BACKGROUND: Our study focuses on the fabrication of appropriate scaffolds for skin wound healing. This research brings valuable insights into the molecular mechanisms of adhesion, proliferation, and control of cell behavior through the extracellular matrix represented by synthetic biodegradable nanofibrous membranes coated by biomolecules. METHODS: Nanofibrous polylactic acid (PLA) membranes were prepared by a needle-less electrospinning technology. These membranes were coated with fibrin according to two preparation protocols, and additionally they were coated with fibronectin in order to increase the cell affinity for colonizing the PLA membranes. The adhesion, growth, and extracellular matrix protein production of neonatal human dermal fibroblasts were evaluated on the nanofibrous membranes. RESULTS: Our results showed that fibrin-coated membranes improved the adhesion and proliferation of human dermal fibroblasts. The morphology of the fibrin nanocoating seems to be crucial for the adhesion of fibroblasts, and consequently for their phenotypic maturation. Fibrin either covered the individual fibers in the membrane (F1 nanocoating), or covered the individual fibers and also formed a fine homogeneous nanofibrous mesh on the surface of the membrane (F2 nanocoating), depending on the mode of fibrin preparation. The fibroblasts on the membranes with the F1 nanocoating remained in their typical spindle-like shape. However, the cells on the F2 nanocoating were spread mostly in a polygon-like shape, and their proliferation was significantly higher. Fibronectin formed an additional mesh attached to the surface of the fibrin mesh, and further enhanced the cell adhesion and growth. The relative gene expression and protein production of collagen I and fibronectin were higher on the F2 nanocoating than on the F1 nanocoating. CONCLUSION: A PLA membrane coated with a homogeneous fibrin mesh seems to be promising for the construction of temporary full-thickness skin tissue substitutes.

Department of Anatomy and Biomechanics Faculty of Physical Education and Sport Charles University Prague Czech Republic

Department of Biomaterials and Tissue Engineering Institute of Physiology of the Czech Academy of Sciences Prague Czech Republic

Zobrazit více v PubMed

Laurens N, Koolwijk P, de Maat MP. Fibrin structure and wound healing. J Thromb Haemost. 2006;4(5):932–939. PubMed

Tracy LE, Minasian RA, Caterson EJ. Extracellular matrix and dermal fibroblast function in the healing wound. Adv Wound Care (New Rochelle) 2016;5(3):119–136. PubMed PMC

Gailit J, Clarke C, Newman D, Tonnesen MG, Mosesson MW, Clark RA. Human fibroblasts bind directly to fibrinogen at RGD sites through integrin alpha(v)beta3. Exp Cell Res. 1997;232(1):118–126. PubMed

Albala DM. Fibrin sealants in clinical practice. Cardiovasc Surg. 2003;11(Suppl 1):5–11. PubMed

Gugerell A, Pasteiner W, Nurnberger S, et al. Thrombin as important factor for cutaneous wound healing: comparison of fibrin biomatrices in vitro and in a rat excisional wound healing model. Wound Repair Regen. 2014;22(6):740–748. PubMed

Tuan TL, Song A, Chang S, Younai S, Nimni ME. In vitro fibroplasia: matrix contraction, cell growth, and collagen production of fibroblasts cultured in fibrin gels. Exp Cell Res. 1996;223(1):127–134. PubMed

Mazzone L, Pontiggia L, Reichmann E, Ochsenbein-Kolble N, Moehrlen U, Meuli M. Experimental tissue engineering of fetal skin. Pediatr Surg Int. 2014;30(12):1241–1247. PubMed

Zeng RX, He JY, Zhang YL, Liu XX, Zhang Y, Tang Q. Experimental study on repairing skin defect by tissue-engineered skin substitute compositely constructed by adipose-derived stem cells and fibrin gel. Eur Rev Med Pharmacol Sci. 2017;21(3 Suppl):1–5. PubMed

Han CM, Zhang LP, Sun JZ, Shi HF, Zhou J, Gao CY. Application of collagen-chitosan/fibrin glue asymmetric scaffolds in skin tissue engineering. J Zhejiang Univ Sci B. 2010;11(7):524–530. PubMed PMC

Han HH, Jun D, Moon SH, Kang IS, Kim MC. Fixation of split-thickness skin graft using fast-clotting fibrin glue containing undiluted high-concentration thrombin or sutures: a comparison study. Springerplus. 2016;5(1):1902. PubMed PMC

Gorodetsky R, Clark RA, An J, et al. Fibrin microbeads (FMB) as biodegradable carriers for culturing cells and for accelerating wound healing. J Invest Dermatol. 1999;112(6):866–872. PubMed

Bacakova M, Musilkova J, Riedel T, et al. The potential applications of fibrin-coated electrospun polylactide nanofibers in skin tissue engineering. Int J Nanomedicine. 2016;11:771–789. PubMed PMC

Bacakova M, Pajorova J, Stranska D, et al. Protein nanocoatings on synthetic polymeric nanofibrous membranes designed as carriers for skin cells. Int J Nanomedicine. 2017;12:1143–1160. PubMed PMC

Murad S, Grove D, Lindberg KA, Reynolds G, Sivarajah A, Pinnell SR. Regulation of collagen synthesis by ascorbic acid. Proc Natl Acad Sci U S A. 1981;78(5):2879–2882. PubMed PMC

Park HJ, Ock SM, Kim HJ, et al. Vitamin C attenuates ERK signalling to inhibit the regulation of collagen production by LL-37 in human dermal fibroblasts. Exp Dermatol. 2010;19(8):E258–E264. PubMed

Bacakova L, Filova E, Parizek M, Ruml T, Svorcik V. Modulation of cell adhesion, proliferation and differentiation on materials designed for body implants. Biotechnol Adv. 2011;29(6):739–767. PubMed

Sun L, Stout DA, Webster TJ. The nano-effect: improving the long-term prognosis for musculoskeletal implants. J Long Term Eff Med Implants. 2012;22(3):195–209. PubMed

Riedel T, Brynda E, Dyr JE, Houska M. Controlled preparation of thin fibrin films immobilized at solid surfaces. J Biomed Mater Res A. 2009;88(2):437–447. PubMed

Schindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676–682. PubMed PMC

Bacakova L, Svorcik V. Cell colonization control by physical and chemical modification of materials. In: Kimura D, editor. Cell Growth Processes: New Research. New York: Nova Science Publishers, Inc; 2008. pp. 5–56.

Vig K, Chaudhari A, Tripathi S, et al. Advances in skin regeneration using tissue engineering. Int J Mol Sci. 2017;18(4):789. PubMed PMC

Engler A, Bacakova L, Newman C, Hategan A, Griffin M, Discher D. Substrate compliance versus ligand density in cell on gel responses. Biophys J. 2004;86(1 Pt 1):617–628. PubMed PMC

Carr ME, Jr, Hermans J. Size and density of fibrin fibers from turbidity. Macromolecules. 1978;11(1):46–50. PubMed

Wolberg AS. Thrombin generation and fibrin clot structure. Blood Rev. 2007;21(3):131–142. PubMed

Dyr JE, Tichý I, Jiroušková M, et al. Molecular arrangement of adsorbed fibrinogen molecules characterized by specific monoclonal antibodies and a surface plasmon resonance sensor. Sens Actuat B Chem. 1998;51(1):268–272.

Lee F, Kurisawa M. Formation and stability of interpenetrating polymer network hydrogels consisting of fibrin and hyaluronic acid for tissue engineering. Acta Biomater. 2013;9(2):5143–5152. PubMed

Bidault L, Deneufchatel M, Vancaeyzeele C, Fichet O, Larreta-Garde V. Self-supported fibrin-polyvinyl alcohol interpenetrating polymer networks: an easily handled and rehydratable biomaterial. Biomacromolecules. 2013;14(11):3870–3879. PubMed

Law JX, Musa F, Ruszymah BH, El Haj AJ, Yang Y. A comparative study of skin cell activities in collagen and fibrin constructs. Med Eng Phys. 2016;38(9):854–861. PubMed

Fernandez-Madrid F, Noonan S, Riddle J. The “spindle-shaped” body in fibroblasts: intracellular collagen fibrils. J Anat. 1981;132(Pt 2):157–166. PubMed PMC

Duval K, Grover H, Han LH, et al. Modeling physiological events in 2D vs. 3D cell culture. Physiology (Bethesda) 2017;32(4):266–277. PubMed PMC

Parizek M, Douglas TE, Novotna K, et al. Nanofibrous poly(lactide-co-glycolide) membranes loaded with diamond nanoparticles as promising substrates for bone tissue engineering. Int J Nanomedicine. 2012;7:1931–1951. PubMed PMC

Novotna K, Zajdlova M, Suchy T, et al. Polylactide nanofibers with hydroxyapatite as growth substrates for osteoblast-like cells. J Biomed Mater Res A. 2014;102(11):3918–3930. PubMed

Mazlyzam AL, Aminuddin BS, Fuzina NH, et al. Reconstruction of living bilayer human skin equivalent utilizing human fibrin as a scaffold. Burns. 2007;33(3):355–363. PubMed

Ruoslahti E, Pierschbacher MD. New perspectives in cell adhesion: RGD and integrins. Science. 1987;238(4826):491–497. PubMed

Sottile J, Hocking DC. Fibronectin polymerization regulates the composition and stability of extracellular matrix fibrils and cell-matrix adhesions. Mol Biol Cell. 2002;13(10):3546–3559. PubMed PMC

Sethi KK, Yannas IV, Mudera V, Eastwood M, McFarland C, Brown RA. Evidence for sequential utilization of fibronectin, vitronectin, and collagen during fibroblast-mediated collagen contraction. Wound Repair Regen. 2002;10(6):397–408. PubMed

The Effect of a Polyester Nanofibrous Membrane with a Fibrin-Platelet Lysate Coating on Keratinocytes and Endothelial Cells in a Co-Culture System

A two-layer skin construct consisting of a collagen hydrogel reinforced by a fibrin-coated polylactide nanofibrous membrane

Fibrin-Modified Cellulose as a Promising Dressing for Accelerated Wound Healing