4 results for Putnam, AJ

  • Interstitial Fibroblasts Enhance Capillary Morphogenesis In Dense 3-D Matrices

    Ghajar, CM; Harris, JW; Suresh, Vinod; Chen, X; Jeon, N; Putnam, AJ; George, SC (2007)

    Conference item
    The University of Auckland Library

    Numerous fibrotic pathologies adversely affect angiogenesis, yet the effects of increased ECM density associated with these pathologies remain unclear. To study this phenomenon, we have utilized a 3-D, fibrin-based in vitro angiogenesis assay in which fibroblasts (FBs) are plated on top of a hydrogel ~2 mm away from HUVEC-coated microcarrier beads polymerized within the matrix. Increasing fibrin density from 2.5 to 10 mg/ml results in a three-fold reduction in capillary network formation. However, distributing FBs throughout the matrix eliminates this inhibitory effect; the number of vessel segments and total network length are enhanced in all conditions independent of matrix density. The interstitial FBs do not adopt an enhanced myofibroblastic phenotype nor extensively remodel the matrix, as shown by insignificant changes in the levels of characteristic markers (ie alpha-SMA, MMP2/9, fibronectin ED-A, collagen). Alternatively, we hypothesized that increasing fibrin density restricts the ability of fibroblast-derived angiogenic factors to reach the HUVECs by diffusion. Using a novel method developed to image bulk diffusion of FITC-dextran (avg. MWs of 10, 40, 70, and 150 kDa) through the matrices, we demonstrate an order of magnitude reduction in the diffusion coefficient with a 4-fold increase in fibrin density. We conclude that diffusion restrictions imposed by fibrotic matrices are a primary factor in inhibiting angiogenesis, a conclusion supported by the enhanced morphogenesis observed when a source of angiogenic regulators is distributed throughout the matrix

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  • The Effect of Matrix Density on the Regulation of 3-D Capillary Morphogenesis

    Ghajar, CM; Chen, X; Harris, JW; Suresh, Vinod; Hughes, CCW; Jeon, N; Putnam, AJ; George, SC (2008)

    Journal article
    The University of Auckland Library

    The means by which extracellular matrix density regulates three-dimensional capillary morphogenesis is unclear. To study this phenomenon, we utilized a fibrin-based in vitro assay in which a fibroblast monolayer is plated atop a fibrin gel approximately 2.5 mm away from endothelial cell-coated beads within the matrix. Increasing fibrin density from 2.5 to 10 mg/ml resulted in a threefold reduction in capillary network formation. However, distributing fibroblasts throughout the matrix completely eliminated this inhibitory effect, resulting in robustly vascularized matrices suitable for in vivo applications, as functional anastomoses formed between the implanted tissues and host vasculature when implanted into immune-compromised mice. Dense matrices did not stimulate fibroblast-mediated matrix remodeling: differentiation into myofibroblasts, matrix production, and protease secretion were not enhanced by the dense condition. Instead, quantifying diffusivity of FITC-dextran (molecular mass 10, 40, 70, and 150 kDa) through fibrin revealed a two- to threefold decrease within the 10 mg/ml matrices. Thus, distributing a proangiogenic source (fibroblasts) throughout the matrix stimulates capillary network formation by overcoming this diffusion restriction due to significantly reduced diffusion distances. Although roles for matrix stiffness and ligand binding density have previously been identified, our results emphasize the importance of diffusion restrictions in limiting capillary morphogenesis.

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  • A novel three-dimensional model to quantify metastatic melanoma invasion

    Ghajar, CM; Suresh, Vinod; Peyton, SR; Raub, CB; Meyskens, FL; George, SC; Putnam, AJ (2007)

    Journal article
    The University of Auckland Library

    Although attempts to develop any viable chemotherapeutic approaches to combat metastatic cancers have largely failed, potential genetic targets to halt metastatic progression continue to be identified. As drugs are developed to address these targets, there is a need for high-throughput systems that accurately reproduce in vivo microenvironments to gauge their efficacy. Accordingly, we have developed a three-dimensional in vitro culture system representative of the environment present upon secondary metastasis to quantitatively measure tumor cell invasion in this setting three-dimensionally. Culturing melanomas of different metastatic capacities within the system showed that each cell type invades the matrix in a manner commensurate to its known metastatic potential in vivo. Moreover, the developed quantitative schemes were put to use to characterize the effect of microenvironmental influences (i.e., matrix components, interstitial cell presence) on planar and vertical melanoma invasion. We propose this novel, quantitative system as a useful tool to assess the effects of pharmacologic and/or microenvironmental influences on tumor cell invasion at a metastatic site.

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  • Noninvasive assessment of collagen gel microstructure and mechanics using multiphoton microscopy

    Raub, CB; Suresh, Vinod; Krasieva, T; Lyubovitsky, J; Mih, JD; Putnam, AJ; Tromberg, BJ; George, SC (2007)

    Journal article
    The University of Auckland Library

    Multiphoton microscopy of collagen hydrogels produces second harmonic generation (SHG) and two-photon fluorescence (TPF) images, which can be used to noninvasively study gel microstructure at depth ( approximately 1 mm). The microstructure is also a primary determinate of the mechanical properties of the gel; thus, we hypothesized that bulk optical properties (i.e., SHG and TPF) could be used to predict bulk mechanical properties of collagen hydrogels. We utilized polymerization temperature (4-37 degrees C) and glutaraldehyde to manipulate collagen hydrogel fiber diameter, space-filling properties, and cross-link density. Multiphoton microscopy and scanning electron microscopy reveal that as polymerization temperature decreases (37-4 degrees C) fiber diameter and pore size increase, whereas hydrogel storage modulus (G', from 23 +/- 3 Pa to 0.28 +/- 0.16 Pa, respectively, mean +/- SE) and mean SHG decrease (minimal change in TPF). In contrast, glutaraldehyde significantly increases the mean TPF signal (without impacting the SHG signal) and the storage modulus (16 +/- 3.5 Pa before to 138 +/- 40 Pa after cross-linking, mean +/- SD). We conclude that SHG and TPF can characterize differential microscopic features of the collagen hydrogel that are strongly correlated with bulk mechanical properties. Thus, optical imaging may be a useful noninvasive tool to assess tissue mechanics.

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