Periodic Reporting for period 1 - REMIX (Regenerative Medicine Innovation Crossing - Research and Innovation Staff Exchange inRegenerative Medicine) Reporting period: 2017-11-01 to 2019-10-31 Summary of the context and overall objectives of the project REMIX works in Tissue Engineering and Regenerative Medicine (TERM), a field of biotechnologies that researches methodologies to trigger the regeneration of damaged human tissues for healing purposes. TERM is a high-potential field thanks to its aim to promote self-healing therapies which are less invasive and more patient-oriented than transplants.The main objective of REMIX is to investigate and develop natural or nature-inspired biomaterials for TERM therapy through collaboration among European and Asian experts with different academic and scientific backgrounds.• exploring innovative applications of Natural materials;• benchmarking results, standards and procedures;• comparing the different approaches and regulations in partners’ countries;• building in vitro models and technologies, and defining agreed procedures aimed at reducing the need for animal testing;• equip post-docs, PhD students and younger researchers with the competence to develop TERM products with the use of natural resources and materials. At the end of the program we expect 24 researchers to be fully trained in the field;• train post-docs and PhD students in research methods and TERM. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far During the first two years, 12 natural materials have been selected and 5 of them are currently undergoing research. Scaffolds have been produced with silk fibroin, alginate based hydrogels and gellan gum and stem cells have been successfully encapsulated. The combination of gellan gum with silk fibroin, processed in different forms has shown adequate properties for TE applications and their properties and well as the response of different cell types on such materials has been investigated. WP1: For the selected materials (silk fibroin and sericin isolated from silk cocoon, keratin isolated from animal hair, collagen isolated from duck foot, and sea buckthorn oil extracted from officinal plants), all the processing, from purification methods optimization using environmental friendly solvent, like ionic liquids, standardization to allow reproducibility, costs and processing time were designed. All the isolated polymers/inorganic phases (calcium phosphate bioceramic obtained from natural resources such as cuttlefish bone) were physico-chemical, mechanically characterized by using well-defined methods that are used by the consortium as quality control system. Isolated materials were then evaluated from biocompatibility point of view, and well-defined and selected assays were used.WP2:For each material, some tissue engineering applications were considered and matrices specifically designed (scaffolds and beads for bone regeneration, hydrogel and films for wound healing and drug release system, and microsphere for cell carriers). Different fabrication methods (novel method for crosslink silk fibroin hydrogel, micro beads and microsphere preparation) of scaffolds were designed, established and optimized according to specific requirements from chemical, biological and physical point of view. WP3:In order to break the bottleneck of applying designed scaffolds into tissue engineering, preliminary biological evaluations were performed on scaffolds. Protocols in vitro were established and optimized for testing the cytotoxicity of the materials and scaffolds, cell metabolic activities, proliferation, differentiation and morphology observation. WP4:The properties of the scaffolds were characterized and tests with stem cells were performed. Properties such as stem cell morphology, differentiation and characteristics on these scaffolds have been evaluated, bone marrow stem cells (BMSCs) behavior after long time in culture on methacrylated silk fibroin (met-SF) scaffold and silk fibroin based hydrogel, loaded with commercially available neural stem cells in order to create a bilayer hydrogel patch as a bypass for spinal cord regeneration is ongoing. The morphology and characteristics of adipose stem cells on alginate based hydrogels has been investigated in detail in order to help in the future to engineer novel substrates that induce targeted cell behavior. The response of adipose stem cells on gellan gum based scaffolds is also ongoing. Namely, their differentiation in the osteogenic lineage has been analysed in order to be used for for bone repair strategies.4 papers in peer-reviewed journals published2 patents submitted15 ESRs have participated in exchange, research and training activities in partner universities abroad. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The project has made progress beyond the current state-of-the-art:- A novel cross-linking method of silk fibroin hydrogel was established by using Au nanoparticles. This method induced Au particles inside hydrogel with antitumor properties.- A new composite system based on millimeter-sized spheres of beta-tricalcium phosphate, hydroxyapatite, alginate and silk fibroin, which could be used for bone tissue engineering applications.- A double controlled dermal delivery system is designed. It can release content by thermal sol-gel transfer and it also has multilayer release by high pressure instrument. The system has tunable porous structure and is built by natural polymer with nontoxic and good biocompatibility. The content includes a wide range of liquid and there is no limitation of the number of contents in a single system. The fabrication method is easy, fast and low cost, as well as the material.- Keratin from camel hair and goat cashmere was extracted by dissolving in an ionic liquid (1-butyl-3-methylimidazolium chloride), and the characteristics (no literature before) of the soluble and insoluble keratin were evaluated. Preliminary in vitro biological properties performed by a lactate dehydrogenase (LDH) assay and scratch test showed good bioactivity in keratin from both sources. - A porous microsphere of silk fibroin and gelatin for cell carrier was designed.- Extraction procedures to obtain calcium phosphate (CaP) from black chicken bone were successfully developed. - CaP from black chicken bone was combined with gellan gum to obtain hybrid scaffolds. It was found that this CaP acts as an extremely effective reinforcement agent and is non cytotoxic. These hybrid scaffolds have been investigated for Osteochondral Regeneration.- New injectable dexamethasone-cyclodextrin complexes-loaded gellan gum hydrogels were developed. The results of their physicochemical characterization and their biological properties (ongoing) showed that these are quite promising materials for cartilage regeneration- New hydrogels based on silk fibroin and gellan gum were successfully produced, which present adequate and tunable properties as a function of their composition, and could be used to regenerate cartilage.- Innovative films based on hyaluronic acid and chitosan, with or without inorganic nanoparticles, were produced by layer-by-layer assembly, with distinct formulations. The produced films could be used in a variety of applications namely for bone regeneration purposes. For instance, these films could be potentially used as adhesive coatings of orthopaedic implants, in order to promote osteogenesis and hydroxyapatite deposition around the implant. Other formulations of the developed fims, which we demonstrated that are highly adhesive, could be used to improve the junction between distinct orthopaedic implants and a variety of hard tissues, in a simple and versatile way. Kaewprasit’s attendance to a training course on Biomaterials and Tissue Engineering (WP1) Sahapaiboonkit, Yamdech and Raggio during labwork at UNITN Wp2-3) Helena Ferreira’s visit to the Stem Cells Laboratory at Chulalongkorn (WP4) PA moment of the round table at TICME conference (WP7) Group photo at TICME Conference in Trento (WP7) Rosasilvia Raggio and Dr. Walter Bonani attended a work shop on spider silk materials (WP1-2) Steering Committee meeting at UNITN (WP6) Steering Committee meeting at Chulalongkorn University (WP6)
