Cellulose Mesh with Charged Nanocellulose Coatings as a Promising Carrier of Skin and Stem Cells for Regenerative Applications.

Engineering artificial skin constructs is an ongoing challenge. An ideal material for hosting skin cells is still to be discovered. A promising candidate is low-cost cellulose, which is commonly fabricated in the form of a mesh and is applied as a wound dressing. Unfortunately, the structure and the topography of current cellulose meshes are not optimal for cell growth. To enhance the surface structure and the physicochemical properties of a commercially available mesh, we coated the mesh with wood-derived cellulose nanofibrils (CNFs). Three different types of mesh coatings are proposed in this study as a skin cell carrier: positively charged cationic cellulose nanofibrils (cCNFs), negatively charged anionic cellulose nanofibrils (aCNFs), and a combination of these two materials (c+aCNFs). These cell carriers were seeded with normal human dermal fibroblasts (NHDFs) or with human adipose-derived stem cells (ADSCs) to investigate cell adhesion, spreading, morphology, and proliferation. The negatively charged aCNF coating significantly improved the proliferation of both cell types. The positively charged cCNF coating significantly enhanced the adhesion of ADSCs only. The number of NHDFs was similar on the cCNF coatings and on the noncoated pristine cellulose mesh. However, the three-dimensional (3D) structure of the cCNF coating promoted cell survival. The c+aCNF construct proved to combine benefits from both types of CNFs, which means that the c+aCNF cell carrier is a promising candidate for further application in skin tissue engineering.

Effects of Bacterial Nanocellulose Loaded with Curcumin and Its Degradation Products on Human Dermal Fibroblasts.

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.

The Influence of Negative Pressure and of the Harvesting Site on the Characteristics of Human Adipose Tissue-Derived Stromal Cells from Lipoaspirates.

BACKGROUND: Adipose tissue-derived stromal cells (ADSCs) have great potential for cell-based therapies, including tissue engineering. However, various factors can influence the characteristics of isolated ADSCs. METHODS: We studied the influence of the harvesting site, i.e., inner thigh (n = 3), outer thigh (n = 3), outer thigh (n = 3), outer thigh (. RESULTS: We revealed higher initial cell yields from the outer thigh region than from the abdomen region. Negative pressure did not influence the cell yields from the outer thigh region, whereas the yields from the abdomen region were higher under high negative pressure than under low negative pressure. In the subsequent passage, in general, no significant relationship was identified between the different negative pressure and ADSC characteristics. No significant difference was observed in the characteristics of thigh ADSCs and abdomen ADSCs. Only on day 1, the diameter was significantly bigger in outer thigh ADSCs than in abdomen ADSCs. Moreover, we noted a tendency of thigh ADSCs (i.e., inner thigh+outer thigh) to reach a higher cell number on day 7. Discussion. The harvesting site and negative pressure can potentially influence initial cell yields from lipoaspirates. However, for subsequent in vitro culturing and for use in tissue engineering, it seems that the harvesting site and the level of negative pressure do not have a crucial or limiting effect on basic ADSC characteristics.in vitro culturing and for use in tissue engineering, it seems that the harvesting site and the level of negative pressure do not have a crucial or limiting effect on basic ADSC characteristics.

Applications of Nanocellulose/Nanocarbon Composites: Focus on Biotechnology and Medicine.

Nanocellulose/nanocarbon composites are newly emerging smart hybrid materials containing cellulose nanoparticles, such as nanofibrils and nanocrystals, and carbon nanoparticles, such as "classical" carbon allotropes (fullerenes, graphene, nanotubes and nanodiamonds), or other carbon nanostructures (carbon nanofibers, carbon quantum dots, activated carbon and carbon black). The nanocellulose component acts as a dispersing agent and homogeneously distributes the carbon nanoparticles in an aqueous environment. Nanocellulose/nanocarbon composites can be prepared with many advantageous properties, such as high mechanical strength, flexibility, stretchability, tunable thermal and electrical conductivity, tunable optical transparency, photodynamic and photothermal activity, nanoporous character and high adsorption capacity. They are therefore promising for a wide range of industrial applications, such as energy generation, storage and conversion, water purification, food packaging, construction of fire retardants and shape memory devices. They also hold great promise for biomedical applications, such as radical scavenging, photodynamic and photothermal therapy of tumors and microbial infections, drug delivery, biosensorics, isolation of various biomolecules, electrical stimulation of damaged tissues (e.g., cardiac, neural), neural and bone tissue engineering, engineering of blood vessels and advanced wound dressing, e.g., with antimicrobial and antitumor activity. However, the potential cytotoxicity and immunogenicity of the composites and their components must also be taken into account.

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

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.

Versatile Application of Nanocellulose: From Industry to Skin Tissue Engineering and Wound Healing.

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

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.

Morphology of a fibrin nanocoating influences dermal fibroblast behavior.

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.

Stem cells: their source, potency and use in regenerative therapies with focus on adipose-derived stem cells - a review.

Stem cells can be defined as units of biological organization that are responsible for the development and the regeneration of organ and tissue systems. They are able to renew their populations and to differentiate into multiple cell lineages. Therefore, these cells have great potential in advanced tissue engineering and cell therapies. When seeded on synthetic or nature-derived scaffolds in vitro, stem cells can be differentiated towards the desired phenotype by an appropriate composition, by an appropriate architecture, and by appropriate physicochemical and mechanical properties of the scaffolds, particularly if the scaffold properties are combined with a suitable composition of cell culture media, and with suitable mechanical, electrical or magnetic stimulation. For cell therapy, stem cells can be injected directly into damaged tissues and organs in vivo. Since the regenerative effect of stem cells is based mainly on the autocrine production of growth factors, immunomodulators and other bioactive molecules stored in extracellular vesicles, these structures can be isolated and used instead of cells for a novel therapeutic approach called "stem cell-based cell-free therapy". There are four main sources of stem cells, i.e. embryonic tissues, fetal tissues, adult tissues and differentiated somatic cells after they have been genetically reprogrammed, which are referred to as induced pluripotent stem cells (iPSCs). Although adult stem cells have lower potency than the other three stem cell types, i.e. they are capable of differentiating into only a limited quantity of specific cell types, these cells are able to overcome the ethical and legal issues accompanying the application of embryonic and fetal stem cells and the mutational effects associated with iPSCs. Moreover, adult stem cells can be used in autogenous form. These cells are present in practically all tissues in the organism. However, adipose tissue seems to be the most advantageous tissue from which to isolate them, because of its abundancy, its subcutaneous location, and the need for less invasive techniques. Adipose tissue-derived stem cells (ASCs) are therefore considered highly promising in present-day regenerative medicine.

Protein nanocoatings on synthetic polymeric nanofibrous membranes designed as carriers for skin cells.

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.

Evaluation of the potential of chitosan/ß-1,3-glucan/hydroxyapatite material as a scaffold for living bone graft production in vitro by comparison of ADSC and BMDSC behaviour on its surface.

The opinion regarding the origin of adult stem cells that should be used for living bone construct generation is strongly divided in the scientific community. In this study, the potential of chitosan/ß-1,3-glucan/hydroxyapatite (chit/glu/HA) material as a scaffold for bone regeneration applications was evaluated by behaviour comparison of adult stem cells derived from both origins-adipose derived mesenchymal stem cell (ADSC) tissue and bone marrow derived mesenchymal stem cells (BMDSCs). In the case of ADSC isolation, low and high negative pressures were applied during a liposuction procedure in order to determine if negative pressure settings may have an impact on subsequent cell behaviour in vitro. The obtained results demonstrated that the chit/glu/HA material is a promising candidate to be used for living bone graft production in vitro as both ADSCs and BMDSCs revealed a satisfactory proliferation and differentiation ability on its surface. Nevertheless, BMDSCs would be a better choice of adult stem cells since they were better spread, more strongly attached and showed a more superior proliferation and differentiation ability than ADSCs when cultured on the chit/glu/HA scaffold. However, if BMDSCs cannot be isolated, ADSCs may be used for bone construct production but lipoaspirate should be collected under low negative pressure (-200 mm Hg), as high negative pressure (-700 mmHg) applied during liposuction surgery may retard subsequent ADSC proliferation and type I collagen production.