Marine collagen-chitosan-fucoidan cryogels as cell-laden biocomposites envisaging tissue engineering.

The combination of marine origin biopolymers for tissue engineering (TE) applications is of high interest, due to their similarities with the proteins and polysaccharides present in the extracellular matrix of different human tissues. This manuscript reports on innovative collagen-chitosan-fucoidan cryogels formed by the simultaneous blending of these three marine polymers in a chemical-free crosslinking approach. The physicochemical characterization of marine biopolymers comprised FTIR, amino acid analysis, circular dichroism and SDS-PAGE, and suggested that the jellyfish collagen used in the cryogels was not denatured (preserved the triple helical structure) and had similarities with type II collagen. The chitosan presented a high deacetylation degree (90.1%) that can strongly influence the polymer physicochemical properties and biomaterial formation. By its turn, rheology, and SEM studies confirmed that these novel cryogels present interesting properties for TE purposes, such as effective blending of biopolymers without visible material segregation, mechanical stability (strong viscoelastic character), as well as adequate porosity to support cell proliferation and exchange of nutrients and waste products. Additionally, in vitro cellular assessments of all cryogel formulations revealed a non-cytotoxic behavior. The MTS test, live/dead assay and cell morphology assessment (phalloidin DAPI) showed that cryogels can provide a proper microenvironment for cell culturing, supporting cell viability and promoting cell proliferation. Overall, the obtained results suggest that the novel collagen-chitosan-fucoidan cryogels herein presented are promising scaffolds envisaging tissue engineering purposes, as both acellular biomaterials or cell-laden cryogels.

Tunable Enzymatically Cross-Linked Silk Fibroin Tubular Conduits for Guided Tissue Regeneration.

Hollow tubular conduits (TCs) with tunable architecture and biological properties are in great need for modulating cell functions and drug delivery in guided tissue regeneration. Here, a new methodology to produce enzymatically cross-linked silk fibroin TCs is described, which takes advantage of the tyrosine groups present in silk structure that are known to allow the formation of a covalently cross-linked hydrogel. Three different processing methods are used as a final step to modulate the properties of the silk-based TCs. This approach allows to virtually adjust any characteristic of the final TCs. The final microstructure ranges from a nonporous to a highly porous network, allowing the TCs to be selectively porous to 4 kDa molecules, but not to human skin fibroblasts. Mechanical properties are dependent both on the processing method and thickness of the TCs. Bioactivity is observed after 30 days of immersion in simulated body fluid only for the TCs submitted to a drying processing method (50 °C). The in vivo study performed in mice demonstrates the good biocompatibility of the TCs. The enzymatically cross-linked silk fibroin TCs are versatile and have adjustable characteristics that can be exploited in a variety of biomedical applications, particularly in guidance of peripheral nerve regeneration.

Dual delivery of hydrophilic and hydrophobic drugs from chitosan/diatomaceous earth composite membranes.

Oral administration of drugs presents important limitations, which are frequently not granted the importance that they really have. For instance, hepatic metabolism means an important drug loss, while some patients have their ability to swell highly compromised (i.e. unconsciousness, cancer…). Sublingual placement of an accurate Pharmaceutical Dosage Form is an attractive alternative. This work explores the use of the ß-chitosan membranes, from marine industry residues, composed with marine sediments for dual sublingual drug delivery. As proof of concept, the membranes were loaded with a hydrophilic (gentamicin) and a hydrophobic (dexamethasone) drug. The physico-chemical and morphological characterization indicated the successful incorporated of diatomaceous earth within the chitosan membranes. Drug delivery studies showed the potential of all formulations for the immediate release of hydrophilic drugs, while diatomaceous earth improved the loading and release of the hydrophobic drug. These results highlight the interest of the herein developed membranes for dual drug delivery.

Gellan gum based physical hydrogels incorporating highly valuable endogen molecules and associating BMP-2 as bone formation platforms.

Physical hydrogels have been designed for a double purpose: as growth factor delivery systems and as scaffolds to support cell colonization and formation of new bone. Specifically, the polysaccharide gellan gum and the ubiquitous endogenous molecules chondroitin, albumin and spermidine have been used as exclusive components of these hydrogels. The mild ionotropic gelation technique was used to preserve the bioactivity of the selected growth factor, rhBMP-2. In vitro tests demonstrated the effective delivery of rhBMP-2 in its bioactive form. In vivo experiments performed in the muscle tissue of Wistar rats provided a proof of concept of the ability of the developed platforms to elicit new bone formation. Furthermore, this biological effect was better than that of a commercial formulation currently used for regenerative purposes, confirming the potential of these hydrogels as new and innovative growth factor delivery platforms and scaffolds for regenerative medicine applications.

Investigation of cell adhesion in chitosan membranes for peripheral nerve regeneration.

Peripheral nerve injuries have produced major concerns in regenerative medicine for several years, as the recovery of normal nerve function continues to be a significant clinical challenge. Chitosan (CHT), because of its good biocompatibility, biodegradability and physicochemical properties, has been widely used as a biomaterial in tissue engineering scaffolding. In this study, CHT membranes were produced with three different Degrees of Acetylation (DA), envisioning its application in peripheral nerve regeneration. The three CHT membranes (DA I: 1%, DA II: 2%, DA III: 5%) were extensively characterized and were found to have a smooth and flat surface, with DA III membrane having slightly higher roughness and surface energy. All the membranes presented suitable mechanical properties and did not show any signs of calcification after SBF test. Biodegradability was similar for all samples, and adequate to physically support neurite outgrowth. The in vitro cell culture results indicate selective cell adhesion. The CHT membranes favoured Schwann cells invasion and proliferation, with a display of appropriate cytoskeletal morphology. At the same time they presented low fibroblast infiltration. This fact may be greatly beneficial for the prevention of fibrotic tissue formation, a common phenomenon impairing peripheral nerve regeneration. The great deal of results obtained during this work permitted to select the formulation with the greatest potential for further biological tests.

Spermidine cross-linked hydrogels as a controlled release biomimetic approach for cloxacillin.

The intrinsic ability of albumin to bind active substances in the physiological fluids has been explored to endow hydrogels with improved capability to regulate drug release. To develop such biomimetic-functional hydrogels, it is critical that albumin conformation is not altered and that the protein remains retained inside the hydrogel keeping its conformational freedom, i.e., it should be not chemically cross-linked. Thus, the hydrogels were prepared with various proportions of albumin by physical cross-linking of anionic polysaccharides (gellan gum and chondroitin sulfate) with the cationic endogen polyamine spermidine under mild conditions in order to prevent albumin denaturation. Texture and swelling properties of hydrogels with various compositions were recorded, and the effect of the preparation variables was evaluated applying neurofuzzy logic tools for hydrogels prepared with and without albumin and associating the antibiotic cloxacillin. Developed hydrogel systems were extensively analyzed by means of nuclear magnetic resonance (NMR) to determine weak-to-medium and strong binding modes and the equilibrium constants of the albumin-cloxacillin association. NMR techniques were also employed to demonstrate the successful modulation of the cloxacillin release from the albumin-containing hydrogels. In vitro microbiological tests carried out with Staphylococcus aureus and Staphylococcus epidermidis confirmed the interest of the albumin-containing hydrogels as efficient platforms for cloxacillin release in its bioactive form.

Progress in the characterization of bio-functionalized nanoparticles using NMR methods and their applications as MRI contrast agents.

Significant progress has been made over the last three decades in the field of NMR, a technique which has proven to have a variety of applications in many scientific disciplines, including nanotechnology. Herein we describe how NMR enables the characterization of nanosystems at different stages of their formation and modification (raw materials, bare or functionalized nanosystems), even making it possible to study in vivo nanoparticle interactions, thereby importantly contributing to nanoparticle design and subsequent optimization. Furthermore, the unique characteristics of nanosystems can open up new prospects for site-targeted, more specific contrast agents, contributing to the development of certain nuclear magnetic resonance applications such as MRI.

Application of NMR spectroscopy in the development of a biomimetic approach for hydrophobic drug association with physical hydrogels.

The clinical application of sparingly soluble drugs is hampered by the wide range of problems associated to their delivery. Herein we present a new physical hydrogel as a delivery system for these drugs. The strategy behind the design of this delivery system involved the incorporation of the protein albumin into the hydrogel with the aim of exploiting its intrinsic capacity to bind small hydrophobic molecules. Prednisolone and ketoconazole were used as model drug molecules. A combination of the saturation transfer difference (STD) spectra and a novel double titration assay followed by NMR was applied to study all of the possible binding modes between albumin and each drug. Finally, the ability of the hydrogel system to release the two model drugs was corroborated. The results of the release studies were in agreement with the drug binding capacities derived from the NMR studies, thus confirming that the potential of the NMR approach as a predictive technique could be useful in evaluating the designs of new drug delivery systems.

Spermidine-cross-linked hydrogels as novel potential platforms for pharmaceutical applications.

Endogen polyamines are known to be molecules of high biological value. Herein, a new generation of physical hydrogels was developed through the mild ionotropic gelantion technique, using the endogen polyamine spermidine as a physical cross-linker. The main negatively charged polymer of the hydrogel is the natural polysaccharide gellan gum. Optionally, interesting endogen molecules, such as chondroitin sulfate and albumin, can be included as part of the formulation. These new hydrogels were characterized and the influence of the different components on their final properties was carefully analyzed, ultimately demonstrating the possibility to modulate these properties as well as the system's versatility in terms of composition. On the contrary, in vitro cell studies showed the absence of cytotoxicity of these hydrogels. Finally, the in vitro-release profiles obtained for different model molecules evidenced the potential of these systems as novel drug delivery platforms.

Chemically modified gelatin as biomaterial in the design of new nanomedicines.

The synthesis of new polymers has led to dramatic enhancements in the medical field. In particular, new chemical entities provided new prospects in tissue engineering, cellular therapy and drug delivery. However, significant efforts still need to be taken in consideration in order to achieve diverse clinical applications in these fields, which is challenging because of the lack of suitable materials with desired microstructure, permeability, degradation rates, products, and suitable mechanical properties. For these reasons some chemical strategies are focused in back to the nature approaches or, in other words, in improving the properties of natural polymers by chemical modifications. We report that by using a simple chemical modification technique we can obtain new biomaterials, specifically suitable for biomedical applications. Concretely, we describe the chemical modification of gelatin and the suitable characteristics of the modified protein to develop new nanomedicines. This protein was selected because of its enormous potential in biomedicine, which is currently limited due to the difficulty of its use without toxic chemical crosslinkers. The modification of the protein was based on the transformation of the carboxylic group into amido groups after their reaction with polyamines, leading to a positively charged biopolymer. To cationize the gelatin two polyamines where used: ethylenediamine and spermine, the latter being one of the endogenous polyamines which has a very positive influence over cells. This modification was monitored by physico-chemical techniques such as NMR, spectrophotometry and potentiometry. With the most promising modified gelatins we were able to develop nanoparticles using the ionotropic gelation technique. In order to determine the ability of these new nanosystems to associate bioactive molecules we selected a model plasmid DNA. The developed nanosystems were characterized corroborating their ability to associate the genetic material. In conclusion, we were able to obtain a semi-synthetic biomaterial with tunable physico-chemical properties, which can be used to develop new nanosystems with the ability to associate genetic material. We therefore propose that the gelatin, with its chemical modification, provide a unique biomaterial for the development of new nanomedicines.