title: Coherent anti-Stokes Raman scattering microscopy of human smooth muscle cells in bioengineered tissue scaffolds.
abstract: The integration of living, human smooth muscle cells in biosynthesized cellulose scaffolds was monitored by nonlinear microscopy toward contractile artificial blood vessels. Combined coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy was applied for studies of the cell interaction with the biopolymer network. CARS microscopy probing CH(2)-groups at 2845 cm(-1) permitted three-dimensional imaging of the cells with high contrast for lipid-rich intracellular structures. SHG microscopy visualized the fibers of the cellulose scaffold, together with a small signal obtained from the cytoplasmic myosin of the muscle cells. From the overlay images we conclude a close interaction between cells and cellulose fibers. We followed the cell migration into the three-dimensional structure, illustrating that while the cells submerge into the scaffold they extrude filopodia on top of the surface. A comparison between compact and porous scaffolds reveals a migration depth of <10 μm for the former, whereas the porous type shows cells further submerged into the cellulose. Thus, the scaffold architecture determines the degree of cell integration. We conclude that the unique ability of nonlinear microscopy to visualize the three-dimensional composition of living, soft matter makes it an ideal instrument within tissue engineering.
authors.affiliation: Chalmers University of Technology, Molecular Microscopy, Department of Chemical and Biological Engineering, SE-412 96 Göteborg, Sweden.
authors.forename: Christian
authors.initials: C
authors.lastname: Brackmann
authors.forename: Maricris
authors.initials: M
authors.lastname: Esguerra
authors.forename: Daniel
authors.initials: D
authors.lastname: Olausson
authors.forename: Dick
authors.initials: D
authors.lastname: Delbro
authors.forename: Alexandra
authors.initials: A
authors.lastname: Krettek
authors.forename: Paul
authors.initials: P
authors.lastname: Gatenholm
authors.forename: Annika
authors.initials: A
authors.lastname: Enejder
mesh_terms: D001698:Biomedical Engineering / Q000295:instrumentation; D001808:Blood Vessels / Q000166:cytology* / Q000254:growth & development*; D002478:Cells, Cultured; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006801:Humans; D008853:Microscopy / Q000295:instrumentation*; D032389:Myocytes, Smooth Muscle / Q000166:cytology* / Q000502:physiology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013059:Spectrum Analysis, Raman / Q000295:instrumentation*; D054457:Tissue Scaffolds