The objective is a first industrial application of the eco-innovative solution ERUTAN (nature backwards), with the intention to reach global replication of the environmentally friendly production process for wool floor coverings. ERUTAN is developed at pilot scale by three SME/s in cooperation with European R&D partners and brings a high added value to the global carpet market. The main objectives and steps beyond the state-of-the-art of this project are: i) up-scaling of an innovative, sustainable enzymatic wool scouring method, ii) up-scaling of a novel enzymatic process for bonding between the yarns and supporting material of the carpet. WP2, realisation of an industrial enzymatic wool scouring process, enables sheep farmers worldwide to scour their own raw wool environmentally responsible. The carpet backing approach brings considerable energy saving and low, if any, carbon footprint using naturally based adhesives and enzymes.
ERUTAN is the first real innovation in manufacturing of textile floor covering since 1960. Although the single production steps remain equal, the environmental impact and production method change greatly. The pilot line for wool scouring, located at partner JMS, will be adapted to reach the industrial standard of scouring 10 tons of raw wool within 6 hours. Intensification is further achieved by optimizing enzyme formulation and conditions for application. Regarding the enzymatic bonding process 4 tasks are planned for WP3: Identification of potential providers for adhesives precursors and enzymes, Up-scaling backing line, Up-scaling adhesive paste, Optimization of process parameters and paste application technology. Within LCA work package, input of ERUTAN carpet after its use phase into a second life such as substrates for the agro and food industry, is taken into account. In WP5, business plan related to the exploitation and commercialization of the industrially developed processes and products. Dissemination activities are in WP6.
Chronic wounds including pressure, venous, arterial and diabetic neuropathic ulcers, represent a significant burden to the healthcare system. These different chronic wound types do not share origin or cause, however they have common features as bacterial infection and continuing influx of polymorphonuclear neutrophils that release high concentrations of matrix metalloproteases (MMPs), myeloperoxidase (MPO) and reactive oxidative species causing excessive degradation of the extracellular matrix (ECM) and the growth factors. Because of the multifactorial nature of virtually all chronic wounds, the therapeutic wound healing approach should emphasize the necessity to investigate wound dressings that possess the ability to directly and indirectly modulate the biochemical environment based on the pathology of chronic to encourage the healing process.
The overall aim of the present work was to develop biopolymer wound dressings capable to improve the management of chronic wounds. The specific research objective was defined in three main targets: i) to balance the proteolytic activity of MMPs, ii) to balance the MPO activity and the oxidative environment, and iii) antimicrobial protection.
The first part of the thesis aimed to provide suitable materials to perform bioactive biopolymer-based wound dressings. The first step was to provide a versatile functionalization of chitosan platform to improve the sequestering ability over metal dependent enzymes (MMPs) in chronic wounds. In order to impart to chitosan the ability to inhibit MMP, chitosan was functionalized with deferent thiol moieties, which can chelate the zinc cation in the active site of the enzyme, modifying MMP activity. In addition the combination of -NH2 and -SH chemistry allowed grafting of other active agents on the chitosan platform.
The second step was the identification and evaluation of natural active agents as chronic wound healing promoters. Polyphenols from bark, twigs and leaf extracts from the medicinal plant Hamamelis virginiana (Witch hazel) were studied for this porpoise and it was found to have both a strong antioxidant activity and an inhibitory effect on MPO and collagenase.
The second part of the thesis was focused in the performance and evaluation of new materials for wound healing applications. To this aim, chitosan and/or thiolated chitosan were modified with H. virginiana extracts following different approaches.
The first approach was focused to develop a new method for covalent functionalization of thiolated chitosan with polyphenols from H. virginiana extracts. The novelty of this approach consists in the use of thiolysis - a common analytical method for proanthocyanidins characterization - to covalently functionalize natural macromolecules such as chitosan with bioactive phenolic moieties. The phenolics-functionalized chitosan showed improved therapeutic properties in vitro.
The second approach focused on the use of laccase-assisted cross-linking between phenolic moieties of H. virginiana with chitosan and gelatin as a functionalization method to obtain stable and bioactive hydrogel wound dressings. H. virginiana extract was oxidized by laccase in a one-step process under mild reaction conditions to covalently crosslink chitosan and gelatin. The physical and mechanical properties of these hydrogels were investigated using different analytical techniques and their potential for chronic wound treatment was evaluated in vitro in terms of antibacterial and inhibitory effect on MPO and collagenase. The results indicated that the polyphenols exerted a dual role in the hydrogel: i) "passive" being a structural element, and ii) "active" modifying the chronic wound environment by attenuating the deleterious MMPs, MPO and ROS activities, and reducing the bacterial load.
This thesis focuses on the development of active multifunctional dressing materials and nanoparticle formulations with suitable exploitation characteristics for chronic wounds treatment. Chronic wounds a growing clinical challenge in the aging and/or reduced mobility population include pressure, venous, arterial and diabetic neuropathic ulcers. Due to the nonhealing character of these ulcers their management requires an intensive medical intervention at huge healthcare costs. The prolonged inflammation and elevated concentrations of oxidative and proteolytic enzymes in all chronic wounds, imposes the need for novel functional dressing materials to actively modulate the wound environment at molecular level and stimulate the healing process. Based on an extensive analysis of the current state-of-the-art in chronic wound healing, the proper dressings should combine both antimicrobial and enzyme inhibitory functions coupled to optimal hydrophilicity. Such integrated approach would allow for the suppression of the persistent inflammation and stimulation of the synthesis of the dermal tissue components. Biopolymers with intrinsic antimicrobial and wound repair properties appear as appropriate matrix materials to be further upgraded with bioactive molecules (therapeutics) that couple high reactivity with the ability to address specific targets in the biochemical environment of chronic wounds. Therapeutic devices can be designed in different forms depending on the particular clinical application, i.e. wound type and its characteristics.
During the thesis realisation biopolymer-based platforms were generated in various designs and functionalised with active agents for controlled inhibition of major chronic wound enzymes. The capacity of all developed materials to inhibit proteolytic (e.g. collagenase) and oxidative (e.g. myeloperoxidase) enzymes involved in chronic inflammation was evaluated in vitro.
In the first approach sponge-like biopolymer matrices were produced via freeze-drying technique and controlled chemical cross-linking. These matrices were further impregnated with natural polyphenolic compounds. Modulation of the deleterious wound enzyme activities was achieved upon release of active agent from the platform. The exploitation characteristics of the sponges, i.e. mechanical properties, biostability, biocompatibility, extent and duration of wound enzymes inhibition, were tuned by: the biopolymer composition, concentration of the cross-linking agent, and the proper selection of the bioactive phenolic compounds.
The second approach aimed at the permanent functionalisation of the biopolymeric platforms with thiol-bearing compounds. In this case the active agent is expected to act from the platform, without being released into the wound. The obtained thiolated biopolymers were further processed into functional nanomaterials of different design: *Nanoscale films/coatings were built using a layer-by-layer approach for alternate deposition of oppositely charged polyelectrolytes. Naturally occurring glycosaminoglycans were used as counterions to cationic thiolated conjugates. *Nanoparticle formulations were obtained from thiolated conjugates in a one-step sonochemical process. In both cases the biopolymer thiolation degree was identified as a key factor for the successful fabrication of the multilayered coatings and nanoparticles, as well as to achieve control of the thickness/size of the functional nanomaterials. In addition, tuneable inhibition/adsorption of the deleterious enzymes coupled to fibroblast attachment/proliferation was observed by ruling the biopolymer modification degree.
Rocasalbas, G.; Francesko, A.; Touriño, S.; Fernandez-Francos, X.; Gübitz, G. M,.; Tzanov, T. Carbohydrate polymers Vol. 92, num. 2, p. 989-996 DOI: 10.1016/j.carbpol.2012.10.045 Data de publicació: 2012-11-02 Article en revista
Laccase-assisted simultaneous cross-linking and functionalization of chitosan/gelatin blends with phenolic compounds from Hamamelis virginiana was investigated for the development of bioactive hydrogel dressings. The potential of these hydrogels for chronic wound treatment was evaluated in vitro, assessing their antibacterial and inhibitory effect on myeloperoxidase and collagenase. Rheological studies revealed that the mechanical properties of the hydrogels were a function of the enzymatic reaction time. Stable hydrogels and resistant to lysozyme degradation were achieved after 2 h laccase reaction. The inhibitory capacity of the hydrogel for myeloperoxidase and collagenase was 32% and 79% respectively after 24 h incubation. Collagenase activity was additionally suppressed by adsorption (20%) of the enzyme onto the hydrogel. Therefore, the bioactive properties of the hydrogels were due to the effect of both released phenolic compounds and the permanently functionalized platform itself. The hydrogels showed antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus.
Francesko, A.; Soares da Costa, D.; Reis, R. L.; Pashkuleva, I.; Tzanov, T. Acta biomaterialia Vol. 9, num. 2, p. 5216-5225 DOI: 10.1016/j.actbio.2012.10.014 Data de publicació: 2012-10-13 Article en revista
Collagen, collagen/hyaluronic acid (HA) and collagen/HA/chitosan (CS) sponges loaded with epigallocatechin gallate (EGCG), catechin (CAT) and gallic acid (GA) were developed and evaluated as active chronic wound dressings. Their physico-mechanical properties, biostability, biocompatibility and ability to inhibit in vitro myeloperoxidase (MPO) and collagenase—major enzymes related with the persistent inflammation in chronic wounds—were investigated as a function of the biopolymer composition and the polyphenolic compound used. The results demonstrated that the molecular weight of HA influences significantly the bulk properties of the obtained materials: higher elastic modulus, swelling ability and biostability against collagenase were measured when HA with higher molecular weights (830 and 2000 kDa) were added to the collagen matrices. The addition of CS and the polyphenols increased further the biostability of the sponges. Preliminary in vitro tests with fibroblasts revealed that the cells were able to adhere to all sponges. Cell viability was not affected significantly by the addition of the polyphenols; however, the presence of CS or high molecular weight HA in the sponge composition was associated with lower cellular viability. Finally, all specimens containing polyphenols efficiently inhibited the MPO activity. The highest inhibition capacity was observed for EGCG (IC50 = 15 ± 1 μM) and it was coupled to the highest extent of binding to the biopolymers (>80%) and optimal release profile from the sponges that allowed for prolonged (up to 3–5 days) effects.
Francesko, A.; Soares da Costa, D.; Lisboa, P.; Reis, R. L.; Tzanov, T.; Pashkuleva, I.H. Journal of materials chemistry B Vol. 22, num. 37, p. 19438-19446 DOI: 10.1039/C2JM31051A Data de publicació: 2012-04-20 Article en revista
Multilayered polyelectrolyte coatings comprising thiolated chitosan (TC) and glycosaminoglycans (GAGs), chondroitin sulphate and hyaluronic acid, were built using a layer-by-layer approach. The surface activity of these coatings for binding and inhibition of enzymes related to chronic inflammation, such as collagenase and myeloperoxidase, was assessed. The build-up of five bi-layers of TC/GAGs onto gold surfaces was monitored in situ by QCM-D. All experimental groups showed exponential growth of the coatings controlled by the degree of chitosan thiolation and the molecular weight of the GAGs. The degree of chitosan modification was also the key parameter influencing the enzyme activity: increasing the thiols content led to more efficient myeloperoxidase inhibition and was inversely proportional to the adsorption of collagenase. Enhanced fibroblast attachment and proliferation were observed when the multilayered polyelectrolyte constructs terminated with GAGs. The possibility to control either the activity of major wound enzymes by the thiolation degree of the coating or the cell adhesion and proliferation by proper selection of the ultimate layer makes these materials potentially useful in chronic wounds treatment and dermal tissue regeneration.
Perelshtein, I.; Ruderman, Y.; Perkas, N.; Traeger, K.; Tzanov, T.; Beddow, J.; Joyce, E.; Mason, T.; Blanes, M.; Mollá, K.; Gedanken, A. Journal of materials chemistry B Vol. 22, p. 10736-10742 DOI: 10.1039/c2jm31054f Data de publicació: 2012-03-15 Article en revista
Zinc oxide nanoparticles (ZnO NP’s) are known for their excellent antibacterial properties. This paper describes a method for enhancing the stability and the antibacterial activity of ZnO NPs synthesized
and embedded sonochemically on cotton fabrics, by pre-treating the fabric surface with cellulase enzyme. The enzymatic pre-treatment resulted in the deposition of smaller sized NPs with improved adhesion. The reduction in particle size brought about better antibacterial performance against several types of bacteria. The sonochemically produced ZnO coating withstood 10 laundry cycles at 92º C
retaining its antibacterial activity.
Díaz, M.; Rocasalbas, G.; Francesko, A.; Touriño, S.; Torres, J.; Tzanov, T. Biocatalysis and biotransformation Vol. 30, num. 1, p. 102-110 DOI: 10.3109/10242422.2012.646676 Data de publicació: 2012-01-18 Article en revista
The chronic wound environment is characterized by high concentrations of reactive oxygen species (ROS) and elevated
levels of myeloperoxidase (MPO) and collagenases, together impairing the healing process. Therefore, the management of chronic wounds at a molecular level requires the synergistic use of antioxidants, MPO and collagenase inhibitors to simultaneously target multiple factors from wound pathogenesis. In this study, a polyphenolic extract from Hamamelis virginiana plant, rich in condensed and hydrolysable oligomeric tannins, was evaluated as an inhibitor of MPO and collagenase.In addition to efficient scavengers of radical and non-radical reactive species, H. virginiana polyphenols were found to act as substrates in the MPO peroxidase cycle, preventing the accumulation of ROS in the chronic wound
site. Furthermore, it was also found that the plant exerts an irreversible inhibitory effect on collagenase activity (IC50 = 75 ± 10 μg/mL)
Perelshtein, I.; Ruderman, Y.; Perkas, N.; Traeger, K.; Tzanov, T.; Beddow, J.; Joyce, E.; Mason, T.; Blanes, M.; Mollá, K.; Gedanken, A. Journal of materials chemistry B Vol. 22, p. 10736-10742 DOI: 10.1039/C2JM31054F Data de publicació: 2012 Article en revista
'The focus of APROPOS is to develop novel eco-efficient bio-mechanical processing solutions to enrich intermediate fractions from industrial high protein and oil-containing process residues originating from agriculture and fisheries. Enzyme-aided modification steps are developed for the intermediate fractions to obtain value-added nutritive and bio-active components, chemical as well as functional bio-materials suitable for exploitation in food, skin care, wound healing, bio-pesticide and soil improvement product applications. Mentioned residues are voluminous in Europe and globally significant. Zero waste concepts to be developed aim at avoidance of unnecessary purification of the components, establishment of local and distributed processing units in connection with the primary production and new business opportunities essentially for SMEs in Europe and beyond. An emphasis is directed to East Africa and India to support their needs to process local residues to components directed to nourish infants and fight against pests, respectively, in rural areas of both regions. The success of technological developments will be assessed in terms of economical feasibility, raw material efficiency and environmental impacts. The assessment will also include study on how the developed residue producer-end use value chain will affect the existing value chain from the residue producer to feed or energy. The multidisciplinary research group and cross-industrial SME group together cover the whole value chain from residue producers and processors to various end-users. The expertises of the partners include crop and fish processing, process hard ware manufacture, mechanical, chemical and biotechnical biomaterial processing, biomaterial up-grading and analytics, enzyme technology, end-product applications, assessment of eco-efficiency and value chains, technology transfer and commercialization. Feasibility of the developed processes is verified by demonstrations. Bio-mechanical processi'
'Biofilms are bacterial communities encased in a self-produced hydrated polymeric matrix. An important characteristic of microbial biofilms is their innate resistance to the immune system and susceptibility to antibiotics. This resistance has made microbial biofilms a common cause of medical infections, and difficult-to-treat infections caused by colonized foreign bodies.
The NOVO project aims at developing novel approaches to prevent and/or degrade biofilms on catheters elongating their usage in humans up to 10 days.
Two complementary approaches for biofilm prophylaxis will be developed:
A. Ultrasonic coating of Inorganic antibiofouling agents (process developed by partner BIU) based on a single step sonochemical process to: a) Produce metal fluorides or metal oxides (e.g. MgF2, ZnO) nanoparticles (NPs) and simultaneously b) Impregnate them as antibacterial factors on the catheters. c) Co-coating with bio-inert polymer layers (containing highly hydrophilic antifouling polyethylene glycol, zwitterionic moieties or sugar-groups) grafted onto NPs of adjusted size to the size of MgF2/ZnO NPs or directly onto MgF2/ZnO NPs; to form a hydrogel layer for the protection of the MgF2/ZnO antibiofouling activity.
B. Bio/organic antibiofouling activation: 1) Novel coating for catheters based on radical catalyzed polymers to yield anti-bacterial activity. An enzymatic reaction will be applied on the phenolic compounds to generate phenolic radicals to be further polymerized on the catheter surface as an antibiofilm agent. 2) Develop and engineer Cellobiose Dehydrogenases (CDH) that actively oxidizes and degrades biofilms polysaccharides concomitantly producing stoichiometrically H2O2 as antibacterial agent. The enzymes will be coated on the catheters via a lubricant or by the Ultrasonic (US) process after their immobilization. Some novel CDH representatives already show very low activity on glucose which should be removed by further genetic engineering.'
'Hospital-acquired (nosocomial) infections are a major financial issue in the European healthcare system. The financial impact of these infections counteract medical advances and expensive medical treatments by increasing the length of hospital stay by at least 8 days on average per affected patient, hence adding more than 10 millions patient days in hospitals in Europe per year. The statistics on patient safety in the EU show alarming tendencies : - 1 in 10 patients are affected by hospital-acquired infections - 3 million deaths are caused by hospital-acquired infections An active infection control program of patients and personnel and hygiene measures, have proven to significantly reduce both the number of infections and hospitalisation costs . The SONO project directly addresses the above problems by developing a pilot line for the production of medical antibacterial textiles. The pilot line will be based on the scale-up of a sonochemical process developed and patented at BIU laboratories. The pilot line will use a sonochemical technique to produce and deposit inorganic, antimicrobial nanoparticles on medical textiles, e.g. hospital sheets, medical coats and bandages. Sonicators are used industrially for heavy and light duty cleaning, for water disinfection and for sewage treatment. It is also used in the food industry for emulsification and drying. The proposed concept based on one step sonochemical process to produce nanoparticles and impregnate them as antibacterial factors on textile is novel and does not exist on an industrial scale. The concept has already been proven (and patented ) on a lab scale where sonochemistry was applied to impregnate nanoparticles in a single-step process. It was demonstrated that due to the special properties of the sonochemical method the antibacterial nanoparticles are adsorbed permanently on the fibres even after 70 “laundry cycles”. The sonochemical impregnation process is a one-step procedure in which the nanopa'