Final Report Summary - ANASTOMOSEAL (Development of a resorbable sealing patch for the prevention of anastomotic leakage after colorectal cancer surgical treatment) Executive Summary:AnastomoSEAL is a EU co-funded project which lasted three years (01/05/2012-30/04/2015) with a Consortium composed of two Universities (University of Trieste, Italy, and University of Maastricht, the Netherlands), three Small-Medium-Enterprises (SIGEA - Italy, Rescoll - France and Impuls - Poland) and one Industry (FMC Biopolymer - Norway). The goal of the AnastomoSEAL project was the development of a biomaterial for preventing leakage of anastomosis following surgical treatment of colorectal cancer (CRC), the second most common form of cancer in Europe.Such biomaterial was prepared in the form of a bioresorbable membrane (patch) based on two polysaccharides: the first polymer (alginate) provides the mechanical support to the patch, while the second polymer (hyaluronan) is the bioactive substance promoting tissue healing.The patch has been developed and optimized throughout the whole project in terms of physical-chemical, mechanical and morphological features and on the basis of the feedback given by biological in vitro and in vivo studies.At the end of the project, the proof on concept was successfully demonstrated since the Consortium has been able to manufacture a biocompatible patch that can be wrapped around the intestinal anastomosis and appears beneficial to the tissue healing in an in vivo model.In addition, the Consortium has developed and injectable solution based on a hyaluronic-acid-butyric-ester derivative that was shown to be beneficial to the intestinal tissue healing in an in vivo model; this such solution can be applied in the form of enema in order to target the colonocytes of the internal part of the intestine.The two devices can be applied simultaneously, depending on the specific operating conditions and the surgeon’s strategy.Overall, the preclinical tests carried out during the project gave support to the conclusion that the developed devices have a great commercial potential. A provisional patent was jointly filed by academic and industrial partners to protect the intellectual property generated by the project regarding the devices. Project Context and Objectives:AnastomoSEAL is a EU co-funded project which lasted three years (01/05/2012-30/04/2015) with a Consortium composed of two Universities (University of Trieste, Italy, and University of Maastricht, the Netherlands), three Small-Medium-Enterprises (SIGEA - Italy, Rescoll - France and Impuls - Poland) and one Industry (FMC Biopolymer - Norway). The goal of the AnastomoSEAL project is the development of a biomaterial for preventing leakage of anastomosis following surgical treatment of colorectal cancer (CRC). CRC is the second most common form of cancer in Europe mostly affecting elderly people. The most frequent post-operative complication occurring at the resected bowel extremities is the Anastomotic Leakage (AL) which occurs when a rapid regeneration and sealing of the intestinal tissue at the resection site is not achieved. Anastomotic Leakage (AL) is one of the most severe complications after surgical treatment of colorectal cancer (CRC) with an incidence rate as high as 21 % and leads to a prolonged hospitalization, possible re-admissions causing a decrease of the quality of life of the patient, repeated surgical operations with a mortality rate in the 10% - 15% range. The AnastomoSEAL project, bringing together six European partners with different expertise, aimed at responding to the widespread clinical need of preventing AL after CRC resection developing an engineered bioresorbable biomaterial, capable to promote a rapid tissue healing after colorectal resection, with the formation of a safe anastomosis. The promotion of tissue regeneration is expected to endow this solution with ideal characteristics for reducing the incidence of AL. As the AnastomoSEAL project targets a so-far unmet medical need of relevant importance, its success could have significant economic and social impact. The concept underlying the project is to prepare medical device(s) based on the combination of:i. two bioresorbable and biodegradable biopolymers;ii. the first polymer is the carrier, providing the mechanical support and adhesiveness to the device;iii. the second polymer is the bioactive substance promoting tissue healing; iv. the expected activity and duration integrity for the device(s) is about two weeks,v. combining effective wound closure with easiness of manipulation for the operating surgeon.A distinctive objective of the project was to maximize the exploitation of in vitro testing and bioassay according to the 3R principles for the reduction of animal testing. The success of this EU project could have a marked economic impact in relation both to the cost of health care and to the global growth potential of the European biomaterials industry. Although a “golden standard” medical device for the prevention of the AL is still lacking, non-European and non-specific products are dominating the market of biomedical devices used for such treatment. A successful medical device sprouting from the project will help the European biomedical industry to keep a forefront position in the global market, at the same time fostering the virtuous cycle of productive collaboration between academia and industry, in particular the SMEs.After the end of the project, some partners have committed to try to use the results for the development of commercial products based on the technologies developed by the project. This commitment also extends to further scientific cooperation with the partners in writing scientific publications and in future research work.Project Results:Selection of materials:The criteria for the specifications of the materials used for patch preparation were defined. A database containing all material specifications of the ingredients was created by the Consortium. The main components selected were alginate, hyaluronan and a butyric ester of hyaluronan (HABut) which allows butyrate release both by chemical and enzymatic hydrolysis owing to the presence of reversible ester bonds.HABut batches covering a wide range of degree of substitution (DS) were prepared, starting from very low esterification levels up to exhaustive substitution on the HA hydroxyls. All technical problems arising from the use of high molecular weight (MW) polysaccharides had been tackled and were solved successfully.The optimization of the synthetic process starting from a low MW HA (300 kDa) to obtain a HABut derivative with DS = 0.3 was performed in view of the technological transfer to an industrial plant. Process parameters for the purification of the products were optimized and the information arising from these tests allowed to compare efficiency and costs of different approaches. This procedure was optimised and successfully applied to laboratory batches. The analytical procedures for quality control and validation of any batch were set up and standardized. Design of custom-made patch manufacturing techniques:Patch manufacturing was developed and optimized according to two types of procedures: • UNITS procedure (freeze-drying technology): the laboratory scale manufacturing of the patches devised by UNITS was based on the temperature-controlled freezing of the polysaccharide-based hydrogels followed by a freeze-drying step. In order to implement the production on a larger scale and to use a more automated process, the procedure was implemented in cooperation with the partner Sigea (SIG) to have a one-step process by using a Single-Chamber Freeze Dryer.• FMC procedure (foaming technology): the manufacturing of the membranes proposed by FMC was based on a foaming technology (FMC proprietary technology) described in the patent EP1663326B1 (Gelled biopolymer based foams). During the project a foam-production equipment has been installed to scale-up the process in a semi-continuous production line.Both processes were successfully developed throughout the project to manufacture patches with desired features. The manufacturing of the patches has been continuously optimized until the very end of the project on the basis of the material characterization and of the feedback given by the biological (in vitro and in vivo) studies. In the second period of the project, a re-design of the patch formulation was needed in order to improve the in vivo biocompatibility. Therefore, a re-design of the manufacturing process as well as of the rehydration protocol of the patch prior to implantation was devised. The Consortium was able to screen different patch formulations and to test them in vitro on cells, in chorioallantoic membrane (CAM) tests and in vivo on small animal models. As described by the process technology evaluation, at the end of the project no selection of a preferred technology for preparation of patches was made; in fact, both technologies, freeze-drying and foam extrusion, have demonstrated high flexibility in optimization of formulation and processing parameters and patches can most likely be made to meet requirements for the intended use. These two technologies are scalable to a commercially relevant size. The members of the Consortium do also believe that the option of two process technologies could benefit the project when aiming for commercialization partners.Material characterization (physical-chemical, mechanical and morphological features):Patches obtained by the two manufacturing techniques were characterized in terms of physical-chemical and mechanical properties as well as in terms of morphological features. Rheological properties of the hydrogels were measured in both liquid and gel phase. These studies pointed out the effect of the concentration of the main components and of the molecular weight of hyaluronan on the visco-elastic properties of the material.The mechanical properties of the freeze-dried patches were tuned in terms of resistance, stiffness and compliance by means or uniaxial tensile tests. Specific common protocols for mechanical tests have been established adapting existing ASTM and ISO standards; the protocols have been made available for the Consortium partners on secure intranet pages. These tests enabled to screen several formulations and to select the best candidates in terms of mechanical performance to withstand the surgical procedures. It should be noticed that these tests were performed both before and after the sterilization procedures. The mechanical performance of the material was also evaluated by UNITS and UNIMA surgeons during preliminary “patch-placement” tests. UNITS surgeons used ex vivo animal explants to test the response of the patches to surgical procedures like cutting, folding/unfolding, insertion through trocar, positioning on the tissue and wrapping with surgical laparoscopic tweezers. UNIMA surgeons performed “patch-placement” studies on non-dedicated pigs, to give a preliminary feedback on the resistance to handling of the patches during a real surgical procedure; UNITS and FMC best candidates were considered to possess sufficient mechanical strength both in laparoscopy and in open procedures. The degradation of the patches has been optimized until the very end of the project, aiming at ensuring good mechanical properties while tailoring the in vivo permanence (which is related with the biocompatibility) of the device; in particular, the material formulation was tailored in order to ensure the desired degradation profile, also in comparison with other commercial products already employed for internal surgery. A common protocol to study the patch degradation in vitro has been prepared with the contribution of all the partners and uploaded on the intranet pages. The studies carried out by UNITS showed that degradation rates depend on the formulation of the patch: the resistance to degradation increased upon increasing alginate concentration and in the presence of supporting salt. By varying the compositional parameters, the in vitro degradation of the patch can be tuned from few days to more than two weeks.Further material characterizations have been performed by UNITS, RC and FMC, by means of techniques like SEM, DMA and profilometry (surface roughness). SEM studies revealed the microscopic arrangement of the interpenetrated polyaccharidic mesh, DMA analysis pointed out the material behaviour under cyclic stress, while profilometry studies enabled to characterize the surface roughness of the patches.A common protocol to study the patch degradation in vitro has been prepared with the contribution of all the partners and uploaded on the intranet pages after the input given during the Consortium Meeting held in Gdansk.In addition, UNITS developed analytical tools for the quantification of the polysaccharide components released from the patch: more in the detail, the possibility to study the release of alginate and hyaluronan from the patch during degradation has been demonstrated by combining 1H-NMR and longitudinal relaxation rate of 23Na. Implementation of patch adhesiveness:One of the key points in the project, pointed out also by the components of the Clinical Advisory Board, relates to the development of an efficient strategy to reach a sufficient long-term adhesion between the tissue and the patch developed by the project. Despite the good initial tackiness of the patch upon contact with the intestine, preliminary “patch-placement” tests performed in vivo by UNIMA on non-dedicated pigs pointed out the need of an additional adhesive component to keep the patch in close contact with the intestine for the whole duration of the anastomotic healing (approx. 2-3 weeks).A considerable effort has been paid to implement the adhesive properties of the membrane; in particular, UNITS, RC and FMC carried out a synergic research activity to evaluate the possible use of commercial glues as well as to devise novel adhesive strategies for the patch. The Consortium focused on two strategies, one based on the exploitation of existing adhesives and another aiming at developing novel approaches specifically designed for the AnastomoSEAL patches (research track). Moreover, patches with and without a dried film of chitosan were prepared. No improved adhesion was seen with the chitosan film, while the presence of PVP was shown to be beneficial.During the process of developing new adhesive techniques, the standard mechanical tests were found not to be fully suitable for the measurement of the real adhesion forces between patches and intestinal tissue since they did not allow efficient ranking of the different adhesives selected. Therefore, a novel method was developed using the patches formulated within this project and ex-vivo colon tissue, which overcame this limitation. Due to a difficulty to achieve a constant daily supply of freshly harvested pig colon, a tissue substitute has been developed by RC to mimic, at the closest, the properties of the colon in terms of surface energy, tensile properties and surface chemistry. Several formulations had been screened before choosing a representative substitute prepared by blending poly(lactic acid), alginate and collagen. The adhesion tests were also performed on the tissue substitute according to a common protocol developed by the partners. As to the research track, two approaches based on the use of dopamine (mussel-glue strategy) showed promising results for the field; in particular, the innovative use of dopamine (grafted to alginate or in the form of nanoparticles) was demonstrated to be successful in improving the in vivo adhesion of the membranes.Evaluation and selection of sterilization techniques:One of the aims of the AnastomoSEAL project was the screening and comparative evaluation of sterilization methods for polysaccharide based materials. In fact, this mandatory technological step can represent a challenge, especially when using natural polymers for the manufacturing of biomaterials.During the project, three different methods of sterilization were compared (gamma radiation, gaseous H2O2 and supercritical carbon dioxide (scCO2)) and selected depending on their acceptance regarding international regulations. The mechanical properties of the patches were measured before and after sterilization in order to evaluate the impact on the material performances. It is important to stress that marked progresses have been made in the use of scCO2, which showed to be the method with the least drawbacks. In fact, results from testing various forms of sterilization showed that they all had detrimental effects on the molecular weight of alginate and therefore on the strength of the patch; supercritical CO2 induced the least effect on the patch and appears to be a good sterilization method. This represents an additional result achieved by the project.Analysis of standards for patch characterizations and regulatory issues:The following standards had been identified as important for AnastomoSEAL (Deliverable D6.4): • ISO 13485 – Medical Devices – Quality Management System• ASTM F2064 – Standard guide for characterization and testing of alginate• ASTM F2103 – Standard guide for characterization and testing of chitosan salts• ASTM F2605 – Standard test method for determining the molar mass of sodium alginate• ASTM F2347 – Standard guide for characterization and testing of hyaluronan• ISO 10993 – Biological evaluation of medical devices• ASTM D638 – Standard test method for tensile properties of plastic• ASTM F2258 – Standard test method for strength properties of tissue adhesive in tension• ASTM F1983 – Standard Practice for Assessment of Compatibility of Absorbable/Resorbable Biomaterials for Implant Applications• ASTM F748 – Standard Practice for Selecting Generic Biological Test Methods for Materials and DevicesMoreover, it should be noticed that ASTM F2064, ASTM F2103 and ASTM F2605 were initiated, contributed and/or published by FMC Biopolymer/Novamatrix. In addition to the work reported on the interim scientific report on the project, partners had worked with ASTM on creating standards for hydrogel characterization as well as draft documents related to testing methods for an alginate-based patch.Referring to the final potential application, the AnastomoSEAL implantable medical device, comprising a patch and an enema solution, can be considered as CE marked Class III medical device for the patch, and as CE marked class II medical device for the enema solution. In order to provide guidelines for the development of a test program to ensure the safety of the AnastomoSEAL medical device, an analysis of the regulatory issues has been performed. The Consortium highlighted that, in 2014, the European commission has adopted two new Regulations of the European Parliament and of the Council on medical devices and on in vitro diagnostic medical devices. They replace the three existing medical devices directives including Directive 93/42/EEC. They will gradually come into effect from 2015 to 2019. They will have a significant impact on the actors (manufacturers, distributors, etc.) and will redefine roles and responsibilities of public and private organizations that operate in the field of medical devices within the European Economic Area. Moreover, it was pointed out that alginate and hyaluronan biopolymers, used for the manufacturing of the patch, are currently marketed as CE marked Class III products with various uses (e.g.: controlled-release drug delivery systems and medical implants). The key features of these polysaccharides (which are positive attributes from a regulatory perspective) are: • Biocompatibility: A potential biocompatibility file of the AnastomoSEAL implantable medical device should include procedures (and/or justification) for cytotoxicity, skin sensitization, dermal irritation and intracutaneous reactivity, acute systemic toxicity, subchronic toxicity, mutagenicity, implantation, chronic toxicity and carcinogenicity.• Biodegradability: The biodegradability of the medical device will have to follow the guidelines of ASTM F2902-12 “Standard guide for assessment of absordable polymeric implants”, ASTM F1635-11 “Standard test method for in vitro degradation”, ISO/TR 37137:2014 “Biological evaluation of medical devices -- Guidance for absorbable implants”, ASTM F2900-11 “Standard Guide for Characterization of Hydrogels used in Regenerative Medicine” and ASTM F1983 - 99(2008) “Standard Practice for Assessment of Compatibility Absorbable/Resorbable Biomaterials for Implant Applications”.• Possibility to be sterilized: The sterilization of the AnastomoSEAL medical devices will need to follow the guidelines of EN 556-1/2002 “Sterilization of medical devices: Requirements for medical devices to be designated “STERILE” – Part 1: Requirements for terminally sterilized medical devices”; EN ISO 11737-1: 2006 “Sterilization of medical devices – Microbiological methods – Part 1: Determination of a population of microorganisms on products”; EN ISO 11737-2: 2010 “Sterilization of medical devices - Microbiological methods – Part 2: Tests of sterility performed in the definition, validation and maintenance of a sterilization process”; ISO 11137-1: 2006 “Sterilization of health care products – radiation – Part 1 - Requirements for development, validation and routine control of sterilization process for medical devices”; ISO 11137-2: 2013 “Sterilization of health care products – radiation – Part 2 – Establishing the sterilization dose”; ISO 11137-3: 2006 “Sterilization of health care products – radiation – Part 3 – Guidance on dosimetric aspects”.• Processability: The polysaccharides used for patch manufacturing should be processed by techniques that comply to GMP standardsInnovation activities:During the different Exploitation task groups, it was discussed the possibility to fill a patent application to protect the knowledge created during the project. Thus, a US provisional patent was filed during the last months of the project. The assignees of the patent are: University of Trieste, Michael Dornish, Therese Andersen, Sigea S.r.l. The claims of the patent cover:• The use of patch a based on polysaccharides for the prevention of the anastomotic leakage and other medical applications (Patch);• The use of HABut as enema treatments for the prevention of the anastomotic leakage (Enema); • The combined use of i) and ii) for the prevention of the anastomotic leakage and other medical applications (Patch + Enema).In vitro biological characterization of the raw materials: An initial important result of the Project has been represented by the in vitro biological characterization of the biomaterials, both native and engineered, selected for the manufacturing of the medical device. Three polysaccharides have been selected for the manufacturing: alginate, hyaluronic acid and the hyaluronic acid derivative (HABut). The activities that were coordinated by the University of Trieste were aimed at the selection of the best performing materials, in terms of molecular weight and, for the hyaluronic acid derivatives, degree of substitution. An alternative use of the hyaluronic acid derivatives has been also proposed and evaluated during the activities of the Project, i.e. the internal administration of a HABut solution by enema for the treatment of the proximal colon anastomosis.A set of techniques has been selected and optimized for the Project needs, by using cell cultures of primary fibroblasts and colonocytes.The biological characterization was based on the study of: i) cell viability, proliferation, and migration; ii) synthesis of extracellular matrix (ECM) components, such as glycosaminoglycans (GAGs) and collagen; iii) biopolymer internalization, by means of flow cytometry analyses.These tests have been combined to the use of in vitro model systems to study the promotion of tissue healing (wound healing assays), and the influence on the acute inflammatory response.The wound healing assay is a laboratory test based on the simulation of a wound (scratch) on a layer of cultured cells, followed by the monitoring of the cellular response. The cells in culture respond just like cells in the body, with an attempt to fill the gap and to close the wound by migration and/or proliferation. The kinetic of the closure of the gap is monitored and measured by using a microscope combined with a camera, and a software for image analysis.The influence of the selected biopolymers on the acute inflammatory response has been studied by evaluating the effects on leucocytes polymorphonuclear (PMNs). Three essential functional responses of the activated cells have been analysed: i) the production of oxygen reactive species (ROS) following the metabolic activation (respiratory burst); ii) the capability to adhere to biological surfaces; iii) the release of granule components. The capability to control these functional responses has been assessed to evaluate the potentiality to induce in vivo a repression of the acute inflammatory response, promoting in this way the initial phases of tissue regeneration.The beneficial and useful properties of hyaluronic acid (HA) in promoting tissue healing have been demonstrated, thus justifying the rationale of the use of HA as bioactive component of the medical device to be developed (surgical patch). Another important result was the demonstration of the hypothesis of the positive effect in tissue healing of specific hyaluronic acid derivatives as soluble system.In vitro biological characterization of the medical devices developed:The manufacturing of the medical devices (surgical patches) has been continuously optimized until the final phase of the Project on the basis of the material characterization and of the feedback given by the biological studies.The aim of the in vitro biological characterization was the selection of the formulations to be used in the animal experiments, by varying the formulation composition (for example biopolymer concentration, presence and type of plasticizer). The in vitro biological characterization of the patches has been carried out by following the procedures described in the International Standard ISO 10993-5, testing the liquid extracts of the devices. The platform of tests developed and optimized for the raw materials has been applied to the study on biopolymeric patches. The patches were studied in vitro in terms of biocompatibility and pro-healing effects by using the cellular models described above. According to the results obtained, the patches to be tested in vivo have been selected.The platform of tests described has been implemented with additional in vitro (laboratory) tests. In particular the CAM (chorioallantoic membrane) assay and further immunological tests, such as the macrophage stimulation studies (TNF-α production), have been carried out.The CAM assay is a method that can be used for the study of many biological processes, which include toxicity and angiogenesis; in the field of biomaterial science it is applied to the study of the reaction of a tissue, in order to evaluate the material biocompatibility.The data obtained by using the CAM assay and macrophage stimulation studies have led to the selection of the best candidates (in terms of formulation composition) for the second phase of the in vivo tests (biocompatibility and efficacy).In addition to the tests performed on the materials addressed to the animal studies, biological studies have been also performed on medical devices (patches) sterilized by different experimental techniques, developed and/or optimized during the Project, and on patches formulated to increase the adhesion forces between the biomaterial and the intestinal tissue. In vivo biological characterization of the medical devices developed:In the organization of the Project, a full Work package was devoted to the preclinical testing (in vivo) of the materials and devices developed. The Work package was coordinated by the Partner University of Maastricht. The choice for the animal models used during the project was based on an extensive literature study. The patches developed were tested in the preclinical setting, articulated in several phases: a biocompatibility rat model (tissue without sutures), and two validated models of healing. The first one in the presence of a non-leaking anastomosis (tissue with 12 sutures), and the second with 4 interrupted sutures that provide a high leakage rate. In addition to the patch studies, animal studies for a research track were also performed. For this purpose solution of hyaluronic acid derivatives were injected in the proximal colon prior to performing the anastomosis. Moreover, in vivo studies were performed, as previously described by Bloemen et al. (Diseases of the Colon & Rectum, July 2010 - Volume 53 - Issue 7 - pp 1069-1075). The preliminary results of this trial suggested a potential beneficial role of the hyaluronic acid derivative. The main results of this part of the Project are reported in the following list.i) Definition of animal models and in vivo testing of the medical devices developed:- Testing of different formulations of Alginate/Hyaluronan patches (“First generation”) on rat models;- Testing of optimized and modified Alginate/Hyaluronan patches on rat models (“Second generation”);- Testing of solutions containing HABut as in situ enema systems; ii) In vivo testing of the patch biocompatibility:- No adverse reactions;iii) In vivo testing of the patch efficacy:- Absence of anastomotic leakage (AL) for patches with alginate and gelatin matrix;- Improvement of anastomotic healing for patches with gelatin matrix;iv) In vivo testing of the enema efficacy- Improvement of anastomotic healing; The most significant results were obtained by tuning the composition of the polymer matrix (alginate-HA patch); the improvement of the anastomotic healing can be achieved by the protein-based patch with HA that degrades quickly, and enhances the healing process in the early phase of wound healing. As an additional study, the effect of the plasticizers employed for the preparation of the patches, in combination with the different biopolymeric matrixes, have been tested in the three different models (biocompatibility and anastomosis with 4 and 12 sutures).The preclinical tests carried out by the project gave a support to the conclusion that the developed devices have a commercial potential. The normal duration of this type of projects is from four to five years: therefore given the time limitation (three years), the expected final result was to provide a “Proof-of-concept” for the successful preparation of a medical device to prevent AL. The availability of a patch for phase 1 clinical trial will require additional efforts for an estimated period of 12 to 18 months. Crucial items to be improved regard, e.g. the setting up of the role of plasticizers in imparting the necessary pliability and softness to the patch, having ascertained in the preclinical study that some low molecular weight compounds commonly used in biomaterials may induce severe adverse effects in the abdomen.Partners are expected to be committed in using the results in further development of commercial products based on the patch and enema technologies. Expectation also extends to further scientific cooperation with the partners in writing scientific public publications and in future research work.Potential Impact:The potential impact of the project is hereafter reported and discussed in terms of socio-economic impact and impact on industries and SMEs. Socio-economic impact:AnastomoSEAL project was focused on responding to the widespread clinical need of preventing anastomotic leakage (AL) after colorectal cancer (CRC) resection. CRC is the second most common form of cancer and the third most cancer cause of death in Europe, with an aged-standardized incidence rate of 48 cases per 100 000 in 2008. CRC is generally a malignancy associated with elderly, with a mean age at diagnosis of 73 years. Anastomotic Leakage (AL) is one of the most severe complications after surgical treatment of colorectal cancer (CRC) with an incidence rate as high as 21 % and leads to a prolonged hospitalization, possible re-admissions causing a decrease of the quality of life of the patient, repeated surgical operations with a mortality rate in the 10% - 15% range. At present, there are no materials capable of satisfy entirely the clinical needs in the prevention and treatment of AL. The divergence between incidence (upward trend) and mortality rates (downward trend from the mid-1990s onwards) suggests an increase of early detection of the disease with a corresponding shift to an earlier stage and improvements in therapy, but at the same time this trend is having important consequences in the disease prevalence. In spite of improvement of the surgical technique and accumulated experiences, AL is still one of the most severe complications, with up to 21 % incidence rate. At present, AL leads to a prolonged initial hospital stay, possible re-admissions and sometimes consequences extending over several years; it requires urgent return to the operating room, repeated surgical operations and death is not uncommon. The mortality rate for an AL in the literature typically is in the 10 % to 15 % range.Apart from the social aspects, the economic impact of the AnastomoSEAL project is significant as it reflects in the cost of health care and the global growth potential of the European biomaterials industry. The management of anastomotic leakage by the health care system is very onerous. AL generates a very considerable demand for hospital resources and diverts these resources from the hospital population at large. This economical cost will increase in a linear fashion because of the aging of the population.Significant contributions to the cost build-up are prolonged hospitalization, radiographic studies during and after the admission, consultation services, more extended use of the operating room, stoma-related complications, re-intervention for ostomy closure, longer nursing care, materials (e.g. antibiotics, ostomy bags). No attempt was made to assess the total cost involved for a sample case of colorectal leak because public hospitals do not have a scale of charges for the individual consultations and services involved and, in any case, charges generally do not reflect actual costs. Moreover, the costs of rehabilitation, physiotherapy, prolonged absence from work of the patients and sometimes of members of the family must be considered. A literature search failed to identify any studies which have quantified the variety and volume of these resources. Nevertheless, it is self-evident that a reduction of the anastomotic leak rates by AnastomoSEAL would produce huge health savings by increasing the cost-effectiveness ratio.AnastomoSEAL project targeted a so-far unmet medical need of relevant importance, its success is expected to have significant economic and social impact. The Consortium has been scrutinizing the market in order to get updates on the products that were proposed. This task was achieved through two dedicated deliverables which revealed that, although many products have efficiently reached the market, none of them was specifically developed for the prevention of AL. Therefore, the development of a biomaterial designed specifically to prevent AL is still sought by the scientific community. The AnastomoSEAL consortium aimed at developing a biomaterial inserted by the surgeon after the anastomotic procedure which wraps around the sutured tissue to promote physiological process of tissue regeneration, to seal the external part of the intestine and increase early mechanical stability, thus preventing anastomotic leakage.This novel biomaterial has the potential to decrease the cost of health care by decreasing the number of CRC patients suffering from this complication thus leading to a substantial socio-economic impact .The normal duration of this type of projects is of four to five years: therefore given the time limitation to three years, the expected final result is to provide a “Proof-of-concept” for the successful preparation of a medical device to prevent AL.The concept underlying the project is to prepare medical device(s) based on the combination of:i. two bioresorbable and biodegradable biopolymers;ii. the first polymer is the carrier, providing the mechanical support and adhesiveness to the device;iii. the second polymer is the bioactive substance promoting tissue healing; iv. the expected activity and duration integrity for the device(s) is about two weeks, combining effective wound closure with easiness of manipulation for the operating surgeon.Successful demonstration of the “Proof-of-Concept” for the specific target of preventing AL after CRC resection will open venue to other applications not only in gastro-intestinal surgery, but also in other areas if internal surgery.A distinctive feature of the project is to maximize the exploitation of in vitro testing and bioassay according to the 3R principles for the reduction of animal testing. An additional result of the project was the accurate scrutiny of the toxicity of chemical components usually employed in the preparation of medical devices to impart desired surgical manipulation properties. This brings about a notable impact from the point of view of the overall costs of the material and reduction of the number of in vivo experiments carried out.Besides the desired impact for the patient, a more general positive social impact of the project will be within the general EU action on “Healthy Aging”. Healthy ageing is about "optimising opportunities for good health, so that older people can take an active part in society and enjoy an independent and high quality of life" (Swedish Institute of Public Health 2006 - A Challenge for Europe). The European Commission has identified active and healthy ageing as a major societal challenge common to all European countries, and an area which presents considerable potential for Europe to lead the world in providing innovative responses to this challenge. As already stated, AL is one of the most severe complications after surgical treatment of CRC, with a relatively high incidence rate, often leading to a prolonged hospitalization, possible re-admissions causing a decrease of the quality of life of the patient. This is particularly true for the elderly persons, seriously threatening their life and certainly heavily reducing their quality of life. Impact on industries and SMEs:The AnastomoSEAL project has represented a direct and indirect economic benefit for the industrial partners present in the Consortium. All industrial and SME partners had the opportunity to increase their competences in the field of biomaterials. In addition, being partners in a Consortium of a European project brought about notable advantages in terms of networking in a very challenging market sector and of providing new customers, and it indirectly generated turnover. In addition, some already existing technologies have been used within the project thereby adding value to the proprietary technology and allowing increasing the existing small scale productions of biomaterial to a prototype level. Furthermore, the work performed in the AnastomoSEAL project has allowed developing novel Standard and Guidelines for biopolymers in medical applications which have generated industry-wide consensus standards also recognized by the U.S. FDA. This aspect has notable impact on the possibility of increasing the market for some of their products. The number and role of industrial partners (and of SMEs in particular) in AnastomoSEAL project clearly show that a special attention has been given to the sector. However, it is expected and desired that the number of those potentially interested in industrialization, expansion to new clinical targets and to wholly different application sectors could be much larger given the proof of concept reached so far in three years’ time. In general terms, the European SMEs dealing with bio-based products will benefit from the application of their core-business products in a high-added value area.Besides the benefits for the industrial and SME partners in the project, the AnastomoSEAL project might lead to a positive impact on the European biomedical device industry in general. Some of the existing focus themes of the public health and industrial challenges in the biomaterial sector are specifically addressed by AnastomoSEAL. The aspects of biomaterial development tackled by AnastomoSEAL could lead to an adequate answer of the European industry to a societal problem due to increase of demographic trends. In addition, a general competitiveness and innovation of the medical device industry might benefit from the R&D innovations brought about by the project towards presently unsolved medical problems in the treatment of AL and other complications connected with general surgery procedures. Within the AnastomoSEAL project, a combination of natural products and biopolymers has been used in a novel assembly at the cross point of nanotechnology and green chemistry. This effort, during the three years of life-time of the project, has led to a convincing proof of concept. It might lead to safe, innovative, useful and affordable products to be launched in the market. AnastomoSEAL is focused on the response to a specific medical need, but at the same time the results and outcomes of the project could be easily and efficiently adapted to answer a much larger range of clinically relevant issues, thereby enormously amplifying the technological life-time of the new medical device. Therefore, the AnastomoSEAL project might have an impact on different and variable clinical applications, thus showing the holistic approach of the project which considers the whole clinical pathway and the full life-time of a product. Dissemination:Dissemination of the results represented an important issue and an opportunity to present the outcomes of the project to the scientific community, policy makers , press and general public. For this reason, a specific work-package was dedicated to this topic. Open, proactive and systematic communication was ensured on the project aims on the results not covered by IPR protection.Dissemination plan has been organized on both an internal and an external level. As to the internal level, the dissemination activities were focused on the consortium members. In particular, the organization of internal scientific seminars and the use of a protected platform available for all the partners involved in the project allowed the dissemination of the scientific and technical results of the work of the consortium members. The use of these approaches allowed fostering the discussion over the achievements and the brainstorming within the project partners on the results. Internal dissemination has been carried out also by means of training seminars and visits. Indeed, this dissemination action was devoted to increase the competence of the young researchers involved in the project. In particular, visits to facilities of some of the beneficiaries were arranged for PhD students who had the chance to improve their technological skills by confronting with other members of the Consortium. In addition, the preparation of the Deliverables allowed confronting between all the beneficiaries involved in the project on the scientific results and on the technical considerations over risk analysis, market analysis and process flow diagram for production to name a few. Overall, sixty Deliverables were produced by the Consortium partners.Within the duration of the AnastomoSEAL project, much effort was devoted to the external dissemination as an efficient mean to raise the awareness over the project and to disseminate the results to the scientific community. A public domain internet page on the project was created to generate an effective flow of information and publicity about the objectives. The web pages were intended to provide the main information on the AnastomoSEAL project to the general public and to the scientific community. Along the same line, a project brochure was realized to reach the scientific community and stakeholders in the field with the basic information on the project aims and targets. The brochure was distributed at public scientific seminars and meetings and to research centers. Another fundamental aspect of the dissemination was related to the diffusion to the competent bodies of the standard methods developed within the project. Along this line, one of the beneficiaries of the project has taken part to the definition of standards for the characterization and use of biopolymer based materials organized by ASTM. An extended dissemination of the results not protected by any IPR has been carried out to define standards applicable to the case of biopolymer based biomaterials. A specific logo for AnastomoSEAL project has been developed in order to increase the awareness and the impact of the project. Attention has also been devoted to the use of large audience media to diffuse the aims of the AnastomoSEAL project. Along this line, press release documents, radio and television broadcastings have been arranged. In order to conveying to the general public the aims and main scopes of the AnastomoSEAL project, video-interviews have been realized and uploaded on the project website. These interviews involved also young researchers to increase the appeal of scientific carriers. In order to keep track of the communication activity and to avoid potential conflicts as to IPR issues, all the communications have been first discussed within an Exploitation Task Group and all the abstracts, poster and oral communications received the clearance by all the beneficiaries prior to publication. The promotion of the aim of the project and of its results when not protected by any IPR was performed with respect to both the scientific community, by means of the participation to conferences for poster or oral presentations, and the industry, by means of participating to workshops and exhibitions. The need of protecting potential intellectual property rights has delayed the publication of the high amount of scientific data that has been collected. However, within the duration of the project three original papers related to the project have been published in peer reviewed journals. The main dissemination target of the AnastomoSEAL project, considering that part of the project was oriented to research, was the external dissemination towards the scientific community on two levels. The first level was the promotion of the AnastomoSEAL project through short communications to conferences and scientific seminars. These communications are intended to detail the scientific achievements of the project only when potential IPR aspects are cleared. Overall, 16 communications (posters and/or oral contributions) have been carried out. The second level involved the dissemination of the scientific results by means of publications in specialized peer reviewed journals. In this case, the aim is to produce the experimental results to the evaluation of the scientific community to evaluate their feasibility and correctnessSpecifically, the three original publications are the following:1) Borgogna, M.; Skjåk-Bræk, G.; Paoletti, S.; Donati, I.; On the initial binding of alginate by calcium ions. The tilted egg-box hypothesis; J. Phys. Chem. B; 2013; 177; 7277-7282.2) Geremia, I.; Borgogna, M; Travan, A.; Marsich, E.; Paoletti, S.; Donati, I.; Determination of the composition for binary mixtures of polyanions: the case of mixed solutions of alginate and hyaluronan; Biomacromolecules; 2014; 15; 1069-1073. 3) Travan, A.; Fiorentino, S.; Grassi, M.; Borgogna, M.; Marsich, E.; Paoletti, S.; Donati, I.; Rheology of mixed alginate-hyaluronan aqueous solutions; Int. J. Biol. Macromol.; 2015; 78; 363-369.In addition, a review on adhesives used in general surgery applications has been published1) Scognamiglio, F.; Travan, A.; Rustighi, I.; Tarchi, P.; Palmisano, S.; Marsich, E.; Borgogna, M.; Donati, I.; de Manzini, N.; Paoletti, S.; Adhesive and sealant interfaces for general surgery applications; J. Biomed. Mater. Res.; 2015; in press.In addition to the reported published manuscripts and after filing a provisional patent in the U.S.A. to ensure proper IP protection, the various beneficiaries of the Consortium started drafting eight more papers, planned to be submitted for publication by mid 2016 at the latest. Exploitation of results:The proper management of the intellectual property was one of the main concerns of the AnastomoSEAL consortium. The protection of the IPR and the analysis of the existing knowledge in the field was a specific task of a work package, whereas the achievement of a patentable result was the general goal of the whole project.Different aspects of the topic were considered. Firstly, a constant scrutiny of the novelty of the fundamental idea underlying the project proposal was carried out. No similar solution to the AL problem appeared neither in the scientific nor in the patent literature during the time course of the project. Albeit conditioned by the limited duration of the project (three years), the final goal was largely achieved, inasmuch the results collected in the duration of the AnastomoSEAL project led to the filing of a provisional patent in the U.S.A. jointly by academic and industrial partners. Two types of medical devices were identified, corresponding to the two initial working hypotheses regarding the healing potential, namely: that of hyaluronan, entrapped within a patch made by calcium alginate and applied by the surgeon externally to the anastomotic site, and that of butyrate, as the ester derivative of hyaluronan, to be applied as an enema. A possible combination of the two methods was also protected in the provisional application. As to the protection issues, a very careful analysis of the dissemination was carried out in order not to disseminate the knowledge prior to suitable IP protections actions. Therefore, all the documents, abstracts and manuscript produced by the Consortium and aimed at being disseminated have been reviewed by the Exploitation Task Group prior to the publication. The careful check on potential IP issue has been guaranteed by the Exploitation Task Group over the scientific reports delivered at the General Assembly. The Task Group, besides evaluating the potential IPR linked to a scientific result, focused also on the check for potential infringement of some potential IP foreground in accordance with the Consortium Agreement. An important outcome of the AnastomoSEAL project is that the work performed and the results collected had also led some of the researchers to set up a new SME (spin-off of the University of Trieste) which will be started in 2016.List of Websites:Public website: www.anastomoseal.euUniversity of Trieste (Coordinator): dr. Ivan Donati, prof. Sergio Paoletti (biomat@units.it) University of Maastricht: Nicole Bouvy (n.bouvy@mumc.nl)Sigea S.r.L.: mr. Luca Stucchi (stucchi@sigea.biz)Rescoll: mr. Konstantin Sipos (konstantin.sipos@rescoll.fr) Marie-Pierre Foulc (mp.foulc@rescoll.fr)FMC: dr. Michael Dornish (michael.dornish@fmc.com dornish@online.no) Therese Andersen (THERESE.ANDERSEN@fmc.com) David Davis (David.Davis@fmc.com)IMPULS: Wladyslaw Fediuk (impulsgdansk@gmail.com)
