Non-destructive label-free monitoring of collagen gel remodeling using optical coherence tomography
Introduction
Cellular remodeling of the extracellular matrix (ECM) plays an important role in developmental biology [1], cell motility [2], aging [3], wound healing [4], atherosclerosis [5], tumorigenesis [6], fibrosis [7], and tissue engineering applications [8], [9], [10]. ECM remodeling involves assembling, degrading, and reorganizing ECM structures [11]. Critical to ECM remodeling is the activity of the matrix metalloproteinases (MMPs) family of enzymes that degrade ECM structural proteins. MMP substrates include collagens, fibronectin, laminin, and proteoglycans [12]. Accordingly, real-time assessment of ECM remodeling would improve our understanding of these physiological and pathological processes.
One common in vitro model of ECM remodeling is a 3D collagen I matrix (collagen gel) populated by fibroblasts or SMCs [13]. Over time, these cells cause gel contraction that decreases the size of the gel matrix [14]. MMP activity has been implicated in this process of collagen gel remodeling [15], [16], [17].
Given the importance of ECM remodeling, there has been a search for methods that could monitor the process in real-time both quantitatively and non-destructively [18]. One promising technology is optical coherence tomography (OCT) [19]. OCT, which measures coherent light backscattered from tissues, has several advantages for imaging collagen gels and tissues, including an axial resolution <10 μm and imaging up to 2 mm depth at video-rate imaging speeds [18]. Importantly, contrast in OCT images is based on endogenous refractive index mismatches between the structures in the imaged sample, and thus no labels need to be added to the sample. OCT data also contain information on the scattering properties of structures within the imaged sample, and recently our group developed an algorithm that fits OCT signals to a theoretical model and determines the optical scattering properties of the imaged sample in local nanoliter volumes [20]. Measuring the scattering properties of collagen–SMC gels from OCT images determined that over a 5 day period, the reflectivity more than doubled, indicating a decrease in the scattering anisotropy [20]. We hypothesized that matrix remodeling, specifically MMP activity, was responsible for the reflectivity increase.
In the present report, OCT was used to show that collagen remodeling caused a 10-fold increase in reflectivity through a decrease in the scattering anisotropy. Specifically, collagen–SMC gels were treated with doxycycline (a non-specific MMP inhibitor), which impeded the decrease in scattering anisotropy over 5 days relative to that measured in untreated gels. Additionally, treatment of acellular collagen gels with collagenase 2 (MMP-8) induced a partial decrease in scattering anisotropy. We propose that the mechanism by which reflectivity is increased is that MMPs break down local collagen fibrils into smaller fragments that scatter light more isotropically, thereby increasing the fraction of backscattered light and the measured reflectivity.
Section snippets
Theory
The theory of light propagation in low-coherence imaging systems [21] that was used in this study was derived from the inverse Monte Carlo method. For a homogeneous turbid medium characterized by scattering coefficient μs, anisotropy factor g, and absorption coefficient μa, the depth-dependent OCT signal R(z) can be described as an exponential decay:with reflectivity ρ and attenuation μ. Specifically,andwhere Δz is the axial resolution and G is a geometry
Cell culture
All cell culture materials were purchased from GIBCO unless otherwise stated. Experiments involving cells used primary SMCs (passage 4–5) isolated from a single baboon carotid artery by enzymatic digestion [22]. SMCs were fed with SMC growth medium (SGM), consisting of minimal essential medium (MEM) supplemented with 10% fetal bovine serum, 1% l-glutamine, and 1% penicillin–streptomycin.
Collagen gel preparation
Soluble calf skin collagen (MP Biomedicals, part # 150026) was dissolved in 0.02 N acetic acid at a
Remodeling increased reflectance intensity in en face confocal mosaics
Confocal mosaics that imaged ∼1 cm2 of collagen gel area at a ∼20 μm depth were acquired to assess the spatial uniformity of the gels. The reflectance signal from each gel, representing the matrix, was false-colored in green, while the eosin fluorescence that represented the cytoplasm was false-colored in red. Fig. 1A and B shows a representative (en face) 0.6 × 0.6 cm confocal mosaic and a mosaic tile at higher magnification (500 × 500 μm) for the day 1 gels; Fig. 1C and D shows 0.6 × 0.6 cm
Discussion
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 (Fig. 2). Blocking MMP activity in the gels by
Conclusion
In this study, OCT quantitatively monitored ECM remodeling in SMC-seeded collagen gels from day 1 to day 5 by measuring the optical properties. Over 5 days, the scattering anisotropy of the collagen–SMC gels decreased from 0.91 to 0.46, while the scattering coefficient stayed the same, corresponding to a 10-fold increase in reflectivity with no change in attenuation. Treatment of the collagen–SMC gels with doxycycline, a non-specific MMP inhibitor, impeded remodeling macroscopically over 5 days
Acknowledgements
The authors would like to thank Dan Gareau of the Department of Biomedical Engineering at OHSU for help aligning the VivaScope. Additionally, the authors would like to thank Fred Grinnell, Kate Phelps, and Miguel Miron of the Cell Biology Department at University of Texas-Southwestern Medical Center for helpful discussions on collagen gel remodeling. This study was funded in part by the National Institutes of Health grant R01-HL084013 (Jacques) and R01-HL095474 (Hanson).
References (35)
ECM and cell surface proteolysis: regulating cellular ecology
Cell
(1997)- et al.
Properties of engineered vascular constructs made from collagen, fibrin, and collagen–fibrin mixtures
Biomaterials
(2004) Fibroblast biology in three-dimensional collagen matrices
Trends Cell Biol
(2003)- et al.
Secreted versus membrane-anchored collagenases: relative roles in fibroblast-dependent collagenolysis and invasion
J Biol Chem
(2009) - et al.
Low coherence interferometry of the cochlear partition
Hear Res
(2006) - et al.
Doxycycline alters vascular smooth muscle cell adhesion, migration, and reorganization of fibrillar collagen matrices
Am J Pathol
(2006) - et al.
Characterization of the mechanisms by which gelatinase A, neutrophil collagenase, and membrane-type metalloproteinase MMP-14 recognize collagen I and enzymatically process the two alpha-chains
J Mol Biol
(2007) - et al.
Matrix metalloproteinases in development and disease
Birth Defects Res C Embryo Today
(2006) - et al.
Regulation of cell invasion and morphogenesis in a three-dimensional type I collagen matrix by membrane-type matrix metalloproteinases 1, 2, and 3
J Cell Biol
(2000) - et al.
Looking older: fibroblast collapse and therapeutic implications
Arch Dermatol
(2008)