Effect of cryopreservation on the integrity of coaxial alginate capsules

Encapsulation of clinically relevant cells, such as multipotent stromal cells (MSCs), in three-dimensional (3D) core-shell alginate structures is a promising method for the treatment of various diseases as well as drug testing and development. By utilizing the alginate capsules, drugs and active substances can be delivered specifically to the desired place within the patients’ body. The aim of this work was to investigate the effect of cryopreservation on the integrity of cell-free coaxial alginate capsules after thawing using slow freezing to ensure their long-term storage. Alginate capsules were produced by electro-spraying and cryopreserved using different cryoprotective agents (CPAs), incubation time and thawing temperature. The results suggest that the capsules loaded with 10% (v/v) dimethyl sulfoxide (DMSO) and 0.3 M sucrose and using high thawing rates demonstrated a beneficial effect on the integrity of capsules, as compared to 10% (v/v) DMSO alone and applying low thawing rates. This study is a further development towards the application of cryopreservation for long-term storage and the emerging cell-based medicine

WYKORZYSTANIE TECHNOLOGII DRUKOWANIA 3D DO MODELOWANIA GÓRNYCH DRÓG ODDECHOWYCH W PEŁNEJ SKALI

The project "Implementation of rapid prototyping for modelling the upper respiratory tract in normal and typical pathologies" investigates the urgent problem of improving the reliability of diagnosis and effectiveness of treatment of disorders of the nasal breathing. Possibilities of modern 3D-printing technology for creation of individual natural anatomical models of the upper respiratory tract and determination of their aerodynamic characteristics are considered. The characteristics of the laminar boundary layer of the air flow in the parietal region of the nasal cavity are investigated under different modes of breathing in normal and with typical disorders of the nasal breathing. The concept of investigation of the aerodynamic indices of the anatomical structures of the respiratory system by the results of test tests of individual full-scale 3D models, obtained by the data of spiral computed tomography, is being developed. Theoretical bases of the method of computer planning of restorative rhinosurgical interventions in patients with chronic diseases of the nasal cavity are grounded, based on the change of the configuration of the anatomical structures of the nasal cavity taking into account the aerodynamic parameters of respiration. Modern distance learning and testing tools are being created to demonstrate the technology developed, to provide theoretical knowledge, practical skills and to solve situational tasks for a wide range of specialists. Development and research of natural patterns of the upper respiratory tract allows for supplementing and expanding the knowledge about the aerodynamic characteristics of the nasal cavity, to make decisions about therapy in a short period of time. Experience of the Laboratory of the Institute for Multiphase Processes (IMP) of the Leibniz Universität Hannover (LUH) in the development and use of rapid prototyping capabilities in biotechnology will provide technical support to the project.Projekt „Wdrożenie szybkiego prototypowania do modelowania górnych dróg oddechowych w normalnych i typowych patologiach” bada pilny problem poprawy wiarygodności diagnozy i skuteczności leczenia zaburzeń oddychania przez nos. Rozważane są możliwości nowoczesnej technologii druku 3D do tworzenia indywidualnych naturalnych modeli anatomicznych górnych dróg oddechowych i określania ich właściwości aerodynamicznych. Charakterystyka laminarnej warstwy granicznej przepływu powietrza w okolicy ciemieniowej jamy nosowej jest badana w różnych trybach oddychania w normalnym i typowym zaburzeniu oddychania przez nos. Opracowywana jest koncepcja badania wskaźników aerodynamicznych struktur anatomicznych układu oddechowego na podstawie wyników testów testowych poszczególnych pełnoskalowych modeli 3D, uzyskanych z danych spiralnej tomografii komputerowej. Podstawy teoretyczne metody komputerowego planowania rekonstrukcyjnych interwencji nosorożców u pacjentów z przewlekłymi chorobami jamy nosowej są oparte na zmianie konfiguracji struktur anatomicznych jamy nosowej z uwzględnieniem parametrów aerodynamicznych oddychania. Tworzone są nowoczesne narzędzia do nauki na odległość i testowania w celu zademonstrowania opracowanej technologii, zapewnienia wiedzy teoretycznej, umiejętności praktycznych i rozwiązywania zadań sytuacyjnych dla szerokiego grona specjalistów. Opracowanie i badanie naturalnych wzorów górnych dróg oddechowych pozwala uzupełnić i poszerzyć wiedzę na temat właściwości aerodynamicznych jamy nosowej w celu podjęcia decyzji o terapii w krótkim okresie czasu. Doświadczenie laboratorium Instytutu Procesów Wielofazowych (IMP) Leibniz Universität Hannover (LUH) w zakresie rozwoju i wykorzystania możliwości szybkiego prototypowania w biotechnologii zapewni wsparcie techniczne dla projektu

Encapsulating Non-Human Primate Multipotent Stromal Cells in Alginate via High Voltage for Cell-Based Therapies and Cryopreservation

Alginate cell-based therapy requires further development focused on clinical application. To assess engraftment, risk of mutations and therapeutic benefit studies should be performed in an appropriate non-human primate model, such as the common marmoset (Callithrix jacchus). In this work we encapsulated amnion derived multipotent stromal cells (MSCs) from Callithrix jacchus in defined size alginate beads using a high voltage technique. Our results indicate that i) alginate-cell mixing procedure and cell concentration do not affect the diameter of alginate beads, ii) encapsulation of high cell numbers (up to 10×106 cells/ml) can be performed in alginate beads utilizing high voltage and iii) high voltage (15–30 kV) does not alter the viability, proliferation and differentiation capacity of MSCs post-encapsulation compared with alginate encapsulated cells produced by the traditional air-flow method. The consistent results were obtained over the period of 7 days of encapsulated MSCs culture and after cryopreservation utilizing a slow cooling procedure (1 K/min). The results of this work show that high voltage encapsulation can further be maximized to develop cell-based therapies with alginate beads in a non-human primate model towards human application.DFG/EXC/62/

Scaffolds with Magnetic Nanoparticles for Tissue Stimulation

Magnetic nanoparticles (MNPs) have been used in several medical applications, including targeted hyperthermia, resonance tomography, diagnostic sensors, and localized drug delivery. Further applications of magnetic field manipulation through MNPs in tissue engineering have been described. The current study aims to develop tissue-engineered polymeric scaffolds with incorporated MNPs for applications that require stimulation of the tissues such as nerves, muscles, or heart. Electrospun scaffolds were obtained using 14%w/v polycaprolactone (PCL) in 2,2,2-Trifluoroethanol (TFE) at concentrations of 5% & 7.5%w/v of dispersed MNPs (iron oxide, Fe3O4, or cobalt iron oxide, CoFe2O4). Scaffolds were analyzed using scanning electron microscopy (SEM), energy dispersive x-ray spectroscopy, uniaxial tensile testing, and cell seeding for biocompatibility. Human bone marrow mesenchymal stem cells (bmMSCs) were seeded on the scaffolds. Biocompatibility was assessed by metabolic activity with Resazurin reduction assay on day 1, 3, 7, 10. Cell-cell and cell-scaffold interactions were analyzed by SEM. Electrospun scaffolds containing MNPs showed a decrease in fiber diameter as compared to scaffolds of pure PCL. The maximum force increases with the inclusion of MNPs, with higher values revealed for iron oxide. The metabolic activity decreased with MNPs, especially for cobalt iron oxide at a higher concentration. On the other hand, the cells developed good cell-scaffold and cell-cell interactions, making the proposed scaffolds good prospects for potential use in tissue stimulation

Solving Biocompatibility Layer by Layer: Designing Scaffolds for Tissues

New opportunities for the design of artificial tissue structures via ice templating and electrospinning are described. Exemplarily, developments of vascular grafts, heart valves and nerve guides will be presented.DFG/SFB/599DFG/SFB/TR 37DFG/ Exc 62/

Validation of a solvent-based process for the smoothing of additively manufactured 3D models of nasal cavities

In order to improve the reliability of diagnosis of nasal breathing disorders, aerodynamic properties have to be analyzed through experiments based on 3D models. The surface properties of the prepared respective 3D models using fused deposition modeling (FDM) should match those of native nasal cavities, thus representing their normal state and typical pathologies. In this work, we validated the smoothing of dual extruded 3D printed samples of PLA (polylactide) and PVA (polyvinyl alcohol) using the solvent TFE (trifluoroethanol). The smoothing was conducted in vapour and liquid phases of TFE. Before and after treatment of the samples in liquid and vapour phases of TFE, mass and surface roughness analysis were performed. The results of this work will help to produce and process a representative model of the human paranasal sinuses, which can be created using CT data from a patient

Human Amniotic Membrane: A review on tissue engineering, application, and storage

Human amniotic membrane (hAM) has been employed as scaffolding material in a wide range of tissue engineering applications, especially as a skin dressing and as a graft for corneal treatment, due to the structure of the extracellular matrix and excellent biological properties that enhance both wound healing and tissue regeneration. This review highlights recent work and current knowledge on the application of native hAM, and/or production of hAM-based tissue-engineered products to create scaffolds mimicking the structure of the native membrane to enhance the hAM performance. Moreover, an overview is presented on the available (cryo) preservation techniques for storage of native hAM and tissue-engineered products that are necessary to maintain biological functions such as angiogenesis, anti-inflammation, antifibrotic and antibacterial activity

Repeated freezing procedures preserve structural and functional properties of amniotic membrane for application in ophthalmology

For decades, the unique regenerative properties of the human amniotic membrane (hAM) have been successfully utilized in ophthalmology. As a directly applied biomaterial, the hAM should be available in a ready to use manner in clinical settings. However, an extended period of time is obligatory for performing quality and safety tests. Hence, the low temperature storage of the hAM is a virtually inevitable step in the chain from donor retrieval to patient application. At the same time, the impact of subzero temperatures carries an increased risk of irreversible alterations of the structure and composition of biological objects. In the present study, we performed a comprehensive analysis of the hAM as a medicinal product; this is intended for a novel strategy of application in ophthalmology requiring a GMP production protocol including double freezing– thawing cycles. We compared clinically relevant parameters, such as levels of growth factors and extracellular matrix proteins content, morphology, ultrastructure and mechanical properties, before and after one and two freezing cycles. It was found that epidermal growth factor (EGF), transforming growth factor beta 1 (TGF-ß1), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), hyaluronic acid, and laminin could be detected in all studied conditions without significant differences. Additionally, histological and ultrastructure analysis, as well as transparency and mechanical tests, demonstrated that properties of the hAM required to support therapeutic efficacy in ophthalmology are not impaired by dual freezing. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

PVDF and P(VDF-TrFE) Electrospun Scaffolds for Nerve Graft Engineering: A Comparative Study on Piezoelectric and Structural Properties, and In Vitro Biocompatibility

Polyvinylidene fluoride (PVDF) and its copolymer with trifluoroethylene (P(VDF-TrFE)) are considered as promising biomaterials for supporting nerve regeneration because of their proven biocompatibility and piezoelectric properties that could stimulate cell ingrowth due to their electrical activity upon mechanical deformation. For the first time, this study reports on the comparative analysis of PVDF and P(VDF-TrFE) electrospun scaffolds in terms of structural and piezoelectric properties as well as their in vitro performance. A dynamic impact test machine was developed, validated, and utilised, to evaluate the generation of an electrical voltage upon the application of an impact load (varying load magnitude and frequency) onto the electrospun PVDF (15–20 wt%) and P(VDF-TrFE) (10–20 wt%) scaffolds. The cytotoxicity and in vitro performance of the scaffolds was evaluated with neonatal rat (nrSCs) and adult human Schwann cells (ahSCs). The neurite outgrowth behaviour from sensory rat dorsal root ganglion neurons cultured on the scaffolds was analysed qualitatively. The results showed (i) a significant increase of the β-phase content in the PVDF after electrospinning as well as a zeta potential similar to P(VDF-TrFE), (ii) a non-constant behaviour of the longitudinal piezoelectric strain constant d33, depending on the load and the load frequency, and (iii) biocompatibility with cultured Schwann cells and guiding properties for sensory neurite outgrowth. In summary, the electrospun PVDF-based scaffolds, representing piezoelectric activity, can be considered as promising materials for the development of artificial nerve conduits for the peripheral nerve injury repair

Coaxial electrospinning as a process to engineer biodegradable polymeric scaffolds as drug delivery systems for anti-inflammatory and anti- thrombotic pharmaceutical agents

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