Patrícia B. Malafaya

Natural origin biodegradable systems in tissue engineering and regenerative medicine: present status and some moving trends

The fields of tissue engineering and regenerative medicine aim at promoting the regeneration of tissues or replacing failing or malfunctioning organs, by means of combining a scaffold/support material, adequate cells and bioactive molecules. Different materials have been proposed to be used as both three-dimensional porous scaffolds and hydrogel matrices for distinct tissue engineering strategies. Among them, polymers of natural origin are one of the most attractive options, mainly due to their similarities with the extracellular matrix (ECM), chemical versatility as well as typically good biological performance. In this review, the most studied and promising and recently proposed naturally derived polymers that have been suggested for tissue engineering applications are described. Different classes of such type of polymers and their blends with synthetic polymers are analysed, with special focus on polysaccharides and proteins, the systems that are more inspired by the ECM. The adaptation of conventional methods or non-conventional processing techniques for processing scaffolds from natural origin based polymers is reviewed. The use of particles, membranes and injectable systems from such kind of materials is also overviewed, especially what concerns the present status of the research that should lead towards their final application. Finally, the biological performance of tissue engineering constructs based on natural-based polymers is discussed, using several examples for different clinically relevant applications.

A new approach based on injection moulding to produce biodegradable starch-based polymeric scaffolds: morphology, mechanical and degradation behaviour.

One of the present challenges in polymer scaffold processing is the fabrication of three-dimensional (3D) architectures with an adequate mechanical performance to be used in the tissue engineering of hard tissues. This paper describes a preliminary study on the development of a new method to produce biodegradable scaffolds from a range of corn-starch-based polymers. In some cases, hydroxlapatite was also used as a reinforcement of the biodegradable polymers. The developed methodology consists of a standard conventional injection moulding process, on which a solid blowing agent based on carboxylic acids is used to generate the foaming of the bulk of the moulded part. The proposed route allows for the production of scaffolds with a compact skin and a porous core, with promising mechanical properties. By using the developed method it is possible to manufacture biodegradable polymer scaffolds in an easy (melt-based processing) and reproducible manner. The scaffolds can be moulded into complex shapes, and the blowing additives do not affect the non-cytotoxic behaviour of the starch-based materials. The materials produced using this method were evaluated with respect to the morphology of the porous structure, and the respective mechanical properties and degradation behaviour. It was demonstrated that it is possible to obtain, by a standard melt based processing route, 3D scaffolds with complex shapes that exhibit an appropriate morphology, without decreasing significantly the mechanical properties of the materials. It is believed that the optimisation of the proposed processing methodology may lead to the production of scaffolds that might be used on the regeneration of load-bearing tissues.

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.

Sodium silicate gel as a precursor for the in vitro nucleation and growth of a bone-like apatite coating in compact and porous polymeric structures.

In the present work, a new methodology to produce bioactive coatings on the surface of starch-based biodegradable polymers or other polymeric biomaterials is proposed. A sodium silicate gel is employed as an alternative nucleating agent to the more typical bioactive glasses for inducing the formation of a calcium-phosphate (Ca-P) layer. The method has the advantage of being able to coat efficiently both compact materials and porous 3D architectures aimed at being used on tissue replacement applications and as tissue engineering scaffolds. By means of this treatment, it is possible to observe the formation of an apatite-like layer, only after 6 hours of simulated body fluid immersion. For the porous materials, this layer could also be observed inside the pores, clearly covering the cell walls. Furthermore, an increase of the surface hydrophilicity (higher amount of polar groups in the surface) might contribute to the formation of silanol groups that also act as apatite inductors. After 30 days of SBF immersion, the apatite-like films exhibit a partially amorphous nature and the Ca/P ratios became much closer to the value attributed to hydroxyapatite (1.67). The obtained results are very promising for the development of cancellous bone replacement materials and for pre-calcifying bone tissue engineering scaffolds.

Bilayered chitosan-based scaffolds for osteochondral tissue engineering: influence of hydroxyapatite on in vitro cytotoxicity and dynamic bioactivity studies in a specific double-chamber bioreactor.

Osteochondral tissue engineering presents a current research challenge due to the necessity of combining both bone and cartilage tissue engineering principles. In the present study, bilayered chitosan-based scaffolds are developed based on the optimization of both polymeric and composite scaffolds. A particle aggregation methodology is proposed in order to achieve an improved integrative bone-cartilage interface needed for this application, since any discontinuity is likely to cause long-term device failure. Cytotoxicity was evaluated by the MTS assay with the L929 fibroblast cell line for different conditions. Surprisingly, in composite scaffolds using unsintered hydroxyapatite, cytotoxicity was observed in vitro. This work reports the investigation that was conducted to overcome and explain this behaviour. It is suggest that the uptake of divalent cations may induce the cytotoxic behaviour. Sintered hydroxyapatite was consequently used and showed no cytotoxicity when compared to the controls. Microcomputed tomography (micro-CT) was carried out to accurately quantify porosity, interconnectivity, ceramic content, particle and pore sizes. The results showed that the developed scaffolds are highly interconnected and present the ideal pore size range to be morphometrically suitable for the proposed applications. Dynamical mechanical analysis (DMA) demonstrated that the scaffolds are mechanically stable in the wet state even under dynamic compression. The obtained elastic modulus was, respectively, 4.21+/-1.04, 7.98+/-1.77 and 6.26+/-1.04 MPa at 1 Hz frequency for polymeric, composite and bilayered scaffolds. Bioactivity studies using both a simulated body fluid (SBF) and a simulated synovial fluid (SSF) were conducted in order to assure that the polymeric component for chondrogenic part would not mineralize, as confirmed by scanning electron microscopy (SEM), inductively coupled plasma-optical emission spectroscopy (ICP) and energy-dispersive spectroscopy (EDS) for different immersion periods. The assays were carried out also under dynamic conditions using, for this purpose, a specifically designed double-chamber bioreactor, aiming at a future osteochondral application. It was concluded that chitosan-based bilayered scaffolds produced by particle aggregation overcome any risk of delamination of both polymeric and composite parts designed, respectively, for chondrogenic and osteogenic components that are mechanically stable. Moreover, the proposed bilayered scaffolds could serve as alternative, biocompatible and safe biodegradable scaffolds for osteochondral tissue engineering applications.

Porous starch-based drug delivery systems processed by a microwave route

A new simple processing route to produce starch-based porous materials was developed based on a microwave baking methodology. This innovative processing route was used to obtain non-loaded controls and loaded drug delivery carriers, incorporating a non-steroid anti-inflammatory agent. This bioactive agent was selected as model drug with expectations that the developed methodology might be used for other drugs and growth factors. The prepared systems were characterized by 1H and 13C NMR spectroscopy which allow the study of the interactions between the starch-based materials and the processing components, i.e. the blowing agents. The porosity of the prepared materials was estimated by measuring their apparent density and studied by comparing drug-loaded and non-loaded carriers. The behaviour of the porous structures, while immersed in aqueous media, was studied in terms of swelling and degradation, being intimately related to their porosity. Finally, in vitro drug release studies were performed showing a clear burst effect, followed by a slow controlled release of the drug over several days (up to 10 days).

Morphology, mechanical characterization and in vivo neo-vascularization of chitosan particle aggregated scaffolds architectures

The present study intended to evaluate the performance of chitosan-based scaffolds produced by a particle aggregation method aimed to be used in tissue engineering applications addressing key issues such as morphological characteristics, mechanical performance and in vivo behaviour. It is claimed that the particle aggregation methodology may present several advantages, such as combine simultaneously a high interconnectivity with high mechanical properties that are both critical for an in vivo successful application. In order to evaluate these properties, micro-Computed Tomography (micro-CT) and Dynamical Mechanical Analysis (DMA) were applied. The herein proposed scaffolds present an interesting morphology as assessed by micro-CT that generally seems to be adequate for the proposed applications. At a mechanical level, DMA has shown that chitosan scaffolds have an elastic behaviour under dynamic compression solicitation, being simultaneously mechanically stable in the wet state and exhibiting a storage modulus of 4.21+/-1.04MPa at 1Hz frequency. Furthermore, chitosan scaffolds were evaluated in vivo using a rat muscle-pockets model for different implantation periods (1, 2 and 12 weeks). The histological and immunohistochemistry results have demonstrated that chitosan scaffolds can provide the required in vivo functionality. In addition, the scaffolds interconnectivity has been shown to be favourable to the connective tissues ingrowth into the scaffolds and to promote the neo-vascularization even in early stages of implantation. It is concluded that the proposed chitosan scaffolds produced by particle aggregation method are suitable alternatives, being simultaneously mechanical stable and in vivo biofunctional that might be used in load-bearing tissue engineering applications, including bone and cartilage regeneration.

Natural origin scaffolds with in situ pore forming capability for bone tissue engineering applications

This work describes the development of a biodegradable matrix, based on chitosan and starch, with the ability to form a porous structure in situ due to the attack by specific enzymes present in the human body (alpha-amylase and lysozyme). Scaffolds with three different compositions were developed: chitosan (C100) and chitosan/starch (CS80-20, CS60-40). Compressive test results showed that these materials exhibit very promising mechanical properties, namely a high modulus in both the dry and wet states. The compressive modulus in the dry state for C100 was 580+/-33MPa, CS80-20 (402+/-62MPa) and CS60-40 (337+/-78MPa). Degradation studies were performed using alpha-amylase and/or lysozyme at concentrations similar to those found in human serum, at 37 degrees C for up to 90 days. Scanning electron micrographs showed that enzymatic degradation caused a porous structure to be formed, indicating the potential of this methodology to obtain in situ forming scaffolds. In order to evaluate the biocompatibility of the scaffolds, extracts and direct contact tests were performed. Results with the MTT test showed that the extracts of the materials were clearly non-toxic to L929 fibroblast cells. Analysis of cell adhesion and morphology of seeded osteoblastic-like cells in direct contact tests showed that at day 7 the number of cells on CS80-20 and CS60-40 was noticeably higher than that on C100, which suggests that starch containing materials may promote cell adhesion and proliferation. This combination of properties seems to be a very promising approach to obtain scaffolds with gradual in vivo pore forming capability for bone tissue engineering applications.

The Role of Lipase and α-Amylase in the Degradation of Starch/Poly(ɛ-Caprolactone) Fiber Meshes and the Osteogenic Differentiation of Cultured Marrow Stromal Cells

The present work studies the influence of hydrolytic enzymes (alpha-amylase or lipase) on the degradation of fiber mesh scaffolds based on a blend of starch and poly(epsilon-caprolactone) (SPCL) and the osteogenic differentiation of osteogenic medium-expanded rat bone marrow stromal cells (MSCs) and subsequent formation of extracellular matrix on these scaffolds under static culture conditions. The biodegradation profile of SPCL fiber meshes was investigated using enzymes that are specifically responsible for the enzymatic hydrolysis of SPCL using concentrations similar to those found in human serum. These degradation studies were performed under static and dynamic conditions. After several degradation periods (3, 7, 14, 21, and 30 days), weight loss measurements and micro-computed tomography analysis (specifically porosity, interconnectivity, mean pore size, and fiber thickness) were performed. The SPCL scaffolds were seeded with rat MSCs and cultured for 8 and 16 days using complete osteogenic media with and without enzymes (alpha-amylase or lipase). Results indicate that culture medium supplemented with enzymes enhanced cell proliferation after 16 days of culture, whereas culture medium without enzymes did not. No calcium was detected in groups cultured with alpha-amylase or without enzymes after each time period, although groups cultured with lipase presented calcium deposition after the eighth day, showing a significant increase at the sixteenth day. Lipase appears to positively influence osteoblastic differentiation of rat MSCs and to enhance matrix mineralization. Furthermore, scanning electron microscopy images showed that the enzymes did not have a deleterious effect on the three-dimensional structure of SPCL fiber meshes, meaning that the scaffolds did not lose their structural integrity after 16 days. Confocal micrographs have shown cells to be evenly distributed and infiltrated within the SPCL fiber meshes up to 410 microm from the surface. This study demonstrates that supplementation of culture media with lipase holds great potential for the generation of bone tissue engineering constructs from MSCs seeded onto SPCL fiber meshes, because lipase enhances the osteoblastic differentiation of the seeded MSCs and promotes matrix mineralization without harming the structural integrity of the meshes over 16 days of culture.

Natural Polymers in Tissue Engineering Applications

Publisher Summary This chapter provides an overview of the natural origin polymers that are commercially available or currently being studied in different laboratories for tissue engineering applications, with some emphasis on the most widely studied systems. It describes their chemical structure, main properties, and potential applications within the field. The chapter helps in understanding the origin, structure, and properties of natural polymers used in tissue engineering applications. It identifies the characteristics that make natural polymers interesting for TE applications. Natural polymers are derived from renewable resources, namely from plants, animals and microorganisms, and are, therefore, widely distributed in nature. These materials exhibit a large diversity of unique rather complex structures. Tissue engineering offers the possibility to help in the regeneration of tissues damaged by disease or trauma and, in some cases, to create new tissues and replace failing or malfunctioning organs. Typically, this is achieved through the use of degradable biomaterials to either induce surrounding tissue and cell ingrowth or to serve as temporary scaffolds for transplanted cells to attach, grow, and maintain differentiated functions.