Skip to main content
SpringerLink
Log in

Microstructural Characteristics of Extracellular Matrix Produced by Stromal Fibroblasts

  • Published:
Annals of Biomedical Engineering Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.
Figure 7.
Figure 8.
Figure 9.

Similar content being viewed by others

References

  1. Aiken-O’Neill P., Mannis M. J. (2002) Summary of corneal transplant activity Eye Bank Association of America. Cornea 21:1–3

    Article  PubMed  Google Scholar 

  2. Alberts, B., A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter. Molecular Biology of the Cell. New York: Garland Science, 2002, pp 1100

  3. Arora P. D., Narani N., McCulloch C.A. (1999) The compliance of collagen gels regulates transforming growth factor-beta induction of alpha-smooth muscle actin in fibroblasts. Am. J. Pathol. 154:871–82

    PubMed  CAS  Google Scholar 

  4. Berthod F., Hayek D., Damour O., Collombel C. (1993) Collagen synthesis by fibroblasts cultured within a collagen sponge. Biomaterials 14:749–754

    Article  PubMed  CAS  Google Scholar 

  5. Borene M. L., Barocas V. H., Hubel A. (2004) Mechanical and cellular changes during compaction of a collagen–sponge-based corneal stromal equivalent. Ann. Biomed. Eng. 32:274–283

    Article  PubMed  Google Scholar 

  6. Cameron, J.D. Corneal Reaction to Injury. In: The Cornea: Fundamentals of Cornea and External Disease. ed. Holland, E.J. Philadelphia: Elsevier Mosby, 1997, Vol. 1, 163-182

  7. Chen L. B., Murray A., Segal R. A., Bushnell A., Walsh M. L. (1978) Studies on intercellular LETS glycoprotein matrices. Cell 14:377–391

    Article  PubMed  CAS  Google Scholar 

  8. Chen Y., Zardi L., Peters D. M. (1997) High-resolution cryo-scanning electron microscopy study of the macromolecular structure of fibronectin fibrils. Scanning 19:349–355

    PubMed  CAS  Google Scholar 

  9. Chu W. (2000) The past twenty-five years in eye banking. Cornea 19:754–765

    Article  PubMed  CAS  Google Scholar 

  10. Chvapil M. (1977) Collagen sponge: theory and practice of medical applications. J Biomed Mater Res 11:721–741

    Article  PubMed  CAS  Google Scholar 

  11. Cintron C., Kublin C. L. (1977) Regeneration of corneal tissue. Dev. Biol. 61:346–357

    Article  PubMed  CAS  Google Scholar 

  12. Crabb, R. A. B., E. P. Chau, M. C. Evans, V. H. Barocas, and A. Hubel, Biomechanical and microstructural characteristics of a collagen film-based corneal stroma equivalent. Tissue Eng. (in press)

  13. Cukierman E., Pankov R., Yamada K. M. (2002) Cell interactions with three-dimensional matrices. Curr. Opin. Cell Biol. 14:633–639

    Article  PubMed  CAS  Google Scholar 

  14. Curtis A., Wilkinson C. (1999) New depths in cell behaviour: reactions of cells to nanotopography. Biochem. Soc. Symp. 65:15–26

    PubMed  CAS  Google Scholar 

  15. Desmouliere A., Chaponnier C., Gabbiani G. (2005) Tissue repair, contraction, and the myofibroblast. Wound Repair Regen. 13:7–12

    Article  PubMed  Google Scholar 

  16. Doillon C. J., Dunn M. G., Bender E., Silver F. H. (1985) Collagen fiber formation in repair tissue: development of strength and toughness. Coll Relat. Res. 5:481–492

    PubMed  CAS  Google Scholar 

  17. Doillon C. J., Dunn M. G., Silver F. H. (1988) Relationship between mechanical properties and collagen structure of closed and open wounds. J. Biomech. Eng. 110:352–356

    Article  PubMed  CAS  Google Scholar 

  18. Doillon, C. J. and F. H. Silver. Collagen-based wound dressing: effects of hyaluronic acid and fibronectin on wound healing. Biomaterials 7:3–8, 1986

    Google Scholar 

  19. Doillon C. J., Watsky M. A., Hakim M., Wang J., Munger R., Laycock N., Osborne R., Griffith M. (2003) A collagen-based scaffold for a tissue engineered human cornea: physical and physiological properties. Int. J. Artif. Organs 26:764–773

    PubMed  CAS  Google Scholar 

  20. Doillon C. J., Whyne C. F., Brandwein S., Silver F. H. (1986) Collagen-based wound dressings: control of the pore structure and morphology. J. Biomed. Mater. Res. 20:1219–1228

    Article  PubMed  CAS  Google Scholar 

  21. E.B.B.A. EBAA releases 2004 statistical report on eye banking. Washington DC: Eye Bank of America, pp. 1–2

  22. Fini M. E. (1999) Keratocyte and fibroblast phenotypes in the repairing cornea. Prog. Retin. Eye Res. 18:529–551

    Article  PubMed  CAS  Google Scholar 

  23. Freyman T. M., Yannas I. V., Pek Y. S., Yokoo R., Gibson L. J. (2001) Micromechanics of fibroblast contraction of a collagen-GAG matrix. Exp. Cell Res. 269:140–153

    Article  PubMed  CAS  Google Scholar 

  24. Grinnell F., Nakagawa S., Ho C. H. (1989) The collagen recognition sequence for fibroblasts depends on collagen topography. Exp. Cell Res. 182:668–672

    Article  PubMed  CAS  Google Scholar 

  25. Hedman K., Vartio T., Johansson S., Kjellen L., Hook M., Linker A., Salonen E. M., Vaheri A. (1984) Integrity of the pericellular fibronectin matrix of fibroblasts is independent of sulfated glycosaminoglycans. Embo J.3:581–584

    PubMed  CAS  Google Scholar 

  26. Heimbach D. M., Warden G. D., Luterman A., Jordan M. H., Ozobia N., Ryan C. M., Voigt D. W., Hickerson W. L., Saffle J. R., DeClement F. A., Sheridan R. L., Dimick A. R.(2003) Multicenter postapproval clinical trial of Integra dermal regeneration template for burn treatment. J. Burn Care Rehabil. 24:42–48

    Article  PubMed  Google Scholar 

  27. Hinz B., Gabbiani G. (2003) Mechanisms of force generation and transmission by myofibroblasts. Curr. Opin. Biotechnol. 14:538–546

    Article  PubMed  CAS  Google Scholar 

  28. Huang Y., Siewe M., Madihally S. V. (2006) Effect of spatial architecture on cellular colonization. Biotechnol. Bioeng. 93:64–75

    Article  PubMed  CAS  Google Scholar 

  29. Jester J. V., Barry-Lane P. A., Cavanagh H. D., Petroll W. M. (1996) Induction of alpha-smooth muscle actin expression and myofibroblast transformation in cultured corneal keratocytes. Cornea 15:505–516

    Article  PubMed  CAS  Google Scholar 

  30. Jester J. V., Ho-Chang J. (2003) Modulation of cultured corneal keratocyte phenotype by growth factors/cytokines control in vitro contractility and extracellular matrix contraction. Exp. Eye Res. 77:581–592

    Article  PubMed  CAS  Google Scholar 

  31. Jester J. V., Huang J., Barry-Lane P. A., Kao W. W., Petroll W. M., Cavanagh H. D. (1999) Transforming growth factor(beta)-mediated corneal myofibroblast differentiation requires actin and fibronectin assembly. Invest. Ophthalmol. Vis. Sci. 40:1959–1967

    PubMed  CAS  Google Scholar 

  32. Johansson S., Svineng G., Wennerberg K., Armulik A., Lohikangas L. (1997) Fibronectin–integrin interactions. Front Biosci. 2:d126–d146

    PubMed  CAS  Google Scholar 

  33. Li F., Carlsson D., Lohmann C., Suuronen E., Vascotto S., Kobuch K., Sheardown H., Munger R., Nakamura M., Griffith M. (2003) Cellular and nerve regeneration within a biosynthetic extracellular matrix for corneal transplantation. Proc. Natl. Acad. Sci. USA 100:15346–15351

    Article  PubMed  CAS  Google Scholar 

  34. Mao Y., Schwarzbauer J. E. (2005) Fibronectin fibrillogenesis, a cell-mediated matrix assembly process. Matrix Biol. 24:389–399

    Article  PubMed  CAS  Google Scholar 

  35. Mao Y., Schwarzbauer J. E. (2005) Stimulatory effects of a three-dimensional microenvironment on cell-mediated fibronectin fibrillogenesis. J. Cell Sci. 118:4427–4436

    Article  PubMed  CAS  Google Scholar 

  36. Masur S. K., Dewal H. S., Dinh T. T., Erenburg I., Petridou S. (1996) Myofibroblasts differentiate from fibroblasts when plated at low density. Proc. Natl. Acad. Sci. USA 93:4219–4223

    Article  PubMed  CAS  Google Scholar 

  37. Menasche M., Robert L., Payrau P., Hamada R., Pouliquen Y. (1988) Comparative biochemical and morphometric studies on corneal wound healing. Pathol. Biol. (Paris) 36:781–789

    CAS  Google Scholar 

  38. Murphy-Ullrich J. E. (2001) The de-adhesive activity of matricellular proteins: is intermediate cell adhesion an adaptive state? J. Clin. Invest. 107:785–790

    Article  PubMed  CAS  Google Scholar 

  39. Narotam, P. K., S. Jose, N. Nathoo, C. Taylon, and Y. Vora, Collagen Matrix (DuraGen®) in Spinal Durotomy: Technique Appraisal and Clinical Results. In 18th Annual Meeting of the North American Spine Society (San Diego, California, October 2003)

  40. Orwin E. J., Borene M. L., Hubel A. (2003) Biomechanical and optical characteristics of a corneal stromal equivalent. J. Biomech. Eng. 125:439–444

    Article  PubMed  Google Scholar 

  41. Orwin E. J., Hubel A. (2000) In vitro culture characteristics of corneal epithelial, endothelial, and keratocyte cells in a native collagen matrix. Tissue Eng. 6:307–319

    Article  PubMed  CAS  Google Scholar 

  42. Ousley P. J., Terry M. A. (2002) Objective screening methods for prior refractive surgery in donor tissue. Cornea 21:181–188

    Article  PubMed  Google Scholar 

  43. Pedersen J. A., Swartz M. A. (2005) Mechanobiology in the third dimension. Ann. Biomed. Eng. 33:1469–1490

    Article  PubMed  Google Scholar 

  44. Pelham R. J. Jr., Wang Y. (1997) Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc. Natl. Acad. Sci. USA 94:13661–13665

    Article  PubMed  CAS  Google Scholar 

  45. Rajnicek A., Britland S., McCaig C. (1997) Contact guidance of CNS neurites on grooved quartz: influence of groove dimensions, neuronal age and cell type. J. Cell Sci. 110(Pt 23):2905–2913

    PubMed  Google Scholar 

  46. Ranucci C. S., Moghe P. V. (2001) Substrate microtopography can enhance cell adhesive and migratory responsiveness to matrix ligand density. J. Biomed. Mater. Res. 54:149–161

    Article  PubMed  CAS  Google Scholar 

  47. Roeder B. A., Kokini K., Sturgis J. E., Robinson J. P., Voytik-Harbin S. L. (2002) Tensile mechanical properties of three-dimensional type I collagen extracellular matrices with varied microstructure. J. Biomech. Eng. 124:214–222

    Article  PubMed  Google Scholar 

  48. Salem A. K., Stevens R., Pearson R. G., Davies M. C., Tendler S. J., Roberts C. J., Williams P. M., Shakesheff K. M. (2002) Interactions of 3T3 fibroblasts and endothelial cells with defined pore features. J. Biomed. Mater. Res. 61:212–217

    Article  PubMed  CAS  Google Scholar 

  49. Suuronen E. J., McLaughlin C. R., Stys P. K., Nakamura M., Munger R., Griffith M. (2004) Functional innervation in tissue engineered models for in vitro study and testing purposes. Toxicol. Sci. 82:525–533

    Article  PubMed  CAS  Google Scholar 

  50. Suuronen E. J., Nakamura M., Watsky M. A., Stys P. K., Muller L. J., Munger R., Shinozaki N., Griffith M. (2004) Innervated human corneal equivalents as in vitro models for nerve-target cell interactions. Faseb. J. 18:170–172

    PubMed  CAS  Google Scholar 

  51. Tomasek J. J., Gabbiani G., Hinz B., Chaponnier C., Brown R. A. (2002) Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat. Rev. Mol. Cell Biol. 3:349–363

    Article  PubMed  CAS  Google Scholar 

  52. Vuento M., Hedman K., Vartio T., Vaheri A. (1988) Fibronectin: a flexible image. Electron Microsc. Rev. 1:341–350

    Article  PubMed  CAS  Google Scholar 

  53. Zeltinger J., Sherwood J. K., Graham D. A., Mueller R., Griffith L. G. (2001) Effect of pore size and void fraction on cellular adhesion, proliferation, and matrix deposition. Tissue Eng. 7:557–572

    Article  PubMed  CAS  Google Scholar 

  54. Zieske J. D., Higashijima S. C., Spurr-Michaud S. J., Gipson I. K. (1987) Biosynthetic responses of the rabbit cornea to a keratectomy wound. Invest. Ophthalmol. Vis. Sci. 28:1668–1677

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors would like to thank the Fight for Sight Foundation and the American Society of Biomechanics for their fellowship support of RABC and the support of NSF for the Research Experience for Undergraduates Program in Mechanical Engineering at the University of Minnesota for DMD. Human corneal tissue for cell isolation was provided by the Minnesota Lions Eye Bank. The authors would also like to thank Audrey Bernstein and Sandra Masur for their discussions about corneal cell phenotype and Ching Yuan for his guidance with Western blotting. Lastly, the authors would like to thank the laboratory of Dr. William R. Kennedy, particularly Gwen Wendelschafer-Crabb, for the use of the confocal microscope and the gifts of Zamboni’s fixative, DAPI, and secondary antibodies.

Author information

Author notes

  1. Eric P. Chau

    Present address: School of Medicine, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS, 66160, USA

  2. Danya M. Decoteau

    Present address: Department of Biomedical Engineering, Worcester Polytechnic Institute, 100 Institute Road, Worcester, MA, 01609, USA

Authors and Affiliations

  1. Department of Biomedical Engineering, University of Minnesota, 7-105 Hasselmo Hall, 312 Church Street SE, Minneapolis, MN, 55455, USA

    Rachael A. B. Crabb & Eric P. Chau

  2. Department of Mechanical Engineering, University of Minnesota, 1100 Mechanical Engineering, 111 Church Street, Minneapolis, MN, 55455, USA

    Danya M. Decoteau & Allison Hubel

Authors
  1. Rachael A. B. Crabb
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eric P. Chau
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Danya M. Decoteau
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Allison Hubel
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Allison Hubel.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Crabb, R.A.B., Chau, E.P., Decoteau, D.M. et al. Microstructural Characteristics of Extracellular Matrix Produced by Stromal Fibroblasts. Ann Biomed Eng 34, 1615–1627 (2006). https://doi.org/10.1007/s10439-006-9181-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10439-006-9181-x

Keywords

Search

Navigation