Chitosan hydrogels incorporating colloids for sustained drug delivery.

In today's biomedical research, a huge effort is being made towards the development of efficient drug delivery systems, achieving sustainable and controlled delivery of drugs. Chitosan (CS) hydrogels are high water content materials with very relevant biological properties to that purpose. Their use for a local and delayed delivery has already been demonstrated for a wide variety of therapeutic agents. One relatively recent strategy to improve these CS-based systems consists in the insertion of colloids, embedding drugs, within their three-dimensional matrix. This provides a second barrier to the diffusion of drugs through the system, and allows to better control their release. The main objective of this review is to report the many existing complex systems composed of CS hydrogels embedding different types of colloids used as drug delivery devices to delay the release of drugs. The various biomedical applications of such final systems are also detailed in this review.

Nanoscale mechanical properties of chitosan hydrogels as revealed by AFM.

In the context of tissue engineering, chitosan hydrogels are attractive biomaterials because they represent a family of natural polymers exhibiting several suitable features (cytocompatibility, bioresorbability, wound healing, bacteriostatic and fungistatic properties, structural similarity with glycosaminoglycans), and tunable mechanical properties. Optimizing the design of these biomaterials requires fine knowledge of its physical characteristics prior to assessment of the cell-biomaterial interactions. In this work, using atomic force microscopy (AFM), we report a characterization of mechanical and topographical properties at the submicron range of chitosan hydrogels, depending on physico-chemical parameters such as their polymer concentration (1.5%, 2.5% and 3.5%), their degree of acetylation (4% and 38.5%), and the conditions of the gelation process. Well-known polyacrylamide gels were used to validate the methodology approach for the determination and analysis of elastic modulus (i.e., Young's modulus) distribution at the gel surface. We present elastic modulus distribution and topographical and stiffness maps for different chitosan hydrogels. For each chitosan hydrogel formulation, AFM analyses reveal a specific asymmetric elastic modulus distribution that constitutes a useful hallmark for chitosan hydrogel characterization. Our results regarding the local mechanical properties and the topography of chitosan hydrogels initiate new possibilities for an interpretation of the behavior of cells in contact with such soft materials.

Dental pulp inflammatory/immune response to a chitosan-enriched fibrin hydrogel in the pulpotomised rat incisor.

Current pulpotomy is limited in its ability to induce regeneration of the dental-pulp (DP) complex. Hydrogels are reported to be well-suited for tissue engineering and are unlikely to induce an inflammatory response that might damage the remaining tissue. The present study investigated the molecular and cellular actors in the early inflammatory/immune response and deciphered M1/M2 macrophage polarisation to a chitosan-enriched fibrin hydrogel in pulpotomised rat incisors. Both fibrin and fibrin-chitosan hydrogels induced a strong increase in interleukin-6 (IL-6) transcript in the DP when compared to the DP of untreated teeth. Gene expression of other inflammatory mediators was not significantly modified after 3 h. In the viable DP cell population, the percentage of leukocytes assessed by flow cytometry was similar to fibrin and fibrin-chitosan hydrogels after 1 d. In this leukocyte population, the proportion of granulocytes increased beneath both hydrogels whereas the antigen-presenting cell, myeloid dendritic cells, T cells and B cells decreased. The natural killer (NK) cell population was significantly decreased only in DPs from teeth treated with fibrin-chitosan hydrogel. Immunolabeling analysis of the DP/hydrogel interface showed accumulation of neutrophil granulocytes in contact with both hydrogels 1 d after treatment. The DP close to this granulocyte area contained M2 but no M1 macrophages. These data collectively demonstrated that fibrin-chitosan hydrogels induced an inflammatory/immune response similar to that of the fibrin hydrogel. The results confirmed the potential clinical use of fibrin-chitosan hydrogel as a new scaffold for vital-pulp therapies.

Chitosan hydrogels for sustained drug delivery.

Sustainable and controlled delivery of drugs is at the centre of a huge amount of undertaken researches. The ability of hydrogels, high water content materials, to achieve a local and delayed-delivery has already been demonstrated for a wide variety of therapeutic agents and various polymer natures. In particular, chitosan, a natural polymer, stands out as a first choice material for hydrogels elaboration in biomedical, cosmetic, and health related applications, owing to its interesting properties (as biocompatibility, biodegradability, antimicrobial capacity, and mucoadhesivity). Moreover, chitosan also allows drugs to go easier through biological barriers. The main objective of this review is to report the various uses of chitosan hydrogels as drug delivery devices to control and/or delay the release of drugs loaded into their three dimensional matrix. A wide spectrum of corresponding biomedical applications of these systems can be encountered in the literature, whatever the physicochemical nature of drugs (hydrophilic, hydrophobic, macromolecular), as detailed in this review.

Embedment of liposomes into chitosan physical hydrogel for the delayed release of antibiotics or anaesthetics, and its first ESEM characterization.

This work describes the characterization of an original liposomes/hydrogel assembly, and its application as a delayed-release system of antibiotics and anaesthetics. This system corresponds to drug-loaded liposomes entrapped within a chitosan (CS) physical hydrogel. To this end, a suspension of pre-formed 1,2-dipalmitoyl-sn-glycero-3-phosphocoline liposomes loaded with an antibiotic (rifampicin, RIF), an anaesthetic (lidocaine, LID), or a model fluorescent molecule (carboxyfluorescein, CF), was added to a CS solution. The CS gelation was subsequently carried out without any trace of chemical cross-linking agent or organic solvent in the final system. Liposomes within the resulting gelled CS matrix were characterized for the first time by environmental scanning electron microscopy. The release of drugs from the assembly was investigated by fluorescence or UV spectroscopy. The cumulative release profiles of RIF and LID (and also CF for comparison) were found to be lower from the "drug-in-liposomes-in-hydrogel" (DLH) assembly in comparison to "drug-in-hydrogel" (DH) system.

Self-crosslinked fibrous collagen/chitosan blends: Processing, properties evaluation and monitoring of degradation by bi-fluorescence imaging.

Porous collagen/chitosan scaffolds with different Collagen:Chitosan (Coll:Ch) ratios were prepared by freeze-drying followed by self-crosslinking via dehydrothermal treatment (DHT) and characterized as biomaterials for tissue engineering. Cy7 and Cy5.5 fluorochromes were covalently grafted to collagen and chitosan, respectively. Thus, it was possible, using optical fluorescence imaging of the two fluorochromes, to simultaneously track their in vivo biodegradation, in a blend scaffold form. The fluorescence signal evolution, due to the bioresorption, corroborated with histological analysis. In vitro cytocompatibility of Coll:Ch blend scaffolds were evaluated with standardized tests. In addition, the scaffolds showed a highly interconnected porous structure. Extent of crosslinking was analyzed by convergent analysis using thermogravimetry, Fourier Transform Infrared Spectroscopy and PBS uptake. The variations observed with these techniques indicate strong interactions between collagen and chitosan (covalent and hydrogen bonds) promoted by the DHT. The mechanical properties were characterized to elucidate the impact of the different processing steps in the sample preparation (DHT, neutralization and sterilization by ß-irradiation) and showed a robust processing scheme with low impact of Coll:Ch composition ratio.

Chitosan-based hydrogels for developing a small-diameter vascular graft: in vitro and in vivo evaluation.

AIMS: Vascular grafts made of synthetic polymers perform poorly in small-diameter applications (cardiac and peripheral bypass). Chitosan is a biocompatible natural polymer that can provide a novel biological scaffold for tissue engineering development. The goal of this study was to demonstrate the biocompatibility of a novel chitosan preparation in vitro and in vivo, and to assess its potential as a scaffold for vascular applications. METHODS AND RESULTS: A series of experiments of increasing complexity, ranging from in vitro biocompatibility and hemocompatibility tests to in vivo studies in small and large animals (rats and sheep), was performed to provide a comprehensive analysis of chitosan hydrogels' biological properties. In vitro studies established that: (i) chitosan supported human endothelial progenitor cells adhesion, proliferation and resistance to physiological shear stress; (ii) chitosan did not activate platelets, the complement system, or the intrinsic coagulation pathway. In vivo results showed: (iii) no resorption of chitosan and no chronic inflammation at 60 days in a rat heterotopic implantation model (magnetic resonance imaging and histology); (iv) no flow obstruction (Doppler ultrasound) and no thrombus formation (histology and scanning electron microscopy) at 2 h after a carotid arteriotomy repair with chitosan patches in sheep. Finally, two chitosan tubes were implanted as carotid interposition grafts for 3 days in sheep showing that chitosan was strong enough to be sutured, to withstand arterial pressure, and no flow obstruction was observed through this short period. CONCLUSION: Chitosan-based hydrogels displayed promising in vitro biocompatibility and hemocompatibility properties as well as in vivo short-term performance.

Polyelectrolyte complexes via desalting mixtures of hyaluronic acid and chitosan-Physicochemical study and structural analysis.

Polyelectrolyte complexes (PECs) were prepared from Chitosan (CS) and Hyaluronic Acid (HYA) homogeneous mixtures of aqueous solutions. The method consisted of preparing a homogeneous mixture of the two polysaccharides via charge screening at high salt concentrations. Then, the mixture was dialyzed, leading to the controlled self-assembly of the two polyelectrolytes. Critical parameters like the chitosan degree of acetylation (DA) and molar mass (Mw), the residual salt concentration and the molar charge ratio r=nNH3(+) (CS)/nCOO(-) (HYA) accounted for the transition from homogeneous aqueous solutions to colloidal suspensions (r=0.1) or gel coacervates (r=0.5). The influence of the DA and Mw of CS was evaluated by visual observations, light scattering and rheological measurements. For low values of r, Small Angle X-ray Scattering (SAXS) experiments revealed that the HYA nanostructure was weakly affected by the presence of PECs. On the contrary, the structure was impacted when increasing r, revealing a heterogeneous aggregate morphology with ladder-like chain interactions.

Chitosan solutions as injectable systems for dermal filler applications: Rheological characterization and biological evidence.

A new generation of dermal filler for wrinkle filler based on chitosan was compared to current hyaluronic acid-based dermal fillers by using a new rheological performance criterion based on viscosity during injection related to Newtonian viscosity. In addition an in vivo evaluation was performed for preclinical evidence of chitosan use as dermal filler. In this way, biocompatibility and dermis reconstruction was evaluated on a pig model.

Particle assemblies: toward new tools for regenerative medicine.

Regenerative medicine is a demanding field in terms of design and elaboration of materials able to meet the specifications that this application imposes. The regeneration of tissue is a multiscale issue, from the signaling molecule through cell expansion and finally tissue growth requiring a large variety of cues that should be delivered in place and time. Hence, the materials should be able to accommodate cells with respect to their phenotypes, to allow cell division to the right tissue, to maintain the integrity of the surrounding sane tissue, and eventually use their signaling machinery to serve the development of the appropriate neo-tissue. They should also present the ability to deliver growth factors and regulate tissue development, to be degraded into safe products, in order not to impede tissue development, and finally be easily implanted/injected into the patients. In this context, colloid-based materials represent a very promising family of products because one can take advantage of their high specific area, their capability to carry/deliver bio-active molecules, and their capacity of assembling (eventually in vivo) into materials featuring other mechanical, rheological, physicochemical properties. Other benefits of great interest would be their ease of production even via high through-put processes and their potential manufacturing from safe, biodegradable and biocompatible parent raw material. This review describes the state-of-the-art of processes leading to complex materials from the assembly of colloids meeting, at least partially, the above-described specifications for tissue engineering and regenerative medicine.

Multi-membrane chitosan hydrogels as chondrocytic cell bioreactors.

We investigated the bioactivity of new chitosan-based multi-membrane hydrogel (MMH) architectures towards chondrocyte-like cells. The microstructure of the hydrogels constituting the membranes precludes any living cell penetration, whereas their lower scale architecture allows the protein diffusion. The biological behavior of chondrocytes implanted within the MMH inter-membrane spaces was studied for 45 days in culture. Chondrocytes formed cell aggregates and proliferated without loosing their chondrogenic phenotype as illustrated by collagen II and aggrecan expressions at the mRNA and protein levels. Cells produced neo-formed alcyan blue matrix proteins filling MMH interspaces. The HiF-2α/SOX9 pattern of expression suggested that the elevated chondrocytic phenotype in MMH could be related to a better hypoxic local environment than in classical culture conditions. Pro-inflammatory markers were not expressed during the period of culture. The low level of nitric oxide accumulation within the inter-membrane spaces and in the incubation medium implied that chitosan consumed nitrites produced by entrapped chondrocytes, in relation with the decrease of its molecular weight of 50%. Our data suggest that MMH structures may be considered as complex chondrocytic cell bioreactors; "active decoys of biological media", potentially promising for various biomedical applications like the inter-vertebral disk replacement.

Polyelectrolyte microstructure in chitosan aqueous and alcohol solutions.

This work deals with chain ordering in aqueous and water-alcohol solutions of chitosan. The so-called polyelectrolyte peak is investigated by small-angle synchrotron X-ray scattering. The polyelectrolyte microstructure was characterized by the position of the maximum of the polyelectrolyte scattering peak qmax, which scales with the polymer concentration cp as qmax approximately cp alpha. An evolution of the power law exponent alpha is observed as a function of the degree of acetylation (DA) of chitosan, which is responsible for changes of both the charge density (f) and the hydrophobicity of the polymer chains. The results highlighted the two organization regimes of the theory of Dobrynin and Rubinstein, investigated here for the first time for a natural polymer. At low DAs, alpha approximately 1/2, in agreement with a pearl necklace organization where the structure is controlled by the string between pearls. For higher DA, alpha approximately 1/3, and the correlation revealed by the polyelectrolyte peak is controlled by the pearls. This analysis offers a way to study quantitatively the balance between solvophobic-solvophilic interactions that play an important role in the solution properties of natural polymers. In addition, the role of several parameters acting on the interaction balance were evidenced, such as the nature of the counterion, the composition of the solvent (amount of alcohol in the aqueous solution), and the screening of Coulombic forces by salt addition. Finally, the nanostructure transition from a polyelectrolyte solution to a physical gel is discussed. The gel state is reached when the solvophobic interactions are favored, but depending on the gelation route the polyelectrolyte ordering could be preserved or not.

A material decoy of biological media based on chitosan physical hydrogels: application to cartilage tissue engineering.

The cartilage tissue has a limited self-regenerative capacity. Tissue-engineering represents a promising trend for cartilage repair. The present study was aimed to develop a biomaterial formulation by combining fragments of chitosan hydrogel with isolated rabbit or human chondrocytes. We first reported the properties of the constructs elaborated with rabbit chondrocytes and pure chitosan physical hydrogels with defined molecular weight, acetylation degree and polymer concentration. Morphological data showed that chondrocytes were not penetrating the hydrogels but tightly bound to the surface of the fragments and spontaneously formed aggregates of combined cell/chitosan. A significant amount of neo-formed cartilage-like extracellular matrix (ECM) was first accumulated in-between cells and hydrogel fragments and furthermore was widely distributed within the neo-construct. The optimal biological response was obtained with hydrogel fragments concentrated at 1.5% (w/w) of polymer made from a chitosan with a degree of acetylation between 30 and 40%. Such hydrogels were then mixed with human chondrocytes. The phenotype of the cells was analyzed by using chondrocytic (mRNA expression of mature type II collagen and aggrecan as well as secretion of proteoglycans of high molecular weight) and non chondrocytic (mRNA expression of immature type II collagen and type I collagen) molecular markers. As compared with human chondrocytes cultured without chitosan hydrogel which rapidly dedifferentiated in primary culture, cells mixed with chitosan rapidly loose the expression of type I and immature type II collagen while they expressed mature type II collagen and aggrecan. In these conditions, chondrocytes maintained their phenotype for as long as 45 days, thus forming cartilage-like nodules. Taken together, these data suggest that a chitosan hydrogel does not work as a scaffold, but could be considered as a decoy of cartilage ECM components, thus favoring the binding of chondrocytes to chitosan. Such a biological response could be described by the concept of reverse encapsulation.

Quality assessment of seven types of fresh-frozen plasma leucoreduced by specific plasma filtration.

BACKGROUND AND OBJECTIVES: A study was undertaken to determine plasma quality after specific filtration. MATERIALS AND METHODS: Seven types of plasma were tested, after filtration of plasma from filtered or non-filtered whole blood. Leucocyte counting was carried out after a 30-fold concentration of the sample. Twenty-nine parameters (including coagulation testing, proteins, coagulation factors and activation markers) were measured before and after filtration, and after 6 months of storage. RESULTS: After specific plasma filtration, the average residual leucocyte counts were less than 2250/l. In spite of small statistically significant changes in proteins, coagulation factors and complement activation, this study showed that plasma filtration did not alter plasma quality. After 6 months of storage at -30 degrees C, factor VIII recovery varied between 91 and 109%. Haemostasis parameters and activation markers remained within the normal range. CONCLUSIONS: Specific plasma filtration reduced the leucocyte number to < 104 leucocytes/l. The quality of plasma was not altered by the additional step of specific plasma filtration.

Frequency of human anti-FVIII antibodies in humanized SCID mice elicited by recombinant deleted factor VIII and by a plasma derived factor VIII.

SCID mice were grafted with human PBL (hu-PBL-SCID) from healthy or haemophilia A donors. Those containing human and no murine Ig in their plasma, were injected with 100 U VIII:Ag of a plasma derived (pd) FVIII or recombinant deleted Factor VIII (FVIII deltaII) and with 10 microg of tetanus toxoid as control immunogen. The frequency and the intensity of the humoral specific responses were measured in 253 mice humanized with PBL from 13 different donors. There was no significant difference in the frequency or intensity of the anti-FVIII immune responses to pd FVIII and FVIII deltaII. Neutralizing antibodies were only detected in the plasma of mice humanized with cells from haemophiliacs having FVIII inhibitors in their blood. The immune responses observed in hu-PBL-SCID mice correlated with the immune status of the corresponding human donor.