不同浓度的葡聚糖/明胶复合水凝胶支架性能表征及其对体外构建组织工程髓核的影响  

Features of dextran / gelatin composite hydrogels with different concentrations and effect of hydrogel concentration on constructing tissue-engineered nucleus pulposus in vitro

作　　者：甘翼搏[1] 李培[1] 周强[1] 莫秀梅[2] 宋磊[1] 徐源[3] 李松涛[1] 李素凯 

机构地区：[1]第三军医大学西南医院全军矫形外科中心,组织工程国家地方联合工程实验室,重庆市再生重建医学工程技术研究中心,重庆市组织工程实验室,重庆400038 [2]东华大学化工与生物工程学院,上海201620 [3]第三军医大学新桥医院骨科,重庆400037

出　　处：《第三军医大学学报》2015年第8期707-712,共6页Journal of Third Military Medical University

基　　金：国家自然科学基金(81272029;81027005);第三军医大学科技成果转化基金(2011XZH006);国家高技术研究发展计划(863计划;2006AA02Z4E3)~~

摘　　要：目的研究不同浓度的葡聚糖/明胶复合水凝胶支架材料对体外构建组织工程髓核的影响,对其性能进行表征,探索适用于组织工程髓核的水凝胶浓度。方法通过Schiff交联反应制备葡聚糖/明胶复合水凝胶支架材料,对不同浓度（6%、8%、10%、12%、14%）水凝胶的内部形态结构、孔径、孔隙率、杨氏模量和细胞相容性进行评估,分离培养猪原代髓核细胞,以P1代细胞在不同浓度的水凝胶内部进行三维培养,MTT染色观察细胞活性,Real-time PCR法检测髓核细胞特异性基因aggrecan和typeⅡcollagen表达。结果葡聚糖/明胶复合水凝胶在电镜扫描下呈现多孔网络结构,随着浓度的升高,水凝胶的孔径（38.182-82.675μm）和孔隙率（95.5%-98.3%）持续下降,杨氏模量不断升高（0.135-11.366 k Pa）;该水凝胶具有较好的细胞相容性,但是6%、8%的水凝胶分别在接种细胞后第4、7天后细胞的黏附和增殖显著下降（P〈0.05）。接种1周后,浓度为10%的水凝胶内部三维培养的髓核细胞活性显著高于其他浓度水凝胶（P〈0.05）,同时aggrecan、typeⅡcollagen基因表达显著高于其他各组（P〈0.05）。结论浓度为10%的葡聚糖/明胶复合水凝胶兼具有适宜的三维多孔网络结构、稳定的力学结构以及较好的细胞相容性,适用于构建组织工程髓核。Objective To investigate the effect of dextran /gelatin composite hydrogels with different concentrations on constructing tissue-engineered nucleus pulposus in vitro and characterize the hydrogels so as to optimize the concentration of dextran / gelatin composite hydrogels for nucleus pulposus tissue engineering.Methods The dextran / gelatin composite hydrogels with different concentrations were fabricated by Schiff base crosslinking reaction, and the interior morphology, pore size, porosity, Young ＇s modulus and biocompatibility of the hydrogels were evaluated. Thereafter,porcine nucleus pulposus cells were isolated and cultured,and the first-passage cells were in situ seeded into the hydrogels,and then the cell viability as well as nucleus pulposus-related gene expression（ aggrecan,type Ⅱ collagen） was tested by MTT assay and RTPCR respectively. Results The interior morphology of the composite hydrogels under a scanning electron microscope was porous network structure. The pore size and porosity（ 38. 182 - 82. 675 μm and 95. 5% -98. 3%,respectively） were decreased along with increase of the hydrogel concentration,but Young ＇s modulus（ 0. 135 - 11. 366 k Pa） was increased. The composite hydrogels showed favorable biocompatility,but the degradation of hydrogels with concentrations of 6% and 8% led to decline of cell proliferation and adhesion on the surface on day 4 and day 7. After 1 week,cell viability and expressions of aggrecan and type Ⅱcollagen in the nucleus pulposus cells seeded in the hydrogel with concentration of 10% were enhanced compared to other groups. Conclusion The dextran / gelatin composite hydrogel with concentration of 10%processes proper three-dimensional porous structure,stable mechanical structure and favorable biocompability,and is suitable for constructing tissue-engineered nucleus pulposus.

关 键 词：支架 复合水凝胶 浓度 组织工程 髓核 

分 类 号：R318.08[医药卫生—生物医学工程] R318.19[医药卫生—基础医学]