Glutaraldehyde-fixed Bovine Pericardium Research Articles

Abstract 11860: Poly-2-Methyl-2-Oxazoline as a Novel Mitigation Strategy in Bioprosthetic Heart Valves Affected by Calcification and Structural Degeneration in Patients With Metabolic Syndrome

I: Bioprosthetic heart valves (BHV) engineered from glutaraldehyde-fixed bovine pericardium (GBP) are widely used for surgical and transcatheter interventions and affected by structural valve degeneration (SVD), often associated with calcification. Risk factors for accelerated SVD are under study. Susceptibility to oxidation, glycation and protein infiltration are key mechanisms. Here we characterize mechanisms of accelerated SVD in patients with metabolic syndrome (MetS) and propose a novel mitigation strategy with poly-2-methyl-2-oxazoline (POZ) coating. M: Retrospective chart review assessed risk factors for accelerated SVD. BHVs affected by SVD were selected (N=24) with/without MetS and next-generation proteomics with enrichment analysis (STRING-Db) performed. Unmodified (U) GBP and POZ GBP were incubated in serum from patients (N=128) with/without MetS to mimic protein absorption. U-GBP and POZ-GBP were subcutaneously implanted in Zucker Diabetic Fatty rats and controls. GBPs were analyzed with immunohistochemistry (IHC), differential scanning calorimetry (DSC), second-harmonic generation (SHG) and scanning electron microscopy (SEM). R: Kaplan Meier log-rank test (N = 251 patients) revealed patients with MetS were at increased risk of accelerated SVD (p = 0.004). Proteomics revealed (N=452) permanently incorporated proteins were differentially expressed (log2FC >0.58, qvalue<0.05) between MetS and nonMetS. Proteins involved in ECM remodeling and immune activation pathways were overrepresented (Fig 1). IHC, DSC, SHG and SEM results from revealed that POZ-GBP reduced protein infiltration, reduced calcification, and preserved structural integrity when compared to U-GBP. C: Accelerated SVD associated with MetS with calcification and serum protein accumulation is risk factor dependent and can be mitigated with POZ. This opens a possibility to chemically modify biomaterials for patients with this highly prevalent comorbidity.

Tissue response, macrophage phenotype, and intrinsic calcification induced by cardiovascular biomaterials: Can clinical regenerative potential be predicted in a rat subcutaneous implant model?

The host immune response to an implanted biomaterial, particularly the phenotype of infiltrating macrophages, is a key determinant of biocompatibility and downstream remodeling outcome. The present study used a subcutaneous rat model to compare the tissue response, including macrophage phenotype, remodeling potential, and calcification propensity of a biologic scaffold composed of glutaraldehyde-fixed bovine pericardium (GF-BP), the standard of care for heart valve replacement, with those of an electrospun polycarbonate-based supramolecular polymer scaffold (ePC-UPy), urinary bladder extracellular matrix (UBM-ECM), and a polypropylene mesh (PP). The ePC-UPy and UBM-ECM materials induced infiltration of mononuclear cells throughout the thickness of the scaffold within 2 days and neovascularization at 14 days. GF-BP and PP elicited a balance of pro-inflammatory (M1-like) and anti-inflammatory (M2-like) macrophages, while UBM-ECM and ePC-UPy supported a dominant M2-like macrophage phenotype at all timepoints. Relative to GF-BP, ePC-UPy was markedly less susceptible to calcification for the 180 day duration of the study. UBM-ECM induced an archetypical constructive remodeling response dominated by M2-like macrophages and the PP caused a typical foreign body reaction dominated by M1-like macrophages. The results of this study highlight the divergent macrophage and host remodeling response to biomaterials with distinct physical and chemical properties and suggest that the rat subcutaneous implantation model can be used to predict in vivo biocompatibility and regenerative potential for clinical application of cardiovascular biomaterials.

Investigation into early stage fatigue-damage accumulation in glutaraldehyde-fixed bovine pericardium using a novel equibiaxial bulge inflation system

A primary cause of bioprosthetic heart valve failure is premature degeneration of the pericardial leaflets, owing specifically to mechanical fatigue. There remains a paucity of experimental data and understanding of the fatigue-damage behaviour of this collagenous tissue under complex loading regimes. To meet this knowledge gap, a novel pressure inflation system was designed and built, to cyclically load circular samples of glutaraldehyde fixed bovine pericardium, under equibiaxial bulge conditions. A study up to 60 million cycles revealed new insights into the fatigue behaviour of pericardial tissue, where a statistically significantly higher level of permanent set was found in samples with high collagen fibre dispersion, in comparison to those with highly aligned fibres. Whilst permanent set is known to occur in the non-collagenous matrix of pericardium, this study demonstrates that at physiological loads, which elicit a matrix dominant mechanical response, permanent set and thus tissue-level damage, is still mediated by the underlying collagen fibres.

Collagen fibre-mediated mechanical damage increases calcification of bovine pericardium for use in bioprosthetic heart valves

In cases of aortic stenosis, bioprosthetic heart valves (BHVs), with glutaraldehyde-fixed bovine pericardium leaflets (GLBP), are often implanted to replace the native diseased valve. Widespread use of BHVs, however, is restricted due to inadequate long-term durability, owing specifically to premature leaflet failure. Mechanical fatigue damage and calcification remain the primary leaflet failure modes, where glutaraldehyde treatment is known to accelerate calcification. The literature in this area is limited, with some studies suggesting mechanical damage increases calcification and others that they are independent degenerative mechanisms. In this study, specimens which were non-destructively pre-sorted according to collagen fibre architecture and uniaxially cyclically loaded until failure or 1 million cycles, were placed in an in vitro calcification solution. The weakest specimen group (those with fibres aligned perpendicular to the load) had statistically significantly higher volumes of calcification when compared to those with a high fatigue life. Moreover, SEM imaging revealed that ruptured and damaged fibres presented calcium binding sites; resulting in 4 times more calcification in fractured samples in comparison to those which did not fail by fatigue. To the authors’ knowledge, this study quantifies for the first time, that mechanical damage drives calcification in commercial-grade GLBP and that calcification varies spatially according to localised damage levels. These findings illustrate that not only is calcification of GLBP exacerbated by fatigue damage, but that both failure phenomena are underpinned by the collagen fibre organisation. Consequently, controlling for GLBP collagen fibre architecture in leaflets could minimise the progression of these primary failure modes in patient BHVs. Statement of significanceMechanical damage and calcification are the primary premature failure modes of glutaraldehyde-fixed bovine pericardial (GLBP) leaflets in bioprosthetic heart valves. In this study, commercial-grade GLBP specimens which were uniaxially cyclically loaded to failure or 1 million cycles, were placed in an in vitro calcification solution. MicroCT and SEM analysis showed that localised calcification levels varied spatially according to damage, where ruptured fibres offered additional calcium binding sites. Furthermore, specimens with a statistically significant lower fatigue life were associated with statistically significant higher calcification. This study revealed that mechanical damage drives calcification of GLBP. Non-destructive pre-screening of collagen fibres demonstrated that both the fatigue life and calcification potential of commercial-grade GLBP, are underpinned by the collagen fibre architecture.

Bovine Pericardium of High Fibre Dispersion Has High Fatigue Life and Increased Collagen Content; Potentially an Untapped Source of Heart Valve Leaflet Tissue.

Bioprosthetic heart valves (BHVs) are implanted in aortic valve stenosis patients to replace the native, dysfunctional valve. Yet, the long-term performance of the glutaraldehyde-fixed bovine pericardium (GLBP) leaflets is known to reduce device durability. The aim of this study was to investigate a type of commercial-grade GLBP which has been over-looked in the literature to date; that of high collagen fibre dispersion (HD). Under uniaxial cyclic loading conditions, it was observed that the fatigue behaviour of HD GLBP was substantially equivalent to GLBP in which the fibres are highly aligned along the loading direction. It was also found that HD GLBP had a statistically significant 9.5% higher collagen content when compared to GLBP with highly aligned collagen fibres. The variability in diseased BHV delivery sites results in unpredictable and complex loading patterns across leaflets in vivo. This study presents the possibility of a shift from the traditional choice of circumferentially aligned GLBP leaflets, to that of high fibre dispersion arrangements. Characterised by its high fatigue life and increased collagen content, in addition to multiple fibre orientations, GLBP of high fibre dispersion may provide better patient outcomes under the multi-directional loading to which BHV leaflets are subjected in vivo.

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 invivo, 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.

Effect of cyclic deformation on xenogeneic heart valve biomaterials.

Glutaraldehyde-fixed bovine pericardium is currently the most popular biomaterial utilized in the creation of bioprosthetic heart valves. However, recent studies indicate that glutaraldehyde fixation results in calcification and structural valve deterioration, limiting the longevity of bioprosthetic heart valves. Additionally, glutaraldehyde fixation renders the tissue incompatible with constructive recipient cellular repopulation, remodeling and growth. Use of unfixed xenogeneic biomaterials devoid of antigenic burden has potential to overcome the limitations of current glutaraldehyde-fixed biomaterials. Heart valves undergo billion cycles of opening and closing throughout the patient’s lifetime. Therefore, understanding the response of unfixed tissues to cyclic loading is crucial to these in a heart valve leaflet configuration. In this manuscript we quantify the effect of cyclic deformation on cycle dependent strain, structural, compositional and mechanical properties of fixed and unfixed tissues. Glutaraldehyde-fixed bovine pericardium underwent marked cyclic dependent strain, resulting from significant changes in structure, composition and mechanical function of the material. Conversely, unfixed bovine pericardium underwent minimal strain and maintained its structure, composition and mechanical integrity. This manuscript demonstrates that unfixed bovine pericardium can withstand cyclic deformations equivalent to 6 months of in vivo heart valve leaflet performance.

Enhanced Vascular Biocompatibility and Remodeling of Decellularized and Secured Xenogeneic/Allogeneic Matrices in a Porcine Model

Background/Purpose: Calcifications and absence of growth potential are the major drawbacks of glutaraldehyde-treated prosthesis. Decellularized and secured xeno-/allogeneic matrices were assessed in a preclinical porcine model for biocompatibility and vascular remodeling in comparison to glutaraldehyde-fixed bovine pericardium (GBP; control). Methods: Native human (fascia lata, pericardium) and porcine tissues (peritoneum) were used and treated. In vitro, biopsies were performed before and after treatment to assess decellularization (hematoxylin and eosin/DAPI). In vivo, each decellularized and control tissue sample was implanted subcutaneously in 4 mini-pigs. In addition, 9 mini-pigs received a patch or a tubularized prosthesis interposition on the carotid artery or abdominal aorta of decellularized (D) human fascia lata (DHFL; n = 4), human pericardium (DHP; n = 9), porcine peritoneum (DPPt; n = 7), and control tissue (GBP: n = 3). Arteries were harvested after 1 month and subcutaneous samples after 15–30 days. Tissues were processed for hematoxylin and eosin/von Kossa staining and immunohistochemistry for CD31, alpha-smooth muscle actin, CD3, and CD68. Histomorphometry was achieved by point counting. Results: A 95% decellularization was confirmed for DHP and DPPt, and to a lower degree for DHFL. In the subcutaneous protocol, CD3 infiltration was significantly higher at day 30 in GBP and DHFL, and CD68 infiltration was significantly higher for GBP (p < 0.05). In intravascular study, no deaths, aneurysms, or pseudoaneurysms were observed. Inflammatory reaction was significantly higher for DHFL and GBP (p < 0.05), while it was lower and comparable for DHP/DPPt. DHP and DPPt showed deeper recellularization, and a new arterial wall was characterized. Conclusions: In a preclinical model, DPPt and DHP offered better results than conventional commercialized GBP for biocompatibility and vascular remodeling.

A novel tissue treatment to reduce mineralization of bovine pericardial heart valves

ObjectiveWith the increasing use of bioprostheses worldwide, continuous efforts have been made to improve tissue durability. We introduce a new treatment for bovine pericardium combining octanediol-ethanol based phospholipid removal with taurine-based glutaraldehyde neutralization and storage in an aldehyde-free solution (FREE). MethodsTreated tissues were evaluated by mechanical and biochemical characterization, phospholipid content, aldehyde levels, cell cultures on pericardial samples (L929 fibroblasts and human umbilical vein endothelial cells), rat subcutaneous implantations, and long-term juvenile sheep mitral valve implantations (n = 3). Comparisons were made to glutaraldehyde-fixed bovine pericardium or to samples from commercially available biological valves (ie, Trifecta [St Jude Medical, Saint Paul, Minn] and Perimount Magna Ease [Edwards Lifesciences, Irvine, Calif]). ResultsFREE-treated pericardium had similar mechanical strength and biochemical properties as commercially available valves. Compared with glutaraldehyde-only samples, FREE-treated samples showed lower phospholipid levels (P < .01), significantly better growth of L929 fibroblasts, and lower calcification levels in rat subcutaneous implants (P < .01). Compared with samples from Linx- (Trifecta) and ThermaFix-treated (Perimount Magna Ease) valves, similar low levels of phospholipids were observed as were similar low calcification levels in subcutaneous implants, but tissue extractions from FREE-treated samples showed the lowest levels of extracted aldehydes (P < .01). Mitral implants of FREE-treated valves in juvenile sheep had excellent hemodynamic behavior without any sign of degeneration or calcification at 5 months. ConclusionsThe new FREE treatment combines an adequate phospholipid reduction and aldehyde neutralization with storage in an aldehyde-free solution. This combination enhances the anticalcification properties and may thereby improve long-term durability of the tissue.

Abstract 14677: Oxidative Damage in Failed Clinical Bioprosthetic Heart Valve Explants

Introduction: Bioprosthetic heart valves (BHVs), fabricated from glutaraldehyde-fixed bovine pericardium or porcine aortic valves, have been widely used in valve replacement surgery. However, BHVs fail over time due to structural degeneration. Despite frequent observations of calcification-associated BHV failure, more than 25% of BHV structural failures occur without calcification. Previously, we reported that oxidative stress may have a pivotal role in noncalcific BHV failure. Moreover, oxidation-induced formation of dityrosine (DT) crosslinks via hydroxyl- and tyrosyl-radical mediated pathways was only detected in clinical BHV explants. Yet the mechanism of DT formation and its role in structural degeneration of clinical BHV is incompletely understood. Hypothesis: Oxidative damage to BHV is due to a specific spatial localization of DT crosslink formation mediated by myeloperoxidase (MPO) and/or iron (Fe) deposition in clinical BHV explants Method &amp; Results: BHVs from 7 human subjects were collected and subjected to mass spectrometry to measure DT in paraffin sections of BHV. 4 different regions of each BHV leaflet (free edge, sewing cushion, commissure, and middle region) were sampled as well as entire cross-sections of each cusp. DT was only detected in the mid–cusp (137.37±49.96 μmol/mol) and was not significantly different from DT quantitated in the cross-sections (191.29±65.98 μmol/mol). DT was not detected in other regions, implying a specific spatial localization of oxidative stress in BHV. MPO, a neutrophil derived enzyme, is responsible for reactive oxygen species (ROS) generation. Immunofluoresence microscopy demonstrated significant presence of MPO in the BHV, implying that MPO may be responsible for the DT formation in BHV. Fe deposition due to intracuspal hematomas and/or Fe-association with calcific deposits can also be responsible for ROS and DT formation. Fe was shown to be present in histology sections in failed BHV by Prussian Blue staining, indicating that Fe deposits may also in part be responsible for the observed DT formation. Conclusion: These results demonstrated a mid-leaflet localization of DT formation in clinically failed BHV explants. This may in part be mediated by MPO and/or Fe-deposits in clinical BHV.

Valved conduit with glutaraldehyde-fixed bovine pericardium treated by anticalcification protocol.

BackgroundA preclinical study was conducted for evaluating a valved conduit manufactured with a glutaraldehyde (GA)-fixed bovine pericardium treated using an anticalcification protocol.MethodsBovine pericardia were decellularized, fixed with GA in an organic solvent, and detoxified. We prepared a valved conduit using these bovine pericardia and a specially designed mold. The valved conduit was placed under in vitro circulation by using a mock circulation model, and the durability under mechanical stress was evaluated for 2 months. The valved conduit was implanted into the right ventricular outflow tract of a goat, and the hemodynamic, radiologic, histopathologic, and biochemical results were obtained for 6 months after the implantation.ResultsThe in vitro mock circulation demonstrated that valve motion was good and that the valved conduit had good gross and microscopic findings. The evaluation of echocardiography and cardiac catheterization demonstrated the good hemodynamic status and function of the pulmonary xenograft valve 6 months after the implantation. According to specimen radiography and a histopathologic examination, the durability of the xenografts was well preserved without calcification at 6 months after the implantation. The calcium and inorganic phosphorus concentrations of the explanted xenografts were low at 6 months after the implantation.ConclusionThis study demonstrated that our synergistic employment of multiple anticalcification therapies has promising safety and efficacy in the future clinical study.

Tissue engineering of regenerative cardiovascular patch material based on decellularized pericardium

Objectives: Autologous pericardium is often used to substitute small or medium sized vascular or myocardial lesions. However, its use is restricted due to its limited availability. Hence, glutaraldehyde-fixed bovine pericardium is currently being employed more extensively despite potential impact of the xenogenic tissue on the host, possible toxicity of residual fixing solution within the graft, and absence of any regenerative and growing potential. We therefore developed a new biological and viable matrix, based on decellularized and reseeded pericardium.

Combined anticalcification treatment of bovine pericardium with decellularization and hyaluronic acid derivative

The objective of this work was to evaluate the effect of decellularization and hyaluronic acid derivative on the improvement of anticalcification of glutaraldehyde fixed bovine pericardium (GFBP) using a rat subcutaneous implantation model A cell extraction process was employed to remove the cells and cellular components from bovine pericardium (BP), leaving a framework of largely insoluble collagen. Then acellular BP was cross-linked by glutaraldehyde solution and treated with hyaluronic acid derivative (HA-ADH) which was obtained by coupling adipic dihydrazide (ADH) on-COOH of hyaluronic acid (HA). The results of in vivo calcification tests showed that the calcium content was decreased dramatically by decellularization alone (from 28.07 ± 18.87 to 2.44 ± 0.55 μg Ca/mg dry tissue after 8 weeks' implantation), and even less concentration was shown by the combination of HA derivative treatment and decellularization (GFaBP-HA group) (0.25 ± 0.08 μg Ca/mg dry tissue after 8 weeks' implantation). In addition, GFaBP-HA group not only presented a lower degree of calcification, but also showed lower ratios of Ca/P molar, which corresponded to amorphous calcium phosphates. The obtained results indicated that GFaBP-HA was a potential candidate for the manufacture of anticalcification bioprostheses.

Cytotoxicity of PVPAC‐treated bovine pericardium: A potential replacement for glutaraldehyde in biological heart valves

Acellular biological tissues, including bovine pericardium (BP), have been proposed as biomaterial for tissue engineering. BP is usually modified chemically to improve mechanical and biological properties using glutaraldehyde, the standard reagent for preservation of fresh bioprosthetic materials. Glutaraldehyde-fixed BP (Glut-BP), the most widely used material in heart valve manufacture, has been associated with calcification in vivo. In an attempt to reduce this issue and maintain its biocompatibility, this study assesses the physical properties and cytotoxicity of lyophilized BP treated with poly (vinylpyrrolidone-co-acrolein) (PVPAC-BP), a novel copolymer, as a substitute for glutaraldehyde. For that, PVPAC-BP surface ultrastructure, elastic function, water uptake and tissue calcification were evaluated. For the analysis of biocompatibility, fibroblasts (3T3-L1) and endothelial cells (HUVEC) were cultured on PVPAC-BP, Untreated-BP and Glut-BP. Nitric oxide (NO) release assay, fluorescence and SEM images of endothelial cells adhered on scaffolds were also performed. As results, the data show some advantages of PVPAC-BP over the Glut-BP. The PVPAC-BP maintains partially the original ultrastructure and elastic properties, improves scaffold hydration, and presents less calcium phosphate deposits. The cells demonstrated strong attachment, high proliferation rate, and formation of a monolayer on PVPAC-BP. Attached cells were also able to release NO de-monstrating regular metabolism. In conclusion, PVPAC may be considered as a promising alternative to BP treatment improving the efficiency of cell attachment and proliferation and also avoid immunogenicity.

Nanocoating with titanium reduces iC3b- and granulocyte-activating immune response against glutaraldehyde-fixed bovine pericardium: A new technique to improve biologic heart valve prosthesis durability?

An IgG and granulocyte-activating immune response with secondary dystrophic calcification might be the reason glutaraldehyde (GA)-fixed xenograft valves fail, especially in young patients, who are more immunocompetent than the elderly. Titanium nanocoating on GA-fixed bovine pericardium was tested for its ability to prevent major immunoreactions. The immune activity of platelets from GA-fixed bovine pericardium with different treatment procedures was evaluated using the blood from 5 human donors: group I (n = 5), GA fixed as the control; group 2 (n = 5), detoxified with 10% citric acid; group 3 (n = 5), 10% citric acid, aldehyde-dehydrogenase, and a physical plasma treatment; and group 4 (n = 5), treated the same as group 3, but with an additional titanium coat 30 nm in thickness. Titanium deposition was visualized using scanning electron microscopy. IgG deposits (iC3b) were shown by immunostaining and documented as colored pixels (red). The pixels were evaluated electronically. Attracted granulocytes (polymorphonuclear leukocytes) were counted in front of the titanium-coated surface. IC3b deposits and polymorphonuclear leukocytes within control group 1 were defined as 100%; in group 2, iC3b was 149% ± 34% and polymorphonuclear leukocytes were 89%, in group 3, IC3b was 102% ± 24% and polymorphonuclear leukocytes were 47%; and in group 4, IC3b had decreased to 38.49% ± 21% (P < .05) and polymorphonuclear leukocyte activation had decreased to 6.3% (P ≤ .01). Titanium coating significantly reduced the iC3b and granulocyte activating immune response of GA-fixed pericardium. Therefore, it might prevent relevant immunorejection and increase the durability of GA-fixed bioprosthetic heart valves.

Aortic valve repair by cusp extension for rheumatic aortic insufficiency in children: Long-term results and impact of extension material

Aortic valve repair has encouraging midterm results in selected patients. However, neither the long-term results of cusp extension nor the durability of different pericardial fixation techniques has been reported. Our goal was to address these issues. Seventy-eight children with severe rheumatic aortic regurgitation (mean age 12 ± 3.5 years) underwent aortic valve repair using cusp extension over a 15-year period, with fresh autologous pericardium in 53 (67.9%), glutaraldehyde-fixed bovine pericardium in 9 (11.5%), and PhotoFix bovine pericardium (Sorin CarboMedics, Milano, Italy) in 16 (20.5%). Fifty-seven children (73.1%) underwent concomitant mitral valve repair, and 8 children (10.3%) underwent tricuspid valve repair. There was 1 operative death from left ventricular failure. During a median follow-up of 10.7 years (range 1 month to 16.4 years), 1 late death occurred and 15 patients (19.7%) required reoperation at a mean of 43 ± 33.7 months (range 1 month to 9 years), 9 within the autologous pericardium group (18%), 3 within the bovine pericardium group (33%), and 3 within the PhotoFix pericardium group (19%). Freedom from reoperation was 96% ± 2.3% at 1 year, 87.5% ± 3.9% at 5 years, 80.7% ± 4.9% at 10 years, and 75.3% ± 6% at 15 years, and was significantly decreased in the bovine pericardium group (P = .039). On multivariable analysis, greater age (hazard ratio 1.25, P < .001) and acute rheumatic carditis (hazard ratio 8.15, P = .001) at operation were significant predictors of reoperation. Aortic cusp extension provides adequate valve repair in a large proportion of children with rheumatic aortic regurgitation. Fresh autologous and PhotoFix pericardium trended toward better durability than glutaraldehyde-fixed bovine pericardium.

The anti-calcification potential of a silsesquioxane nanocomposite polymer under in vitro conditions: Potential material for synthetic leaflet heart valve☆

Calcification currently represents a major cause of failure of biological tissue heart valves. It is a complex phenomenon influenced by a number of biochemical and mechanical factors. Recent advances in material science offer new polymers with improved properties, potentially suitable for synthetic leaflets heart valves manufacturing. In this study, the calcification-resistance efficacy and mechanical and surface properties of a new nanocomposite polymeric material (polyhedral oligomeric silsesquioxane-poly(carbonate-urea)urethane; POSS-PCU) which has been developed by our group are assessed by means of in vitro testing. In particular, thin sheets of nanocomposite, glutaraldehyde-fixed bovine pericardium (BP) and polyurethane (PU) were exposed to a calcium solution into a specially designed in vitro accelerated physiological pulsatile pressure system for a period of 31days and a total of 4×10(7) cycles. The samples were investigated for signs of calcification after exposure to calcium solution by means of X-ray, microscopic and chemical inspections. Mechanical and surface properties were also studied using stress-strain behaviour and surface morphology and hydrophobicity. Comparison shows that, in the experimental conditions, the level of calcification for the nanocomposite is considerably lower than for the fixed BP (p=0.008) and PU samples (p=0.015). Also, mechanical properties were unchanged in POSS-PCU, while there was a significant deterioration in PU samples (p<0.05). Hydrophobicity was significantly reduced in both the POSS-PCU and PU samples (p<0.0001). However, the POSS-PCU nanocomposite remained more hydrophobic than the PU sample (p<0.0001). Less platelet adhered to the POSS-PCU compared to the PU (p<0.0001). These results indicate that the use of this nanocomposite in synthetic leaflets heart valves may lead to potential advantages in terms of long-term performances and durability.

Detoxification and Endothelialization of Glutaraldehyde-Fixed Bovine Pericardium With Titanium Coating

Endothelial cell seeding of glutardialdehyde-fixed biological heart valves is hypothesized to improve biocompatibility and durability; however, the toxicity of glutardialdehyde prevents its use as a biological coating. Therefore, different detoxification strategies are applied, including surface coating with titanium, before in vitro endothelialization of glutaraldehyde-fixed bovine pericardium as the base material for prosthetic heart valves. Bovine pericardium was fixed with 0.25% glutardialdehyde. Detoxification was performed with citric acid, aldehyde dehydrogenase, and plasma deposition with titanium at low temperatures of 30 degrees C to 35 degrees C. Toxic glutaraldehyde ligands were quantified photometrically, and the vitality of seeded cells was tested to validate detoxification methods. Detoxification agents and titanium coating were applied before seeding with human endothelial cells. Endothelial cells were visualized by electron microscopic surface scanning. To evaluate cell adhesion, shear stress was applied by a flow of 5 L/min over 24 hours. Compared with untreated glutaraldehyde-fixed samples, treatment with the different agents reduced free aldehyde groups gradually (citric acid 5% < citric acid 10% < titanium < aldehyde dehydrogenase). A combination of citric acid 10%, aldehyde dehydrogenase, and titanium coating resulted in a reduction of free aldehyde ligands to 17.3+/-4.6% (P < or = 0.05) and demonstrated a vitality of seeded cells of 94+/-6.7% (P < or = 0.05). This procedure yielded a completely confluent layer of regular human endothelial cells (n=5). After application of shear stress for 24 hours on these endothelial layers, cell vitality was 81%. Titanium coating combined with chemical procedures yielded significant detoxification and complete endothelialization of conventional glutaraldehyde-fixed pericardium. This new technique might improve glutardialdehyde-fixed cardiovascular bioimplants for better biocompatibility and longer durability.

Mitigated calcification of glutaraldehyde-fixed bovine pericardium by tannic acid in rats

Bioprosthetic heart valves derived from glutaraldehyde (Glut)-fixed xenografts have been widely used to replace diseased cardiac valves. However, calcification and degeneration are common following their implantation. Inflammation is closely associated with calcification of Glut-fixed xenografts via macrophage infiltration. Tannic acid (TA) possesses anti-inflammatory effects. This study was designed to investigate the anti-calcification of TA treatment on the Glut-fixed bovine pericardium (BP) in a rat subdermal model. Fresh BP was divided into two groups (10 in each group) and separately subjected to different fixation procedures as follows: (1) Glut group: fixation with 0.6% Glut alone; (2) Glut/TA group: fixation with 0.6% Glut and subsequent 0.3% TA. Then the BP samples were subdermally implanted in juvenile male Sprague-Dawley rats and explanted 21 days after implantation. Each explanted BP sample was divided into three parts for calcium content analysis, von Kossa's staining and immunohistochemical staining, and reverse transcription-polymerase chain reaction study. The data from quantitative calcium analysis and von Kossa's staining showed that Glut-fixed BP developed significantly more calcification than Glut/TA-fixed BP ((90.3 +/- 32.5) mg/g dry weight vs (6.4 +/- 1.3) mg/g dry tissue, P < 0.01). Immunostaining demonstrated lower matrix metalloproteinase-9 and tenascin-C expression as well as macrophage infiltration into Glut/TA-fixed BP than in its Glut-fixed counterpart (P < 0.01 for all). Additionally, the reverse transcription-polymerase chain reaction study showed that higher levels of expression of matrix metalloproteinase-9 and tenascin-C mRNA occurred within Glut-fixed BP than within the Glut/TA-fixed ones (P < 0.01 for all). TA exerts excellent anti-calcification effects on Glut-fixed BP via inhibiting macrophage infiltration and expression of matrix metalloproteinase-9 and tenascin-C.

Recellularization of biological heart valves with human vascular cells: In vitro hemocompatibility assessment

Coverage of cardiovascular bioprostheses with autologous endothelium is used for the purpose of improving blood compatibility. The aim of our study was to analyze endothelialization potential of glutaraldehyde-fixed heart valves, cellular functions of seeded endothelial cells (EC), and the impact of a two-stage seeding protocol using human vascular fibroblasts (FB) and EC from saphenous veins (HSVEC) on cellular functional properties in vitro. Adherence and morphology of adhered cells were assessed by scanning electronic microscopy and immunohistochemistry. Reproducible, complete surface coverage with EC was established on decellularized and glutaraldehyde-fixed bovine pericardium. Analyzing functional properties of cells directly adhered to biomaterial revealed nonproliferative cells, which were capable of inflammatory stimulation in terms of TNF-induced increase in interleukin-6 secretion and adhesion of inflammatory cells. Furthermore, EC showed sustained antithrombotic properties quantified by platelet adhesion onto EC and prostacyclin secretion by EC. Preseeding with vascular fibroblasts using a two-stage seeding protocol induced EC proliferation and improved inflammatory and anti-thrombotic functions. Cardiovascular biomaterials differ significantly in their potential to allow for adhesion of human EC. Successfully endothelialized biomaterial, however, revealed cellular properties which are likely to be favorable to improving performance of biomaterials. Two-stage seeding adds regenerative potential and improves cell functions of adherent EC.