Nanocellulose is cellulose in the form of nanostructures, i.e., features not exceeding 100 nm at least in one dimension. These nanostructures include nanofibrils, found in bacterial cellulose; nanofibers, present particularly in electrospun matrices; and nanowhiskers, nanocrystals, nanorods, and nanoballs. These structures can be further assembled into bigger two-dimensional (2D) and three-dimensional (3D) nano-, micro-, and macro-structures, such as nanoplatelets, membranes, films, microparticles, and porous macroscopic matrices. There are four main sources of nanocellulose: bacteria (Gluconacetobacter), plants (trees, shrubs, herbs), algae (Cladophora), and animals (Tunicata). Nanocellulose has emerged for a wide range of industrial, technology, and biomedical applications, namely for adsorption, ultrafiltration, packaging, conservation of historical artifacts, thermal insulation and fire retardation, energy extraction and storage, acoustics, sensorics, controlled drug delivery, and particularly for tissue engineering. Nanocellulose is promising for use in scaffolds for engineering of blood vessels, neural tissue, bone, cartilage, liver, adipose tissue, urethra and dura mater, for repairing connective tissue and congenital heart defects, and for constructing contact lenses and protective barriers. This review is focused on applications of nanocellulose in skin tissue engineering and wound healing as a scaffold for cell growth, for delivering cells into wounds, and as a material for advanced wound dressings coupled with drug delivery, transparency and sensorics. Potential cytotoxicity and immunogenicity of nanocellulose are also discussed.

Fibrin plays an important role during wound healing and skin regeneration. It is often applied in clinical practice for treatment of skin injuries or as a component of skin substitutes. We prepared electrospun nanofibrous membranes made from poly(L-lactide) modified with a thin fibrin nanocoating. Fibrin surrounded the individual fibers in the membrane and also formed a thin fibrous mesh on several places on the membrane surface. The cell-free fibrin nanocoating remained stable in the cell culture medium for 14 days and did not change its morphology. On membranes populated with human dermal fibroblasts, the rate of fibrin degradation correlated with the degree of cell proliferation. The cell spreading, mitochondrial activity, and cell population density were significantly higher on membranes coated with fibrin than on nonmodified membranes, and this cell performance was further improved by the addition of ascorbic acid in the cell culture medium. Similarly, fibrin stimulated the expression and synthesis of collagen I in human dermal fibroblasts, and this effect was further enhanced by ascorbic acid. The expression of beta₁-integrins was also improved by fibrin, and on pure polylactide membranes, it was slightly enhanced by ascorbic acid. In addition, ascorbic acid promoted deposition of collagen I in the form of a fibrous extracellular matrix. Thus, the combination of nanofibrous membranes with a fibrin nanocoating and ascorbic acid seems to be particularly advantageous for skin tissue engineering.

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.

Bacterial nanocellulose has found applications in tissue engineering, in skin tissue repair, and in wound healing. Its large surface area enables the adsorption of various substances. Bacterial nanocellulose with adsorbed substances can serve as a substrate for drug-delivery of specific bioactive healing agents into wounds. In this study, we loaded a bacterial nanocellulose hydrogel with curcumin, i.e., an important anti-bacterial and healing agent, and its degradation products. These products were prepared by thermal decomposition of curcumin (DC) at a temperature of 180 °C (DC 180) or of 300 °C (DC 300). The main thermal decomposition products were tumerone, vanillin, and feruloylmethane. Curcumin and its degradation products were loaded into the bacterial nanocellulose by an autoclaving process. The increased temperature during autoclaving enhanced the solubility and the penetration of the agents into the nanocellulose. The aim of this study was to investigate the cytotoxicity and the antimicrobial activity of pure curcumin, its degradation products, and finally of bacterial nanocellulose loaded with these agents. In vitro tests performed on human dermal fibroblasts revealed that the degradation products of curcumin, i.e., DC 180 and DC 300, were more cytotoxic than pure curcumin. However, if DC 300 was loaded into nanocellulose, the cytotoxic effect was not as strong as in the case of DC 300 powder added into the culture medium. DC 300 was found to be the least soluble product in water, which probably resulted in the poor loading of this agent into the nanocellulose. Nanocellulose loaded with pure curcumin or DC 180 exhibited more antibacterial activity than pristine nanocellulose.

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.

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.