New vegetable-waste biomaterials by Lupin albus L. as cellular scaffolds for applications in biomedicine and food.

The reprocessing of vegetal-waste represents a new research field in order to design novel biomaterials for potential biomedical applications and in food industry. Here we obtained a biomaterial from Lupinus albus L. hull (LH) that was characterized micro-structurally by scanning electron microscopy and for its antimicrobial and scaffolding properties. A good adhesion and proliferation of human mesenchymal stem cells (hMSCs) seeded on LH scaffold were observed. Thanks to its high content of cellulose and beneficial phytochemical substances, LH and its derivatives can represent an available source for fabrication of biocompatible and bioactive scaffolds. Therefore, a reprocessing protocol of LH was optimized for producing a new LH bioplastic named BPLH. This new biomaterial was characterized by chemico-physical analyses. The water uptake, degradability and antimicrobial properties of BPLH were evaluated, as well as the mechanical properties. A good adhesion and proliferation of both fibroblasts and hMSCs on BPLH were observed over 2 weeks, and immunofluorescence analysis of hMSCs after 3 weeks indicates an initial commitment toward muscle differentiation. Our work represents a new approach toward the recovery and valorization of the vegetal waste showing the remarkable properties of LH and BPLH as cellular waste-based scaffold with potential applications in cell-based food field as well as in medicine for topical patches in wound healing and bedsores treatment.

Photo-Polymerization Damage Protection by Hydrogen Sulfide Donors for 3D-Cell Culture Systems Optimization.

Photo-polymerized hydrogels are ideally suited for stem-cell based tissue regeneration and three dimensional (3D) bioprinting because they can be highly biocompatible, injectable, easy to use, and their mechanical and physical properties can be controlled. However, photo-polymerization involves the use of potentially toxic photo-initiators, exposure to ultraviolet light radiation, formation of free radicals that trigger the cross-linking reaction, and other events whose effects on cells are not yet fully understood. The purpose of this study was to examine the effects of hydrogen sulfide (H2S) in mitigating cellular toxicity of photo-polymerization caused to resident cells during the process of hydrogel formation. H2S, which is the latest discovered member of the gasotransmitter family of gaseous signalling molecules, has a number of established beneficial properties, including cell protection from oxidative damage both directly (by acting as a scavenger molecule) and indirectly (by inducing the expression of anti-oxidant proteins in the cell). Cells were exposed to slow release H2S treatment using pre-conditioning with glutathione-conjugated-garlic extract in order to mitigate toxicity during the photo-polymerization process of hydrogel formation. The protective effects of the H2S treatment were evaluated in both an enzymatic model and a 3D cell culture system using cell viability as a quantitative indicator. The protective effect of H2S treatment of cells is a promising approach to enhance cell survival in tissue engineering applications requiring photo-polymerized hydrogel scaffolds.

Hydrogen Sulfide-Releasing Fibrous Membranes: Potential Patches for Stimulating Human Stem Cells Proliferation and Viability under Oxidative Stress.

The design of biomaterial platforms able to release bioactive molecules is mandatory in tissue repair and regenerative medicine. In this context, electrospinning is a user-friendly, versatile and low-cost technique, able to process different kinds of materials in micro- and nano-fibers with a large surface area-to-volume ratio for an optimal release of gaseous signaling molecules. Recently, the antioxidant and anti-inflammatory properties of the endogenous gasotramsmitter hydrogen sulfide (H2S), as well as its ability to stimulate relevant biochemical processes on the growth of mesenchymal stem cells (MSC), have been investigated. Therefore, in this work, new poly(lactic) acid fibrous membranes (PFM), doped and functionalized with H2S slow-releasing donors extracted from garlic, were synthetized. These innovative H2S-releasing mats were characterized for their morphological, thermal, mechanical, and biological properties. Their antimicrobial activity and effects on the in vitro human cardiac MSC growth, either in the presence or in the absence of oxidative stress, were here assessed. On the basis of the results here presented, these new H2S-releasing PFM could represent promising and low-cost scaffolds or patches for biomedical applications in tissue repair.

Trichormus variabilis (Cyanobacteria) Biomass: From the Nutraceutical Products to Novel EPS-Cell/Protein Carrier Systems.

A native strain of the heterocytous cyanobacterium Trichormus variabilis VRUC 168 was mass cultivated in a low-cost photobioreactor for a combined production of Polyunsaturated Fatty Acids (PUFA) and Exopolymeric Substances (EPS) from the same cyanobacterial biomass. A sequential extraction protocol was optimized leading to high yields of Released EPS (REPS) and PUFA, useful for nutraceutical products and biomaterials. REPS were extracted and characterized by chemical staining, Reversed Phase-High-Performance Liquid Chromatography (RP-HPLC), Fourier Transform Infrared Spectroscopy (FT-IR) and other spectroscopic techniques. Due to their gelation property, REPS were used to produce a photo-polymerizable hybrid hydrogel (REPS-Hy) with addition of polyethylene glycol diacrylated (PEGDa). REPS-Hy was stable over time and resistant to dehydration and spontaneous hydrolysis. The rheological and functional properties of REPS-Hy were studied. The enzyme carrier ability of REPS-Hy was assessed using the detoxification enzyme thiosulfate:cyanide sulfur transferase (TST), suggesting the possibility to use REPS-Hy as an enzymatic hydrogel system. Finally, REPS-Hy was used as a scaffold for culturing human mesenchymal stem cells (hMSCs). The cell seeding onto the REPS-Hy and the cell embedding into 3D-REPS-Hy demonstrated a scaffolding property of REPS-Hy with non-cytotoxic effect, suggesting potential applications of cyanobacteria REPS for producing enzyme- and cell-carrier systems.

Injectable silk fibroin hydrogels functionalized with microspheres as adult stem cells-carrier systems.

Hydrogels are good candidate materials for cell delivery scaffolds because they can mimic the physical, chemical, electrical and biological properties of most of the native tissues. In this study, composite biosynthetic hydrogels were produced by combining the bio-functionality of silk fibroin (SF) with the structural versatility of polyethylene-glycol-diacrylated (PEGDa). The formation of a photopolymerizable PEGDa-SF hydrogel (PSFHy) was optimized for 3D-cell culture. Functionalization of the 3D-PSFHy with protein microspheres (MS) was required to increase the porosity and cell-adhesive properties of the material. Cardiac mesenchymal stem cells, which were cultured within the MS-embedding PSFHy, exhibited good viability and expression of proteins that are characteristic of the initial phases of the cardiac muscle differentiation process. Further, the addition of chondroitin sulfate into the scaffolds improved the cell viability. A cell-preconditioning of the scaffold was also performed, suggesting a potential application of these sponge-like scaffolds for analysing the effects of several extracellular microenvironments, produced by different kinds of cells, on the stem cells fate. The results presented herein highlight on the possibility to use the PSFHys functionalized with MS as stem cell-carrier systems with sponge-like properties, potential ultrasound-imaging contrast agents and controlled biochemical factor delivery.

Scaffold-in-Scaffold Potential to Induce Growth and Differentiation of Cardiac Progenitor Cells.

The design of reliable biocompatible and biodegradable scaffolds remains one of the most important challenges for tissue engineering. In fact, properly designed scaffolds must display an adequate and interconnected porosity to facilitate cell spreading and colonization of the inner layers, and must release physical signals concurring to modulate cell function to ultimately drive cell fate. In this study, a combination of optimal mechanical and biochemical properties has been considered to design a one-component three-dimensional (3D) multitextured hydrogel scaffold to favor cell-scaffold interactions. A polyethylene glycol diacrylate woodpile (PEGDa-Wp) structure of the order of 100 µm has been manufactured using a microstereolithography process. Subsequently, the PEGDa-Wp has been embedded in a PEGDa hydrogel to obtain a 3D scaffold-in-scaffold (3D-SS) system. Finally, the 3D-SS capability to address cell fate has been assessed using human Lin- Sca-1+ cardiac progenitor cells (hCPCs). Results have shown that a multitextured 3D scaffold represents a favorable microenvironment to promote hCPC differentiation and orientation. In fact, while cultured on 3D-SS, hCPCs adopt an ordered 3D spatial orientation and activate the expression of structural proteins, such as the α-sarcomeric actinin, a specific marker of the cardiomyocyte phenotype, and connexin 43, the principal gap junction protein of the heart. Although preliminary, this study demonstrates that complex multitextured scaffolds closely mimicking the extracellular matrix structure and function are efficient in driving progenitor cell fate. A leap forward will be determined by the use of advanced 3D printing technologies that will improve multitextured scaffold manufacturing and their biological efficiency.

H2S-releasing nanoemulsions: a new formulation to inhibit tumor cells proliferation and improve tissue repair.

The improvement of solubility and/or dissolution rate of poorly soluble natural compounds is an ideal strategy to make them optimal candidates as new potential drugs. Accordingly, the allyl sulfur compounds and omega-3 fatty acids are natural hydrophobic compounds that exhibit two important combined properties: cardiovascular protection and antitumor activity. Here, we have synthesized and characterized a novel formulation of diallyl disulfide (DADS) and α-linolenic acid (ALA) as protein-nanoemulsions (BAD-NEs), using ultrasounds. BAD-NEs are stable over time at room temperature and show antioxidant and radical scavenging property. These NEs are also optimal H2S slow-release donors and show a significant anti-proliferative effect on different human cancer cell lines: MCF-7 breast cancer and HuT 78 T-cell lymphoma cells. BAD-NEs are able to regulate the ERK1/2 pathway, inducing apoptosis and cell cycle arrest at the G0/G1 phase. We have also investigated their effect on cell proliferation of human adult stem/progenitor cells. Interestingly, BAD-NEs are able to improve the Lin- Sca1+ human cardiac progenitor cells (hCPC) proliferation. This stem cell growth stimulation is combined with the expression and activation of proteins involved in tissue-repair, such as P-AKT, α-sma and connexin 43. Altogether, our results suggest that these antioxidant nanoemulsions might have potential application in selective cancer therapy and for promoting the muscle tissue repair.