Isabel F. Amaral

Chemical modification of chitosan by phosphorylation: an XPS, FT-IR and SEM study

In the present work, the surface of chitosan membranes was modified using a phosphorylation method carried out at room temperature. Phosphorylation may be of particular interest in materials for orthopaedic applications, due to the cation-exchange properties of phosphate functionalities. Phosphate groups chelate calcium ions, thus inducing the deposition of an apatite-like layer known to improve the osteoconduction of polymer-based implants. Additionally, the negatively charged phosphate functionalities, together with the positively charged amine groups from chitosan, are expected to provide chitosan with an amphoteric character, which may be useful as a combinatorial therapeutic strategy, by simultaneously allowing the immobilization of signalling molecules like growth factors. Phosphorylation was carried out at room temperature using the H3PO4/Et3PO4/P2O5/butanol method. Surface characterization was performed by XPS, ATR–FT-IR, and SEM. Cross-sections were analyzed by SEM fitted with EDS. The phosphate content increased with the reaction time, as shown by XPS and ATR–FT-IR, a P/N atomic ratio of 0.73 being obtained after 48 h of treatment. High-resolution XPS spectra regarding C1s, O1s, N1s and P2p are discussed. The introduction of a neutralization step led to a reduction of P content, which pointed out to the presence of phosphates ionically bound to protonated amines, in addition to phosphate esters. EDS analysis of cross-sections revealed a gradual P reduction up to 50% towards the inner part of the membrane.

Engineering endochondral bone: in vitro studies.

Chitosan scaffolds have been shown to possess biological and mechanical properties suitable for tissue engineering and clinical applications. In the present work, chitosan sponges were evaluated regarding their ability to support cartilage cell proliferation and maturation, which are the first steps in endochondral bone formation. Chitosan sponges were seeded with chondrocytes isolated from chicken embryo sterna. Chondrocyte/chitosan constructs were cultured for 20 days, and treated with retinoic acid (RA) to induce chondrocyte maturation and matrix synthesis. At different time points, samples were collected for microscopic, histological, biochemical, and mechanical analyses. Results show chondrocyte attachment, proliferation, and abundant matrix synthesis, completely obliterating the pores of the sponges. RA treatment caused chondrocyte hypertrophy, characterized by the presence of type X collagen in the extracellular matrix and increased alkaline phosphatase activity. In addition, hypertrophy markedly changed the mechanical properties of the chondrocyte/chitosan constructs. In conclusion, we have developed chitosan sponges with adequate pore structure and mechanical properties to serve as a support for hypertrophic chondrocytes. In parallel studies, we have evaluated the ability of this mature cartilage scaffold to induce endochondral ossification.

Attachment, spreading and short-term proliferation of human osteoblastic cells cultured on chitosan films with different degrees of acetylation.

Chitosan (Ch) is being actively investigated as a non-protein template for the growth of an increasing number of anchorage-dependent cells, including chondrocytes and bone cells. In the present work, Ch films with degrees of N-acetylation (DAs) in the range of 4 to 49% were evaluated with respect to the attachment, spreading and short-term proliferation of osteoblasts, using human osteoblastic MG-63 cells. The films were characterized in terms of surface morphology and surface charge by atomic force microscopy and streaming potential measurements, respectively. Cell attachment was assessed after 3 and 24 h of cell culture. After 24 h of incubation, cell attachment was found to be dependent on the DA, lower DAs favouring cell adhesion. With time, cell spreading and cytoskeleton organization were only attained for DAs ≤ 13%. Regarding cell proliferation, cells grown on films with the lowest DA (4%) revealed a higher specific growth rate as compared to those grown on films with higher DAs. Films with a DA of 49% failed to sustain cell proliferation. In addition, a longer lag-phase was observed on Ch, as compared to TCPS, in accordance to an observed delay of cell spreading. The present findings revealed that differences in the DA as small as 9% may be critical in terms of the osteoblast response to two-dimensional Ch-based matrices.

Evaluation of the effect of the degree of acetylation on the inflammatory response to 3D porous chitosan scaffolds

The effect of the degree of acetylation (DA) of 3D chitosan (Ch) scaffolds on the inflammatory reaction was investigated. Chitosan porous scaffolds with DAs of 4 and 15% were implanted using a subcutaneous air-pouch model of inflammation. The initial acute inflammatory response was evaluated 24 and 48 h after implantation. To characterize the initial response, the recruitment and adhesion of inflammatory cells to the implant site was studied. The fibrous capsule formation and the infiltration of inflammatory cells within the scaffolds were evaluated for longer implantation times (2 and 4 weeks). Chitosan with DA 15% attracted the highest number of leukocytes to the implant site. High numbers of adherent inflammatory cells were also observed in this material. For longer implantation periods Ch scaffolds with a DA of 15% induced the formation of a thick fibrous capsule and a high infiltration of inflammatory cells within the scaffold. Our results indicate that the biological response to implanted Ch scaffolds was influenced by the DA. Chitosan with a DA of 15% induce a more intense inflammatory response when compared with DA 4% Ch. Because inflammation and healing are interrelated, this result may provide clues for the relative importance of acetyl and amine functional groups in tissue repair and regeneration.

Macrophage polarization following chitosan implantation

Macrophages are a key cell in the host response to implants and can be polarized into different phenotypes capable of inducing both detrimental and beneficial outcomes in tissue repair and remodeling, being important in tissue engineering and regenerative medicine. The objective of this study was to evaluate the macrophage response to 3D porous chitosan (Ch) scaffolds with different degrees of acetylation (DA, 5% and 15%). The M1/M2 phenotypic polarization profile of macrophages was investigated in vivo using a rodent air-pouch model. Our results show that the DA affects the macrophage response. Ch scaffolds with DA 5% induced the adhesion of lower numbers of inflammatory cells, being the M2 the predominant phenotypic profile among the adherent macrophages. In the inflammatory exudates F4/80(+)/CD206(+) cells (M2 macrophages) appeared in higher numbers then F4/80(+)/CCR7(+) cells (M1 macrophages), in addition, lower levels of pro-inflammatory cytokines together with higher levels of anti-inflammatory cytokines were found. Ch scaffolds with DA 15% showed opposite results, since M1 were the predominant macrophages both adherent to the scaffold and in the exudates, together with high levels of pro-inflammatory cytokines. In conclusion, Ch scaffolds with DA 5% induced a benign M2 anti-inflammatory macrophage response, whereas Ch scaffolds with DA 15% caused a macrophage M1 pro-inflammatory response.

Fibronectin-mediated endothelialisation of chitosan porous matrices

Chitosan (Ch) porous matrices were investigated regarding their ability to be colonized by human microvascular endothelial cells (HPMEC-ST1.6R cell line) and macrovascular endothelial cells namely HUVECs. Specifically we assessed if previous incubation of Ch in a fibronectin (FN) solution was effective in promoting endothelial cell (EC) adhesion to Ch matrices with different degrees of acetylation (DAs). Upon FN physiadsorption, marked differences were found between the two DAs investigated, namely DA 4% and 15%. While cell adhesion was impaired on Ch with DA 15%, ECs were able to not only adhere to Ch with DA 4%, but also to spread and colonize the scaffolds, with retention of the EC phenotype and angiogenic potential. To explain the observed differences between the two DAs, protein adsorption studies using (125)I-FN and immunofluorescent labelling of FN cell-binding domains were carried out. In agreement with the higher cell numbers found, scaffolds with DA 4% revealed a higher number of exposed FN cell-binding domains as well as greater ability to adsorb FN and to retain and exchange adsorbed FN in the presence of competitive proteins. These findings suggest that the DA is a key parameter modulating EC adhesion to FN-coated Ch by influencing the adsorbed protein layer.

Modulation of stability and mucoadhesive properties of chitosan microspheres for therapeutic gastric application

Chitosan microspheres have been explored for pharmaceutical applications, namely as a drug delivery systems for Helicobacter pylori gastric infection treatment, due to their mucoadhesive capacity. In this study, a different application of chitosan microspheres is proposed aiming the creation of an H. pylori-binding system where, after oral administration, microspheres will capture and remove these bacteria from infected patients, taking advantage of their muco/bacterial adhesive process. However, mucoadhesion is influenced by the degree of crosslinking necessary to avoid microspheres dissolution in the acidic gastric environment. During this work, the effect of genipin crosslinking on the stability, size, charge and mucoadhesive properties of chitosan microspheres under acidic pH was studied. Chitosan microspheres with ∼170 μm were produced by ionotropic gelation and subsequently covalently crosslinked with genipin in different degrees. The crosslinking reaction was followed by infrared spectroscopy and time-lapse fluorescence microscopy, since we have demonstrated that the fluorescence intensity of chitosan microspheres increases with genipin chemical bonding to chitosan. Results showed that both the zeta potential and the swelling capacity of chitosan microspheres decrease with increasing crosslinking. When immersed in simulated gastric fluid (SGF) with pepsin for 7 days, chitosan microspheres crosslinked with 10mM of genipin for 1h did not dissolve and doubled their size to approximately 345 μm. Furthermore, they maintained their in vitro mucoadhesion to soluble gastric mucins at both pH tested (3.6 and 6.5) and presented an in vivo retention time of around 2h in the stomach of C57BL/6 mice.

Preparation and Characterization of Injectable Chitosan-Hydroxyapatite Microspheres

The combination of chitosan and hydroxyapatite (HAp) in the form of injec table, porous and biodegradable structures seems to be an interesting route to promot e localized bone regeneration, especially with the incorporation of cells or cell-targeted molecules. In the present work, chitosan-HAp microspheres were prepared and charac terized in terms of size, morphology, water sorption and structure. Chitosan-HAp porous microspheres were successfully prepared using tripolyphosphate as coagulating agent. The size increas ed and the water sorption decreased with increasing HAp contents. The ceramic particles w ere well embedded and homogeneously distributed within the polymer matrix.

Endothelialization of chitosan porous conduits via immobilization of a recombinant fibronectin fragment (rhFNIII7-10).

The present study aimed to develop a pre-endothelialized chitosan (CH) porous hollowed scaffold for application in spinal cord regenerative therapies. CH conduits with different degrees of acetylation (DA; 4% and 15%) were prepared, characterized (microstructure, porosity and water uptake) and functionalized with a recombinant fragment of human fibronectin (rhFNIII(7-10)). Immobilized rhFNIII(7-10) was characterized in terms of amount ((125)I-radiolabelling), exposure of cell-binding domains (immunofluorescence) and ability to mediate endothelial cell (EC) adhesion and cytoskeletal rearrangement. Functionalized conduits revealed a linear increase in immobilized rhFNIII(7-10) with rhFNIII(7-10) concentration, and, for the same concentration, higher amounts of rhFNIII(7-10) on DA 4% compared with DA 15%. Moreover, rhFNIII(7-10) concentrations as low as 5 and 20μg ml(-1) in the coupling reaction were shown to provide DA 4% and 15% scaffolds, respectively, with levels of exposed cell-binding domains exceeding those observed on the control (DA 4% scaffolds incubated in a 20μg ml(-1) human fibronectin solution). These grafting conditions proved to be effective in mediating EC adhesion/cytoskeletal organization on CH with DA 4% and 15%, without affecting the endothelial angiogenic potential. rhFNIII(7-10) grafting to CH could be a strategy of particular interest in tissue engineering applications requiring the use of endothelialized porous matrices with tunable degradation rates.

In Vitro Mineralisation of Chitosan Membranes Carrying Phosphate Functionalities

Squid chitosan membranes were phosphorylated through the H3PO 4/P 2O5/Et 3PO 4/butanol method. P-chitosan membranes were immersed in Ca(OH) 2 or NaOH solutions, in order to obtain the Na or the Ca salts, respectively. These materials were investigated regarding their ability to nucleate calcium phosphates, under simulated physiologic conditions. SEM-EDS studies revealed the presence of a calcium phosphate mineral layer all over the sur face of P-chitosan membranes, after incubation in Ca(OH) 2 solution. The release of ionically bound phosphate functionalities under alkaline conditions, possibly contributed to the formation of calcium phosphate precursor sites, due to the chelation of calcium ions from solution. During the immersion in Simulated Body Fluid (SBF), a multilayer porous mineral structure composed of poorly crys talline carbonated apatite was formed on the surface of these membranes, as shown by EDS and ATR-FTIR analysis. Unmodified membranes and P-chitosan membranes pre-incubated in NaOH solution did not mineralise.