Effect of the ex situ physical and in situ chemical modification of bacterial nanocellulose on mechanical properties in the context of its potential applications in heart valve design.

Bacterial nanocellulose (BNC) is a promising material for heart valve prostheses. However, its low strength properties limit its applicability in cardiovascular surgery. To overcome these limitations, the mechanical properties of BNC can be improved through modifications. The aim of the research was to investigate the extent to which the mechanical properties of BNC can be altered by modifying its structure during its production and after synthesis. The study presents the results of various analyses, including tensile tests, nanoindentation tests, X-ray diffraction (XRD) tests, scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR) and Raman spectroscopy, conducted on BNC chemically modified in situ with hyaluronic acid (BNC/HA) and physically modified ex situ through a dehydration/rehydration process (BNC 25DR, BNC105DR, BNC FDR and BNC/HA 25DR, BNC/HA 105DR, BNC/HA FDR). The results demonstrate that both chemical and physical modifications can effectively shape the mechanical properties of BNC. These modifications induce changes in the crystalline structure, pore size and distribution, and residual stresses of BNC. Results show the effect of the crystalline structure of BNC on its mechanical properties. There is correlation between hardness and Young's modulus and Iα/Iß index for BNC/HA and between creep rate of BNC/HA, and Young's modulus for BNC vs Iα/Iß index.

Usefulness of the N-Terminal of the Prohormone Brain Natriuretic Peptide in Predicting Acute Kidney Injury Requiring Renal Replacement Therapy in Patients Undergoing Heart Valve Surgery.

Background and Objectives: By definition, acute kidney injury (AKI) is a clinical syndrome diagnosed when the increase in serum creatinine concentration is >0.3 mg/dL in 48 h or >1.5-fold in the last seven days or when diuresis < 0.5 mL/kg/h for a consecutive 6 h. AKI is one of the severe complications that may occur in the early postoperative period in patients undergoing heart valve surgery, significantly increasing the risk of death. Early implementation of renal replacement therapy increases the chances of improving treatment results in patients with postoperative AKI. The study assessed the predictive ability of selected preoperative and perioperative parameters for the occurrence of postoperative AKI requiring renal replacement therapy in the early postoperative period in a group of patients with severe valvular heart disease. Materials and Methods: A prospective study was conducted on a group of patients undergoing consecutive heart valve surgeries. The primary endpoint was postoperative AKI requiring renal replacement therapy. AKI was diagnosed with an increase in serum creatinine > 0.3 mg/dL in 48 h or >1.5-fold in the previous 7 days and/or a decrease in diuresis < 0.5 mL/kg/h for 6 h. The observation period was until the patient was discharged home or death occurred. Logistic regression analysis was used to assess which variables were predictive of primary endpoint, and odds ratios (OR) were calculated with a 95% confidence interval (CI). Multivariate analysis was based on the result of single factor logistic regression, i.e., to further steps, all statistically significant variables were taken into consideration. Results: A total of 607 patients were included in the study. The primary endpoint occurred in 50 patients. At multivariate analysis: NT-proBNP (OR 1.406; 95% CI 1.015-1.949; p = 0.04), CRP (OR 1.523; 95% CI 1.171-1.980; p = 0.001), EuroSCORE II (OR 1.090; 95% CI 1.014-1.172; p = 0.01), age (OR 1.037; 95% CI 1.001-1.075; p = 0.04) and if they stayed in the intensive care unit longer than 2 days (OR 9.077; 95% CI 2.026-40.663; p = 0.004) remained the independent predictors of the primary endpoint. The mean preoperative NT-proBNP level was 2063 pg/mL (±1751). Thirty-eight patients with AKI requiring renal replacement therapy died in intrahospital follow-up. Conclusions: The results of the presented study indicate that a high preoperative level of NT-proBNP and postoperative hemodynamic instability may be associated with a significant risk of a postoperative AKI requiring renal replacement therapy. The results of the study may also suggest that qualifying for heart valve surgery earlier may be associated with improved prognosis in this group of patients.

Vitrification of Heart Valve Tissues.

Application of the original vitrification protocol used for pieces of heart valves to intact heart valves has evolved over time. Ice-free cryopreservation by Protocol 1 using VS55 is limited to small samples (1-3 mL total volume) where relatively rapid cooling and warming rates are possible. VS55 cryopreservation typically provides extracellular matrix preservation with approximately 80% cell viability and tissue function compared with fresh untreated tissues. In contrast, ice-free cryopreservation using VS83, Protocols 2 and 3, permits preservation of large samples (80-100 mL total volume) with several advantages over conventional cryopreservation methods and VS55 preservation, including long-term preservation capability at -80 °C; better matrix preservation than freezing with retention of material properties; very low cell viability, reducing the risks of an immune reaction in vivo; reduced risks of microbial contamination associated with use of liquid nitrogen; improved in vivo functions; no significant recipient allogeneic immune response; simplified manufacturing process; increased operator safety because liquid nitrogen is not used; and reduced manufacturing costs. More recently, we have developed Protocol 4 in which VS55 is supplemented with sugars resulting in reduced concerns regarding nucleation during cooling and warming. This method can be used for large samples resulting in retention of cell viability and permits short-term exposure to -80 °C with long-term storage preferred at or below -135 °C.

Effects of perioperative oral care on postoperative inflammation following heart valve surgery.

OBJECTIVE: Whether oral health care during the perioperative period can lead to a better outcome after heart valve surgery has not been adequately elucidated. We examined the effects of perioperative oral care on postoperative inflammation response in patients who underwent heart valve surgery. MATERIALS AND METHODS: In this retrospective cohort study, 223 patients scheduled for single valve heart surgery were divided into the oral care, who underwent professional teeth cleaning or scaling within 3 days prior to surgery, and also following surgery at least twice a week (n = 111), and non-oral care (n = 112) groups. After propensity score matching, records of both groups (80:80) were examined after surgery to evaluate inflammation markers (white blood cell count [WBC], neutrophil/white blood cell ratio [NWR], C-reactive protein [CRP] level, body temperature [BT]). RESULTS: WBC, NWR, CRP level, and BT were increased in both groups the day following surgery. Thereafter, CRP level, WBC, NWR, and BT on various days after surgery in the oral care group showed greater decreases as compared to the non-oral care group. CONCLUSIONS: Perioperative oral health care can decrease postoperative inflammation in patients undergoing heart valve surgery and may be important to ensure a better outcome in those patients.

Human Heart Explant-Derived Extracellular Vesicles: Characterization and Effects on the In Vitro Recellularization of Decellularized Heart Valves.

Extracellular vesicles (EVs) are particles released from different cell types and represent key components of paracrine secretion. Accumulating evidence supports the beneficial effects of EVs for tissue regeneration. In this study, discarded human heart tissues were used to isolate human heart-derived extracellular vesicles (hH-EVs). We used nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) to physically characterize hH-EVs and mass spectrometry (MS) to profile the protein content in these particles. The MS analysis identified a total of 1248 proteins. Gene ontology (GO) enrichment analysis in hH-EVs revealed the proteins involved in processes, such as the regulation of cell death and response to wounding. The potential of hH-EVs to induce proliferation, adhesion, angiogenesis and wound healing was investigated in vitro. Our findings demonstrate that hH-EVs have the potential to induce proliferation and angiogenesis in endothelial cells, improve wound healing and reduce mesenchymal stem-cell adhesion. Last, we showed that hH-EVs were able to significantly promote mesenchymal stem-cell recellularization of decellularized porcine heart valve leaflets. Altogether our data confirmed that hH-EVs modulate cellular processes, shedding light on the potential of these particles for tissue regeneration and for scaffold recellularization.

Spectral fingerprinting of decellularized heart valve scaffolds.

Decellularized heart valves hold promise for their use as bioscaffolds in cardiovascular surgery. Quality assessment of heart valves after decellularization processing and/or storage is time consuming and destructive. Fourier transform infrared spectroscopy (FTIR) allows rapid non-invasive assessment of biomolecular structures in tissues. In this study, IR-spectra taken from different layers of the pulmonary artery trunk and leaflet tissues of decellularized porcine heart valves were compared with those of pure collagen and elastin, the main protein components in these tissues. In addition, spectral changes associated with aging and oxidative damage were investigated. Infrared absorbance spectra of the arteria intima and media layer were found to be very similar, whereas distinct differences were observed when compared with spectra of the externa layer. In the latter, the shape of the CH-stretching vibration region (3050-2800 cm-1) resembled that of pure collagen. Also, pronounced νCOOH and amide-II bands and a relatively high content of α-helical structures in the externa layer indicated the presence of collagen in this layer. The externa layer of the artery appeared to be sensitive to collagenase treatment, whereas the media and intima layer were particularly affected by elastase and not by collagenase treatment. Protein conformational changes after treatment with collagenase were observed in all three layers. Collagenase treatment completely degraded the leaflet tissue sections. Spectra were also collected from scaffolds after 2 and 12 weeks storage at 37 °C, and after induced oxidative damage. Spectral changes related to aging and oxidative damage were particularly evident in the CH-stretching region, whereas the shape of the amide-I band, reflecting the overall protein secondary structure, remained unaltered.

Use of sucrose to diminish pore formation in freeze-dried heart valves.

Freeze-dried storage of decellularized heart valves provides easy storage and transport for clinical use. Freeze-drying without protectants, however, results in a disrupted histoarchitecture after rehydration. In this study, heart valves were incubated in solutions of various sucrose concentrations and subsequently freeze-dried. Porosity of rehydrated valves was determined from histological images. In the absence of sucrose, freeze-dried valves were shown to have pores after rehydration in the cusp, artery and muscle sections. Use of sucrose reduced pore formation in a dose-dependent manner, and pretreatment of the valves in a 40% (w/v) sucrose solution prior to freeze-drying was found to be sufficient to completely diminish pore formation. The presence of pores in freeze-dried valves was found to coincide with altered biomechanical characteristics, whereas biomechanical parameters of valves freeze-dried with enough sucrose were not significantly different from those of valves not exposed to freeze-drying. Multiphoton imaging, Fourier transform infrared spectroscopy, and differential scanning calorimetry studies revealed that matrix proteins (i.e. collagen and elastin) were not affected by freeze-drying.

Improving the biological function of decellularized heart valves through integration of protein tethering and three-dimensional cell seeding in a bioreactor.

Decellularized xenogeneic heart valves (DHVs) are promising products for valve replacement. However, the widespread clinical application of such products is limited due to the risk of immune reaction, progressive degeneration, inflammation, and calcification. Here, we have developed an optimized decellularization protocol for a xenogeneic heart valve. We improved the biological function of DHVs by protein tethering onto DHV and three-dimensional (3D) cell seeding in a bioreactor. Our results showed that heart valves treated with a Triton X-100 and sodium deoxycholate-based protocol were completely cell-free, with preserved biochemical and biomechanical properties. The immobilization of stromal derived factor-1α (SDF-1α) and basic fibroblast growth factor on DHV significantly improved recellularization with endothelial progenitor cells under the 3D culture condition in the bioreactor compared to static culture conditions. Cell phenotype analysis showed higher fibroblast-like cells and less myofibroblast-like cells in both protein-tethered DHVs. However, SDF-DHV significantly enhanced recellularization both in vitro and in vivo compared to basic fibroblast growth factor DHV and demonstrated less inflammatory cell infiltration. SDF-DHV had less calcification and platelet adhesion. Altogether, integration of SDF-1α immobilization and 3D cell seeding in a bioreactor might provide a novel, promising approach for production of functional heart valves.

Complete Static Repopulation of Decellularized Porcine Tissues for Heart Valve Engineering: An in vitro Study.

To date, a completely in vitro repopulated tissue-engineered heart valve has not been developed. This study focused on sequentially seeding 2 cell populations onto porcine decellularized heart valve leaflets (HVL) and pericardia (PER) to obtain fully repopulated tissues. For repopulation of the interstitium, porcine valvular interstitial cells (VIC) and bone marrow-derived mesenchymal stem cells (BM-MSC) or adipose tissue-derived stem cells (ADSC) were used. In parallel, the culture medium was supplemented with ascorbic acid 2-phosphate (AA) and its effect on recolonization was investigated. Subsequently and in order to obtain an endothelial surface layer similar to those in native HVL, valvular endothelial cells (VEC) were seeded onto the scaffolds. It was shown that VIC efficiently recolonized HVL and partially also PER. On the other hand, stem cells only demonstrated limited or no subsurface cell infiltration of HVL and PER. Interestingly, the addition of AA increased the migratory capacity of both stem cell populations. However, this was more pronounced for BM-MSC, and recolonization of HVL appeared to be more efficient than that of PER tissue. VEC were demonstrated to generate a new endothelial layer on HVL and PER. However, scanning microscopy revealed that these endothelial cells were not allowed to fully spread onto PER. This study provided a proof of concept for the future generation of a bioactive tissue-engineered heart valve by showing that bioactive HVL could be generated in vitro within 14 days via complete repopulation of the interstitium with BM-MSC or VIC and subsequent generation of an entirely new endothelium.

[Investigation of the structure of a functionally intact xenopericardial bioconduit after long-term implantation]. / Issledovanie struktury funktsional'no sokhrannogo ksenoperikardial'nogo bioproteza posle prodolzhitel'nogo perioda implantatsii.

AIM: to investigate the cellular composition of a functionally intact xenopericardial valve in a recipient with acquired mitral defect after long-term implantation. MATERIAL AND METHODS: A Uniline bioconduit (BC) ('Neocor', Kemerovo) removed from the heart in the mitral position at 7.2 years after implantation was investigated. Heart valve leaflets were fixed in a buffered 4% paraformaldehyde solution and imbedded in paraffin or epoxy resin. Slices made from the paraffin samples were stained with hematoxylin and eosin or underwent immunohistochemical (IHC) examination for typing endothelial cells, smooth muscle cells, macrophages, fibroblasts, and T and B lymphocytes. The epoxy resin-embedded samples were examined using light and scanning electron microscopy according to the original procedure. For this, the samples were ground and polished, then stained with toluidine blue and basic fuchsin or contrasted with uranyl acetate and lead citrate. RESULTS: Different cell types were found in the outer layers of heart valve leaflets. IHC showed that endothelial cells, macrophages, smooth muscle cells, and fibroblasts were present in the samples. A relationship was found between the degree of degenerative changes in the BC surface and the magnitude of cellular infiltration in xenotissue. This paper debates whether impaired integrity of the surface leaflet layers plays a trigger role in structural dysfunctions of the implanted valves and whether BC endothelialization has a protective effect, which can considerably reduce the immunogenicity of xenotussie and prevent the penetration of recipient cells. CONCLUSION: The paper shows that it is expedient to modify the surface of the heart valve leaflets in order to create favorable conditions for the attachment and function of endothelial progenitor cells.

Valve interstitial cell culture: Production of mature type I collagen and precise detection.

Collagen often acts as an extracellular and intracellular marker for in vitro experiments, and its quality defines tissue constructs. To validate collagen detection techniques, cardiac valve interstitial cells were isolated from pigs and cultured under two different conditions; with and without ascorbic acid. The culture with ascorbic acid reached higher cell growth and collagen deposition, although the expression levels of collagen gene stayed similar to the culture without ascorbic acid. The fluorescent microscopy was positive for collagen fibers in both the cultures. Visualization of only extracellular collagen returned a higher correlation coefficient when comparing the immunolabeling and second harmonic generation microscopy images in the culture with ascorbic acid. Lastly, it was proved that the hydroxyproline strongly contributes to the second-order susceptibility tensor of collagen molecules, and therefore the second harmonic generation signal is impaired in the culture without ascorbic acid.

Relative Effects of Fluid Oscillations and Nutrient Transport in the In Vitro Growth of Valvular Tissues.

Engineered valvular tissues are cultured dynamically, and involve specimen movement. We previously demonstrated that oscillatory shear stresses (OSS) under combined steady flow and specimen cyclic flexure (flex-flow) promote tissue formation. However, localized efficiency of specimen mass transport is also important in the context of cell viability within the growing tissues. Here, we investigated the delivery of two essential species for cell survival, glucose and oxygen, to 3-dimensional (3D) engineered valvular tissues. We applied a convective-diffusive model to characterize glucose and oxygen mass transport with and without valve-like specimen flexural movement. We found the mass transport effects for glucose and oxygen to be negligible for scaffold porosities typically present during in vitro experiments and non-essential unless the porosity was unusually low (<40%). For more typical scaffold porosities (75%) however, we found negligible variation in the specimen mass fraction of glucose and oxygen in both non-moving and moving constructs (p > 0.05). Based on this result, we conducted an experiment using bone marrow stem cell (BMSC)-seeded scaffolds under Pulsatile flow-alone states to permit OSS without any specimen movement. BMSC-seeded specimen collagen from the pulsatile flow and flex-flow environments were subsequently found to be comparable (p > 0.05) and exhibited some gene expression similarities. We conclude that a critical magnitude of fluid-induced, OSS created by either pulsatile flow or flex-flow conditions, particularly when the oscillations are physiologically-relevant, is the direct, principal stimulus that promotes engineered valvular tissues and its phenotype, whereas mass transport benefits derived from specimen movement are minimal.

Evaluation of bioprosthetic heart valve failure using a matrix-fibril shear stress transfer approach.

A matrix-fibril shear stress transfer approach is devised and developed in this paper to analyse the primary biomechanical factors which initiate the structural degeneration of the bioprosthetic heart valves (BHVs). Using this approach, the critical length of the collagen fibrils l c and the interface shear acting on the fibrils in both BHV and natural aortic valve (AV) tissues under physiological loading conditions are calculated and presented. It is shown that the required critical fibril length to provide effective reinforcement to the natural AV and the BHV tissue is l c  = 25.36 µm and l c  = 66.81 µm, respectively. Furthermore, the magnitude of the required shear force acting on fibril interface to break a cross-linked fibril in the BHV tissue is shown to be 38 µN, while the required interfacial force to break the bonds between the fibril and the surrounding extracellular matrix is 31 µN. Direct correlations are underpinned between these values and the ultimate failure strength and the failure mode of the BHV tissue compared with the natural AV, and are verified against the existing experimental data. The analyses presented in this paper explain the role of fibril interface shear and critical length in regulating the biomechanics of the structural failure of the BHVs, for the first time. This insight facilitates further understanding into the underlying causes of the structural degeneration of the BHVs in vivo.

Mesothelial/monocytic incidental cardiac excrescence mimicking cardiac tumor.

Mesothelial incidental cardiac excrescence is a non-neoplastic tumor-like lesion commonly occurring in the intracardiac region. The exact etiology is unclear. A 32-year-old woman presented with respiratory distress on exertion. Echocardiography showed severe aortic, mitral, and tricuspid regurgitation, for which triple-valve replacement was performed. A small cardiac excrescence was found over the aortic valve, measuring 0.6 × 0.3 × 0.3-cm, which on microscopy showed features of mesothelial/monocytic incidental cardiac excrescence. This condition is very rare but it must be recognized because it mimics a metastatic malignancy.

Functional Heart Valve Scaffolds Obtained by Complete Decellularization of Porcine Aortic Roots in a Novel Differential Pressure Gradient Perfusion System.

There is a great need for living valve replacements for patients of all ages. Such constructs could be built by tissue engineering, with perspective of the unique structure and biology of the aortic root. The aortic valve root is composed of several different tissues, and careful structural and functional consideration has to be given to each segment and component. Previous work has shown that immersion techniques are inadequate for whole-root decellularization, with the aortic wall segment being particularly resistant to decellularization. The aim of this study was to develop a differential pressure gradient perfusion system capable of being rigorous enough to decellularize the aortic root wall while gentle enough to preserve the integrity of the cusps. Fresh porcine aortic roots have been subjected to various regimens of perfusion decellularization using detergents and enzymes and results compared to immersion decellularized roots. Success criteria for evaluation of each root segment (cusp, muscle, sinus, wall) for decellularization completeness, tissue integrity, and valve functionality were defined using complementary methods of cell analysis (histology with nuclear and matrix stains and DNA analysis), biomechanics (biaxial and bending tests), and physiologic heart valve bioreactor testing (with advanced image analysis of open-close cycles and geometric orifice area measurement). Fully acellular porcine roots treated with the optimized method exhibited preserved macroscopic structures and microscopic matrix components, which translated into conserved anisotropic mechanical properties, including bending and excellent valve functionality when tested in aortic flow and pressure conditions. This study highlighted the importance of (1) adapting decellularization methods to specific target tissues, (2) combining several methods of cell analysis compared to relying solely on histology, (3) developing relevant valve-specific mechanical tests, and (4) in vitro testing of valve functionality.

Biaxial stress relaxation of semilunar heart valve leaflets during simulated collagen catabolism: Effects of collagenase concentration and equibiaxial strain state.

Heart valve leaflet collagen turnover and remodeling are innate to physiological homeostasis; valvular interstitial cells routinely catabolize damaged collagen and affect repair. Moreover, evidence indicates that leaflets can adapt to altered physiological (e.g. pregnancy) and pathological (e.g. hypertension) mechanical load states, tuning collagen structure and composition to changes in pressure and flow. However, while valvular interstitial cell-secreted matrix metalloproteinases are considered the primary effectors of collagen catabolism, the mechanisms by which damaged collagen fibers are selectively degraded remain unclear. Growing evidence suggests that the collagen fiber strain state plays a key role, with the strain-dependent configuration of the collagen molecules either masking or presenting proteolytic sites, thereby protecting or accelerating collagen proteolysis. In this study, the effects of equibiaxial strain state on collagen catabolism were investigated in porcine aortic valve and pulmonary valve tissues. Bacterial collagenase (0.2 and 0.5 mg/mL) was utilized to simulate endogenous matrix metalloproteinases, and biaxial stress relaxation and biochemical collagen concentration served as functional and compositional measures of collagen catabolism, respectively. At a collagenase concentration of 0.5 mg/mL, increasing the equibiaxial strain imposed during stress relaxation (0%, 37.5%, and 50%) yielded significantly lower median collagen concentrations in the aortic valve (p = 0.0231) and pulmonary valve (p = 0.0183), suggesting that relatively large strain magnitudes may enhance collagen catabolism. Collagen concentration decreases were paralleled by trends of accelerated normalized stress relaxation rate with equibiaxial strain in aortic valve tissues. Collectively, these in vitro results indicate that biaxial strain state is capable of affecting the susceptibility of valvular collagens to catabolism, providing a basis for further investigation of how such phenomena may manifest at different strain magnitudes or in vivo.

Bioprinting a cardiac valve.

Heart valve tissue engineering could be a possible solution for the limitations of mechanical and biological prostheses, which are commonly used for heart valve replacement. In tissue engineering, cells are seeded into a 3-dimensional platform, termed the scaffold, to make the engineered tissue construct. However, mimicking the mechanical and spatial heterogeneity of a heart valve structure in a fabricated scaffold with uniform cell distribution is daunting when approached conventionally. Bioprinting is an emerging technique that can produce biological products containing matrix and cells, together or separately with morphological, structural and mechanical diversity. This advance increases the possibility of fabricating the structure of a heart valve in vitro and using it as a functional tissue construct for implantation. This review describes the use of bioprinting technology in heart valve tissue engineering.

Mesothelial/monocytic incidental cardiac excrescences of the heart - a report of three cases.

Mesothelial/monocytic incidental cardiac excrescences (MICE) are uncommon pseudotumours and may histologically mimic metastatic adenocarcinoma. They consist of nonneoplastic proliferations of mesothelial cells intermingled with foamy macrophages enmeshed in fibrin. There are only around 40 cases reported in literature, and it is important that the pathologists should be aware of this lesion especially while dealing with cardiac surgery specimens. We report a series of three cases of MICE that were incidentally discovered during valve replacement surgeries.

Freeze-drying of decellularized heart valve tissues.

Decellularized xeno-antigen-depleted porcine pulmonary heart valves tissues may be used as matrix implants for patients with malfunctioning heart valves. Decellularized tissues are biological scaffolds composed of extracellular matrix components. Biological scaffolds closely resemble properties of native tissue, but lack immunogenic factors of cellular components. Decellularized heart valve scaffolds need to be stored to be readily available whenever needed. Scaffolds can be stored at reduced supra-zero temperatures, cryopreserved or freeze-dried. The advantage of freeze-drying is that it allows long-term storage at room temperature. This chapter outlines the entire process from decellularization to freeze-drying to obtain dry decellularized porcine heart valve scaffolds.