Thayer School of Engineering, Dartmouth College, Hanover, NH 03755, United States of America; NanoString Technologies, Inc., Seattle, WA, 98109, United States of America.
NanoString Technologies, Inc., Seattle, WA, 98109, United States of America.
Acta Biomater. 2022 Jan 15;138:342-350. doi: 10.1016/j.actbio.2021.10.018. Epub 2021 Oct 19.
Surprisingly little clarity exists concerning effects of biomaterial properties on spatially localized protein expression, which drives implant success. Wound healing and tissue regeneration must be optimally supported by the implant, adsorbed proteins, immune cells, and fibroblasts; cells determine repair and functional recovery through protein production and regulation. However, not yet fully understood is how implants differentially drive spatial quantities of individual proteins both within the implant interior and the tissue surrounding it. Here we apply GeoMx digital spatial profiling to site-specifically investigate protein production in porous implants. Data is collected on the location and quantity of 40+ proteins from formalin-fixed, paraffin-embedded tissue slides of anisotropic tissue scaffolds (n = 18) with differing pore sizes (35 µm, 53 µm) and implantation durations (2, 14, 28 days); matching bulk gene expression data (700+ genes) is measured for identical implants. Notably, we discover fundamental spatial relationships in protein localization that in both the implant interior and the exterior are either uniquely independent or dependent of implant microstructure: dendritic cell marker CD11c and fibronectin significantly dominate the scaffold interior, while cell-to-cell adhesion marker CD34 and anti-inflammatory M2 polarization marker CD163 localize in the exterior. Lastly, collating spatial and bulk information, unique spatiotemporal expression patterns are identified for markers such as fibronectin, which are only uncoverable through spatial profiling and are otherwise hidden in bulk expression results. Together, these discoveries illustrate the critical importance of quantifying spatial expression patterns for implants, facilitating a paradigm shift in the iterative design, mechanistic understanding, and rapid assessment of biomaterials. STATEMENT OF SIGNIFICANCE: Spatial localization and expression of proteins, which determine implant success, are not fully understood because quantitative high-plex profiling is challenging. Applying GeoMx digital spatial profiling to site-specifically investigate protein production in porous implants, data is collected on the location and quantity of 40+ protein targets from tissue scaffolds with differing pore sizes (35 µm, 53 µm) and implantation durations (2, 14, 28 days). Collecting in parallel matched bulk gene expression data (700+ genes) for identical implants, we discover significant spatiotemporal expression patterns that remain otherwise hidden in differential bulk results. This new approach for the rapid assessment of biomaterials offers an enhanced mechanistic understanding and enables the tailoring of implants for superior regenerative outcomes.
令人惊讶的是，对于生物材料特性如何影响驱动植入物成功的空间定位蛋白质表达，目前还没有明确的认识。植入物必须通过吸附蛋白、免疫细胞和成纤维细胞来优化地支持伤口愈合和组织再生；细胞通过蛋白质的产生和调节来决定修复和功能恢复。然而，人们还不完全了解植入物如何在植入物内部和周围组织中，以不同的方式驱动个体蛋白质的空间数量。在这里，我们应用 GeoMx 数字空间分析技术来特异性地研究多孔植入物中的蛋白质产生。我们从具有不同孔径（35 µm、53 µm）和植入时间（2、14、28 天）的各向异性组织支架的福尔马林固定、石蜡包埋组织切片上收集了 40 多种蛋白质的位置和数量的数据；对于相同的植入物，还测量了匹配的大量基因表达数据（700 多个基因）。值得注意的是，我们发现了蛋白质定位中的基本空间关系，这些关系在植入物内部和外部都是独立的或依赖于植入物的微观结构的：树突状细胞标记物 CD11c 和纤维连接蛋白显著占据支架内部，而细胞间黏附标记物 CD34 和抗炎 M2 极化标记物 CD163 则定位于外部。最后，综合空间和大量信息，我们确定了纤维连接蛋白等标记物的独特时空表达模式，这些模式只有通过空间分析才能揭示，否则在大量表达结果中是隐藏的。总之，这些发现说明了定量空间表达模式对于植入物的重要性，促进了对生物材料的迭代设计、机制理解和快速评估的范式转变。
