Carbon nanotubes reinforced chitosan films: mechanical properties and cell response of a novel biomaterial for cardiovascular tissue engineering.

Carbon nanotubes have been proposed as fillers to reinforce polymeric biomaterials for the strengthening of their structural integrity to achieve better biomechanical properties. In this study, a new polymeric composite material was introduced by incorporating various low concentrations of multiwalled carbon nanotubes (MWCNTs) into chitosan (CS), aiming at achieving a novel composite biomaterial with superior mechanical and biological properties compared to neat CS, in order to be used in cardiovascular tissue engineering applications. Both mechanical and biological characteristics in contact with the two relevant cell types (endothelial cells and vascular myofibroblasts) were studied. Regarding the mechanical behavior of MWCNT reinforced CS (MWCNT/CS), 5 and 10 % concentrations of MWCNTs enhanced the mechanical behavior of CS, with that of 5 % exhibiting a superior mechanical strength compared to 10 % concentration and neat CS. Regarding biological properties, MWCNT/CS best supported proliferation of endothelial and myofibroblast cells, MWCNTs and MWCNT/CS caused no apoptosis and were not toxic of the examined cell types. Conclusively, the new material could be suitable for tissue engineering (TE) and particularly for cardiovascular TE applications.

A polyconvex anisotropic strain-energy function for soft collagenous tissues.

Polyconvexity of a strain-energy function is a very important mathematical condition, especially in the context of a boundary-value problem. In the present paper, we propose an exponential polyconvex anisotropic strain-energy function. It is given by a series with an arbitrary number of terms and associated material constants. Each term of this series a priori satisfies the condition of the energy- and stress-free natural state so that no additional restrictions have to be imposed. Due to the exponential form, the proposed hyperelastic model is suitable for soft biological tissues. Thus, a good agreement with experimental data on different types of tissues is achieved.

Dynamic mechanical characteristics of intact and structurally modified bovine pericardial tissues.

Bovine pericardium (BP) is a source of natural biomaterials with a wide range of clinical applications. In the present work we studied the dynamic mechanical behavior of BP in native form and under specific enzymatic degradation with chondroitinase ABC extracted a 17% of the total glycosaminoglycans (GAGs). The GAGs content of native BP was composed mainly from hyaluronan, chondroitine sulfate and dermatan sulfate. Dynamic tensile mechanical testing of BP in the frequency range 0.1-20 Hz demonstrated its viscoelastic nature. The storage modulus was equal to 6.5 (native) and 5.5 (degraded) MPa initially, increased in the region nearby 1 Hz by about 15%. This was related with physical resonance mechanisms activated in this frequency region. The high modulus (modulus of the high linear phase of stress-strain) was equal to 14 (native) and 10 (degraded) MPa, dropped at high frequencies to 7 and 5 Mpa, respectively. The damping, expressed by the hysteresis, was equal to 20% of the loading energy, changed exponentially with the frequency to 30% at 20 Hz. It seemed that of the elastic mechanical parameters, the storage modulus and the high modulus were even slightly dropped as a result of degradation. As a final conclusion, there was evident that GAGs may play a non-negligible role in the dynamic mechanical behavior of BP and, probably in other soft tissue biomechanics. It is suggested that the GAGs content may be considered during the design and chemical modification of biomaterials based on BP and other soft tissues.

Screening biomaterials with a new in vitro method for potential calcification: porcine aortic valves and bovine pericardium.

Calcification is still a major cause of failure of implantable biomaterials. A fast and reliable in vitro model could contribute to the study of its mechanisms and to testing different anticalcification techniques. In this work, we attempted to investigate the potential calcification of biomaterials using an in vitro model. We purposed to test the ability of this model to screening possible anticalcification efficacy of different biomaterials. Porcine heart valve (PAV) and bovine pericardial (BP) tissues, fixed with glutaraldehyde were immersed into biological mimicking solution, where the pH and the initial concentrations of calcium and phosphoric ions were kept stable by the addition of precipitated ions during calcification. Kinetics of calcification was continuously monitored. The evaluation of biomaterials was carried out by comparing the kinetic rates of formation of calcific deposits. After 24 h, the calcific deposits on PAVs were found to be developed at significant higher rates (ranged from 0.81 x 10(-4)-2.18 x 10(-4)mol/min m2) than on BP (0.19 x 10(-4)-0.52 x 10(-4)mol/min m2) (one-way ANOVA, p < 0.05) depending on the experimental conditions (supersaturation of the solution). Parallel tests for similar biomaterials implanted subcutaneously in animal (rat) model showed after 49 days that significant higher amounts of total minerals deposited on PAV (236.73+/-139.12, 9 animals mg minerals/g dry net tissue) (mean+/-standard deviation) compared with that formed on BP (104.36+/-79.21, #9 mg minerals/g dry net tissue) (ANOVA, p < 0.05). There is evidence that in vitro calcification was correlated well with that of animal model and clinical data.

Physicochemical and microscopical study of calcific deposits from natural and bioprosthetic heart valves. Comparison and implications for mineralization mechanism.

Natural and bioprosthetic heart valves suffer from calcification, despite their differences in etiology and tissue material. The mechanism of developing calcific deposits in valve tissue is still not elucidated. The calcific deposits developed on human natural and bioprosthetic heart valves have been investigated and compared by physicochemical studies and microscopy investigations and the results were correlated with possible mechanisms of mineral crystal growth. Deposits from 16 surgically excised calcified valves (seven natural aortic and nine bioprosthetic porcine aortic valves) were examined by chemical analysis, FTIR, XRD, and SEM-EDS. The Ca/P molar ratio of the deposits from bioprosthetic valves (1.52+/-0.06) was significantly lower compared to that of the natural valves (1.83+/-0.03) (p=0.05, 1-way ANOVA). SEM-EDS examination of the two types of valve deposits revealed the coexistence of large (>20 microm) and medium (5-20 microm) plate-like crystals as well as microcrystalline (<5 microm) calcium phosphate mineral formations. The results confirmed the hypothesis that the mineral salt of calcified valves is a mixture of calcium phosphate phases such as dicalcium phosphate dihydrate (DCPD), octacalcium phosphate (OCP) and hydroxyapatite (HAP). DCPD and OCP are suggested to be precursor phases transformed to HAP by hydrolysis. The lower value of the Ca/P molar ratio found in the bioprostheses, in comparison with that corresponding in natural valves, was ascribed to the higher content in these deposits in precursor phases DCPD and OCP which were subsequently transformed into HAP. On the basis of chemical composition of the deposits and their morphology it is suggested that crystal growth proceeds in both types of valves by the same mechanism (hydrolysis of precursor phases to HAP) in spite of their differences in etiology, material, and possible initiation pathways.

Model experimental system for investigation of heart valve calcification in vitro.

A model system was developed for the in vitro quantitative investigation of the calcification process occurring in heart valves. The process of heart valve calcification consists of the formation of calcium phosphates at the heart valve-biological fluid interface. Calcium phosphate deposits may consist of more than one calcium phosphate mineral phase, differing with respect to their physical and chemical properties. The kinetics of the formation of hydroxyapatite, the model inorganic compound for the calcified deposits, was precisely monitored in a reactor containing supersaturated calcium phosphate solutions in which the heart valves were immersed after being treated with glutaraldehyde and mounted on special racks. The precipitation process, accompanied with proton release in the solution, was monitored by a pair of glass-saturated calomel electrodes. Upon initiation of the formation of calcium phosphate deposits, the supersaturation in the working solution was reestablished through the addition of titrant solutions made with the appropriate concentration to compensate for the ions precipitated. With this methodology, not only the rates were measured very precisely but also the nucleation capability of the various substrates could be evaluated. Moreover, it was possible to identify the formation of intermediate calcium phosphate phases formed during the calcification process. Valves previously treated with glutaraldehyde were shown to nucleate octacalcium phosphate, which at lower supersaturations converted to the thermodynamically more stable hydroxyapatite. The rates measured were found to depend on the solution supersaturation, while the apparent order of the precipitation process was found to be 1.

Test methodology for characterizing in vitro biodegradation.

In this paper mechanical tests for the characterization of the time-dependent behaviour of absorbable osteosynthetic materials are described. The tensile test is performed according to International Standard Organization (ISO) 3268/1978 and provides Young's modulus, tensile strength, elongation at tensile strength, and rupture force. The bending test is performed according to ISO 178/1975 and gives the flexural stress at various deflections, the force and deflection at break and the initial bending modulus. The torsional test is carried out according to ISO 458-1 and gives torsional load. The bending and torsional stress relaxation are measured. Relaxation modulus and the creep compliance are then determined. The parameters to be recorded in a cyclic bending test include the number of cycles or, in the case of sample survival, the remaining force at break at a bending test. The cyclic torsion test is carried out according to ISO 537/1989. The parameters to be recorded are the number of cycles or, in case of sample survival, the remaining force at a torsional test at 45 deg. Simulation tests provide a comparative assessment of the degradation phenomena under cyclic mechanical loading in bending or in torsion at elevated temperatures.

An approach to the optimization of preparation of bioprosthetic heart valves.

The stress and strain states of the valve leaflets during fixation with glutaraldehyde affect their final mechanical parameters. Comparative studies of the stress-strain relationships of aortic valve leaflet strips from fresh, statically and dynamically fixed porcine and human valves were made. Static pressures of 5 mmHg, 16 mmHg, and 95 mmHg result in stress-strain relationships which are in a region between that of fresh porcine and fresh human leaflet strips in the circumferential direction, while they are far from that of fresh porcine tissue (larger strains) in the radial direction. Leaflet strips, fixed under dynamic loading between zero and a predefined maximum load, set at an early post-transition state, give parameters not significantly different from those of human valves.