Development and Evaluation of Biodegradable Core-Shell Microfibrous and Nanofibrous Scaffolds for Tissue Engineering Applications.

Tissue engineering scaffolds as three-dimensional substrates may serve as ideal templates for tissue regeneration by simulating the structure of the extracellular matrix (ECM). Many biodegradable synthetic polymers, either hydrophobic, like Poly-ε-caprolactone (PCL), or hydrophilic, like Poly(Vinyl Alcohol) (PVA), are widely used as candidate bioactive materials for fabricating tissue engineering scaffolds. However, a combination of good cytocompatibility of hydrophilic polymers with good biomechanical performance of hydrophobic polymers could be beneficial for the in vivo performance of the scaffolds. In this study, we aimed to fabricate biodegradable fibrous scaffolds by combining the properties of hydrophobic PCL with those of hydrophilic PVA and evaluate their properties in comparison with pristine PCL scaffolds. Therefore, single-layered PCL scaffolds, sequential tri-layered (PVA/PCL/PVA), and core-shell (PVA as shell and PCL as core) composite scaffolds were developed utilizing the electrospinning technique. The material structural and biomechanical properties of the electrospun scaffolds, before and after their hydrolytic degradation over a seven-month period following storage in phosphate-buffered saline (PBS) at 37 °C, were comprehensively compared. In addition, human embryonic kidney cells (HEK-293) were cultured on the scaffolds to investigate potential cell attachment, infiltration, and proliferation. The results demonstrated the long-term efficacy of core-shell biodegradable fibrous scaffolds in comparison to single-layers PCL and tri-layers PVA/PCL/PVA, not only due to its superior morphological characteristics and mechanical properties, but also due to its ability to promote homogeneous cell distribution and proliferation, without any external chemical or physical stimuli.

A novel polymeric fibrous microstructured biodegradable small-caliber tubular scaffold for cardiovascular tissue engineering.

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).

Decellularized tissue-engineered heart valves calcification: what do animal and clinical studies tell us?

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.

Calcification Assessment of Bioprosthetic Heart Valve Tissues Using an Improved In Vitro Model.

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.

Correction to: The effect of heparin hydrogel embedding on glutaraldehyde fixed bovine pericardial tissues: Mechanical behavior and anticalcification potential.

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.

The effect of heparin hydrogel embedding on glutaraldehyde fixed bovine pericardial tissues: Mechanical behavior and anticalcification potential.

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.

A sterilization method for decellularized xenogeneic cardiovascular scaffolds.

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.

Inhibition of Atherosclerosis Progression, Intimal Hyperplasia, and Oxidative Stress by Simvastatin and Ivabradine May Reduce Thoracic Aorta's Stiffness in Hypercholesterolemic Rabbits.

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.

Biomechanical and structural changes following the decellularization of bovine pericardial tissues for use as a tissue engineering scaffold.

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.

Structural and biomechanical alterations in rabbit thoracic aortas are associated with the progression of atherosclerosis.

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.

Aldosterone receptor blockade inhibits degenerative processes in the early stage of calcific aortic stenosis.

Calcific aortic valve disease is associated with increased morbidity and mortality, especially in the elderly. To date, pharmacological therapies have not proven as effective as surgical intervention. Here, we used a hyperlipidemic rabbit model to investigate the potential effects of selective aldosterone inhibition on the early stages of aortic valve calcification, a pharmacological strategy that has not yet been tested. Forty New Zealand male rabbits fed a standard diet for 4 weeks were separated into three groups: (1) control (n=10), fed a standard diet; (2) vehicle (n=15), fed a hyperlipidemic diet (cholesterol 1%) plus vehicle; and (3) eplerenone (n=15), fed a hyperlipidemic diet plus 100 mg/kg/d eplerenone (last 4 weeks). After 8 weeks, animals were sacrificed and prepared aortic valve sections were examined with Von Kossa silver stain and by immunostaining for mineralocorticoid receptor, macrophages and angiotensin-converting enzyme. The presence of calcium deposits was confirmed by scanning electron microscopy. Eplerenone increased aldosterone levels but did not affect blood pressure, cholesterol or potassium levels. Hyperlipidemia induced macrophage accumulation and angiotensin-converting enzyme expression, as well as calcium deposition in the leaflets. All markers were decreased by eplerenone treatment. Immunohistochemistry for mineralocorticoid (aldosterone) receptors revealed similar expression in the leaflets of both control and hyperlipidemic groups. Collectively, these results indicate that aldosterone receptors are present in rabbit aortic valve leaflets and their selective blockade with eplerenone inhibits formation of the sclerotic lesions induced by a high fat diet.

Development of a new combined test setup for accelerated dynamic pH-controlled in vitro calcification of porcine heart valves.

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 mux-ray, and by conventional, clinical and micro-computer tomography (CT, muCT). 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.

In vitro evaluation for potential calcification of biomaterials used for staple line reinforcement in lung surgery.

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 degrees C +/- 0.1 degrees 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(+), Ca(2+), and PO(4)(3-) ions from titrant solutions, the concentrations of which were based on the stoichiometry 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 (sigma) 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 sigma 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 sigma 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.