The temporal and spatial dynamics of microscale collagen scaffold remodeling by smooth muscle cells.

Smooth muscle cells (SMCs) and collagen scaffolds are widely used in vascular tissue engineering but their interactions in remodeling at the microscale level remained unclear. We characterized microscale morphologic alterations of collagen remodeled by SMCs in six dimensions: three spatial, time, multichannel and multi-position dimensions. In live imaging assays, computer-assisted cell tracking showed locomotion characteristics of SMCs; reflection and fluorescent confocal microscopy and spatial reconstruction images of each time point showed detailed morphologic changes of collagen fibers and spatial collagen-SMC interactions. The density of the collagen around the SMCs was changed dynamically by the leading edges of the cells. The density of the collagen following 24h of cell-induced remodeling increased 51.61+/-9.73% compared to unremodeled collagen containing cells for 1h (P<0.0001, n=40) (NS vs. collagen without cells). Fast Fourier transform analysis showed that the collagen fibers' orientation changed from random (alignment index=0.047+/-0.029, n=40) after 1h into concordant with that of the SMCs (alignment index=0.379+/-0.098, P<0.0001, n=40) after 24h. Mosaic imaging extended the visual field from a single cell to a group of cells in one image without loss of optical resolution. Direct visualization of alignment of actin fibers and collagen fibers showed the molecular machinery of the process of scaffold remodeling. This is a new approach to better understanding the mechanism of scaffold remodeling and our techniques represent effective tools to investigate the interactions between cells and scaffold in detail at the microscale level.

[1]  Athanassios Sambanis,et al.  A biological hybrid model for collagen-based tissue engineered vascular constructs. , 2003, Biomaterials.

[2]  Rothman,et al.  Automatic image acquisition, calibration and montage assembly for biological X‐ray microscopy , 2000, Journal of microscopy.

[3]  E Bell,et al.  A blood vessel model constructed from collagen and cultured vascular cells. , 1986, Science.

[4]  P. Friedl,et al.  Migration of dendritic cells in 3D-collagen lattices. Visualisation of dynamic interactions with the substratum and the distribution of surface structures via a novel confocal reflection imaging technique. , 1997, Advances in experimental medicine and biology.

[5]  R. L. Marks,et al.  Real-time video mosaicking of the ocean floor , 1995 .

[6]  K. Zänker,et al.  CD4+ T lymphocytes migrating in three‐dimensional collagen lattices lack focal adhesions and utilize β1 integrin‐independent strategies for polarization, interaction with collagen fibers and locomotion , 1998, European journal of immunology.

[7]  S. J. Jones,et al.  Mapping and Measuring Surfaces Using Reflection Confocal Microscopy , 1995 .

[8]  Sean P. Palecek,et al.  Erratum: Integrin–ligand binding properties govern cell migration speed through cell–substratum adhesiveness , 1997, Nature.

[9]  C. McCulloch,et al.  Dependence of collagen remodelling on α‐smooth muscle actin expression by fibroblasts , 1994 .

[10]  R. Nerem,et al.  Altered response of vascular smooth muscle cells to exogenous biochemical stimulation in two- and three-dimensional culture. , 2003, Experimental cell research.

[11]  J. Garrels,et al.  Identification and characterization of multiple forms of actin , 1976, Cell.

[12]  Peter Friedl,et al.  Compensation mechanism in tumor cell migration , 2003, The Journal of cell biology.

[13]  Michael Unser,et al.  User‐friendly semiautomated assembly of accurate image mosaics in microscopy , 2007, Microscopy research and technique.

[14]  Steve Paddock,et al.  Confocal reflection microscopy: the "other" confocal mode. , 2002, BioTechniques.

[15]  L. Griffith,et al.  Tissue Engineering--Current Challenges and Expanding Opportunities , 2002, Science.

[16]  M. Sacks,et al.  Quantification of vertical-fiber defect in cattle hide by small-angle light scattering. , 1991, Connective tissue research.

[17]  G. Hou,et al.  Doxycycline alters vascular smooth muscle cell adhesion, migration, and reorganization of fibrillar collagen matrices. , 2006, The American journal of pathology.

[18]  Debby Gawlitta,et al.  Properties of engineered vascular constructs made from collagen, fibrin, and collagen-fibrin mixtures. , 2004, Biomaterials.

[19]  E. J. Miller,et al.  Preparation and characterization of the different types of collagen. , 1982, Methods in enzymology.

[20]  P. Friedl.  Dynamic imaging of cellular interactions with extracellular matrix , 2004, Histochemistry and Cell Biology.

[21]  Stephen F Badylak,et al.  The extracellular matrix as a biologic scaffold material. , 2007, Biomaterials.

[22]  J. McPherson,et al.  A comparative study of the biologic and immunologic response to medical devices derived from dermal collagen. , 1986, Journal of biomedical materials research.

[23]  H. Greisler,et al.  Selective stimulation of endothelial cell proliferation with inhibition of smooth muscle cell proliferation by fibroblast growth factor-1 plus heparin delivered from fibrin glue suspensions. , 1995, Surgery.

[24]  John M. Tarbell,et al.  Effect of Fluid Flow on Smooth Muscle Cells in a 3-Dimensional Collagen Gel Model , 2000, Arteriosclerosis, thrombosis, and vascular biology.

[25]  Michal Irani,et al.  Representation of scenes from collections of images , 1995, Proceedings IEEE Workshop on Representation of Visual Scenes (In Conjunction with ICCV'95).

[26]  M. Harmsen,et al.  Vascular smooth muscle cells for use in vascular tissue engineering obtained by endothelial-to-mesenchymal transdifferentiation (EnMT) on collagen matrices. , 2008, Biomaterials.

[27]  W Matthew Petroll,et al.  Differential interference contrast and confocal reflectance imaging of collagen organization in three-dimensional matrices. , 2007, Scanning.

[28]  Jan P. Stegemann,et al.  Phenotype Modulation in Vascular Tissue Engineering Using Biochemical and Mechanical Stimulation , 2003, Annals of Biomedical Engineering.

[29]  B. Chen,et al.  Signal Transduction in Matrix Contraction and the Migration of Vascular Smooth Muscle Cells in Three-Dimensional Matrix , 2003, Journal of Vascular Research.

[30]  Wolf Malkusch.  User-oriented image processing throughout the changing times , 2002 .

[31]  D. Fox,et al.  Optimizing fluorescent labeling of endothelial cells for tracking during long-term studies of autologous transplantation. , 1999, The Journal of surgical research.