Evaluation of cell and matrix mechanics using fluorescence excitation spectroscopy: Feasibility study in collagen gels containing fibroblasts

Background and ObjectiveCollagen gels containing cells are commonly used in tissue engineering, wound healing, and cancer research to investigate the interplay between cells and the extracellular matrix (ECM), as changes in the density and stiffness of the microenvironment are known to play a role in many diseases or pathological conditions. In these gels, the stiffness is regularly determined using destructive methods, such as indentation and tensile tests. Certain molecules native to cells and the ECM display fluorescence upon irradiation with ultraviolet light. The objective of the present study was to investigate the feasibility of using the endogenous, or innate, fluorescence of collagen gels containing fibroblasts as an optical marker to evaluate changes in the mechanical properties of the ECM.Materials and MethodsHuman foreskin fibroblasts cells at concentrations of 50,000 and 100,000 cells/ml were cultured in three-dimensional gels of type I collagen for 16 days. Fibroblast cells remodeled the ECM, contracting and increasing the stiffness of the gel. During this remodeling process, changes in mechanical properties and fluorescence were measured with an indentation test and a spectrofluorometer, respectively. Force and displacement measurements from the indentation test were used to calculate the elastic modulus of the gel. Maps of fluorescence intensity, at excitation/emission of 240-520/290-530nm, were used to identify the wavelengths of interest.ResultsFluorescence excitation/emission maps exhibited two distinct excitation/emission bands whose intensities increased as the fibroblasts remodeled and increased the stiffness of the ECM: The 290/340nm band ascribed to tryptophan and the 330/390nm band ascribed to cross-links of collagen (pepsin-digestible cross-links). A Spearman correlation analysis, between the elastic modulus of the gel containing fibroblasts and the fluorescence of cross-links of collagen, resulted in R=0.95 (P

背景与目的 含细胞的胶原凝胶常用于组织工程、伤口愈合和癌症研究中，以探究细胞与细胞外基质（ECM）之间的相互作用，因为已知微环境的密度和硬度变化在许多疾病或病理状况中起作用。在这些凝胶中，硬度通常使用破坏性方法测定，如压痕和拉伸试验。细胞和ECM固有的某些分子在紫外线照射下会显示荧光。本研究的目的是探讨利用含成纤维细胞的胶原凝胶的内源性（或固有）荧光作为一种光学标记来评估ECM机械性能变化的可行性。 材料与方法 将浓度为50000和100000个细胞/毫升的人包皮成纤维细胞在I型胶原三维凝胶中培养16天。成纤维细胞重塑ECM，使凝胶收缩并增加其硬度。在此重塑过程中，分别用压痕试验和荧光分光光度计测量机械性能和荧光的变化。压痕试验中的力和位移测量用于计算凝胶的弹性模量。在激发/发射波长为240 - 520/290 - 530nm的荧光强度图谱用于确定感兴趣的波长。 结果 荧光激发/发射图谱显示出两个不同的激发/发射带，其强度随着成纤维细胞重塑并增加ECM硬度而增加：归属于色氨酸的290/340nm带和归属于胶原交联（胃蛋白酶可消化交联）的330/390nm带。对含成纤维细胞的凝胶弹性模量与胶原交联荧光之间进行斯皮尔曼相关分析，结果R = 0.95（P（此处似乎内容不完整）