Biomedical Engineering Reference
In-Depth Information
11.4 SHG imaging of cellularized collagen Gels
11.4.1 introduction
The studies described below use multiphoton microscopy (MPM) imaging and mechanical testing to study
floating cellularized collagen gels, which are contracting to <5% of their original volume, attain physi-
ological collagen and cell densities. During the contraction process, cellularized gels exhibit microstruc-
ture-dependent trends in mechanical and optical properties, and these dynamic changes may be captured
through MPM imaging and analysis of the SHG signal. The analysis of microstructure-mechanics rela-
tionships in these simple engineered tissues is important to understand how engineered tissues develop
and how cell-induced matrix remodeling occurs
in vitro
. Cellularized collagen gels were prepared in a
method similar to that used to make acellular gels at pH 6.5 (coarse-structured gels) and 8.5 (fine-struc-
tured gels), except that 50,000 normal human lung fibroblasts (NHLFs), passages 3-7 were added per mil-
liliter of a 4 mg/mL collagen solution. The gels were polymerized in 24-well plates at room temperature
(24°C) for 1 h and were then pH equilibrated with excess culture media. After overnight tissue culture to
allow fibroblasts to adhere and spread within the collagen gels, the constructs were released from the wells
and were placed in floating culture in Petri dishes half-filled with culture media. These floating gels were
cultured in standard conditions for up to 15 days, during which time the gels were periodically removed
for imaging and mechanical testing. To monitor the cell location and interactions with collagen, the imag-
ing wavelength was set to 780 nm, and SHG signal from collagen was collected at 390 nm, while TPF signal
from endogenous fluorophores [56,96] within the fibroblasts was collected at 500-550 nm.
11.4.2 effect of Gel contraction on SHG images
Fine and coarse gels retained distinct microstructures, revealed in MPM images by SHG signal, even
after significant cell-induced contraction (Figures 11.9a through 11.9c, fine-structured gels; 11.9d
through 11.9f, coarse-structured gels). Matrix defects and holes appear in the SHG images from denser
cellularized gels, typically adjacent to areas with cellular TPF signal (Figure 11.9f). The final collagen
concentration approached approximately 200 mg/mL for the most contracted gels, with a final volume
of ~11 μL (Figure 11.9g). Collagen mass content and total cell content increased during the culture
period, in a trend clearly visible from the coregistered SHG and TPF images.
Interestingly, the mean SHG signal intensity from both fine- and coarse-structured gels increased
linearly with collagen concentration (Figure 11.9h, signal and concentration normalized), identically
to acellular gels (Figure 11.4g). The linear trend of SHG signal with bulk collagen concentration may
be interpreted as a function of increased collagen volume fraction and multiple backscattering of SHG
signal within the gel.
11.4.3 SHG image texture Simulation
To determine whether SHG image parameters could accurately assess the collagen network microstruc-
ture from cellularized gels varying in concentration over two orders of magnitude (~4-200 mg/mL),
simulated textural images with well-defined numbers of overlapping “collagen fibers” were constructed
to recreate structural features from the SHG images of cellularized gels. Using the simulations of image
texture, the relationship between image parameters and collagen fiber number density could be deter-
mined and trends could be compared between the simulation and SHG images.
The textural features of images of the SHG signal from coarse-structured collagen gels were simulated
using a MATLAB
®
routine. The constructed images were meant to simulate the images of a randomly
oriented collagen fiber network, to determine the precise relationship of robust, gain-independent
image parameters of fiber number density. Collagen fiber segments within the MPM image plane were
simulated as two-dimensional elliptical Gaussian functions. The length and width of the fiber segments
