Caterina Cristallini

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.

Bioartificial materials based on blends of collagen and poly(acrylic acid)

The interactions between soluble collagen (C) from calf skin and poly(acrylic acid) (PAA) were studied. Mixing aqueous solutions of collagen and PAA, at various pH values (2.5-4), leads to the formation of complexes that precipitate in the form of insoluble aggregates. The effects of mixture composition, pH, and ionic strength on C/PAA complex formation were investigated by gravimetric, turbidimetric, and conductometric analysis. The experimental results indicate that the complexes form through electrostatic interactions. Homogeneous solid films with variable C/PAA ratios were obtained by casting from solutions in which the pH was adjusted just over the isoelectric point of collagen, thus avoiding the attractive ionic interactions responsible for the complexation of collagen and PAA molecules. A relevant result obtained is related to the possibility of restoring the ionic interactions between the two polymers inside the solid films. Mixture composition and pH appear to influence the thermal properties of both complexes and films.

Kinetics and reaction mechanism of template polymerization investigated by conductimetric measurements. Part 3. Radical polymerization of acrylic acid in the presence of poly(N-vinylpyrrolidone)

Conductimetry was used for monitoring the radical polymerization of a weak electrolyte (acrylic acid) in aqueous solution and for determination of the kinetic parameters of the reaction (reaction order with respect to monomer, activation energy). The results obtained were consistent with those determined by other techniques (such as dilatometry) or expected from theory. Dilatometric and conductimetric measurements were also used to study the template polymerization of acrylic acid onto poly(N-vinylpyrrolidone). Results indicate that the reaction proceeds according to a pick-up mechanism. Complexes between poly(acrylic acid) and poly(N-vinylpyrrolidone) were always isolated in equimolar composition of the two polymers, regardless of the polymerization mixture composition. Spectroscopic evidence of the existence of strong interaction and intimate mixing of the two polymers in the complexes was found. An influence of the template molecular weight on the chain length of the forming poly(acrylic acid) was detected by means of viscometry. © 2000 Society of Chemical Industry

Hydroxyapatite/gelatin/gellan sponges as nanocomposite scaffolds for bone reconstruction

The aim of this work was the morphological, physicochemical, mechanical and biological characterization of a new composite system, based on gelatin, gellan and hydroxyapatite, and mimicking the composition of natural bone. Porous scaffolds were prepared by freeze–drying technique, under three different conditions of freezing. The morphological analysis showed a homogeneous porosity, with well interconnected pores, for the sample which underwent a more rapid freezing. The elastic modulus of the same sample was close to that of the natural bone. The presence of interactions among the components was demonstrated through the physicochemical investigation. In addition, the infrared chemical imaging analysis pointed out the similarity among the composite scaffold and the natural bone, in terms of chemical composition, homogeneity, molecular interactions and structural conformation. Preliminary biological characterization showed a good adhesion and proliferation of human mesenchymal stem cells.

New biomedical devices with selective peptide recognition properties. Part 1: Characterization and cytotoxicity of molecularly imprinted polymers

Molecular imprinting is a technique for the synthesis of polymers capable to bind target molecules selectively. The imprinting of large proteins, such as cell adhesion proteins or cell receptors, opens the way to important and innovative biomedical applications. However, such molecules can incur into important conformational changes during the preparation of the imprinted polymer impairing the specificity of the recognition cavities. The “epitope approach” can overcome this limit by adopting, as template, a short peptide sequence representative of an accessible fragment of a larger protein. The resulting imprinted polymer can recognize both the template and the whole molecule thanks to the specific cavities for the epitope. In this work two molecularly imprinted polymer formulations (a macroporous monolith and nanospheres) were obtained using the protected peptide Z‐Thr‐Ala‐Ala‐OMe, as template, and Z‐Thr‐Ile‐Leu‐OMe, as analogue for the selectivity evaluation, methacrylic acid, as functional monomer, and trimethylolpropane trimethacrylate and pentaerythritol triacrylate (PETRA), as cross‐linkers. Polymers were synthesized by precipitation polymerization and characterized by standard techniques. Polymerization and rebinding solutions were analyzed by high performance liquid chromatography. The highly cross‐linked polymers retained about 70% of the total template amount, against (20% for the less cross‐linked ones). The extracted template amount and the rebinding capacity decreased with the cross‐linking degree, while the selectivity showed the opposite behaviour. The PETRA cross‐linked polymers showed the best recognition (MIP 2−, α= 1.71) and selectivity (MIP 2+, α′= 5.58) capabilities. The cytotoxicity tests showed normal adhesion and proliferation of fibroblasts cultured in the medium that was put in contact with the imprinted polymers.

The biodegradation of poly (ester-ether-ester) block copolymers in a cellular environmentin vitro

In this paper we report about the biodegradation of tri-block poly (ester-ether-ester) copolymers obtained by reaction of preformed poly (ethylene glycol) (PEG) with two different lactone monomers, i.e. ε-caprolactone (CL) and L-lactide (LA). The two series of copolymers will be indicated as PCL-POE-PCL and PLA-POE-PLA. We identified and measured by HPLC analysis the amount of degradation products of the poly (ester-ether-ester) copolymers; three copolymers were tested for each series during 3–8 week experiments. The experiments were carried out both in the presence and absence of fibroblast cell populations. We evaluated at the same time the decrease of copolymer molecular mass after degradation by means of intrinsic viscosity [η] measurements. From the [η] measurements we can conclude that the higher the hydrophilicity of the material, the faster the rate of decrease of its intrinsic viscosity with time. The HPLC results indicate that the amount of the degradation products, i.e., respectively, the monomers 6-hydroxyhexanoic acid and L-lactic acid, is a function of both hydrophilicity of the molecule and the lateral block length. When the fibroblast cell populations were present in the same wells together with the biodegradable copolymers, signs of cellular metabolism of the degraded monomers were detected.

Polymerisation onto Biological Templates. A New Way to Obtain Bioartificial Polymeric Materials

The radical polymerisation of synthetic monomers onto biological templates can be recognised as a technique for the straightforward preparation of bioartificial polymeric materials, polymer blends based materials in which interactions at the molecular level lead to enhanced properties. In the first part the polymerisations of different acrylic monomers in the presence of synthetic templates are presented as study models. The second part deals with preliminary experiments carried out using natural polymers as templates. Dilatometric and conductimetric measurements were used to study the kinetics of the template polymerisation reactions. Results indicate that the reactions proceed according the different mechanisms depending on the interactions between functionla groups present in the systems. In many instances the results of the characterisation analyses have shown that the polymer complexes obtained by template polymerisation have a more ordered structure than the complexes prepared by simple mixing the two polymers. In addition conductimetry has revealed as a very simple diagnostic tool for identifying the template reaction mechanism.

Copolymerization of acrylic acid and 2‐hydroxyethyl methacrylate onto poly(N‐vinylpyrrolidone): Template influence on comonomer reactivity

The copolymerization of acrylic acid and 2-hydroxyethyl methacrylate in aqueous solution is investigated by means of High Performance Liquid Chromatography (HPLC). The reactivity ratios of the two monomers are determined by the method of Kelen-Tudos. Acrylic acid showed a preferential tendency to cross-polymerisation while 2-hydroxyethyl methacrylate tends to auto-propagate. The characterisation of the copolymer obtained in the absence of the template indicates that comonomeric units are distributed along the polymeric chain with a higher 2-hydroxyethyl methacrylate/acrylic acid ratio with respect to the feed composition. In the presence of the template (poly(N-vinylpyrrolidone)), the reaction rate and the reactivity ratio of acrylic acid increase while the same quantities decrease for the other comonomer. This experimental evidence suggests the presence of a stronger interaction betweers acrylic acid and the template. The approach used to study the system could be an useful model to investigate the mechanism of molecular recognition in aqueous (natural) environments.

The effect of bioartificial constructs that mimic myocardial structure and biomechanical properties on stem cell commitment towards cardiac lineage

Despite the enormous progress in the treatment of coronary artery diseases, they remain the most common cause of heart failure in the Western countries. New translational therapeutic approaches explore cardiomyogenic differentiation of various types of stem cells in combination with tissue-engineered scaffolds. In this study we fabricated PHBHV/gelatin constructs mimicking myocardial structural properties. Chemical structure and molecular interaction between material components induced specific properties to the substrate in terms of hydrophilicity degree, porosity and mechanical characteristics. Viability and proliferation assays demonstrated that these constructs allow adhesion and growth of mesenchymal stem cells (MSCs) and cardiac resident non myocytic cells (NMCs). Immunofluorescence analysis demonstrated that stem cells cultured on these constructs adopt a distribution mimicking the three-dimensional cell alignment of myocardium. qPCR and immunofluorescence analyses showed the ability of this construct to direct initial MSC and NMC lineage specification towards cardiomyogenesis: both MSCs and NMCs showed the expression of the cardiac transcription factor GATA-4, fundamental for early cardiac commitment. Moreover NMCs also acquired the expression of the cardiac transcription factors Nkx2.5 and TBX5 and produced sarcomeric proteins. This work may represent a new approach to induce both resident and non-resident stem cells to cardiac commitment in a 3-D structure, without using additional stimuli.