António M. Cunha

Alternative tissue engineering scaffolds based on starch: processing methodologies, morphology, degradation and mechanical properties

Abstract An ideal tissue engineering scaffold must be designed from a polymer with an adequate degradation rate. The processing technique must allow for the preparation of 3-D scaffolds with controlled porosity and adequate pore sizes, as well as tissue matching mechanical properties and an appropriate biological response. This communication revises recent work that has been developed in our laboratories with the aim of producing 3-D polymeric structures (from starch-based blends) with adequate properties to be used as scaffolds for bone tissue engineering applications. Several processing methodologies were originally developed and optimised. Some of these methodologies were based on conventional melt-based processing routes, such as extrusion using blowing agents (BA) and compression moulding (combined with particulate leaching). Other developed technologies included solvent casting and particle leaching and an innovative in situ polymerization method. By means of using the described methodologies, it is possible to tailor the properties of the different scaffolds, namely their degradation, morphology and mechanical properties, for several applications in tissue engineering. Furthermore, the processing methodologies (including the blowing agents used in the melt-based technologies) described above do not affect the biocompatible behaviour of starch-based polymers. Therefore, scaffolds obtained from these materials by means of using one of the described methodologies may constitute an important alternative to the materials currently used in tissue engineering.

The thermomechanical environment and the microstructure of an injection moulded polypropylene copolymer

The microstructure of an injection moulding propylene copolymer is varied through systematic changes on the processing conditions (melt and mould temperatures and injection flow rate). The skin-core structure was characterised by several experimental techniques. The skin ratio was assessed by polarised light microscopy. The morphological features of the skin layer (level of crystalline phase orientation, degree of crystallinity, β-phase content and double texture) were evaluated by wide-angle X-ray diffraction. The core features (degree of crystallinity and lamella thickness) were assessed by differential scanning calorimetry. The thermomechanical environment imposed during processing was characterised by mould filling simulations. The thermal and shear stress levels were evaluated by a cooling index and the wall shear stress. The results show the relationship between these and the microstructural features. The microstructure development is then interpreted considering the constrictions imposed during processing, being assessed by thermomechanical indices. Furthermore, the direct connections between these indices and the degree of crystallinity of the core and the level of orientation of the skin are verified.

Novel starch thermoplastic/Bioglass® composites: Mechanical properties, degradation behavior and in-vitro bioactivity

The present research aims to evaluate the possibility of creating new degradable, stiff and highly bioactive composites based on a biodegradable thermoplastic starch-based polymeric blend and a Bioglass® filler. Such combination should allow for the development of bioactive and degradable composites with a great potential for a range of temporary applications. A blend of starch with ethylene–vinyl alcohol copolymer (SEVA-C) was reinforced with a 45S5 Bioglass® powder presenting a granulometric distribution between 38 and 53 μm. Composites with 10 and 40 wt % of 45S5 Bioglass® were compounded by twin-screw extrusion (TSE) and subsequently injection molded under optimized conditions. The mechanical properties of the composites were evaluated by tensile testing, and their bioactivity assessed by immersion in a simulated body fluid (SBF) for different periods of time. The biodegradability of these composites was also monitored after several immersion periods in an isotonic saline solution. The tensile tests results obtained indicated that SEVA-C/Bioglass® composites present a slightly higher stiffness and strength (a modulus of 3.8 GPa and UTS of 38.6 MPa) than previously developed SEVA-C/Hydroxylapatite (HA) composites. The bioactivity of SEVA-C composites becomes relevant for 45S5 amounts of only 10 wt %. This was observed by scanning electron microscopy (SEM) and confirmed for immersion periods up to 30 days by both thin-film X-ray diffraction (TF-XRD) (where HA typical peaks are clearly observed) and induced coupled plasma emission (ICP) spectroscopy used to follow the elemental composition of the SBF as function of time. Additionally, it was observed that the composites are biodegradable being the results correlated with the correspondent materials composition.

Mechanical, dynamic-mechanical, and thermal properties of soy protein-based thermoplastics with potential biomedical applications

In this study the tensile and the dynamic-mechanical behavior of injection-molded samples of various soy protein thermoplastic compounds were evaluated as a function of the amount of glycerol, type and amount of ceramic reinforcement, and eventual incorporation of coupling agents. The incorporation of glycerol into a soy-based matrix resulted in its plasticization, as confirmed by the drop in stiffness (storage and elastic modulus) above 20°C and a decrease in the protein glass transition temperature. Differential scanning calorimetric thermograms proved the occurrence of conformational changes in the soy protein during processing. Furthermore, the developed soy protein-based thermoplastics showed a thermal stability up to 100°C, as confirmed by thermogravimetric analysis. The reinforcement of the soy protein matrix with a ceramic filler (tricalcium phosphate) was shown to be effective for amounts above 10% w/w. The introduction of an amino-coupling agent led to a plasticizing effect, detected in the mechanical and dynamic-mechanical properties of the resulting materials. The results also show a good qualitative agreement between the properties obtained from quasi-static and dynamic experiments. The materials present a range of properties that might allow for their use eventually in a range of biomedical applications.

Degradation model of starch-EVOH+HA composites

Abstract Composites of starch based blends (starch-EVOH) reinforced with bioactive bone-like hydroxyapatite (HA) have been recently proposed for temporary biomedical implants. Very promising mechanical results were obtained so far, both by the introduction of coupling agents (titanates, zirconates and silanes) or by optimizing the respective processing route. In this study coupled and non-coupled composites were aged up to 30 days in two types of simulated physiological solutions (with and without added proteins/enzymes) and the respective property variation was evaluated by means of: weight loss, water-uptake and mechanical performance (strength, stiffness and ductility). The interfacial attack generated by the solutions was observed by scanning electron microscopy (SEM) and quantified (calcium and phosphorous amounts in the solution) by atomic emission spectrometry (ICP). The in-vitro degradation process of starch-EVOH+HA composites consists apparently of three main stages: i) for short periods (0–6 days) it is characterized by a high degradation rate due to the leaching of plasticizers, low molecular weight polymeric chains and some dissolution of HA; ii) for longer periods (7–15 days), the major extraction of the plasticizers occurs and the material becomes brittle and; iii) from the 15th immersion day on, the degradation rate is lower and, eventually chemical attack on the polymer structure takes place, mainly in the presence of enzymes/proteins.The confirmation of this type of behavior will support the potential use of these composites, already tested for their non-cytotoxic character, in temporary applications where the retention of mechanical properties for 3 to 6 weeks is required.

Relationship between processing and mechanical properties of injection molded high molecular mass polyethylene + hydroxyapatite composites

Abstract We apply a macromolecular-orientation approach to produce high molecular weight polyethylene (HMWPE) + hydroxyapatite (HA) ductile composites with the stiffness and strength within the range of human cortical bone. Our composites are produced with different amounts (10 to 50% by weight) of the reinforcement by two procedures: bi-axial rotating drum and twin screw extrusion (TSE). The processing is by conventional injection molding and by Scorim (shear controlled orientation in injection molding) under a wide range of processing windows. Tensile testing is performed and the corresponding performance related to the morphology evaluated by polarized light microscopy and scanning electron microscopy. The control of the processing parameters led to significant improvements of the tensile properties. Compounding by TSE and then processing by Scorim produces the maximum modulus of 7.4 GPa and the ductility as high as 19%, for the HA weight fraction of 30%. These mechanical properties match those of bone, and were obtained with much smaller amounts of HA reinforcement then has been previously reported in literature. Our PE + HA composites present the additional benefit of being ductile even for 50% HA amounts. The use Scorim is a unique way of inducing anisotropy to thick sections and to produce very stiff composites that may be used in biomedical applications with important mechanical loads. This fact, combined with the bioactive behavior of the HA phase, makes our composite usable for orthopedic load-bearing implants.

Proteolytic enzyme engineering: a tool for wool.

One of the goals of protein engineering is to tailor the structure of enzymes to optimize industrial bioprocesses. In the present work, we present the construction of a novel high molecular weight subtilisin, based on the fusion of the DNA sequences coding for Bacillus subtilis prosubtilisin E and for an elastin-like polymer (ELP). The resulting fusion protein was biologically produced in Escherichia coli , purified and used for wool finishing assays. When compared to the commercial protease Esperase, the recombinant subtilisinE-VPAVG(220) activity was restricted to the cuticle of wool, allowing a significant reduction of pilling, weight loss and tensile strength loss of wool fibers. Here we report, for the first time, the microbial production of a functionalized high molecular weight protease for controlled enzymatic hydrolysis of wool surface. This original process overcomes the unrestrained diffusion and extended fiber damage which are the major obstacles for the use of proteases for wool finishing applications.

Preparation, processing and characterization of biodegradable wood flour/starch-cellulose acetate compounds

A novel biodegradable material was prepared by compounding, in different proportions, pine wood flour (WF) and a commercial starch-cellulose acetate blend on a configurable co-rotating twin screw extruder. After pelletizing, the compounds were injection moulded and the mechanical and rheological properties of the mouldings determined. As the content of wood flour increases up to 50% (wt/wt), the tensile strength and the modulus improve significantly, whereas the toughness drops gradually. The effect of the wood flour content on the shear viscosity is complex, being impossible to establish a linear relationship between the two. The shear viscosity decreases with shear rate, but for a level of 40% and 50% of WF there is evidence of a quasi-Newtonian behaviour, irrespective of the test temperature. Compounds with 50% WF present the highest tensile strength and modulus but are difficult to process. However, the processability can be improved by using glycerol as plasticizer, without paying a too severe penalty in mechanical properties. In fact, by adding 15% glycerol (wt/wt), compounds with 50% WF can be successfully injection moulded into specimens with good mechanical properties.

The effect of the skin thickness and spherulite size on the mechanical properties of injection mouldings

In this work are studied the relationships between the microstructure and the mechanical properties of an injection moulded propylene-ethylene copolymer. Distinct microstructures were obtained by processing, through a moulding programme that includes the variation of the injection and the mould temperatures and the injection flow rate. They were characterized by the skin ratio (measured by polarised light microscopy) and the spherulite size (evaluated by small angle light scattering system). Tensile tests were carried out at two different constant loading velocities: 2 mm/min (3.33 × 10−5 m/s) and 3 m/s, in order to assess the initial modulus, the yield stress, the strain and the energy at break. The results are presented in terms of the relationships between the chosen microstructural parameters and the selected tensile properties. The skin thickness is evidenced as an important microstructural feature. The role of the core spherulite size is secondary or even negligible. The results also show that other microstructural parameters must be considered to establish more general microstructure-properties relationships.

Dynamic Mechanical Analysis in Polymers for Medical Applications

The Dynamic Mechanical Analysis (DMA) is a powerful thermal analysis technique, which allows to detect phase transitions and relaxation processes in a variety of materials. With this technique, the solid-state rheological properties of viscoelastic materials can be characterised over a wide range of temperature and frequencies. This chapter summarizes the principles and the capabilities of the DMA technique focusing on its uses on polymeric-based systems aimed to medical and environmental applications. The examples presented include the materials that have been investigated in our research group in the last few years, such as starch-based blends, proteins, polyethylenes, and composites thereof, among other materials. These newly developed biomaterials are being proposed for a range of biomedicai applications that go from fracture replacement/fixation and tissue engineering scaffolding, to new partially degradable bone cements and hydrogels, carriers for controlled release of drugs and growth factors and new wound dressings and membranes.