Cation-exchange performance of a citric-acid esterified cellulose nanofibrous membrane for highly-selective proteins' permeability and adsorption capacity.

The usage of low-cost, readily available, or even disposable, single-use membranes in macromolecules' purification and separation is still in the development phase. In this research, highly porous (>95 %), water- and compression stable cation-exchange membranes were prepared by freeze-casting using cellulose nanofibrils (CNF) and citric acid (CA) acting as a crosslinker and source of weak anionic (carboxylic) surface groups arising from the mono-esterified CA. The membranes were characterized by different analytical techniques, and evaluated for the ionic adsorption efficacy of different proteins in dead-end filtration mode using a Tri-buffer of pH 8. The membrane's internal microstructure (porosity and density) with the available (quantity and access) carboxylic groups was confirmed, to determine not only the proteins' specific (related to the net charged and molecular weight) adsorption dynamic (>52 % of positive Lysozyme/Cytochrome, <8 % of negative BSA/Myoglobin; ≤0.5 g/L) at extremely high flow rates (>3.000 hL/h*MPa*m2), but also their desorption (>97 %) and re-equilibration (using NaCl) with flux recovery (>80 %). Such efficiency was achieved with up to 5 consecutive filtering cycles. The high permeability (>87 %) of the spherical and negatively surface charged microparticles (used as models) also suggests the likelihood of removing larger microbial species, which, while retaining relatively smaller and positively charged proteins, further increases their potential in biopharma applications.

Antibacterial and degradation properties of dialdehyded and aminohexamethylated nanocelluloses.

Dialdehyde cellulose nanofibrils (CNF) and nanocrystals (CNC) were prepared via periodate oxidation (CNF/CNC-ox) and subsequently functionalized with hexamethylenediamine (HMDA) via a Schiff-base reaction, resulting in partially crosslinked micro-sized (0.5-10 µm) particles (CNF/CNC-ox-HMDA) with an aggregation and sedimentation tendency in an aqueous media, as assessed by Dynamic Light Scattering and Scanning Electron Microscopy. The antibacterial efficacy, aquatic in vivo (to Daphnia magna) and human in vitro (to A594 lung cells) toxicities, and degradation profiles in composting soil of all forms of CNF/CNC were assessed to define their safety profile. CNF/CNC-ox-HMDA exhibited higher antibacterial activity than CNF/CNC-ox and higher against Gram-positive S. aureus than Gram-negative E. coli, yielding a bacteria reduction of >90 % after 24 h of exposure at the minimum (≤2 mg/mL), but potentially moderately/aquatic and low/human toxic concentrations (≥50 mg/L). The presence of anionic, un/protonated amino-hydrophobized groups in addition to unconjugated aldehydes of hydrodynamically smaller (<1 µm) CNC-ox-HMDA increased the reduction of both bacteria to log 9 at ≥4 mg/mL and their bactericidal activity. While only CNF/CNC-ox can be considered as biosafe and up to >80 % biodegradable within 24 weeks, this process was inhibited for the CNF/CNC-ox-HMDA. This indicated their different stability, application and disposal after use (composting vs. recycling).

Using Different Surface Energy Models to Assess the Interactions between Antiviral Coating Films and phi6 Model Virus.

High molecular weight chitosan (HMWCh), quaternised cellulose nanofibrils (qCNF), and their mixture showed antiviral potential in liquid phase, while this effect decreased when applied to facial masks, as studied in our recent work. To gain more insight into material antiviral activity, spin-coated thin films were prepared from each suspension (HMWCh, qCNF) and their mixture with a 1:1 ratio. To understand their mechanism of action, the interactions between these model films with various polar and nonpolar liquids and bacteriophage phi6 (in liquid phase) as a viral surrogate were studied. Surface free energy (SFE) estimates were used as a tool to evaluate the potential adhesion of different polar liquid phases to these films by contact angle measurements (CA) using the sessile drop method. The Fowkes, Owens-Wendt-Rabel-Kealble (OWRK), Wu, and van Oss-Chaudhury-Good (vOGC) mathematical models were used to estimate surface free energy and its polar and dispersive contributions, as well as the Lewis acid and Lewis base contributions. In addition, the surface tension SFT of liquids was also determined. The adhesion and cohesion forces in wetting processes were also observed. The estimated SFE of spin-coated films varied between mathematical models (26-31 mJ/m2) depending on the polarity of the solvents tested, but the correlation between models clearly indicated a significant dominance of the dispersion components that hinder wettability. The poor wettability was also supported by the fact that the cohesive forces in the liquid phase were stronger than the adhesion to the contact surface. In addition, the dispersive (hydrophobic) component dominated in the phi6 dispersion, and since this was also the case in the spin-coated films, it can be assumed that weak physical van der Waals forces (dispersion forces) and hydrophobic interactions occurred between phi6 and the polysaccharide films, resulting in the virus not being in sufficient contact with the tested material during antiviral testing of the material to be inactivated by the active coatings of the polysaccharides used. Regarding the contact killing mechanism, this is a disadvantage that can be overcome by changing the previous material surface (activation). In this way, HMWCh, qCNF, and their mixture can attach to the material surface with better adhesion, thickness, and different shape and orientation, resulting in a more dominant polar fraction of SFE and thus enabling the interactions within the polar part of phi6 dispersion.

Cationised Fibre-Based Cellulose Multi-Layer Membranes for Sterile and High-Flow Bacteria Retention and Inactivation.

Low-cost, readily available, or even disposable membranes in water purification or downstream biopharma processes are becoming attractive alternatives to expensive polymeric columns or filters. In this article, the potential of microfiltration membranes prepared from differently orientated viscose fibre slivers, infused with ultrafine quaternised (qCNF) and amino-hydrophobised (aCNF) cellulose nanofibrils, were investigated for capturing and deactivating the bacteria from water during vacuum filtration. The morphology and capturing mechanism of the single- and multi-layer structured membranes were evaluated using microscopic imaging and colloidal particles. They were assessed for antibacterial efficacy and the retention of selected bacterial species (Escherichia coli, Staphylococcus aureus, Micrococcus luteus), differing in the cell envelope structure, hydrodynamic biovolume (shape and size) and their clustering. The aCNF increased biocidal efficacy significantly when compared to qCNF-integrated membrane, although the latter retained bacteria equally effectively by a thicker multi-layer structured membrane. The retention of bacterial cells occurred through electrostatic and hydrophobic interactions, as well as via interfibrous pore diffusion, depending on their physicochemical properties. For all bacterial strains, the highest retention (up to 100% or log 6 reduction) at >50 L/h∗bar∗m2 flow rate was achieved with a 4-layer gradient-structured membrane containing different aCNF content, thereby matching the performance of industrial polymeric filters used for removing bacteria.

Enzymatic, Antioxidant, and Antimicrobial Activities of Bioactive Compounds from Avocado (Persea americana L.) Seeds.

The aim of this research was to identify and quantify biologically active compounds from avocado (Persea americana L.) seeds (AS) utilizing different techniques with the use of ultrasound (US), ethanol (EtOH), and supercritical carbon dioxide (scCO2) for possible applications in (bio)medicine, pharmaceutical, cosmetic, or other relevant industries. Initially, a study of the process efficiency (η) was carried out, which revealed yields in the range of 2.96-12.11 wt%. The sample obtained using scCO2 was found to be the richest in total phenols (TPC) and total proteins (PC), while the sample obtained with the use of EtOH resulted in the highest content of proanthocyanidins (PAC). Phytochemical screening of AS samples, quantified by the HPLC method, indicated the presence of 14 specific phenolic compounds. In addition, the activity of the selected enzymes (cellulase, lipase, peroxidase, polyphenol oxidase, protease, transglutaminase, and superoxide dismutase) was quantified for the first time in the samples from AS. Using DPPH radical scavenging activity, the highest antioxidant potential (67.49%) was detected in the sample obtained with EtOH. The antimicrobial activity was studied using disc diffusion method against 15 microorganisms. Additionally, for the first time, the antimicrobial effectiveness of AS extract was quantified by determination of microbial growth-inhibition rates (MGIRs) at different concentrations of AS extract against three strains of Gram-negative (Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas fluorescens) bacteria, three strains of Gram-positive (Bacillus cereus, Staphylococcus aureus, and Streptococcus pyogenes) bacteria, and fungi (Candida albicans). MGIRs and minimal inhibitory concentration (MIC90) values were determined after 8 and 24 h of incubation, thus enabling the screening of antimicrobial efficacy for possible further applications of AS extracts as antimicrobial agents in (bio)medicine, pharmaceutical, cosmetic, or other industries. For example, the lowest MIC90 value was determined for B. cereus after 8 h of incubation in the case of UE and SFE extracts (70 µg/mL), indicating an outstanding result and the potential of AS extracts, as the MIC values for B. cereus have not been investigated so far.

Screen-printing of chitosan and cationised cellulose nanofibril coatings for integration into functional face masks with potential antiviral activity.

Masks proved to be necessary protective measure during the COVID-19 pandemic, but they provided a physical barrier rather than inactivating viruses, increasing the risk of cross-infection. In this study, high-molecular weight chitosan and cationised cellulose nanofibrils were screen-printed individually or as a mixture onto the inner surface of the first polypropylene (PP) layer. First, biopolymers were evaluated by various physicochemical methods for their suitability for screen-printing and antiviral activity. Second, the effect of the coatings was evaluated by analysing the morphology, surface chemistry, charge of the modified PP layer, air permeability, water-vapour retention, add-on, contact angle, antiviral activity against the model virus phi6 and cytotoxicity. Finally, the functional PP layers were integrated into face masks, and resulting masks were tested for wettability, air permeability, and viral filtration efficiency (VFE). Air permeability was reduced for modified PP layers (43 % reduction for kat-CNF) and face masks (52 % reduction of kat-CNF layer). The antiviral potential of the modified PP layers against phi6 showed inhibition of 0.08 to 0.97 log (pH 7.5) and cytotoxicity assay showed cell viability above 70 %. VFE of the masks remained the same (~99.9 %), even after applying the biopolymers, confirming that these masks provided high level of protection against viruses.

Phosphonate-Modified Cellulose Nanocrystals Potentiate the Th1 Polarising Capacity of Monocyte-Derived Dendritic Cells via GABA-B Receptor.

Purpose: Phosphonates, like 3-AminoPropylphosphonic Acid (ApA), possess a great potential for the therapy of bone tumours, and their delivery via cellulose nanocrystals (CNCs) seems a promising approach for their increased efficacy in target tissues. However, the immunological effects of CNC-phosphonates have not been investigated thoroughly. The main aim was to examine how the modification of CNCs with phosphonate affects their immunomodulatory properties in human cells. Methods: Wood-based native (n) CNCs were modified via oxidation (ox-CNCs) and subsequent conjugation with ApA (ApA-CNCs). CNCs were characterised by atomic force microscopy (AFM) and nanoindentation. Cytotoxicity and immunomodulatory potential of CNCs were investigated in cultures of human peripheral blood mononuclear cells (PBMCs) and monocyte-derived dendritic cells (MoDCs)/T cells co-cultures by monitoring phenotype, cytokines production, allostimulatory and Th/Treg polarisation capacity. Results: AFM showed an increase in CNCs' thickens, elasticity modulus and hardness during the modification with ApA. When applied at non-toxic doses, nCNCs showed a tolerogenic potential upon internalisation by MoDCs, as judged by their increased capacity to up-regulate tolerogenic markers and induce regulatory T cells (Treg), especially when present during the differentiation of MoDCs. In contrast, ox- and ApA-CNCs induced oxidative stress and autophagy in MoDCs, which correlated with their stimulatory effect on the maturation of MoDCs, but also inhibition of MoDCs differentiation. ApA-CNC-treated MoDCs displayed the highest allostimulatory and Th1/CTL polarising activity in co-cultures with T cells. These effects of ApA-CNCs were mediated via GABA-B receptor-induced lowering of cAMP levels in MoDCs, and they could be blocked by GABA-B receptor inhibitor. Moreover, the Th1 polarising and allostimulatory capacity of MoDCs differentiated with ApA-CNC were largely preserved upon the maturation of MoDCs, whereas nCNC- and ox-CNC-differentiated MoDCs displayed an increased tolerogenic potential. Conclusion: The delivery of ApA via CNCs induces potent DC-mediated Th1 polarisation, which could be beneficial in their potential application in tumour therapy.

Solidification of Gelatine Hydrogels by Using a Cryoplatform and Its Validation through CFD Approaches.

In this work, we developed a numerical approach based on an experimental platform to determine the working conditions on a cryoplatform and to predict and evaluate the cryogenic printing of hydrogels. Although hydrogels have good biocompatibility, their material properties make it difficult to print them with high precision and shape fidelity. To overcome these problems, a cryogenic cooling platform was introduced to accelerate the physical stabilisation of each deposited layer during the printing process. By precisely controlling solidification (crystallisation), each printed material can withstand its own weight to maintain shape fidelity, and the porosity of the scaffolds can also be controlled more selectively. The thermophysical properties of gelatine hydrogels were investigated to gain a better understanding of the phase change upon freezing. The corresponding material properties and experimental observations of gelatine solidification served as the basis for developing a computational fluid model (CFD) to mimic the solidification of gelatine hydrogels using a cryoplatform at different process conditions and extruder speeds. The goal was to develop a tool simple enough to predict acceptable process conditions for printing gelatine hydrogels using a cryoplatform.

Synergistic Effect of Screen-Printed Single-Walled Carbon Nanotubes and Phosphorylated Cellulose Nanofibrils on Thermophysiological Comfort, Thermal/UV Resistance, Mechanical and Electroconductive Properties of Flame-Retardant Fabric.

Single-walled carbon nanotubes (SWCNTs) and phosphorylated nanocellulose fibrils (PCNFs) were used as functional screen-print coatings on flame-retardant (FR) fabric, to improve its thermal resistance and thermophysiological comfort (wetting, water vapour and heat transmission) properties, while inducing it with electrical conductivity and UV protection. The effect of PCNF printing, followed by applying a hydrophobic polyacrylate (AP), on the same (back/B, turned outwards) or other (front/F, turned towards skin) side of the fabric, with and without the addition of 0.1-0.4 wt% SWCNTs, was studied by determining the amount of applied coating and its distribution (microscopic imaging), and measuring the fabric's colour, air permeability, thickness, mechanical, flame and abrasion resistance properties. Due to the synergistic effect of PCNF and SWCNTs, both-sided printed fabric (front-side printed with PCNF and back-side with SWCNTs within AP) resulted in an increased heat transfer (25%) and an improved thermal resistance (shift of degradation temperature by up to 18 °C towards a higher value) and UV protection (UPF of 109) without changing the colour of the fabric. Such treatment also affected the moisture management properties with an increased water-vapour transfer (17%), reduced water uptake (39%) and asymmetric wettability due to the hydrophilic front (Contact Angle 46°) and hydrophobic back (129°) side. The increased tensile (16%) and tear (39%) strengths were also assessed in the warp direction, without worsening the abrasion resistance of the front-side. A pressure-sensing electrical conductivity (up to 4.9∙10-4 S/cm with an increase to 12.0∙10-4 S/cm at 2 bars) of the SWCNT-printed side ranks the fabric among the antistatic, electrostatic discharge (ESD) or electromagnetic interference (EMI) shielding protectives.

Nanocellulose in Drug Delivery and Antimicrobially Active Materials.

In recent years, nanocellulose (NC) has also attracted a great deal of attention in drug delivery systems due to its unique physical properties, specific surface area, low risk of cytotoxicity, and excellent biological properties. This review is focused on nanocellulose based systems acting as carriers to be used in drug or antimicrobial delivery by providing different but controlled and sustained release of drugs or antimicrobial agents, respectively, thus showing potential for different routes of applications and administration. Microorganisms are increasingly resistant to antibiotics, and because, generally, the used metal or metal oxide nanoparticles at some concentration have toxic effects, more research has focused on finding biocompatible antimicrobial agents that have been obtained from natural sources. Our review contains the latest research from the last five years that tested nanocellulose-based materials in the field of drug delivery and antimicrobial activity.

Mechanical Properties and Cytotoxicity of Differently Structured Nanocellulose-hydroxyapatite Based Composites for Bone Regeneration Application.

The nanocomposites were prepared by synthesizing (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO)-oxidized cellulose nanofibrils (TCNFs) or cellulose nanocrystals (CNCs) with hydroxyapatite (HA) in varying composition ratios in situ. These nanocomposites were first obtained from eggshell-derived calcium and phosphate of ammonium dihydrogen orthophosphate as precursors at a stoichiometric Ca/P ratio of 1.67 with ultrasonication and compressed further by a uniaxial high-pressure technique. Different spectroscopic, microscopic, and thermogravimetric analyses were used to evaluate their structural, crystalline, and morphological properties, while their mechanical properties were assessed by an indentation method. The contents of TCNF and CNC were shown to render the formation of the HA crystallites and thus influenced strongly on the composite nanostructure and further on the mechanical properties. In this sense, the TCNF-based composites with relatively higher contents (30 and 40 wt %) of semicrystalline and flexible TCNFs resulted in smoother and more uniformly distributed HA particles with good interconnectivity, a hardness range of 550-640 MPa, a compression strength range of 110-180 MPa, an elastic modulus of ~5 GPa, and a fracture toughness value of ~6 MPa1/2 in the range of that of cortical bone. Furthermore, all the composites did not induce cytotoxicity to human bone-derived osteoblast cells but rather improved their viability, making them promising for bone tissue regeneration in load-bearing applications.

Rapid Water Softening with TEMPO-Oxidized/Phosphorylated Nanopapers.

Water hardness not only constitutes a significant hazard for the functionality of water infrastructure but is also associated with health concerns. Commonly, water hardness is tackled with synthetic ion-exchange resins or membranes that have the drawbacks of requiring the awkward disposal of saturated materials and being based on fossil resources. In this work, we present a renewable nanopaper for the purpose of water softening prepared from phosphorylated TEMPO-oxidized cellulose nanofibrils (PT-CNF). Nanopapers were prepared from CNF suspensions in water (PT-CNF nanopapers) or low surface tension organic liquids (ethanol), named EPT-CNF nanopapers, respectively. Nanopaper preparation from ethanol resulted in a significantly increased porosity of the nanopapers enabling much higher permeances: more than 10,000× higher as compared to nanopapers from aqueous suspensions. The adsorption capacity for Ca2+ of nanopapers from aqueous suspensions was 17 mg g-1 and 5 mg g-1 for Mg2+; however, EPT-CNF nanopapers adsorbed more than 90 mg g-1 Ca2+ and almost 70 mg g-1 Mg2+. The higher adsorption capacity was a result of the increased accessibility of functional groups in the bulk of the nanopapers caused by the higher porosity of nanopapers prepared from ethanol. The combination of very high permeance and adsorption capacity constitutes a high overall performance of these nanopapers in water softening applications.

Effect of Selected Comonomers on the Transition Temperature of Thermosensitive NIPA Derivatives Synthesized with an Anionic Initiator.

The present study evaluates the effect of the cationic initiator on the hydrodynamic diameter of copolymers of N-isopropylacrylamide nanogels synthesized via a surfactant-free precipitation polymerization at 70 °C in the presence of the cationic initiator 2,2'-azobis[2- methylpropionamidine] dihydrochloride. Three types of polymeric nanoparticles were synthesized using N, N'-methylenebisacrylamide as a crosslinker. The first batch was used as a reference. The second type of particles included a poly(ethylene glycol) methyl ether-acrylate monomer, while the third type used an N-tert-butylacrylamide comonomer. The hydrodynamic diameters of the synthesized particles were between 160 and 970 nm at 18 °C. The chemical composition and morphology of the synthesized co-polymeric nanoparticles were confirmed using infrared spectroscopy, nuclear magnetic resonance and scanning electron microscopy. The zeta potentials measured via dynamic light scattering were 20.0, 17.0, -0.1 mV for the three types, respectively. The volume phase transition temperature was between 22 and 41 °C. The polydispersity index of particles synthesized with N-tert-butylacrylamide varied depending on the measurement temperature.

Functionalization-dependent effects of cellulose nanofibrils on tolerogenic mechanisms of human dendritic cells.

BACKGROUND: Cellulose nanofibrils (CNF) are attractive nanomaterials for various biomedical applications due to their excellent biocompatibility and biomimetic properties. However, their immunoregulatory properties are insufficiently investigated, especially in relation to their functionalization, which could cause problems during their clinical application. METHODS: Using a model of human dendritic cells (DC), which have a central role in the regulation of immune response, we investigated how differentially functionalized CNF, ie, native (n) CNF, 2,2,6,6-tetramethylpiperidine 1-oxyl radical-oxidized (c) CNF, and 3-aminopropylphosphoric acid-functionalized (APAc) CNF, affect DC properties, their viability, morphology, differentiation and maturation potential, and the capacity to regulate T cell-mediated immune response. RESULTS: Nontoxic doses of APAcCNF displayed the strongest inhibitory effects on DC differentiation, maturation, and T helper (Th) 1 and Th17 polarization capacity, followed by cCNF and nCNF, respectively. These results correlated with a specific pattern of regulatory cytokines production by APAcCNF-DC and their increased capacity to induce suppressive CD8+CD25+IL-10+ regulatory T cells in immunoglobulin-like transcript (ILT)-3- and ILT-4- dependent manner. In contrast, nCNF-DC induced predominantly suppressive CD4+CD25hiFoxP3hi regulatory T cells in indolamine 2,3-dioxygenase-1-dependent manner. Different tolerogenic properties of CNF correlated with their size and APA functionalization, as well as with different expression of CD209 and actin bundles at the place of contact with CNF. CONCLUSION: The capacity to induce different types of DC-mediated tolerogenic immune responses by functionalized CNF opens new perspectives for their application as well-tolerated nanomaterials in tissue engineering and novel platforms for the therapy of inflammatory T cell-mediated pathologies.

Biocompatible antimicrobial electrospun nanofibers functionalized with ε-poly-l-lysine.

The antimicrobial polypeptide ε-poly(l-lysine) (ε-PL) was electrostatically incorporated to poly(acrylic acid) (PAA)/poly(vinyl alcohol) (PVA) electrospun nanofibers. ε-PL loading and distribution was assessed by infrared spectra, ζ-potential measurements and the primary amino reactive dye fluorescamine. Functionalized fibers with 485 ±â€¯140 nm diameter, could be loaded with 0.57-0.74 g ε-PL (g dressing)-1 that released at a constant rate of 5.4 ±â€¯2.8 mg ε-PL (g dressing day)-1. Such a dressings resulted in two orders of magnitude lower bacterial colonization than non-functionalized PAA-PVA after 14 days of incubation. Bacterial impairment was attributed to the damage of cell membranes and the formation of intracellular reactive oxygen species. ε-PL functionalized nanofibers did not display cytotoxicity to human corneal epithelial cells, HCEpC, in 24 h MTT assays. However, the viability of rapidly growing tumoral HeLa cells decreased >50% under the same conditions. The prepared biocompatible nanofibrous dressings with durable antibacterial activity show potential application as wound dressings and other biomedical uses.

Autofluorescence-aided assessment of integration and µ-structuring in chitosan/gelatin bilayer membranes with rapidly mineralized interface in relevance to guided tissue regeneration.

Beyond providing barrier function, the advanced materials in guided tissue regeneration (GTR) concept are further prompt to foster regeneration of distinct interfacing tissues. Herein we develop chitosan (CHT)/gelatin (GEL) bilayer membranes via successive solvent- and freeze-casting procedures and genipin (GEN) cross-linking chemistry. By utilizing the autofluorescence signal from GEN cross-linking products (i.e. the secondary CHT (GEL) amines and GEN esters), the Confocal Fluorescent Microscopy (CFM) identifies the chemical inter-linking as well as physical integration between interface layers. The presence of non- and highly µ-porous and pore-interconnecting regions is demonstrated within cross-sections of membranes with (by weight) prevalent GEL contribution in contrast to the sheet-like organization in membrane with equal presence of components. The constant processing conditions on variable compositions did not significantly affect the pore size distributions (in 1-230 µm range), while pore wall thickness increase up to 220 µm with GEL increase, which also improves the yield stress at compression (from 10 kPa to 19 kPa) and elastic modulus (from 26 kPa to 34 kPa). The rapid mineralization procedure resulted in deposition of non-regular to spherical minerals, containing nonstoichiometric carbonated apatite with Ca/P ration in 1.7-2 range, which demonstrates formation of osseointegrative interface. The fast and high (up to 580%), composition-dependent swelling, as well as 67% to 100% weight loss in 4 weeks in vitro degradation experiment point on membranes' relevance in GTR.

Incorporation of antimicrobial peptides on electrospun nanofibres for biomedical applications.

The aim of this work was to immobilize antimicrobial peptides onto a fibrous scaffold to create functional wound dressings. The scaffold was produced by electrospinning from a mixture of the water soluble polymers poly(acrylic acid) and poly(vinyl alcohol) and subsequently heat cured at 140 °C to produce a stable material with fibre diameter below micron size. The peptides were incorporated into the negatively charged scaffold by electrostatic interaction. The best results were obtained for lysozyme impregnated at pH 7, which rendered a loading of up to 3.0 × 10-4 mmol mg-1. The dressings were characterized using SEM, ATR-FTIR, elemental analysis, ζ-potential and confocal microscopy using fluorescamine as an amine-reactive probe. The dressings preserved their fibrous structure after impregnation and peptides were distributed homogeneously throughout the fibrous network. The antibacterial activity was assessed by solid agar diffusion tests and growth inhibition in liquid cultures using Staphylococcus aureus, a pathogenic strain generally found in infected wounds. The antibacterial activity caused clear halo inhibition zones for lysozyme-loaded dressings and a 4-fold decrease in S. aureus viable colonies after two weeks of contact of dressings with bacterial liquid cultures. The release profile in different media showed sustained release in acidic environments, and a rapid discharge at high pH values. The incorporation of lysozyme resulted in dressing surfaces essentially free of microbial growth after 14 days of contact with bacteria at pH 7.4 attributed to the peptide that remained attached to the dressing surface.

Antibacterial activity and cytotoxycity of gelatine-conjugated lysine-based peptides.

The effect of the coupling approach (chemical by using carbodiimide chemistry, and enzymatic by using transglutaminase) of a hydrophilic ɛ-poly-L-lysine (ɛPL) and a structurally-hydrophobic oligo-acyl-lysyl (OAK) to a gelatine (GEL) macromolecule, and their antibacterial activity against Gram-negative E. coli and Gram-positive S. aureus bacteria, as well as cytotoxicity to human osteoblast cells was studied as potential macromolecules for biomedical applications. Different spectroscopic (ultraviolet-visible, infrared, fluorescence, and electron paramagnetic resonance) and separation (size-exclusion chromatography and capillary zone electrophoresis) techniques, as well as zeta-potential analysis were performed to confirm the ɛPL/OAK covalent coupling and to determine their amount and orientation of the immobilization. The highest and kinetically the fastest reduction of bacteria (≥77% against E. coli vs. ≥82% against S. aureus) was achieved with GEL functionalized with ɛPL/OAK by the chemical grafting-to approach being correlated with conformationally the highly-flexible ˝brush-like˝ orientation linkage of peptides, enable its targeted and rapid interactions with bacteria membrane. The up to 400-fold lower yield of OAKs being immobilized may be related also to its cationic charge and hydrophobic alkyl chain moieties, compared to more hydrophilic ɛPL easily causing random polymerization and self-conjugation. The ɛPL/OAK-functionalized GEL did not induce citotoxicity to osteoblasts, even at ∼25-fold higher concentration than bacterial minimum inhibitory (MIC) concentration of ɛPL/OAK. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3110-3126, 2017.

Ultrasound-assisted green economic synthesis of hydroxyapatite nanoparticles using eggshell biowaste and study of mechanical and biological properties for orthopedic applications.

Nanostructured hydroxyapatite (HAp) is the most favorable candidate biomaterial for bone tissue engineering because of its bioactive and osteoconductive properties. Herein, we report for the first time ultrasound-assisted facile and economic approach for the synthesis of nanocrystalline hydroxyapatite (Ca10 (PO4 )6 (OH)2 ) using recycled eggshell biowaste referred as EHAp. The process involves the reaction of eggshell biowaste as a source of calcium and ammonium dihydrogen orthophosphate as a phosphate source. Ultrasound-mediated chemical synthesis of hydroxyapatite (HAp) is also carried out using similar approach wherein commercially available calcium hydroxide and ammonium dihydrogen orthophosphate were used as calcium and phosphate precursors, respectively and referred as CHAp for better comparison. The prepared materials were characterized by X-ray diffraction, field emission scanning electron microscopy, Fourier transform infrared spectroscopy, and Raman spectroscopy to determine crystal structure, particle morphology, and the presence of chemical functional groups. The nanocrystalline EHAp and CHAp were observed to have spherical morphology with uniform size distribution. Furthermore, mechanical properties such as Vickers hardness, fracture toughness, and compression tests have been studied of the EHAp and CHAp samples showing promising results. Mechanical properties show the influence of calcination at 600°C EHAp and CHAp material. After calcination, in the case of EHAp material an average hardness, mechanical strength, elastic modulus, and fracture toughness were found 552 MPa, 46.6 MPa, 2824 MPa, and 3.85 MPa m1/2 , respectively, while in the case of CHAp 618 MPa, 47.5 MPa, 2071 MPa, and 3.13 MPa m1/2 . In vitro cell studies revealed that the EHAp and CHAp nanoparticles significantly increased the attachment and proliferation of the hFOB cells. Here, we showed that EHAp and CHAp provide promising biocompatible materials that do not affect the cell viability and proliferation with enhancing the osteogenic activity of osteoblasts. Moreover, hFOB cells are found to express Osteocalcin, Osteopontin, Collagen I, Osteonectin, BMP-2 on the EHAp and CHAp bone graft. This study demonstrates the formation of pure nanocrystalline HAp with promising properties justifying the fact that the eggshell biowaste could be successfully used for the synthesis of HAp with good mechanical and osteogenic properties. These findings may have significant implications for designing of biomaterial for use in orthopedic tissue regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2935-2947, 2017.

Mineralization potential of cellulose-nanofibrils reinforced gelatine scaffolds for promoted calcium deposition by mesenchymal stem cells.

Cellulose-nanofibrils (CNFs) enriched gelatine (GEL) scaffolds were fabricated in-situ by the combined freeze-thawing process and carbodiimide crosslinking chemistry. The original- and variously surface anionised CNFs (carboxylated/CNF-COOH/, and phosphonated with 3-AminoPropylphosphoric Acid/CNF-COOH-ApA/) were used in order to tune the scaffolds' biomimetic structure towards a more intensive mineralization process. The pore size reduction (from 208±35µm to 91±35µm) after 50% v/v of CNFs addition to GEL was identified, while separated pore-walls' alignment vs. shorter, dense and elongated pores are observed when using 80% v/v of original-CNFs vs. anionised-CNFs, all of them possessed osteoid-like compressive strength (0.025-0.40MPa) and elasticity (0.04-0.15MPa). While randomly distributed Ca2+-deficient hydroxyapatite/HAp/(Ca/P≈1.4) aggregates were identified in the case of original-CNF prevalent scaffolds after four weeks of incubation in SBF, the more uniform and intensified deposition with HAp-like (Ca/P≈1.69) structures were established using CNF-COOH-Apa. The growth of Mesenchymal Stem Cells (MSCs) was observed on all CNF-containing scaffolds, resulting in more extensive Ca2+ deposition compared to the positive control or pure GEL scaffold. Among them, the scaffold prepared with the 50% v/v CNF-COOH-ApA showed significantly increased mineralization kinetic as well as the capacity for bone-like patterning in bone tissue regeneration.