title: Evaluation of cell and matrix mechanics using fluorescence excitation spectroscopy: Feasibility study in collagen gels containing fibroblasts.
abstract: OBJECTIVE
  Collagen 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.
  
  
  METHODS
  Human 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 240-520/290-530 nm, were used to identify the wavelengths of interest.
  
  
  RESULTS
  Fluorescence excitation/emission maps exhibited two distinct excitation/emission bands whose intensities increased as the fibroblasts remodeled and increased the stiffness of the ECM: The 290/340 nm band ascribed to tryptophan and the 330/390 nm 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 < 0.05) and R = 0.77 (P = 0.12) for 50,000 and 100,000 cells/ml, respectively.
  
  
  CONCLUSIONS
  The endogenous fluorescence intensity ascribed to pepsin-digestible cross-links of collagen may serve as an optical marker to evaluate changes in the mechanical properties of the ECM; this is relevant to collagenous tissues for which pathological states are related to mechanical alterations, such as keratoconus in cornea and osteoarthritis in articular cartilage.
authors.affiliation: Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts.|Department of Dermatology, Harvard Medical School, Boston, Massachusetts.
authors.forename: Juan Pablo
authors.initials: JP
authors.lastname: Padilla-Martinez
authors.affiliation: Department of Biomedical Engineering, Cornell University, Ithaca, New York.
authors.forename: Ruisheng
authors.initials: R
authors.lastname: Wang
authors.affiliation: Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts.|Department of Dermatology, Harvard Medical School, Boston, Massachusetts.
authors.forename: Walfre
authors.initials: W
authors.lastname: Franco
mesh_terms: D015415:Biomarkers; D001696:Biomechanical Phenomena; D002478:Cells, Cultured; D024042:Collagen Type I / Q000502:physiology*; D055119:Elastic Modulus; D005109:Extracellular Matrix / Q000502:physiology*; D005240:Feasibility Studies; D005347:Fibroblasts / Q000502:physiology*; D006801:Humans; D061848:Optical Imaging / Q000379:methods*; D013050:Spectrometry, Fluorescence
chemical_list: D015415:Biomarkers; D024042:Collagen Type I
keywords: autofluorescence; collagen cross-links; collagen gel; elastic modulus; extracellular matrix; fibroblast; indentation; tryptophan