Observations on the Tritiated Water and TEWL Skin Integrity Tests: Relevance to In Vitro Permeation Testing (IVPT).

BACKGROUND: The in vitro permeation test (IVPT) using ex vivo human skin is a sensitive and robust model system that has been vital in elucidating the fundamental parameters surrounding the absorption of both therapeutic agents and industrial chemicals through skin. FDA and OECD IVPT Guidances recommend that each skin section selected for study should be screened prior to use to ensure that the stratum corneum integrity is retained. Three methods are currently considered acceptable: 1) transepidermal water loss (TEWL), 2) electrical resistance, and 3) tritiated water (3H2O) absorption. METHODS: A retrospective analysis of data from the authors' laboratory has been performed with the objective of addressing a number of questions regarding the 3H2O and TEWL integrity tests, and the population attributes of a large database consisting of 17,330 individual skin sections obtained from 459 skin donors. The applicability and usefulness of these tests, when compared to companion permeation data obtained from 25 topical drug products, has also been examined. RESULTS: Both integrity tests found water permeability to be equal in White and Hispanic races but higher than in Blacks, 3H2O being more discriminating than TEWL. Male skin is more permeable than female and there is a slight decrease in permeability with advancing age in both groups. Correlation between 3H2O absorption and drug absorption revealed a minimal relationship between the two in most cases, the Pearson correlation coefficient ranging from -0.417 to 0.953. Additionally, drug outliers were not always identified with a failing integrity test. CONCLUSION: The results call for a critical reexamination of the value of the 3H2O integrity test, and by extension, TEWL, for use in IVPT studies.

Mathematical model for force and energy of virion-cell interactions during full engulfment in HIV: Impact of virion maturation and host cell morphology.

Viral endocytosis involves elastic cell deformation, driven by chemical adhesion energy, and depends on physical interactions between the virion and cell membrane. These interactions are not easy to quantify experimentally. Hence, this study aimed to develop a mathematical model of the interactions of HIV particles with host cells and explore the effects of mechanical and morphological parameters during full virion engulfment. The invagination force and engulfment energy were described as viscoelastic and linear-elastic functions of radius and elastic modulus of virion and cell, ligand-receptor energy density and engulfment depth. The influence of changes in the virion-cell contact geometry representing different immune cells and ultrastructural membrane features and the decrease in virion radius and shedding of gp120 proteins during maturation on invagination force and engulfment energy was investigated. A low invagination force and high ligand-receptor energy are associated with high virion entry ability. The required invagination force was the same for immune cells of different sizes but lower for a local convex geometry of the cell membrane at the virion length scale. This suggests that localized membrane features of immune cells play a role in viral entry ability. The available engulfment energy decreased during virion maturation, indicating the involvement of additional biological or biochemical changes in viral entry. The developed mathematical model offers potential for the mechanobiological assessment of the invagination of enveloped viruses towards improving the prevention and treatment of viral infections.

Intracellular mechanics and TBX3 expression jointly dictate the spreading mode of melanoma cells in 3D environments.

Cell stiffness and T-box transcription factor 3 (TBX3) expression have been identified as biomarkers of melanoma metastasis in 2D environments. This study aimed to determine how mechanical and biochemical properties of melanoma cells change during cluster formation in 3D environments. Vertical growth phase (VGP) and metastatic (MET) melanoma cells were embedded in 3D collagen matrices of 2 and 4 mg/ml collagen concentrations, representing low and high matrix stiffness. Mitochondrial fluctuation, intracellular stiffness, and TBX3 expression were quantified before and during cluster formation. In isolated cells, mitochondrial fluctuation decreased and intracellular stiffness increased with increase in disease stage from VGP to MET and increased matrix stiffness. TBX3 was highly expressed in soft matrices but diminished in stiff matrices for VGP and MET cells. Cluster formation of VGP cells was excessive in soft matrices but limited in stiff matrices, whereas for MET cells it was limited in soft and stiff matrices. In soft matrices, VGP cells did not change the intracellular properties, whereas MET cells exhibited increased mitochondrial fluctuation and decreased TBX3 expression. In stiff matrices, mitochondrial fluctuation and TBX3 expression increased in VGP and MET, and intracellular stiffness increased in VGP but decreased in MET cells. The findings suggest that soft extracellular environments are more favourable for tumour growth, and high TBX3 levels mediate collective cell migration and tumour growth in the earlier VGP disease stage but play a lesser role in the later metastatic stage of melanoma.

Effect of biomaterial stiffness on cardiac mechanics in a biventricular infarcted rat heart model with microstructural representation of in situ intramyocardial injectate.

Intramyocardial delivery of biomaterials is a promising concept for treating myocardial infarction. The delivered biomaterial provides mechanical support and attenuates wall thinning and elevated wall stress in the infarct region. This study aimed at developing a biventricular finite element model of an infarcted rat heart with a microstructural representation of an in situ biomaterial injectate, and a parametric investigation of the effect of the injectate stiffness on the cardiac mechanics. A three-dimensional subject-specific biventricular finite element model of a rat heart with left ventricular infarct and microstructurally dispersed biomaterial delivered 1 week after infarct induction was developed from ex vivo microcomputed tomography data. The volumetric mesh density varied between 303 mm-3 in the myocardium and 3852 mm-3 in the injectate region due to the microstructural intramyocardial dispersion. Parametric simulations were conducted with the injectate's elastic modulus varying from 4.1 to 405,900 kPa, and myocardial and injectate strains were recorded. With increasing injectate stiffness, the end-diastolic median myocardial fibre and cross-fibre strain decreased in magnitude from 3.6% to 1.1% and from -6.0% to -2.9%, respectively. At end-systole, the myocardial fibre and cross-fibre strain decreased in magnitude from -20.4% to -11.8% and from 6.5% to 4.6%, respectively. In the injectate, the maximum and minimum principal strains decreased in magnitude from 5.4% to 0.001% and from -5.4% to -0.001%, respectively, at end-diastole and from 38.5% to 0.06% and from -39.0% to -0.06%, respectively, at end-systole. With the microstructural injectate geometry, the developed subject-specific cardiac finite element model offers potential for extension to cellular injectates and in silico studies of mechanotransduction and therapeutic signalling in the infarcted heart with an infarct animal model extensively used in preclinical research.

An analytical model describing the mechanics of erythrocyte membrane wrapping during active invasion of a plasmodium falciparum merozoite.

The invasion of a merozoite into an erythrocyte by membrane wrapping is a hallmark of malaria pathogenesis. The invasion involves biomechanical interactions whereby the merozoite exerts actomyosin-based forces to push itself into and through the erythrocyte membrane while concurrently inducing biochemical damage to the erythrocyte membrane. Whereas the biochemical damage process has been investigated, the detailed mechanistic understanding of the invasion mechanics remains limited. Thus, the current study aimed to develop a mathematical model describing the mechanical factors involved in the merozoite invasion into an erythrocyte and explore the invasion mechanics. A shell theory model was developed comprising constitutive, equilibrium and governing equations of the deformable erythrocyte membrane to predict membrane mechanics during the wrapping of an entire non-deformable ellipsoidal merozoite. Predicted parameters include principal erythrocyte membrane deformations and stresses, wrapping and indentation forces, and indentation work. The numerical investigations considered two limits for the erythrocyte membrane deformation during wrapping (4% and 51% areal strain) and erythrocyte membrane phosphorylation (decrease of membrane elastic modulus from 1 to 0.5 kPa). For an intact erythrocyte, the maximum indentation force was 1 and 8.5 pN, and the indentation work was 1.92 × 10-18 and 1.40 × 10-17 J for 4% and 51% areal membrane strain. Phosphorylation damage in the erythrocyte membrane reduced the required indentation work by 50% to 0.97 × 10-18 and 0.70 × 10-17 J for 4% and 51% areal strain. The current study demonstrated the developed model's feasibility to provide new knowledge on the physical mechanisms of the merozoite invasion process that contribute to the invasion efficiency towards the discovery of new invasion-blocking anti-malaria drugs.

Mathematical model of mechano-sensing and mechanically induced collective motility of cells on planar elastic substrates.

Cells mechanically interact with their environment to sense, for example, topography, elasticity and mechanical cues from other cells. Mechano-sensing has profound effects on cellular behaviour, including motility. The current study aims to develop a mathematical model of cellular mechano-sensing on planar elastic substrates and demonstrate the model's predictive capabilities for the motility of individual cells in a colony. In the model, a cell is assumed to transmit an adhesion force, derived from a dynamic focal adhesion integrin density, that locally deforms a substrate, and to sense substrate deformation originating from neighbouring cells. The substrate deformation from multiple cells is expressed as total strain energy density with a spatially varying gradient. The magnitude and direction of the gradient at the cell location define the cell motion. Cell-substrate friction, partial motion randomness, and cell death and division are included. The substrate deformation by a single cell and the motility of two cells are presented for several substrate elasticities and thicknesses. The collective motility of 25 cells on a uniform substrate mimicking the closure of a circular wound of 200 µm is predicted for deterministic and random motion. Cell motility on substrates with varying elasticity and thickness is explored for four cells and 15 cells, the latter again mimicking wound closure. Wound closure by 45 cells is used to demonstrate the simulation of cell death and division during migration. The mathematical model can adequately simulate the mechanically induced collective cell motility on planar elastic substrates. The model is suitable for extension to other cell and substrates shapes and the inclusion of chemotactic cues, offering the potential to complement in vitro and in vivo studies.

Effect of paclitaxel treatment on cellular mechanics and morphology of human oesophageal squamous cell carcinoma in 2D and 3D environments.

During chemotherapy, structural and mechanical changes in malignant cells have been observed in several cancers, including leukaemia and pancreatic and prostate cancer. Such cellular changes may act as physical biomarkers for chemoresistance and cancer recurrence. This study aimed to determine how exposure to paclitaxel affects the intracellular stiffness of human oesophageal cancer of South African origin in vitro. A human oesophageal squamous cell carcinoma cell line WHCO1 was cultured on glass substrates (2D) and in collagen gels (3D) and exposed to paclitaxel for up to 48 h. Cellular morphology and stiffness were assessed with confocal microscopy, visually aided morpho-phenotyping image recognition and mitochondrial particle tracking microrheology at 24 and 48 h. In the 2D environment, the intracellular stiffness was higher for the paclitaxel-treated than for untreated cells at 24 and 48 h. In the 3D environment, the paclitaxel-treated cells were stiffer than the untreated cells at 24 h, but no statistically significant differences in stiffness were observed at 48 h. In 2D, paclitaxel-treated cells were significantly larger at 24 and 48 h and more circular at 24 but not at 48 h than the untreated controls. In 3D, there were no significant morphological differences between treated and untreated cells. The distribution of cell shapes was not significantly different across the different treatment conditions in 2D and 3D environments. Future studies with patient-derived primary cancer cells and prolonged drug exposure will help identify physical cellular biomarkers to detect chemoresistance onset and assess therapy effectiveness in oesophageal cancer patients.

Fast and highly sensitive determination of tetrahydrocannabinol (THC) metabolites in hair using liquid chromatography-multistage mass spectrometry (LC-MS3 ).

In hair analysis, identification of 11-nor-9-carboxy-∆9 -tetrahydrocannabinol (THC-COOH), one of the major endogenously formed metabolites of the psychoactive cannabinoid tetrahydrocannabinol (THC), is considered unambiguous proof of cannabis consumption. Due to the complex hair matrix and low target concentrations of THC-COOH in hair, this kind of investigation represents a great analytical challenge. The aim of this work was to establish a fast, simple, and reliable LC-MS3 routine method for sensitive detection of THC-COOH in hair samples. Furthermore, the LC-MS3 method developed also included the detection of derivatized 11-hydroxy-∆9 -THC (11-OH-THC) as an additional marker of cannabis use. Hair sample preparation prior to detection of the two THC metabolites was based on digestion of the hair matrix under alkaline conditions followed by an optimized liquid-liquid extraction (LLE) procedure. Sample preparation by LLE proved to be more suitable than solid-phase extraction (SPE) due to less laborious and time-consuming steps while still yielding satisfactory results. A significant improvement in analytical detection was introduced by multistage fragmentation (MS3 ), which led to enhanced sensitivity and selectivity and thus low limits of quantification (0.1 pg/mg hair). The MS3 method included two transitions for THC-COOH (m/z 343 → 299 → 245 and m/z 343 → 299 → 191) encompassing the quantifier (m/z 245) and the qualifier ion (m/z 191). The method was fully validated, and successful application to authentic toxicology case samples was demonstrated by the analysis of more than 2000 hair samples from cannabis users with THC-COOH concentrations determined ranging from 0.1 to >15 pg/mg hair.

Cutaneous Pharmacokinetic Approaches to Compare Bioavailability and/or Bioequivalence for Topical Drug Products.

The evaluation of bioequivalence (BE) involves comparing the test product to its reference product in a study whose fundamental scientific principles allow inferring of the clinical performance of the products. Several test methods have been discussed and developed to evaluate topical bioavailability (BA) and BE. Pharmacokinetics-based approaches characterize the rate and extent to which an active ingredient becomes available at or near its site of action in the skin. Such methodologies are considered to be among the most accurate, sensitive, and reproducible approaches for determining the BA or BE of a product.

In silico stress fibre content affects peak strain in cytoplasm and nucleus but not in the membrane for uniaxial substrate stretch.

Existing in silico models for single cell mechanics feature limited representations of cytoskeletal structures that contribute substantially to the mechanics of a cell. We propose a micromechanical hierarchical approach to capture the mechanical contribution of actin stress fibres. For a cell-specific fibroblast geometry with membrane, cytoplasm and nucleus, the Mori-Tanaka homogenization method was employed to describe cytoplasmic inhomogeneities and constitutive contribution of actin stress fibres. The homogenization was implemented in a finite element model of the fibroblast attached to a substrate through focal adhesions. Strain in cell membrane, cytoplasm and nucleus due to uniaxial substrate stretch was assessed for different stress fibre volume fractions and different elastic modulus of the substrate. A considerable decrease of the peak strain with increasing stress fibre content was observed in cytoplasm and nucleus but not the membrane, whereas the peak strain in cytoplasm, nucleus and membrane increased for increasing elastic modulus of the substrate. Finite element mesh of reconstructed human fibroblast and intracellular strain distribution in cell subjected to substrate stretch.

Step-by-Step Sample Preparation of Proteins for Mass Spectrometric Analysis.

Nowadays identification and quantification of proteins from biological samples by mass spectrometry are widely used. For the identification of proteins, there are two scenarios. Proteins are either pre-fractionated in some way, e.g., by gel electrophoresis or chromatography, or analyzed as complex mixture (shotgun). Because of technological developments of mass spectrometry, the identification of several thousand proteins from complex biological matrix becomes possible. However, in many cases, it is still useful to separate proteins first in a gel. For quantifying proteins, label-free, isotopic labeling, and data-independent acquisition (DIA) library are widely used. Not only mass spectrometry technology made progress. This is also true for the sample preparation. Protocols and techniques developed recently not only make the analysis of starting material in the low microgram range possible but also simplify the whole procedure. Here, we will describe some detailed protocols of preparing samples for mass spectrometry-based protein identification and protein quantification, as in-gel digestion, in-solution digestion, peptide cleaning, and TMT labeling. This will allow also inexperienced beginners to get good results.

Alopecia in Harlequin mutant mice is associated with reduced AIF protein levels and expression of retroviral elements.

We investigated the contribution of apoptosis-inducing factor (AIF), a key regulator of mitochondrial biogenesis, in supporting hair growth. We report that pelage abnormalities developed during hair follicle (HF) morphogenesis in Harlequin (Hq) mutant mice. Fragility of the hair cortex was associated with decreased expression of genes encoding structural hair proteins, though key transcriptional regulators of HF development were expressed at normal levels. Notably, Aifm1 (R200 del) knockin males and Aifm1(R200 del)/Hq females showed minor hair defects, despite substantially reduced AIF levels. Furthermore, we cloned the integrated ecotropic provirus of the Aifm1Hq allele. We found that its overexpression in wild-type keratinocyte cell lines led to down-regulation of HF-specific Krt84 and Krtap3-3 genes without altering Aifm1 or epidermal Krt5 expression. Together, our findings imply that pelage paucity in Hq mutant mice is mechanistically linked to severe AIF deficiency and is associated with the expression of retroviral elements that might potentially influence the transcriptional regulation of structural hair proteins.

Tissue Ingrowth Markedly Reduces Mechanical Anisotropy and Stiffness in Fibre Direction of Highly Aligned Electrospun Polyurethane Scaffolds.

PURPOSE: The lack of long-term patency of synthetic vascular grafts currently available on the market has directed research towards improving the performance of small diameter grafts. Improved radial compliance matching and tissue ingrowth into the graft scaffold are amongst the main goals for an ideal vascular graft. METHODS: Biostable polyurethane scaffolds were manufactured by electrospinning and implanted in subcutaneous and circulatory positions in the rat for 7, 14 and 28 days. Scaffold morphology, tissue ingrowth, and mechanical properties of the scaffolds were assessed before implantation and after retrieval. RESULTS: Tissue ingrowth after 24 days was 96.5 ± 2.3% in the subcutaneous implants and 77.8 ± 5.4% in the circulatory implants. Over the 24 days implantation, the elastic modulus at 12% strain decreased by 59% in direction of the fibre alignment whereas it increased by 1379% transverse to the fibre alignment of the highly aligned scaffold of the subcutaneous implants. The lesser aligned scaffold of the circulatory graft implants exhibited an increase of the elastic modulus at 12% strain by 77% in circumferential direction. CONCLUSION: Based on the observations, it is proposed that the mechanism underlying the softening of the highly aligned scaffold in the predominant fibre direction is associated with scaffold compaction and local displacement of fibres by the newly formed tissue. The stiffening of the scaffold, observed transverse to highly aligned fibres and for more a random fibre distribution, represents the actual mechanical contribution of the tissue that developed in the scaffold.

Cytoskeletal tubulin competes with actin to increase deformability of metastatic melanoma cells.

The formation of membrane protrusions during migration is reliant upon the cells' cytoskeletal structure and stiffness. It has been reported that actin disruption blocks protrusion and decreases cell stiffness whereas microtubule disruption blocks protrusion but increases stiffness in several cell types. In melanoma, cell migration is of concern as this cancer spreads unusually rapidly during early tumour development. The aim of this study was to characterise motility, structural properties and stiffness of human melanoma cells at radial growth phase (RGP), vertical growth phase (VGP), and metastatic stage (MET) in two-dimensional in vitro environments. Wound assays, western blotting and mitochondrial particle tracking were used to assess cell migration, cytoskeletal content and intracellular fluidity. Our results indicate that cell motility increase with increasing disease stage. Despite their different motility, RGP and VGP cells exhibit similar fluidity, actin and tubulin levels. MET cells, however, display increased fluidity which was associated with increased actin and tubulin content. Our findings demonstrate an interplay between actin and microtubule activity and their role in increasing motility of cells while minimizing cell stiffness at advanced disease stage. In earlier disease stages, cell stiffness may however not serve as an indicator of migratory capabilities.

Intra-myocardial alginate hydrogel injection acts as a left ventricular mid-wall constraint in swine.

Despite positive initial outcomes emerging from preclinical and early clinical investigation of alginate hydrogel injection therapy as a treatment for heart failure, the lack of knowledge about the mechanism of action remains a major shortcoming that limits the efficacy of treatment design. To identify the mechanism of action, we examined previously unobtainable measurements of cardiac function from in vivo, ex vivo, and in silico states of clinically relevant heart failure (HF) in large animals. High-resolution ex vivo magnetic resonance imaging and histological data were used along with state-of-the-art subject-specific computational model simulations. Ex vivo data were incorporated in detailed geometric computational models for swine hearts in health (n = 5), ischemic HF (n = 5), and ischemic HF treated with alginate hydrogel injection therapy (n = 5). Hydrogel injection therapy mitigated elongation of sarcomere lengths (1.68 ± 0.10µm [treated] vs. 1.78 ± 0.15µm [untreated], p<0.001). Systolic contractility in treated animals improved substantially (ejection fraction = 43.9 ± 2.8% [treated] vs. 34.7 ± 2.7% [untreated], p<0.01). The in silico models realistically simulated in vivo function with >99% accuracy and predicted small myofiber strain in the vicinity of the solidified hydrogel that was sustained for up to 13 mm away from the implant. These findings suggest that the solidified alginate hydrogel material acts as an LV mid-wall constraint that significantly reduces adverse LV remodeling compared to untreated HF controls without causing negative secondary outcomes to cardiac function. STATEMENT OF SIGNIFICANCE: Heart failure is considered a growing epidemic and hence an important health problem in the US and worldwide. Its high prevalence (5.8 million and 23 million, respectively) is expected to increase by 25% in the US alone by 2030. Heart failure is associated with high morbidity and mortality, has a 5-year mortality rate of 50%, and contributes considerably to the overall cost of health care ($53.1 billion in the US by 2030). Despite positive initial outcomes emerging from preclinical and early clinical investigation of alginate hydrogel injection therapy as a treatment for heart failure, the lack of knowledge concerning the mechanism of action remains a major shortcoming that limits the efficacy of treatment design. To understand the mechanism of action, we combined high-resolution ex vivo magnetic resonance imaging and histological data in swine with state-of-the-art subject-specific computational model simulations. The in silico models realistically simulated in vivo function with >99% accuracy and predicted small myofiber strain in the vicinity of the solidified hydrogel that was sustained for up to 13 mm away from the implant. These findings suggest that the solidified alginate hydrogel material acts as a left ventricular mid-wall constraint that significantly reduces adverse LV remodeling compared to untreated heart failure controls without causing negative secondary outcomes to cardiac function. Moreover, if the hydrogel can be delivered percutaneously rather than via the currently used open-chest procedure, this therapy may become routine for heart failure treatment. A minimally invasive procedure would be in the best interest of this patient population; i.e., one that cannot tolerate general anesthesia and surgery, and it would be significantly more cost-effective than surgery.

Customized stent-grafts for endovascular aneurysm repair with challenging necks: A numerical proof of concept.

Endovascular aortic repair (EVAR) is a challenging intervention whose long-term success strongly depends on the appropriate stent-graft (SG) selection and sizing. Most off-the-shelf SGs are straight and cylindrical. Especially in challenging vessel morphologies, the morphology of off-the-shelf SGs is not able to meet the patient-specific demands. Advanced manufacturing technologies facilitate the development of highly customized SGs. Customized SGs that have the same morphology as the luminal vessel surface could considerably improve the quality of the EVAR outcome with reduced likelihoods of EVAR related complications such as endoleaks type I and SG migration. In this contribution, we use an in silico EVAR methodology that approximates the deployed state of the elastically deformable SG in a hyperelastic, anisotropic vessel. The in silico EVAR results of off-the-shelf SGs and customized SGs are compared qualitatively and quantitatively in terms of mechanical and geometrical parameters such as stent stresses, contact tractions, SG fixation forces and the SG-vessel attachment. In a numerical proof of concept, eight different vessel morphologies, such as a conical vessel, a barrel shaped vessel and a curved vessel, are used to demonstrate the added value of customized SGs compared to off-the-shelf SGs. The numerical investigation has shown large benefits of the highly customized SGs compared to off-the-shelf SGs with respect to a better SG-vessel attachment and a considerable increase in SG fixation forces of up to 50% which indicate decreased likelihoods of EVAR related complications. Hence, this numerical proof of concept motivates further research and development of highly customized SGs for the use in challenging vessel morphologies.

Cell focal adhesion clustering leads to decreased and homogenized basal strains.

The subendothelial matrix of the artery is a complex mechanical environment where endothelial cells respond to and affect changes upon the underlying substrate. Our recent work has demonstrated that endothelial cell strain heterogeneity increases on a more heterogeneous underlying subendothelial matrix, and these cells display increased focal adhesion presence on stiffer substrate areas. However, the impact of these grouped focal adhesions on endothelial cell strains has not been explored. Here, we use finite element modeling to investigate the effects of microscale stiffness heterogeneities and focal adhesion location and stiffness on endothelial cell strains. Shear stress applied to the apical cell layer demonstrated a minimal effect on cell strain values, while substrate stretch had a greater effect on cell strain in the cell-substrate model. The addition of focal adhesions into the computational model (cell-FA-substrate model) predicted a decrease and homogenization of the cell strains. For simulations including focal adhesions, stiffer and more distributed adhesions caused increased and more heterogeneous endothelial cell strains. Overall, our data indicate that cells may group focal adhesions to minimize and homogenize their basal strains.

A Preliminary Computational Investigation Into the Flow of PEG in Rat Myocardial Tissue for Regenerative Therapy.

Myocardial infarction (MI), a type of cardiovascular disease, affects a significant proportion of people around the world. Traditionally, non-communicable chronic diseases were largely associated with aging populations in higher income countries. It is now evident that low- to middle-income countries are also affected and in these settings, younger individuals are at high risk. Currently, interventions for MI prolong the time to heart failure. Regenerative medicine and stem cell therapy have the potential to mitigate the effects of MI and to significantly improve the quality of life for patients. The main drawback with these therapies is that many of the injected cells are lost due to the vigorous motion of the heart. Great effort has been directed toward the development of scaffolds which can be injected alongside stem cells, in an attempt to improve retention and cell engraftment. In some cases, the scaffold alone has been seen to improve heart function. This study focuses on a synthetic polyethylene glycol (PEG) based hydrogel which is injected into the heart to improve left ventricular function following MI. Many studies in literature characterize PEG as a Newtonian fluid within a specified shear rate range, on the macroscale. The aim of the study is to characterize the flow of a 20 kDa PEG on the microscale, where the behavior is likely to deviate from macroscale flow patterns. Micro particle image velocimetry (µPIV) is used to observe flow behavior in microchannels, representing the gaps in myocardial tissue. The fluid exhibits non-Newtonian, shear-thinning behavior at this scale. Idealized two-dimensional computational fluid dynamics (CFD) models of PEG flow in microchannels are then developed and validated using the µPIV study. The validated computational model is applied to a realistic, microscopy-derived myocardial tissue model. From the realistic tissue reconstruction, it is evident that the myocardial flow region plays an important role in the distribution of PEG, and therefore, in the retention of material.

Kinematic boundary conditions substantially impact in silico ventricular function.

Computational cardiac mechanical models, individualized to the patient, have the potential to elucidate the fundamentals of cardiac (patho-)physiology, enable non-invasive quantification of clinically significant metrics (eg, stiffness, active contraction, work), and anticipate the potential efficacy of therapeutic cardiovascular intervention. In a clinical setting, however, the available imaging resolution is often limited, which limits cardiac models to focus on the ventricles, without including the atria, valves, and proximal arteries and veins. In such models, the absence of surrounding structures needs to be accounted for by imposing realistic kinematic boundary conditions, which, for prognostic purposes, are preferably generic and thus non-image derived. Unfortunately, the literature on cardiac models shows no consistent approach to kinematically constrain the myocardium. The impact of different approaches (eg, fully constrained base, constrained epi-ring) on the predictive capacity of cardiac mechanical models has not been thoroughly studied. For that reason, this study first gives an overview of current approaches to kinematically constrain (bi) ventricular models. Next, we developed a patient-specific in silico biventricular model that compares well with literature and in vivo recorded strains. Alternative constraints were introduced to assess the influence of commonly used mechanical boundary conditions on both the predicted global functional behavior of the in-silico heart (cavity volumes, stroke volume, ejection fraction) and local strain distributions. Meaningful differences in global functioning were found between different kinematic anchoring strategies, which brought forward the importance of selecting appropriate boundary conditions for biventricular models that, in the near future, may inform clinical intervention. However, whilst statistically significant differences were also found in local strain distributions, these differences were minor and mostly confined to the region close to the applied boundary conditions.