We demonstrate a method for imaging the wound healing process with near infrared fluorescent fibrinogen. Wound healing studies were performed on a rat punch biopsy model. Fibrinogen was conjugated with a near infrared fluorescent dye and injected into the tail vein. Fibrinogen is a useful protein for tracking wound healing because it is involved in fibrin clot formation and formation of new provisional matrix through transglutaminase’s crosslinking activity. Strong fluorescence specific to the wound was observed and persisted for several days, indicating that the fibrinogen is converted to crosslinked fibrin. Administration of contrast agent simultaneously with wound creation led to primary labeling of the fibrin clot, indicating that the wound was in its early phase of healing. Administration on the following day showed labeling on the wound periphery, indicating location of formation of a new provisional matrix. This method may prove to be useful as a diagnostic for basic studies of the wound healing process, in drug development, or in clinical assessment of chronic wounds.

© 2010 OSA

1. L. Khaodhiar, T. Dinh, K. T. Schomacker, S. V. Panasyuk, J. E. Freeman, R. Lew, T. Vo, A. A. Panasyuk, C. Lima, J. M. Giurini, T. E. Lyons, and A. Veves, “The use of medical hyperspectral technology to evaluate microcirculatory changes in diabetic foot ulcers and to predict clinical outcomes,” Diabetes Care 30(4), 903–910 (2007). [CrossRef] [PubMed]
2. A. J. Singer and R. A. F. Clark, “Mechanisms of disease - Cutaneous wound healing,” N. Engl. J. Med. 341(10), 738–746 (1999). [CrossRef] [PubMed]
3. R. Gillitzer and M. Goebeler, “Chemokines in cutaneous wound healing,” J. Leukoc. Biol. 69(4), 513–521 (2001). [PubMed]
4. T. Kisseleva and D. A. Brenner, “Mechanisms of fibrogenesis,” Exp. Biol. Med. (Maywood) 233(2), 109–122 (2008). [CrossRef] [PubMed]
5. D. Telci and M. Griffin, “Tissue transglutaminase (TG2)--a wound response enzyme,” Front. Biosci. 11(1), 867–882 (2006). [CrossRef] [PubMed]
6. E. A. Verderio, T. S. Johnson, and M. Griffin, “Transglutaminases in wound healing and inflammation,” Prog. Exp. Tumor Res. 38, 89–114 (2005). [CrossRef] [PubMed]
7. M. Griffin, R. Casadio, and C. M. Bergamini, “Transglutaminases: nature’s biological glues,” Biochem. J. 368(2), 377–396 (2002). [CrossRef] [PubMed]
8. C. S. Greenberg, P. J. Birckbichler, and R. H. Rice, “Transglutaminases: multifunctional cross-linking enzymes that stabilize tissues,” FASEB J. 5(15), 3071–3077 (1991). [PubMed]
9. S. Kojima, K. Nara, and D. B. Rifkin, “Requirement for transglutaminase in the activation of latent transforming growth factor-beta in bovine endothelial cells,” J. Cell Biol. 121(2), 439–448 (1993). [CrossRef] [PubMed]
10. M. Siegel, P. Strnad, R. Watts, K. Choi, B. Jabri, G. Adler, B. Omary, and C. Khosla, “Extracellular transglutaminase 2 is catalytically inactive, but is transiently activated upon tissue injury in the small intestine,” Gastroenterology 134(4), A151 (2008). [CrossRef]
11. E. A. Zemskov, A. Janiak, J. Hang, A. Waghray, and A. M. Belkin, “The role of tissue transglutaminase in cell-matrix interactions,” Front. Biosci. 11(1), 1057–1076 (2006). [CrossRef] [PubMed]
12. C. S. Greenberg, K. E. Achyuthan, M. J. Borowitz, and M. A. Shuman, “The transglutaminase in vascular cells and tissues could provide an alternate pathway for fibrin stabilization,” Blood 70(3), 702–709 (1987). [PubMed]
13. D. C. Sane, T. L. Moser, A. M. M. Pippen, C. J. Parker, K. E. Achyuthan, and C. S. Greenberg, “Vitronectin is a substrate for transglutaminases,” Biochem. Biophys. Res. Commun. 157(1), 115–120 (1988). [CrossRef] [PubMed]
14. D. H. Keast, C. K. Bowering, A. W. Evans, G. L. Mackean, C. Burrows, and L. D’Souza, “MEASURE: A proposed assessment framework for developing best practice recommendations for wound assessment,” Wound Repair Regen. 12(3Suppl), s1–s17 (2004). [CrossRef] [PubMed]
15. R. Salcido, “The future of wound measurement,” Adv. Skin Wound Care 13(2), 54, 56 (2000). [PubMed]
16. J. W. Griffin, E. A. Tolley, R. E. Tooms, R. A. Reyes, and J. K. Clifft, “A comparison of photographic and transparency-based methods for measuring wound surface area,” Phys. Ther. 73(2), 117–122 (1993). [PubMed]
17. D. J. Leaper, “Angiography as an index of healing in experimental laparotomy wounds and colonic anastomoses,” Ann. R. Coll. Surg. Engl. 65(1), 20–23 (1983). [PubMed]
18. M. J. Cobb, Y. C. Chen, R. A. Underwood, M. L. Usui, J. Olerud, and X. D. Li, “Noninvasive assessment of cutaneous wound healing using ultrahigh-resolution optical coherence tomography,” J. Biomed. Opt. 11(6), 064002 (2006). [CrossRef] [PubMed]
19. T. Gambichler, G. Moussa, M. Sand, D. Sand, P. Altmeyer, and K. Hoffmann, “Applications of optical coherence tomography in dermatology,” J. Dermatol. Sci. 40(2), 85–94 (2005). [CrossRef] [PubMed]
20. A. T. Yeh, B. S. Kao, W. G. Jung, Z. P. Chen, J. S. Nelson, and B. J. Tromberg, “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 (2004). [CrossRef] [PubMed]
21. M. Dyson, S. Moodley, L. Verjee, W. Verling, J. Weinman, and P. Wilson, “Wound healing assessment using 20 MHz ultrasound and photography,” Skin Res. Technol. 9(2), 116–121 (2003). [CrossRef] [PubMed]
22. H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Imaging acute thermal burns by photoacoustic microscopy,” J. Biomed. Opt. 11(5), 054033 (2006). [CrossRef] [PubMed]
23. G. Mazooz, T. Mehlman, T. S. Lai, C. S. Greenberg, M. W. Dewhirst, and M. Neeman, “Development of magnetic resonance imaging contrast material for in vivo mapping of tissue transglutaminase activity,” Cancer Res. 65(4), 1369–1375 (2005). [CrossRef] [PubMed]
24. F. A. Jaffer, D. E. Sosnovik, M. Nahrendorf, and R. Weissleder, “Molecular imaging of myocardial infarction,” J. Mol. Cell. Cardiol. 41(6), 921–933 (2006). [CrossRef] [PubMed]
25. F. A. Jaffer, C. H. Tung, J. J. Wykrzykowska, N. H. Ho, A. K. Houng, G. L. Reed, and R. Weissleder, “Molecular imaging of factor XIIIa activity in thrombosis using a novel, near-infrared fluorescent contrast agent that covalently links to thrombi,” Circulation 110(2), 170–176 (2004). [CrossRef] [PubMed]
26. J. M. Hettasch, N. Bandarenko, J. L. Burchette, T. S. Lai, J. R. Marks, Z. A. Haroon, K. Peters, M. W. Dewhirst, J. D. Iglehart, and C. S. Greenberg, “Tissue transglutaminase expression in human breast cancer,” Lab. Invest. 75(5), 637–645 (1996). [PubMed]
27. Z. A. Haroon, T. S. Lai, J. M. Hettasch, R. A. Lindberg, M. W. Dewhirst, and C. S. Greenberg, “Tissue transglutaminase is expressed as a host response to tumor invasion and inhibits tumor growth,” Lab. Invest. 79(12), 1679–1686 (1999). [PubMed]
28. J. M. Kollman, L. Pandi, M. R. Sawaya, M. Riley, and R. F. Doolittle, “Crystal structure of human fibrinogen,” Biochemistry 48(18), 3877–3886 (2009). [CrossRef] [PubMed]
29. Z. A. Haroon, J. M. Hettasch, T. S. Lai, M. W. Dewhirst, and C. S. Greenberg, “Tissue transglutaminase is expressed, active, and directly involved in rat dermal wound healing and angiogenesis,” FASEB J. 13(13), 1787–1795 (1999). [PubMed]
30. S. A. Yuan, C. A. Roney, J. Wierwille, C. W. Chen, B. Y. Xu, G. Griffiths, J. Jiang, H. Z. Ma, A. Cable, R. M. Summers, and Y. Chen, “Co-registered optical coherence tomography and fluorescence molecular imaging for simultaneous morphological and molecular imaging,” Phys. Med. Biol. 55(1), 191–206 (2010). [CrossRef] [PubMed]
31. H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging,” Nat. Biotechnol. 24(7), 848–851 (2006). [CrossRef] [PubMed]
32. D. P. Pan, M. Pramanik, A. Senpan, X. M. Yang, K. H. Song, M. J. Scott, H. Y. Zhang, P. J. Gaffney, S. A. Wickline, L. V. Wang, and G. M. Lanza, “Molecular photoacoustic tomography with colloidal nanobeacons,” Angew. Chem. Int. Ed. Engl. 48(23), 4170–4173 (2009). [CrossRef] [PubMed]
33. L. V. Wang, “Prospects of photoacoustic tomography,” Med. Phys. 35(12), 5758–5767 (2008). [CrossRef] [PubMed]
34. K. H. Song, E. W. Stein, J. A. Margenthaler, and L. V. Wang, “Noninvasive photoacoustic identification of sentinel lymph nodes containing methylene blue in vivo in a rat model,” J. Biomed. Opt. 13(5), 054033 (2008). [CrossRef] [PubMed]
35. H. Thangarajah, D. Yao, E. I. Chang, Y. Shi, L. Jazayeri, I. N. Vial, R. D. Galiano, X. L. Du, R. Grogan, M. G. Galvez, M. Januszyk, M. Brownlee, and G. C. Gurtner, “The molecular basis for impaired hypoxia-induced VEGF expression in diabetic tissues,” Proc. Natl. Acad. Sci. U.S.A. 106(32), 13505–13510 (2009). [CrossRef] [PubMed]
36. L. F. Brown, K. T. Yeo, B. Berse, T. K. Yeo, D. R. Senger, H. F. Dvorak, and L. van de Water, “Expression of vascular permeability factor (vascular endothelial growth factor) by epidermal keratinocytes during wound healing,” J. Exp. Med. 176(5), 1375–1379 (1992). [CrossRef] [PubMed]
37. Y. Sugimura, M. Hosono, F. Wada, T. Yoshimura, M. Maki, and K. Hitomi, “Screening for the preferred substrate sequence of transglutaminase using a phage-displayed peptide library: identification of peptide substrates for TGASE 2 and Factor XIIIA,” J. Biol. Chem. 281(26), 17699–17706 (2006). [CrossRef] [PubMed]

Adv. Skin Wound Care

• R. Salcido, “The future of wound measurement,” Adv. Skin Wound Care 13(2), 54, 56 (2000). [PubMed]

Angew. Chem. Int. Ed. Engl.

• D. P. Pan, M. Pramanik, A. Senpan, X. M. Yang, K. H. Song, M. J. Scott, H. Y. Zhang, P. J. Gaffney, S. A. Wickline, L. V. Wang, and G. M. Lanza, “Molecular photoacoustic tomography with colloidal nanobeacons,” Angew. Chem. Int. Ed. Engl. 48(23), 4170–4173 (2009). [CrossRef] [PubMed]

Ann. R. Coll. Surg. Engl.

• D. J. Leaper, “Angiography as an index of healing in experimental laparotomy wounds and colonic anastomoses,” Ann. R. Coll. Surg. Engl. 65(1), 20–23 (1983). [PubMed]

Biochem. Biophys. Res. Commun.

• D. C. Sane, T. L. Moser, A. M. M. Pippen, C. J. Parker, K. E. Achyuthan, and C. S. Greenberg, “Vitronectin is a substrate for transglutaminases,” Biochem. Biophys. Res. Commun. 157(1), 115–120 (1988). [CrossRef] [PubMed]

Biochem. J.

• M. Griffin, R. Casadio, and C. M. Bergamini, “Transglutaminases: nature’s biological glues,” Biochem. J. 368(2), 377–396 (2002). [CrossRef] [PubMed]

Biochemistry

• J. M. Kollman, L. Pandi, M. R. Sawaya, M. Riley, and R. F. Doolittle, “Crystal structure of human fibrinogen,” Biochemistry 48(18), 3877–3886 (2009). [CrossRef] [PubMed]

Blood

• C. S. Greenberg, K. E. Achyuthan, M. J. Borowitz, and M. A. Shuman, “The transglutaminase in vascular cells and tissues could provide an alternate pathway for fibrin stabilization,” Blood 70(3), 702–709 (1987). [PubMed]

Cancer Res.

• G. Mazooz, T. Mehlman, T. S. Lai, C. S. Greenberg, M. W. Dewhirst, and M. Neeman, “Development of magnetic resonance imaging contrast material for in vivo mapping of tissue transglutaminase activity,” Cancer Res. 65(4), 1369–1375 (2005). [CrossRef] [PubMed]

Circulation

• F. A. Jaffer, C. H. Tung, J. J. Wykrzykowska, N. H. Ho, A. K. Houng, G. L. Reed, and R. Weissleder, “Molecular imaging of factor XIIIa activity in thrombosis using a novel, near-infrared fluorescent contrast agent that covalently links to thrombi,” Circulation 110(2), 170–176 (2004). [CrossRef] [PubMed]

Diabetes Care

• L. Khaodhiar, T. Dinh, K. T. Schomacker, S. V. Panasyuk, J. E. Freeman, R. Lew, T. Vo, A. A. Panasyuk, C. Lima, J. M. Giurini, T. E. Lyons, and A. Veves, “The use of medical hyperspectral technology to evaluate microcirculatory changes in diabetic foot ulcers and to predict clinical outcomes,” Diabetes Care 30(4), 903–910 (2007). [CrossRef] [PubMed]

Exp. Biol. Med. (Maywood)

• T. Kisseleva and D. A. Brenner, “Mechanisms of fibrogenesis,” Exp. Biol. Med. (Maywood) 233(2), 109–122 (2008). [CrossRef] [PubMed]

FASEB J.

• C. S. Greenberg, P. J. Birckbichler, and R. H. Rice, “Transglutaminases: multifunctional cross-linking enzymes that stabilize tissues,” FASEB J. 5(15), 3071–3077 (1991). [PubMed]
• Z. A. Haroon, J. M. Hettasch, T. S. Lai, M. W. Dewhirst, and C. S. Greenberg, “Tissue transglutaminase is expressed, active, and directly involved in rat dermal wound healing and angiogenesis,” FASEB J. 13(13), 1787–1795 (1999). [PubMed]

Front. Biosci.

• D. Telci and M. Griffin, “Tissue transglutaminase (TG2)--a wound response enzyme,” Front. Biosci. 11(1), 867–882 (2006). [CrossRef] [PubMed]
• E. A. Zemskov, A. Janiak, J. Hang, A. Waghray, and A. M. Belkin, “The role of tissue transglutaminase in cell-matrix interactions,” Front. Biosci. 11(1), 1057–1076 (2006). [CrossRef] [PubMed]

Gastroenterology

• M. Siegel, P. Strnad, R. Watts, K. Choi, B. Jabri, G. Adler, B. Omary, and C. Khosla, “Extracellular transglutaminase 2 is catalytically inactive, but is transiently activated upon tissue injury in the small intestine,” Gastroenterology 134(4), A151 (2008). [CrossRef]

J. Biol. Chem.

• Y. Sugimura, M. Hosono, F. Wada, T. Yoshimura, M. Maki, and K. Hitomi, “Screening for the preferred substrate sequence of transglutaminase using a phage-displayed peptide library: identification of peptide substrates for TGASE 2 and Factor XIIIA,” J. Biol. Chem. 281(26), 17699–17706 (2006). [CrossRef] [PubMed]

J. Biomed. Opt.

• K. H. Song, E. W. Stein, J. A. Margenthaler, and L. V. Wang, “Noninvasive photoacoustic identification of sentinel lymph nodes containing methylene blue in vivo in a rat model,” J. Biomed. Opt. 13(5), 054033 (2008). [CrossRef] [PubMed]
• H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Imaging acute thermal burns by photoacoustic microscopy,” J. Biomed. Opt. 11(5), 054033 (2006). [CrossRef] [PubMed]
• A. T. Yeh, B. S. Kao, W. G. Jung, Z. P. Chen, J. S. Nelson, and B. J. Tromberg, “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 (2004). [CrossRef] [PubMed]
• M. J. Cobb, Y. C. Chen, R. A. Underwood, M. L. Usui, J. Olerud, and X. D. Li, “Noninvasive assessment of cutaneous wound healing using ultrahigh-resolution optical coherence tomography,” J. Biomed. Opt. 11(6), 064002 (2006). [CrossRef] [PubMed]

J. Cell Biol.

• S. Kojima, K. Nara, and D. B. Rifkin, “Requirement for transglutaminase in the activation of latent transforming growth factor-beta in bovine endothelial cells,” J. Cell Biol. 121(2), 439–448 (1993). [CrossRef] [PubMed]

J. Dermatol. Sci.

• T. Gambichler, G. Moussa, M. Sand, D. Sand, P. Altmeyer, and K. Hoffmann, “Applications of optical coherence tomography in dermatology,” J. Dermatol. Sci. 40(2), 85–94 (2005). [CrossRef] [PubMed]

J. Exp. Med.

• L. F. Brown, K. T. Yeo, B. Berse, T. K. Yeo, D. R. Senger, H. F. Dvorak, and L. van de Water, “Expression of vascular permeability factor (vascular endothelial growth factor) by epidermal keratinocytes during wound healing,” J. Exp. Med. 176(5), 1375–1379 (1992). [CrossRef] [PubMed]

J. Leukoc. Biol.

• R. Gillitzer and M. Goebeler, “Chemokines in cutaneous wound healing,” J. Leukoc. Biol. 69(4), 513–521 (2001). [PubMed]

J. Mol. Cell. Cardiol.

• F. A. Jaffer, D. E. Sosnovik, M. Nahrendorf, and R. Weissleder, “Molecular imaging of myocardial infarction,” J. Mol. Cell. Cardiol. 41(6), 921–933 (2006). [CrossRef] [PubMed]

Lab. Invest.

• J. M. Hettasch, N. Bandarenko, J. L. Burchette, T. S. Lai, J. R. Marks, Z. A. Haroon, K. Peters, M. W. Dewhirst, J. D. Iglehart, and C. S. Greenberg, “Tissue transglutaminase expression in human breast cancer,” Lab. Invest. 75(5), 637–645 (1996). [PubMed]
• Z. A. Haroon, T. S. Lai, J. M. Hettasch, R. A. Lindberg, M. W. Dewhirst, and C. S. Greenberg, “Tissue transglutaminase is expressed as a host response to tumor invasion and inhibits tumor growth,” Lab. Invest. 79(12), 1679–1686 (1999). [PubMed]

Med. Phys.

• L. V. Wang, “Prospects of photoacoustic tomography,” Med. Phys. 35(12), 5758–5767 (2008). [CrossRef] [PubMed]

N. Engl. J. Med.

• A. J. Singer and R. A. F. Clark, “Mechanisms of disease - Cutaneous wound healing,” N. Engl. J. Med. 341(10), 738–746 (1999). [CrossRef] [PubMed]

Nat. Biotechnol.

• H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging,” Nat. Biotechnol. 24(7), 848–851 (2006). [CrossRef] [PubMed]

Phys. Med. Biol.

• S. A. Yuan, C. A. Roney, J. Wierwille, C. W. Chen, B. Y. Xu, G. Griffiths, J. Jiang, H. Z. Ma, A. Cable, R. M. Summers, and Y. Chen, “Co-registered optical coherence tomography and fluorescence molecular imaging for simultaneous morphological and molecular imaging,” Phys. Med. Biol. 55(1), 191–206 (2010). [CrossRef] [PubMed]

Phys. Ther.

• J. W. Griffin, E. A. Tolley, R. E. Tooms, R. A. Reyes, and J. K. Clifft, “A comparison of photographic and transparency-based methods for measuring wound surface area,” Phys. Ther. 73(2), 117–122 (1993). [PubMed]

Proc. Natl. Acad. Sci. U.S.A.

• H. Thangarajah, D. Yao, E. I. Chang, Y. Shi, L. Jazayeri, I. N. Vial, R. D. Galiano, X. L. Du, R. Grogan, M. G. Galvez, M. Januszyk, M. Brownlee, and G. C. Gurtner, “The molecular basis for impaired hypoxia-induced VEGF expression in diabetic tissues,” Proc. Natl. Acad. Sci. U.S.A. 106(32), 13505–13510 (2009). [CrossRef] [PubMed]

Prog. Exp. Tumor Res.

• E. A. Verderio, T. S. Johnson, and M. Griffin, “Transglutaminases in wound healing and inflammation,” Prog. Exp. Tumor Res. 38, 89–114 (2005). [CrossRef] [PubMed]

Skin Res. Technol.

• M. Dyson, S. Moodley, L. Verjee, W. Verling, J. Weinman, and P. Wilson, “Wound healing assessment using 20 MHz ultrasound and photography,” Skin Res. Technol. 9(2), 116–121 (2003). [CrossRef] [PubMed]

Wound Repair Regen.

• D. H. Keast, C. K. Bowering, A. W. Evans, G. L. Mackean, C. Burrows, and L. D’Souza, “MEASURE: A proposed assessment framework for developing best practice recommendations for wound assessment,” Wound Repair Regen. 12(3Suppl), s1–s17 (2004). [CrossRef] [PubMed]

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