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

Effect of applied encapsulation methods and incubation on proliferation of MSCs post-encapsulation and cryopreservation.

<p>Proliferation of MSCs: (<b>A1)</b> after encapsulation in alginate; (<b>A2</b>) after recovery post-encapsulation; (<b>B1</b>) cryopreserved in alginate; (<b>B2</b>) after recovery post-thawing. “Recovery” – incubation of cells in culture (37°C, 5% CO₂ in a humidified incubator) for 5 days after encapsulation and/or cryopreservation; “Immediate” – immediately after encapsulation; “Incubated” – after 24 h of incubation inside alginate beads in culture; AF – air flow; CF – frozen not encapsulated cells; CN – not encapsulated and not frozen MSCs. The next process parameters were kept constant during encapsulation: both for AF and electro-spraying – spraying distance 10 cm, concentration of gelling solution 100 mM CaCl₂, alginate flow rate 10 ml/h, and alginate concentration 1.6% (w/v); for AF – air flow 150 l/h. In B1–B2 freezing parameters included cooling rate 1 K/min down to −80°C and freezing medium with 10% DMSO and 10% FBS. The data are shown as a mean±SD (n = 3, N = 5). One-way ANOVA: NS – not significant (p = 0.05); *, **, *** - significantly different with p<0.05, p<0.01, p<0.001, respectively.</p

Schematic presentation of alginate high voltage encapsulation.

<p>(<b>A</b>) Application of encapsulation of cells in alginate using high voltage (<b>B</b>) in cell-based therapy for immunoisolation, controllable drug release through semi-permeable membrane (SPM) and long-term storage of cells. Scale bar is 100 µm.</p

Characterization of amnion derived multipotent stromal cells.

<p>(<b>A</b>) FACS analysis with characteristic markers CD105 and 73 (mean±SD, n = 4). (<b>B–E</b>) Immunohistochemistry stainings with MSC markers CD105, CD90, Bra and Snail1. (<b>F</b>) RT-PCR analysis displays presence of characteristic MSC markers CD90, ITGA6, GFRa1, CD73, CD105, ALCAMm CD44, whereas CD34 as marker for hematopoietic progenitors is absent (housekeeper RPS29). Scale bars are 50 µm.</p

Viability of encapsulated MSCs <i>vs.</i> applied voltage and incubation time.

<p>Live-dead staining with CalceinAM (2 µM) and Ethidium Homodymer (4 µM). Note: beads obtained under 15 kV and using air flow (AF) had slightly bigger diameter as compared to the others. Viable cells stained in green, cells with massive membrane damages – in red. No visible difference in viability of encapsulated cells depending on applied voltage can be observed. The next process parameters were kept constant: both for AF and electro-spraying – spraying distance 10 cm, concentration of gelling solution 100 mM CaCl₂, alginate flow rate 10 ml/h, and alginate concentration 1.6% (w/v); for AF – air flow 150 l/h. Scale bar is 100 µm.</p

Effect of encapsulation method and period of incubation of MSCs inside beads on proliferation of cells.

<p>MSCs were encapsulated in alginate beads using air flow (AF) and high voltage (15, 20, 25 kV) and incubated for 0, 1, 3, 5 and 7 days inside beads in culture (37°C, 5% CO₂ in a humidified incubator). Culture medium was replaced each second day to a fresh one. The next process parameters were kept constant: both for AF and electro-spraying – spraying distance 10 cm, concentration of gelling solution 100 mM CaCl₂, alginate flow rate 10 ml/h, and alginate concentration 1.6% (w/v); for AF – air flow 150 l/h. The data are shown as a mean±SD (n = 2, N = 5). One-way ANOVA: NS – not significant (p = 0.05); *** – significantly different (p<0.001).</p

Optimization of cell concentration and cell number.

<p>(<b>A</b>) Effect of applied voltage, initial cell concentration and alginate-cell mixing procedure on diameter of alginate beads (mean±SD). (<b>B</b>) Shrinking of alginate in presence of 100 mM CaCl₂ depending on gelling time for different initial surface-to-volume ratio (S/V) and (<b>C</b>) calculation of encapsulation efficiency of MSCs in alginate beads. Shrinking rate was analyzed based on decrease in volume during gelling caused by water release for: MD – manual dropping of alginate solution, AF – using air-flow method and ES – using electro-spraying; dotted lines show the tendency of shrinking outside the studied cross-linking time. (<b>C</b>) Effect of initial cell concentration on number of encapsulated cells and respective photographs of alginate encapsulated MSCs at initial concentration of 1×10⁶ (<b>C1</b>), 5×10⁶ (<b>C2</b>) and 10×10⁶ (<b>C3</b>) cells/ml. The next process parameters were kept constant: both for AF and ES – spraying distance 10 cm, concentration of gelling solution 100 mM CaCl₂, alginate flow rate 10 ml/h, and alginate concentration from 1.2% to 1.6% (w/v); for AF – air flow 150 l/h; for ES – applied voltage 20 kV. The data are presented as a mean±SD (n = 3, N = 10). Scale bars are 200 µm.</p

‘In air’ cryopreservation of mesenchymal stromal cells on 3d collagen-hydroxyapatite scaffolds

Abstract: ‘In air’ cryopreservation of mesenchymal stromal cells on 3d collagen-hydroxyapatite scaffolds

Effect of ‘in air’ freezing on post-thaw recovery of <i>Callithrix jacchus</i> mesenchymal stromal cells and properties of 3D collagen-hydroxyapatite scaffolds

Through enabling an efficient supply of cells and tissues in the health sector on demand, cryopreservation is increasingly becoming one of the mainstream technologies in rapid translation and commercialization of regenerative medicine research. Cryopreservation of tissue-engineered constructs (TECs) is an emerging trend that requires the development of practically competitive biobanking technologies. In our previous studies, we demonstrated that conventional slow-freezing using dimethyl sulfoxide (Me2SO) does not provide sufficient protection of mesenchymal stromal cells (MSCs) frozen in 3D collagen-hydroxyapatite scaffolds. After simple modifications to a cryopreservation protocol, we report on significantly improved cryopreservation of TECs. Porous 3D scaffolds were fabricated using freeze-drying of a mineralized collagen suspension and following chemical crosslinking. Amnion-derived MSCs from common marmoset monkey Callithrix jacchus were seeded onto scaffolds in static conditions. Cell-seeded scaffolds were subjected to 24 h pre-treatment with 100 mM sucrose and slow freezing in 10% Me2SO/20% FBS alone or supplemented with 300 mM sucrose. Scaffolds were frozen ‘in air’ and thawed using a two-step procedure. Diverse analytical methods were used for the interpretation of cryopreservation outcome for both cell-seeded and cell-free scaffolds. In both groups, cells exhibited their typical shape and well-preserved cell-cell and cell-matrix contacts after thawing. Moreover, viability test 24 h post-thaw demonstrated that application of sucrose in the cryoprotective solution preserves a significantly greater portion of sucrose-pretreated cells (more than 80%) in comparison to Me2SO alone (60%). No differences in overall protein structure and porosity of frozen scaffolds were revealed whereas their compressive stress was lower than in the control group. In conclusion, this approach holds promise for the cryopreservation of ‘ready-to-use’ TECs