-4228.
[9]陈海霞,谢志刚.骨组织工程支架材料:脱矿骨基质[J].中国组织工程研究,2014,18(3):426-431.
[10]Zheng Z,Ning M,Li D.Silicification of silk fibroin and their application in bone tissue engineering.Sheng Wu Yi Xue Gong Cheng Xue Za Zhi.2018;35(4):643-646.
[11]高欣欣,许燕,周建平,等.丝素蛋白-聚乙烯醇复合凝胶对骨组织工程支架性能的影响[J].燕山大学学报,2017,41(2):127-132.
[12]Mandal BB,Park SH,Gil ES,et al.Multilayered silk scaffolds for meniscus tissue engineering.Biomaterials.2011;32(2):639-651.
[13]陆史俊,左保齐,刘洪臣.丝素蛋白生物支架材料在骨组织工程中的应用进展[J].中国修复重建外科杂志,2014,28(10):1307-1310.
[14]Meinel L,Karageorgiou V,Hofmann S,et al.Engineering bone-like tissue in vitro using human bone marrow stem cells and silk scaffolds.J Biomed Mater Res A.2004;71(1):25-34.
[15]余文,孟昊业,孙逊,等.3D打印生物陶瓷在骨组织工程中的研究现状[J].中国矫形外科杂志,2018,26(14):1306-1310.
[16]赵呈智,张悠,付睿捷,等.骨组织工程中纳米羟基磷灰石复合材料现状、评价及展望[J].全科口腔医学电子杂志,2018,5(17):4-5.
[17]Bhumiratana S,Grayson WL,Castaneda A,et al.Nucleation and growth of mineralized bone matrix on silk-hydroxyapatite composite scaffolds.Biomaterials.2011;32(11):2812-2820.
[18]赵呈智,张悠,付睿捷,等.骨组织工程中纳米羟基磷灰石复合材料现状、评价及展望[J].全科口腔医学电子杂志,2018,5(17):4-5.
[19]Jo YY,Kim S,Kwon K,et al.Silk Fibroin-Alginate-Hydroxyapatite Composite Particles in Bone Tissue Engineering Applications In Vivo.Int J Mol Sci.2017;18(4):E858.
[20]Zhou J,Xu C,Wu G,et al.In vitro generation of osteochondral differentiation of human marrow mesenchymal stem cells in novel collagen-hydroxyapatite layered scaffolds.Acta Biomater.2011;7(11):3999-4006.
[21]Lee CM,Yang SW,Jung SC,et al.Oxygen Plasma Treatment on3D-Printed Chitosan/Gelatin/Hydroxyapatite Scaffolds for Bone Tissue Engineering.J Nanosci Nanotechnol.2017;17(4):2747-2750.
[22]Zeng C,Yang Q,Zhu M,et al.Silk fibroin porous scaffolds for nucleus pulposus tissue engineering.Mater Sci Eng C Mater Biol Appl.2014;37:232-240.
[23]杨强,丁晓明,徐宝山,等.取向性丝素蛋白仿生软骨支架的制备及其生物相容性评估[J].天津医药,2015,43(7):709-712,834.
[24]Bunnell BA,Flaat M,Gagliardi C,et al.Adipose-derived stem cells:isolation,expansion and differentiation.Methods.2008;45(2):115-120.
[25]Liew LJ,Ong HT,Dilley RJ.Isolation and Culture of Adipose-Derived Stromal Cells from Subcutaneous Fat.Methods Mol Biol.2017;1627:193-203.
[26]Shafieian R,Matin MM,Rahpeyma A,et al.Effects of Human Adipose-derived Stem Cells and Platelet-Rich Plasma on Healing Response of Canine Alveolar Surgical Bone Defects.Arch Bone JSurg.2017;5(6):406-418.
[27]Angele P,Kujat R,Nerlich M,et al.Engineering of osteochondral tissue with bone marrow mesenchymal progenitor cells in a derivatized hyaluronan-gelatin composite sponge.Tissue Eng.1999;5(6):545-554.
[28]Moriguchi Y,Mojica-Santiago J,Grunert P,et al.Total disc replacement using tissue-engineered intervertebral discs in the canine cervical spine.PLoS One.2017;12(10):e0185716.
[29]Mohan N,Dormer NH,Caldwell KL,et al.The Tissue-Engineered Tendon-Bone Interface:In Vitro and In Vivo Synergistic Effects of Adipose-Derived Stem Cells,Platelet-Rich Plasma,and Extracellular Matrix Hydrogel.Plast Reconstr Surg.2017;140(6):1169-1184.
[30]Wang Y,Wang K,Li X,et al.3D fabrication and characterization of phosphoric acid scaffold with a HA/β-TCP weight ratio of 60:40 for bone tissue engineering applications.PLoS One 2017;12(4):e0174870.
[31]Campbell GM,Ominsky MS,Boyd SK.Bone quality is partially recovered after the discontinuation of RANKL administration in rats by increased bone mass on existing trabeculae:an in vivo micro-CTstudy.Osteoporos Int.2011;22(3):931-942.