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The lifespan of biological heart valve prostheses available in the market is limited due to structural alterations caused by calcium phosphate deposits formed from blood plasma in contact with the tissues. The objective of this work is to present a comparative methodology for the investigation of the formation of calcium phosphate deposits on bioprosthetic and tissue-engineered scaffolds in vitro and the influence of mechanical forces on tissue mineralization. Based on earlier investigations on biological mineralization at constant supersaturation, a circulatory loop simulating dynamic blood flow and physiological pressure conditions was developed. The system was appropriately adapted to evaluate the calcification potential of decellularized (DCV) and glutaraldehyde-fixed (GAV) porcine aortic valves. Results indicated that DCV calcified at higher, statistically nonsignificant, rates in comparison with GAV. This difference was attributed to the tissue surface modifications and cell debris leftovers from the decellularization process. Morphological analysis of the solids deposited after 20 h by scanning electron microscopy in combination with chemical microanalysis electron-dispersive spectroscopy identified the solid formed as octacalcium phosphate (Ca8(PO4)6H2·5H2O, OCP). OCP crystallites were preferentially deposited in high mechanical stress areas of the test tissues. Moreover, GAV tissues developed a significant transvalvular pressure gradient increase past 36 h with a calcium deposition distribution similar to the one found in explanted prostheses. In conclusion, the presented in vitro circulatory model serves as a valuable prescreening methodology for the investigation of the calcification process of bioprosthetic and tissue-engineered valves under physiological mechanical load.

Cardiovascular diseases are the first cause of death worldwide. Among different heart malfunctions, heart valve failure due to calcification is still a challenging problem. While drug-dependent treatment for the early stage calcification could slow down its progression, heart valve replacement is inevitable in the late stages. Currently, heart valve replacements involve mainly two types of substitutes: mechanical and biological heart valves. Despite their significant advantages in restoring the cardiac function, both types of valves suffered from serious drawbacks in the long term. On the one hand, the mechanical one showed non-physiological hemodynamics and the need for the chronic anticoagulation therapy. On the other hand, the biological one showed stenosis and/or regurgitation due to calcification. Nowadays, new promising heart valve substitutes have emerged, known as decellularized tissue-engineered heart valves (dTEHV). Decellularized tissues of different types have been widely tested in bioprosthetic and tissue-engineered valves because of their superior biomechanics, biocompatibility, and biomimetic material composition. Such advantages allow successful cell attachment, growth and function leading finally to a living regenerative valvular tissue in vivo. Yet, there are no comprehensive studies that are covering the performance of dTEHV scaffolds in terms of their efficiency for the calcification problem. In this review article, we sought to answer the question of whether decellularized heart valves calcify or not. Also, which factors make them calcify and which ones lower and/or prevent their calcification. In addition, the review discussed the possible mechanisms for dTEHV calcification in comparison to the calcification in the native and bioprosthetic heart valves. For this purpose, we did a retrospective study for all the published work of decellularized heart valves. Only animal and clinical studies were included in this review. Those animal and clinical studies were further subcategorized into 4 categories for each depending on the effect of decellularization on calcification. Due to the complex nature of calcification in heart valves, other in vitro and in silico studies were not included. Finally, we compared the different results and summed up all the solid findings of whether decellularized heart valves calcify or not. Based on our review, the selection of the proper heart valve tissue sources (no immunological provoking residues), decellularization technique (no damaged exposed residues of the decellularized tissues, no remnants of dead cells, no remnants of decellularizing agents) and implantation techniques (avoiding suturing during the surgical implantation) could provide a perfect anticalcification potential even without in vitro cell seeding or additional scaffold treatment.

Increasing morbidity of cardiovascular diseases in modern society has made it crucial to develop artificial small-caliber cardiovascular grafts for surgical intervention of diseased natural arteries, as alternatives to the gold standard autologous implants. Synthetic small-caliber grafts are still not in use due to increased risk of restenosis, lack of lumen re-endothelialization and mechanical mismatch, leading sometimes either to graft failure or to unsuccessful remodeling and pathology of the distal parts of the anastomosed healthy vascular tissues. In this work, we aimed to synthesize small-caliber polymeric (polycaprolactone) tissue-engineered vascular scaffolds that mimic the structure and biomechanics of natural vessels. Electrospinning was implemented to prepare microstructured polymeric membranes with controlled axis-parallel fiber alignment. Consequently, we designed small-caliber multilayer anisotropic biodegradable nanofibrous tubular scaffolds, giving attention to their radial compliance. Polycaprolactone scaffold morphology and mechanical properties were assessed, quantified, and compared with those of native vessels and commercial synthetic grafts. Results showed a highly hydrophobic scaffold material with a three-layered tubular morphology, 4-mm internal diameter/0.25 ± 0.09-mm thickness, consisting of predominantly axially aligned thin (1.156 ± 0.447 μm), homogeneous and continuous microfibers, with adequate (17.702 ± 5.369 μm) pore size, potentially able to promote cell infiltration in vivo. In vitro accelerated degradation showed a 5% mass loss within 17–25 weeks. Mechanical anisotropy was attained as a result, almost one order of magnitude difference of the elastic modulus (18 ± 3 MPa axially/1 ± 0.3 MPa circumferentially), like that of natural arterial walls. Furthermore, a desirable radial compliance (5.04 ± 0.82%, within the physiological pressure range) as well as cyclic stability of the tubular scaffold was achieved. Finally, cytotoxicity evaluation of the polymeric scaffolds revealed that the materials were nontoxic and did not release substances harmful to living cells (over 80% cell viability achieved).

Calcification is among the principal causes of biological heart valve substitute failure. Glycosaminoglcans (GAGs) are negatively charged molecules, possessing anticoagulation and anti-inflammatory activity that make them a potential solution against calcification. In the present work, the anticalcification potential of heparin was investigated under constant supersaturation conditions with respect to hydroxyapatite (Ca 5 (PO 4 ) 3 OH; HAP). Heparin concentration in the supersaturated solutions was in the range between 0.25 and 3 ppm, at pH 7.40 and 37 °C. Heparin showed inhibitory activity, which was attributed to adsorption at the active crystal growth centres. Heparin concentration as low as ca. 50 μM, reduced the rate of HAP crystal growth by more than 80%, while further increase (up to 200 μM) failed to completely inhibit the process beyond 90%. Heparin uptake studies at equilibrium conditions and analysis of the kinetics data in the presence of heparin, strongly suggest that the inhibition is due to the adsorption of heparin onto the HAP crystals, which resulted in the poisoning of the active growth sites.

Decellularized xenogeneic scaffolds have shown promise to be employed as compatible and functional cardiovascular biomaterials. However, one of the main barriers to their clinical exploitation is the lack of appropriate sterilization procedures. This study investigated the efficiency of a two-step sterilization method, antibiotics/antimycotic (AA) cocktail and peracetic acid (PAA), on porcine and bovine decellularized pericardium. In order to assess the efficiency of the method, a sterilization assessment protocol was specifically designed, comprising: i) controlled contamination with a known amount of bacteria; ii) sterility test; iii) identification of contaminants through MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) mass spectrometry and iv) quantification by the Most Probable Number (MPN) method. This sterilization assessment protocol proved to be a successful tool to monitor and optimize the proposed sterilization method. The treatment with AA + PAA method provided sterile scaffolds while preserving the structural integrity and biocompatibility of the decellularized porcine and bovine tissues. However, surface properties and cellular adhesion resulted slightly impaired on porcine pericardium. This work developed a sterilization method suitable for decellularized pericardial scaffolds that could be adopted for in vivo tissue engineering. Together with the proposed sterilization assessment protocol, this decontamination method will foster the clinical translation of decellularized xenogeneic substitutes. Statement of Significance Clinical application of functional and compatible xenogeneic decellularized scaffolds has been delayed due to the lack of appropriate sterilization methodologies. In this study, it was investigated an effective sterilization method optimized for porcine and bovine decellularized pericardia, based on the use of antibiotics/antimycotics followed by peracetic acid treatment. This treatment effectively sterilizes both species scaffolds, proves to maintain tissue overall structure and components, preserves biocompatibility and biomechanical properties. Furthermore, it was also developed a sterilization assessment protocol used to monitor and validate the previous method, consisting in three main parts: i) controlled contamination; ii) sterility test, and iii) identification and quantification of contaminants. Both methodologies were optimized for the tissues in study but can be applied to other scaffolds and accelerate their clinical translation.

Calcification is a recurrent problem in patients suffering from heart valve disease and it is the main cause of failure in biological heart valve prostheses. The development of reliable calcification tests that consider both the material properties of the prostheses and the fluid composition is of paramount importance for the effective testing and subsequent selection of new cardiovascular implants. In this article, a fast, reliable, and highly reproducible method for the assessment of the calcification potential of biomaterials was developed. The developed method simulated closely the chemical environment in vivo , where the supersaturation levels of calcium and phosphate remain constant. Seeded hydroxyapatite (HAP) crystal growth experiments were used as the reference system and compared to the mineralization kinetics and extent of frozen untreated bovine and porcine pericardium, and glutaraldehyde-fixed bovine pericardium. Untreated pericardial patches did not calcify in the supersaturated calcium phosphate solutions whereas glutaraldehyde-fixed bovine pericardial patches mineralized at the same conditions. The present work suggested that the loose collagenous serosa side of the pericardium mineralized at lower rates compared to its dense collagenous fibrous side. Concordant with these findings, the mineralization of bioprostheses may also be attributed, to the structural deterioration of collagen-rich tissues, induced by chemical treatment used to control in vivo structural stability and immunomodulation of the implants.

The original version of this article unfortunately contained a few errors. The captions of Figs. 4, 5, 6, 7, and 8 were mixed up and they were misreferred in the text. The correct captions and their references in text are given below.

Heart valve diseases remain common in industrialized countries. Bioprosthetic heart valves, introduced as free of anticoagulation therapy alternatives to mechanical substitutes. Still they suffer from long term failure due to calcification. Different treatment methods introduced to inhibit calcification, have so far been limited in success. Glycosaminoglycans (GAGs) possess properties including high negative charge, anticoagulation and anti-inflammatory activity that make them a potential solution for calcification problem. In this study, heparin hydrogel was prepared and characterized both chemically and mechanically. After that, heparin hydrogel embedded bovine pericardial tissues, fixed with glutaraldehyde, were produced and tested for their mechanical behavior and anticalcifcation potential in vitro using the constant composition model. In the calcification experiments, tissues were divided into three groups: a) Controls without treatment, b) Hydrogel treated tissues and c) Tissues with raw heparin dissolved in the calcification solution. The results showed that embedding of tissue with hydrogel had no stiffening effect on its mechanical behavior. Calcification assessment showed a significant efficacy on inhibition of calcium phosphate deposition of hydrogel treated (second group) in comparison to untreated tissues (control, first group). Calcification inhibition potential was very similar in both the second and raw heparin (third group). Histological data confirmed the obtained results, suggesting that heparin treatment is a promising anticalcification agent.

Background: Soft tissues are characterized by strong mechanical anisotropy, as a result of internal fiber architecture, matching the needs of mechanical function in each body part. Polymeric grafts, used for diseased tissues replacement, suffer from mechanical mismatch with the tissues replaced and the remaining healthy tissues to be connected. Electrospinning is an attractive technique by which we can produce biodegradable polymeric scaffolds for tissue engineering applications. Fiber characteristics and structural architecture has to be tuned to match mechanically the tissues to be replaced. Furthermore, for the design of fibrous scaffolds, other characteristics, like fiber diameter, porosity and hydrophilicity play an important role as far as cell atraction, function and tissue regeneration are concerned.Objective: In the present work, we aimed to produce polymeric membranous scaffolds with specific architecture, giving attention to fibers’ orientation and hence, controlling the final mechanical behavior to match that of the physiological tissues to be replaced.Methods: To this end, we used a specifically designed drum collector, with accurate velocity control, and tested different electrospinning parameters (polymeric solution concentrations, transfer rates, rotational speed, etc) to obtain design optimization.Results: Scanning Electron Microscopy on scaffolds showed a good morphology quality. Fiber orientation was directly related to the drum speed. Tensile testing showed mechanical anisotropy in higher speeds. Young’s modulus and Ultimate tensile strength demonstrated strong anisotropy (one order of magnitude larger) in parallel to transverse direction, with regard to drum speed, similar to that of physiologic soft cardiovascular tissues. Scaffold hydrophilicity, expressed by contact angle measurements remained high, although a relation to fiber architecture has been recorded. Conclusion: Enhancement of membranous anisotropy was attained, one order of magnitude greater for the parallel fibers’ direction compared to the transverse one. A similar anisotropy can be found in cardiovascular soft tissues, like human and porcine aortic heart valve leaflets.

Instrumented lumbar fusion with posterior pedicle screw systems enhances fusion rate. Although rigid pedicle-screw instrumentation increases fusion rate, recently many adverse effects have been reported in lumbar spinal fusion augmented with rigid instrumentation. This study compares static, non-destructive mechanical “in vitro” properties of three different systems [segmental contouring system (SCS), Claris and Twinflex], to investigate the inherent stiffness of these devices. Forty-eight identical lumbar synthetic models, each including three vertebral bodies and intervertebral discs, were instrumented with each of the three instrumentations Twinflex, Claris and SCS. Constructs with instrumented vertebrae as well as non-instrumented models were used in this study. The constructs underwent non-destructive static torsional and compressive-bending loading. Rigid instrumentation provided the highest torsional and compression stiffness. Torsional stiffness differed significantly (P Claris > Twinflex). Compressive stiffness also demonstrated statistically significant (P Claris, SCS > Twinflex). Both Claris and Twinflex provided similar compressive stiffness. In torsion, all three instrumentations equally reduced at 80% the amount of relative torsional displacement between adjacent instrumented vertebrae (P

Aims: This study aims to evaluate atherosclerosis, oxidative stress, and arterial stiffness attenuation by simvastatin and ivabradine in hyperlipidemic rabbits. Methods and Results: Forty rabbits were randomly divided into 4 groups: atherogenic diet (group C), atherogenic diet plus simvastatin (group S), atherogenic diet plus ivabradine (group I), and atherogenic diet plus simvastatin and ivabradine (group S + I). After 9 weeks, rabbits were euthanized and descending aortas excised for mechanical testing. Atherogenic diet induced the development of significant atherosclerotic lesions in group C animals but in none of groups S, I, and S + I. RAM-11 and HHF-35–positive cells were significantly reduced in groups S, I, and S + I compared with group C ( P < .001). A significant neointimal hyperplasia and intima–media ratio reduction was demonstrated in groups S ( P = .015 and P < .001), I ( P = .021 and P < .001), and S + I ( P = .019 and P < .001) compared with group C. Protein nitrotyrosine levels were significantly decreased in group S compared with group C ( P = .009), and reactive oxygen species levels were decreased in group I compared with group C ( P = .011). Aortic stiffness was significantly reduced in groups S, I, and S + I compared with group C ( P = .003, P = .011, and P = .029). Conclusion: Simvastatin and ivabradine significantly inhibited intimal hyperplasia and oxidative stress contributing to aortic stiffness reduction in hyperlipidemic rabbits.

Background: Bioprosthetic heart valves still suffer from calcification. In the department of Cryobiology & Biomaterials, we have established an in vitro calcification test method which is suitable for the investigation of calcification patterns of heart valves. In a modified test protocol we use the pH-dependency of the calcification process to control and stabilise the process by keeping the calcification ions more constant than in the established test protocol. Methods: Five glutaraldehyde-fixed porcine heart valve bioprostheses underwent accelerated dynamic in vitro calcification individually. Three valves were tested according to the established test protocol. Two valves were studied with the new pH-controlled loop system. pH drop was used as an index for the consumption of the calcification ions. The degree of calcification was determined by means of μX-ray (high resolution x-ray on mammography machine), conventional and micro computer tomography (CT, μ-CT). Results: We determined the average degree of calcification after 12 million cycles. The value was calculated to be 12 % vs. 67 % (established vs. pH-controlled group) using μX-ray (2D), 3 % vs. 16 % using CT, and 3 % vs. 33 % using μ-CT. Conclusion: A new faster and more potent in vitro calcification method has been developed. Optimisation of this pH-controlled method in order to implement an additional direct calcification ion concentration control is under construction.

Biological fluids are supersaturated with respect to various sparingly soluble salts including phosphates, oxalates, and in some cases carbonates. The presence of surfaces provided by tissues (valves, aortas, or other membranes) provides substrates favoring heterogeneous nucleation of insoluble salts that adhere tenaciously. The investigation of the kinetics of crystal growth at sustained conditions is of paramount importance for understanding the role of the various substrates and of the mechanism underlying the formation of the various salts. Artificial tissues for heart or implant cements for bone restoration are substrates that favor the formation of metastable calcium phosphates like octacalcium phosphate. Crystal lattice compatibility results in higher rates of crystallization. Calcium oxalate may also form in supersaturated solutions, resulting in encrustations buildup in metallic stents by heterogeneous nucleation and growth.

In this work we studied the frequency dependence of the dynamic mechanical characteristic s of saphenous vein (SV) and internal mammary artery (IMA) grafts. Rectangular longitudinal strips from 14 patients were tested under cyclic uniaxial tensile loading in the frequency range of 0.1–20 Hz, at 37°C in wet conditions. The dynamic mechanical parameters (the storage modulus ES and the hysteresis ratio h (loading/loop area)) together with the collagen phase modulus EH were computed as a function of frequency. The results showed that in all graft types ES and EH varied with frequency in the range 0.5–10 Hz, presenting a maximum in the neighboring of 1 Hz. The hysteresis ratio h was increased in the frequency range 1–20 Hz. It seems from the results that the physical resonance frequency of the components of the tissue responsible for their elastic behavior may lay in the range around 1 Hz, while that for the viscous behavior in the range of 20 Hz or more. Early clinical outcomes of both grafting were studied in parallel. In a one-year postoperative period the follow-up (clinical examination, electrocardiography, echocardiography and stress test) did not reveal any sign of graft occlusion or severe stenosis except one perioperative infraction but without any correlation to the graft quality.

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 (

While calcification of bioprosthetic valves presents a clinical problem, the mechanism of formation of calcific deposits in the leaflets remains unclear. A new method for in vitro calcification is employed, in parallel with an in vivo (subcutaneous animal) model. The nature of crystal phases grown by both methods on porcine bioprosthetic heart valves was investigated. Light microscopy, scanning electron microscopy (SEM), X-ray energy dispersive spectroscopy (SEM-EDS) and stoichiometric chemical analysis were used as tools for the qualitative and quantitative assessment of the calcification process. Photomicrographs from leaflets calcified early in vitro (24–48 h) and in vivo (5–10 days) confirmed that calcification initiated in the central region of the tissue, mainly in fibrosa, and extended with time (after 28 and 56 days) in vivo towards the outer surface of the tissue. SEM micrographs from tissue sections of early in vivo and in vitro calcified leaflets showed coexistence of different sizes (1–30 μm) of crystal phases in contact with tissue fibers. EDS analysis confirmed that the deposits were calcium phosphate salts. Chemical analysis of samples calcified in vivo for longer time periods, showed that the content of the salts was mainly calcium and phosphate with a Ca/P molar ratio 1.78 and 1.94 after 28 and 56 days, respectively. These results suggested that the calcification process is followed by a sequential hydrolytic transformation of CaHPO 4 ·2H 2 O (DCPD) to Ca 4 H(PO 4 ) 3 ·2.5H 2 O (OCP) and to Ca 5 (PO 4 ) 3 OH (HAP). Calcium phosphate crystal phases grown in vitro under controlled conditions and at constant supersaturation provide a successful simulation of the calcification process in the animal model and may be applied in screening new biomaterials with respect to their potential for calcification.

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.

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.

The need for replacement of damaged or malfunctioning organs or tissues in the human body has led through intense and innovative research during the past century to the development of materials and devices which are compatible with living tissues. Materials’ compatibility consists in their capability to be accepted by the body when implanted and in contact with other tissues and body fluids. These materials, known as biomaterials, are the fundamental tools for engineering implantable devices dedicated to function in a specific way that substitutes corresponding function of the tissues or organs replaced due to malfunction, in synergism with the surrounding biological environment. Bone fracture healing by the incorporation of plates was known as early as the beginning of the century. Implants to replace heart valves and hip joints have been reported in the early 60s (Park & Lakes, 1992). Among the problems recorded when the first implants were employed were corrosion, mechanical failure and rejection by the body. The latter remains the main problem in the development of novel biomaterials which can be used as implants. Biomaterials now play a major role in replacing or improving the function of every major body system (skeletal, circulatory, nervous, etc.). Commonly employed implants include orthopedic devices such as total knee and hip joint replacements, spinal implants, and bone fixators; cardiac implants such as artificial heart valves and pacemakers; soft tissue implants such as breast implants and injectable collagen for soft tissue augmentation; and dental implants to replace teeth/root systems and bony tissue in the oral cavity. When a man-made material is placed in the human body, tissue reacts to the implant in a variety of ways depending on the material type and function. The mechanism of tissue attachment depends on the tissue response to the implant surface. In general, materials can be placed into three classes that represent the tissue response they elicit: inert, bioresorbable, and bioactive. Inert materials such as titanium and alumina (Al2O3) are nearly chemically inert in the body and exhibit minimal chemical interaction with adjacent tissue. A fibrous tissue capsule will normally form around inert implants. Tissue attachment with inert materials can be through tissue growth into surface irregularities, by bone cement, or by press fitting into a defect. This morphological fixation is not ideal for the long-term stability of permanent implants and often becomes a problem with orthopedic and dental implant applications. Bioresorbable materials, such as tricalcium phosphate and polylactic

To achieve natural scaffolds for tissue engineering applications we decellularized bovine pericardial (BP) tissues according to two different protocols: a novel treatment based on Triton(®) X-100 (12 h, 4 °C) (BP1) and a trypsin/EDTA treatment (37 °C, 48 h) (BP2). Results were compared with commercially available acellular xenogeneic biomaterials, Veritas(®) and Collamed(®). Biomechanical characteristics, high (E(h)) and low (E(l)) modulus of elasticity, of the fresh untreated tissue varied with the anatomical direction (apex to base (T) to transverse (L)) (mean ± SDEV): (41.63 ± 14.65-48.12 ± 10.19 MPa and 0.27 ± 0.05-0.30 ± 0.12 MPa respectively). BP1 had no mechanical effect (44.65 ± 19.73-52.67 ± 7.59 MPa and 0.37 ± 0.14-0.37 ± 0.11 MPa, respectively) but BP2 resulted in significant decrease in E(h) and E(l) (20.96 ± 8.17-36.82 ± 3.23 MPa and 0.20 ± 0.06-0.23 ± 0.06 MPa). Hysteresis ratio (h) varied (19-26 % of the loading energy) independently of anatomical direction. Glycosaminoglycans content was unaffected by BP1, while 22 % of chondroitin/dermatan sulphate and 60 % of hyaluronan were removed after BP2 treatment. Endothelial cell adhesion was achieved after 24 h and 3 days cell culture.

Background Atherosclerosis is a diffuse and highly variable disease of arteries that alters the mechanical properties of the vessel wall through highly variable changes in its cellular composition and histological structure. We have analyzed the effects of acute atherosclerotic changes on the mechanical properties of the descending thoracic aorta of rabbits fed a 4% cholesterol diet. Methods Two groups of eight male New Zealand White rabbits were randomly selected and fed for 8 weeks either an atherogenic diet (4% cholesterol plus regular rabbit chow), or regular chow. Animals were sacrificed after 8 weeks, and the descending thoracic aortas were excised for pressure-diameter tests and histological evaluation to examine the relationship between aortic elastic properties and atherosclerotic lesions. Results All rabbits fed the high-cholesterol diet developed either intermediate or advanced atherosclerotic lesions, particularly American Heart Association-type III and IV, which were fatty and contained abundant lipid-filled foam cells (RAM 11-positive cells) and fewer SMCs with solid-like actin staining (HHF-35-positive cells). In contrast, rabbits fed a normal diet had no visible atherosclerotic changes. The atherosclerotic lesions correlated with a statistically significant decrease in mean vessel wall stiffness in the cholesterol-fed rabbits (51.52 ± 8.76 kPa) compared to the control animals (68.98 ± 11.98 kPa), especially in rabbits with more progressive disease. Conclusions Notably, stiffness appears to decrease with the progression of atherosclerosis after the 8-week period.

Fifty years after their first implantation, bioprosthetic heart valves still suffer from tissue rupture and calcification. Since new bioprostheses exhibit a lower risk of calcification, fast and reliable in vitro methods need to be evaluated for testing the application of new anti-calcification techniques. This report describes a modification of the well-known in vitro dynamic calcification test method (Glasmacher et al, Leibniz University Hannover (LUH)), combined with the pH-controlled, constant solution supersaturation (CSS) method (University of Patras (UP)). The CSS method is based on monitoring the pH of the solution and the addition of calcium and phosphate ion solutions through the implementation of two syringe pumps. The pH and the activities of all ions in the solutions are thus kept constant, resulting in higher calcification rates compared to conventional in vitro methods in which solution supersaturation is allowed to decrease without any further control. To verify this hypothesis, five glutaraldehyde preserved porcine aortic valves were tested. Three of the valves were tested according to a free-drift methodology: the valves were immersed in a supersaturated calcification solution, with an initial total calcium times total phosphate product of (CaxP)=10.5 (mmol/L)2 renewed weekly. Two valves were tested by the new pH-controlled loop system, implementing the CSS methodology. All valves were tested for a 4-week period, loaded at 300 cycles per minute, resulting in a total of 12 million cycles at the end of the testing period. The degree of calcification was determined weekly by means of μx-ray, and by conventional, clinical and micro-computer tomography (CT, μCT). The results showed that the valves mineralizing at constant solution supersaturation in vitro yielded higher rates of calcification compared to the valves tested at conditions of decreasing solution supersaturation without any control, indicating the development of a new, accelerated, controllable in vitro calcification method.

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.

Bovine pericardium (BPC) and polytetrafluoroethylene (PTFE) have been widely used to reinforce staple lines in lung resection. Since limited Information regarding the calcification of these biomaterials is available, we undertook an In vitro study to evaluate their calcification potential. Commercially available BPC and PTFE biomaterials were evaluated and compared with custom-prepared BPC tissue. In vitro calcification was performed via submersion in supersaturated solution In a double-walled glass reactor at 37.0°C ± 0.1°C, pH 7.4 ± 0.1, mimicking most ion concentrations of human blood plasma. In processing of calcification, the pH decrease of the solution simulated the addition of consumed H+, Ca2+, and PO43– ions from titrant solutions, the concentrations of which were based on the stolchiometry of octacalcium phosphate. The molar ion addition with time was recorded, and the initial slope of the curve was computed for each experiment. The rate of calcification developed (molar calcium phosphate ion addition rate per time and total surface area) (R) was computed after that with respect to the relative supersaturation (σ) used in each experiment. R for custom-prepared BPC tissues was found to be in the range of 0.19 ± 0.08 to 0.52 ± 0.19 (n = 17) in σ range of 0.72 to 1.42. Commercial BPC was found to be 0.016 to 0.052 (n = 4), and PTFE was 0.005 to 0.05 (n = 8) in the same σ range. Both clinically applied biomaterials, BPC and PTFE, seemed to be calcified with rates of at least one order of magnitude lower than the custom-prepared BPC tissue. This data suggested that BPC and PTFE biomaterials showed a similar, relatively very low tendency for calcification compared with custom-prepared BPC tissue. Although further studies are necessary, staple line reinforcement by these two biomaterials should be considered safe from the calcification point of view.

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.

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.

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.