Hostname: page-component-848d4c4894-p2v8j Total loading time: 0 Render date: 2024-06-10T20:30:39.876Z Has data issue: false hasContentIssue false
  • English
  • Français

Visualization of Morphological and Molecular Features Associated with Chronic Ischemia in Bioengineered Human Skin

Published online by Cambridge University Press:  04 March 2010

Erin M. Gill ,
Joely A. Straseski ,
Cathy A. Rasmussen ,
Sara J. Liliensiek ,
Kevin W. Eliceiri ,
Nirmala Ramanujam ,
John G. White  and
B. Lynn Allen-Hoffmann
Erin M. Gill
Affiliation:
Department of Biomedical Engineering, University of Wisconsin, Madison, WI 53706, USA Laboratory for Optical and Computational Instrumentation, University of Wisconsin, Madison, WI 53706, USA
Joely A. Straseski
Affiliation:
Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD 21287, USA
Cathy A. Rasmussen
Affiliation:
Department of Pathology and Laboratory Medicine, University of Wisconsin Medical School, Madison, WI 53706, USA Stratatech Corporation, Research and Development, Madison, WI, USA
Sara J. Liliensiek
Affiliation:
School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706, USA
Kevin W. Eliceiri
Affiliation:
Laboratory for Optical and Computational Instrumentation, University of Wisconsin, Madison, WI 53706, USA
Nirmala Ramanujam
Affiliation:
Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA
John G. White
Affiliation:
Laboratory for Optical and Computational Instrumentation, University of Wisconsin, Madison, WI 53706, USA
B. Lynn Allen-Hoffmann*
Affiliation:
Department of Pathology and Laboratory Medicine, University of Wisconsin Medical School, Madison, WI 53706, USA Stratatech Corporation, Research and Development, Madison, WI, USA
*
Corresponding author. E-mail: blallenh@wisc.edu
Get access

Abstract

We present an in vitro model of human skin that, together with nonlinear optical microscopy, provides a useful system for characterizing morphological and structural changes in a living skin tissue microenvironment due to changes in oxygen status and proteolytic balance. We describe for the first time the effects of chronic oxygen deprivation on a bioengineered model of human interfollicular epidermis. Histological analysis and multiphoton imaging revealed a progressively degenerating ballooning phenotype of the keratinocytes that manifested after 48 h of hypoxic exposure. Multiphoton images of the dermal compartment revealed a decrease in collagen structural order. Immunofluorescence analysis showed changes in matrix metalloproteinase (MMP)-2 protein spatial localization in the epidermis with a shift to the basal layer, and loss of Ki67 expression in proliferative basal cells after 192 h of hypoxic exposure. Upon reoxygenation MMP-2 mRNA levels showed a biphasic response, with restoration of MMP-2 levels and localization. These results indicate that chronic oxygen deprivation causes an overall degeneration in tissue architecture, combined with an imbalance in proteolytic expression and a decrease in proliferative capacity. We propose that these tissue changes are representative of the ischemic condition and that our experimental model system is appropriate for addressing mechanisms of susceptibility to chronic wounds.

Keywords

hypoxiaischemiaulcerationbioengineered skinmultiphotonMMP-2
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 16 , Issue 2 , April 2010 , pp. 117 - 131
Copyright
Copyright © Microscopy Society of America 2010

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Abramoff, M., Magelhaes, P. & Ram, S. (2004). Image processing with ImageJ. Biophotonics Int 11(7), 36–42.Google Scholar
Agren, M.S. (1994). Gelatinase activity during wound healing. Br J Dermatol 131(5), 634–640.CrossRefGoogle ScholarPubMed
Allen, T.D. & Schor, S.L. (1983). The contraction of collagen matrices by dermal fibroblasts. J Ultrastruct Res 83(2), 205–219.CrossRefGoogle ScholarPubMed
Allen-Hoffmann, B.L., Schlosser, S.J., Ivarie, C.A., Sattler, C.A., Meisner, L.F. & O'Connor, S.L. (2000). Normal growth and differentiation in a spontaneously immortalized near-diploid human keratinocyte cell line, NIKS. J Invest Dermatol 114(3), 444–455.CrossRefGoogle Scholar
Brown, E., McKee, T., diTomaso, E., Pluen, A., Seed, B., Boucher, Y. & Jain, R.K. (2003). Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation. Nat Med 9(6), 796–800.CrossRefGoogle ScholarPubMed
Campagnola, P.J. & Loew, L.M. (2003). Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms. Nat Biotechnol 21(11), 1356–1360.CrossRefGoogle ScholarPubMed
Centonze, V.E. & White, J.G. (1998). Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging. Biophys J 75(4), 2015–2024.CrossRefGoogle ScholarPubMed
Dalton, S.J., Mitchell, D.C., Whiting, C.V. & Tarlton, J.F. (2005). Abnormal extracellular matrix metabolism in chronically ischemic skin: A mechanism for dermal failure in leg ulcers. J Invest Dermatol 125(2), 373–379.CrossRefGoogle ScholarPubMed
Dalton, S.J., Whiting, C.V., Bailey, J.R., Mitchell, D.C. & Tarlton, J.F. (2007). Mechanisms of chronic skin ulceration linking lactate, transforming growth factor-beta, vascular endothelial growth factor, collagen remodeling, collagen stability, and defective angiogenesis. J Invest Dermatol 127(4), 958–968.CrossRefGoogle ScholarPubMed
Denk, W., Strickler, J.H. & Webb, W.W. (1990). Two-photon laser scanning fluorescence microscopy. Science 248(4951), 73–76.CrossRefGoogle ScholarPubMed
Diegelmann, R.F. & Evans, M.C. (2004). Wound healing: An overview of acute, fibrotic and delayed healing. Front Biosci 9, 283–289.CrossRefGoogle ScholarPubMed
Grinnell, F. (2003). Fibroblast biology in three-dimensional collagen matrices. Trends Cell Biol 13(5), 264–269.CrossRefGoogle ScholarPubMed
Herman, I.M. & Leung, A. (2009). Creation of human skin equivalents for the in vitro study of angiogenesis in wound healing. Methods Mol Biol 467, 241–248.CrossRefGoogle ScholarPubMed
Jansen, P.L., Rosch, R., Jansen, M., Binnebosel, M., Junge, K., Alfonso-Jaume, A., Klinge, U., Lovett, D.H. & Mertens, P.R. (2007). Regulation of MMP-2 gene transcription in dermal wounds. J Invest Dermatol 127(7), 1762–1767.CrossRefGoogle ScholarPubMed
Konig, K. (2000). Multiphoton microscopy in life sciences. J Microsc 200(Pt 2), 83–104.CrossRefGoogle ScholarPubMed
Konig, K., Ehlers, A., Stracke, F. & Riemann, I. (2006). In vivo drug screening in human skin using femtosecond laser multiphoton tomography. Skin Pharmacol Physiol 19(2), 78–88.CrossRefGoogle ScholarPubMed
Lee, J.H., Chen, S.Y., Yu, C.H., Chu, S.W., Wang, L.F., Sun, C.K. & Chiang, B.L. (2009). Noninvasive in vitro and in vivo assessment of epidermal hyperkeratosis and dermal fibrosis in atopic dermatitis. J Biomed Opt 14(1), 014008.CrossRefGoogle ScholarPubMed
Masters, B.R. & So, P.T. (1999). Multi-photon excitation microscopy and confocal microscopy imaging of in vivo human skin: A comparison. Microsc Microanal 5(4), 282–289.CrossRefGoogle ScholarPubMed
Mehra, T.D., Ghosh, K., Shu, X.Z., Prestwich, G.D. & Clark, R.A. (2006). Molecular stenting with a crosslinked hyaluronan derivative inhibits collagen gel contraction. J Invest Dermatol 126(10), 2202–2209.CrossRefGoogle ScholarPubMed
Mohler, W., Millard, A.C. & Campagnola, P.J. (2003). Second harmonic generation imaging of endogenous structural proteins. Methods 29(1), 97–109.CrossRefGoogle ScholarPubMed
Nagase, H., Visse, R. & Murphy, G. (2006). Structure and function of matrix metalloproteinases and TIMPs. Cardiovasc Res 69(3), 562–573.CrossRefGoogle ScholarPubMed
Pavlova, I., Sokolov, K., Drezek, R., Malpica, A., Follen, M. & Richards-Kortum, R. (2003). Microanatomical and biochemical origins of normal and precancerous cervical autofluorescence using laser-scanning fluorescence confocal microscopy. Photochem Photobiol 77(5), 550–555.2.0.CO;2>CrossRefGoogle ScholarPubMed
Pawley, J.B. (Ed.) (2006). Handbook of Biological Confocal Microscopy. New York: Springer.CrossRefGoogle Scholar
Pena, A.M., Strupler, M., Boulesteix, T. & Schanne-Klein, M.C. (2005). Spectroscopic analysis of keratin endogenous signal for skin multiphoton microscopy. Optics Express 13(16), 6268–6274.CrossRefGoogle ScholarPubMed
Rajadhyaksha, M., Grossman, M., Esterowitz, D., Webb, R.H. & Anderson, R.R. (1995). In vivo confocal scanning laser microscopy of human skin: Melanin provides strong contrast. J Invest Dermatol 104(6), 946–952.CrossRefGoogle ScholarPubMed
Rasmussen, C.A., Gibson, A.L., Schlosser, S.J., Schurr, M.J. & Allen-Hoffmann, B.L. (2010). Chimeric composite skin substitutes for delivery of autologous keratinocytes to promote tissue regeneration. Ann Surg 251(2), 368–376.CrossRefGoogle ScholarPubMed
Raub, C.B., Suresh, V., Krasieva, T., Lyubovitsky, J., Mih, J.D., Putnam, A.J., Tromberg, B.J. & George, S.C. (2007). Noninvasive assessment of collagen gel microstructure and mechanics using multiphoton microscopy. Biophys J 92(6), 2212–2222.CrossRefGoogle ScholarPubMed
Riches, K., Morley, M.E., Turner, N.A., O'Regan, D.J., Ball, S.G., Peers, C. & Porter, K.E. (2009). Chronic hypoxia inhibits MMP-2 activation and cellular invasion in human cardiac myofibroblasts. J Mol Cell Cardiol 47(3), 391–399.CrossRefGoogle ScholarPubMed
Rueden, C., Eliceiri, K. & White, J. (2004). VisBio: A computational tool for visualization of multidimensional biological image data. Traffic 5, 1–7.CrossRefGoogle ScholarPubMed
Smith, G. & Melhuish, W. (1985). Fluorescence and phosphorescence of wool keratin excited by UV-A radiation. Textile Res J 55, 304–307.CrossRefGoogle Scholar
Squirrell, J.M., Wokosin, D.L., White, J.G. & Bavister, B.D. (1999). Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability. Nat Biotechnol 17(8), 763–767.CrossRefGoogle ScholarPubMed
Straseski, J.A., Gibson, A.L., Thomas-Virnig, C.L. & Allen-Hoffmann, B.L. (2009). Oxygen deprivation inhibits basal keratinocyte proliferation in a model of human skin and induces regio-specific changes in the distribution of epidermal adherens junction proteins, aquaporin-3, and glycogen. Wound Repair Regen 17(4), 606–616.CrossRefGoogle Scholar
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(5), 349–363.CrossRefGoogle ScholarPubMed
Torkian, B.A., Yeh, A.T., Engel, R., Sun, C.H., Tromberg, B.J. & Wong, B.J. (2004). Modeling aberrant wound healing using tissue-engineered skin constructs and multiphoton microscopy. Arch Facial Plast Surg 6(3), 180–187.CrossRefGoogle ScholarPubMed
White, J.G., Amos, W.B. & Fordham, M. (1987). An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy. J Cell Biol 105(1), 41–48.CrossRefGoogle ScholarPubMed
Wokosin, D., Squirrell, J., Eliceiri, K. & White, J. (2003). Optical workstation with concurrent, independent multiphoton imaging and experimental laser microbeam capabilities. Rev Sci Instrum 74(1), 193–201.CrossRefGoogle ScholarPubMed
Yeh, A.T., Kao, B., Jung, W.G., Chen, Z., Nelson, J.S. & Tromberg, B.J. (2004). Imaging wound healing using optical coherence tomography and multiphoton microscopy in an in vitro skin-equivalent tissue model. J Biomed Opt 9(2), 248–253.CrossRefGoogle Scholar
Zipfel, W.R., Williams, R.M., Christie, R., Nikitin, A.Y., Hyman, B.T. & Webb, W.W. (2003a). Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proc Natl Acad Sci USA 100(12), 7075–7080.CrossRefGoogle ScholarPubMed
Zipfel, W.R., Williams, R.M. & Webb, W.W. (2003b). Nonlinear magic: Multiphoton microscopy in the biosciences. Nat Biotechnol 21(11), 1369–1377.CrossRefGoogle ScholarPubMed
Zoumi, A., Yeh, A. & Tromberg, B.J. (2002). Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence. Proc Natl Acad Sci USA 99(17), 11014–11019.CrossRefGoogle ScholarPubMed