Maria Cristina Tanzi

A coating with biomacromolecules can improve the performance of electrospun 3D scaffolds and 2D matrices that show morphology similar to that of native ECM, but their mechanical and biological properties are often inadequate, particularly in applications in contact with blood. In this work, a gelatin coating was applied to electrospun silk fibroin (ESF) mats and tubes intended for the regeneration of cardiovascular tissues. The crosslinking reaction used is based on a Michael-type addition in water that promotes the formation of covalent bonds between gelatin amino groups and β-carbons of N-N′-methylene bis-acrylamide (MBA). ESF mats and tubes were coated with gelatin MBA crosslinked in situ by loading or dipping the ESF samples with the crosslinking solution by use of static or dynamic homemade systems. SEM analysis on coated samples showed a homogeneous coating with gelatin penetrating the whole thickness of the SF matrix (≈120 µm for mats and ≈212 µm for tubes), with an increase of thickness of about 40% in wet conditions. Water uptake tests indicated for coated samples a faster and higher swelling (1600% after 14 days) than not coated ones (500%), due to the presence of gelatin. Tensile mechanical tests showed higher values of ultimate stress and elastic modulus for silk fibroin samples (_b = 2.4, E = 1.82 MPa) compared to gelatin-coated ones (_b = 1.2, E = 0.58 MPa), with not significant differences in the ultimate deformation (≈150%). Indirect cytocompatibility tests, performed by culturing L929 cells in the presence of eluates obtained by immersing coated and uncoated samples up to 7 days in culture medium, demonstrated a cell viability higher than the control. For direct contact tests, primary human umbilical vein endothelial cell (HUVEC), obtained by enzymatic digestion, and cell adhesion and growth on the MBA-crosslinked gelatin was analyzed by OM after fixing with formalin and staining with toluidine blue. HUVEC were seeded onto ESF and ESF-coated samples and cultured under standard tissue culture conditions. After 7 days from seeding, cell proliferation was evaluated from the quantitation of total proteins in cell lysates (BCA protein assay) and the obtained results indicated a significantly higher (p < 0.05) cell growth on gelatin-coated ESF samples. Overall, these results point out that the described gelatin coating allows for the production of a structure with adequate mechanical properties for cardio-vascular applications and biological characteristics even better than those of silk fibroin.

The regeneration of injured or damaged tissues by cell delivery approaches requires the fabrication of cell carriers (e.g., microspheres, MS) that allow for cell delivery to limit cells spreading from the injection site. Ideal MS for cell delivery should allow for cells adhesion and proliferation on the MS before the injection, while they should allow for viable cells release after the injection to promote the damaged tissue regeneration. We optimized a water-in-oil emulsion method to obtain gelatin MS crosslinked by methylenebisacrylamide (MBA). The method we propose allowed obtaining spherical, chemically crosslinked MS characterized by a percentage crosslinking degree of 74.5 ± 2.1%. The chemically crosslinked gelatin MS are characterized by a diameter of 70.9 ± 17.2 μm in the dry state and, at swelling plateau in culture medium at 37 °C, by a diameter of 169.3 ± 41.3 μm. The MS show dimensional stability up to 28 days, after which they undergo complete degradation. Moreover, during their degradation, MS release gelatin that can improve the engraftment of cells in the injured site. The produced MS did not induce any cytotoxic effect in vitro and they supported viable L929 fibroblasts adhesion and proliferation. The MS released viable cells able to colonize and proliferate on the tissue culture plastic, used as release substrate, potentially proving their ability in supporting a simplified in vitro wound healing process, thus representing an optimal tool for cell delivery applications.

In this work an innovative method to obtain hybrid bio-functional scaffolds has been developed. Polyether urethane (PU) foam scaffolds were synthetized by one-step gas foaming process. PU foams were coated with crosslinked gelatin hydrogel to promote cell adhesion and proliferation for the regeneration of soft tissues (e.g., adipose tissue). PU foams were coated with inorganic coating (i.e., CaPs) to improve the interaction with osteoblasts for bone tissue regeneration. The functionalized 3D PU porous scaffolds have been characterized investigating morphological properties by SEM and microCT, water uptake and coating stability, and compressive mechanical properties. Adipose tissue derived stem cells (ADSCs), endothelial cells (MS1), amnion mesenchymal cells (AMCs) and chorion mesenchymal cells (CMCs) isolated from human placenta were in vitro cultured on the hybrid functionalized 3D scaffolds. Mechanical properties showed elastic modulus ranging between 15.75 ± 2.14 and 22.9 ± 3.1 kPa; in vitro biological studies showed good cell adhesion, proliferation, and differentiation. In particular, compared to the results with uncoated PU, when cells where differentiated into adipocytes, Oil red O staining confirmed a higher presence of lipid droplets; in case of osteoblasts differentiation, inorganic extracellular matrix deposition was evidenced on CaPs coated PU. The obtained results suggest the important role of an adequate coating on the scaffold to stimulate a better interaction with cells, promoting the differentiation into different cells phenotypes.

The design of scaffolding materials that mimic the properties of the target tissue to be regenerated is a mandatory requirement to engineer a successful scaffold; however, the heterogeneous properties of adipose tissue (AT), strictly dependent on the AT depot, are often underestimated when engineering AT scaffolds. Moreover, a scaffolding material with versatile properties, suitable for the regeneration of different AT depots, is currently missing. Chemically crosslinked gelatin hydrogels are here prepared, and their properties tuned by varying gelatin concentration and reaction stoichiometry to obtain hydrogels suitable for AT regeneration. All hydrogel formulations are stable in water at 37 °C, showing swelling behavior dependent on synthesis parameters. The mechanical compressive response mimics the viscoelastic response typical of native AT, with elastic modulus values covering the range of breast and heel pad AT. The rheological properties vary among the hydrogel formulations, showing a typical shear thinning response, comparable to other AT scaffolds described in literature. In vitro cytotoxicity tests using 3T3‐L1 preadipocytes show no cytotoxic effects up to 7 days. 3T3‐L1 cells seeded on the hydrogels show good adhesion, proliferation, and adipogenic differentiation, confirmed by an increase in peroxisome proliferator‐activated receptor gamma gene expression and lipid droplets accumulation observed by Oil Red O staining. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019, 136, 47104.

In this work an innovative method to obtain natural and biocompatible small diameter tubular structures is proposed. The biocompatibility and good mechanical properties of electrospun silk fibroin tubular matrices (SFts), extensively studied for tissue engineering applications, have been coupled with the excellent cell interaction properties of gelatin. In fact, an innovative non-cytotoxic gelatin gel, crosslinked in mild conditions via a Michael-type addition reaction, has been used to coat SFt matrices and obtain SFt/gel structures (I.D. = 6 mm). SFts/gel exhibited homogeneous gelatin coating on the electrospun fibrous tubular structure. Circumferential tensile tests performed on SFts/gel showed mechanical properties comparable to those of natural blood vessels in terms of UTS, compliance and viscoelastic behavior. Finally, SFt/gel in vitro cytocompatibility was confirmed by the good viability and spread morphology of L929 fibroblasts up to 7 days. These results demonstrated that SFt/gel is a promising off-the-shelf graft for small diameter blood vessel regeneration.