Experimental Trauma Surgery, Department of Orthopedics and Trauma Surgery, University Hospital of Schleswig-Holstein, Campus Kiel, Arnold-Heller-Straße 3, 24105 Kiel, Germany.
Functional Nanomaterials, Institute for Materials Science, Kiel University, Kaiserstr. 2, 24143 Kiel, Germany.
Int J Mol Sci. 2022 Mar 21;23(6):3379. doi: 10.3390/ijms23063379.
Graphene oxide (GO) is a promising material for bone tissue engineering, but the validation of its molecular biological effects, especially in the context of clinically applied materials, is still limited. In this study, we compare the effects of graphene oxide framework structures (F-GO) and reduced graphene oxide-based framework structures (F-rGO) as scaffold material with a special focus on vascularization associated processes and mechanisms in the bone. Highly porous networks of zinc oxide tetrapods serving as sacrificial templates were used to create F-GO and F-rGO with porosities >99% consisting of hollow interconnected microtubes. Framework materials were seeded with human mesenchymal stem cells (MSC), and the cell response was evaluated by confocal laser scanning microscopy (CLSM), deoxyribonucleic acid (DNA) quantification, real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and alkaline phosphatase activity (ALP) to define their impact on cellular adhesion, osteogenic differentiation, and secretion of vascular growth factors. F-GO based scaffolds improved adhesion and growth of MSC as indicated by CLSM and DNA quantification. Further, F-GO showed a better vascular endothelial growth factor (VEGF) binding capacity and improved cell growth as well as the formation of microvascular capillary-like structures in co-cultures with outgrowth endothelial cells (OEC). These results clearly favored non-reduced graphene oxide in the form of F-GO for bone regeneration applications. To study GO in the context of a clinically used implant material, we coated a commercially available xenograft (Bio-Oss® block) with GO and compared the growth of MSC in monoculture and in coculture with OEC to the native scaffold. We observed a significantly improved growth of MSC and formation of prevascular structures on coated Bio-Oss®, again associated with a higher VEGF binding capacity. We conclude that graphene oxide coating of this clinically used, but highly debiologized bone graft improves MSC cell adhesion and vascularization.
氧化石墨烯（GO）是一种很有前途的骨组织工程材料，但它的分子生物学效应，尤其是在临床应用材料方面的验证仍然有限。在这项研究中，我们比较了氧化石墨烯框架结构（F-GO）和基于还原氧化石墨烯的框架结构（F-rGO）作为支架材料的效果，特别关注与骨血管化相关的过程和机制。使用氧化锌四足体的高度多孔网络作为牺牲模板来制备具有>99%孔隙率的 F-GO 和 F-rGO，其由中空互连的微管组成。将框架材料接种人骨髓间充质干细胞（MSC），通过共聚焦激光扫描显微镜（CLSM）、脱氧核糖核酸（DNA）定量、实时聚合酶链反应（RT-PCR）、酶联免疫吸附测定（ELISA）和碱性磷酸酶活性（ALP）评估细胞反应，以定义它们对细胞黏附、成骨分化和血管生长因子分泌的影响。F-GO 基支架通过 CLSM 和 DNA 定量表明可改善 MSC 的黏附和生长。此外，F-GO 显示出更好的血管内皮生长因子（VEGF）结合能力，并在与血管生成内皮细胞（OEC）共培养时改善细胞生长和微血管样毛细血管结构的形成。这些结果明显有利于非还原氧化石墨烯形式的 F-GO 用于骨再生应用。为了研究 GO 在临床应用植入材料中的情况，我们用 GO 涂覆一种市售的异种移植物（Bio-Oss® 块），并将 MSC 在单核培养和与 OEC 共培养中的生长情况与天然支架进行了比较。我们观察到在涂覆的 Bio-Oss® 上 MSC 的生长和前血管结构的形成明显得到改善，这与更高的 VEGF 结合能力有关。我们得出结论，这种临床应用的、高度去生物化的骨移植物的氧化石墨烯涂层可改善 MSC 细胞黏附和血管化。
