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Targeting Hernia Operation Using Sustainable Resources and Green Nanotechnologies. An Integrated Pan-European Approach

Targeting Hernia Operation Using Sustainable Resources and Green Nanotechnologies. An Integrated Pan-European Approach

Final Report Summary - GREEN NANO-MESH (Targeting Hernia Operation Using Sustainable Resources and Green Nanotechnologies. An Integrated Pan-European Approach)

Executive Summary:
In Green Nano-Mesh, we proposed a novel approach that employs recent advances in green nanotechnology and sustainable raw materials for scaffold fabrication to not only eliminate toxic chemicals from the processes, but also enhance functional repair due to superior biological properties.
Specifically, we aim to fabricate a nano-fibrous mesh with well-defined nano-topography using cellulose; human recombinant collagen, derived from transgenic tobacco plants; and biodegradable poly-ε-caprolactone or polylactic/polyglycolic acid as raw materials. The green credentials of this innovative approach are based on the use of sustainable eco-friendly raw materials that will produce biodegradable waste products and therefore replacing hazardous chemicals currently in use.
To-date, at the end of the project, the Green Nano Mesh consortium:
(a) has developed environmental friendly raw materials (e.g. cellulose nano-crystals, pepsin soluble animal extracted collagen; human recombinant collagen, poly-ε-caprolactone) that would be part of the ultimate green hernia mesh;
(b) has developed numerous nano-textured scaffolds using electro-spinning, evaporation at room temperature, freeze-drying, and supercritical CO2 technologies;
(c) has extruded PLA and PCL fibres without using organic solvents and coated them with pepsin extracted bovine collagen and human recombinant collagen using dip coating;
(d) has stabilised materials with cellulose nano-crystals and green cross-linking methods based on CNC and plant extracts respectively. The produced lab-based prototypes have mechanical properties similar to commercially available synthetic meshes;
(e) has functionalised the produced scaffolds with biophysical (e.g. porosity) and biological (e.g. collagen coatings) signals;
(f) has demonstrated the in vitro cytocompatibility of the produced prototypes;
(g) has demonstrated in vivo compatibility
(h) has demonstrated environmental friendliness of the materials used;
(i) has scientifically contributed with numerous conference and peer-reviewed publications;
(j) has developed new technologies (IDFs are under preparation);
(k) has engaged with key stakeholders (e.g. companies, patients, clinicians, funding agencies; policy makers) for appropriate dissemination of the developed technologies;
(l) has trained numerous researchers to PhD level, whilst elements of the cutting edge work developed were used in lectures of under- and post- graduate students at the institutes of academic partners;
(m) has created employment, as partner organisation will take developed technologies further;
(n) has enhanced European competiveness in the field, as numerous products are expected to be launched within the years to come as direct result of this project.
The Green Nano Mesh Consortium has brought several platforms technologies (e.g. nano-scaffold fabrication; functionalization) to Technology Readiness Level 4 and we expect to reach level 5-9 within 4 years post project, which will enable clinical trials and commercialisation of the developed technologies. Further, the novel green products and processing methods will be game-changers in the field of medical device, enabling European-based industries to excel in the competitive field of medical devices.

Project Context and Objectives:
Green Nano Mesh will fabricate a clinical relevant prototype for hernia repair employing distinct advantages of biodegradable polymers and recent developments in green nanotechnology. Whilst Green Nano Mesh is specific for hernia repair, the developed platform-technologies can be successfully implemented in other tissue engineering applications (e.g. tendon, nerve, skin and bone regeneration and delivery of bioactive or therapeutic molecules applications) and thus will represent a breakthrough in the biomedical field.
The Clinical Need: Over 20 million hernia operations take place worldwide annually; 4.5 million is the prevalence in US with associated expenditure exceeding US $48 billion 1-6. Proportional is the situation in Europe, with prevalence in Germany and UK exceeding the 300,000 7-9. Over 18 million hernia operations involve the use of synthetic, non-degradable mesh prosthesis 4,8,9. Despite the early success of non-degradable meshes 10-15, there is still no efficacious therapy for hernia repair. In fact, non-degradable meshes are characterised by poor healing response, unfavourable foreign body reaction and in vivo erosion that lead to over 10% failure rate and an estimated 42% of recurrent hernias 16-22. Recurring hernias, not only cause further distress to the patients and compromise their quality of life, but also put an additional financial strain on healthcare systems. Moreover, the use of petroleum-based non-renewable non-degradable synthetic polymers not only does not align with the European Commission commitment for a low carbon economy 23,24, but also (a) leads to increased wear of processing equipment; (b) gives rise to problems with respect to disposal at the end of the materials’ lifespan since they cannot be thermally recycled; and (c) is associated with health and safety toxicity issues such as skin and lung irritation / toxicity 25-29. Green Nano Mesh will use biodegradable polymers and green nano-processes to fabricate clinically relevant hernia meshes.
The Raw Materials: The first generation of biomaterials was aiming to imitate structural characteristics and the mechanical properties of native tissues. However, modern biomedicine requires the use of materials designed to interact with the body and encourage functional tissue regeneration 30-32. To this end, biodegradable materials are widely used in medical devices with market size expecting to exceed US $252.7 billion by 2014 annually with a 15% CAGR 33-35. Degradable biopolymers are preferred candidates for developing implantable devices and means of therapeutics delivery since: (a) they minimally activate the innate and acquired host immune response; (b) their inherent biological properties, such as cell recognition signals, modulate cellular functions and enhance functional morphogenesis; (c) their degradation products are not toxic since they are subjected to natural biodegradation processes (e.g. hydrolysis and enzymatic activity); and (d) a second operation for removal is not required 36-39.
The Technology: Nanotechnology currently enjoys significant funding support from the private and public sectors. It is estimated that the world nano-material demand will reach US $4.1 billion by 2011 and by 2014 nanotechnology will be incorporated in manufactured goods worth US $2.6 trillion and will provide 10 million jobs 40,41. Nano-scale manufacturing technologies are emerging as powerful tools for tissue engineering and biological studies since they provide topographical, chemical and immunological control over cellular functions and therefore promote functional tissue regeneration, whilst they promote overall quality of life and avoid toxicity 42-50. It is therefore of paramount importance to develop green nanotechnology processes that will allow us to fabricate safer nano-composites without any negative impacts to human health using efficient and environmental friendly processes 51,52 and align with the EU’s target for chemical substitution 53 and reduction by 2020 emission levels by 20% lower than those of 1990 54. Only then biomedical nanotechnology will be able to play a pivotal role in the development of sophisticated, high-value nano-structures for niche applications that will facilitate disease prevention, early diagnosis and effective treatment.

The driving hypothesis of Green Nano Mesh is that using green nano-processes and sustainable raw materials such as (a) cellulose nano-crystals (CNC); (b) human recombinant collagen (rhCol) from transgenic tobacco plants; and (c) biodegradable polyesters, such as polylactic acid (PLLA), polyglycolic acid (PLGA) or poly-ε-caprolactone (PCL), not only we will eliminate hazardous chemicals from processes, but we will also fabricate a clinically relevant nano-mesh-like structure suitable for hernia repair. To this end, Green Nano Mesh is organised in eight in total RTD and horizontal Work Packages as outlined below along with their respective Specific Objectives (SO) and bring Europe in a leading technological position within this field.
Work Package 1: Preparation and optimisation of the basic ingredients for the Green Nano Mesh using sustainable resources.
➢ SO 1.1: Development and optimisation of the raw materials for the Green Nano Mesh (e.g. rhCol; pepsin extracted collagen produced from human cells; PLLA / PLGA / PCL; and cellulose).
➢ SO 1.2: Development and optimisation of green stabilisation methods based on plant extracts (e.g. oleuropein).
➢ SO 1.3: Development and optimisation of biological functionalisation methods (e.g. transglutaminase).
➢ SO 1.4: Cytotoxicity evaluation of the final raw materials.
Work Package 2: Fabrication and preliminary in vitro and in vivo evaluation of Green Nano Mesh prototypes using different green nano-processes suitable for hernia repair.
➢ SO 2.1: Fabrication of green nano-composites with distinct nano-architecture using green nano-processes.
➢ SO 2.2: Development of means for controlled and localised delivery of bioactive or therapeutic molecules using green nano-processes.
➢ SO 2.3: Initial screening of the produced prototypes in vitro (e.g. structural, mechanical and, thermal properties; release profile of bioactive / therapeutic molecules; in vitro cell seeding biological evaluation).
➢ SO 2.4: Initial screening of successful in in vitro set up prototypes, in small animals.
Work Package 3: Preclinical in vitro and small animal in vivo study.
➢ SO 3.1: Evaluation of the optimally stabilised and effectively functionalised Green Nano Mesh Prototypes in vitro under GLP conditions.
➢ SO 3.2: Preclinical in vivo trials in small animals under GLP conditions.
Work Package 4: Assessment of the risks, the safety and the benefits of the proposed green substitution.
➢ SO 4.1: Evaluation of the environmental toxicity and safety of raw materials; processes; and Green Nano Mesh prototypes that successfully passed rigorous in vitro and in vivo testing under GLP conditions.
Work Package 5: Scaling up the most successful Green Nano Mesh prototypes.
➢ SO 5.1: Scaling up the most environmentally friendly and least toxic Green Nano Mesh prototypes for hernia repair.
The Consortium has also organised further three Work Packages that deal with Scientific Coordination (Work Package 6), Training and Dissemination (Work Package 7) and Technical and Administrative Management (Work Package 8) that aim to:
➢ Foster expertise exchange and training among the partners;
➢ Enable smooth operation practices during the life-time of the Green Nano Mesh project;
➢ Enable appropriate dissemination and maximum exposure of the technologies developed by the consortium within and outside of the Consortium during the life-time of the Green Nano Mesh project;
➢ Facilitate commercialisation of the developed platform technologies following the end of the project.
A concerted collaborative effort between key Academic Institutes (3), Research Centres (1), non-profit Research Organisations (1) and Industrial partners (6) (Figure 1) will facilitate meeting our goals, namely:
(a) Substitution of hazardous chemicals, using sustainable, eco-friendly raw materials;
(b) Substitution of hazardous processes, using advancements in green nanotechnology; and
(c) Fabrication of clinically relevant prototypes for hernia repair, using eco-friendly raw materials and advancements in green nanotechnology.
Thus, Green Nano Mesh addresses therapeutic intervention for hernia repair and fits within the scope of the call for the substitution of materials or components utilising ‘green nanotechnology’. Moreover, although the Green Nano Mesh is specific for hernia repair, the developed platform-technologies can be successfully implemented in other tissue engineering applications (e.g. tendon regeneration, nerve regeneration, skin regeneration and delivery of therapeutic molecules applications) and as such they will be a breakthrough in the biomedical field. Table 1.1 summarises how Green Nano Mesh closely matches the criteria of the call.

Project Results:
WP1 - Preparation and optimisation of basic ingredients for the green nano mesh using sustainable resource.
WP1 aim was to research and develop critical raw materials necessary to produce a hernia mesh that has, in addition to all of the required mechanical properties, higher biocompatibility and biological performance. Moreover, these raw materials will be used to replace chemicals and processes that can be deleterious to the environment and are currently used in the production of hernia repair meshes.
Collagen
Enzymatic extracted collagen
Soluble collagen type I has been extracted and purified from bovine, porcine and rat tendons. Analysis by NUI Galway and Vornia using SDS-PAGE showed purity of over 90-95%. Cryo-milling of bovine and porcine tendons resulted in tendon powder free of metallic particles that dissolved fast and homogenously in dilute acidic conditions. In addition, the adjustment of acid, salt precipitation and temperature and time during pepsin treatment was critical for an improved extraction. Although hydrochloric acid extraction had higher dissociation capacity than acetic acid, hydrochloric acid-extracted collagen reduced macrophage population and increased release of pro-inflammatory cytokines. By contrast, acetic acid-extracted collagen produced a macrophage response similar to that of tissue culture plastic.
Following adjustments, the production was scaled up at Vornia facilities. The final collagen demonstrated properties similar or even superior to commercially available collagen preparations (e.g. purity, solubility). The produced collagen has become available to consortium members for the development of various products under the GNM project.
Human recombinant collagen
CP accomplished the production of human recombinant type I collagen from transgenic tobacco plants. High quality monomeric collagen was produced according to the company’s established process that included a series of acid and salt precipitation steps. The resulting collagen is virgin and therefore does not have higher molecular weight cross-links. Further, the produced collagen is of high purity and consistent characteristics, due to the recombinant nature of the protein. Certain amounts of the produced collagen are available to the consortium for GNM product development, due to its high production price.
Cellulose
Cellulose nano crystals was one of the approaches of the consortium to enhance the mechanical properties of the produced fibres / meshes. The initial raw material is microcrystalline cellulose, sourced from Avicell that has a particle size of 200 microns. Nano-crystalline cellulose (NCC) was prepared by HUJI for the production of fibres. The NCC was characterised in a foam formulation. The foam was produced and structured into nano-metric scale laminated planes. The resulting planes were nano-meter in size. Mechanical tests indicated high tensile strength. The modulus of pure NCC foam was 16 KPa. Initial tests indicated that in order for NCC to adhere to the biopolymer matrices the NCC needs to undergo chemical modifications.
Synthetic materials
A comprehensive review of synthetic materials and vendors among them biocompatibility, degradation rate, low bearing ability, knittable conformable and compliant were taken under consideration. The review resulted in narrowing to 2 families of materials: PLLA and PCL, based on material required characteristics and PB’s previous design experience. Following key industrial and academic partners meetings (VN, PB, NUI Galway and LI), PCL was chosen as the most desired polymer for the needs of the project, due its relative low production cost, acceptable in vivo degradation rate and superior mechanical properties.
Fabrication methods
The consortium tested various fabrication methods for scaffold development, including:
- A new polymerisation method based on supercritical CO2 was developed by CISC to produce high quality PCL by using stannous octoate as catalysis for the ring-opening step. This resulted in PCL membranes with controlled hierarchical porosity. Analysis of the resulting polymers using NMR and GPC indicate the production of controlled molecular weight polymers and higher purity than conventional non-green routes. However, problems were found by using this technique when scaling up samples for hernia repair. Unfortunately, due to budget limitations, scalability to meet the requirements of the clinical target is not possible. A new reactor system is required, which is in the hundred of thousand euros range.
- PCL fibres produced from different sources (Purac or Vornia) were coated with rhCollagen (HUJI) or bovine collagen (VN). Wrap knitting was chosen as the method to form a suitable mesh (LI, PB). In addition, electro-spun PCL (NUIG) material was examined as an alternative method with very promising results. PB characterised these meshes for its mechanical properties and pore size. Since these methods were suitable for scale up, an initial process development in terms of finishing processes (i.e. sterilisation method) was further investigated by PB.
Evaluation of cytotoxicity for the final raw material
A preliminary biological evaluation of the bovine collagen type I solution and porous PCL membranes developed by CISC was carried out by NUIG with a view to being applied as meshes in tissue engineering. Preliminary cell culture experiments showed that collagen extraction protocol did not alter fibroblasts response. When fibroblasts were cultured on the macro-porous membrane samples, they were found to be metabolically active and maintained their adhesion, spreading and proliferation capacity. Therefore, PCL and collagen are suitable for future studies in the development of clinical alternatives to current soft tissue repair materials.
Preliminary cell culture experiments showed that collagen extraction protocol did not alter fibroblast response. Regarding PCL macroporous films, fibroblasts were metabolically active and maintained adhesion, spreading and proliferation capacity, when cultured on the prepared PCL macroporous membrane samples.

WP2 - Fabrication and preliminary in vitro and in vivo evaluation of green nano mesh prototypes using different green nano-processes suitable for hernia repair.
Results to-date have shown that collagen scaffolds, such as films and sponges, can be obtained and stabilised using green processes (evaporation and freeze-drying) and chemical (CNC, alternative synthetic or plant-extracted cross-linkers) without compromising in vitro cell viability and modulating in vitro macrophage response. Furthermore, the novel incorporation of methacrylamide to rhCollagen (rhCollagen-MA) permitted gel formation when solutions were irradiated with UV light.
With regards to synthetic fibres, melt processing has been demonstrated to be an efficient alternative to organic solvent-based process to produce polylactic acid (PLA) and PCL fibres. Additionally, CNC incorporation was explored during the fibres extrusion; its incorporation increased mechanical resistance of PCL fibres, although any positive effects were observed for PLA fibres due to a low compatibility between CNC and polymer matrix. In addition, rhcollagen coating of fibres was performed using a dip coating method and a special pilot coating line. Both methods showed a high efficiency coating PLA and PCL fibres when fibres were treated with plasma in order to improve coating adhesion. In spite of light sensitivity of rhCollagen-MA, homogenously coated fibres were obtained. Basically, collagen coating demonstrated to improve in vitro cell response.
Moreover, PCL films were produced using solvent casting and electro-spinning. During each producing technology, different ways to induce controlled porosity were assessed. Supercritical CO2 produced hierarchically structured porous PCL films when combined with solvent casting. On the other hand, pattern collectors allowed producing electro-spun films with different porosity degree and shape porous. Preliminary cell culture experiments of PCL films showed that fibroblasts are metabolically active and maintain adhesion, spreading and proliferation capacity, indicating their potential in tissues repair.
Material reinforce using nanocellulose crystals
At the request of HUJI, Vornia incorporated NCC into PCL during the polymerisation process, as the acetylated version of NCC does not readily mix with the monomer. The reaction was carried out largely as normal with some modifications to heat and pressure in the reactor to produce an off white granulated powder. The materials were dispatched to Centexbel for further analyses. Concurrently, the butrylated version of NCC was produced at HUJI by simple mixing in the polymer and extrusion, which yielded very encouraging results. The acetylated version of NCC was not examined further to date.
Acetylated NCC was dissolved at various concentrations with 200 mg/ml poly caprolactone dissolved in 2,2,2-trifluoroethanol (TFE). Butylated CNC at 0.01 0.05 0.1 0.5 and 1% was unsuccessfully dissolved in TFE and chloroform at which point we contacted our colleagues in Israel who advised us that toluene would work but this failed to dissolve PCL sufficiently to electro-spin. Therefore all results are with acetylated NCC. A solution of PCL at 200mg/ml and NCC at various concentrations was electro-spun to create a randomly orientated mesh randomly orientated meshes. However, NCC reduced the tensile properties of electro-spun PCL for all concentrations and, therefore, NCC was discarded for further investigation. We also tried with butylated CNC but this failed to dissolve in any solvent, which would dissolve PCL and so was dropped.
Supercritical CO2
Supercritical CO2 (SCCO2) treatment was used to prepare hernia meshes from biodegradable polymers (PCL). Samples were prepared from polymer solutions and casting films using tetrahydrofurane (THF) and ethyl acetate (EtOAc) as solvents and adjusting processing parameters (pressure, P, temperature, T, and depressurization rate, DPR) in the SCCO2 reactor to attain the desired porosity.
Basically, PCL membranes with controlled hierarchical macroporosity have been prepared by SCCO2 treatment. The hierarchical porosity consists of two pores types, type 1 with an average pore size of 300-500 microns and type 2 of 15-50 microns. Indeed, hierarchical porosity was achieved in SCCO2 conditions by using pressures of > 100 bars and < 300 bars, temperatures lower than PCL Tm (< 40 ºC), and depressurization over several steps. Processing time and CO2 flow did not have a strong influence on the final porosity. CO2 absorption was found to take place only in the amorphous phase of PCL with a slight reduction in crystallinity observed after the SCCO2 treatment. Moreover, preliminary cell culture experiments showed that fibroblasts are metabolically active and maintain adhesion, spreading and proliferation capacity, when cultured on the prepared PCL macroporous membranes indicating their potential for applications such as meshes that could be suitable for use in tissues repair. However, when scaling-up sample size, problems were found by using this technique.
Comparing the samples fabricated in our laboratory with the commercially available meshes, the laboratory prepared samples are reproducible, non-cytotoxic, biodegradable and have a suitable thickness (between 250-450 µm), good porosity percentages (70-80%), uniformly distributed and homogeneous. In addition, they are made by an environmentally friendly process (no solvent residues).
Freeze-drying
Freeze-drying was used to fabricate sponges that were cross-linked with different cross-linking agents. We first explored the use of genipin because of its greenness features. Genipin needed the cross-linking process to be carried out in solution. Thus, the scaffold was soaked in an aqueous solution of genipin and then, crosslinking took place. Unfortunately, the scaffold was partially dissolved before crosslinking and despite the lateral dimensions were preserved – those where there were no pores, the in-height dimension shrunk so that, ultimately, the material became dense in every dimension. Thus, the particular porous structure resulting from freeze-casting allowed noticing that wherever the structure was porous, there was a densification process as consequence of the collapse of the porous structure that takes place upon soaking in the aqueous solution of genipin.
In this case, we explore a cross-linker-less process based on the Maillard reaction. The starting aqueous solution contained collagen – as in the previous cases – and ball-milled microcrystalline cellulose – so that we reduced the dimensions of the original crystallites to ca. 100 nm. After freeze-drying, the scaffolds was submitted to a soft thermal treatment so that the carbonyl group of the cellulose reacts with the amino group of the collagen, producing N-substituted glycosylamine and water. Afterwards, the unstable glycosylamine undergoes Amadori rearrangement, forming ketosamines. It is worth noting that, given the novelty of the process, we decided to apply the ISISA process rather than by the simple freeze casting used in previous cases to confirm the formation of a micro-channelled structure. We performed the in vivo culture of endothelial progenitor cells on the cross-linked scaffolds obtained upon the use of HDMI and the Maillard reaction. We found that, in biocompatibility terms, the scaffolds cross-linked via Maillard reaction provided better viabilities and less apoptosis that those cross-linked with HDMI.
Dip coating
In order to allow direct coating of NCC (contain sulphate group, therefore it is negatively charged) on fibre surface we used the plasma pre-treatment on PLA fibres surface. Plasma technique is a convenient method for modifying polymeric materials without altering their bulk properties surface properties of a material. This treatment results in positively charged groups that have been incorporated in the PLA fibre surface. Finally, fibres were coated by immersion in 4.5% (w/v) Rhcollagen solution for an hour and, finally, the resulted fibres were air-dried.
Collagen photo-cross-linking
Cross-linked rhcollagen gels were prepared according the followed procedure. 8.3 µl of photoinitiator solution (50 mg/ml) was added to 50 µl of 0.1 wt % rhCollagen-MA in DDW. Then the solution was gently mixed and irradiated with UV light with a wavelength of 254 nm for 5 min using a Duo-UV-Source for thin layer and column chromatography. After an optimization of crosslinking conditions a stable homogenous rhCollagen gel was obtained.
Chemical cross-linking
Chemical cross-linking is frequently used to enhance enzymatic and mechanical stability of collagen-based devices. However, chemical cross-linking has been associated with non-green process and adverse effects on host response. Within this context, alternative cross-linking methods are required and better understanding of the mechanism behind the inflammatory response elicited by collagen cross-linking is crucial. This research part investigates the human macrophage response in contact with cross-linked collagen films and the stabilisation capacity of synthetic (glutaraldehyde, carbodiimide, 4-arm branched-PEG) and natural (genipin, oleuropein) cross-linking agents. The addition of carbodiimide and oleuropein before collagen self-assembling decreased free amine groups of collagen, but it did not increase enzymatic stability. Only 4-arm branched-PEG and genipin provided enzymatic stability equivalent to glutaraldehyde cross-linked collagen films without increasing toxicity and inducing a similar release profile of pro-inflammatory cytokines to non-cross-linked films. Moreover, cell culture assays using pre-conditioned media strongly suggest that potential released sub-products from cross-linked collagen films are not responsible for the macrophage alteration. This is most likely associated with modifications in the collagen surface by chemical cross-linking. Overall, this study advocates 4-arm branched-PEG and genipin as alternative cross-linkers for tissue engineering scaffolds.
Electro-spinning
There are numerous methods to create a nano-fibrous construct drawing, self-assembly, and electro-spinning. However, the most commonly used by far is electro-spinning. Electro-spinning is a simplistic, inexpensive yet highly versatile method of fabricating materials with fibres diameters ranging from 1.2 nm to several micro-meters. While cells have been shown to respond to nano features as small as 13nm, the threshold below which the cells ignore the under lying topography is ≈500nm. This is true for fibrous materials also and while the diameter varies with cell type, in neural cells elongation is inhibited by fibres less than 750nm and for endothelial cells fibre diameters of 2µm have shown to have minimal collagen deposition compared with larger diameter fibres.
Electro-spinning was used to obtain porous meshes using chemically etched collectors with 2mm pores with a distribution of 30, 50 and 70% porosities and rhomboid (45/135 degrees), squares and circles pore shapes. While it was possible to create well-defined pores in all condition the definition of the circular pores practically at 30 and 50% are superior to the other conditions. This was due to the fibre deposition occurring adjacent to the collector as was seen in the SEM analysis. The uniform angle of the circle did not force the fibre to alter its trajectory rapidly to conform to a corner and therefore enhanced clarity in the pore periphery was maintained. However, all samples have a noticeable reduction in tensile properties compared to the control sample and as expected the trend for a decrease in the tensile properties associated with increased porosity is obtained. On the other hand, the multiple fabrication method, combining thermal compression and laser cutting, allowed obtaining porous meshes with sufficient mechanical properties.

WP3 - Preclinical in vitro and small animal in vivo study
In conformity with ISO 10993-1 (2009/Cor 1: 2010) requirements, a Biological Safety Evaluation Plan (BSEP) has been written and shared with all partners during the meeting in Gent (25th/26th November 2013). The goal of this BSEP was to document the strategy for confirming the biological safety profile of the green nano mesh (final finished product, categorized as an implant in permanent contact (> 30 days) with tissues) for CE marking and USFDA registration.
This assessment focussed on the requirements of ISO 10993-1 (2009/Cor 1: 2010), ISO 14971 (2007), ISO/TR 15499 (2012) and FDA General Program Memorandum #G95-1.
All information available at that time on raw materials and overall manufacturing processes were considered to help defining which biological tests should be considered on the green nano mesh and which ones could be avoided and justified by a risk assessment.
In parallel, some details in vitro studies were performed which demonstrated that the addition of 4-arm branched-PEG and genipin before collagen self-assembling were the only methods that provided an enzymatic stability equivalent to GTA treated collagen films without increasing toxicity and inducing a similar release profile of pro-inflammatory cytokines to non-cross-linked films. However, mechanical properties were only improved by genipin cross-linking. Moreover, the pro-inflammatory macrophage response associated with cross-linked collagen-based devices is most likely associated with modifications in the collagen surface by chemical cross-linking, not via solubilised products.
Moreover, the introduction of porosity does not appear to have any detrimental effect on the fibroblasts and the reduction of surface area in the increasingly porous meshes equally appears to have minimal effect of the quantity of cells present. An interesting correlation between the electrospun meshes, which underwent the inflammatory response analysis indicates that all meshes elicit similar metabolic responses even when the sample is positively primed with a lipopolysaccharide to induce the response. This inhibition of the inflammatory response could potentially lead to a significant reduction of chronic foreign body responses, which have sever repercussions for patient wellbeing and outcomes following hernia repair.
Based on the information acquired from WP1 and WP2 the electrospun meshes were coated with both BAT and hr Col I. Samples of the coated meshes in both the non crosslinked and crosslinked phases. And, in an attempt to identify the final finished product that should be submitted to biological safety evaluation as described in the BSEP for marketing approval, in vivo screening studies have been performed.
Full thickness parietal defect model in the rat
The evaluation of the performance, local tissue effects, degradation and tissue ingrowth of an abdominal repair mesh was performed in a full thickness parietal defect model in the rat. The purpose of this nonclinical screening study was to evaluate the performance (prevention of hernia), local tissue effects, tissue integration and degradation of abdominal repair mesh prototypes associated with a collagen based film of human recombinant collagen (rHCol) (Test article). Two meshes without film were defined as control (Control articles 1 and 2).
After creation of a full thickness defect in the right abdominal muscle, articles were implanted so as to cover the defect. The local tissue effects, tissue integration, degradation and performance were evaluated at 4 weeks (test article versus control article 1) and 12 weeks (test article versus control article 2) through macroscopic observations, qualitative and semi-qualitative histopathologic analysis.
No hernias were observed in test and control groups at both time periods. Based on histopathologic qualitative and semi-quantitative evaluation, both the test and control articles appeared entirely colonized by newly-formed connective tissue (complete tissue ingrowth). The article individual textile filaments elicited a foreign body-type reaction (macrophages and giant cells) of moderate intensity. The only difference was the slightly lower amount of mast cells infiltrating the test sites in comparison with the controls 1.
After the 4-week implantation period, there were no signs of degradation and the tissue integration was graded moderate to complete and did not show relevant difference between the test and control article 1. The relevant differences after 12 weeks were the lower mean score of fibrosis and the slightly lower amount of polymorphonuclear cells (neutrophils) and mast cells infiltrating the test sites in comparison with the controls 2. After the 12-week implantation period, there were no signs of degradation when compared with Week 4 and the tissue integration was graded marked to complete and did not show relevant difference between the test and control article 2.
The newly-formed connective tissue showed varying proportions of fibrosis and fibroplasia, and a time-related decrease in the fibroplasia mean score was observed for test article, demonstrating an on-going integration process. The mesh associated with a collagen-based film (rHCol – test article) did not show relevant differences when compared with uncoated meshes (control articles 1 and 2) in terms of performance (prevention of hernia), degradation (no degradation) and tissue ingrowth after the 4 week and 12 week implantation period. However, the local tissue effects of the test article were characterized by a foreign body-type reaction of moderate intensity, lower mean score of fibrosis and less abundant neutrophils and mast cells compared to the control articles 1 and 2 after 4 and 12 weeks.
Local tissue effects, tissue integration and degradation screening
Local tissue effects, tissue integration and degradation screening was studied in rats following subcutaneous implantation for 10 days, 4 and 8 weeks. The purpose of this non clinical screening study was to evaluate the local tissue effects, tissue integration and degradation after implantation of hernia meshes prototypes. Each type of mesh was associated with a collagen based film of human recombinant collagen (hR Col) or Bovine Achilles Tendon Collagen (BAT Col) (Test articles 1, 2, 3 and 4). Two uncoated meshes were defined as control (Control articles 1 and 2).
Two different types of meshes were evaluated following the manufacturing process PCL or E spun technologies:
- meshes manufactured using polycaprolactone (PCL) technology: Test Article 1 (associated with BAT Col) and Test Article 2 (associated with hR Col)
- meshes manufactured using E-spun technology: Test Article 3 (associated with BAT Col) and Test Article 4 (associated with hR Col).
The coated meshes were compared to uncoated mesh of each manufacturing process Control Article 1 (PCL technology) or Control article 2 (E-spun technology), in order to assess the tissue reaction associated to the coatings.
Articles were implanted in direct contact with subcutaneous tissue in rats. The local tissue effects, tissue integration and degradation were evaluated at 10 days, 4 and 8 weeks through macroscopic observations and qualitative and semi-qualitative histopathologic analysis. The performance was also evaluated using quantitative morphometric and histomorphometric analysis (n= 5 sites) per article and per time-period).
Macroscopically, neither macroscopic local signs of adverse tissue effect nor degradation were observed in any of the four test groups and of the two control groups after 10 days, 4 weeks and 8 weeks of subcutaneous implantation. All the articles appeared similarly integrated into the subcutaneous tissue along the three time periods.
Based on histopathologic qualitative, semi-quantitative and quantitative evaluation, Test Article 1 and Test Article 2 did not show relevant differences when compared with Control Article 1, in terms of local tissue effects and performance, after the 10-day, 4-week and 8-week implantation periods. Test Article 3 and Test Article 4 did not show relevant differences when compared with Control Article 2, in terms of local tissue effects after the 10-day, 4-week and 8-week implantation periods. Test Articles 3 and 4 had slightly higher integration mean scores in comparison with Control Article 2 after 8 weeks. However, this was not confirmed by the histomorphometric analysis and was therefore considered equivocal.

WP4 - Assessment of the risks, the safety and the benefits of the proposed green substitution
The biodegradation along time of PCL mesh and spun layer without coating and with two different collagen coatings, hR collagen and BAT collagen, was tested in sewage sludge and in standard soils with different physico-chemical characteristic.
In the sludge the non-coated and hR coated test materials were fully degraded after 24 days. In the soil approx. 20% of the test materials was degraded after 28 days. Toxicity to soil organisms was tested with the Enchytraeid reproduction test; no negative effect on the reproduction of enchytraeids was observed.
The work performed within Green NanoMesh, has also been used to support the general development of the international risk assessment work through meetings, ensuring the focus in such risk assessment also cover materials as used (the PI is leading the European Risk work in Nanosafety Cluster and Community of Research).

WP5 - Scaling up the most successful green nano mesh prototypes
The work has been done as planned. The coating of rh-collagen on the selected PCL yarns was successful. The coated samples showed a good collagen adhesion and were uniform and stable coated.
The coating technology and plasma technology are still very green and environmental friendly technologies. On top of that due to the changes on nano-level at the surface of the yarn a very performing thin layer of collagen could be deposed. However the extrusion of NCC/PCL was not satisfactory. Li will not continue to work on this.
The up scaling of the coating line to an industrial pilot line has been done successfully. As well the output, line speed and curing are more performing now and enable us to start industrialisation when biocompatibility study will be completed. The cooperation between the different partners was excellent, fruitful and will continue even after the end of the project. The results are meeting our expectations. Initial contacts have been made to start a successful commercialisation. The project and results have been noticed at conferences with positive feedback.

WP6 – Scientific coordination
Scientific and technical coordination are progressing according to plan. Several strategies have been performed to guarantee a high quality and relevance of the project during this period. Regular data transfer and dissemination were carried out in monthly meetings and telephone calls. Further discussions were had in coordination meetings and general assemblies. Additional workshops within the project members were arranged on scaling up of the most optimal technologies, the exploitation of IP and the commercialisation of research outcomes.
Progress and scheduling of each work package are monitoring regularly at each meeting; there are some slight delays on the animal study to-date. Some of the outcomes of this projects are numerous international conference papers (posters, oral presentations and invited keynote talks) and peer-reviewed publications (reviews and research paper). It is worth pointing out that technologies with new IP potential have not publicised as yet (2 research papers).
Finally, some members are in on-going training within the project about ISO standards, CE Mark, collagen production and characterisation, methacrylation of collagen, polymer production, in vitro and in vivo sample analysis. In addition, some of the principal investigators of GNM project were invited to 4 plenary lectures and workshops outside the project about the medical technologies and solutions in medical device industry.

WP7 – Training and dissemination
The consortium has succeeded in disseminating the results of this project on a large scale. The workshops held within the consortium were very effective and there have been a significant number of peer-reviewed publications associated with the project’s academic partners in the last, with more likely to be published after the completion of the project.
The development and sharing of protocols arising directly from the project is seen as an important foundation for the development of a sustainable mesh based product with green credentials and a commercial future. All information is posted on the consortium website www.greennanomesh.com which is updated regularly by the scientific co-ordinator. In addition, documents relating to the project and partners are shared through the project file server, which is managed by European Research Services (Partner 11).

Potential Impact:

Strategic Impact
Given that in excess of 206 Kt of medical plastics (primarily PVC and propylene) were produced in Europe alone with production forecast of almost 300 Kt for 2015, ecological concerns have resulted in a renewed interest for natural materials and environmental friendly processes. The Green Nano Mesh project successfully produced sustainable raw materials to replace such products from processes. Green Nano Mesh is therefore timely, given that the global market for ‘green’ materials was estimated at US $6.1 billion in 2005 and is expected to reach US $8.7 billion by 2010 with an average annual growth rate (AAGR) of 7.4%.
In meeting the scientific and technological objectives of the call, a multidisciplinary approach was taken herein facilitating the minimisation of non-environmental friendly raw materials from processes; maximising the use of sustainable resources in biomedical field due to increased biocompatibility; and promotion of eco-friendly green nano-processes. In addition, the platform-based technologies developed can be successfully applied to other tissue engineering applications and as such facilitates further reduction in non-environmental friendly processes and generation of more revenue. For example, the developed scaffold can find applications in wound healing management, a market with revenues in excess of US $653.5 million in Europe and over US $517 million in US.
Moreover, very few life cycle assessments comparing the sustainability of conventional and nanotechnology-based materials are as yet available. By achieving this impact, Green Nano Mesh provided a Life Cycle Analysis (LCA) that quantifies the environmental impacts of the new products all along its life: from the production of the different material it is using, integrating all impacts during the production and assembly of all subparts, through their efficiency during their use phase and what happens after end of life.
Specifically, Green Nano Mesh aimed to fabricate a nano-fibrous mesh with well-defined nano-topography using recent advancements in green nano-processing technologies utilising sustainable and environmental friendly raw materials such as: (a) cellulose nano-crystals as a reinforcement as opposed to potentially toxic carbon nanotubes; (b) human recombinant collagen, derived from transgenic tobacco plants as opposed to animal extracted collagen that harbours risks of interspecies transmission of disease; (c) biodegradable polylactic/polyglycolic acid as opposed to non-sustainable and non-degradable plastics such as polypropylene, polytetrafluoroethylene and nylon; (d) natural stabilisation methods based on plant extracts as opposed to chemical stabilisation based on toxic glutaraldehyde or epoxides; and (e) biological functionalisation methods as opposed to chemical ones based on polyamidoamine dendrimers.
Overall, Green Nano Mesh through this innovative Industry-Academia collaboration contributed to the reduction of non-environmentally friendly raw materials; eliminated the use of hazardous substances in production processes utilising green nanotechnology; and promoted the use of sustainable eco-friendly materials for biomedical and pharmaceutical applications due to their superior biocompatibility and environmental friendly degradation process.

Economic Impact
Nanotechnology Market: The Nanotechnology Market Forecast to 2013, predicted that the global nanotechnology market was projected to grow rapidly and that the market for nanotechnology incorporated in manufactured goods would be worth US $1.6 trillion, representing a CAGR of more than 49% in the forecast period (2009-2013). To illustrate the current drive toward developing these capacities, the Lux Research estimated that by the year 2015, US $3.1 trillion worth of products will be using nanotechnology across the value chain which is comprised of nano-materials, nano-intermediates and nano-enabled final products and provide 10 million jobs 41. While this project was initially launched in a period of extensive media coverage focusing on the advent of a global recession and its effects on investing, industrial growth, employment, and trade, even back then indicators of a revival were present such as the 2009 Global R&D Funding Forecast (compiled by Battelle, Columbus, Ohio, and R&D Magazine) notes that global R&D spending reached US $1.143 trillion in 2009, 3.2% higher than in 2008.
The breakdown of this market by category is Nanobiotechnology (52%); Medical devices (32%); Materials (12%); and Tools (4%). Various government initiatives, including the US government's National Nanotechnology Initiative and the EU's Work plan 2008 for the European Technology Platform (ETP) nanomedicine, developed opportunities for this growth. Biotechnology is considered one of the key enabling technologies of the 21st century to support the EU’s Lisbon strategy and sustainable development for Europe. Modern biotechnology has widespread applications in human medicine and health care which make a significant contribution to the EU economy. In fact, wherever industrial biotechnology is applied, it has positive economic and environmental implications: industrial biotechnology increases labour productivity by 10% to 20% compared with conventional processes. In 2008, North America was the largest market for healthcare nanotechnology applications with revenue of US $197 billion and had predicted revenue of US $643.21 billion by 2014 with a CAGR of 21.8% from 2009-2014. However, the European market for healthcare nanotechnology applications had the highest CAGR of 22.3% from 2009-2014, which was worth US $187.5 billion in 2008 and US $627.4 billion in 2014.
Green Nano Mesh focused on delivering long term effective nano-technological solutions to 3 of the largest segments of the market: (a) The European Market of nano-fibres in the medical and life science sector for tissue repair and drug delivery applications with market share of US $122.3 billion in 2007 and which was projected to increase to US $439.7 billion by 2014 growing at a CAGR of 15.3 %; (b) The European healthcare nanotech drug formulation and delivery market that in 2007 was worth US $2.02 billion and was expected to reach US $10.9 billion in 2014 at CAGR of 20 % for the same period; and (c) The European healthcare nanotech drug delivery market that had market share of US $1.37 billion in 2007 and was expected to reach US $5.6 billion in 2014 at CAGR of 21.7 % for the same period.
In addition, it has to be stressed that through the project, Green Nano Mesh’s industrial partners (both MNCs, and SMEs) gained leadership positions in the highest revenue and growth-rate European Healthcare Nanotechnology segments. These are translating into increased market competition, which, to the lead users and stakeholders, means cheaper and more cost-effective medical products and technologies.
Hernia market
Over 20 million hernia operations take place worldwide annually; 4.5 million is the prevalence in US with associated expenditure exceeding US $48 billion. Proportional is the situation in Europe, with prevalence in Germany and UK exceeding the 300,000. Non-degradable meshes are characterised by poor healing response, unfavourable foreign body reaction and in vivo erosion that lead to over 10% failure rate and an estimated 42% of recurrent hernias. Recurring hernias, not only cause further distress to the patients and compromise their quality of life, but also put an additional financial strain on healthcare systems. In addition, 30-70% of the EU population was classified as overweight or obese in latest estimates. The obesity epidemic and the growing elderly population demographics present in many Western countries, indicate the growing need for the procedure of hernia repair.
Frost & Sullivan also estimated that the 2005 European market revenue for minimally invasive surgical devices was US $2.48 billion at a compound annual growth rate (CAGR) of 6.2% for the period 2006 to 2013. At the end of the forecast period, the market was expected to be worth US $3.78 billion Overall, the European Market for Hernia Repair Devices to be US $300 million and it was expected to reach US $520 million by 2013 at a CAGR of 5.2% for the period. Manufacturers in Europe constitute a limited pool of participants that include large global participants as well as regional European and country-specific local participants. In terms of technologies and products, the market mainly focuses on two or three main types of procedures that are most common, safe and have a well-established reputation. The Market Competitors are classified according to their Market Share: Tier 1 companies: Johnson & Johnson; Tier II companies: Allergen and Tyco (Covidien); Tier III companies are smaller participants in different parts of Europe such as Helioscopie, Cousin Biotech, AMI and Hospimedical. Proxy Biomedical has already established a worldwide distribution of their hernia meshes and is expected to commercialise the Green Nano Mesh upon completion of the project. The distinct advantages of the Green Nano Mesh beyond the state-of-art facilitates establishing the product in the market.
Raw materials market
Over 18 million hernia operations involve the use of synthetic, non-degradable mesh prosthesis. Despite the early success of non-degradable meshes, there is still no efficacious therapy for hernia repair. In fact, non-degradable meshes lead to over 10% failure rate and an estimated 42% of recurrent hernias. Given that in excess of 206 Kt of medical plastics (primarily PVC and propylene) were produced in Europe alone with production forecast of almost 300 Kt for 2015 and associate market share of €686.6 million by 2015 growing at a CAGR of 6.2%, ecological concerns have resulted in a renewed interest for natural materials and environmental friendly processes. Green Nano Mesh is therefore timely, given that the global market for ‘green’ materials was estimated at US $6.1 billion in 2005 and was predicated to reach US $8.7 billion by 2010 with an average annual growth rate (AAGR) of 7.4%.
Other markets
Although Green Nano Mesh is intended to provide a clinical valuable solution for hernia repair, the developed nano-fibrous structure, with only slight modifications, can be successfully adopted for other tissue engineering burdens. For example, aligned fibrous meshes can be used for musculoskeletal repair. Recent estimates suggest that the total health care costs in European countries attributable to musculoskeletal conditions amount to €70 to €150 billion per year. Typically, around 50% of the European population report musculoskeletal pain at one or more sites for at least one week in the last month. Musculoskeletal conditions are chronic diseases involving the joints, causing inflammation and affecting the surrounding ligaments and tendons. These are chronic diseases involving the joints, causing inflammation and affecting the surrounding ligaments and tendons. There is currently no efficacious therapy for enhancing the rate and/or ability of these tissues to heal 237. Similarly, nano-fibrous meshes can be used for wound healing management, a market with revenues in excess of US $653.5 million in Europe and over US $517 million in US.

Social and Economic Impact
Green Nano Mesh brought together a highly skilled and complementary grouping of European researchers from academic and industrial arenas, some of which directly participated in FP6 and FP7 initiatives. Within the context of this programme, there are and were multiple intermediate and long-term benefits for the scientific researchers, the principal investigators, the academic institutions and the SMEs. Green Nano Mesh synergised the collaborative efforts made by all those involved and facilitated integration of research capabilities, within both public and private institutions, in order to increase coherence and critical mass in the study of hernia repair. In 2009, the EU Commission estimated that in excess of 3,000,000 European schoolchildren are obese and some 85,000 more children become obese every year. Obesity-related medical conditions include high blood pressure, high cholesterol, cardiovascular disease (heart attack and stroke), type 2 diabetes, cancer (most commonly breast cancer) and hernia incidents resulting in a higher risk of mortality. Even at a conservative estimate, obesity related illnesses are estimated to account for as much as 7% of total healthcare costs in the EU. Obesity related diseases account for and this chronic disease the cause of approximately 2.5 million deaths in Europe annually. The obesity epidemic and the growing elderly population, demographics present in many Western countries, indicate the growing need for the procedure of hernia repair. Hernia operations are among the most common surgical procedures performed today with over 20 million procedures worldwide annually. It is therefore of paramount importance to develop therapeutic strategies with clinical relevance. Such efforts must be addressed in international level in order to ensure development of common valuable platforms to address the problem. Our approach successfully fabricated the world’s first biomimetic biomaterial with clinical relevance for hernia repair using sustainable materials and green nanotechnologies. Frost & Sullivan estimated the 2005 European market revenue for implants to be US $2.48 billion at a compound annual growth rate (CAGR) of 6.2 per cent for the period 2006 to 2013. At the end of the forecast period, the market was expected to be worth US $3.78 billion. Biotechnology is considered one of the key enabling technologies of the 21st century to support the EU’s Lisbon strategy and sustainable development for Europe. About 40 products are currently on the market, mainly autologous skin replacements, cartilage and bone products, generating sales of about €60 million/year.
Modern biotechnology has widespread applications in human medicine and health care, which make a significant contribution to the EU economy. In fact, it has been postulated that wherever industrial biotechnology is applied, it has positive economic and environmental implications: industrial biotechnology increases labour productivity by 10% to 20% compared with conventional processes. Nanotechnology currently enjoys significant funding support both from the private and public sectors of the economy in most of the industrialised world. In 2005, total investment in nanotechnology was estimated to be in excess of €5 billion.
Nano-scale technologies are emerging as powerful tools for tissue engineering and biological studies. The supremacy of nanomedicine rests in its ability to operate on the same small scale as all functions involved in the growth, development and ageing of the human body. Biomedical nanotechnology is therefore expected to play a pivotal role in disease prevention, early diagnosis and effective treatment. It was estimated that the world nanomaterial demand will reach US $4.1 billion by 2011. Moreover Lux Research predicated that by 2014 nanotechnology will have been incorporated in manufactured goods worth US $2.6 trillion and will provide 10 million jobs. However, world RTD investment forecasts indicate that Europe’s investment in manufacturing, environmental technologies, and health is significantly lower than Asia and North America. The US is investing in research into the environment, health and safety risks of engineered nano-materials at an annual rate that is 3 times that of the EU. The European spend on environmental remediation and low carbon sectors was worth US $1.2 billion in 2008 and was projected to increase to US $1.64 billion by 2014 growing at a CAGR of 5.1%.
This compares with a global market of US $4.5 billion, which was projected to increase to US $6.3 billion by 2014 growing at a CAGR of 6.3 %. This was expected to increase at a compound annual growth rate (CAGR) of 61.8% to reach US $21.8 billion in 2014. It is therefore of paramount importance to develop strategies to facilitate the smooth transmission of nanotechnology from the discovery phase to translation without any negative impacts to human health that will become barriers to future development of the field. Initial efforts for the implementation of nano-technological entities were aimed at environmental applications, which focus on cleaning up the billions of tons of contaminants and waste materials that now permeate our air, water, and soil. The utilisation of robust and effective environmental remedial nanotechnologies may be considered as being positioned at the most critical of importance and demand levels however in order to truly address this challenge Europe needs to take on board sustainable development. This requires the development of green nanotechnology-based products and processes to eradicate the use and production of the wide range of pathogens, reactive chemicals, and toxins that currently contaminate our environment. Green Nano Mesh through this innovative Pan-European Industry-Academia collaboration contributed to the reduction of non-environmentally friendly raw materials; eliminated the use of hazardous substances in production processes utilising green nanotechnology; and promoted the use of sustainable eco-friendly materials for biomedical and pharmaceutical applications due to their superior biocompatibility and environmental friendly degradation process. Thus, Green Nano Mesh directly aligned with the European Commission commitment for a low carbon economy and the European Parliament Scientific Technology Options Assessment report for chemical substitution. As such, this Pan-European approach contributed to establishing a Green EU economy; increased the competiveness of European based biomedical industries; generated new Intellectual Property that will lead to generation of revenue and employment.

List of Websites:

http://www.greennanomesh.com/

Project information

Grant agreement ID: 263289

Status

Closed project

  • Start date

    1 June 2011

  • End date

    31 May 2015

Funded under:

FP7-NMP

  • Overall budget:

    € 3 617 820,20

  • EU contribution

    € 2 692 666

Coordinated by:

NATIONAL UNIVERSITY OF IRELAND GALWAY