Valvulogenesis of a living, innervated pulmonary root induced by an acellular scaffold.

Heart valve disease is a major cause of mortality and morbidity worldwide with no effective medical therapy and no ideal valve substitute emulating the extremely sophisticated functions of a living heart valve. These functions influence survival and quality of life. This has stimulated extensive attempts at tissue engineering "living" heart valves. These attempts utilised combinations of allogeneic/ autologous cells and biological scaffolds with practical, regulatory, and ethical issues. In situ regeneration depends on scaffolds that attract, house and instruct cells and promote connective tissue formation. We describe a surgical, tissue-engineered, anatomically precise, novel off-the-shelf, acellular, synthetic scaffold inducing a rapid process of morphogenesis involving relevant cell types, extracellular matrix, regulatory elements including nerves and humoral components. This process relies on specific material characteristics, design and "morphodynamism".

Multiscale Structure and Function of the Aortic Valve Apparatus.

While studying the aortic valve in isolation has facilitated the development of life-saving procedures and technologies, the dynamic interplay of the aortic valve and its surrounding structures is vital to preserving their function across the wide range of conditions encountered in an active lifestyle. Our view is that these structures should be viewed as an integrated functional unit, herein referred to as the aortic valve apparatus (AVA). The coupling of the aortic valve and root, left ventricular outflow tract, and blood circulation is crucial for AVA's functions: unidirectional flow out of the left ventricle, coronary perfusion, reservoir function, and supporting left ventricular function. In this review, we explore the multiscale biological and physical phenomena that underly the simultaneous fulfilment of these functions. A brief overview of the tools used to investigate the AVA is included, such as: medical imaging modalities, experimental methods, and computational modelling, specifically fluid-structure interaction (FSI) simulations, is included. Some pathologies affecting the AVA are explored, and insights are provided on treatments and interventions that aim to maintain quality of life. The concepts explained in this paper support the idea of AVA being an integrated functional unit and help identify unanswered research questions. Incorporating phenomena through the molecular, micro, meso and whole tissue scales is crucial for understanding the sophisticated normal functions and diseases of the AVA.

How to test adhesive strength: a biomechanical testing for aortic glue used in type a dissection repair.

OBJECTIVES: The aim of this study was to develop a method to quantify the peel force in an in vitro model simulating repair of ascending aortic dissections with tissue glue (Bioglue). METHODS: This study adapted an adhesive T-Peel test for the determination of the peel strength of adhesives by measuring the peeling force of a T-shaped bonded tissue. Measurements were performed on iatrogenic dissected ascending porcine aorta, which has been repaired with Bioglue using different pressure levels. Four conditions were tested: zero sample pressure according to the manufacturer's recommendation (n = 10), low (504 Pa; n = 11), moderate pressure (1711 Pa; n = 24) and pressure applied by a round shaped vascular 'Borst clamp' (1764 Pa; n = 23). Non-parametric one-way analysis of variance was applied for statistical significance. RESULTS: The median peel force (lower quartile, upper quartile) of aortic samples increased depending on the applied pressure: [no pressure 0.030 N/mm (0.016, 0.057), low pressure 0.040 N/mm (0.032, 0.070) and moderate pressure 0.214 N/mm (0.050, 0.304)]. Samples pressurized with the Borst clamp reached 0.078 N/mm (0.046, 0.152), which was comparable to the peel force of the unpeeled controls [0.107 N/mm (0.087, 0.124)]. Compared to samples without pressure, Bioglue with the application of the Borst clamp (P = 0.021) and with moderate pressure (P = 0.0007) performed significantly better. CONCLUSIONS: The novel T-Peel test offers an attractive method to test tissue glues in defined in vitro environments. Bioglue peel force increased with pressure on the aortic sample in contrast to low or no pressure as per the manufacturer's recommendation. Modifying current recommended use may aid in increasing effectiveness of this approach.

A New Dissipation Function to Model the Rate-Dependent Mechanical Behavior of Semilunar Valve Leaflets.

A new dissipation function Wv is devised and presented to capture the rate-dependent mechanical behavior of the semilunar heart valves. Following the experimentally-guided framework introduced in our previous work (Anssari-Benam et al., 2022 "Modelling the Rate-Dependency of the Mechanical Behaviour of the Aortic Heart Valve: An Experimentally Guided Theoretical Framework," J. Mech. Behav. Biomed. Mater., 134, p. 105341), we derive our proposed Wv function from the experimental data pertaining to the biaxial deformation of the aortic and pulmonary valve specimens across a 10,000-fold range of deformation rate, exhibiting two distinct rate-dependent features: (i) the stiffening effect in σ-λ curves with increase in rate; and (ii) the asymptotic effect of rate on stress levels at higher rates. The devised Wv function is then used in conjunction with a hyperelastic strain energy function We to model the rate-dependent behavior of the valves, incorporating the rate of deformation as an explicit variable. It is shown that the devised function favorably captures the observed rate-dependent features, and the model provides excellent fits to the experimentally obtained σ-λ curves. The proposed function is thereby recommended for application to the rate-dependent mechanical behavior of heart valves, as well as other soft tissues that exhibit a similar rate-dependent behavior.

Modelling the rate-dependency of the mechanical behaviour of the aortic heart valve: An experimentally guided theoretical framework.

A theoretical framework, based on extant experimental findings, is presented to devise a novel viscous dissipation function Wv in order to model the rate-dependent mechanical behaviour of the aortic heart valve. The experimental data encompasses Cauchy stress-stretch (σ-λ) curves obtained across a 10,000-fold range of stretch rates (λ˙), from quasi-static (λ˙= 0.001 s-1) to upper-range of physiological (λ˙= 12.4 s-1) deformation rates. The analysis of the data elicits two important trends: (i) the mechanical behaviour of the aortic valve across the tested rates is rate-dependent, with specimens becoming stiffer by increasing rate; and (ii) there appears to be a plateau in the rate-effects on the σ-λ curves; i.e. the rate-effects approach an asymptote with increase in the stretch rate λ˙. Guided by these empirical observations, we devise our new Wv function and demonstrate that the well-known form of the dissipation function commonly used in the literature is a special case of our proposed Wv. The ensuing model is then compared against the experimental σ-λ curves and is shown to provide favourable predictions. An important advantage of the employed modelling framework is that it allows the incorporation of the rate of deformation, which is a direct experimental control parameter, as an explicit modelling variable. The application of the proposed model is thereby recommended for heart valves and other soft tissues that exhibit similar rate-dependent features.

Profiling Genome-Wide DNA Methylation Patterns in Human Aortic and Mitral Valves.

Cardiac valves exhibit highly complex structures and specialized functions that include dynamic interactions between cells, extracellular matrix (ECM) and their hemodynamic environment. Valvular gene expression is tightly regulated by a variety of mechanisms including epigenetic factors such as histone modifications, RNA-based mechanisms and DNA methylation. To date, methylation fingerprints of non-diseased human aortic and mitral valves have not been studied. In this work we analyzed the differential methylation profiles of 12 non-diseased aortic and mitral valve tissue samples (in matched pairs). Analysis of methylation data [reduced representation bisulfite sequencing (RRBS)] of 16,101 promoters genome-wide revealed 584 differentially methylated (DM) promoters, of which 13 were reported in endothelial mesenchymal trans-differentiation (EMT), 37 in aortic and mitral valve disease and 7 in ECM remodeling. Both functional classification as well as network analysis showed that the genes associated with the DM promoters were enriched for WNT-, Cadherin-, Endothelin-, PDGF-, HIF-1 and VEGF- signaling implicated in valvular physiology and pathophysiology. Additional enrichment was detected for TGFB-, NOTCH- and Integrin- signaling involved in EMT as well as ECM remodeling. This data provides the first insight into differential regulation of human aortic and mitral valve tissue and identifies candidate genes linked to DM promoters. Our work will improve the understanding of valve biology, valve tissue engineering approaches and contributes to the identification of relevant drug targets.

Biocompatibility and Application of Carbon Fibers in Heart Valve Tissue Engineering.

The success of tissue-engineered heart valves rely on a balance between polymer degradation, appropriate cell repopulation, and extracellular matrix (ECM) deposition, in order for the valves to continue their vital function. However, the process of remodeling is highly dynamic and species dependent. The carbon fibers have been well used in the construction industry for their high tensile strength and flexibility and, therefore, might be relevant to support tissue-engineered hearts valve during this transition in the mechanically demanding environment of the circulation. The aim of this study was to assess the suitability of the carbon fibers to be incorporated into tissue-engineered heart valves, with respect to optimizing their cellular interaction and mechanical flexibility during valve opening and closure. The morphology and surface oxidation of the carbon fibers were characterized by scanning electron microscopy (SEM). Their ability to interact with human adipose-derived stem cells (hADSCs) was assessed with respect to cell attachment and phenotypic changes. hADSCs attached and maintained their expression of stem cell markers with negligible differentiation to other lineages. Incorporation of the carbon fibers into a stand-alone tissue-engineered aortic root, comprised of jet-sprayed polycaprolactone aligned carbon fibers, had no negative effects on the opening and closure characteristics of the valve when simulated in a pulsatile bioreactor. In conclusion, the carbon fibers were found to be conducive to hADSC attachment and maintaining their phenotype. The carbon fibers were sufficiently flexible for full motion of valvular opening and closure. This study provides a proof-of-concept for the incorporation of the carbon fibers into tissue-engineered heart valves to continue their vital function during scaffold degradation.

A New Technique for Shaping the Aortic Sinuses and Conserving Dynamism in the Remodeling Operation.

BACKGROUND: Preserving dynamism and recreating the sinuses in the Dacron graft are thought to be important for optimizing results of aortic valve-conserving operations. METHODS: We describe a novel technique that preserves dynamism and recreates the sinotubular junction. In addition, it tailors 3 sinuses of defined longitudinal and transverse curvatures in a straight Dacron tube during the operation. The technique has been used in 6 patients with varied aortic root pathology. We performed preoperative and postoperative multimodality imaging using computerized image analysis as well as 3-dimensional models. RESULTS: There was no early or midterm death. Upon discharge, patients were clinically well, with echocardiographic evidence of minimal (3 patients) or mild (3 patients) aortic regurgitation. Computed tomography and cardiac magnetic resonance imaging with extensive image analysis of the aortic root size, shape, and function showed partial or complete normalization of these parameters. This included the shape and dynamism of the aortic annulus and the size and shape of the geometric (effective) orifice. The 4-dimensional magnetic resonance imaging pattern of flow in the sinuses and ascending aorta showed favorable vortices in the sinuses, right-handed helical flow, and marked diminution of energy loss in the ascending aorta. CONCLUSIONS: The novel technique described here is simple, practical, and cost-effective because it uses a widely available straight Dacron tube. The technique does not use rigid internal or external support. The early results are encouraging. Larger series with longer follow-up are required.

Rate-dependent mechanical behaviour of semilunar valves under biaxial deformation: From quasi-static to physiological loading rates.

In this study we investigate the rate-dependency of the mechanical behaviour of semilunar heart valves under biaxial deformation, from quasi-static to physiological loading rates. This work extends and complements our previous undertaking, where the rate-dependency in the mechanical behaviour of semilunar valve specimens was documented in sub-physiological rate domains (Acta Biomater. 2019; https://doi.org/10.1016/j.actbio.2019.02.008). For the first time we demonstrate herein that the stress-stretch curves obtained from specimens under physiological rates too are markedly different to those at sufficiently lower rates and at quasi-static conditions. The results importantly underline that the mechanical behaviour of semilunar heart valves is rate dependent, and the physiological mechanical behaviour of the valves may not be correctly obtained via material characterisation tests at arbitrary low deformation rates. Presented results in this work provide an inclusive dataset for material characterisation and modelling of semilunar heart valves across a 10,000 fold deformation rate, both under equi-biaxial and 1:3 ratio deformation rates. The important application of these results is to inform the development of appropriate mechanical testing protocols, as well as devising new models, for suitable determination of the rate-dependent constitutive mechanical behaviour of the semilunar valves.

Microstructure of the juvenile sheep aortic valve hinge region and the trilamellar sliding hypothesis.

Background: The aortic valve mechanism performs extremely sophisticated functions which depend on the microstructure of its component parts. The hinge mechanism of the aortic leaflets plays a crucial part in the overall function. However, the detailed microstructure and its relation to function has not been adequately studied. Methods: The aortic roots of juvenile sheep were fixed under physiologic pressure. Sections through all three sinuses were then performed to illustrate the microstructure of the hinge mechanism in different regions of the aortic root. Results: The hinge region in the different sinuses showed unique microstructure with a trilamellar topology with a dominant core consisting of glycosaminoglycans. The exact arrangement of the trilamellar structures varies around the aortic sinuses, which could have functional implications. These features allow the hinge to perform its complex functions through what we have described as "the trilamellar sliding hypothesis". Conclusion: The microstructure of the hinge mechanism is unique and enables it to perform it sophisticated functions.

Rate-dependency of the mechanical behaviour of semilunar heart valves under biaxial deformation.

This paper presents an experimental investigation and evidence of rate-dependency in the planar mechanical behaviour of semilunar heart valves. Samples of porcine aortic and pulmonary valves were subjected to biaxial deformations across 1000-fold stretch rate, ranging from λ̇=0.001 to 1 s-1. The experimental campaign encompassed protocols covering (i) tests on samples without preconditioning, (ii) preconditioning immediately followed by tensile tests; and (iii) tensile tests at different rates performed on the same preconditioned specimen. Our results indicate that under all employed loading protocols, heart valve samples exhibit a marked rate-dependency in their deformation behaviour. This rate-dependency is reflected in stress-stretch curves and the calculated ensuing gradients, where samples typically show stiffening with increased rate. These results underpin one conclusive outcome: the in-plane mechanical behaviour of semilunar valves is rate-dependent (p<0.05 for Cauchy stress levels ≥50 kPa). This outcome implies that the rate of deformation for characterising the mechanical behaviour of semilunar heart valves may not be chosen arbitrarily low, and models that incorporate rate-effects may be more appropriate for better capturing the mechanical behaviour of heart valves. STATEMENT OF SIGNIFICANCE: This study presents for the first time a comprehensive set of results and evidence of rate-dependency in the mechanical behaviour of semilunar heart valves under biaxial deformation. Our results challenge the widely-applied assumption in the bulk of the existing literature, where an implicit rate-independency is assumed in both experimental and modelling propositions related to the biomechanics of the aortic and pulmonary valves. This study therefore creates a solid platform for future research in heart valve biomechanics with two important implications. First, experimental campaigns have to be carried out at high stretch rates; ideally as close to the physiological rate as possible. Second, new continuum/computational models are required to address the rate-dependent mechanical behaviour of the semilunar valves.

A transverse isotropic constitutive model for the aortic valve tissue incorporating rate-dependency and fibre dispersion: Application to biaxial deformation.

This paper presents a continuum-based transverse isotropic model incorporating rate-dependency and fibre dispersion, applied to the planar biaxial deformation of aortic valve (AV) specimens under various stretch rates. The rate dependency of the mechanical behaviour of the AV tissue under biaxial deformation, the (pseudo-) invariants of the right Cauchy-Green deformation-rate tensor C associated with fibre dispersion, and a new fibre orientation density function motivated by fibre kinematics are presented for the first time. It is shown that the model captures the experimentally observed deformation of the specimens, and characterises a shear-thinning behaviour associated with the dissipative (viscous) kinematics of the matrix and the fibres. The application of the model for predicting the deformation behaviour of the AV under physiological rates is illustrated and an example of the predicted σ-λ curves is presented. While the development of the model was principally motivated by the AV biomechanics requisites, the comprehensive theoretical approach employed in the study renders the model suitable for application to other fibrous soft tissues that possess similar rate-dependent and structural attributes.

Fabrication and In Vitro Characterization of a Tissue Engineered PCL-PLLA Heart Valve.

Heart valve diseases are among the leading causes of cardiac failure around the globe. Nearly 90,000 heart valve replacements occur in the USA annually. Currently, available options for heart valve replacement include bioprosthetic and mechanical valves, both of which have severe limitations. Bioprosthetic valves can last for only 10-20 years while patients with mechanical valves always require blood-thinning medications throughout the remainder of the patient's life. Tissue engineering has emerged as a promising solution for the development of a viable, biocompatible and durable heart valve; however, a human implantable tissue engineered heart valve is yet to be achieved. In this study, a tri-leaflet heart valve structure is developed using electrospun polycaprolactone (PCL) and poly L-lactic acid (PLLA) scaffolds, and a set of in vitro testing protocol has been developed for routine manufacturing of tissue engineered heart valves. Stress-strain curves were obtained for mechanical characterization of different valves. The performances of the developed valves were hemodynamically tested using a pulse duplicator, and an echocardiography machine. Results confirmed the superiority of the PCL-PLLA heart valve compared to pure PCL or pure PLLA. The developed in vitro test protocol involving pulse duplicator and echocardiography tests have enormous potential for routine application in tissue engineering of heart valves.

A Strategy to Enhance Secretion of Extracellular Matrix Components by Stem Cells: Relevance to Tissue Engineering.

The ability of cells to secrete extracellular matrix proteins is an important property in the repair, replacement, and regeneration of living tissue. Cells that populate tissue-engineered constructs need to be able to emulate these functions. The motifs, KTTKS or palmitoyl-KTTKS (peptide amphiphile), have been shown to stimulate production of collagen and fibronectin in differentiated cells. Molecular modeling was used to design different forms of active peptide motifs to enhance the efficacy of peptides to increase collagen and fibronectin production using terminals KTTKS/SKTTK/SKTTKS connected by various hydrophobic linkers, V4A3/V4A2/A4G3. Molecular dynamic simulations showed SKTTKS-V4A3-SKTTKS (P3), with palindromic (SKTTKS) motifs and SKTTK-V4A2-KTTKS (P5), maintained structural integrity and favorable surface electrostatic distributions that are required for functionality. In vitro studies showed that peptides, P3 and P5, showed low toxicity to human adipose-derived stem cells (hADSCs) and significantly increased the production of collagen and fibronectin in a concentration-dependent manner compared with the original active peptide motif. The 4-day treatment showed that stem cell markers of hADSCs remained stable with P3. The molecular design of novel peptides is a promising strategy for the development of intelligent biomaterials to guide stem cell function for tissue engineering applications.

Extracellular matrix production by adipose-derived stem cells: implications for heart valve tissue engineering.

A key challenge in tissue engineering a heart valve is to reproduce the major tissue structures responsible for native valve function. Here we evaluated human adipose-derived stem cells (ADSCs) as a source of cells for heart valve tissue engineering investigating their ability to synthesize and process collagen and elastin. ADSCs were compared with human bone marrow mesenchymal stem cells (BmMSCs) and human aortic valve interstitial cells (hVICs). ADSCs and BmMSCs were stretched at 14% for 3 days and collagen synthesis determined by [(3)H]-proline incorporation. Collagen and elastin crosslinking was assessed by measuring pyridinoline and desmosine respectively, using liquid chromatography/mass spectrometry. Three-dimensional culture was obtained by seeding cells onto bovine collagen type I scaffolds for 2-20 days. Expression of matrix proteins and processing enzymes was assessed by Real Time-PCR, immunofluorescence and transmission electron microscopy. Stretch increased the incorporation of [(3)H]-proline in ADSCs and BmMSCs, however only ADSCs and hVICs upregulated COL3A1 gene. ADSCs produced collagen and elastin crosslinks. ADSCs uniformly populated collagen scaffolds after 2 days, and fibrillar-like collagen was detected after 20 days. ADSCs sense mechanical stimulation and produce and process collagen and elastin. These novel findings have important implications for the use of these cells in tissue engineering.