Cláudia M. Vaz

In vitro degradation and cytocompatibility evaluation of novel soy and sodium caseinate-based membrane biomaterials.

Soy- and casein-based membranes are newly proposed materials disclosing a combination of properties that might allow for their use in a range of biomedical applications. Two of the most promising applications are drug delivery carrier systems and wound dressing membranes. As for all newly proposed biomaterials, a cytotoxic scanning must be performed as a preliminary step in the process of the determination of the compatibility with biological systems (biocompatibility). In this study, the cytotoxicity of both soy- and casein-based protein biomaterials has been evaluated and correlated with the materials degradation behavior. It was possible to show, through morphological and biochemical tests that these natural origin materials do not exert any cytotoxic effect over cells, and in some cases can in fact enhance cell proliferation. The different treatments to which the membranes were subjected during their processing (that include crosslinking with glyoxal and tannic acid, and physical modification by thermal treatment) seemed to have a clear effect both on the materials mechanical properties and on their in vitro biological behavior.

Use of coupling agents to enhance the interfacial interactions in starch-EVOH/hydroxylapatite composites

Different zirconate, titanate and silane coupling agents were selected in an effort to improve the mechanical properties of starch and ethylene-vinyl alcohol copolymer (EVOH) hydroxylapatite (HA) composites, through the enhancement of the filler particles-polymer matrix interactions and the promotion of the interfacial adhesion between these two phases. The mechanical performance was assessed by tensile tests and discussed on the basis of the respective interfacial morphology (evaluated by scanning electron microscopy). The main relevant parameters were found to be the surface properties and reactivity of the filler (non-sintered HA) and the chemical nature (pH and type of metallic centre) of the added coupling agent. Significant improvements in the stiffness were achieved (about 30% increase in the modulus) when using the acidic zirconate coupling agents. The acidic zirconate combined the capability of crosslinking the polymer matrix with the establishment of donor-acceptor interactions and hydrogen bonding between it and the ceramic particles, leading to very good interfacial adhesion. The optimization of these coupling processes associated with the introduction of higher amounts of filler, may be an effective way to produce composites with mechanical properties analogous to those of the human cortical bone.

In vitro degradation behaviour of biodegradable soy plastics: effects of crosslinking with glyoxal and thermal treatment

In-vitro degradation of soy-derived protein materials, non-crosslinked (SItp), crosslinked with glyoxal (X-SItp) or submitted to heat treatment (24TT-SItp), was studied with either an isotonic saline solution without enzymatic activity or containing bacterial collagenase. The changes in weight of the samples during the in-vitro degradation were studied and compared with the variations of the mechanical properties. The weight loss of SItp, X-SItp and 24TT-SItp were more pronounced when using collagenase. After 24 h of immersion, SItp lost 10.6% of its initial weight whereas 0.6X-SItp and 24TT-SItp lost 1.7 and 5.7%, respectively. In every case, the weight loss was found to be directly proportional to the respective crosslinking degree: 2.4% for SItp, 44% for 0.6X-SItp and 27.8% for 24TT-SItp. Consequently, the susceptibility of the soy materials towards enzymatic degradation could be controlled by varying the degree of crosslinking of the samples. The mechanical properties proved to be more sensitive to the loss of plasticiser (glycerol) during immersion than to the degradation of the polymeric matrices. After 24 h of immersion all the materials presented an increase in stiffness and brittleness due to the complete leaching of glycerol from the matrices. SItp, X-SItp and 24TT-SItp proved to be suitable materials for either load-bearing applications or temporary applications such as tissue engineering scaffolds or drug delivery systems. # 2003 Elsevier Science Ltd. All rights reserved.

Dynamic mechanical properties of hydroxyapatite-reinforced and porous starch-based degradable biomaterials

It has been shown that blends of starch with a poly(ethylene-vinyl-alcohol) copolymer, EVOH, designated as SEVA-C, present an interesting combination of mechanical, degradation and biocompatible properties, specially when filled with hydroxyapatite (HA). Consequently, they may find a range of applications in the biomaterials field. This work evaluated the influence of HA fillers and of blowing agents (used to produce porous architectures) over the viscoelastic properties of SEVA-C polymers, as seen by dynamic mechanical analysis (DMA), in order to speculate on their performances when withstanding cyclic loading in the body. The composite materials presented a promising performance under dynamic mechanical solicitation conditions. Two relaxations were found being attributed to the starch and EVOH phases. The EVOH relaxation process may be very useful in vivo improving the implants performance under cyclic loading. DMA results also showed that it is possible to produce SEVA-C compact surface/porous core architectures with a mechanical performance similar to that of SEVA-C dense materials. This may allow for the use of these materials as bone replacements or scaffolds that must withstand loads when implanted.

Effect of crosslinking, thermal treatment and UV irradiation on the mechanical properties and in vitro degradation behavior of several natural proteins aimed to be used in the biomedical field

Gelatine (GEL), soy (SI), casein (CAS) and sodium-caseinate (NaCAS) solutions were cast to produce protein films. All the proteins were chemically modified by adding glyoxal to the film-forming solutions in amounts varying from 0 to 0.9% (w/w based on the protein content). After casting, the same films were also submitted to a heat treatment performed at 80 °C or UV irradiation. The effect of those chemical/physical modifications on the mechanical properties and on the hydrolytic stability of the protein films was evaluated. As a result, a large variety of protein films with different mechanical properties and degradation profiles were developed. CAS and NaCAS even when chemically/physically modified do not resist to hydrolysis longer than 2 weeks. GEL, only when chemically modified with glyoxal, become water resistant. Due to its hydrolytic stability, SI become a very attractive material for biomedical applications where long term treatments are a requisite.

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.

Development and design of double-layer co-injection moulded soy protein based drug delivery devices

Novel double-layer delivery devices based on soy protein derived materials were designed and produced using an innovative two material co-injection moulding technique. It was demonstrated that the viscosity ratio between core and skin layer materials played an important role in the formation of the interfacial shape, namely the skin thickness and uniformity of the bi-materials. The adequate selection of the materials used and the optimisation of the respective processing conditions enabled an accurate control of the relative thickness of the layers of the device. The preliminary results confirmed the potential of these systems to achieve a controlled drug delivery.

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.

Soy Protein-Based Systems for Different Tissue Regeneration Applications

With the main aim of developing soy protein drug delivery systems for different tissue regeneration applications, various processing techniques are being studied in our research group. A wide range of shapes and processing methods could already be developed. This includes membrane, microparticles and thermoplastic systems. The resulting soy membranes, microparticles, and thermoplastics are intensively characterised and their potential use as drug delivery systems, especially as pH-sensitive systems, is discussed. The use of protein modifications, namely crosslinking, for the improvement of functional properties relevant for each specific application is also addressed in this chapter.

ph-sensitive soy protein films for the controlled release of an anti-inflammatory drug

pH-responsive delivery systems based on biodegradable polymers represent one of the most rapidly advancing areas of science. Such delivery systems offer numerous advantages compared to conventional dosage fonns including improved efficacy, localized delivery of the drug to a particular part of the body, improved patient compliance, and convenience [I]. However, only a reduced number ofbiodegradable drug delivery systems exhibiting such behavior are known [2, 3, 4]. Proteins present, by nature, interesting characteristics to be used in the