京尼平对壳聚糖/藻酸盐组织工程支架制备的影响
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摘要
背景：美国的学者在20世纪80年代提出了“组织工程”的概念,组织工程是一门交叉学科,包括了生命科学、材料学等学科知识。组织工程研究主要包括：种子细胞、支架材料、细胞外基质。其中,支架材料是该领域近年来研究的热点。
本实验通过冷冻干燥法制备壳聚糖/藻酸盐复合支架材料,并采用新型天然交联剂京尼平对材料进行交联,探究京尼平、紫外线两种交联方法对支架材料制备的影响。
目的：1.比较京尼平和紫外线交联后的壳聚糖/藻酸盐复合支架材料的降解率、孔隙率、含水量、细胞毒性以及生物力学等特性；2.探究交联温度对京尼平交联壳聚糖/藻酸盐组织工程支架的影响。
方法：取1只成年健康Wistar大鼠骨髓,分离、培养BMSCs.采用冷冻干燥法制备壳聚糖/藻酸盐复合支架。1.①按照交联方法不同分为：京尼平组、紫外线组。②扫描电镜下观察材料的表面结构以及检测材料的降解率、孔隙率、含水量、细胞毒性以及生物力学。2.运用冷冻干燥法制备壳聚糖/藻酸盐支架材料,分别在4℃、25℃、37℃条件,于0.5%京尼平溶液中交联24h,作为实验组。以未用京尼平交联的支架材料作为对照组,评价支架材料降解率、细胞毒性、孔隙率、含水量以及生物力学性能的特点。
结果：1.①紫外线组与京尼平组均表现为多孔隙结构,无明显差异。②紫外线组降解率高于京尼平组。③京尼平组与紫外线组的孔隙率差异无统计学意义,含水量差异比较有统计学意义。④两组均表现为较低的细胞毒性,良好的生物相容性。⑤京尼平交联组的生物力学特性较紫外线组显著提高。2.①伴随交联温度升高,支架材料的颜色从浅紫色变为紫黑色,未交联的支架材料为白色；②降解率4周后在4℃组为21.54%±3.07%、25℃组为9.9%±1.2%、37℃组为8.98%±0.79%。其中,37℃和25℃组抗降解能力优于4℃组(P<0.05)。各实验组均优于对照组(P<0.01)。③弹性模量在4℃组为0.84±0.55,25℃组为1.44±0.06,37℃组为1.53±0.02,对照组为0.79±0.16。对照组与25℃组与37℃组之间差异计较有统计学意义(P<0.05)。④孔隙率、含水量、细胞毒性各实验组间未见明显差异。
结论：1.京尼平交联的壳聚糖／藻酸盐复合支架材料,具有良好的生物学特性,为组织工程脊髓领域提供了非常具有潜力的材料。2.随着交联温度的提高,支架材料的抗降解能力、抗拉伸性能增加,且对支架材料的结构、细胞毒性、孔隙率以及含水量无明显影响。
Background American scholars proposed "tissue engineering" concept in the1980s, tissue engineering is an interdisciplinary subject, including life science, materials science and other disciplines of knowledge. Tissue engineering research includes: seed cells, scaffolds, extracellular matrix. Among them, the scaffold material is a hot research topic in recent years in this field.
Chitosan/alginate composite scaffolds was produced by freeze drying.In this study,we adopt a new natural crosslinking agent genipin crosslinking of the materials, to explore the effect of preparation of scaffold by genipin, UV two crosslinking methods.
Objective1. To investigate genipin(GP) and ultraviolet rays(UV) crosslinked effects of chitosan/alginate scaffolds for tissue engineering by detecting the change of the chitosan/alginate scaffolds material degradation rate, porosity, water content, cytotoxicity and biomechanics properties.2. we try to explore cross-linking temperature of genipin crosslinked effects of chitosan/alginate scaffolds for tissue engineering.
Methods BMSCs were separated and cultured from one Wistar rat.Chitosan/alginate scaffolds was produced via freeze drying.1.①ccording to different cross-linking methods, biomaterials were divided into (GP)group,(UV)group.②he surface structure of two groups was detected by scanning electron microscope(SEM). The material degradation rate, porosity, water content, cytotoxicity, and biomechanics properties were detected.2.We make the chitosan/alginate scaffolds of tissue engineering by freeze-drying, respectively, in4℃,25℃,37℃conditions, at0.5%solution of genipin crosslinked24h, as the experimental group. The scaffolds of not cross-linked with genipin as a control group to evaluate the degradation rate of scaffolds, cytotoxicity, porosity, water content and biomechanics properties.
Rssults1.①UV group and GP group showed more pore structure, and there was no difference.②Degradation rate of UV group was higher than that in GP group.③The porosity for GP group and UV group was no difference. The difference of water content is meaningful in
Statistics.④GP group and UV group showed low cytotoxicity, good biocompatibility.⑤CP group of biomechanics properties were significantly inproved compared with UV group.2.①The color of scaffolds material from light purple to purplish black, according with the rise of the cross-linking temperature, however, not cross-linked scaffold material is white;⑤The degradation rate after4weeks at4℃group was21.54%±3.07%,25℃group was9.9%±1.2%,37℃group was8.98%±0.79%. Among them, the37℃group of degradation resistant capacity is better than the4℃group (P<0.05). The experimental groups were better than the control group (P<0.01).③Elastic modulus at4℃group was0.84±0.55,25"C group was1.44±0.06,37℃group was1.53±0.02.37℃group and25℃group of tensile properties better than control group (P<0.05).④Forosity,water content,cytotoxicity between the experimental groups had no significant difference.
Conclusion1. Genipin crosslinked chitosan/alginate composite scaffold material has good biological characteristics. It is a very potential material in tissue engineering spinal cord field.2. With the cross-linking temperature increasing, scaffold degradation resistant capacity and tensile properties to strengthen, but on the scaffold material structure, cytotoxicity, porosity and water content were not altered significantly.
本实验通过冷冻干燥法制备壳聚糖/藻酸盐复合支架材料,并采用新型天然交联剂京尼平对材料进行交联,探究京尼平、紫外线两种交联方法对支架材料制备的影响。
目的：1.比较京尼平和紫外线交联后的壳聚糖/藻酸盐复合支架材料的降解率、孔隙率、含水量、细胞毒性以及生物力学等特性；2.探究交联温度对京尼平交联壳聚糖/藻酸盐组织工程支架的影响。
方法：取1只成年健康Wistar大鼠骨髓,分离、培养BMSCs.采用冷冻干燥法制备壳聚糖/藻酸盐复合支架。1.①按照交联方法不同分为：京尼平组、紫外线组。②扫描电镜下观察材料的表面结构以及检测材料的降解率、孔隙率、含水量、细胞毒性以及生物力学。2.运用冷冻干燥法制备壳聚糖/藻酸盐支架材料,分别在4℃、25℃、37℃条件,于0.5%京尼平溶液中交联24h,作为实验组。以未用京尼平交联的支架材料作为对照组,评价支架材料降解率、细胞毒性、孔隙率、含水量以及生物力学性能的特点。
结果：1.①紫外线组与京尼平组均表现为多孔隙结构,无明显差异。②紫外线组降解率高于京尼平组。③京尼平组与紫外线组的孔隙率差异无统计学意义,含水量差异比较有统计学意义。④两组均表现为较低的细胞毒性,良好的生物相容性。⑤京尼平交联组的生物力学特性较紫外线组显著提高。2.①伴随交联温度升高,支架材料的颜色从浅紫色变为紫黑色,未交联的支架材料为白色；②降解率4周后在4℃组为21.54%±3.07%、25℃组为9.9%±1.2%、37℃组为8.98%±0.79%。其中,37℃和25℃组抗降解能力优于4℃组(P<0.05)。各实验组均优于对照组(P<0.01)。③弹性模量在4℃组为0.84±0.55,25℃组为1.44±0.06,37℃组为1.53±0.02,对照组为0.79±0.16。对照组与25℃组与37℃组之间差异计较有统计学意义(P<0.05)。④孔隙率、含水量、细胞毒性各实验组间未见明显差异。
结论：1.京尼平交联的壳聚糖／藻酸盐复合支架材料,具有良好的生物学特性,为组织工程脊髓领域提供了非常具有潜力的材料。2.随着交联温度的提高,支架材料的抗降解能力、抗拉伸性能增加,且对支架材料的结构、细胞毒性、孔隙率以及含水量无明显影响。
Background American scholars proposed "tissue engineering" concept in the1980s, tissue engineering is an interdisciplinary subject, including life science, materials science and other disciplines of knowledge. Tissue engineering research includes: seed cells, scaffolds, extracellular matrix. Among them, the scaffold material is a hot research topic in recent years in this field.
Chitosan/alginate composite scaffolds was produced by freeze drying.In this study,we adopt a new natural crosslinking agent genipin crosslinking of the materials, to explore the effect of preparation of scaffold by genipin, UV two crosslinking methods.
Objective1. To investigate genipin(GP) and ultraviolet rays(UV) crosslinked effects of chitosan/alginate scaffolds for tissue engineering by detecting the change of the chitosan/alginate scaffolds material degradation rate, porosity, water content, cytotoxicity and biomechanics properties.2. we try to explore cross-linking temperature of genipin crosslinked effects of chitosan/alginate scaffolds for tissue engineering.
Methods BMSCs were separated and cultured from one Wistar rat.Chitosan/alginate scaffolds was produced via freeze drying.1.①ccording to different cross-linking methods, biomaterials were divided into (GP)group,(UV)group.②he surface structure of two groups was detected by scanning electron microscope(SEM). The material degradation rate, porosity, water content, cytotoxicity, and biomechanics properties were detected.2.We make the chitosan/alginate scaffolds of tissue engineering by freeze-drying, respectively, in4℃,25℃,37℃conditions, at0.5%solution of genipin crosslinked24h, as the experimental group. The scaffolds of not cross-linked with genipin as a control group to evaluate the degradation rate of scaffolds, cytotoxicity, porosity, water content and biomechanics properties.
Rssults1.①UV group and GP group showed more pore structure, and there was no difference.②Degradation rate of UV group was higher than that in GP group.③The porosity for GP group and UV group was no difference. The difference of water content is meaningful in
Statistics.④GP group and UV group showed low cytotoxicity, good biocompatibility.⑤CP group of biomechanics properties were significantly inproved compared with UV group.2.①The color of scaffolds material from light purple to purplish black, according with the rise of the cross-linking temperature, however, not cross-linked scaffold material is white;⑤The degradation rate after4weeks at4℃group was21.54%±3.07%,25℃group was9.9%±1.2%,37℃group was8.98%±0.79%. Among them, the37℃group of degradation resistant capacity is better than the4℃group (P<0.05). The experimental groups were better than the control group (P<0.01).③Elastic modulus at4℃group was0.84±0.55,25"C group was1.44±0.06,37℃group was1.53±0.02.37℃group and25℃group of tensile properties better than control group (P<0.05).④Forosity,water content,cytotoxicity between the experimental groups had no significant difference.
Conclusion1. Genipin crosslinked chitosan/alginate composite scaffold material has good biological characteristics. It is a very potential material in tissue engineering spinal cord field.2. With the cross-linking temperature increasing, scaffold degradation resistant capacity and tensile properties to strengthen, but on the scaffold material structure, cytotoxicity, porosity and water content were not altered significantly.
引文
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[1]汪大彬,文益民,蓝旭,等.壳聚糖-藻酸盐复合BMSCs修复急性脊髓损伤的实验研究[J].中国修复重建杂志外科杂志,2010,24(2)：190-196.
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[4]谢德明.生命科学新领域—组织工程[J].生物学通报,2001,36(10)：7.
[5]Todd C Holems.Novel peptide-based biomaterial scaffolds for tissue engineering[J].Trend in biotechnology,2002,20 (1):16.
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[7]Terada S, Sato M, Sevy A, et al.Tissue engineering in the twenty first century[J].Yonsei Med J,2000,14:685.
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