Abstract
Molecular transport in avascular collagenous tissues such as articular cartilage occurs primarily via diffusion. The presence of ordered structures in the extracellular matrix may influence the local transport of macromolecules, leading to anisotropic diffusion depending on the relative size of the molecule and that of extracellular matrix structures. Here we present what we believe is a novel photobleaching technique for measuring the anisotropic diffusivity of macromolecules in collagenous tissues. We hypothesized that macromolecular diffusion is anisotropic in collagenous tissues, depending on molecular size and the local organization of the collagen structure. A theoretical model and experimental protocol for fluorescence imaging of continuous point photobleaching was developed to measure diffusional anisotropy. Significant anisotropy was observed in highly ordered collagenous tissues such as ligament, with diffusivity ratios >2 along the fiber direction compared to the perpendicular direction. In less-ordered tissues such as articular cartilage, diffusional anisotropy was dependent on site in the tissue and size of the diffusing molecule. Anisotropic diffusion was also dependent on the size of the diffusing molecule, with greatest anisotropy observed for larger molecules. These findings suggest that diffusional transport of macromolecules is anisotropic in collagenous tissues, with higher rates of diffusion along primary orientation of collagen fibers.
| Original language | English |
|---|---|
| Pages (from-to) | 311-316 |
| Number of pages | 6 |
| Journal | Biophysical Journal |
| Volume | 91 |
| Issue number | 1 |
| DOIs | |
| State | Published - 2006 |
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In: Biophysical Journal, Vol. 91, No. 1, 2006, p. 311-316.
Research output: Contribution to journal › Article › peer-review
TY - JOUR
T1 - Diffusional anisotropy in collagenous tissues
T2 - Fluorescence imaging of continuous point photobleaching
AU - Leddy, Holly A.
AU - Haider, Mansoor A.
AU - Guilak, Farshid
N1 - Funding Information: We have applied what we believe is a new microscopy imaging technique, FICOPP, to demonstrate the presence of significant anisotropy in the diffusion coefficients of macromolecules in oriented collagenous tissues. Diffusion coefficients were significantly greater in a direction parallel to the collagen fibers, and diffusion anisotropy was greater at higher molecular weights. No anisotropy was observed in agarose gel, which was expected to be isotropic and homogeneous. Our experimental findings are consistent with a previous theoretical model kDa dextran in ligament. Our measured value of 2.2 is slightly lower than this prediction, but given that the ligament is not solely comprised of perfectly oriented fibers, a slightly lower value might be expected. The model also predicts a dramatic decrease in anisotropy as the size of the diffusing molecule decreases relative to the fibers through which it moves, consistent with our observations on the 3 kDa dextran as compared to the 500 kDa dextran. However, the technique may be slightly biased toward underestimating anisotropy when high levels of noise are present in the images. This technique may also underestimate anisotropy when measurements are made too close to the surface of the tissue, due to changes in the boundary conditions at the tissue edge. Ultimately, the maximum penetration distance from the cut edge will depend on the power of the laser and the working distance of the objective to allow bleaching and confocal imaging of the region of interest. (10) that predicts an anisotropic diffusivity ratio of ∼3 for a molecule of similar size to the fiber diameter of the matrix, and a fiber volume fraction of 0.6, as would be the case for 500 An important point in the application of this method is that FICOPP allows measurement of the anisotropy without quantification of the individual diffusion coefficients. This point represents both an advantage and a potential disadvantage, as the measurement process is easier but does not provide absolute values for the diffusion coefficients in different directions, only their ratio in principal directions. We have used FICOPP to examine diffusional anisotropy in a structure with known fiber directions where we are interested in transport across a broad zone. FICOPP can also be used to examine how closely diffusional anisotropy correlates with fiber direction by measuring the direction of the long axis of the ellipse relative to fiber direction. Alternatively, this method could be used to determine the principal directions of the diffusion tensor, which presumably are aligned with the local fiber orientation in the tissue. However, it is important to note that the anisotropy measurements are made within a single plane, and therefore three-dimensional diffusion anisotropy cannot be determined using this method. Articular cartilage is a highly stratified tissue that exhibits significant collagen fiber orientation in the superficial-most and deep-most zones of the tissue, with decreased fiber orientation in the middle zone of the tissue. Consistent with this structure, we observed significant diffusional anisotropy of 500 kDa dextran in the superficial zone, but not in the middle zone. By the Han and Herzfeld model kDa dextran would exhibit little interaction with the widely spaced collagen fibers in the deep zone (200 nm apart kDa dextran is too small to interact strongly with the collagen fibers, even in the superficial zone (60 nm apart (10) , the 500 (29) ), whereas the 3 (29) ). Thus, these molecules may not be expected to show significant diffusional anisotropy in these settings. The presence of such diffusional anisotropy appears to reflect the underlying collagenous structure of the tissue. Although the overall implications of this property are not clear, anisotropic diffusion properties may have significant implications on the transport of matrix macromolecules or solutes. The size-dependence of diffusional anisotropy may allow nutrients and other small molecules to move easily into cartilage from the synovial fluid, but prevent diffusion of larger structural molecules out of the cartilage. In addition, it is likely that compression, which has been shown to decrease overall diffusivity (30) , could further increase the anisotropy by increasing the packing and alignment of superficial zone collagen fibers (31,32) . Conversely, loss or degradation of the superficial zone as occurs with osteoarthritis could eradicate this diffusional anisotropy, allowing further loss of structural molecules from the cartilage surface. The molecules used for this study were uncharged, inert dextrans. The large, 500 kDa molecule is similar in size to some matrix macromolecules such as hyaluronic acid, cartilage oligomeric protein, or fibronectin, whereas the small dextran, 3 kDa, is similar in size to signaling molecules such as insulin or epidermal growth factor (33,34) . A charged or bioactive molecule that interacts directly with the matrix or with cells could have different diffusion characteristics. The dextran molecules are also linear, and a molecule with a more globular configuration may also have different diffusive characteristics (35) . Using other techniques, there have been limited reports of anisotropic diffusion in cells. In neurons, the diffusion of a 10 kDa protein has been found to be faster along the length of the axon rather than across the axon (21) , likely due to the dense microtubules oriented parallel to the length of the axon. On the cell surface, diffusion in the cell membrane has been shown to be anisotropic over oriented stress fibers of the cytoskeleton (23) . This phenomenon could facilitate formation of membrane signaling complexes, such as focal adhesions, in a manner analogous to the formation of lipid rafts (36) . In muscle, diffusion of ATP and phosphocreatine is faster along muscle fibers, but the need for these molecules is to move radially, across the muscle fiber (14) . At a larger tissue scale, the formation of stripe patterns on fishes, crucial for finding a mate or hiding from predators, are thought to form from anisotropic diffusion, possibly due to the structure of the scales (37,38) . Measurements of diffusional anisotropy at a tissue scale (19,20) have largely focused on the movement of water, which interacts differently with collagen fibers than larger molecules because the water can move within the fibers. In summary, FICOPP provides a relatively straightforward and robust method for measuring the diffusional anisotropy of molecules. The method can be applied on a standard confocal laser scanning microscope, and the diffusion of any fluorescently labeled macromolecule can be determined. This method has potential implications for the study of diffusional transport at the tissue level, and may provide further insight on the role of molecular structure in governing solute diffusion. This study was supported by the American Association of University Women, National Institutes of Health (AG15768, AR48182, AR50245, and GM08555), National Aeronautics and Space Administration (NNJ04HC72G), the Whitaker Foundation (RG-020933), and the National Science Foundation (DMS-0211154).
PY - 2006
Y1 - 2006
N2 - Molecular transport in avascular collagenous tissues such as articular cartilage occurs primarily via diffusion. The presence of ordered structures in the extracellular matrix may influence the local transport of macromolecules, leading to anisotropic diffusion depending on the relative size of the molecule and that of extracellular matrix structures. Here we present what we believe is a novel photobleaching technique for measuring the anisotropic diffusivity of macromolecules in collagenous tissues. We hypothesized that macromolecular diffusion is anisotropic in collagenous tissues, depending on molecular size and the local organization of the collagen structure. A theoretical model and experimental protocol for fluorescence imaging of continuous point photobleaching was developed to measure diffusional anisotropy. Significant anisotropy was observed in highly ordered collagenous tissues such as ligament, with diffusivity ratios >2 along the fiber direction compared to the perpendicular direction. In less-ordered tissues such as articular cartilage, diffusional anisotropy was dependent on site in the tissue and size of the diffusing molecule. Anisotropic diffusion was also dependent on the size of the diffusing molecule, with greatest anisotropy observed for larger molecules. These findings suggest that diffusional transport of macromolecules is anisotropic in collagenous tissues, with higher rates of diffusion along primary orientation of collagen fibers.
AB - Molecular transport in avascular collagenous tissues such as articular cartilage occurs primarily via diffusion. The presence of ordered structures in the extracellular matrix may influence the local transport of macromolecules, leading to anisotropic diffusion depending on the relative size of the molecule and that of extracellular matrix structures. Here we present what we believe is a novel photobleaching technique for measuring the anisotropic diffusivity of macromolecules in collagenous tissues. We hypothesized that macromolecular diffusion is anisotropic in collagenous tissues, depending on molecular size and the local organization of the collagen structure. A theoretical model and experimental protocol for fluorescence imaging of continuous point photobleaching was developed to measure diffusional anisotropy. Significant anisotropy was observed in highly ordered collagenous tissues such as ligament, with diffusivity ratios >2 along the fiber direction compared to the perpendicular direction. In less-ordered tissues such as articular cartilage, diffusional anisotropy was dependent on site in the tissue and size of the diffusing molecule. Anisotropic diffusion was also dependent on the size of the diffusing molecule, with greatest anisotropy observed for larger molecules. These findings suggest that diffusional transport of macromolecules is anisotropic in collagenous tissues, with higher rates of diffusion along primary orientation of collagen fibers.
UR - http://www.scopus.com/inward/record.url?scp=33745758519&partnerID=8YFLogxK
U2 - 10.1529/biophysj.105.075283
DO - 10.1529/biophysj.105.075283
M3 - Article
C2 - 16603503
AN - SCOPUS:33745758519
SN - 0006-3495
VL - 91
SP - 311
EP - 316
JO - Biophysical Journal
JF - Biophysical Journal
IS - 1
ER -