Taylor Adam J, Ratner Buddy D, Buttery Lee D K, Alexander Morgan R
School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK; Department of Chemical Engineering, University of Washington, Seattle, WA 98195, USA; National ESCA and Surface Analysis Center for Biomedical Problems, University of Washington, Seattle, WA 98195, USA.
National ESCA and Surface Analysis Center for Biomedical Problems, University of Washington, Seattle, WA 98195, USA; Department of Bioengineering, University of Washington, Seattle, WA 98195, USA.
Acta Biomater. 2015 Mar;14:70-83. doi: 10.1016/j.actbio.2014.12.005. Epub 2014 Dec 15.
Cell-secreted matrices (CSMs), where extracellular matrix (ECM) deposited by monolayer cell cultures is decellularized, have been increasingly used to produce surfaces that may be reseeded with cells. Such surfaces are useful to help us understand cell-ECM interactions in a microenvironment closer to the in vivo situation than synthetic substrates with adsorbed proteins. We describe the production of CSMs from mouse primary osteoblasts (mPObs) exposed to cytokine challenge during matrix secretion, mimicking in vivo inflammatory environments. Time-of-flight secondary ion mass spectrometry data revealed that CSMs with cytokine challenge at day 7 or 12 of culture can be chemically distinguished from one another and from untreated CSM using multivariate analysis. Comparison of the differences with reference spectra from adsorbed protein mixtures points towards cytokine challenge resulting in a decrease in collagen content. This is supported by immunocytochemical and histological staining, demonstrating a 44% loss of collagen mass and a 32% loss in collagen I coverage. CSM surfaces demonstrate greater cell adhesion than adsorbed ECM proteins. When mPObs were reseeded onto cytokine-challenged CSMs they exhibited reduced adhesion and elongated morphology compared to untreated CSMs. Such changes may direct subsequent cell fate and function, and provide insights into pathological responses at sites of inflammation.
细胞分泌基质（CSM）是通过单层细胞培养沉积的细胞外基质（ECM）脱细胞后得到的，已越来越多地用于制备可重新接种细胞的表面。与吸附有蛋白质的合成基质相比，此类表面有助于我们在更接近体内情况的微环境中理解细胞与ECM的相互作用。我们描述了在基质分泌过程中暴露于细胞因子刺激的小鼠原代成骨细胞（mPOb）产生CSM的过程，模拟体内炎症环境。飞行时间二次离子质谱数据显示，使用多变量分析可以在化学上区分培养第7天或第12天受到细胞因子刺激的CSM以及未处理的CSM。将这些差异与吸附蛋白混合物的参考光谱进行比较，表明细胞因子刺激导致胶原蛋白含量降低。免疫细胞化学和组织学染色支持了这一点，显示胶原蛋白质量损失44%，I型胶原蛋白覆盖率损失32%。CSM表面显示出比吸附的ECM蛋白更强的细胞黏附性。当将mPOb重新接种到受到细胞因子刺激的CSM上时，与未处理的CSM相比，它们表现出黏附性降低和形态拉长。这些变化可能会引导后续的细胞命运和功能，并为炎症部位的病理反应提供见解。
