Evaluation of bacterial nanocellulose-based uniform wound dressing for large area skin transplantation

Bacterial nanocellulose (BNC) was biosynthesized by Gluconacetobacter xylinus. The surface area, physicochemical structure and morphology of the materials were characterized. Here provides a method for an efficient production of uniform BNC, which is beneficial for the fast characterization and evaluation of BNC. In vitro cytotoxicity of the materials was evaluated by the proliferation, the adhesion, the viability and the morphology of NIH/3T3 cells. Low cytotoxicity of the BNC was observed, and micrographs demonstrate a good proliferation and adhesion of NIH/3T3 cells on BNC. Large area full-thickness skin defects were made on the back of C57BL/6 mice in animal surgery. The wounds were transplanted with BNC films and the results compared to those in a control group. The rehabilitation of the wound surfaces and the pathological sections of mice were investigated and are discussed. Histological examinations demonstrated faster and better healing effect and lower inflammatory response in the BNC group than those in the control group. Preliminary results on wound dressings from BNC show a curative effect promoting the healing of epithelial tissue. BNC is a promising natural polymer with medical applications in wound dressings.     

Isolation and characterisation of locally isolated Gluconacetobacter xylinus BCZM sp. with nanocellulose producing potentials

Recently, attention has been given to nanocellulose produced by bacteria due to its unique properties and environmentally friendly nature when compared with plant cellulose. Bacterial nanocellulose (BNC) producing isolate was successfully isolated from rotten fruits via dilution and spread plates method. Based on the biochemical characterisation and molecular analysis of the 16S rDNA gene, the isolate was identified as Gluconacetobacter xylinus BCMZ sp. Nanocellulose productivity was confirmed by the formation of the white gelatinous layer between air/liquid surfaces when the culture was cultivated under a stationary condition at 30°C. Successful purification of nanocellulose was achieved using alkaline treatment method. The Fourier transformed infrared spectrum showed a characteristics band signature of pure nanocellulose, by displaying strong absorption peaks at 3335.36 and 2901.40 cm⁻¹ representing carbonyl and carbon–hydrogen bonding, respectively. Morphological characteristics of the BNC were determined by scanning electron microscopy (SEM). Elemental analysis of BNC was determined by energy dispersive X‐ray (SEM/EDX) analysis. The isolates BCZM showed significant nanocellulose production ability with a high degree of purity when compared with plant nanocellulose. BNC purification using 1 M NaOH solution is effective and eco‐friendly with no indication of recalcitrant formation as commonly found in plant nanocellulose purification steps.Inspec keywords: microorganisms, purification, scanning electron microscopy, X‐ray chemical analysis, Fourier transform infrared spectroscopy, biotechnologyOther keywords: locally isolated Gluconacetobacter xylinus BCZM sp, nanocellulose producing potentials, bacterial nanocellulose producing isolate, BNC producing isolate, rotten fruits, dilution, spread plates method, biochemical characterisation, molecular analysis, white gelatinous layer, air/liquid surfaces, nanocellulose purification, alkaline treatment method, Fourier transformed infrared spectrum, characteristics band signature, scanning electron microscopy, elemental analysis, energy dispersive X‐ray analysis, SEM analysis, EDX analysis, plant nanocellulose, BNC purification, recalcitrant formation     

In vitro real-time characterization of cell attachment and spreading

A method based on the piezoelectric quartz crystal microbalance (QCM) technique for in vitro real-time characterization of cell attachment and spreading on surfaces has been developed. The method simultaneously measures the resonant frequency, f, and the dissipation energy, D, of the oscillating system. The QCM responses are sensitive to very small amounts (a few hundreds) of cells and highly specific to surface chemical properties. The first results from deposition of cells on two polystyrene surfaces of different wettability in serum-containing medium are reported. It has previously been shown that a decrease in f is related to the degree of cell spreading. In our data it appears that the extent or quality of cell attachment is reflected in an increase in D caused by adhering cells. The combined information from f and D measured by this technique might therefore be useful to probe cell–surface interactions for biomaterials. © 1998 Kluwer Academic Publishers     

Fibrin-mediated endothelial cell adhesion to vascular biomaterials resists shear stress due to flow

In vitro endothelial cell (EC) seeding onto biomaterials for blood-contacting applications can improve the blood compatibility of materials. Adhesive proteins adsorbed from serum that is supplemented with the culture medium intercede the initial cell adhesion and subsequent spreading on material surface during culture. Nevertheless, physical and chemical properties of vascular biomaterial surface fluctuate widely between materials resulting in dissimilarity in protein adsorption characteristics. Thus, a variation is expected in cell adhesion, growth and the ability of cell to resist shear stress when tissue engineering on to vascular biomaterials is attempted. This study was carried out with an objective to determine the significance of a matrix coating on cell adhesion and shear stress resistance when cells are cultured on materials such as polytetrafluoroethylene (PTFE, Teflon) and polyethyleneterephthalate (Dacron), ultra high molecular weight polyethylene (UHMWPE) and titanium (Ti), that are used for prosthetic devices. The study illustrates the distinction of EC attachment and proliferation between uncoated and matrix-coated surfaces. The cell attachment and proliferation on uncoated UHMWPE and titanium surfaces were not significantly different from matrix-coated surfaces. However, shear stress resistance of the cells grown on composite coated surfaces appeared superior compared to the cells grown on uncoated surface. On uncoated vascular graft materials, the cell adhesion was not supported by serum alone and proliferation was scanty as compared to matrix-coated surface. Therefore, coating of implant devices with a composite of adhesive proteins and growth factors can improve EC attachment and resistance of the cells to the forces of flow.     

Ion-implanted polytetrafluoroethylene enhances Saccharomyces cerevisiae biofilm formation for improved immobilization

The surface of polytetrafluoroethylene (PTFE) was modified using plasma immersion ion implantation (PIII) with the aim of improving its ability to immobilize yeast. The density of immobilized cells on PIII-treated and -untreated PTFE was compared as a function of incubation time over 24 h. Rehydrated yeast cells attached to the PIII-treated PTFE surface more rapidly, with higher density, and greater attachment strength than on the untreated surface. The immobilized yeast cells were removed mechanically or chemically with sodium hydroxide and the residues left on the surfaces were analysed with Fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR) and X-ray photoelectron spectroscopy (XPS). The results revealed that the mechanism of cell attachment on both surfaces differs and a model is presented for each. Rapid attachment on the PIII-treated surface occurs through covalent bonds of cell wall proteins and the radicals on the treated surface. In contrast, on the untreated surface, only physisorbed molecules were found in the residue and lipids were more highly concentrated than proteins. The presence of lipids in the residue was found to be a consequence of damage to the plasma membrane during the rehydration process and the increased cell stress was also apparent by the amount of Hsp12 in the protein residue. The immobilized yeast cells on PIII-treated PTFE were found to be as active as yeast cells in suspension.     

Catalytically Active Bacterial Nanocellulose‐Based Ultrafiltration Membrane

Large quantities of highly toxic organic dyes in industrial wastewater is a persistent challenge in wastewater treatment processes. Here, for highly efficient wastewater treatment, a novel membrane based on bacterial nanocellulose (BNC) loaded with graphene oxide (GO) and palladium (Pd) nanoparticles is demonstrated. This Pd/GO/BNC membrane is realized through the in situ incorporation of GO flakes into BNC matrix during its growth followed by the in situ formation of palladium nanoparticles. The Pd/GO/BNC membrane exhibits highly efficient methylene orange (MO) degradation during filtration (up to 99.3% over a wide range of MO concentrations, pH, and multiple cycles of reuse). Multiple contaminants (a cocktail of 4‐nitrophenol, methylene blue, and rhodamine 6G) can also be effectively treated by Pd/GO/BNC membrane simultaneously during filtration. Furthermore, the Pd/GO/BNC membrane demonstrates stable flux (33.1 L m^?2 h^?1) under 58 psi over long duration. The novel and robust membrane demonstrated here is highly scalable and holds a great promise for wastewater treatment.     

纳米纤维素在功能纳米材料中的应用进展

纳米纤维素分为纤维素纳米纤维(CNF)、纤维素纳米晶体(CNC)、细菌纳米纤维素(BNC)。CNF主要由机械法和2,2,6,6-四甲基哌啶-1-氧基(TEMPO)介导氧化法制备,呈微纤丝状。CNC主要由酸水解法制备,呈棒状或针状颗粒。BNC由细菌合成,呈纳米纤维网络状。文中综述了纳米纤维素在凝胶、仿生复合材料、导电材料、电极材料、导热材料、电磁屏蔽材料、压电材料及传感器材料领域的应用现状,并对其功能纳米材料未来发展的方向进行了展望。     

Interactions of Fibroblasts with Different Morphologies Made of an Engineered Spider Silk Protein

Spider silk has been investigated for decades due to the intriguing mechanical and also biomedical properties of the silk fibers. Previously, it has been shown that recombinant silk proteins can also be processed into other morphologies. Here, we characterized scaffolds made of the recombinant spider silk protein eADF4(C16) concerning their surface interactions with fibroblasts. Studies of BALB/3T3 cells on hydrogels and films made of eADF4(C16) showed low cell adhesion without observable duplication. Electro‐spun non‐woven scaffolds made of eADF4(C16), however, enabled both their adhesion and proliferation. Since eADF4(C16) lacks specific motifs for cell attachment, fibroblasts cannot generate focal adhesions with the material's surface, and, therefore, other cell–interface interactions such as topographical anchorage or cell attachment mediated by adhesion of extracellular matrix proteins are discussed in this paper. On non‐woven meshes protrusion of filopodia and/or lamellipodia between individual fibers increase the surface contact area, which depends on the diameter of the fibers of the non‐woven meshes. In contrast, at flat (film) or microstructured surfaces (hydrogels) such interactions seem to be precluded.     

Porous nanocellulose gels and foams: Breakthrough status in the development of scaffolds for tissue engineering

We report on the latest scientific advances related to the use of porous foams and gels prepared with cellulose nanofibrils (CNF) and nanocrystals (CNC) as well as bacterial nanocellulose (BNC) – collectively nanocelluloses – as biomedical materials for application in tissue regeneration. Interest in such applications stems from the lightweight and strong structures that can be efficiently produced from these nanocelluloses. Dried nanocellulose foams and gels, including xerogels, cryogels, and aerogels have been synthesized effortlessly using green, scalable, and cost-effective techniques. Methods to control structural features (e.g., porosity, morphology, and mechanical performance) and biological interactions (e.g., biocompatibility and biodegradability) are discussed in light of specific tissues of interest. The state-of-the-art in the field of nanocellulose-based scaffolds for tissue engineering is presented, covering physicochemical and biological properties relevant to these porous systems that promise groundbreaking advances. Specifically, these materials show excellent performance for in vitro cell culturing and in vivo implantation. We report on recent efforts related to BNC scaffolds used in animal and human implants, which furthermore support the viability of CNF- and CNC-based scaffolds in next-generation biomedical materials.     

Cytocompatible and multifunctional polymeric nanoparticles for transportation of bioactive molecules into and within cells

Multifunctional polymeric nanoparticles are materials with great potential for a wide range of biomedical applications. For progression in this area of research, unfavorable interactions of these nanoparticles with proteins and cells must be avoided in biological environments, for example, through treatment of the nanoparticle surfaces. Construction of an artificial cell membrane structure based on polymers bearing the zwitterionic phosphorylcholine group can prevent biological reactions at the surface effectively. In addition, certain bioactive molecules can be immobilized on the surface of the polymer to generate enough affinity to capture target biomolecules. Furthermore, entrapment of inorganic nanoparticles inside polymeric matrices enhances the nanoparticle functionality significantly. This review summarizes the preparation and characterization of cytocompatible and multifunctional polymeric nanoparticles; it analyzes the efficiency of their fluorescence function, the nature of the artificial cell membrane structure, and their performance as in-cell devices; and finally, it evaluates both their chemical reactivity and effects in cells.     

Cell/surface interactions on laser micro-textured titanium-coated silicon surfaces

This paper examines the effects of nano-scale titanium coatings, and micro-groove/micro-grid patterns on cell/surface interactions on silicon surfaces. The nature of the cellular attachment and adhesion to the coated/uncoated micro-textured surfaces was elucidated by the visualization of the cells and relevant cytoskeletal & focal adhesion proteins through scanning electron microscopy and immunofluorescence staining. Increased cell spreading and proliferation rates are observed on surfaces with 50 nm thick Ti coatings. The micro-groove geometries have been shown to promote contact guidance, which leads to reduced scar tissue formation. In contrast, smooth surfaces result in random cell orientations and the increased possibility of scar tissue formation. Immunofluorescence cell staining experiments also reveal that the actin stress fibers are aligned along the groove dimensions, with discrete focal adhesions occurring along the ridges, within the grooves and at the ends of the cell extensions. The implications of the observed cell/surface interactions are discussed for possible applications of silicon in implantable biomedical systems.     

Dextran‐based coating system for the immobilization of cell adhesion promoting molecules on titanium surfaces

Immobilization of adhesive peptides interacting with cellular integrin receptors onto metallic implant surfaces represents a promising approach to improve osseointegration of implants into the surrounding tissue. In the present study, a functional dextran‐based coating system consisting of an amino titanate adhesion promoter with dendritic structure and a carboxymethyl dextran was established to bind an RGD‐containing adhesive peptide via a selective coupling methodology onto titanium surfaces. The three‐step reaction procedure was characterized by X‐ray photoelectron spectroscopy. In cell adhesion experiments it could be demonstrated that dextran coatings containing immobilized RGD promote attachment and spreading of fibroblast and pre‐osteoblastic cells compared to native as well as CMD‐coated titanium surfaces without RGD. The direct attachment of the RGD sequence to the metal surface via the amino titanate adhesion promoter did not increase pre‐osteoblastic cell spreading, whereas coupling of RGD to the polymeric carboxy­methyl dextran layer slightly enhanced spreading of the cells.     

Biocompatibility assessment of next generation materials for brain implantable microelectrodes

Proper interfacing between the microelectrode device and the cells is very important for functional stimulation of brain; the microelectrodes need to be in close proximity to the cells. The materials currently being used in device or research (silicon, polymer, platinum) do not support neuronal attachment on their surfaces. Here, we investigated primary neuronal cell attachment on Pyrex glass, sapphire, and iridium oxide as promising alternative materials. Sapphire was found to be an excellent substrate to support neuron attachment in serum free condition. Using micro-patterning, sapphire was shown to have great potential as a substrate for next generation neural implants.     

Human fibroblast attachment on fibrous parylene-C thin-film substrates

Although the polymeric form of parylene-C is used in many medical devices, the mechanistic nature of cellular attachment to polymeric parylene-C is not clear. We examined the effects of (i) substrate morphology, (ii) surface wettability and (iii) presence of serum proteins on fibroblast attachment. A physicochemical vapor deposition technique was implemented to deposit flat parylene-C substrates as well as fibrous substrates of three different morphologies: slanted columnar, chevronic and chiral. Flat parylene-C surfaces were moderately hydrophobic while fibrous substrates were superhydrophobic. Pretreatment with oxygen plasma changed the substrate surfaces from hydrophobic to superhydrophilic. The attachment efficiency of human fibroblast cells to the flat and three fibrous thin-film parylene-C substrates was investigated. Fibroblast attachment was better on fibrous substrates than on flat substrates, and oxygen plasma pretreatment facilitated fibroblast attachment on all four morphologies. Serum proteins also facilitated cell attachment on all substrates. The combination of oxygen plasma pre-treatment and serum proteins increased fibroblast adhesion in an additive manner on flat, but not on fibrous parylene-C substrates. The morphology of cell–substrate interactions differed between fibrous and flat parylene-C substrates.     

Comparison of selective attachment and growth of smooth muscle cells on gelatin- and fibronectin-coated micropatterns

Tissue engineering research has been on going for many years, people are making all the effort to explore the cell functions in cellular level and even in molecular level. Making the cells functional in an in vitro environment is a preliminary goal for the implantation and repair of complicated tissues/organs. Fabricating artificial ECM to mimic the in vivo environment is an essential approach in tissue engineering. The work in this paper is to study how rat aorta smooth muscle cells (RASMCs) behave in two engineered cell culture scaffolds: gelatin- and fibronectin (FN)-coated micropatterns. The investigation on the initial attachment and further growth of SMCs cultured on gelatin- and FN-coated micropatterns was addressed. This study focused on both the characterization of gelatin and fibronectin assembly properties and cell responses to these two protein-coated micropatterns. Thin film patterns with gelatin and fibronectin coatings were fabricated on microscope glass slides using photolithography, electrostatic layer-by-layer self-assembly and lift-off (LbL-LO) technologies. In this work, the scaffolds were built up by commonly used polyelectrolyte materials and proteins through LbL process, containing cationic poly(diallyldimethylammonium chloride) (PDDA), poly(allylamine hydrochloride) (PAH), anionic poly(sodium 4-styrenesulfonate) (PSS), gelatin and fibronectin. The resulting polyelectrolyte thin films were characterized by contact angle (CA), quartz crystal microbalance (QCM), atomic force microscopy (AFM), and fluorescence microscopy. CA measurement shows the consistent hydrophylicity of gelatin surfaces in different number of layers with LbL deposition method. Different from our previous QCM measurement of gelatin, fibronectin does not show high electrostatic attraction to either positively or negatively charged polyelectrolytes, although it can be weakly assembled to both polyelectrolyte surfaces. AFM images show Gelatin- and FN-coated micropatterns are around 50-60 nm thick. RASMCs were cultured on these gelatin- and FN-coated micropatterns. It was observed that, for the cells cultured on gelatin-coated micropatterns, they initially landed on the gelatin-coated surface, not on the PDDA-coated surface in between. But further growth of the cells was affected by the shape of the patterns: strip pattern limited cell growth beyond the patterns, but square patterns could not. While, it was found interestingly, for the cells cultured on FN-coated micropatterns, SMCs initially landed on PDDA-coated surface, and then migrated to FN-coated both square and strip patterns. These findings indicate that both gelatin and fibronectin are adhesive proteins, but they have different effects on the initial attachment and later growth for SMCs.     

Surface modification of poly(dimethylsiloxane) for controlling biological cells’ adhesion using a scanning radical microjet

A scanning radical microjet (SRMJ) equipment using oxygen microplasma has been developed and successfully applied for controlling biological cells’ attachment on biocompatible polymer material, poly(dimethylsiloxane) (PDMS). The radical microjet has advantages in localized and high-rate surface treatment. Moreover, maskless hydrophilic patterning using SRMJ has been demonstrated to be applicable to patterned cell cultivation which is useful in emerging biotechnological field such as tissue engineering and cell-based biosensors. Since control of PDMS surface properties is an indispensable prerequisite for cells’ attachment, effects of oxygen flow rates and treatment time on localized hydrophilic patterning of PDMS surfaces were first investigated for controlling HeLa cells’ (human epitheloid carcinoma cell line) attachment. Relationships between surface conditions of treated PDMS films and attached cell density are also discussed based on surface properties analyzed using atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS).     

Plasma treated polyethylene grafted with adhesive molecules for enhanced adhesion and growth of fibroblasts

The cell–material interface plays a crucial role in the interaction of cells with synthetic materials for biomedical use. The application of plasma for tailoring polymer surfaces is of abiding interest and holds a great promise in biomedicine. In this paper, we describe polyethylene (PE) surface tuning by Ar plasma irradiating and subsequent grafting of the chemically active PE surface with adhesive proteins or motives to support cell attachment. These simple modifications resulted in changed polymer surface hydrophilicity, roughness and morphology, which we thoroughly characterized. The effect of our modifications on adhesion and growth was tested in vitro using mouse embryonic fibroblasts (NIH 3T3 cell line). We demonstrate that the plasma treatment of PE had a positive effect on the adhesion, spreading, homogeneity of distribution and moderately on proliferation activity of NIH 3T3 cells. This effect was even more pronounced on PE coated with biomolecules.     

Microfluidic devices modulate tumor cell line susceptibility to NK cell recognition

This study aims to adoptively reduce the major histocompatibility complex class I (MHC-I) molecule surface expression of cancer cells by exposure to microfluid shear stress and a monoclonal antibody. A microfluidic system is developed and tumor cells are injected at different flow rates. The bottom surface of the microfluidic system is biofunctionalized with antibodies (W6/32) specific for the MHC-I molecules with a simple method based on microfluidic protocols. The antibodies promote binding between the bottom surface and the MHC-I molecules on the tumor cell membrane. The cells are injected at an optimized flow rate, then roll on the bottom surface and are subjected to shear stress. The stress is localized and enhanced on the part of the membrane where MHC-I proteins are expressed, since they stick to the antibodies of the system. The localized stress allows a stripping effect and consequent reduction of the MHC-I expression. It is shown that it is possible to specifically treat and recover eukaryotic cells without damaging the biological samples. MHC-I molecule expression on treated and control cell surfaces is measured on tumor and healthy cells. After the cell rolling treatment a clear reduction of MHC-I levels on the tumor cell membrane is observed, whereas no changes are observed on healthy cells (monocytes). The MHC-I reduction is investigated and the possibility that the developed system could induce a loss of these molecules from the tumor cell surface is addressed. The percentage of living tumor cells (viability) that remain after the treatment is measured. The changes induced by the microfluidic system are analyzed by fluorescence-activated cell sorting and confocal microscopy. Cytotoxicity tests show a relevant increased susceptibility of natural killer (NK) cells on microchip-treated tumor cells.     

Preparation of protein- and cell-resistant surfaces by hyperthermal hydrogen induced cross-linking of poly(ethylene oxide)

The functionalization of surfaces with poly(ethylene oxide) (PEO) is an effective means of imparting resistance to the adsorption of proteins and the attachment and growth of cells, properties that are critical for many biomedical applications. In this work, a new hyperthermal hydrogen induced cross-linking (HHIC) method was explored as a simple one-step approach for attaching PEO to surfaces through the selective cleavage of C-H bonds and subsequent cross-linking of the resulting carbon radicals. In order to study the effects of the process on the polymer, PEO-coated silicon wafers were prepared and the effects of different treatment times were investigated. Subsequently, using an optimized treatment time and a modified butyl polymer with increased affinity for PEO, the technique was applied to butyl rubber surfaces. All of the treated surfaces exhibited significantly reduced protein adsorption and cell growth relative to control surfaces and compared favorably with surfaces that were functionalized with PEO using conventional chemical methods. Thus HHIC is a simple and effective means of attaching PEO to non-functional polymer surfaces.     

Development of a surface to increase retinal pigment epithelial cell (ARPE-19) proliferation under reduced serum conditions

Age related macular degeneration of the eye is brought about by damage to the retinal pigment epithelium (RPE) and is a major cause of adult blindness. One potential treatment method is transplantation of RPE cells grown in vitro. Maintaining RPE cell viability and physiological function in vitro is a challenge, and this must also be achieved using materials that can be subsequently used to deliver an intact cell sheet into the eye. In this paper, plasma polymerisation has been used to develop a chemically modified surface for maintaining RPE cells in vitro. Multiwell plates modified with a plasma copolymer of allylamine and octadiene maintained RPE cell growth at a level similar to that of TCPS. However, the addition of bound glycosaminoglycans (GAGs) to the plasma polymerised surface significantly enhanced RPE proliferation. Simply adding GAG to the culture media had no positive effect. It is shown that a combination of plasma polymer and GAG is a promising method for developing suitable surfaces for cell growth and delivery, that can be applied to any substrate material.