Periodic Reporting for period 1 - BIOeSPUN scaffolds (Bioactive glass in electrospun matrices: functionalised smart scaffolds for interface tissue engineering applications) Reporting period: 2015-06-01 to 2017-05-31 Summary of the context and overall objectives of the project “BIOeSPUN scaffolds” project aims the fabrication and characterization of novel types of multilayered scaffolds suitable for interface tissue engineering (ITE) applications, in particular for osteochondral segment regeneration. Osteochondral lesions are defects or diseases which usually involve the cartilage, bone or the interface cartilage-to-bone tissue and they can occur in all the joints of the human body, like knee, shoulder, elbow, wrist, and hip. The causes of these articular defects could be traumatic or due to aging-related disease, involving young, adult and older patients. For this reason an effective and long lasting treatment is needed. It is fundamental to mention that articular joint disease (and generally musculoskeletal conditions) is included in the list of major and chronic disease (MCDs), defined as pathologies affecting at least 50 per 100000 people, causing together 87% deaths in EU. In this framework, interface tissue engineering (ITE) offers an innovative approach for the regeneration of interface tissues, like osteochondral segment. ITE aims the fabrication of scaffolds able to mimic the complexity and anisotropy of the native tissues. “BIOeSPUN scaffolds” project reported the successful fabrication and characterization of multilayered graded scaffolds for the osteochondral segment regeneration. The electrospinning technique was selected for the fabrication of fibrous scaffolds, mimicking the morphology of the native extra cellular matrix. Benign solvents were used for the electrospinning process to avoid the use of toxic harsh solvents, having advantages in terms of avoidance of toxic solvents residuals in the obtained scaffolds. The use of benign solvents for the electrospinning is innovative and challenging because often these solvents require additional process parameters optimization. The electrospinning technique was combined with foam replica method and dip-coating for the fabrication of scaffolds having morphological and compositional gradients among the different layers. Cell biology studies were performed on the obtained multilayered scaffolds. The additional value of this project was represented by the training activities which allow the enrolled researcher to improve her skills and to complete her interdisciplinary profile. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far At the beginning of the project a bibliographic research was performed on the state-of-the-art about the use of benign solvents for the fabrication of scaffolds for tissue engineering and in particular for the combined use with other scaffolds fabrication techniques for the development of multilayered graded scaffolds. After the characterization of the single layer scaffolds obtained from the electrospinning with acetic acid and formic acid, composites fibers were obtained after the addition of bioactive glass (BG) particles in the solution before the electrospinning. BG particles were selected because of their well-known effects on osteogenesis, angiogenesis and their antibacterial activity. For the selection of the polymers, a synthetic polymer, poly(epsilon-caprolactone) (PCL), and a natural polymer, chitosan, and their blends were chosen as biomaterials for the scaffolds fabrication, because of their biocompatibility and documented use for scaffold fabrication. The focus for the replication of subchondral bone was oriented on the development of composites fibers, having a polymer matrix (constituted by PCL and PCL/chitosan blends) with the addition of BG particles having different glass compositions and particles size. Positive results were obtained, confirming the incorporation of the BG particles inside the polymeric matrix and the preservation of the BG bioactivity, confirmed after the immersion of the samples in simulated body fluid (SBF) solution. The mineralization observed on the composite fibers confirmed their suitability in mimicking the subchondral bone side in multilayered scaffolds. For mimicking the cartilage side a polymeric electrospun matrix was selected. Positive results were obtained by using neat PCL microfibers and nanofibers and PCL/chitosan blended nanofibers with acetic acid and formic acid as solvents for this layer. Electrospun fibrous mats could present the disadvantage of lack or reduced cell infiltration inside the scaffolds because of the high density of the fiber nets with scare porosity and pore size not compatible with complete scaffold colonization. For this reason, electrospun PCL fibers with a regular macropattern, improving cell infiltration in the scaffold, were fabricated. This layer was used for the fabrication of stratified multilayered scaffolds. Another typology of multilayered scaffold was fabricated by integrating the electrospinning process with foam replica method and dip-coating. In fact, BG porous scaffolds, used as substrate, resembling the bone side, were fabricated by sponge replica method and reinforced by dip-coating in a solution of PCL in acetic acid. These BG-based scaffolds were used as target for fiber collection during the electrospinning process. PCL monolayer or bilayer of composite and polymers electrospun fibers were deposited on the BG-based scaffolds. Positive results were obtained from the characterization of these samples. In particular, a selective bioactivity was reported on the multilayered electrospun scaffolds, demonstrating high potential for the osteochondral segment regeneration. On the contrary, all the multilayered scaffolds based on BG porous scaffolds showed mineralization even in the neat polymeric layers. Cell biology tests were relevant because they were the focus of the researcher´s training during “BIOeSPUN scaffolds” project, completing the interdisciplinarity of her profile. Positive and promising results were obtained in terms of cell viability and morphology on the seeded multilayered scaffolds. 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 fabrication of multilayered scaffolds for interface tissue engineering is challenging and the obtainment of different functional gradients in complex structures is a relevant topic among the scientific community, reported by an increased number of publications in the last years on this topic. In the framework of “BIOeSPUN scaffolds” project, complex stratified scaffolds with gradients in porosity and composition were fabricated and investigated for the regeneration of osteochondral segment. Promising results were obtained in terms of selective mineralization on the different layers and cells response. Further in vivo studies (not planned in the framework of this project) are still needed to validate the results obtained with the in vitro characterization. Their potential applications in clinical practice could represent a valuable alternative to the actual gold standard in the treatment of the joint defects which affect young and older patients, having a relevant social impact. In this project another important issue was the use of benign solvents (like acetic acid and formic acid) during the electrospinning and dip-coating processes. In fact, the avoidance of toxic solvents has positive impact not only concerning the final application in biomedical field, eliminating the possibility to denature proteins and biomolecules and the presence of toxic solvent traces in the fabricated samples. Moreover, it enhances also lab workers safety and environmental impact, reducing the waste management of toxic solvents. Overview of the main activities performed in the framework of “BIOeSPUN scaffolds” project.
