Total RNA-sequencing Detects Transcriptome Differences in Mouse Preosteoblast Response to Bulk Nanostructured Titanium

Titanium is the most widely used alloy family in dental and orthopedic implants due to its natural ability to integrate into bone, but cytotoxic alloying elements are required in titanium for its mechanical properties to match the functionality of natural bone in high-load bearing applications. Recent advances in nanostructuring, such as Equal Channel Angular Pressing-Conform (ECAP-C), reduce the grain size and increase the strength of pure grades of titanium, thereby eliminating the need for cytotoxic elements and increasing cytocompatibilty as measured by traditional cell biology techniques. Transcriptomic profiling of cells grown on conventional coarse grain (CG) versus nanostructured ultrafine grain (UG) surfaces can simultaneously enhance our understanding of genomics and biomaterials, facilitating the development of a new generation of implantable materials. Sterile Ti discs, 13.5 mm in diameter and polished to a 2 nm average surface roughness were arrayed in a non-tissue culture treated 24-well cell culture plate using a block design, with 12 CG and 12 UG disks. Wells were re-fed after 24 hours and after 72 hours Ti discs were transferred to new plates for standard cell harvesting protocols. The cells from four wells were consolidated into one sample and were stored at -80°C in RNAlater. All subsequent steps were identical for all samples. Samples on CG were used as the control for UG-CG comparisons.钛是牙科与骨科植入物中应用最为广泛的合金体系，其凭借天然的骨整合能力成为主流选择，但在高负载承重应用场景中，为使钛的力学性能匹配天然骨的功能需求，必须添加具备细胞毒性的合金元素。近年来，等通道转角挤压-适配（Equal Channel Angular Pressing-Conform，ECAP-C）等纳米结构化技术可细化纯钛品级的晶粒尺寸并提升其强度，从而无需再添加细胞毒性元素，且经传统细胞生物学技术检测证实，其细胞相容性得到改善。 相较于传统粗晶（CG）表面，对在纳米结构化超细晶（UG）表面培养的细胞开展转录组谱分析，可同时深化我们对基因组学与生物材料的认知，助力新一代可植入材料的研发。 将直径13.5 mm、抛光至平均表面粗糙度为2 nm的无菌钛圆盘，以区组设计排布于未经过细胞培养处理的24孔细胞培养板中，其中包含12个粗晶圆盘与12个超细晶圆盘。 分别在细胞接种24小时后与培养72小时后更换培养液，待培养至72小时时将钛圆盘转移至新培养板，以执行标准细胞收获流程。 将来自4个孔的细胞合并为一个样本，并用RNAlater储存于-80℃环境中。所有样本的后续操作步骤均完全一致。 以粗晶组样本作为对照，用于超细晶与粗晶组间的对比分析。