Galli C, Parisi L, Piergianni M, Smerieri A, Passeri G, Guizzardi S, Costa F, Lumetti S, Manfredi E, Macaluso G M
Dep. Biomedical, Biotechnological and Translational Sciences, University of Parma, Parma, Italy; Centro Universitario di Odontoiatria, University of Parma, Parma, Italy; Istituto Materiali per l'Elettronica ed il Magnetismo IMEM-CNR, Parma, Italy.
Dep. Biomedical, Biotechnological and Translational Sciences, University of Parma, Parma, Italy; Centro Universitario di Odontoiatria, University of Parma, Parma, Italy.
Acta Biomater. 2016 Sep 15;42:147-156. doi: 10.1016/j.actbio.2016.07.035. Epub 2016 Jul 20.
Protein adsorption is the first and decisive step to define cell-biomaterial interaction. Guiding the adsorption of desired protein species may represent a viable approach to promote cell activities conducive to tissue regeneration. The aim of the present study was to investigate whether immobilized anti-Fibronectin aptamers could promote the attachment and growth of osteoblastic cells. Polyethyleneglycole diacrylate/thiolated Hyaluronic Acid hydrogels (PEGDA/tHA) were coated with anti-Fibronectin aptamers. Hydrogel loading and Fibronectin bonding were investigated, through spectrophotometry and Bradford assay. Subsequently, human osteoblasts (hOBs) were cultured on hydrogels for 10days in 2D and 3D cultures. Cells were monitored through microscopy and stained for focal adhesions, microfilaments and nuclei using fluorescence microscopy. Samples were also included in paraffin and stained with Hematoxylin-Eosin. Cell number on hydrogels was quantitated over time. Cell migration into the hydrogels was also studied through Calcein AM staining. Aptamers increased the number of adherent hOBs and their cytoplasm appeared more spread and richer in adhesion complexes than on control hydrogels. Viability assays confirmed that significantly more cells were present on hydrogels in the presence of aptamers, already after 48h of culture. When hOBs were encapsulated into hydrogels, cells were more numerous on aptamer-containing PEGDA-tHA. Cells migrated deeper in the gel in the presence of DNA aptamers, appearing on different focus planes. Our data demonstrate that anti-Fibronectin aptamers promote scaffold enrichment for this protein, thus improving cell adhesion and scaffold colonization.
We believe aptamer coating of biomaterials is a useful and viable approach to improve the performance of scaffold materials for both research and possibly clinical purposes, because different medical devices could be envisaged able to capture bioactive mediators from the patients' blood and concentrate them where they are needed, on the biomaterial itself. At the same time, this technology could be used to confer 3D cell culture scaffold with the ability to store proteins, such as Fibronectin, taking it from the medium and capture what is produced by cells. This is an improvement of traditional biomaterials that can be enriched with exogenous molecules but are not able to selectively capture a desired molecule.
蛋白质吸附是定义细胞与生物材料相互作用的第一步且具有决定性作用。引导所需蛋白质种类的吸附可能是促进有利于组织再生的细胞活动的一种可行方法。本研究的目的是调查固定化抗纤连蛋白适体是否能促进成骨细胞的附着和生长。用抗纤连蛋白适体包被聚乙二醇二丙烯酸酯/硫醇化透明质酸水凝胶（PEGDA/tHA）。通过分光光度法和Bradford测定法研究水凝胶负载和纤连蛋白结合情况。随后，在二维和三维培养中，将人成骨细胞（hOBs）在水凝胶上培养10天。通过显微镜监测细胞，并用荧光显微镜对粘着斑、微丝和细胞核进行染色。样品也制成石蜡切片并用苏木精 - 伊红染色。对水凝胶上的细胞数量随时间进行定量。还通过钙黄绿素AM染色研究细胞向水凝胶中的迁移。与对照水凝胶相比，适体增加了贴壁hOBs的数量，并且其细胞质看起来更伸展，粘着复合物更丰富。活力测定证实，在培养48小时后，在存在适体的情况下，水凝胶上存在的细胞明显更多。当hOBs被封装到水凝胶中时，含适体的PEGDA - tHA上的细胞更多。在DNA适体存在的情况下，细胞在凝胶中迁移得更深，出现在不同的焦平面上。我们的数据表明，抗纤连蛋白适体促进了该蛋白质在支架上的富集，从而改善了细胞粘附和支架定植。
我们认为生物材料的适体包被是一种有用且可行的方法，可用于改善支架材料在研究和可能的临床应用中的性能，因为可以设想不同的医疗设备能够从患者血液中捕获生物活性介质并将它们集中在生物材料自身所需的位置。同时，该技术可用于赋予三维细胞培养支架储存蛋白质（如纤连蛋白）的能力，从培养基中获取并捕获细胞产生的物质。这是对传统生物材料的一种改进，传统生物材料可以用外源分子富集，但不能选择性地捕获所需分子。
