Pigs as Models to Test Cardiovascular Devices.

Pigs as laboratory animals are used in preclinical studies aimed at developing medical devices for cardiac surgery. The anatomy of the cardiovascular system of these animals has been well studied and acknowledged as suitable for use and the testing of new cardiovascular devices developed for humans. However, there are no morphometric characteristics of the aortic root and thoraco-abdominal part of porcine aorta. This can lead to difficulties in experimental surgery and even result in the death of experimental animals due to the mismatch in the size of the implantable devices. Thus, such information is essential to enhance the efficiency of surgical technologies used for eliminating aortic pathologies in their various sections. The purpose of our research is to study the anatomy of the aorta in mini pigs and to assess whether the size, age, and sex of the animals affect the size of the main structures in their aortas. In addition, we attempted to compare the results obtained by transesophageal echocardiography (TEE) and angiography. We studied 28 laboratory mini pigs, dividing them into three groups by body weight (40-70 kg, 71-90 kg, and 90 kg). We did not find any relationship between the external somatometric characteristics of the animals and the size of their aortas. Animals have individual anatomical variability in their cardiovascular systems, which means that they need to be examined in terms of preoperative planning by any available method-echocardiography, angiography, or multispiral computed tomography (CT).

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.

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.

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.

Allografts and xenografts for right ventricular outflow tract reconstruction in Ross patients.

OBJECTIVES: Pulmonary allografts (AG) are the gold standard for right ventricular outflow tract (RVOT) reconstruction during the Ross procedure. However, there is limited availability of AG in some countries, and the use of alternative grafts for RVOT reconstruction remains controversial. This study aimed to compare the rates of freedom from RVOT graft dysfunction for AG and diepoxide-treated pericardial xenografts (DPXG). METHODS: Between 1998 and 2015, 793 adult patients underwent the Ross procedure in our centre. Using propensity score matching, the clinical outcomes and echocardiographic results of AG and DPXG were compared. RESULTS: Propensity score matching resulted in 2 groups (AG and DPXG) of 122 patients each. No difference was found in early mortality (2.5%) in both groups. The freedom from RVOT graft dysfunction curves were comparable between the AG and DPXG groups (P = 0.186) and the 8-year rates of freedom from graft dysfunction were 91.8% and 82.2%, respectively. The survival rates at 8 years were 90.5% and 90.1%, and the rates of freedom from RVOT reintervention at 8 years were 100% and 96.8% for the AG and DPXG groups, respectively. At discharge and follow-up, transprosthetic gradients were significantly higher in the DPXG group. The rate of the RVOT gradient progression was also higher in the DPXG group than in the AG group (1.80 ± 0.06 vs 1.39 ± 0.04 mmHg/year, P < 0.001). CONCLUSIONS: There was no difference in freedom from RVOT graft dysfunction by 8 years when using AG and DPXG in adult Ross patients, nor in survival and freedom from RVOT conduit reintervention. Long-term results need further evaluation.

Treatment with bisphosphonates to mitigate calcification of elastin-containing bioprosthetic materials.

This study evaluated the ability of bisphosphonates (BPAs) of different molecular structures to mitigate the calcification of porcine aortic wall (PAW) and bovine jugular vein wall (BJVW). Tissues cross-linked with glutaraldehyde (GA) or diepoxide (DE) were modified with pamidronic acid (PAM), alendronic acid (ALE), neridronic acid (NER) (type 1 BPAs); 2-(2'-carboxyethylamino)ethylidene-1,1-bisphosphonic acid (CEABA), 2-(5-carboxypentylamino)ethylidene-1,1-bisphosphonic acid (CPABA) (type 2); and zoledronic acid (ZOL) (type 3). After implanting the tissue samples subcutaneously in 100 rats, calcification was examined using atomic absorption spectrophotometry (60-day explants) and light microscopy after von Kossa staining (10- and 30-day explants). The calcium contents in GA-BJVW and GA- and DE-PAW increased up to 100-120 mg/g after 60 days, while being 3 times lower in DE-BJVW. In modified and nonmodified PAW samples, calcium phosphates appeared by day 10 and were associated with elastic fibers and devitalized cellular elements. In all groups of BJVW samples, mineralization began in elastic fibers near the subendothelial layer. In addition, calcified collagen was found in the GA-BJVW samples. Minimal calcification was found in GA-PAW treated with type 1 BPAs and CEABA. For DE-PAW and GA-BJVW, the calcium level significantly decreased with PAM and CEABA. Meanwhile, ALE and NER were effective for DE-BJVW.

Diepoxy- Versus Glutaraldehyde-Treated Xenografts: Outcomes of Right Ventricular Outflow Tract Reconstruction in Children.

BACKGROUND: Xenografts used for right ventricular outflow tract (RVOT) reconstruction are typically treated with glutaraldehyde. However, potential benefit of epoxy treatment was demonstrated in experimental studies. We aimed to compare diepoxy-treated bovine pericardial valved conduits (DE-PVCs) and glutaraldehyde-treated bovine pericardial valved conduits (GA-PVCs) for RVOT reconstruction in pediatric patients. METHODS: Between 2002 and 2017, 117 patients underwent RVOT reconstruction with PVC in single center: DE-PVC group, n = 39; and GA-PVC group, n = 78. After performing propensity score analysis (1:1) for the entire sample, 29 patients from the DE-PVC group were matched with 29 patients from the GA-PVC group. RESULTS: There were no conduit-related deaths. In the DE-PVC group, the freedom from conduit failure was 90.9% at four years and 54.3% at eight years postoperatively. In the GA-PVC group, it was 46.3% and 33.1%, respectively. The difference was significant (P = .037). Conduit failure was typically caused by stenosis in both groups. In the DE-PVC group, the main cause of stenosis was xenograft calcification (27.6%); while in the GA-PVC group, it was mostly due to neointimal proliferation (25.0%) and, less often, calcification (14.3%). Conduit thrombosis was the cause of replacement in 6.9% of patients from the GA-PVC group. CONCLUSIONS: Diepoxy-treated bovine pericardial valved conduit is a suitable alternative to GA-PVC for RVOT reconstruction in pediatric patients. Diepoxy-treated bovine pericardial valved conduits may be less prone to conduit failure and more resistant to neointimal proliferation and conduit thrombosis than GA-PVCs.

In search of the best xenogeneic material for a paediatric conduit: an analysis of clinical data.

OBJECTIVES: In this study, we aimed to determine the incidence of reintervention and calcification of xenografts in paediatric patients who underwent placement of the right ventricle-to-pulmonary artery valved conduits. METHODS: We retrospectively analysed clinical data of paediatric patients (1 day-18 years) who underwent right ventricular outflow tract reconstruction using xenograft from 2000 to 2016 at a single centre. RESULTS: A total of 301 patients underwent the placement of 337 xenografts, including glutaraldehyde-treated bovine jugular vein (n = 171, 50.7%), glutaraldehyde-treated bovine pericardial valved conduit (n = 75, 22.3%), diepoxy-treated porcine aortic conduit (n = 58, 17.2%) and diepoxy-treated bovine pericardial valved conduit (DE-PVC) (n = 33, 9.8%). There were 284 (84.3%) primary implantations and 53 (15.7%) reimplantations. The median follow-up was 4.2 years (range 1.5 months-14.5 years). The multivariate regression analysis did not reveal statistically significant associations of the first reintervention with the type of xenograft (P = 0.78). At reintervention, calcification of the wall and/or cusps was the main cause of conduit dysfunction in 66.4% of cases. On the basis of the multivariate Cox regression analysis, xenograft types were significant predictors of reintervention caused by conduit calcification (P = 0.012). The diepoxy-treated porcine aortic conduit group had the risk of calcification 3 times higher than the glutaraldehyde-treated bovine jugular vein group (P < 0.001).The glutaraldehyde-treated bovine pericardial valved conduit and diepoxy-treated bovine pericardial valved conduit groups had the risk of calcification comparable with the glutaraldehyde-treated bovine jugular vein group in multivariate proportional hazards model (P = 0.36 and P = 0.59, respectively). CONCLUSIONS: We have not revealed significant difference in the freedom from first reintervention among types of conduit. Calcification leading to the conduit dysfunction was present in all groups; however, diepoxy-treated porcine aortic conduits demonstrated suboptimal results in terms of calcification at follow-up.

In search of the best xenogeneic material for a paediatric conduit: an experimental study.

OBJECTIVES: The development of calcification-resistant bioprosthetic materials is a very important challenge for paediatric surgery. The subcutaneous implantation in rats is the well-known first-stage model for this kind of research. Using this model, we aimed to compare calcification of the porcine aortic wall and bovine pericardium and jugular vein wall cross-linked with glutaraldehyde (GA) and ethylene glycol diglycidyl ether (DE). We also determined the efficacy of DE-preserved tissue modification with 2-(2-carboxyethylamino)ethylidene-1,1-bisphosphonic acid (CEABA). METHODS: Three groups of each biomaterial were evaluated: GA-treated, DE-treated and DE + CEABA-treated. The microstructure of non-implanted biomaterials was assessed by light microscopy after Picro Mallory staining; the phosphorus content of the DE and DE + CEABA samples was assessed by atomic emission spectrometry. Samples were implanted subcutaneously into young rats for 10 and 60 days. The explant end-point included quantitative calcification assessment by atomic absorption spectrophotometry and light microscopy examination after von Kossa staining. RESULTS: All GA-treated biomaterials had a high calcium-binding capacity (>100 µg/mg dry tissue). DE preservation decreased the vein wall and pericardium calcium content by 4- and 40-fold, respectively, but was ineffective for the aortic wall. The calculated CEABA content was almost equal in the vein wall and pericardium (17.7 and 18.5 µM/g) and slightly less in the aortic wall (15 µM/g) (P = 0.011). CEABA effectively reduced mineralization in the DE aortic wall and DE pericardium to 10.1 (7.8-21.1) and 0.95 (0.57-1.38) µg/mg but had no effect in the DE vein wall. Mineralization in the GA- and DE-treated aortic and vein walls was predominantly associated with elastin. CEABA modification decreased elastin calcification but did not block it completely. CONCLUSIONS: Each xenogeneic material requires individual anticalcification strategy. DE + CEABA pretreatment demonstrates a high mineralization-blocking efficacy for the bovine pericardium and should be employed to further develop the paediatric pericardial conduit. Aortic wall calcification cannot be blocked completely using this strategy.

Right Ventricular Outflow Tract Replacement With Xenografts in Ross Patients Older Than 60 Years.

BACKGROUND: Because of the limited availability of pulmonary allografts, stentless pericardial xenografts have been proposed as an alternative for right ventricular outflow tract (RVOT) reconstruction during the Ross procedure. However, the durability of the bioprostheses for RVOT reconstruction in older patients is unknown. In the present study, we evaluated the use of pericardial xenografts for RVOT reconstruction during the Ross procedure in patients aged more than 60 years. METHODS: Between 1998 and 2014, 710 consecutive adult patients underwent the Ross procedure. We analyzed the results of the operation in 102 patients aged more than 60 years. The mean patient age was 63.4 ± 2.8 years (range, 60 to 68). The total root replacement technique was used in all patients. The RVOT reconstruction was performed with stentless pericardial xenografts. The mean size of the xenografts was 26.8 ± 1.3 mm. Xenograft calcification was assessed by computed tomography in 39 patients. RESULTS: At the time of discharge, the RVOT peak gradient was 10.3 ± 3.5 mm Hg. The mean follow-up duration was 52.3 ± 23.6 months. None of the cases required reoperation for xenograft dysfunction. At the final follow-up, the RVOT peak gradient was 17.9 ± 4.3 mm Hg. Computed tomography scan analyses showed that calcification was localized mainly at the graft wall, and that the valve was relatively free of calcium. CONCLUSIONS: Stentless diepoxide-treated pericardial xenografts are an acceptable alternative to pulmonary allografts for RVOT reconstruction with the Ross procedure in patients aged more than 60 years, and yield good midterm results.

Degradation effect of diepoxide fixation on porcine endogenous retrovirus DNA in heart valves: molecular aspects.

PURPOSE: Xenotransplantations of porcine cells, tissues, and organs involve a risk of zoonotic viral infections in recipients, including by porcine endogenous retroviruses (PERVs), which are embedded the genome of all pigs. An appropriate preparation of porcine heart valves for transplantation can prevent retroviral infection. Therefore, the present study focuses on the effect of epoxy compounds and glutaraldehyde on the PERV presence in porcine heart valves prepared for clinical use. METHODS: Porcine aortic heart valves were fixed with ethylene glycol diglycidyl ether (EDGE) at 5 °C and 25 °C as well as with glutaraldehyde (GA) for 4 weeks. Salting out was used to isolate genomic DNA from native as well as EDGE- and GA-fixed fragments of valves every week. Quantification of PERV-A, PERV-B, and PERV-C DNA was performed by real-time quantitative polymerase chain reaction (QPCR). RESULTS: All subtypes of PERVs were detected in native porcine aortic heart valves. The reduction of the PERV-A, PERV-B, and PERV-C DNA copy numbers was observed in the heart valves which were EDGE-fixed at both temperatures, and in GA-fixed ones in the following weeks. After 7 and 14 days of EDGE cross-linking, significant differences between the investigated temperatures were found for the number of PERV-A and PERV-B copies. PERV DNA was completely degraded within the first week of EDGE fixation at 25 °C. CONCLUSIONS: EDGE fixation induces complete PERV genetic material degradation in porcine aortic heart valves. This suggests that epoxy compounds may be alternatively used in the preparation of bioprosthetic heart valves in future.

Smoking and associated factors among adolescents in Tallinn, Helsinki and Moscow: a multilevel analysis.

AIMS: To investigate variations in smoking and associated factors on individual and aggregate level among adolescents in Tallinn (Estonia), Helsinki (Finland) and Moscow (the Russian Federation). METHODS: The data was collected in Tallinn (N=1268 in Estonian and N=901 in Russian schools), in Helsinki (N=1396) and in Moscow (N=618) from pupils aged 13-18 by self-administered questionnaire. Multilevel analysis was used to estimate the possible effects of background characteristics, of the proximity of other smokers and the school context on the tobacco use of pupils. RESULTS: The prevalence rate of smoking among girls in Russian schools in Tallinn (34.6%), among girls in Helsinki (39.5%), and in Moscow (32.1%) was higher than that among girls in Estonian schools in Tallinn (17.6%). Smoking was slightly less prevalent among boys in Helsinki (27.5%), compared to boys in Estonian (33.6%) and Russian (35.6%) schools in Tallinn, and to boys in Moscow (32.8%). Smoking increased with age. Multilevel analysis showed that smoking differed by school among pupils. Individual determinants of smoking as study site, grade, friends', siblings' and parental smoking behaved differently depending upon school. Friends and siblings' smoking showed interaction with study site to the smoking among girls. CONCLUSIONS: Strategies aimed at influencing smoking behaviour need to be directed not only towards the individual, but also towards the influences within the child's school environment.