The 3D bioprinting of cell-incorporated gels is a promising direction in tissue engineering applications. Collagen-based hydrogels, due to their similarity to extracellular matrix tissue, can be a good candidate for bioink and 3D bioprinting applications. However, low hydrogel concentrations of hydrogel (<10 mg/mL) provide insufficient structural support and, in highly concentrated gels, cell proliferation is reduced. In this study, we showed that it is possible to print highly concentrated collagen hydrogels with incorporated cells, where the viability of the cells in the gel remains very good. This can be achieved simply by optimizing the properties of the bioink, particularly the gel composition and pH modification, as well as by optimizing the printing parameters. The bioink composed of porcine collagen hydrogel with a collagen concentration of 20 mg/mL was tested, while the final bioink collagen concentration was 10 mg/mL. This bioink was modified with 0, 5, 9, 13, 17 and 20 μL/mL of 1M NaOH solution, which affected the resulting pH and gelling time. Cylindrical samples based on the given bioink, with the incorporation of porcine adipose-derived stromal cells, were printed with a custom 3D bioprinter. These constructs were cultivated in static conditions for 6 h, and 3 and 5 days. Cell viability and morphology were evaluated. Mechanical properties were evaluated by means of a compression test. Our results showed that optimal composition and the addition of 13 μL NaOH per mL of bioink adjusted the pH of the bioink enough to allow cells to grow and divide. This modification also contributed to a higher elastic modulus, making it possible to print structures up to several millimeters with sufficient mechanical resistance. We optimized the bioprinter parameters for printing low-viscosity bioinks. With this experiment, we showed that a high concentration of collagen gels may not be a limiting factor for cell proliferation.

• Klíčová slova
• biofabrication, bioink, bioprinting, collagen hydrogels, compressive elastic modulus, stromal cells,
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• collagen hydrogel, fibrin, fibroblast and keratinocyte co-cultivation, full-thickness skin substitutes, nanostructure,
• MeSH
• fibrin farmakologie   MeSH
• fibroblasty cytologie   účinky léků   MeSH
• hydrogely farmakologie   MeSH
• keratinocyty cytologie   účinky léků   MeSH
• kolagen farmakologie   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• membrány umělé * MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• nanovlákna chemie   MeSH
• novorozenec MeSH
• pohyb buněk účinky léků   MeSH
• polyestery farmakologie   MeSH
• proliferace buněk účinky léků   MeSH
• škára cytologie   MeSH
• umělá kůže * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• novorozenec MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fibrin MeSH
• hydrogely MeSH
• kolagen MeSH
• membrány umělé * MeSH
• poly(lactide) MeSH Prohlížeč
• polyestery MeSH