Sotirios Korossis

Coaxial Electrospinning as a Process to Engineer Biodegradable PolymericScaffolds as Drug Delivery Systems for Anti-Inflammatory and Anti-Thrombotic Pharmaceutical Agents

Abstract Objective: Blend electrospinning has been acknowledged as a cost-effective technique for the production of fibrous scaffolds, suitable for various biomedical applications. Coaxial electrospinning is a method variant that results in core-shell structures with advantages, such as delayed diffusion and protection of sensitive biomolecules. The aim of this work was to evaluate how different process and solution parameters affect the structural, mechanical and physical properties of the fibers, created by polycaprolactone (PCL). In addition, acetylsalicylic acid (ASA) that was used as a model anti-inflammatory and anti-thrombotic agent, was loaded within the fiber meshes in order to compare release kinetics between fibers produced by conventional blend and coaxial electrospinning. Methods: Scanning electron microscopy (SEM) was used to investigate the structural and morphological characteristics of the fibers. The fibers’ hydrophilicity was investigated using contact angle measurements while the electrical conductivity of the polymeric solutions and the thermal properties of the fibers were also evaluated. Differential scanning calorimetry (DSC) was used to determine the fibers’ melting point and mechanical tensile tests were performed in order to study the mechanical properties of the fibers. Moreover, UV-vis spectroscopy was used to determine the release kinetics of ASA. Results: The results indicated that increasing the concentration of PCL led to thicker and less aligned fibers. Furthermore, the physicochemical characterization did not reveal significant changes during the process. Coaxially electrospun fibers that were loaded with ASA exhibited a slower and sustained, biphasic release profile compared to blend electrospun fibers with 34% of ASA released during the first 8h and 97% in total after 3 months. Conclusion: Taken together, fibrous meshes created by coaxial electrospinning using PCL, can be tailor-made by a careful optimization of all the process and solution parameters, in order to fit the scope of specific applications in the fields of biomedical engineering and drug delivery.

Effects of combined cryopreservation and decellularization on the biomechanical, structural and biochemical properties of porcine pulmonary heart valves.

UNLABELLED Non-fixed, decellularized allogeneic heart valve scaffolds seem to be the best choice for heart valve replacement, their availability, however, is quite limited. Cryopreservation could prolong their shelf-life, allowing for their ideal match to a recipient. In this study, porcine pulmonary valves were decellularized using detergents, either prior or after cryopreservation, and analyzed. Mechanical integrity was analyzed by uniaxial tensile testing, histoarchitecture by histological staining, and composition by DNA, collagen (hydroxyproline) and GAG (chondroitin sulfate) quantification. Residual sodium dodecyl sulfate (SDS) in the scaffold was quantified by applying a methylene blue activation assay (MBAS). Cryopreserved decellularized scaffolds (DC) and scaffolds that were decellularized after cryopreservation (CD) were compared to fresh valves (F), cryopreserved native valves (C), and decellularized only scaffolds (D). The E-modulus and tensile strength of decellularized (D) tissue showed no significant difference compared to DC and CD. The decellularization resulted in an overall reduction of DNA and GAG, with DC containing the lowest amount of GAGs. The DNA content in the valvular wall of the CD group was higher than in the D and DC groups. CD valves showed slightly more residual SDS than DC valves, which might be harmful to recipient cells. In conclusion, cryopreservation after decellularization was shown to be preferable over cryopreservation before decellularization. However, in vivo testing would be necessary to determine whether these differences are significant in biocompatibility or immunogenicity of the scaffolds. STATEMENT OF SIGNIFICANCE Absence of adverse effects on biomechanical stability of acellular heart valve grafts by cryopreservation, neither before nor after decellularization, allows the identification of best matching patients in a less time pressure dictated process, and therefore to an optimized use of a very limited, but best-suited heart valve prosthesis.

Prevention of rejection of allogeneic endothelial cells in a biohybrid lung by silencing HLA-class I expression.

Variability in Human Leukocyte Antigens (HLA) remains a hurdle to the application of allogeneic cellular products. Due to insufficient autologous endothelial cell harvesting for the biohybrid lung, allogeneic human cord blood derived endothelial cells (HCBEC) were used for the endothelialization of poly-4-methyl-1-pentene (PMP) gas exchange membranes. Therefore, HLA class I expression was silenced stably in HCBECs to prevent rejection. The capacity of HLA class I-silenced HCBEC to abrogate allogeneic immune responses, their functional properties and suitability for endothelialization of PMP membranes were investigated. Delivery of β2-microglobulin (β2m)-specific shRNAs reduced β2m mRNA levels by up to 90% and caused a knockdown of HLA class I expression by up to 85%. HLA-silenced HCBEC abrogated T-cell responses and escaped antibody-mediated complement-dependent cytotoxicity. The EC phenotype and cytokine secretion profiles between HLA-expressing or -silenced HCBEC remained unaltered. EC specific activation (e.g. ICAM) and thrombogenic markers (e.g. thrombomodulin) remained unaffected by HLA-silencing, but their expression was upregulated by TNFα-stimulation. Furthermore, HLA-silenced HCBECs showed high proliferation rates and built an EC monolayer onto PMP membranes. This study represents a new therapeutic concept in the field of cell and organ transplantation and may bring the bioartificial lung as an alternative to lung transplantation closer to reality.

Tissue-engineered mitral valve: morphology and biomechanics †

OBJECTIVES The present study aimed at developing tissue-engineered mitral valves based on cell-free ovine mitral allografts. METHODS The ovine mitral valves (OMVs) (n = 46) were harvested in the local slaughter house. They were decellularized using detergent solutions and DNase. The effectiveness of decellularization was assessed by histological (haematoxylin-eosin, Movats pentachrome) and immunofluorescent staining (for DNA and α-Gal), and DNA-quantification. To reveal the receptiveness of decellularized tissue to endothelial cells (ECs), the valve leaflets were reseeded with ovine ECs, derived from endothelial progenitor cells in vitro. For assessment of biomechanical properties, uniaxial tensile tests were carried out. RESULTS Histology and immunofluorescent staining revealed absence of cell nuclei in decellularized leaflets, chordae and papillary muscles. According to the software for immunofluorescence analysis, reduction in DNA and α-Gal was 99.9 and 99.6%, respectively. DNA-quantification showed 71.2% reduction in DNA content without DNase and 96.4% reduction after DNase treatment. Decellularized leaflets were comparable with native in ultimate tensile strain (native, 0.34 ± 0.09 mm/mm, vs decellularized, 0.44 ± 0.1 mm/mm; P = 0.09), and elastin modulus (native, 0.39 ± 0.27, vs decellularized, 0.57 ± 0.55, P = 0.46), had increased ultimate tensile stress (native, 1.23 ± 0.35 MPa, vs decellularized 2.12 ± 0.43 MPa; P = 0.001) and collagen modulus (native, 5.5 ± 1.26, vs decellularized, 8.29 ± 2.9; P = 0.04). After EC seeding, immunofluorescent staining revealed a monolayer of CD31-, eNOS- and vWF-positive cells on the surface of the leaflet, as well as a typical cobble-stone morphology of those cells. CONCLUSIONS Decellularization of ovine mitral valve results in a mitral valves scaffold with mechanical properties comparable with native tissue, and a graft surface, which can be repopulated by endothelial cells.

Development and Characterization of a Porcine Mitral Valve Scaffold for Tissue Engineering

Decellularized scaffolds represent a promising alternative for mitral valve (MV) replacement. This work developed and characterized a protocol for the decellularization of whole MVs. Porcine MVs were decellularized with 0.5% (w/v) SDS and 0.5% (w/v) SD and sterilized with 0.1% (v/v) PAA. Decellularized samples were seeded with human foreskin fibroblasts and human adipose-derived stem cells to investigate cellular repopulation and infiltration, and with human colony-forming endothelial cells to investigate collagen IV formation. Histology revealed an acellular scaffold with a generally conserved histoarchitecture, but collagen IV loss. Following decellularization, no significant changes were observed in the hydroxyproline content, but there was a significant reduction in the glycosaminoglycan content. SEM/TEM analysis confirmed cellular removal and loss of some extracellular matrix components. Collagen and elastin were generally preserved. The endothelial cells produced newly formed collagen IV on the non-cytotoxic scaffold. The protocol produced acellular scaffolds with generally preserved histoarchitecture, biochemistry, and biomechanics.

Developing a biohybrid lung – sufficient endothelialization of poly-4-methly-1-pentene gas exchange hollow-fiber membranes

Working towards establishing a biohybrid lung with optimized hemocompatibility, this study analyzed the feasibility of establishing flow-resistant endothelium on heparin/albumin coated poly-4-methly-1-pentene hollow fiber gas exchange membranes (PMP-HFs). The seeding efficiency and proliferation of human cord blood derived endothelial cells (HCBEC) on PMP-HFs were analyzed under static conditions by WST-8 cell proliferation assay and fluorescence microscopy. The HCBEC monolayer integrity under different flow conditions was also assessed. Endothelial-specific phenotype verification, expression activation levels and thrombogenic state markers were quantified by real-time RT-PCR for cell-to-PMP-HF contact under static and dynamic conditions. The results demonstrated the feasibility of establishing a viable, confluent, and flow-resistant endothelial monolayer on the blood-contact surface of PMP-HFs, which maintained a physiological response to TNFα-stimulation and flow conditions. The endothelial phenotype, expression levels of adhesion molecules and thrombogenic state markers were unaffected by cell-to-PMP-HFs contact. These results represent a significant step towards establishing a biohybrid lung.

Identifying an optimal seeding protocol and endothelial cell substrate for biohybrid lung development

Several key prerequisites need to be fulfilled for the development of a biohybrid lung, which can offer an actual alternative to lung transplantation. A major aspect is an optimized haemocompatibility of the devices artificial surfaces via endothelial cell seeding. In this study, four different types of polymeric gas exchange hollow fibre membranes (HFMs) were analysed utilizing four different seeding protocols in order to identify the ideal combination for sufficient long‐term endothelialization. Human cord blood‐derived endothelial cells (HCBECs) were used for the endothelialization of polypropylene HFMs with two different pore sizes and poly‐4‐methyl‐1‐pentene HFMs, both with and without heparin/albumin coating. The qualitative and quantitative impact of four different rotational seeding protocols regarding long‐term HFM endothelialization and the impact of inflammatory stimulation on the seeded HCBECs were examined by fluorescence microscopy, cell counting, and analysis of relative expression levels of activation, shear stress, and thrombogenic state markers. Optimized endothelial cell seeding and long‐term cultivation were only achieved using heparin/albumin‐coated poly‐4‐methyl‐1‐pentene HFMs, applying 24 hr of rotational speed at 1 rpm followed by 120 hr of static culture. Neither cell‐to‐HFM contact nor the rotational cultivation procedure showed an impact on the physiological anti‐thrombogenic and anti‐inflammatory HCBEC activation status. Additionally, the cells maintained their physiological responsiveness towards inflammatory stimulation. Rotational seeding strategies and a seamless heparin/albumin coating of the HFMs are crucial requirements for a sufficient and long‐lasting endothelialization and thus a key element in the future development and in vivo application of the biohybrid lung.

Preservation strategies for decellularized pericardial scaffolds for off-the-shelf availability

Decellularized biological scaffolds hold great promise in cardiovascular surgery. In order to ensure off-the-shelf availability, routine use of decellularized scaffolds requires tissue banking. In this study, the suitability of cryopreservation, vitrification and freeze-drying for the preservation of decellularized bovine pericardial (DBP) scaffolds was evaluated. Cryopreservation was conducted using 10% DMSO and slow-rate freezing. Vitrification was performed using vitrification solution (VS83) and rapid cooling. Freeze-drying was done using a programmable freeze-dryer and sucrose as lyoprotectant. The impact of the preservation methods on the DBP extracellular matrix structure, integrity and composition was assessed using histology, biomechanical testing, spectroscopic and thermal analysis, and biochemistry. In addition, the cytocompatibility of the preserved scaffolds was also assessed. All preservation methods were found to be suitable to preserve the extracellular matrix structure and its components, with no apparent signs of collagen deterioration or denaturation, or loss of elastin and glycosaminoglycans. Biomechanical testing, however, showed that the cryopreserved DBP displayed a loss of extensibility compared to vitrified or freeze-dried scaffolds, which both displayed similar biomechanical behavior compared to non-preserved control scaffolds. In conclusion, cryopreservation altered the biomechanical behavior of the DBP scaffolds, which might lead to graft dysfunction in vivo. In contrast to cryopreservation and vitrification, freeze-drying is performed with non-toxic protective agents and does not require storage at ultra-low temperatures, thus allowing for a cost-effective and easy storage and transport. Due to these advantages, freeze-drying is a preferable method for the preservation of decellularized pericardium. STATEMENT OF SIGNIFICANCE: Clinical use of DBP scaffolds for surgical reconstructions or substitutions requires development of a preservation technology that does not alter scaffold properties during long-term storage. Conclusive investigation on adverse impacts of the preservation methods on DBP matrix integrity is still missing. This work is aiming to close this gap by studying three potential preservation technologies, cryopreservation, vitrification and freeze-drying, in order to achieve the off-the-shelf availability of DBP patches for clinical application. Furthermore, it provides novel insights for dry-preservation of decellularized xenogeneic scaffolds that can be used in the routine clinical cardiovascular practice, allowing the surgeon the opportunity to choose an ideal implant matching with the needs of each patient.

A sterilization method for decellularized xenogeneic cardiovascular scaffolds

Decellularized xenogeneic scaffolds have shown promise to be employed as compatible and functional cardiovascular biomaterials. However, one of the main barriers to their clinical exploitation is the lack of appropriate sterilization procedures. This study investigated the efficiency of a two-step sterilization method, antibiotics/antimycotic (AA) cocktail and peracetic acid (PAA), on porcine and bovine decellularized pericardium. In order to assess the efficiency of the method, a sterilization assessment protocol was specifically designed, comprising: i) controlled contamination with a known amount of bacteria; ii) sterility test; iii) identification of contaminants through MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) mass spectrometry and iv) quantification by the Most Probable Number (MPN) method. This sterilization assessment protocol proved to be a successful tool to monitor and optimize the proposed sterilization method. The treatment with AA + PAA method provided sterile scaffolds while preserving the structural integrity and biocompatibility of the decellularized porcine and bovine tissues. However, surface properties and cellular adhesion resulted slightly impaired on porcine pericardium. This work developed a sterilization method suitable for decellularized pericardial scaffolds that could be adopted for in vivo tissue engineering. Together with the proposed sterilization assessment protocol, this decontamination method will foster the clinical translation of decellularized xenogeneic substitutes. STATEMENT OF SIGNIFICANCE Clinical application of functional and compatible xenogeneic decellularized scaffolds has been delayed due to the lack of appropriate sterilization methodologies. In this study, it was investigated an effective sterilization method optimized for porcine and bovine decellularized pericardia, based on the use of antibiotics/antimycotics followed by peracetic acid treatment. This treatment effectively sterilizes both species scaffolds, proves to maintain tissue overall structure and components, preserves biocompatibility and biomechanical properties. Furthermore, it was also developed a sterilization assessment protocol used to monitor and validate the previous method, consisting in three main parts: i) controlled contamination; ii) sterility test, and iii) identification and quantification of contaminants. Both methodologies were optimized for the tissues in study but can be applied to other scaffolds and accelerate their clinical translation.