João T. Oliveira

Genipin-cross-linked collagen/chitosan biomimetic scaffolds for articular cartilage tissue engineering applications

In this study, genipin-cross-linked collagen/chitosan biodegradable porous scaffolds were prepared for articular cartilage regeneration. The influence of chitosan amount and genipin concentration on the scaffolds physicochemical properties was evaluated. The morphologies of the scaffolds were characterized by scanning electron microscope (SEM) and cross-linking degree was investigated by ninhydrin assay. Additionally, the mechanical properties of the scaffolds were assessed under dynamic compression. To study the swelling ratio and the biostability of the collagen/chitosan scaffold, in vitro tests were also carried out by immersion of the scaffolds in PBS solution or digestion in collagenase, respectively. The results showed that the morphologies of the scaffolds underwent a fiber-like to a sheet-like structural transition by increasing chitosan amount. Genipin cross-linking remarkably changed the morphologies and pore sizes of the scaffolds when chitosan amount was less than 25%. Either by increasing the chitosan ratio or performing cross-linking treatment, the swelling ratio of the scaffolds can be tailored. The ninhydrin assay demonstrated that the addition of chitosan could obviously increase the cross-linking efficiency. The degradation studies indicated that genipin cross-linking can effectively enhance the biostability of the scaffolds. The biocompatibility of the scaffolds was evaluated by culturing rabbit chondrocytes in vitro. This study demonstrated that a good viability of the chondrocytes seeded on the scaffold was achieved. The SEM analysis has revealed that the chondrocytes adhered well to the surface of the scaffolds and contacted each other. These results suggest that the genipin-cross-linked collagen/chitosan matrix may be a promising formulation for articular cartilage scaffolding.

Modified Gellan Gum hydrogels with tunable physical and mechanical properties.

Gellan Gum (GG) has been recently proposed for tissue engineering applications. GG hydrogels are produced by physical crosslinking methods induced by temperature variation or by the presence of divalent cations. However, physical crosslinking methods may yield hydrogels that become weaker in physiological conditions due to the exchange of divalent cations by monovalent ones. Hence, this work presents a new class of GG hydrogels crosslinkable by both physical and chemical mechanisms. Methacrylate groups were incorporated in the GG chain, leading to the production of a methacrylated Gellan Gum (MeGG) hydrogel with highly tunable physical and mechanical properties. The chemical modification was confirmed by proton nuclear magnetic resonance (1H NMR) and Fourier transform infrared spectroscopy (FTIR-ATR). The mechanical properties of the developed hydrogel networks, with Youngs modulus values between 0.15 and 148 kPa, showed to be tuned by the different crosslinking mechanisms used. The in vitro swelling kinetics and hydrolytic degradation rate were dependent on the crosslinking mechanisms used to form the hydrogels. Three-dimensional (3D) encapsulation of NIH-3T3 fibroblast cells in MeGG networks demonstrated in vitro biocompatibility confirmed by high cell survival. Given the highly tunable mechanical and degradation properties of MeGG, it may be applicable for a wide range of tissue engineering approaches.

Gellan gum: A new biomaterial for cartilage tissue engineering applications

Gellan gum is a polysaccharide manufactured by microbial fermentation of the Sphingomonas paucimobilis microorganism, being commonly used in the food and pharmaceutical industry. It can be dissolved in water, and when heated and mixed with mono or divalent cations, forms a gel upon lowering the temperature under mild conditions. In this work, gellan gum hydrogels were analyzed as cells supports in the context of cartilage regeneration. Gellan gum hydrogel discs were characterized in terms of mechanical and structural properties. Transmissionelectron microscopy revealed a quite homogeneous chain arrangement within the hydrogels matrix, and dynamic mechanical analysis allowed to characterize the hydrogels discs viscoelastic properties upon compression solicitation, being the compressive storage and loss modulus of approximately 40 kPa and 3 kPa, respectively, at a frequency of 1 Hz. Rheological measurements determined the sol-gel transition started to occur at approximately 36 degrees C, exhibiting a gelation time of approximately 11 s. Evaluation of the gellan gum hydrogels biological performance was performed using a standard MTS cytotoxicity test, which showed that the leachables released are not deleterious to the cells and hence were noncytotoxic. Gellan gum hydrogels were afterwards used to encapsulate human nasal chondrocytes (1 x 10(6) cells/mL) and culture them for total periods of 2 weeks. Cells viability was confirmed using confocal calcein AM staining. Histological observations revealed normal chondrocytes morphology and the obtained data supports the claim that this new biomaterial has the potential to serve as a cell support in the field of cartilage regeneration.

Gellan gum-based hydrogels for intervertebral disc tissue-engineering applications

Intervertebral disc (IVD) degeneration is a challenging clinical problem that urgently demands viable nucleus pulposus (NP) implant materials. The best suited biomaterial for NP regeneration has yet to be identified, but it is believed that biodegradable hydrogel‐based materials are promising candidates. In this work, we have developed ionic‐ and photo‐crosslinked methacrylated gellan gum (GG–MA) hydrogels to be used in acellular and cellular tissue‐engineering strategies for the regeneration of IVDs. The physicochemical properties of the developed hydrogels were investigated by Fourier‐transform infrared spectroscopy, 1H nuclear magnetic resonance and differential scanning calorimetry. The swelling ability and degradation rate of hydrogels were also analysed in phosphate‐buffered saline solution at physiological pH for a period of 30 days. Additionally, the morphology and mechanical properties of the hydrogels were assessed under a scanning electron microscope and dynamic compression, respectively. An in vitro study was carried out to screen possible cytotoxicity of the gellan gum‐based hydrogels by culturing rat lung fibroblasts (L929 cells) with hydrogel leachables up to 7 days. The results demonstrated that gellan gum was successfully methacrylated. We observed that the produced GG–MA hydrogels possess improved mechanical properties and lower water uptake ability and degradation rate as compared to gellan gum. This work also revealed that GG–MA hydrogels are non‐cytotoxic in vitro, thus being promising biomaterials to be used in IVD tissue‐engineering strategies. Copyright

Injectable gellan gum hydrogels with autologous cells for the treatment of rabbit articular cartilage defects

In this work, the ability of gellan gum hydrogels coupled with autologous cells to regenerate rabbit full‐thickness articular cartilage defects was tested. Five study groups were defined: (a) gellan gum with encapsulated chondrogenic predifferentiated rabbit adipose stem cells (ASC + GF); (b) gellan gum with encapsulated nonchondrogenic predifferentiated rabbit adipose stem cells (ASC); (c) gellan gum with encapsulated rabbit articular chondrocytes (AC) (standard control); (d) gellan gum alone (control); (e) empty defect (control). Full‐thickness articular cartilage defects were created and the gellan gum constructs were injected and left for 8 weeks. The macroscopic aspect of the explants showed a progressive increase of similarity with the lateral native cartilage, stable integration at the defect site, more pronouncedly in the cell‐loaded constructs. Tissue scoring showed that ASC + GF exhibited the best results regarding tissue quality progression. Alcian blue retrieved similar results with a better outcome for the cell‐loaded constructs. Regarding real‐time PCR analyses, ASC + GF had the best progression with an upregulation of collagen type II and aggrecan, and a downregulation of collagen type I. Gellan gum hydrogels combined with autologous cells constitute a promising approach for the treatment of articular cartilage defects, and adipose derived cells may constitute a valid alternative to currently used articular chondrocytes.

Performance of new gellan gum hydrogels combined with human articular chondrocytes for cartilage regeneration when subcutaneously implanted in nude mice

Gellan gum is a polysaccharide that has been recently proposed by our group for cartilage tissue‐engineering applications. It is commonly used in the food and pharmaceutical industry and has the ability to form stable gels without the use of harsh reagents. Gellan gum can function as a minimally invasive injectable system, gelling inside the body in situ under physiological conditions and efficiently adapting to the defect site. In this work, gellan gum hydrogels were combined with human articular chondrocytes (hACs) and were subcutaneously implanted in nude mice for 4 weeks. The implants were collected for histological (haematoxylin and eosin and Alcian blue staining), biochemical [dimethylmethylene blue (GAG) assay], molecular (real‐time PCR analyses for collagen types I, II and X, aggrecan) and immunological analyses (immunolocalization of collagen types I and II). The results showed a homogeneous cell distribution and the typical round‐shaped morphology of the chondrocytes within the matrix upon implantation. Proteoglycans synthesis was detected by Alcian blue staining and a statistically significant increase of proteoglycans content was measured with the GAG assay quantified from 1 to 4 weeks of implantation. Real‐time PCR analyses showed a statistically significant upregulation of collagen type II and aggrecan levels in the same periods. The immunological assays suggest deposition of collagen type II along with some collagen type I. The overall data shows that gellan gum hydrogels adequately support the growth and ECM deposition of human articular chondrocytes when implanted subcutaneously in nude mice. Copyright

Polysaccharide-based materials for cartilage tissue engineering applications

Tissue engineering was proposed approximately 15 years ago as an alternative and innovative way to address tissue regeneration problems. During the development of this field, researchers have proposed a variety of ways of looking into the regeneration and engineering of tissues, using different types of materials coupled with a wide range of cells and bioactive agents. This trilogy is commonly considered the basis of a tissue‐engineering strategy, meaning by this the use of a support material, cells and bioactive agents. Different researchers have been adding to these basic approaches other parameters able to improve the functionality of the tissue‐engineered construct, such as specific mechanical environments and conditioned gaseous atmospheres, among others. Nowadays, tissue‐engineering principles have been applied, with different degrees of success, to almost every tissue lacking efficient regeneration ability and the knowledge and intellectual property produced since then has experienced an immense growth. Materials for regenerating tissues, namely cartilage, have also been continuously increasing and most of the theoretical requirements for a tissue engineering support have been addressed by a single material or a mixture of materials. Due to their intrinsic features, polysaccharides are interesting for cartilage tissue‐engineering approaches and as a result their exploitation for this purpose has been increasing. The present paper intends to provide an overview of some of the most relevant polysaccharides used in cartilage tissue‐engineering research. Copyright

Ambivalence resolution in emotion-focused therapy: The successful case of Sarah

Abstract Ambivalence can be understood as a cyclical movement between two opposing positions of the self: one expressed in a novelty—an innovative moment (IM)—and another one conveyed by a return to the maladaptive pattern. If not properly addressed and resolved during therapy, ambivalence can prevent change and lead to psychotherapeutic failure. Two processes of ambivalence resolution have been suggested: (1) the dominance of the innovative position and consequent inhibition of the problematic position and (2) the negotiation between both positions. Objectives: To empirically study both processes of ambivalence resolution in a successful case of emotion-focused therapy. Method: Sessions were independently coded with three coding systems—the IMs, the return to the problem and the ambivalence resolution. Results: Ambivalence tended to be resolved from the initial to the final sessions. Although resolutions through dominance tended to decrease and resolutions through negotiation seemingly increased along treatment, dominance was, nonetheless, the most prominent process of resolution along the whole treatment. Conclusions: Although it has been suggested that integrating opposing parts of the self is a necessary process for psychotherapeutic success, a less integrative process of ambivalence resolution may also be an important resource along the process.

Ambivalence resolution in brief psychotherapy for depression

Ambivalence in the process of psychotherapeutic change should be addressed and resolved if we are to avoid psychotherapeutic failure and promote sustained change. In this context, ambivalence can be defined as the cyclical conflictual relation between two opposed positions of the self: one expressed as an innovation, and a subsequent one expressed in a trivialization or rejection of the innovation (problematic position). This conflict may be resolved in two different ways: (a) the dominance of the innovative position and the consequent inhibition of the problematic one and (b) the negotiation between the innovative and the problematic positions. In this study, we sought to study the evolution of the dominance and the negotiation processes in recovered and unchanged cases; to analyse if different therapeutic models produce different results on the evolution of the dominance and negotiation processes, and finally, to study if these processes are predictive of ambivalence resolution. The complete sessions of 22 clinical cases of depression (6 cognitive-behavioural therapy, 10 narrative therapy, and 6 emotion-focused therapy cases) were independently coded for innovative moments, ambivalence, and ambivalence resolution. Results revealed that recovered cases had a progressively higher proportion of negotiation along treatment, whereas in unchanged cases, negotiation was virtually absent throughout treatment. Both dominance and negotiation were significant predictors of ambivalence reduction, however, negotiation had a higher impact than dominance. Overall, these results did not significantly differ for the 3 therapeutic models. The theoretical implications of these findings are discussed, and theoretical derived suggestions for clinicians are presented.