Microstructural Characteristics of Extracellular Matrix Produced by Stromal Fibroblasts

The overall objective of this investigation was to characterize the extracellular matrix deposited by the stromal fibroblasts as a function of time in culture and matrix microstructure. Stromal fibroblasts were seeded onto collagen matrices and cultured for up to 5 weeks. The collagen matrices contained collagen fibrils with an average diameter of 215 ± 20 nm. When cultured on a collagen film, an average fibril diameter of 62 ± 39 nm was observed for single layer films with only slight variations with time in culture, and after 1 week of culture between two film layers 67 ± 47 nm fibrils were observed after 1 week. When the film surface was molded into 1 and 2 μm microgrooves, the initial average fibril diameter of the extracellular matrix was 73 ± 21 and 73 ± 31 nm respectively. When cultured on a collagen sponge, an average fibril diameter of 107 ± 20 nm was initially observed and decreased to 47.5 ± 17 nm after 1 week in culture. For cells cultured on a collagen sponge, Western blotting showed an increase in myofibroblast phenotype expression with time in culture. Shifts in phenotype were less distinct for cells cultured on collagen films. The microstructure, rather than geometry, of the matrix substrate appeared to influence the newly synthesized extracellular matrix and cell phenotype.

[1]  John A. Pedersen,et al.  Mechanobiology in the Third Dimension , 2005, Annals of Biomedical Engineering.

[2]  Xiu‐da Shen,et al.  Fibronectin-α4β1 integrin interactions modulate p42/44 MAPK phosphorylation in steatotic liver cold ischemia-reperfusion injury , 2005 .

[3]  F H Silver,et al.  Relationship between mechanical properties and collagen structure of closed and open wounds. , 1988, Journal of biomechanical engineering.

[4]  V. Barocas,et al.  Mechanical and Cellular Changes During Compaction of a Collagen-Sponge-Based Corneal Stromal Equivalent , 2004, Annals of Biomedical Engineering.

[5]  Fini Me Keratocyte and fibroblast phenotypes in the repairing cornea. , 1999 .

[6]  S. Britland,et al.  Contact guidance of CNS neurites on grooved quartz: influence of groove dimensions, neuronal age and cell type. , 1997, Journal of cell science.

[7]  M. Terry,et al.  Objective Screening Methods for Prior Refractive Surgery in Donor Tissue , 2002, Cornea.

[8]  C. McCulloch,et al.  The compliance of collagen gels regulates transforming growth factor-β induction of α-smooth muscle actin in fibroblasts , 1999 .

[9]  F. Silver,et al.  Collagen-based wound dressings: control of the pore structure and morphology. , 1986, Journal of biomedical materials research.

[10]  T. T. Dinh,et al.  Myofibroblasts differentiate from fibroblasts when plated at low density. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[11]  E. Suuronen,et al.  Innervated human corneal equivalents as in vitro models for nerve‐target cell interactions , 2004 .

[12]  J Wang,et al.  A Collagen-Based Scaffold for a Tissue Engineered Human Cornea: Physical and Physiological Properties , 2003, The International journal of artificial organs.

[13]  Y. Pouliquen,et al.  Comparative biochemical and morphometric studies on corneal wound healing. , 1988, Pathologie-biologie.

[14]  K. Hedman,et al.  Fibronectin: a flexible image. , 1988, Electron microscopy reviews.

[15]  C. Kublin,et al.  Regeneration of corneal tissue. , 1977, Developmental biology.

[16]  William L Hickerson,et al.  Multicenter postapproval clinical trial of Integra dermal regeneration template for burn treatment. , 2003, The Journal of burn care & rehabilitation.

[17]  M C Davies,et al.  Interactions of 3T3 fibroblasts and endothelial cells with defined pore features. , 2002, Journal of biomedical materials research.

[18]  Y. Wang,et al.  Cell locomotion and focal adhesions are regulated by substrate flexibility. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[19]  C. Wilkinson,et al.  New depths in cell behaviour: reactions of cells to nanotopography. , 1999, Biochemical Society symposium.

[20]  E. Suuronen,et al.  Cellular and nerve regeneration within a biosynthetic extracellular matrix for corneal transplantation , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[21]  M. Fini Keratocyte and fibroblast phenotypes in the repairing cornea , 1999, Progress in Retinal and Eye Research.

[22]  F. Silver,et al.  Collagen fiber formation in repair tissue: development of strength and toughness. , 1985, Collagen and related research.

[23]  B. Hinz,et al.  Myofibroblasts and mechano-regulation of connective tissue remodelling , 2002, Nature Reviews Molecular Cell Biology.

[24]  Y. Pek,et al.  Micromechanics of fibroblast contraction of a collagen-GAG matrix. , 2001, Experimental cell research.

[25]  K. Hedman,et al.  Integrity of the pericellular fibronectin matrix of fibroblasts is independent of sulfated glycosaminoglycans. , 1984, The EMBO journal.

[26]  C. McCulloch,et al.  The compliance of collagen gels regulates transforming growth factor-beta induction of alpha-smooth muscle actin in fibroblasts. , 1999, The American journal of pathology.

[27]  Y. Huang,et al.  Effect of spatial architecture on cellular colonization. , 2006, Biotechnology and bioengineering.

[28]  J. Zieske,et al.  Biosynthetic responses of the rabbit cornea to a keratectomy wound. , 1987, Investigative ophthalmology & visual science.

[29]  L. Zardi,et al.  High-resolution cryo-scanning electron microscopy study of the macromolecular structure of fibronectin fibrils. , 1997, Scanning.

[30]  F. Silver,et al.  Collagen-based wound dressing: effects of hyaluronic acid and fibronectin on wound healing. , 1986, Biomaterials.

[31]  Giulio Gabbiani,et al.  Perspective Article: Tissue repair, contraction, and the myofibroblast , 2005, Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society.

[32]  Mark J Mannis,et al.  Summary of Corneal Transplant Activity: Eye Bank Association of America , 2002, Cornea.

[33]  M Chvapil,et al.  Collagen sponge: theory and practice of medical applications. , 1977, Journal of biomedical materials research.

[34]  Kenneth M. Yamada,et al.  Cell interactions with three-dimensional matrices. , 2002, Current opinion in cell biology.

[35]  E. Orwin,et al.  Biomechanical and optical characteristics of a corneal stromal equivalent. , 2003, Journal of biomechanical engineering.

[36]  R. Segal,et al.  Studies on intercellular LETS glycoprotein matrices , 1978, Cell.

[37]  W. Chu,et al.  The Past Twenty-five Years in Eye Banking , 2000, Cornea.

[38]  J. Schwarzbauer,et al.  Fibronectin fibrillogenesis, a cell-mediated matrix assembly process. , 2005, Matrix biology : journal of the International Society for Matrix Biology.

[39]  J. Paul Robinson,et al.  Tensile mechanical properties of three-dimensional type I collagen extracellular matrices with varied microstructure. , 2002, Journal of biomechanical engineering.

[40]  J. Jester,et al.  Modulation of cultured corneal keratocyte phenotype by growth factors/cytokines control in vitro contractility and extracellular matrix contraction. , 2003, Experimental eye research.

[41]  O Damour,et al.  Collagen synthesis by fibroblasts cultured within a collagen sponge. , 1993, Biomaterials.

[42]  P. Moghe,et al.  Substrate microtopography can enhance cell adhesive and migratory responsiveness to matrix ligand density. , 2001, Journal of biomedical materials research.

[43]  Fengfu Li,et al.  Cellular and nerve regeneration within a biosynthetic extracellular matrix for corneal transplantation. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[44]  E. Suuronen,et al.  Innervated human corneal equivalents as in vitro models for nerve‐target cell interactions , 2004, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[45]  W. Kao,et al.  Transforming growth factor(beta)-mediated corneal myofibroblast differentiation requires actin and fibronectin assembly. , 1999, Investigative ophthalmology & visual science.

[46]  E. Orwin,et al.  In vitro culture characteristics of corneal epithelial, endothelial, and keratocyte cells in a native collagen matrix. , 2000, Tissue engineering.

[47]  Giulio Gabbiani,et al.  Mechanisms of force generation and transmission by myofibroblasts. , 2003, Current opinion in biotechnology.

[48]  J. Murphy-Ullrich The de-adhesive activity of matricellular proteins: is intermediate cell adhesion an adaptive state? , 2001, The Journal of clinical investigation.

[49]  Victor H Barocas,et al.  Biomechanical and microstructural characteristics of a collagen film-based corneal stroma equivalent. , 2006, Tissue engineering.

[50]  Rejean Munger,et al.  Functional innervation in tissue engineered models for in vitro study and testing purposes. , 2004, Toxicological sciences : an official journal of the Society of Toxicology.

[51]  L G Griffith,et al.  Effect of pore size and void fraction on cellular adhesion, proliferation, and matrix deposition. , 2001, Tissue engineering.

[52]  J. Schwarzbauer,et al.  Stimulatory effects of a three-dimensional microenvironment on cell-mediated fibronectin fibrillogenesis , 2005, Journal of Cell Science.

[53]  F. Grinnell,et al.  The collagen recognition sequence for fibroblasts depends on collagen topography. , 1989, Experimental cell research.

[54]  H. D. Cavanagh,et al.  Induction of alpha-smooth muscle actin expression and myofibroblast transformation in cultured corneal keratocytes. , 1996, Cornea.