Impact of Composition and Autoclave Sterilization on the Mechanical and Biological Properties of ECM-Mimicking Cryogels.

Cryogels represent a valid strategy as scaffolds for tissue engineering. In order to adequately support adhesion and proliferation of anchorage-dependent cells, different polymers need to be combined within the same scaffold trying to mimic the complex features of a natural extracellular matrix (ECM). For this reason, in this work, gelatin (Gel) and chondroitin sulfate (CS), both functionalized with methacrylic groups to produce CSMA and GelMA derivatives, were selected to prepare cryogel networks. Both homopolymer and heteropolymer cryogels were produced, via radical crosslinking reactions carried out at -12 °C for 2 h. All the scaffolds were characterized for their mechanical, swelling and morphological properties, before and after autoclave sterilization. Moreover, they were evaluated for their biocompatibility and ability to support the adhesion of human gingival fibroblasts and tenocytes. GelMA-based homopolymer networks better withstood the autoclave sterilization process, compared to CSMA cryogels. Indeed, GelMA cryogels showed a decrease in stiffness of approximately 30%, whereas CSMA cryogels of approximately 80%. When GelMA and CSMA were blended in the same network, an intermediate outcome was observed. However, the hybrid scaffolds showed a general worsening of the biological performance. Indeed, despite their ability to withstand autoclave sterilization with limited modification of the mechanical and morphological properties, the hybrid cryogels exhibited poor cell adhesion and high LDH leakage. Therefore, not only do network components need to be properly selected, but also their combination and ability to withstand effective sterilization process should be carefully evaluated for the development of efficient scaffolds designed for tissue engineering purposes.

Liposome Encapsulation of the Palmitoyl-KTTKS Peptide: Structural and Functional Characterization.

In this study, the amphiphilic N-palmitoyl-KTTKS peptide was integrated in the bilayer of egg-derived phosphatidylcholine (PC) vesicles using two different preparation methods, namely thin-film evaporation (TLE) and reverse-phase evaporation (REV). Both the REV and TLE methods allowed for the formation of homogeneous liposome dispersions (PdI < 0.20) with mean hydrodynamic diameters of <100 nm and <200 nm, respectively, a net negative surface charge and a percentage of structured phospholipids higher than 90%. The inclusion of the amphiphilic N-palmitoyl-KTTKS peptide within phospholipid-based vesicles could improve peptide stability and skin delivery. Therefore, the obtained liposomes were evaluated via experiments assessing the synthesis of collagen and the ECM in 3T3-NIH fibroblasts. The obtained results showed that, when delivered with PC liposomes, pal-KTTKS stimulated collagen production more than free pentapeptide and 1 mM ascorbic acid, used as a positive control.

Long-Term Stability of Lavandula x intermedia Essential Oil Nanoemulsions: Can the Addition of the Ripening Inhibitor Impact the Biocidal Activity of the Nanoformulations?

In this work, Lavandula x intermedia essential oil (LEO) was encapsulated in lipid-based nanoemulsions (NanoLEO) using the solvent-displacement technique. In order to preserve the colloidal stability of the formulation, LEO was appropriately doped with the incorporation of different levels of a water-insoluble oil used as a ripening inhibitor. All the nanoemulsion samples were evaluated in terms of the impact of the water-insoluble oil on the nanoemulsion formation, physical-chemical properties, and antibacterial effectiveness against E. coli (Gram-negative) and B. cereus (Gram-positive). The presence of the inert oil added benefits to the formulations in terms of appearance, colloidal stability, and loss of volatile components. However, the antimicrobial activity of the nanoemulsions dramatically decreased with the ripening inhibitor addition, probably because it hampered the internalization of the antimicrobial components of LEO within the bacterial cell membranes, thus nullifying the delivery ability of the nanoemulsion formulation. On the contrary, the undoped NanoLEO formulation showed unaltered antibacterial activity in both E. coli and B. cereus up to 40 weeks from the preparation.

Cryogel Scaffolds for Tissue-Engineering: Advances and Challenges for Effective Bone and Cartilage Regeneration.

Critical-sized bone defects and articular cartilage injuries resulting from trauma, osteonecrosis, or age-related degeneration can be often non-healed by physiological repairing mechanisms, thus representing a relevant clinical issue due to a high epidemiological incidence rate. Novel tissue-engineering approaches have been proposed as an alternative to common clinical practices. This cutting-edge technology is based on the combination of three fundamental components, generally referred to as the tissue-engineering triad: autologous or allogenic cells, growth-stimulating factors, and a scaffold. Three-dimensional polymer networks are frequently used as scaffolds to allow cell proliferation and tissue regeneration. In particular, cryogels give promising results for this purpose, thanks to their peculiar properties. Cryogels are indeed characterized by an interconnected porous structure and a typical sponge-like behavior, which facilitate cellular infiltration and ingrowth. Their composition and the fabrication procedure can be appropriately tuned to obtain scaffolds that match the requirements of a specific tissue or organ to be regenerated. These features make cryogels interesting and promising scaffolds for the regeneration of different tissues, including those characterized by very complex mechanical and physical properties, such as bones and joints. In this review, state-of-the-art fabrication and employment of cryogels for supporting effective osteogenic or chondrogenic differentiation to allow for the regeneration of functional tissues is reported. Current progress and challenges for the implementation of this technology in clinical practice are also highlighted.

Insights into the reaction of chondroitin sulfate with glycidyl methacrylate: 1D and 2D NMR investigation.

Chondroitin sulfate methacrylate (CS-MA) is a semisynthetic biopolymer increasingly used for the fabrication of chemical hydrogels. In this study, the methacrylation reaction of native CS was carried out with glycidyl methacrylate in dimethyl sulfoxide and optimized to obtain tunable and reproducible methacrylation degrees in a short reaction time. The methacrylation reaction was deeply characterized by mono- and bi-dimensional (1D, 2D) NMR spectroscopy of CS-MA derivatives with different methacrylation degrees. In contrast to what previously reported in the literature, HSQC, HMBC and TOCSY analyses revealed that the methacrylation reaction proceeds via both epoxy ring-opening and transesterification, involving predominantly the primary hydroxyl groups of CS, while preserving sulfate and carboxyl groups of the biopolymer. These findings are of fundamental importance for appropriate and rational design of CS-MA-based biomaterials.

Solvent Casting and UV Photocuring for Easy and Safe Fabrication of Nanocomposite Film Dressings.

The aim of this work was to optimize and characterize nanocomposite films based on gellan gum methacrylate (GG-MA) and silver nanoparticles (AgNPs) for application in the field of wound dressing. The films were produced using the solvent casting technique coupled with a photocuring process. The UV irradiation of GG-MA solutions containing glycerol as a plasticizer and different amounts of silver nitrate resulted in the concurrent crosslinking of the photocurable polymer and a reduction of Ag ions with consequent in situ generation of AgNPs. In the first part of the work, the composition of the films was optimized, varying the concentration of the different components, the GG-MA/glycerol and GG-MA/silver nitrate weight ratios as well as the volume of the film-forming mixture. Rheological analyses were performed on the starting solutions, whereas the obtained films were characterized for their mechanical properties. Colorimetric analyses and swelling studies were also performed in order to determine the AgNPs release and the water uptake capacity of the films. Finally, microbiological tests were carried out to evaluate the antimicrobial efficacy of the optimized films, in order to demonstrate their possible application as dressings for the treatment of infected hard-to-heal wounds, which is a demanding task for public healthcare.