Geometry influences inflammatory host cell response and remodeling in tissue-engineered heart valves in-vivo.

Regenerative tissue-engineered matrix-based heart valves (TEM-based TEHVs) may become an alternative to currently-used bioprostheses for transcatheter valve replacement. We recently identified TEM-based TEHVs-geometry as one key-factor guiding their remodeling towards successful long-term performance or failure. While our first-generation TEHVs, with a simple, non-physiological valve-geometry, failed over time due to leaflet-wall fusion phenomena, our second-generation TEHVs, with a computational modeling-inspired design, showed native-like remodeling resulting in long-term performance. However, a thorough understanding on how TEHV-geometry impacts the underlying host cell response, which in return determines tissue remodeling, is not yet fully understood. To assess that, we here present a comparative samples evaluation derived from our first- and second-generation TEHVs. We performed an in-depth qualitative and quantitative (immuno-)histological analysis focusing on key-players of the inflammatory and remodeling cascades (M1/M2 macrophages, α-SMA+- and endothelial cells). First-generation TEHVs were prone to chronic inflammation, showing a high presence of macrophages and α-SMA+-cells, hinge-area thickening, and delayed endothelialization. Second-generation TEHVs presented with negligible amounts of macrophages and α-SMA+-cells, absence of hinge-area thickening, and early endothelialization. Our results suggest that TEHV-geometry can significantly influence the host cell response by determining the infiltration and presence of macrophages and α-SMA+-cells, which play a crucial role in orchestrating TEHV remodeling.

Hydrogel hybrid porcine pericardium for the fabrication of a pre-mounted TAVI valve with improved biocompatibility.

Transcatheter aortic valve implantation (TAVI) has been developed years ago for patients who cannot undergo a surgical aortic valve replacement (SAVR). Although TAVI possesses the advantages of lower trauma and simpler manipulation compared to SAVR, the need for storage in glutaraldehyde (GLU) and a tedious intraoperative assembly process have caused great inconvenience for its further application. A pre-mounted TAVI valve assembled by mounting a dry valve frame to a delivery system is expected to address these problems. However, the currently used GLU treated leaflet cannot unfold normally after being crimped for a long-term and loses its function when the BHV is assembled to the catheter. Besides, its cytotoxicity and immune response after implantation are still problems to be solved. In the present study, a hydrogel hybrid porcine pericardium (HHPP) approach was developed to endow the BHVs with a favorable unfolding property and good biocompatibility. Three monomers with different charge characteristics (sodium acrylate, 2-methacryloyloxyethyl phosphorylcholine, and acryloyloxyethyltrimethyl ammonium chloride) were complexed with GLU treated PP (GLU-PP) to form three kinds of HHPPs (SAAH-PP, MPCH-PP, and DACH-PP). The results of the crimping simulation experiment showed that all HHPPs could quickly recover in PBS after being folded for 10 days, while the traditional BHVs (GLU-PP) could not recover under the same conditions. Bovine serum albumin adsorption and platelet adhesion test showed that SAAH-PP and MPCH-PP had good anti-adhesion abilities. A cell culture study indicated that all the three HHPPs promoted HUVEC growth and proliferation. In vivo biocompatibility studies showed that the immune response induced by MPCH-PP was reduced compared to that by GLU-PP. These studies demonstrated that the strategy of MPC hydrogel hybridization may be an effective approach to prepare a pre-mounted TAVI valve with improved biocompatibility.

Effects on human heart valve immunogenicity in vitro by high concentration cryoprotectant treatment.

It has been shown previously that cryopreservation, using an ice-free cryopreservation method with the cryoprotectant formulation VS83, beneficially modulated immune reactions in vivo and in vitro when compared with conventionally frozen tissues. In this study, we assessed the impact of a VS83 post-treatment of previously conventionally frozen human tissue on responses of human immune cells in vitro. Tissue punches of treated and non-treated (control) aortic heart valve tissue (leaflets and associated aortic root) were co-cultured for 7 days with peripheral blood mononuclear cells or enriched CD14+ monocytes. Effects on cellular activation markers, cytokine secretion and immune cell proliferation were analysed by flow cytometry. Flow cytometry studies showed that VS83 treatment of aortic root tissue promoted activation and differentiation of CD14+ monocytes, inducing both up-regulation of CD16 and down-regulation of CD14. Significantly enhanced expression levels for the C-C chemokine receptor (CCR)7 and the human leukocyte antigen (HLA)-DR on monocytes co-cultured with VS83-treated aortic root tissue were measured, while the interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1 release was suppressed. However, the levels of interferon (IFN)γ and tumour necrosis factor (TNF)α remained undetectable, indicating that complete activation into pro-inflammatory macrophages did not occur. Similar, but non-significant, changes occurred with VS83-treated leaflets. Additionally, in co-cultures with T cells, proliferation and cytokine secretion responses were minimal. In conclusion, post-treatment of conventionally cryopreserved human heart valve tissue with the VS83 formulation induces changes in the activation and differentiation characteristics of human monocytes, and thereby may influence long-term performance following implantation. Copyright © 2017 John Wiley & Sons, Ltd.

The Immune Responses and Calcification of Bioprostheses in the α1,3-Galactosyltransferase Knockout Mouse.

BACKGROUND: The study aim was to evaluate the immune reaction, difference of degenerative calcification, and anti-calcification effect of decellularization with or without α-galactosidase in bovine pericardium and porcine heart valves, using an α1,3-galactosyltransferase (α-Gal) knockout (KO) mouse model. METHODS: In order to elucidate the anti-calcification effect of decellularization with or without α-galactosidase, bovine pericardium and porcine heart valve tissues were assigned to four groups according to the tissue preparation method: (i) glutaraldehyde (GA) fixation only; (ii) decellularization + GA fixation (Decell); (iii) α-galactosidase + GA fixation (α-galactosidase); and (iv) decellularization +α-galactosidase + GA fixation (Decell + α-galactosidase). Each prepared tissue was implanted subcutaneously into α-Gal KO mice. Anti-α-Gal immunoglobulin (Ig) G and IgM antibody titers were monitored prior to implantation and at four, eight and 12 weeks after implantation using an enzyme-linked immunosorbent assay. Calcium contents of explanted tissues were measured at 12 weeks after implantation. RESULTS: There were no significant differences in the anti-α-Gal IgG antibody titers according to the type of bioprosthetic material or tissue preparation method (p >0.05). The calcium content was significantly lower in porcine heart valves than in bovine pericardium when implanted in α-Gal-KO mice (p <0.001). Calcium contents in bovine pericardium and porcine heart valves were significantly lower in the Decell, α-galactosidase and Decell + α-galactosidase groups than in the GA group (all p <0.05). CONCLUSIONS: The porcine heart valve induced lower levels of calcium deposition than did the bovine pericardium, but the anti-α-Gal IgG antibody titers did not differ significantly between the bioprosthetic tissues. Decellularization had significant anticalcification effects in both the bovine pericardium and porcine heart valves, though there was no significant difference in the anti-α-Gal IgG antibody titers among tissue preparation methods.

Predicted Coverage and Immuno-Safety of a Recombinant C-Repeat Region Based Streptococcus pyogenes Vaccine Candidate.

The C-terminal region of the M-protein of Streptococcus pyogenes is a major target for vaccine development. The major feature is the C-repeat region, consisting of 35-42 amino acid repeat units that display high but not perfect identity. SV1 is a S. pyogenes vaccine candidate that incorporates five 14mer amino acid sequences (called J14i variants) from differing C-repeat units in a single recombinant construct. Here we show that the J14i variants chosen for inclusion in SV1 are the most common variants in a dataset of 176 unique M-proteins. Murine antibodies raised against SV1 were shown to bind to each of the J14i variants present in SV1, as well as variants not present in the vaccine. Antibodies raised to the individual J14i variants were also shown to bind to multiple but different combinations of J14i variants, supporting the underlying rationale for the design of SV1. A Lewis Rat Model of valvulitis was then used to assess the capacity of SV1 to induce deleterious immune response associated with rheumatic heart disease. In this model, both SV1 and the M5 positive control protein were immunogenic. Neither of these antibodies were cross-reactive with cardiac myosin or collagen. Splenic T cells from SV1/CFA and SV1/alum immunized rats did not proliferate in response to cardiac myosin or collagen. Subsequent histological examination of heart tissue showed that 4 of 5 mice from the M5/CFA group had valvulitis and inflammatory cell infiltration into valvular tissue, whereas mice immunised with SV1/CFA, SV1/alum showed no sign of valvulitis. These results suggest that SV1 is a safe vaccine candidate that will elicit antibodies that recognise the vast majority of circulating GAS M-types.

Xeno-immunogenicity of ice-free cryopreserved porcine leaflets.

BACKGROUND: Undesirable processes of inflammation, calcification, or immune-mediated reactions are limiting factors in long-term survival of heart valves in patients. In this study, we target the modulatory effects of ice-free cryopreservation (IFC) of xenogeneic heart valve leaflet matrices, without decellularization, on the adaptive human immune responses in vitro. METHODS: We tested porcine leaflet matrices from fresh untreated, conventionally cryopreserved (CFC), and IFC pulmonary valves by culturing them with human blood mononuclear cells for 5 d in vitro. No other tissue treatment protocols to modify possible immune responses were used. Matrices alone or in addition with a low-dose second stimulus were analyzed for induction of proliferation and cytokine release by flow cytometry-based techniques. Evaluation of the α-Gal epitope expression was performed by immunohistochemistry with fluorochrome-labeled B4 isolectin. RESULTS: None of the tested leaflet treatment groups directly triggered the proliferation of immune cells. But when tested in combination with a second trigger by anti-CD3, IFC valves showed significantly reduced proliferation of T cells, especially effector memory T cells, in comparison with fresh or CFC tissue. Moreover, the cytokine levels for interferon-γ (IFNγ), tumor necrosis factor α, and interleukin-10 were reduced for the IFC-treated group being significantly different compared with the CFC group. However, no difference between treatment groups in the expression of the α-Gal antigen was observed. CONCLUSIONS: IFC of xenogeneic tissue might be an appropriate treatment method or processing step to prevent responses of the adaptive immune system.

Inflammatory response assessment of a hybrid tissue-engineered heart valve leaflet.

Despite substantial research in the past few decades, only slight progress has been made toward developing biocompatible, tissue-engineered scaffolds for heart valve leaflets that can withstand the dynamic pressure inside the heart. Recent progress on the development of hybrid scaffolds, which are composed of a thin metal mesh enclosed by multi-layered tissue, appear to be promising for heart valve engineering. This approach retains all the advantages of biological scaffolds while developing a strong extracellular matrix backbone to withstand dynamic loading. This study aims to test the inflammatory response of hybrid tissue-engineered leaflets based on characterizing the activation of macrophage cells cultured on the surfaces of the tissue construct. The results indicate that integration of biological layers around a metal mesh core-regardless of its type-may reduce the evoked inflammatory responses by THP-1 monocyte-like cells. This observation implies that masking a metal implant within a tissue construct prior to implantation can hide it from the immune system and may improve the implant's biocompatibility.

Análisis numérico de una prótesis endobronquial utilizada para el tratamiento de cáncer pulmonar / Numerical analysis of intrabronchial prosthesis used for the treatment of lung cancer

En México, la mortalidad debido a enfermedades bronco-respiratorias se ubica en el sexto lugar según datos estadísticos dados por el Instituto Nacional de Enfermedades Respiratorias (INER). Esto genera la necesidad de incrementar la eficiencia en la aplicación de los tratamientos usados para este tipo de patología. Algunos de los métodos utilizados con mayor frecuencia para el tratamiento de estas dolencias hacen uso de micro dispositivos, también conocidos como válvulas endobronquiales. Este es un sistema alternativo que evita cirugías invasivas y logra prolongar e incrementar la calidad de vida de los pacientes. En este trabajo se presenta el análisis del desempeño de la válvula IBV®. Para el desarrollo del estudio numérico se determinaron las dimensiones y propiedades mecánicas del modelo a partir de catálogos del fabricante. Se desarrolló un modelo para el cual se consideraron las propiedades del Nitinol® y Silastic®. Asimismo, se propusieron dos condiciones de operación para la válvula, una anclada en el bronquio y la otra en la condición en la que se encuentra plegada dentro del broncoscopio. Se utilizó el Método del elemento finito (MEF) para simular las condiciones de trabajo de la válvula. Los resultados encontrados muestran el funcionamiento estructural y el nivel de los esfuerzos generados en el implante durante el ciclo de respiración forzada del individuo. Además, se proporcionan las bases para generar un nuevo dispositivo que pueda emular el funcionamiento de este tipo de implantes y aumente la eficiencia del tratamiento de dicha patología.

In Mexico, the mortality rate due to bronchial respiratory sickness is placed in the sixth position, according to statistics from the National Institute of Breathing Sickness (INER), so it is convenient to increment the efficiency of treatments for those pathologies. The intrabronchial valve is a recommended alternative method; being it main objective to avoid invasive surgery and increase the time and quality of patient´s life. Within this work a biomechanical analysis of an IBV® valve is carried out. Regarding the numerical analysis, the dimensions and mechanical properties of the valve were proposed based on catalogues published by the manufacturer as more reliable information was not available in the open literature. As a result, a new model was developed in which both materials Nitinol® and Silastic® are considered as the main valve materials. The proposed working conditions assume that the valve is implanted in folded form at the bronchus and then anchored when it is unfolded. Finite Element Method (FEM) was used to simulate the proposed working conditions. Results obtained show the structural performance and the level of stress generated in the implant during the breathing cycle. In addition, it provides the knowledge to generate a new device that could emulate the performance of these implants and develop a more efficient treatment this disease.

Human or animal homograft: could they have a future as a biological scaffold for engineered heart valves?

Tissue-engineered heart valves (TEHVs) promise to be the ideal heart valve replacement: they have the potential to grow and repair within the host, to minimise inflammatory and immunological responses and to limit thromboembolism. Viable cells included in TEHVs can theoretically adapt to a growing and changing environment exactly as a native biological structure. This could be extremely important in case of paediatric applications, where reoperations are frequently required to replace failed valve substitutes or to accommodate the growth of the patient. At present time the biological matrix from allogenic or xenogenic decellularized valves represents an appropriate valve scaffold in TEHVs, showing theoretically an ability to grow and repair within the host. Viable cells included in extracellular valve matrix can theoretically adapt to a growing and changing environment like the native biological structure. The aim of this paper is to present a review concerning the use of homograft and allograft valves as an ideal substrate for cardiac engineered tissue valves that represent an exciting possibility for in situ regeneration and repair of heart valves.

Prosthetic heart valves: catering for the few.

Prosthetic heart valves epitomize both the triumphant advance of cardiac surgery in its early days and its stagnation into a retrospective, exclusive first world discipline of late. Fifty-two years after the first diseased heart valve was replaced in a patient, prostheses largely represent the concepts of the 1960s with many of their design-inherent complications. While the sophisticated medical systems of the developed world may be able to cope with sub-optimal replacements, these valves are poorly suited to the developing world (where the overwhelming majority of potential valve recipients reside), due to differences in age profiles and socio-economic circumstances. Therefore, it is the latter group which suffered most from the sluggish pace of developments. While it previously took less than 7 years for mechanical heart valves to develop from the first commercially available ball-in-cage valve to the tilting pyrolytic-carbon disc valve, and another 10 years to arrive at the all-carbon bi-leaflet design, only small incremental improvements have been achieved since 1977. Similarly, bioprosthetic valves saw their last major break-through development in the late 1960s when formalin fixation was replaced by glutaraldehyde cross linking. Since then, poorly understood so-called 'anti-calcification' treatments were added and the homograft concept rediscovered under the catch-phrase 'stentless'. Still, tissue valves continue to degenerate fast in younger patients, making them unsuitable for developing countries. Yet, catheter-delivered prostheses almost exclusively use bioprosthetic tissue, thereby reducing one of the most promising developments for patients of the developing world into a fringe product for the few first world recipients. With tissue-engineered valves aiming at the narrow niche of congenital malformations and synthetic flexible leaflet valves being in their fifth decade of low-key development, heart valve prostheses seem to be destined to remain an unsatisfying and exclusive first world solution for a long time to come.

Human mesenchymal stem cells induce T cell anergy and downregulate T cell allo-responses via the TH2 pathway: relevance to tissue engineering human heart valves.

To generate an ''off the shelf'' tissue-engineered heart valve, the cells would need to be of allogeneic origin. Here, we report the possibility of using human bone marrow-derived mesenchymal stem cells (MSCs) as a suitable allogeneic cell source for tissue-engineered heart valves. Proliferative responses of primary and primed CD4+ T cells to allogeneic MSCs were examined. A protein microarray system was used to detect soluble factors from supernatants collected from the T cell assays. MSCs are poor stimulators of primary and primed CD4+ T cell proliferation, despite provision of B7-1 trans-co-stimulation. MSCs not only directly inhibited primary and primed T cell responses to allogeneic peripheral blood mononuclear cells (PBMCs), but 24-h pre-culture of T cells with MSCs suppressed subsequent T cell proliferative responses to allogeneic PBMCs in a contact-dependent manner. Analysis of supernatants revealed a distinctly different cytokine profile after co-culture of T cells with MSCs than with PBMCs or endothelial cells. Pro-inflammatory Th1 cytokines interleukin (IL)-1alpha and beta, interferon (IFN)gamma, and tumor necrosis factor (TNF)alpha were downregulated, whereas, anti-inflammatory Th2 cytokines IL-3, IL-5, IL-10, and IL-13 and the Th2 chemokine I-309, a chemoattractant for regulatory T cells, were upregulated. Further analysis revealed that after co-culture with MSCs, the T cells exhibited a regulatory phenotype (CD4+ CD25(lo) CD69(lo) FoxP3+). MSCs downregulate T cell responses through direct contact and secretion of anti-inflammatory and tolerogenic cytokines, which may involve the recruitment of regulatory T cells. This implies that allogeneic MSCs could be a suitable cell source for tissue engineering a heart valve.

CD117-positive cells and mast cells in adult human cardiac valves--observations and implications for the creation of bioengineered grafts.

BACKGROUND: There is no report to date of stem cells in human cardiac valves. We examined their possible presence, number, and distribution in valves removed at cardiac surgery from patients with a variety of underlying valve pathologies. METHODS: Grossly normal aortic and mitral valves were obtained from live heart transplant patients. Surgically excised valves with rheumatic mitral stenosis, aortic valve age-related degeneration, aortic valve changes of aortoannular ectasia, and mitral valves with myxomatous degeneration were studied. Immunohistochemical and histochemical studies were performed on sequential valve sections, including hematoxylin and eosin, hematoxylin phloxine saffron, Movat pentachrome, toluidine blue, CD31, CD34, and CD117. RESULTS: There were small clusters of CD117-positive cells in the fibrosa and spongiosa of mitral and aortic valves from all groups of valves. Sequential sectioning and staining showed that almost all of these cells were mast cells. However, in the mitral myxomatous valves and the mitral rheumatic valves, there were rare CD117-positive cells that did not have corresponding toluidine blue staining and thus could be valve mesenchymal stem cells. CONCLUSIONS: Most of the CD117-positive cells in normal and diseased adult heart valves are mast cells. These valve cells could play a role in valve pathology and injury. A very small number of possible valve stem cells were also identified. It is unlikely that these valve stem cells are sufficient in number to allow isolation and expansion for tissue engineering purposes.

[Spinal osteochondrosis, mesenchymal dysplasia and herpes infection].

36 intervertebrate disks (IVD) were studied in spinal osteochondrosis concurrent with herniation. Expression of herpes simplex types 1 and 2 (HSV-1 and HSV-2) antigens, which was absent in IVD of the control group (autopsy cases without disk hernia). The similarity of herniation in osteochondrosis and cardiac mesenchymal dysplasia, a frequent concomitance of these processes and the presence of HSV-1 and HSV-2 antigens in the IVD cells and cardiac valves may indicate the same nature of these diseases.

Dysregulation of cytokines in acute Q fever: role of interleukin-10 and tumor necrosis factor in chronic evolution of Q fever.

Q fever manifests as primary infection or acute Q fever and may become chronic in patients with underlying valvulopathy. Because Coxiella burnetii infection depends on host response, we measured tumor necrosis factor (TNF), interleukin (IL)-6, IL-12, and IL-10 in patients with different clinical presentations of acute Q fever. Compared with control subjects, patients with uncomplicated acute Q fever exhibited increased release of the 4 cytokines. Their amounts were higher in patients with hepatitis than in patients with fever or pneumonia. In patients with valvulopathy, who exhibited the highest risk of chronic evolution, the amounts of TNF and IL-10 were higher than in patients without valvulopathy. TNF production was specifically enhanced in patients who developed Q fever endocarditis. These results show that acute Q fever is associated with cytokine overproduction. Persistent TNF amounts were associated with the occurrence of endocarditis in patients with valvulopathy, and that may be a marker of chronic evolution of Q fever.

Restriction in the usage of variable beta regions in T-cells infiltrating valvular tissue from rheumatic heart disease patients.

Rheumatic Heart Disease (RHD) is a delayed consequence of a pharyngeal infection with group A streptococcus (GAS), usually ascribed to a cross-reactive immune response to the host's cardiac tissues. Several GAS proteins have been reported to be superantigens, also raising the possibility that T cells in RHD could be driven by superantigens. We therefore analysed the variable beta (V beta) repertoire of T cells infiltrating heart valves from chronic RHD patients undergoing elective valvular surgery. We analysed 15 valve specimens from patients with longstanding quiescent RHD and control valves from four non-rheumatic individuals. Total RNA was extracted from fresh valve tissue and employed to amplify 22 V beta genes by RT-PCR. In valvular tissue, a restricted number of only 2 to 9 V beta regions were detected as opposed to the findings in control valves. In 8 RHD valves, the expression of V beta1, 2, 3, 5.1, 7, 8, 9 or 14 was marked. These V beta regions have been related to GAS superantigens. Our results evidence the presence of a restricted set of T lymphocytes in valvular tissue from a majority of patients with chronic RHD and suggest that valvular sequelae in these patients might be related to a local antigen or superantigen driven inflammatory process that persists even many years after the initial triggering event.

Valve interstitial cells induce donor-specific T-cell anergy.

OBJECTIVES: Valve allografts produce an immune response, which can influence their performance. The exact role of the interaction between recipient T cells and the different cellular components of the donor valve in stimulating an immune response is not known. Therefore the T-cell response to valve endothelial and interstitial cells was investigated in vitro. METHODS: Valve endothelial and interstitial cells were characterized for cell-surface molecules before and after interferon gamma treatment by means of a panel of specific monoclonal antibodies and flow cytometry. The proliferative response of highly purified T lymphocytes was used to assess the immunogenicity of cultured valve endothelial and interstitial cells. This was further investigated by using a 2-step tolerance-induction protocol. RESULTS: Valve endothelial and interstitial cells express similar levels of human leukocyte antigens and adhesion and costimulatory molecules, which are either induced or upregulated after interferon gamma treatment. T-cell responses to endothelial cells were detected after interferon gamma treatment, but responses to interferon gamma-treated interstitial cells were not detected. This lack of response resulted in the induction of T-cell anergy, which was reversed by the presence of the costimulatory molecule B7-1. CONCLUSIONS: Although valve endothelial and interstitial cells express a similar range of cell-surface molecules, it is only the endothelial cells that are immunogenic. In addition, we have shown that these 2 cell types interact in a donor-specific manner to orchestrate the immune response and therefore may have clinical relevance in the allogeneic response of the heart valve recipients.

Immunological aspects of fresh and cryopreserved aortic valve transplantation in rats.

BACKGROUND: The influence of immune activation on valve allograft degeneration remains unclear. We studied the combined effect of major histocompatibility complex (MHC)-incompatibility and cryopreservation on valve performance, histomorphology, and tissue antigenicity in rats. METHODS: Fresh or cryopreserved allogeneic aortic valves from WAG (RT1u) rats were transplanted to DA (RT1a) recipients and syngenic transplants served as controls. After 7 or 21 days, valves were examined for competence and morphology. Immune reactivity of the recipient was measured by concanavalin A (conA) stimulation and analysis of donor-reactive Helper T-lymphocyte frequencies (HTLf) in peripheral blood and spleen. RESULTS: Syngenic grafts demonstrated normal competence and structure. Allografts lost their competence over time caused by destruction of the leaflets combined with cellular infiltration in the vascular wall. Cryopreservation induces early loss of competence and retrovalvular thrombosis. Cryopreserved allografts were also heavily infiltrated. ConA stimulation indices and HTLf were higher in allogeneic recipients compared to syngenic recipients (p < 0.03). Cryopreserved allografts elicited a lower immune response compared with fresh allografts (p < 0.03). CONCLUSIONS: Aortic valve allografts are able to induce a donor-reactive immune response that is related to early graft destruction and incompetence. Cryopreservation appears to diminish but not eliminate the antigenicity of the allograft.