真空冷冻干燥法制备聚乳酸/生物玻璃支架及性能研究
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摘要
背景:有文献指出,向聚乳酸中加入生物活性玻璃颗粒可提高材料的力学性能。目的:采用真空冷冻干燥法制备聚乳酸/生物活性玻璃骨组织工程支架材料,并分析其性能。方法:以1,4-二氧六环与二氯甲烷为致孔剂,采用真空冷冻干燥技术制备含生物活性玻璃质量分数分别为10%,20%,30%的聚乳酸/生物活性玻璃复合材料,检测复合材料的孔隙率、抗压强度。将含10%,20%,30%生物活性玻璃复合材料浸泡于模拟体液中2周,观察支架浸泡前后的微观结构与元素变化。分别以聚乳酸浸提液、含10%,20%,30%生物活性玻璃复合材料浸提液与苯酚溶液(阳性对照)培养L929成纤维细胞,以常规培养的细胞为对阴性对照,培养1,3,5 d,采用MTT法检测细胞增殖。结果与结论:①含10%,20%生物活性玻璃复合材料的孔隙率高于聚乳酸材料(P <0.05);②各聚乳酸/生物活性玻璃复合材料的抗压强度均高于聚乳酸材料(P<0.05),并且含20%生物活性玻璃复合支架组的抗压强度高于含10%,30%生物活性玻璃复合支架组(P<0.05);③扫描电镜显示,复合材料孔隙内壁上均有大量微孔结构,生物活性玻璃分散在材料中,孔隙大小不均,孔隙之间相互沟通,随着生物活性玻璃含量的增加,孔隙有堵塞现象;在模拟体液中浸泡2周后,复合材料有明显的羟基磷灰石生成,并且钙、磷、硅元素比例升高;④培养1,3,5d,含10%,20%,30%生物活性玻璃复合支架组表面的细胞增殖快于阳性对照组(P <0.05),与阴性对照组、聚乳酸组比较差异无显著性意义(P> 0.05);⑤结果表明,采用真空冷冻干燥法制备可制备具有良好孔隙率、抗压强度与细胞相容性的聚乳酸/生物活性玻璃复合支架。
BACKGROUND: Bioactive glass added into polylactic acid can improve the mechanical property of the material.OBJECTIVE: To prepare the tissue-engineered scaffold of polylactic acid/bioactive glass composites by vacuum freeze-drying technique and study its performance.METHODS: For 1,4-dioxane and dichloromethane as pore-forming agents, polylactic acid containing 10%, 20% and 30% bioactive glass were prepared by vacuum freeze-drying technique. The porosity and compressive strength were detected. In order to observe the microstructure and element changes of composites before and after soaked into the simulated body fluid for 2 weeks. L929 fibroblasts were cultured with polylactic acid leach liquor, polylactic acid/bioactive glass composites leaching liquor and phenol solution, and the cells of being cultured in routine culture were used as control, respectively. After culture for 1, 3 and 5 days, MTT was used to detect cell proliferation.RESULTS AND CONCLUSION:(1) The porosity of polylactic acid containing 10% and 20% bioactive glass was higher than that of polylactic acid(P < 0.05).(2) The compressive strength of polylactic acid/bioactive glass composite was higher than that of polylactic acid(P < 0.05),and the compressive strength of 20% bioactive glass composite was higher than that of 10% and 30% bioactive glass composites(P < 0.05).(3) Scanning electron microscopy showed that the inner pore wall of polylactic acid materials has a large amount of micropore structures,bioactive glass distributed in the composite, the pore size was uneven and the pores communicated with each other. As the increasing of bioactive glass contents, there were lots of blocked pores. After immersion in simulated body fluid for 2 weeks, polylactic acid/bioactive glass containing composites showed obvious hydroxyapatite formation, but there was no hydroxyapatite formation in polylactic acid. The immersion of polylactic acid was only the change of PH value, but the ratio of calcium and phosphorus was higher than that before soaking in composite materials. After soaking, there was a large amount of hydroxyapatite formation, and the proportion of calcium and phosphorus was reduced accordingly. At the same time, the Si in bioactive glass was released.(4) The cell proliferation in the polylactic acid/bioactive glass composite containing 10% and 20% bioactive glass was significantly faster than that of positive control group after 1, 3 and 5 days of culture(P < 0.05),which showed no significant difference compared with the negative control and polylactic acid groups(P > 0.05)(5) These results suggest that polylactic acid/bioactive glass containing composite scaffolds with good porosity, compressive strength and cellular compatibility can be prepared by vacuum freeze-drying technique.
BACKGROUND: Bioactive glass added into polylactic acid can improve the mechanical property of the material.OBJECTIVE: To prepare the tissue-engineered scaffold of polylactic acid/bioactive glass composites by vacuum freeze-drying technique and study its performance.METHODS: For 1,4-dioxane and dichloromethane as pore-forming agents, polylactic acid containing 10%, 20% and 30% bioactive glass were prepared by vacuum freeze-drying technique. The porosity and compressive strength were detected. In order to observe the microstructure and element changes of composites before and after soaked into the simulated body fluid for 2 weeks. L929 fibroblasts were cultured with polylactic acid leach liquor, polylactic acid/bioactive glass composites leaching liquor and phenol solution, and the cells of being cultured in routine culture were used as control, respectively. After culture for 1, 3 and 5 days, MTT was used to detect cell proliferation.RESULTS AND CONCLUSION:(1) The porosity of polylactic acid containing 10% and 20% bioactive glass was higher than that of polylactic acid(P < 0.05).(2) The compressive strength of polylactic acid/bioactive glass composite was higher than that of polylactic acid(P < 0.05),and the compressive strength of 20% bioactive glass composite was higher than that of 10% and 30% bioactive glass composites(P < 0.05).(3) Scanning electron microscopy showed that the inner pore wall of polylactic acid materials has a large amount of micropore structures,bioactive glass distributed in the composite, the pore size was uneven and the pores communicated with each other. As the increasing of bioactive glass contents, there were lots of blocked pores. After immersion in simulated body fluid for 2 weeks, polylactic acid/bioactive glass containing composites showed obvious hydroxyapatite formation, but there was no hydroxyapatite formation in polylactic acid. The immersion of polylactic acid was only the change of PH value, but the ratio of calcium and phosphorus was higher than that before soaking in composite materials. After soaking, there was a large amount of hydroxyapatite formation, and the proportion of calcium and phosphorus was reduced accordingly. At the same time, the Si in bioactive glass was released.(4) The cell proliferation in the polylactic acid/bioactive glass composite containing 10% and 20% bioactive glass was significantly faster than that of positive control group after 1, 3 and 5 days of culture(P < 0.05),which showed no significant difference compared with the negative control and polylactic acid groups(P > 0.05)(5) These results suggest that polylactic acid/bioactive glass containing composite scaffolds with good porosity, compressive strength and cellular compatibility can be prepared by vacuum freeze-drying technique.
引文
[1]刘相杰,宋科官.生物支架材料及间充质干细胞在骨组织工程中的研究与应用[J].中国组织工程研究,2018,22(10):1618-1624.
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[4]杜瑞林,倪似愚,常江,等.含Zn生物玻璃、玻璃陶瓷与聚酯复合骨组织工程支架的制备及性能[J].复合材料学报, 2008,25(6):123-129.
[5] Jie W,Cai Y,Shen H,et al.Mesoporous calcium–silicon xerogels with mesopore size and pore volume influence h MSC behaviors by load and sustained release of rh BMP-2.Int J Nanomedicine. 2015;10:1715-1726.
[6] Wu Z,Tang T,Guo H,et al.In vitro dejgradability, bioactivity and cell responses to mesoporous magnesium silicate for the induction of bone regeneration.Colloids Surf B Biointerfaces.2014;120(120):38-46.
[7] Zheng K, Lu M, Liu Y,et al.Monodispersed lysozyme-functionalized bioactive glass nanoparticles with antibacterial and anticancer activities.Biomed Mater.2016;11(3):035012.
[8] Liu T,Ding X,Lai D,et al.Enhancing in vitro bioactivity and in vivo osteogenesis of organic-inorganic nanofibrous biocomposites with novel bioceramics.J Mater Chem B.2014;2(37):6293-6305.
[9]葛建华,王迎军,陈晓峰.生物活性玻璃/聚乳酸组织工程支架在SBF溶液中的降解和矿化性能[J].复合材料学报, 2011,28(5):90-93.
[10] Eqtesadi S,Motealleh A,Perera FH,et al. Poly-(lactic acid)infiltration of 45S5 Bioglass?robocast scaffolds:Chemical interaction and its deleterious effect in mechanical enhancement.Mater Lett.2016;163:196-200.
[11] Mohammadkhah A,Day DE.Mechanical properties of bioactive glass/polymer composite scaffolds for repairing load bearing bones.Int J Appl Glass Sci.2018;9(2):188-197.
[12] Eqtesadi S,Motealleh A,Hugo Perera F,et al. Poly-(lactic acid)infiltration of 45S5 Bioglass?robocast scaffolds:Chemical interaction and its deleterious effect in mechanical enhancement. Mater Lett.2016;163(15):196-200.
[13] Qazi TH,Mooney DJ,Pumberger M,et al.Biomaterials based strategies for skeletal muscle tissue engineering:existing technologies and future trends.Biomaterials. 2015;53:502-521.
[14] Zhou H,Lee J, Sun F.Various preparation methods of highly porous hydroxyapatite/polymer nanoscale biocomposites forbone regeneration.Acta Biomater.2011;7(11):3813-3828.
[15] Wu S, Liu X,Yeung KWK,et al.Biomimetic porous scaffolds for bone tissue engineering.Mater Sci Eng R.2014;80(1):1-36.
[16] Fu Q,Saiz E,Rahaman MN,et al.Toward Strong and Tough Glass and Ceramic Scaffolds for Bone Repair. Adv Funct Mater.2013;23(44):5461-5476.
[17] Iafisco M,Sandri M,Panseri S,et al.Magnetic Bioactive and Biodegradable Hollow Fe-Doped Hydroxyapatite Coated Poly(L-lactic)Acid Micro-nanospheres.Chem Mater. 2013;25(13):2610-2617.
[18] Locatelli E,Franchini MC.Biodegradable PLGA-b-PEG polymeric nanoparticles:synthesis, properties, and nanomedical applications as drug delivery system.J Nanopart Res.2012;14(12):1316.
[19] Zia S,Mozafari M,Natasha G,et al.Hearts beating through decellularized scaffolds:whole-organ engineering for cardiac regeneration and transplantation. Crit Rev Biotechnol. 2016;36(4):705-715.
[20] Zhang C,Mcadams DA,Grunlan JC.Bioinspired Materials:Nano/Micro-Manufacturing of Bioinspired Materials:a Review of Methods to Mimic Natural Structures.Adv Mater.2016;28(30):6265-6265.
[21] Liu Z,Ji J,Tang S,et al.Biocompatibility, degradability,bioactivity and osteogenesis of mesoporous/macroporous scaffolds of mesoporous diopside/poly(L-lactide)composite.J Royal Soc Interface.2015;12(111):20150507.
[22] Wu C,Chen Z,Yi D,et al.Multidirectional Effects of Sr-, Mg-,and Si-Containing Bioceramic Coatings with High Bonding Strength on Inflammation, Osteoclastogenesis, and Osteogenesis.ACS Appl Mater Interfaces. 2014;6(6):4264-4276.
[23] Parizek M,Douglas TE,Novotna K,et al.Nanofibrous poly(lactide-co-glycolide)membranes loaded with diamond nanoparticles as promising substrates for bone tissue engineering. Int J Nanomedicine. 2012;7:1931-1951.
[24] Zhong S,Teo WE,Zhu X,et al.An aligned nanofibrous collagen scaffold by electrospinning and its effects on in vitro fibroblast culture.J Biomed Mater Res A. 2010;79A(3):456-463.
[25] Aravamudhan A,Ramos DM,Jenkins NA,et al.Collagen nanofibril self-assembly on a natural polymeric material for the osteoinduction of stem cells in vitro and biocompatibility in vivo. RSC Adv.2016; 6(84):80851-80866.
[26] Novajra G,Perdika P,Pisano R,et al.Tailoring of Bone Scaffold Properties Using Silicate/Phosphate Glass Mixtures.Key Eng Mater.2015;631:283-288.
[27] Novajra G,Perdika P,Pisano R,et al.Structure optimisation and biological evaluation of bone scaffolds prepared by co-sintering of silicate and phosphate glasses.Adv Appl Ceramic. 2015;114(sup1):S48-S55.
[28] Ger?ek I,Tigli RS,Gümü?derelioglu M.A novel Scaffold Based on Formation and Agglomeration of PCL Microbeads by Freeze-Drying.J Biomed Mater Res A. 2008;86(4):1012-1022.
[29] Narayanan G,Gupta BS,Tonelli AE.Enhanced Mechanical properties of Poly(ε-caprolactone)Nanofibers produced by the Addition of Non-stoichiometric Inclusion Complexes of Poly(ε-caprolactone)andα-Cyclodextrin.Polymer. 2015;75(12):321-330.
[2]徐桂文,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.
[3]李建伟,郭全义,张景春,等.组织工程骨软骨一体化多相支架的制备与体外检测评估[J].中国修复重建外科杂志,2018,32(4):1-7.
[4]杜瑞林,倪似愚,常江,等.含Zn生物玻璃、玻璃陶瓷与聚酯复合骨组织工程支架的制备及性能[J].复合材料学报, 2008,25(6):123-129.
[5] Jie W,Cai Y,Shen H,et al.Mesoporous calcium–silicon xerogels with mesopore size and pore volume influence h MSC behaviors by load and sustained release of rh BMP-2.Int J Nanomedicine. 2015;10:1715-1726.
[6] Wu Z,Tang T,Guo H,et al.In vitro dejgradability, bioactivity and cell responses to mesoporous magnesium silicate for the induction of bone regeneration.Colloids Surf B Biointerfaces.2014;120(120):38-46.
[7] Zheng K, Lu M, Liu Y,et al.Monodispersed lysozyme-functionalized bioactive glass nanoparticles with antibacterial and anticancer activities.Biomed Mater.2016;11(3):035012.
[8] Liu T,Ding X,Lai D,et al.Enhancing in vitro bioactivity and in vivo osteogenesis of organic-inorganic nanofibrous biocomposites with novel bioceramics.J Mater Chem B.2014;2(37):6293-6305.
[9]葛建华,王迎军,陈晓峰.生物活性玻璃/聚乳酸组织工程支架在SBF溶液中的降解和矿化性能[J].复合材料学报, 2011,28(5):90-93.
[10] Eqtesadi S,Motealleh A,Perera FH,et al. Poly-(lactic acid)infiltration of 45S5 Bioglass?robocast scaffolds:Chemical interaction and its deleterious effect in mechanical enhancement.Mater Lett.2016;163:196-200.
[11] Mohammadkhah A,Day DE.Mechanical properties of bioactive glass/polymer composite scaffolds for repairing load bearing bones.Int J Appl Glass Sci.2018;9(2):188-197.
[12] Eqtesadi S,Motealleh A,Hugo Perera F,et al. Poly-(lactic acid)infiltration of 45S5 Bioglass?robocast scaffolds:Chemical interaction and its deleterious effect in mechanical enhancement. Mater Lett.2016;163(15):196-200.
[13] Qazi TH,Mooney DJ,Pumberger M,et al.Biomaterials based strategies for skeletal muscle tissue engineering:existing technologies and future trends.Biomaterials. 2015;53:502-521.
[14] Zhou H,Lee J, Sun F.Various preparation methods of highly porous hydroxyapatite/polymer nanoscale biocomposites forbone regeneration.Acta Biomater.2011;7(11):3813-3828.
[15] Wu S, Liu X,Yeung KWK,et al.Biomimetic porous scaffolds for bone tissue engineering.Mater Sci Eng R.2014;80(1):1-36.
[16] Fu Q,Saiz E,Rahaman MN,et al.Toward Strong and Tough Glass and Ceramic Scaffolds for Bone Repair. Adv Funct Mater.2013;23(44):5461-5476.
[17] Iafisco M,Sandri M,Panseri S,et al.Magnetic Bioactive and Biodegradable Hollow Fe-Doped Hydroxyapatite Coated Poly(L-lactic)Acid Micro-nanospheres.Chem Mater. 2013;25(13):2610-2617.
[18] Locatelli E,Franchini MC.Biodegradable PLGA-b-PEG polymeric nanoparticles:synthesis, properties, and nanomedical applications as drug delivery system.J Nanopart Res.2012;14(12):1316.
[19] Zia S,Mozafari M,Natasha G,et al.Hearts beating through decellularized scaffolds:whole-organ engineering for cardiac regeneration and transplantation. Crit Rev Biotechnol. 2016;36(4):705-715.
[20] Zhang C,Mcadams DA,Grunlan JC.Bioinspired Materials:Nano/Micro-Manufacturing of Bioinspired Materials:a Review of Methods to Mimic Natural Structures.Adv Mater.2016;28(30):6265-6265.
[21] Liu Z,Ji J,Tang S,et al.Biocompatibility, degradability,bioactivity and osteogenesis of mesoporous/macroporous scaffolds of mesoporous diopside/poly(L-lactide)composite.J Royal Soc Interface.2015;12(111):20150507.
[22] Wu C,Chen Z,Yi D,et al.Multidirectional Effects of Sr-, Mg-,and Si-Containing Bioceramic Coatings with High Bonding Strength on Inflammation, Osteoclastogenesis, and Osteogenesis.ACS Appl Mater Interfaces. 2014;6(6):4264-4276.
[23] Parizek M,Douglas TE,Novotna K,et al.Nanofibrous poly(lactide-co-glycolide)membranes loaded with diamond nanoparticles as promising substrates for bone tissue engineering. Int J Nanomedicine. 2012;7:1931-1951.
[24] Zhong S,Teo WE,Zhu X,et al.An aligned nanofibrous collagen scaffold by electrospinning and its effects on in vitro fibroblast culture.J Biomed Mater Res A. 2010;79A(3):456-463.
[25] Aravamudhan A,Ramos DM,Jenkins NA,et al.Collagen nanofibril self-assembly on a natural polymeric material for the osteoinduction of stem cells in vitro and biocompatibility in vivo. RSC Adv.2016; 6(84):80851-80866.
[26] Novajra G,Perdika P,Pisano R,et al.Tailoring of Bone Scaffold Properties Using Silicate/Phosphate Glass Mixtures.Key Eng Mater.2015;631:283-288.
[27] Novajra G,Perdika P,Pisano R,et al.Structure optimisation and biological evaluation of bone scaffolds prepared by co-sintering of silicate and phosphate glasses.Adv Appl Ceramic. 2015;114(sup1):S48-S55.
[28] Ger?ek I,Tigli RS,Gümü?derelioglu M.A novel Scaffold Based on Formation and Agglomeration of PCL Microbeads by Freeze-Drying.J Biomed Mater Res A. 2008;86(4):1012-1022.
[29] Narayanan G,Gupta BS,Tonelli AE.Enhanced Mechanical properties of Poly(ε-caprolactone)Nanofibers produced by the Addition of Non-stoichiometric Inclusion Complexes of Poly(ε-caprolactone)andα-Cyclodextrin.Polymer. 2015;75(12):321-330.