INTRODUCTION: The formation of diabetic ulcers (DU) is a common complication for diabetic patients resulting in serious chronic wounds. There is therefore, an urgent need for complex treatment of this problem. This study examines a bioactive wound dressing of a biodegradable electrospun nanofibrous blend of poly(L-lactide-co-ε-caprolactone) and poly(ε-caprolactone) (PLCL/PCL) covered by a thin fibrin layer for sustained delivery of bioactive molecules. METHODS: Electrospun PLCL/PCL nanofibers were coated with fibrin-based coating prepared by a controlled technique and enriched with human platelet lysate (hPL), fibroblast growth factor 2 (FGF), and vascular endothelial growth factor (VEGF). The coating was characterized by scanning electron microscopy and fluorescent microscopy. Protein content and its release rate and the effect on human saphenous vein endothelial cells (HSVEC) were evaluated. RESULTS: The highest protein amount is achieved by the coating of PLCL/PCL with a fibrin mesh containing 20% v/v hPL (NF20). The fibrin coating serves as an excellent scaffold to accumulate bioactive molecules from hPL such as PDGF-BB, fibronectin (Fn), and α-2 antiplasmin. The NF20 coating shows both fast and a sustained release of the attached bioactive molecules (Fn, VEGF, FGF). The dressing significantly increases the viability of human saphenous vein endothelial cells (HSVECs) cultivated on a collagen-based wound model. The exogenous addition of FGF and VEGF during the coating procedure further increases the HSVECs viability. In addition, the presence of α-2 antiplasmin significantly stabilizes the fibrin mesh and prevents its cleavage by plasmin. DISCUSSION: The NF20 coating supplemented with FGF and VEGF provides a promising wound dressing for the complex treatment of DU. The incorporation of various bioactive molecules from hPL and growth factors has great potential to support the healing processes by providing appropriate stimuli in the chronic wound.

• Keywords
• bioactive dressing, diabetic ulcer, fibrin coating, growth factors, human platelet lysate, nanofibers,
• MeSH
• alpha-2-Antiplasmin MeSH
• Endothelial Cells MeSH
• Wound Healing MeSH
• Humans MeSH
• Nanofibers * MeSH
• Bandages MeSH
• Polyesters pharmacology   MeSH
• Vascular Endothelial Growth Factor A * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• alpha-2-Antiplasmin MeSH
• Polyesters MeSH
• Vascular Endothelial Growth Factor A * MeSH

Background: Repairs to deep skin wounds continue to be a difficult issue in clinical practice. A promising approach is to fabricate full-thickness skin substitutes with functions closely similar to those of the natural tissue. For many years, a three-dimensional (3D) collagen hydrogel has been considered to provide a physiological 3D environment for co-cultivation of skin fibroblasts and keratinocytes. This collagen hydrogel is frequently used for fabricating tissue-engineered skin analogues with fibroblasts embedded inside the hydrogel and keratinocytes cultivated on its surface. Despite its unique biological properties, the collagen hydrogel has insufficient stiffness, with a tendency to collapse under the traction forces generated by the embedded cells. Methods: The aim of our study was to develop a two-layer skin construct consisting of a collagen hydrogel reinforced by a nanofibrous poly-L-lactide (PLLA) membrane pre-seeded with fibroblasts. The attractiveness of the membrane for dermal fibroblasts was enhanced by coating it with a thin nanofibrous fibrin mesh. Results: The fibrin mesh promoted the adhesion, proliferation and migration of the fibroblasts upwards into the collagen hydrogel. Moreover, the fibroblasts spontaneously migrating into the collagen hydrogel showed a lower tendency to contract and shrink the hydrogel by their traction forces. The surface of the collagen was seeded with human dermal keratinocytes. The keratinocytes were able to form a basal layer of highly mitotically-active cells, and a suprabasal layer. Conclusion: The two-layer skin construct based on collagen hydrogel with spontaneously immigrated fibroblasts and reinforced by a fibrin-coated nanofibrous membrane seems to be promising for the construction of full-thickness skin substitute.

• Keywords
• collagen hydrogel, fibrin, fibroblast and keratinocyte co-cultivation, full-thickness skin substitutes, nanostructure,
• MeSH
• Fibrin pharmacology   MeSH
• Fibroblasts cytology   drug effects   MeSH
• Hydrogels pharmacology   MeSH
• Keratinocytes cytology   drug effects   MeSH
• Collagen pharmacology   MeSH
• Rats MeSH
• Humans MeSH
• Membranes, Artificial * MeSH
• Mitochondria drug effects   metabolism   MeSH
• Nanofibers chemistry   MeSH
• Infant, Newborn MeSH
• Cell Movement drug effects   MeSH
• Polyesters pharmacology   MeSH
• Cell Proliferation drug effects   MeSH
• Dermis cytology   MeSH
• Skin, Artificial * MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Infant, Newborn MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Fibrin MeSH
• Hydrogels MeSH
• Collagen MeSH
• Membranes, Artificial * MeSH
• poly(lactide) MeSH Browser
• Polyesters MeSH

Dermal injuries and chronic wounds usually regenerate with scar formation. Successful treatment without scarring might be achieved by pre-seeding a wound dressing with cells. We aimed to prepare a wound dressing fabricated from sodium carboxymethylcellulose (Hcel® NaT), combined with fibrin and seeded with dermal fibroblasts in vitro. We fabricated the Hcel® NaT in a porous and homogeneous form (P form and H form, respectively) differing in structural morphology and in the degree of substitution of hydroxyl groups. Each form of Hcel® NaT was functionalized with two morphologically different fibrin structures to improve cell adhesion and proliferation, estimated by an MTS assay. Fibrin functionalization of the Hcel® NaT strongly enhanced colonization of the material with human dermal fibroblasts. Moreover, the type of fibrin structures influenced the ability of the cells to adhere to the material and proliferate on it. The fibrin mesh filling the void spaces between cellulose fibers better supported cell attachment and subsequent proliferation than the fibrin coating, which only enwrapped individual cellulose fibers. On the fibrin mesh, the cell proliferation activity on day 3 was higher on the H form than on the P form of Hcel® NaT, while on the fibrin coating, the cell proliferation on day 7 was higher on the P form. The Hcel® NaT wound dressing functionalized with fibrin, especially when in the form of a mesh, can accelerate wound healing by supporting fibroblast adhesion and proliferation.

• Keywords
• dermal fibroblasts, fibrin, skin, sodium carboxymethylcellulose, wound dressing, wound healing,
• Publication type
• Journal Article MeSH

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.

• Keywords
• dermal fibroblasts, extracellular matrix synthesis, fibrin, nanocoating, nanofibers, polylactic acid, skin substitute,
• MeSH
• Cell Adhesion physiology   MeSH
• Cell Culture Techniques instrumentation   methods   MeSH
• Extracellular Matrix metabolism   MeSH
• Fibrin chemistry   pharmacology   MeSH
• Fibroblasts cytology   drug effects   MeSH
• Fibronectins metabolism   MeSH
• Collagen Type I metabolism   MeSH
• Cells, Cultured MeSH
• Skin cytology   MeSH
• Humans MeSH
• Membranes, Artificial MeSH
• Nanostructures chemistry   MeSH
• Nanotechnology methods   MeSH
• Polyesters chemistry   MeSH
• Cell Proliferation physiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Fibrin MeSH
• Fibronectins MeSH
• Collagen Type I MeSH
• Membranes, Artificial MeSH
• poly(lactide) MeSH Browser
• Polyesters MeSH

Protein-coated resorbable synthetic polymeric nanofibrous membranes are promising for the fabrication of advanced skin substitutes. We fabricated electrospun polylactic acid and poly(lactide-co-glycolic acid) nanofibrous membranes and coated them with fibrin or collagen I. Fibronectin was attached to a fibrin or collagen nanocoating, in order further to enhance the cell adhesion and spreading. Fibrin regularly formed a coating around individual nanofibers in the membranes, and also formed a thin noncontinuous nanofibrous mesh on top of the membranes. Collagen also coated most of the fibers of the membrane and randomly created a soft gel on the membrane surface. Fibronectin predominantly adsorbed onto a thin fibrin mesh or a collagen gel, and formed a thin nanofibrous structure. Fibrin nanocoating greatly improved the attachment, spreading, and proliferation of human dermal fibroblasts, whereas collagen nanocoating had a positive influence on the behavior of human HaCaT keratinocytes. In addition, fibrin stimulated the fibroblasts to synthesize fibronectin and to deposit it as an extracellular matrix. Fibrin coating also showed a tendency to improve the ultimate tensile strength of the nanofibrous membranes. Fibronectin attached to fibrin or to a collagen coating further enhanced the adhesion, spreading, and proliferation of both cell types.

• Keywords
• collagen, fibrin, nanocoating, nanofibers, skin cells, skin-tissue engineering,
• MeSH
• Cell Adhesion MeSH
• Extracellular Matrix metabolism   MeSH
• Fibrin metabolism   MeSH
• Fibroblasts cytology   metabolism   MeSH
• Fibronectins metabolism   MeSH
• Keratinocytes cytology   metabolism   MeSH
• Collagen metabolism   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Nanofibers chemistry   MeSH
• Tensile Strength MeSH
• Polymers chemistry   MeSH
• Cell Proliferation MeSH
• Tissue Engineering MeSH
• Tissue Scaffolds chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Fibrin MeSH
• Fibronectins MeSH
• Collagen MeSH
• Polymers MeSH