P. Giusti

Bioartificial polymeric materials based on polysaccharides

Bioartificial polymeric materials, based on blends of polysaccharides with synthetic polymers such as poly(vinyl alcohol) (PVA) and poly(acrylic acid) (PAA), were prepared as films or hydrogels. The physico-chemical, mechanical, and biological properties of these materials were investigated by different techniques such as differential scanning calorimetry, dynamic mechanical thermal analysis, scanning electron microscopy, and in vitro release tests, with the aim of evaluating the miscibility of the polymer blends and to establish their potential applications. The results indicate that while dextran is perfectly miscible with PAA, dextran/PVA, chitosan/PVA, starch/PVA, and gellan/PVAblends behave mainly as two-phase systems, although interactions can occur between the components. Cross-linked starch/PVAfilms could be employed as dialysis membranes: they showed transport properties comparable to, and in some cases better than, those of currently used commercial membranes. Hydrogels based on dextran/PVA and chitosan/PVA blends could find applications as delivery systems. They appeared able to release physiological amounts of human growth hormone, offering the possibility to modulate the release of the drug by varying the content of the biological component.

Preparation and characterization of alginate/gelatin blend films for cardiac tissue engineering

The aim of this work was the preparation of blends based on alginate and gelatin, with different weight ratio, to combine the advantages of these two natural polymers for application in cardiac tissue engineering. The physicochemical characterization, performed by Fourier transform infrared spectroscopy, differential scanning calorimetry and thermogravimetric analysis, revealed a good miscibility and the presence of interactions among the functional groups of pure biopolymers. Concerning the swelling and degradation tests, performed in different solutions simulating body fluids, both swelling degree and weight losses were higher in phosphate buffer saline (PBS) and for the blends with a higher content of gelatin. These results indicated a better stability of the blends in cell culture medium than in PBS and suggested a mainly hydrolytic degradation process. Cell culture tests, carried out using C2C12 myoblasts, showed a good cell proliferation for all the blends containing more than 60% of gelatin, with the alginate/gelatin 20:80 showing the best response. The same blend was the only one on which cell differentiation was observed. The results obtained in the biological characterization allow to select the alginate/gelatin 20:80 blend as a suitable material to prepare scaffolds for myocardial tissue engineering.

Collagen-based new bioartificial polymeric materials

Bioartificial polymeric materials, based on blends of biological and synthetic polymers, have been proposed as new materials for applications in the biomedical field. They should usefully combine the biocompatibility of the biological component with the physical and mechanical properties of the synthetic component. Blends of collagen with either poly(vinyl alcohol) or poly(acrylic acid) have been prepared by mixing aqueous solutions of the two polymers. Differential scanning calorimetry and dynamic mechanical thermal analysis has been carried out to investigate the miscibility properties of the polymers and the mechanical behaviour of the blends.

Block copolymers of L-lactide and poly(ethylene glycol) for biomedical applications

Poly (L-lactide)-poly (oxyethylene)-poly (L-lactide) block copolymers obtained in bulk, by a ring opening mechanism, from poly (ethylene glycol)s (PEG)s and L-lactide (LA), at 120–140°C, in the absence of added catalysts are described. By using PEGs with different molecular masses, 3000 and 35000, respectively, and varying the initial molar ratio LA to PEG, two series of copolymers with different molecular masses, relative length of blocks and hydrophilicity were obtained. Physico-chemical characterization of the copolymers had been previously performed. The morphological characteristics of the copolymers were investigated by means of X-ray diffractometry, optical and scanning electron microscopy. The biological properties of the materials were determined by evaluating their cytotoxicity, cytocompatibility, hemocompatibility and degradability using different standard tests. The results obtained indicate that the block copolymers synthesized may be useful for biomedical applications, in particular as resorbable drug vehicles. The materials are brittle and their mechanical properties are not appropriate for implant devices.

Poly(ester-ether-ester) block copolymers as biomaterials

Poly(ester-ether-ester) block copolymers, belonging to a class of biodegradable materials, were synthesized from poly(ethylene glycol) and ε-caprolactone by a simple ring-opening mechanism, which avoids the use of potentially toxic inorganic or organometallic initiators. The morphological and mechanical properties of such materials were investigated by gelpermeation chromatography, vapour pressure osmometry, proton magnetic resonance, infrared spectroscopy, differential scanning calorimetry, X-ray diffractometry and stress-strain tensile tests. The biocompatibility was investigated by cytotoxicity and hemocompatibility tests; the cytotoxicity was tested by the Neutral Red uptake assay, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, the Kenacid Blue R-binding method, and by the cell proliferation test on polymer films; the hemocompatibility was tested by the contact activation both of the coagulation cascade (intrinsic pathway), by the plasma prekallikrein activation test, and of the thrombocytes, by measuring the release of platelet factor 4 and β-thromboglobulin. The experimental results show that such a polymerization process permits high-molecular mass block copolymers with relatively good tensile and mechanical properties to be obtained. Their cyto- and hemo-compatibility makes them suitable for employment as biomaterials.

Enzymatically crosslinked porous composite matrices for bone tissue regeneration

Three-dimensional porous hydroxyapatite/collagen (HA/Coll) composites with a random pore structure were obtained by freeze-drying and crosslinked by an enzymatic treatment using microbial transglutaminase (mTGase). The procedure resulted in improved mechanical strength and thermal stability of the scaffolds. The scaffolds were characterized in terms of their stability (Coll release, swelling, collagenase-mediated degradation), thermal properties (thermogravimetric analysis, differential scanning calorimetry), mechanical behavior under compression and cell compatibility. Enzymatic treatment stabilized the sponges to water vapors, with measurable swelling ratio between 100% for HA/Coll/mTGase 0/100 to 5% for HA/Coll/mTGase 80/20. Weight loss in water due to Coll release was between 2 and 10% in mTGase-crosslinked samples and decreased with increasing HA content. Cultures of MG63 osteoblast-like cells and human umbilical vein endothelial cells (HUVEC) showed good adhesion and proliferation on the scaffolds, good viability (through MTT test, 100-150% of control), and good differentiation (alkaline phosphatase, up to 40 UI/L with respect to 35 UI/L for control).

Transglutaminase Reactivity with Gelatine: Perspective Applications in Tissue Engineering

Gelatine was crosslinked by means of an enzymatic treatment using tissue transglutaminase (tTGase) (Sigma) and microbial transglutaminase (mTGase) (Ajinomoto) which catalyses the formation of isopeptide bonds between the γ-carbonyl group of a glutamine residue and the ε-amino group of a lysine residue. The reaction is an interesting alternative to the traditional glutaraldehyde crosslinking, which has several drawbacks (e.g., in medical application) due to the toxicity of the chemical reagent. To further investigate the possibility to utilize the modified protein for tissue engineering application, TGase crosslinked gelatine was incorporated in a gellan matrix, a polysaccharide, to enhance the stability in aqueous media. Films obtained by casting were characterized by thermal analysis, chemical imaging, swelling behaviour and cell adhesion.

Hydrogels of poly(vinyl alcohol) and collagen as new bioartificial materials Part I Physical and morphological characterization

Poly(vinyl alcohol) was used to make hydrogels containing various amounts of collagen. These “bioartificial materials”, made of synthetic and biological polymers, were studied to investigate the effect of the presence of the collagen on the structural properties of the hydrogels. A comparison between thermal and morphological properties of collagen-containing hydrogels and hydrogels of pure poly(vinyl alcohol) was made.

Physico-chemical and mechanical characterization of hydrogels of poly (vinyl alcohol) and hyaluronic acid

Hydrogels are three-dimensional polymeric networks very similar to biological tissues and potentially useful as soft tissue substitutes and drug delivery systems. Many synthetic polymers can be used to make hydrogels: poly (vinyl alcohol) is widely employed to make hydrogels for biomedical applications. Improvements in the biocompatibility characteristics of synthetic materials could be achieved by the addition of biological macromolecules. The resulting materials named “bioartificial polymeric materials” could possess the good mechanical properties of the synthetic component and adequate biocompatibility due to the biological component. We have used poly (vinyl alcohol) to make hydrogels containing various amounts of hyaluronic acid. These bioartificial materials were studied to investigate the effect of the presence of the hyaluronic acid on the structural properties of the hydrogels. Thermal, mechanical, morphological and X-ray analyses were performed. A close correspondence between the network consistency and the degree of crystallinity developed in the matrix suggested that the hyaluronic acid, when its content is about 20%, could provide heterogeneous crystallization nuclei for poly (vinyl alcohol) thus increasing the crystallization degree, and consequently, the storage modulus.

In vitro biocompatibility of fluorinated polyurethanes

The in vitro biocompatibility of fluorinated polyurethanes (FPUs), labelled as FPU 42, 52, 58, and 60, was evaluated by means of thrombogenicity, cytoxicity and cytocompatibility tests. Cardiothane® was taken as control material. The thrombogenicity was tested on thin material films by measuring the activation of prekallikrein (PKK) to kallikrein (KK). Level I cytoxicity tests of the bulk materials, i.e. Neutral Red (NR) uptake, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and Kenacid Blue (KB) assays, were performed to assess the influence of the polymer extracts on, respectively, lysosomes, mitochondria and cell proliferation. The cytocompatibility was evaluated, on thin membranes made by a spraying phase-inversion process, by measuring the area of the polymer surface covered by human umbilical vein endothelial cells (HUVEC) 1 week after seeding. The results indicate that all the polymers are not thrombogenic, and not cytotoxic. The FPUs that contain polycaprolactone glycol (PCLG) (FPU 52 and 60) instead of poly (tetramethylene ether) glycol (PTMEG) (FPU 42 and 58) as soft segment show the lowest thrombogenicity and the best cytocompatibility.