Advertisement

Cytotechnology

, Volume 68, Issue 5, pp 1873–1884 | Cite as

Secretion of wound healing mediators by single and bi-layer skin substitutes

  • Manira Maarof
  • Jia Xian Law
  • Shiplu Roy Chowdhury
  • Khairul Anuar Khairoji
  • Aminuddin Bin Saim
  • Ruszymah Bt. Hj IdrusEmail author
Original Article

Abstract

Limitations of current treatments for skin loss caused by major injuries leads to the use of skin substitutes. It is assumed that secretion of wound healing mediators by these skin substitutes plays a role in treating skin loss. In our previous study, single layer keratinocytes (SK), single layer fibroblast (SF) and bilayer (BL; containing keratinocytes and fibroblasts layers) skin substitutes were fabricated using fibrin that had shown potential to heal wounds in preclinical studies. This study aimed to quantify the secretion of wound healing mediators, and compare between single and bi-layer skin substitutes. Skin samples were digested to harvest fibroblasts and keratinocytes, and expanded to obtain sufficient cells for the construction of skin substitutes. Acellular fibrin (AF) construct was used as control. Substitutes i.e. AF, SK, SF and BL were cultured for 2 days, and culture supernatant was collected to analyze secretion of wound healing mediators via multiplex ELISA. Among 19 wound healing mediators tested, BL substitute secreted significantly higher amounts of CXCL1 and GCSF compared to SF and AF substitute but this was not significant with respect to SK substitute. The BL substitute also secreted significantly higher amounts of CXCL5 and IL-6 compared to other substitutes. In contrast, the SK substitute secreted significantly higher amounts of VCAM-1 compared to other substitutes. However, all three skin substitutes also secreted CCL2, CCL5, CCL11, GM-CSF, IL8, IL-1α, TNF-α, ICAM-1, FGF-β, TGF-β, HGF, VEGF-α and PDGF-BB factors, but no significant difference was seen. Secretion of these mediators after transplantation may play a significant role in promoting wound healing process for the treatment of skin loss.

Keywords

Keratinocytes Fibroblasts Bi-layer skin substitute Fibrin Tissue engineering 

Notes

Acknowledgments

The research was funded by research grants from Science Fund 02-01-02-SF0964, Arus Perdana AP-2013-015 and Tissue Engineering Centre, UKM Medical Centre.

Supplementary material

Supplementary material 1 (MPG 684 kb)

References

  1. Balasubramani M, Kumar TR, Babu M (2001) Skin substitutes: a review. Burns 27:534–544CrossRefGoogle Scholar
  2. Barrientos S, Stojadinovic O, Golinko MS, Brem H, Tomic-Canic M (2008) Growth factors and cytokines in wound healing. Wound Repair Regen 16:585–601CrossRefGoogle Scholar
  3. Bello YM, Falabella AF, Eaglstein WH (2001) Tissue-engineered skin. Am J Clin Dermatol 2:305–313CrossRefGoogle Scholar
  4. Collins RG, Velji R, Guevara NV, Hicks MJ, Chan L, Beaudet AL (2000) P-selectin or intercellular adhesion molecule (ICAM)-1 deficiency substantially protects against atherosclerosis in apolipoprotein E-deficient mice. J Exp Med 191:189–194CrossRefGoogle Scholar
  5. Costin G-E, Hearing VJ (2007) Human skin pigmentation: melanocytes modulate skin color in response to stress. FASEB J 21:976–994CrossRefGoogle Scholar
  6. Donnez J, Smoes P, Gillerot S, Casanas-Roux F, Nisolle M (1998) Vascular endothelial growth factor (VEGF) in endometriosis. Hum Reprod 13:1686–1690CrossRefGoogle Scholar
  7. Fivenson DP, Faria DT, Nickoloff BJ, Poverini PJ, Kunkel S, Burdick M, Strieter RM (1997) Chemokine and inflammatory cytokine changes during chronic wound healing. Wound Repair Regen 5:310–322CrossRefGoogle Scholar
  8. Garcia-Zepeda EA, Rothenberg ME, Ownbey RT, Celestin J, Leder P, Luster AD (1996) Human eotaxin is a specific chemoattractant for eosinophil cells and provides a new mechanism to explain tissue eosinophilia. Nat Med 2:449–456CrossRefGoogle Scholar
  9. Geer DJ, Andreadis ST (2003) A novel role of fibrin in epidermal healing: plasminogen-mediated migration and selective detachment of differentiated keratinocytes. J Invest Dermatol 121:1210–1216CrossRefGoogle Scholar
  10. Huang S, Deng T, Wang Y, Deng Z, He L, Liu S, Yang J, Jin Y (2008) Multifunctional implantable particles for skin tissue regeneration: preparation, characterization, in vitro and in vivo studies. Acta Biomater 4:1057–1066CrossRefGoogle Scholar
  11. Hutmacher DW, Vanscheidt W (2002) Matrices for tissue-engineered skin. Drugs Today 38:113–133CrossRefGoogle Scholar
  12. Ishigame H, Nakajima A, Saijo S, Komiyama Y, Nambu A, Matsuki T, Nakae S, Horai R, Kakuta S, Iwakura Y (2006) The role of TNFα and IL-17 in the development of excess IL-1 signaling-induced inflammatory diseases in IL-1 receptor antagonist-deficient mice. E Schering Res Fdn W 56:129–153Google Scholar
  13. Knebelmann B, Ananth S, Cohen HT, Sukhatme VP (1998) Transforming growth factor α is a target for the von Hippel-Lindau tumor suppressor. Cancer Res 58:226–231Google Scholar
  14. Kubo K, Kuroyanagi Y (2005) A study of cytokines released from fibroblasts in cultured dermal substitute. Artif Organs 29:845–849CrossRefGoogle Scholar
  15. Lee KH (2000) Tissue-engineered human living skin substitutes: development and clinical application. Yonsei Med J 41:774–779CrossRefGoogle Scholar
  16. Liang C-C, Park AY, Guan J-L (2007) In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2:329–333CrossRefGoogle Scholar
  17. Libermann TA, Nusbaum HR, Razon N, Kris R, Lax I, Soreq H, Whittle N, Waterfield MD, Ullrich A, Schlessinger J (1985) Amplification, enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumours of glial origin. Nature 313:144–147CrossRefGoogle Scholar
  18. Manira M, Anuar KK, Seet WT, Irfan AWA, Ng MH, Chua KH, Heikal MYM, Aminuddin BS, Ruszymah BHI (2013) Comparison of the effects between animal-derived trypsin and recombinant trypsin on human skin cells proliferation, gene and protein expression. Cell Tissue Bank 15:41–49CrossRefGoogle Scholar
  19. Matsuguchi T, Lilly MB, Kraft AS (1998) Cytoplasmic domains of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor β chain (hβc) responsible for human GM-CSF-induced myeloid cell differentiation. J Biol Chem 273:19411–19418CrossRefGoogle Scholar
  20. Mazlyzam AL, Aminuddin BS, Fuzina NH, Norhayati MM, Fauziah O, Isa MR, Saim L, Ruszymah BHI (2007) Reconstruction of living bilayer human skin equivalent utilizing human fibrin as a scaffold. Burns 33:355–363CrossRefGoogle Scholar
  21. Metcalfe AD, Ferguson MW (2007) Tissue engineering of replacement skin: the crossroads of biomaterials, wound healing, embryonic development, stem cells and regeneration. J R Soc Interface 4:413–437CrossRefGoogle Scholar
  22. Monteiro-Riviere NA, Inman AO, Snider TH, Blank JA, Hobson DW (1997) Comparison of an in vitro skin model to normal human skin for dermatological research. Microsc Res Tech 37:172–179CrossRefGoogle Scholar
  23. Peschen M, Lahaye T, Hennig B, Weyl A, Simon J, Vanscheidt W (1999) Expression of the adhesion molecules ICAM-1, VCAM-1, LFA-1 and VLA-4 in the skin is modulated in progressing stages of chronic venous insufficiency. Acta Dermatovenerol 79:27–32Google Scholar
  24. Põld M, Zhu LX, Sharma S, Burdick MD, Lin Y, Lee PP, Põld A, Luo J, Krysan K, Dohadwala M (2004) Cyclooxygenase-2-dependent expression of angiogenic CXC chemokines ENA-78/CXC Ligand (CXCL) 5 and interleukin-8/CXCL8 in human non-small cell lung cancer. Cancer Res 64:1853–1860CrossRefGoogle Scholar
  25. Raines EW, Dower SK, Ross R (1989) Interleukin-1 mitogenic activity for fibroblasts and smooth muscle cells is due to PDGF-AA. Science 243:393–396CrossRefGoogle Scholar
  26. Raman D, Sobolik-Delmaire T, Richmond A (2011) Chemokines in health and disease. Exp Cell Res 317:575–589CrossRefGoogle Scholar
  27. Rodriguez L, Wu X, Guan J-L (2005) Wound-Healing Assay. Methods Mol Biol 294:23–29Google Scholar
  28. Ruszymah BHI, Mohd Adha PR, Low KC, Law JX, Chua KH, Mazlyzam AL, Aminuddin BS (2014) Full-thickness skin wound healing using autologous keratinocytes and dermal fibroblasts with fibrin: bilayered versus single-layered substitute. Adv Skin Wound Care 27:171–180CrossRefGoogle Scholar
  29. Seet WT, Maarof M, Anuar KK, Chua KH, Irfan AWA, Ng MH, Aminuddin BS, Ruszymah BHI (2012) Shelf-life evaluation of bilayered human skin equivalent, MyDerm™. PLoS One 7:e40978CrossRefGoogle Scholar
  30. Shakespeare PG (2005) The role of skin substitutes in the treatment of burn injuries. Clin Dermatol 23:413–418CrossRefGoogle Scholar
  31. Sheridan WP, Fox RM, Begley CG, Maher D, McGrath KM, Begley CG, Juttner CA, To LB, Szer J, Mostyn G (1992) Effect of peripheral-blood progenitor cells mobilised by filgrastim (G-CSF) on platelet recovery after high-dose chemotherapy. Lancet 339:640–644CrossRefGoogle Scholar
  32. Spiekstra SW, Breetveld M, Rustemeyer T, Scheper RJ, Gibbs S (2007) Wound-healing factors secreted by epidermal keratinocytes and dermal fibroblasts in skin substitutes. Wound Repair Regen 15:708–717CrossRefGoogle Scholar
  33. Werner S, Grose R (2003) Regulation of wound healing by growth factors and cytokines. Physiol Rev 83:835–870Google Scholar
  34. Werner S, Krieg T, Smola H (2007) Keratinocyte-fibroblast interactions in wound healing. J Invest Dermatol 127:998–1008CrossRefGoogle Scholar
  35. Wong T, McGrath J, Navsaria H (2007) The role of fibroblasts in tissue engineering and regeneration. Br J Dermatol 156:1149–1155CrossRefGoogle Scholar
  36. Xian LJ, Roy Chowdhury S, Bin Saim A, Bt Hj Idrus R (2015) Concentration-dependent effect of platelet-rich plasma on keratinocyte and fibroblast wound healing. Cytotherapy 17:293–300CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Manira Maarof
    • 1
  • Jia Xian Law
    • 1
  • Shiplu Roy Chowdhury
    • 1
  • Khairul Anuar Khairoji
    • 1
  • Aminuddin Bin Saim
    • 3
  • Ruszymah Bt. Hj Idrus
    • 1
    • 2
    Email author
  1. 1.Tissue Engineering CentreUniversiti Kebangsaan Malaysia Medical CentreKuala LumpurMalaysia
  2. 2.Department of Physiology, Faculty of MedicineUniversiti Kebangsaan MalaysiaKuala LumpurMalaysia
  3. 3.Ear Nose and Throat Consultant ClinicAmpang Puteri Specialist HospitalAmpangMalaysia

Personalised recommendations