Amaral Isabel F, Sousa Susana R, Neiva Ismael, Marcos-Silva Lara, Kirkpatrick Charles J, Barbosa Mário A, Pêgo Ana P
INEB-Instituto de Engenharia Biomédica; Universidade do Porto; Porto, Portugal.
INEB-Instituto de Engenharia Biomédica; Universidade do Porto; Porto, Portugal; REQUIMTE; Instituto Superior de Engenharia do Porto; Instituto Politécnico do Porto; Porto, Portugal.
Biomatter. 2013 Apr-Jun;3(2). doi: 10.4161/biom.24791. Epub 2013 Apr 29.
In this study, (125)I-radiolabelling was explored to follow the kinetics and isotherm of fibronectin (FN) adsorption to porous polymeric scaffolds, as well as to assess the elution and exchangeability of pre-adsorbed FN following incubation in serum-containing culture medium. Chitosan (CH) porous scaffolds with two different degrees of acetylation (DA 4% and 15%) were incubated in FN solutions with concentrations ranging from 5 to 50 µg/mL. The kinetic and isotherm of FN adsorption to CH were successfully followed using (125)I-FN as a tracer molecule. While on DA 4% the levels of adsorbed FN increased linearly with FN solution concentration, on DA 15% a saturation plateau was attained, and FN adsorbed amounts were significantly lower. These findings were supported by immunofluorescent studies that revealed, for the same FN solution concentration, higher levels of exposed cell-binding domains on DA 4% as compared with DA 15%. Following incubation in serum containing medium, DA 4% also revealed higher ability to exchange pre-adsorbed FN by new FN molecules from serum than DA 15%. In accordance, when assessing the efficacy of passively adsorbed FN to promote endothelial cell (EC) adhesion to CH, ECs were found to adhere at higher levels to DA 4% as compared with DA 15%, 5 µg/mL of FN being already efficient in promoting cell adhesion and cytoskeletal organization on CH with DA 4%. Taken together the results show that protein radiolabelling can be used as an effective tool to study protein adsorption to porous polymeric scaffolds, both from single and complex protein solutions.
在本研究中，探索了用¹²⁵I进行放射性标记以追踪纤连蛋白（FN）吸附到多孔聚合物支架上的动力学和等温线，以及评估在含血清培养基中孵育后预吸附FN的洗脱和交换能力。将具有两种不同乙酰化程度（脱乙酰度4%和15%）的壳聚糖（CH）多孔支架在浓度范围为5至50μg/mL的FN溶液中孵育。以¹²⁵I-FN作为示踪分子成功追踪了FN吸附到CH上的动力学和等温线。在脱乙酰度4%时，吸附的FN水平随FN溶液浓度呈线性增加，而在脱乙酰度15%时达到饱和平台期，且吸附的FN量显著更低。免疫荧光研究支持了这些发现，该研究表明，对于相同的FN溶液浓度，脱乙酰度4%上暴露的细胞结合结构域水平高于脱乙酰度15%。在含血清培养基中孵育后，脱乙酰度4%还显示出比脱乙酰度15%更高的能力，能够用血清中的新FN分子交换预吸附的FN。相应地，在评估被动吸附的FN促进内皮细胞（EC）黏附到CH的效果时，发现与脱乙酰度15%相比，EC在内皮细胞在脱乙酰度4%上的黏附水平更高，5μg/mL的FN已足以有效促进脱乙酰度4%的CH上的细胞黏附和细胞骨架组织。综合来看，结果表明蛋白质放射性标记可作为一种有效工具，用于研究蛋白质从单一和复杂蛋白质溶液吸附到多孔聚合物支架上的情况。
