Dynamic multicomponent engineered tissue reorganization and matrix deposition measured with an integrated nonlinear optical microscopy-optical coherence microscopy system

Abstract

Multicomponent tissue models are viable tools to better understand cell responses in complex environments, but present challenges when investigated with live cell microscopy noninvasively. In this study, integrated nonlinear optical microscopy-optical coherence microscopy (NLOM-OCM) was used to characterize cell interactions within three-dimensional (3-D), multicomponent extracellular matrices. In fibrin-collagen mixtures, 3T3 fibroblasts were observed to recruit both fibrin and collagen fibers while remodeling matrices. Also, NLOM-OCM was used to observe collagen deposition by neonatal human dermal fibroblasts within originally fibrin matrices over an extended time. It was observed that preferentially aligned collagen deposition could be achieved with aligned fibroblasts but that cell alignment could be achieved without aligning the extant extracellular matrix. In summary, this multimodel imaging system has potential for both real-time and longitudinal imaging of living 3-D cultures, which is particularly important for evaluating cell microenvironments in composite scaffolds or serial characterization of engineered tissue constructs during culture.

Fig. 1

Cruciform-shaped fibroblast seeded fibrin matrices were cultured under three static stretch conditions, anchored at $1.0:1.0$ (a), strip biaxial at $1.0:1.1$ (b), and equibiaxial at $1.1:1.1$ (c), where $\lambda$ is the global stretch ratio. Images were acquired from the central region of the cruciform gel.

Fig. 2

Longitudinal NLOM-OCM images from cell populated (top) and acellular regions (bottom) of a fibroblast seeded collagen-fibrin matrix. Images rendered by cell-specific two-photon excited autofluorescence (a), second harmonic generation in collagen (b), and nonspecific OCM (c) are overlaid in false color (d) showing relative spatiotemporal distributions of cells (blue), collagen (red), and fibrin (green). NLOM–OCM images were acquired from $\sim 100\mu \text{m}$ below the surface.

Fig. 3

Representative NLOM-OCM images of fibroblast seeded fibrin matrix anchored at static $1.0:1.0$ stretch ratio during 17-day culture. Cellular GFP fluorescence by TPF (a). SHG in collagen (b). Overlay of TPF and SHG, false colored green and red, showing fibroblasts and collagen, respectively (c). Nonspecific OCM images showing dense fibrin matrix (d).

Fig. 4

Representative NLOM-OCM images of fibroblast seeded fibrin matrix cultured for 17 days under static strip biaxial stretch at $1.0:1.1$ stretch ratio. Cellular GFP fluorescence by TPF (a). SHG in collagen (b). Overlay of TPF and SHG, false colored green and red, showing fibroblasts and collagen, respectively (c). Nonspecific OCM images showing dense fibrin matrix (d).

Fig. 5

Representative optical coherence microscopy (OCM) images of $5\mathrm{mg}/\mathrm{ml}$ (a), $7.5\mathrm{mg}/\mathrm{ml}$ (b), $10\mathrm{mg}/\mathrm{ml}$ (c), and $15\mathrm{mg}/\mathrm{ml}$ acellular fibrin matrices (d). Porosity, as indicated by void regions in images, decreased with increasing fibrin concentration.

Fig. 6

Fiber orientation distribution analyses of initial fibrin matrix on day 1 and deposited collagen on days 3, 10, and 17 from three-dimensional cultures anchored at $1.0:1.0$ stretch ratio (a), equibiaxial $1.1:1.1$ stretch (b), and strip biaxial at $1.0:1.1$ stretch ratio (c). Relative intensity represents fraction of total fibrin or collagen fibers at orientation angle. Error bars are standard errors ($n=60$ images for each plot).