In this paper, we showed that OCT is capable of measuring and quantifying changes in optical scattering properties caused by SMC remodeling of collagen fibrils. By measuring these optical properties from OCT data, we showed that from day 1 to day 5, there was a 10-fold increase in reflectivity ρ with no change in attenuation μ, which corresponded to a decrease in anisotropy g from 0.91 to 0.46, with little change to scattering coefficient μs
(). Blocking MMP activity in the gels by treating them with doxycycline for 5 days impeded both collagen gel remodeling macroscopically (, supplementary Fig. 1
) and the corresponding decrease in scattering anisotropy (). The effect that the presence (or absence) of MMPs had on collagen reflectance could be seen in the confocal mosaics (, supplementary Fig. 2
), where the collagen matrix in the day 5 dox- gels had a much higher reflectance signal than the day 1 gels and both doses of the day 5 dox+ gels. Treating acellular collagen gels with MMP-8 for 3 hr partially mimicked the increase in reflectivity that occurs over 5 days (). Taken together, the data presented here demonstrate that: (A) a relationship exists between MMP activity and the optical properties of collagen gels, and (B) that measuring optical properties from OCT data provides an non-destructive assessment of remodeling in local nanoliter volumes.
Using OCT to measure optical properties is advantageous for several reasons. The optical scattering properties of a sample, particularly the scattering phase function P(θ) and scattering anisotropy g, are very sensitive to the molecular microstructure of the sample [32
]. The decrease in scattering anisotropy between the day 1 and day 5 gels illustrates that the optical properties are sensitive to ECM degradation (among other things). Moreover, measuring optical properties is non-destructive, allowing for repeated measurements on the same sites and on the same sample. This OCT technique is based on endogenous contrast between the collagen, the cells, and the surrounding medium, making it label-free, which reduces both the costs and complexity associated with adding exogenous fluorescent labels. As a fiber-based technology, OCT probes can be readily integrated into the incubator for continuous, time-lapse monitoring of construct uniformity. Finally, since the sample arm of the OCT system is essentially a confocal microscope, an OCT system can be designed to acquire mosaics of data (analogous to confocal mosaics in , supplementary Fig. 2
), that cover centimeters of area and millimeters of depth, compared to the ~100 μm depth limit on confocal mosaic imaging. Thus, such OCT-based techniques that continuously and non-destructively monitor milliliter volumes of a sample at micron resolution, and detect changes in the ECM in nanoliter volumes without any exogenous labels, could prove to be powerful tools for surveying engineered tissues.
Recently, we demonstrated that over 5 days SMC remodeling of collagen gels decreased both anisotropy g and scattering coefficient μs
, thereby causing the reflectivity to double and the attenuation to remain the same [20
]. Moreover, the measured reflectivity statistically followed a bimodal distribution, with a sizeable subpopulation of data points with 10-fold reflectivity relative to that from the first 24 hr. Although the trends are similar, this earlier data contrasts with , where nearly all the day 5 reflectivity data was 10-fold higher than the day 1 reflectivity data. Subsequent experiments showed that this discrepancy was caused both by the cell source (i.e., from different baboons) and the serum lot. Moreover, the gel compaction time dynamics from the previous study also contrasted with those in the present study: Earlier, the gel volume reduced in incremental steps every day, while in this study most of the compaction happened between days 1 and 2.
In trying to dissect the optical property data and identify what caused these observed decreases in scattering anisotropy (increases in reflectivity), 4 possibilities were hypothesized: increased collagen density, increased cell density, SMC contraction, and ECM remodeling. In the SMC gels, there was no correlation between anisotropy and either collagen density or cell density [20
]. To assess whether SMC contraction, i.e., a change in cell morphology, caused the measured increase in reflectivity, collagen gels were prepared and seeded with endothelial cells (ECs) instead of SMCs. The ECs and SMCs were determined to have different morphologies in 3D collagen gels by F-actin staining (data not shown). At day 1, the EC gels and SMC gels differed in μs
(P<0.05), but not in g (P>0.05) [33
]. Since the primary changes in the remodeling of SMC gels were in g, the EC data suggests such changes were not caused by a change in cell shape, which further supports the hypothesis that ECM remodeling caused the changes in the optical properties of the collagen gels.
Mie theory [32
], which calculates the angular distribution of light energy scattered off a sphere, can be used to interpret the optical properties of the collagen gels. Mie theory states that in general, as the sphere diameter increases, light is scattered more anisotropically, that is, mostly in the forward direction (g→1); conversely, as the sphere diameter decreases, light is scattered more isotropically, that is, relatively equally in all directions (g→0). The OCT signal depends on light backscattered from the sample, specifically, the fraction of light scattered back towards the lens relative to the total scattered energy. The fraction of light scattered in the backwards direction increases as the scattering anisotropy decreases, forming an inverse relationship between scattering anisotropy and reflectivity, as has been described in our theoretical model [21
]. Applying these principles to collagen-SMC gels, scattering by the collagen fibril network is intrinsically very forward-directed, as shown by the high scattering anisotropy/low reflectivity measured from these samples (, ). Proteolytic degradation of the collagen matrix by local MMP activity breaks down the fibrillar collagen network into small fibril fragments. These fibril fragments constitute a dense collection of local isotropic scatterers that would scatter more light back to the objective lens, thereby increasing the reflectivity.
Collagen gels prepared in our lab and seeded with fibroblasts, cells that contract collagen fibrils as well as express and activate MMPs, showed both similar macroscopic remodeling and a similar decrease in scattering anisotropy/increase in reflectivity over 5 days (data not shown). However, collagen gels seeded with ECs, a cell type that generates little secreted MMP activity in 3D collagen matrices [34
], did not produce comparable remodeling at 5 days, neither macroscopically nor microscopically [33
]. Considering the fibroblast gels as positive controls and the EC gels as negative controls further confirms our hypothesis that ECM remodeling is causing the measured changes in optical properties.
In general, there are 2 types of remodeling – mechanical remodeling, in which the collagen matrix is pressed down and fluid is expelled, and biochemical remodeling, which involves proteolytic enzymes such as MMPs breaking down collagen [15
]. Several studies measuring MMP activity in collagen-SMC gels using gelatin zymography showed that secreted MMPs, specifically MMP-2 (gelatinase A), play a critical role in matrix remodeling [8
]. In the present study, blocking MMP activity with doxycycline impeded the decrease in scattering anisotropy () and impeded gel compaction (supplementary Fig. 1
]), but did not eliminate it. Similarly, treating acellular gels with collagenase partially mimicked the increase in reflectivity, but did not reproduce it in full. In both the doxycycline and collagenase experiments, the dose response of anisotropy was nonlinear. Thus, it is likely that other mechanisms contribute to the measured changes in optical properties. More research is needed to identify such mechanisms.