The present study examined the effects of a bilayered fibrin/poly(ether)urethane scaffold loaded with platelet lysate by a combination of electrospinning and spray, phase-inversion method for wound healing. In particular, the poly(ether)urethane layer was obtained using by a spray phase-inversion method and the fibrin fibers network were loaded with platelet lysate by electrospinning. The kinetics release and the bioactivity of growth factors released from platelet lysate-scaffold were investigated by ELISA and cell proliferation test using mouse fibroblasts, respectively. The in-vitro experiments demonstrated that a bilayered fibrin/poly(ether)urethane scaffold loaded with platelet lysate provides a sustained release of bioactive platelet-derived growth factors. The effect of a bilayered fibrin/poly(ether)urethane scaffold loaded with platelet lysate on wound healing in diabetic mouse (db/db) was also investigated. The application of the scaffold on full-thickness skin wounds significantly accelerated wound closure at day 14 post-surgery when compared to scaffold without platelet lysates or commercially available polyurethane film, and at the same level of growth factor-loaded scaffold. Histological analysis demonstrated an increased re-epithelialization and collagen deposition in platelet lysate and growth factor loaded scaffolds. The ability of bilayered fibrin/poly(ether)urethane scaffold loaded with platelet lysate to promote in-vivo wound healing suggests its usefulness in clinical treatment of diabetic ulcers.

Platelets contain abundant growth factors and cytokines that have a positive influence on the migration and proliferation of different cell types by modulating its physiopathological processes. As it is known that human umbilical cord blood platelet lysate (UCB-PL) contains a supraphysiological concentration of growth factors, in the present study, we investigated its effectiveness in wound-healing processes. Human UCB-PL was obtained by the freeze/thaw of platelet concentrate (1.1 × 10 9 platelets/L), and its effect was evaluated on human or mouse endothelial cells, monocytes, fibroblasts, and keratinocytes in different concentrations. Human UCB-PL was observed to have high levels of pro-angiogenic growth factor than peripheral blood platelet-rich plasma. Among the cell lines, different concentrations of human UCB-PL were necessary to influence their viability and proliferation. For L929 cells, 5% of total volume was necessary, while for human umbilical vein endothelial cell, it was 10%. Cell migration on monocytes was increased with respect to the positive control, and scratch closure on keratinocytes was increased with respect to serum-free medium with only 10% of human UCB-PL. We concluded that the human UCB-PL may be useful to produce a large amount of standard platelet concentrates sufficient for several clinical-scale expansions avoiding inter-individual variability, which can also be used as a functional tool for clinical regenerative application for wound healing. K E Y W O R D S human umbilical cord blood, in vitro characterisation, platelet lysate, wound healing

Marine organisms (i.e., fish, jellyfish, sponges or seaweeds) represent an abundant and eco-friendly source of collagen. Marine collagen, compared to mammalian collagen, can be easily extracted, is water-soluble, avoids transmissible diseases and owns anti-microbial activities. Recent studies have reported marine collagen as a suitable biomaterial for skin tissue regeneration. The aim of this work was to investigate, for the first time, marine collagen from basa fish skin for the development of a bioink for extrusion 3D bioprinting of a bilayered skin model. The bioinks were obtained by mixing semi-crosslinked alginate with 10 and 20 mg/mL of collagen. The bioinks were characterised by evaluating the printability in terms of homogeneity, spreading ratio, shape fidelity and rheological properties. Morphology, degradation rate, swelling properties and antibacterial activity were also evaluated. The alginate-based bioink containing 20 mg/mL of marine collagen was selected for 3D bioprinting of skin-like constructs with human fibroblasts and keratinocytes. The bioprinted constructs showed a homogeneous distribution of viable and proliferating cells at days 1, 7 and 14 of culture evaluated by qualitative (live/dead) and qualitative (XTT) assays, and histological (H&E) and gene expression analysis. In conclusion, marine collagen can be successfully used to formulate a bioink for 3D bioprinting. In particular, the obtained bioink can be printed in 3D structures and is able to support fibroblasts and keratinocytes viability and proliferation.

Plasminogen is a protein involved in intravascular and extravascular fibrinolysis, as well as in wound healing, cell migration, tissue formation and angiogenesis. In recent years its role in healing of tympanic perforations has been demonstrated in plasminogen deficient mice. The aim of this work was to fabricate a fibrin-based drug delivery system able to provide a local and sustained release of plasminogen at the wound site. Initially, the biological activity of plasminogen was evaluated by in vitro experiments on cell cultures. A metabolic assay (MTT) was carried out on L929 mouse fibroblast to determine the concentration that does not affect cell viability, which turned out to be 64 nM. The effect of plasminogen on cell migration was evaluated through a scratch test on human keratinocytes: cells treated with 64 nM plasminogen showed faster scratch closure than in complete medium. Fibrin scaffold loaded with plasminogen was fabricated by a spray process. SEM analysis showed the typical nano-fibrillar structure of a fibrin scaffold. Tensile tests highlighted significantly higher value of the ultimate stress and strain of fibrin scaffold with respect to fibrin clot. The in-vitro release kinetic showed an initial plasminogen burst, after that the release slowed, reaching a plateau at 7 days. Plasminogen-loaded fibrin scaffold applied in full-thickness diabetic mouse lesions showed a significantly higher closure rate at 14 days than scaffold used as a reference material. Histological analysis demonstrated an improved reepithelization and collagen deposition in granulation tissue in mouse treated with plasminogen-loaded fibrin scaffold in comparison to unloaded fibrin scaffold. The obtained results demonstrated the suitability of the fibrin scaffold loaded with plasminogen as drug delivery system and suggest its use in wound healing applications, such as for the treatment of chronic diabetic ulcers.

At the present, no wearable device is available to continuously monitor the ulcer status. For chronic or infected wounds, the literature reports a pH interval between 6.5 and 9. This study aims to produce an innovative scaffold capable of monitoring ulcers healing. The scaffold constituted by a synthetic biocompatible material, poly(ether)urethane-polydimethylsiloxane (PU-PDMS), was manufactured by spray, phase-inversion technique. A micro-fibrillar tubular scaffold was obtained using a 2% polymer solution and H2O as non-solvent (I layer) and 0.2% and H2O (II layer) and was lyophilized. Morphological analysis of PU-PDMS scaffold surfaces was performed using a stereo-microscope after Sudan Black B staining. The biocompatible scaffold was functionalized by inkjet-printing of a biocompatible conductive polymer (PEDOT:PSS), used as active material in a biosensor, to develop organic electrochemical transistor (OECT) architecture. This polymer presents a high sensitivity to positive ions in liquid environment and allows to determine ions concentration in easy and stable way. The fiber textile electrochemical device was prepared by inkjet-printing process and connected with electric contacts to create a channel and a gate electrode to control the modulation changes of the sensor. The device functionality was proved on human serum at different pH (between 4 and 10). The morphological analysis showed a dense, non-porous surface obtained with the 2% solution, while a porous surface was obtained with the 0.2% solution where the PEDOT:PSS was positioned. This feature was maintained after lyophilization and re-hydratation. The characteristic of the device was tested showing the sensitivity to saline concentration and the effective functionality of the device. Moreover, the device response shows a dependence to pH variations and also transconductance presents substantial changes in presence of pH variation. These data suggest the possibility of using this sensorised scaffold as a wearable detector for wound healing monitoring in patients affected by chronic lesions.

The non-selective β-blocking (±)-Propranolol Hydrochloride was demonstrated to improve the progression-free survival of patients and to reduce the incidence of different cancer types. Since the expression of β-adrenoceptors (β-AR) in the A431 squamous cell carcinoma (cSCC) human cells was described, we had suggested that cSCC proliferation may be controlled by using β-AR-blockers. Thus, we hypothesized that the topical application of a β-AR-blocker over the tumor lesion may decrease/restrain its extension before the surgical excision becoming an adjuvant\therapy against cSCC. However, it is known that β-AR-blocker anti-cancer activity as a single agent is limited. Hence, we suggested that the combination of Propranolol with the glucose analog 2-Deoxy-D-glucose (2-DG) could improve its antiproliferative effect through the induction of metabolic stress. Our results have demonstrated that the addition of 2DG to (±)-Propranolol Hydrochloride therapy can improve its effect on A431 cells metabolism and proliferation, suggesting that the combination of (±)-Propranolol Hydrochloride with low dose of 2DG could be a promising treatment to be topically applied as an adjuvant pre-surgical therapy against cSCC, aiming to decrease the size of the injury before the surgical procedure, avoiding systemic adverse effects to the patient.