Epoxy- versus Glutaraldehyde-Treated Bovine Jugular Vein Conduit for Pulmonary Valve Replacement: A Comparison of Morphological Changes in a Pig Model.

Valved conduits are often required to replace pulmonary arteries (PA). A widely used Contegra device is made of bovine jugular vein (BJV), preserved with glutaraldehyde (GA) and iso-propanol. However, it has several drawbacks that may be attributed to its chemical treatment. We hypothesized that the use of an alternative preservation compound may significantly improve BJV conduit performance. This study aimed to compare the macroscopic and microscopic properties of the BJV treated with diepoxide (DE) and GA in a porcine model. Twelve DE-BJVs and four Contegra conduits were used for PA replacement in minipigs. To assess the isolated influence of GA, we included an additional control group-BJV treated with 0.625% GA (n = 4). The animals were withdrawn after 6 months of follow-up and the conduits were examined. Explanted DE-BJV had a soft elastic wall with no signs of thrombosis or calcification and good conduit integration, including myofibroblast germination, an ingrowth of soft connective tissue formations and remarkable neoangiogenesis. The inner surface of DE-BJVs was covered by a thin neointimal layer with a solid endothelium. Contegra grafts had a stiffer wall with thrombosis on the leaflets. Calcified foci, chondroid metaplasia, and hyalinosis were observed within the wall. The distal anastomotic sites had hyperplastic neointima, partially covered with the endothelium. The wall of GA-BJV was stiff and rigid with degenerative changes, a substantial amount of calcium deposits and dense fibrotic formations in adventitia. An irregular neointimal layer was presented in the anastomotic sites without endothelial cover in the GA BJV wall. These results demonstrate that DE treatment improves conduit integration and the endothelialization of the inner surface while preventing the mineralization of the BJV, which may reduce the risk of early conduit dysfunction.

Remote Outcomes with Poly-ε-Caprolactone Aortic Grafts in Rats.

Poly-ε-caprolactone ((1,7)-polyoxepan-2-one; PCL) is a biodegradable polymer widely used in various fields of bioengineering, but its behavior in long-term studies appears to depend on many conditions, such as application specificity, chemical structure, in vivo test systems, and even environmental conditions in which the construction is exploited in. In this study, we offer an observation of the remote outcomes of PCL tubular grafts for abdominal aorta replacement in an in vivo experiment on a rat model. Adult Wistar rats were implanted with PCL vascular matrices and observed for 180 days. The results of ultrasound diagnostics and X-ray tomography (CBCT) show that the grafts maintained patency for the entire follow-up period without thrombosis, leakage, or interruptions, but different types of tissue reactions were found at this time point. By the day of examination, all the implants revealed a confluent endothelial monolayer covering layers of hyperplastic neointima formed on the luminal surface of the grafts. Foreign body reactions were found in several explants including those without signs of stenosis. Most of the scaffolds showed a pronounced infiltration with fibroblastic cells. All the samples revealed subintimal calcium phosphate deposits. A correlation between chondroid metaplasia in profound cells of neointima and the process of mineralization was supported by immunohistochemical (IHC) staining for S100 proteins and EDS mapping. Microscopy showed that the scaffolds with an intensive inflammatory response or formed fibrotic capsules retain their fibrillar structure even on day 180 after implantation, but matrices infiltrated with viable cells partially save the original fibrillary network. This research highlights the advantages of PCL vascular scaffolds, such as graft permeability, revitalization, and good surgical outcomes. The disadvantages are low biodegradation rates and exceptionally high risks of mineralization and intimal hyperplasia.

Influence of Single-Wall Carbon Nanotube Suspension on the Mechanical Properties of Polymeric Films and Electrospun Scaffolds.

Carbon nanotubes (CNTs) are used in applications ranging from electrical engineering to medical device manufacturing. It is well known that the addition of nanotubes can influence the mechanical properties of various industrial materials, including plastics. Electrospinning is a popular method for fabricating nanomaterials, widely suggested for polymer scaffold manufacturing. In this study, we aimed to describe the influence of single-walled carbon nanotube (SWCNT) suspensions on polymeric poured films and electrospun scaffolds and to investigate their structural and mechanical properties obtained from various compositions. To obtain films and electrospun scaffolds of 8 mm diameter, we used poly-ε-caprolactone (PCL) and poly(cyclohexene carbonate) (PCHC) solutions containing several mass fractions of SWCNT. The samples were characterized using tensile tests, atomic force and scanning electronic microscopy (AFM and SEM). All the studied SWCNT concentrations were shown to decrease the extensibility and strength of electrospun scaffolds, so SWCNT use was considered unsuitable for this technique. The 0.01% mass fraction of SWCNT in PCL films increased the polymer strength, while fractions of 0.03% and more significantly decreased the polymer strength and extensibility compared to the undoped polymer. The PHCH polymeric films showed a similar behavior with an extremum at 0.02% concentration for strength at break.

Calcification of Various Bioprosthetic Materials in Rats: Is It Really Different?

The causes of heart valve bioprosthetic calcification are still not clear. In this paper, we compared the calcification in the porcine aorta (Ao) and the bovine jugular vein (Ve) walls, as well as the bovine pericardium (Pe). Biomaterials were crosslinked with glutaraldehyde (GA) and diepoxide (DE), after which they were implanted subcutaneously in young rats for 10, 20, and 30 days. Collagen, elastin, and fibrillin were visualized in non-implanted samples. Atomic absorption spectroscopy, histological methods, scanning electron microscopy, and Fourier-transform infrared spectroscopy were used to study the dynamics of calcification. By the 30th day, calcium accumulated most intensively in the collagen fibers of the GA-Pe. In elastin-rich materials, calcium deposits were associated with elastin fibers and localized differences in the walls of Ao and Ve. The DE-Pe did not calcify at all for 30 days. Alkaline phosphatase does not affect calcification since it was not found in the implant tissue. Fibrillin surrounds elastin fibers in the Ao and Ve, but its involvement in calcification is questionable. In the subcutaneous space of young rats, which are used to model the implants' calcification, the content of phosphorus was five times higher than in aging animals. We hypothesize that the centers of calcium phosphate nucleation are the positively charged nitrogen of the pyridinium rings, which is the main one in fresh elastin and appears in collagen as a result of GA preservation. Nucleation can be significantly accelerated at high concentrations of phosphorus in biological fluids. The hypothesis needs further experimental confirmation.

In Vivo Evaluation of PCL Vascular Grafts Implanted in Rat Abdominal Aorta.

Electrospun tissue-engineered grafts made of biodegradable materials have become a perspective search field in terms of vascular replacement, and more research is required to describe their in vivo transformation. This study aimed to give a detailed observation of hemodynamic and structural properties of electrospun, monolayered poly-ε-caprolactone (PCL) grafts in an in vivo experiment using a rat aorta replacement model at 10, 30, 60 and 90 implantation days. It was shown using ultrasound diagnostic and X-ray tomography that PCL grafts maintain patency throughout the entire follow-up period, without stenosis or thrombosis. Vascular compliance, assessed by the resistance index (RI), remains at the stable level from the 10th to the 90th day. A histological study using hematoxylin-eosin (H&E), von Kossa and Russell-Movat pentachrome staining demonstrated the dynamics of tissue response to the implant. By the 10th day, an endothelial monolayer was forming on the graft luminal surface, followed by the gradual growth and compaction of the neointima up to the 90th day. The intense inflammatory cellular reaction observed on the 10th day in the thickness of the scaffold was changed by the fibroblast and myofibroblast penetration by the 30th day. The cellularity maximum was reached on the 60th day, but by the 90th day the cellularity significantly (p = 0.02) decreased. From the 60th day, in some samples, the calcium phosphate depositions were revealed at the scaffold-neointima interface. Scanning electron microscopy showed that the scaffolds retained their fibrillar structure up to the 90th day. Thus, we have shown that the advantages of PCL scaffolds are excellent endothelialization and good surgical outcome. The disadvantages include their slow biodegradation, ineffective cellularization, and risks for mineralization and intimal hyperplasia.

Effects of Electrospinning Parameter Adjustment on the Mechanical Behavior of Poly-ε-caprolactone Vascular Scaffolds.

Electrospinning is a perspective method widely suggested for use in bioengineering applications, but the variability in currently available data and equipment necessitates additional research to ascertain the desirable methodology. In this study, we aimed to describe the effects of electrospinning technique alterations on the structural and mechanical properties of (1,7)-polyoxepan-2-one (poly-ε-caprolactone, PCL) scaffolds, such as circumferential and longitudinal stress/strain curves, in comparison with corresponding properties of fresh rat aorta samples. Scaffolds manufactured under different electrospinning modes were analyzed and evaluated using scanning electronic microscopy as well as uniaxial longitudinal and circumferential tensile tests. Fiber diameter was shown to be the most crucial characteristic of the scaffold, correlating with its mechanical properties.

Bovine jugular vein conduit: What affects its elastomechanical properties and thermostability?

The aim of this study was to compare the mechanical properties and thermal stability of the venous wall depending on the treatment method used, and, accordingly, on those structural changes in the tissue that this treatment causes. Bovine jugular vein walls (BJVWs) cross-linked with glutaraldehyde (GA), ethylene glycol diglycidyl ether (DE), and Contegra commercial conduit were evaluated using uniaxial stretching [with and without pre-conditioning (PreC)], differential scanning calorimetry, amino acid analysis, and attenuated total reflection infrared spectroscopy. Fresh BJVW was used as a control. It was shown that failure stress in non-PreC GA-treated and DE-treated materials was lower than that in fresh and Contegra counterparts. Contegra samples were the stiffest among the tested materials. Cyclic preloading leads to distortion of the mechanical behavior of this material, which is heterogeneous in composition and structure. The denaturation temperatures (Td ) of all cross-linked BJVWs were higher than the Td of the fresh vein. The microstructures of the tested BJVWs did not exhibit any differences, but the cross-linking density and hydration of the DE-vein were the highest. GA-cross-linking or DE-cross-linking and isopropanol exposure (Contegra) changed the protein secondary structures of the tested materials in different ways. We hypothesized that the protein secondary structure and hydration degree are the main causes of differences in the mechanical properties and thermal stability of BJVW.

Immobilized Bisphosphonates as Potential Inhibitors of Bioprosthetic Calcification: Effects on Various Xenogeneic Cardiovascular Tissues.

Calcification is the major factor limiting the clinical use of bioprostheses. It may be prevented by the immobilization of bisphosphonic compounds (BPs) on the biomaterial. In this study, we assessed the accumulation and structure of calcium phosphate deposits in collagen-rich bovine pericardium (Pe) and elastin-rich porcine aortic wall (Ao) and bovine jugular vein wall (Ve) cross-linked with glutaraldehyde (GA) or diepoxy compound (DE). These tissues were then modified with pamidronic (PAM) acid or 2-(2'-carboxyethylamino)ethylidene-1,1-bisphosphonic (CEABA) acid. Tissue transformations were studied using Fourier-transform infrared spectroscopy. After subcutaneous implantation of the biomaterials in 220 rats, calcification dynamics were examined using atomic absorption spectrophotometry, light microscopy after von Kossa staining, and scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy The calcium content in all GA-cross-linked tissues and DE-cross-linked Ao increased to 100-160 mg/g on day 60 after implantation. BPs prevented the accumulation of phosphates on the surface of all materials and most effectively inhibited calcification in GA-cross-linked Ao and DE-cross-linked Pe. PAM containing -OH in the R1 group was more effective than CEABA containing -H in R1. The calcification-inhibitory effect of BPs may be realized through their ability to block nucleation and prevent the growth of hydroxyapatite crystals.