On the Automatic Decellularisation of Porcine Aortae: A Repeatability Study Using a Non-Enzymatic Approach.

OBJECTIVE: To investigate the repeatability of automatic decellularisation of porcine aortae using a non-enzymatic approach, addressing current limitations associated with other automatic decellularisation processes. MATERIALS AND METHODS: Individual porcine aortae (n = 3) were resected and every third segment (n = 4) was allocated to one of three different groups: a control or a manually or automatically decellularised group. Manual and automatic decellularisation was performed using Triton X-100 (2% v/v) and sodium deoxycholate. Protein preservation and the elimination of a galactosyl-α(1,3)galactose (GAL) epitope were measured using immunohistochemistry and protein binding assays. The presence of residual DNA was determined with gel electrophoresis and spectrophotometry. Scaffold integrity was characterised with scanning electron microscopy and uni-axial tensile testing. Manual and automatic results were compared to one another, to control groups and to current gold standards. RESULTS: The results were comparable to those of current gold standard decellularisation techniques. Successful repeatability was achieved, both manually and automatically, with little effect on mechanical characteristics. Complete acellularity was not confirmed in either decellularisation group. Protein preservation was consistent in both the manually and automatically decellularised groups and between each individual aorta. Elimination of GAL was not achieved. CONCLUSION: Repeatable automatic decellularisation of porcine aortae is feasible using a Triton X-100-sodium deoxycholate protocol. Protein preservation was satisfactory; however, gold standard thresholds for permissible residual DNA levels were not achieved. Future research will focus on addressing this issue by optimisation of the existing protocol for thick tissues.

Automatic decellularization of ovine aorta-derived extracellular matrix offers reduced processing and attendee times while being as effective as manual techniques.

OBJECTIVE: To construct an automatic decellularization platform (ADP) for preparing xenogenic extracellular matrices (ECMs), and to demonstrate that automatic decellularization for preparing xenogenic ECMs reduces processing time, requires fewer attendee hours, and is as effective as the manual gold standard preparation protocols. MATERIALS AND METHODS: A soft tissue ADP was constructed and ovine aorta was harvested (n=9). Manual and automatic decellularization was performed on aortic tissue specimens and both groups were compared. The presence of acellularity was assessed with viability/cytotoxicity assays, and the presence of residual ovine DNA was determined with gel electrophoresis and spectrophotometry. Scaffold integrity was characterized with scanning electron microscopy (SEM) and uniaxial tensile testing. RESULTS: Acellularity was confirmed with both preparation techniques and DNA concentrations measuring 540±130 and 590±270 ng/mg wet weight and the control measuring 6690±1210 ng/mg wet weight (p<0.05). SEM demonstrated no differences in the surface architecture of ECMs prepared by both techniques. Uniaxial testing demonstrated no significant differences in the incremental elastic moduli E below a stretch ratio of 2.70λ in both groups and a large reduction in E was recorded when both groups were compared with control samples above a stretch ratio of 1.7. CONCLUSION: Automatic decellularization of ovine aorta is as effective as gold standard manual decellularization protocols. Future research will focus on optimizing the automated decellularization technique and on upscaling protocols.

Regions of high wall stress can predict the future location of rupture of abdominal aortic aneurysm.

Predicting the wall stress in abdominal aortic aneurysm (AAA) using computational modeling may be a useful adjunct to traditional clinical parameters that indicate the risk of rupture. Maximum diameter has been shown to have many limitations, and using current technology it is possible to provide a patient-specific computational risk assessment using routinely acquired medical images. We present a case of AAA rupture where the exact rupture point was clearly visible on the computed tomography (CT) images. A blind computational study based on CT scans acquired 4 months earlier predicted elevated wall stresses in the same region that later experienced rupture.

Optimizing the fat and water content of impaction bone allograft.

Fresh morselized impacted bone graft usually fails due to shear forces. The presence of fat, water, and marrow particles act as interparticle lubricants, reducing the interlocking of particles and allowing the graft to move more freely. Furthermore, the presence of this incompressible fluid damps and resists compressive forces during impaction, preventing the graft particles from moving into a closer formation. We believe there exists an ideal concentration of fat and water that will maximize resistance to shear forces. We performed mechanical shear testing in vitro on morselized human femoral heads, varying the amount of fat and water to determine their optimum concentrations. Level of fat and water were determined that increased strength by 36% over unaltered bone graft. This is most closely approximated in an operating room by washing and subsequently squeezing the bone graft. Optimizing the fat and water content of bone graft produces a stronger graft that is more resistant to shear stresses, protecting the surgical construct until bone growth can occur.

Mechanical characterisation of unidirectional and cross-directional multilayered urinary bladder matrix (UBM) scaffolds.

Multilayered biological scaffolds derived from mammalian extracellular matrix (ECM) have shown promising long-term clinical results when reconstructing damaged tissues and organs. Despite their established clinical applicability, experimental studies that describe the effects of alternate manufacturing protocols on an ECM's mechanical properties are lacking. In the present study the mechanical properties of multilayered 'unidirectional' porcine urinary bladder matrix (UBM) scaffolds were determined in favour of its longitudinal and circumferential axes. The scaffold's unidirectional mechanical properties were then compared with 'cross-directional' UBM scaffolds. The results showed significant variations when alternate manufacturing protocols for multilayered UBM were applied. Unidirectional longitudinal UBM remained the strongest biomaterial on a consistent basis. Its failure strength occurred at 4.79±0.85MPa compared to 3.36±0.53MPa for unidirectional circumferential and 2.91±1.05MPa for cross-directional UBM respectively (p<0.0001). Distensibility was greatest in unidirectional circumferential UBM with failure extension occurring at 14.77±1.66mm. In comparison, failure extension occurred at 12.88±0.94mm and 13.04±4.35mm for unidirectional longitudinal and cross-directional UBM respectively (p=0.0024). The present study demonstrates that predefined manufacturing protocols for UBM should be considered when reconstructing anatomical structures with specific mechanical requirements.

Use of regional mechanical properties of abdominal aortic aneurysms to advance finite element modeling of rupture risk.

PURPOSE: To investigate the use of regional variations in the mechanical properties of abdominal aortic aneurysms (AAA) in finite element (FE) modeling of AAA rupture risk, which has heretofore assumed homogeneous mechanical tissue properties. METHODS: Electrocardiogram-gated computed tomography scans from 3 male patients with known infrarenal AAA were used to characterize the behavior of the aneurysm in 4 different segments (posterior, anterior, and left and right lateral) at maximum diameter and above the infrarenal aorta. The elasticity of the aneurysm (circumferential cyclic strain, compliance, and the Hudetz incremental modulus) was calculated for each segment and the aneurysm as a whole. The FE analysis inclusive of prestress (pre-existing tensile stress) produced a detailed stress pattern on each of the aneurysm models under pressure loading. The 4 largest areas of stress in each region were considered in conjunction with the local regional properties of the segment to define a specific regional prestress rupture index (RPRI). RESULTS: In terms of elasticity, there were average reductions of 68% in circumferential cyclic strain and 63% in compliance, with a >5-fold increase in incremental modulus, between the healthy and the aneurysmal aorta for each patient. There were also regional variations in all elastic properties in each individual patient. The average difference in total stress inclusive of prestress was 59%, 67%, and 15%, respectively, for the 3 patients. Comparing the strain from FE models with the CT scans revealed an average difference in strain of 1.55% for the segmented models and 3.61% for the homogeneous models, which suggests that the segmented models more accurately reflect in vivo behavior. RPRI values were calculated for each segment for all patients. CONCLUSION: A greater understanding of the local material properties and their use in FE models is essential for greater accuracy in rupture prediction. Quantifying the regional behavior will yield insight into the changes in patient-specific aneurysms and increase understanding about the progression of aneurysmal disease.

In vivo feasibility case study for evaluating abdominal aortic aneurysm tissue properties and rupture potential using acoustic radiation force impulse imaging.

An abdominal aortic aneurysm (AAA) is defined as a permanent and irreversible localized dilatation of the abdominal aorta. A reliable, non-invasive method to assess the wall mechanics of an aneurysm may provide additional information regarding their susceptibility to rupture. Acoustic radiation force impulse (ARFI) imaging is a phenomenon associated with the propagation of acoustic waves in attenuating media. This study was a preliminary evaluation to explore the feasibility of using ARFI imaging to examine an AAA in vivo. A previously diagnosed in vivo aneurysm case study was imaged to demonstrate the viability of excitation of the abdominal aorta using ARFI imaging. Ex vivo experiments were used to assess an artificially induced aneurysm to establish its development and whether ARFI was able to capture the mechanical changes during artificial aneurysm formation. A combination of in vivo and ex vivo results demonstrated a proposed hypothesis of estimation of the tissue's stiffness properties. The study details a method for non-invasive rupture potential prediction of AAAs using patient-specific moduli to generate a physiological stiffness rupture potential index (PSRPI) of the AAA. Clinical feasibility of ARFI imaging as an additional surgical tool to interrogate AAAs was verified and methods to utilize this data as a diagnostic tool was demonstrated with the PSRPI.

Experimental validation of convection-diffusion discretisation scheme employed for computational modelling of biological mass transport.

BACKGROUND: The finite volume solver Fluent (Lebanon, NH, USA) is a computational fluid dynamics software employed to analyse biological mass-transport in the vasculature. A principal consideration for computational modelling of blood-side mass-transport is convection-diffusion discretisation scheme selection. Due to numerous discretisation schemes available when developing a mass-transport numerical model, the results obtained should either be validated against benchmark theoretical solutions or experimentally obtained results. METHODS: An idealised aneurysm model was selected for the experimental and computational mass-transport analysis of species concentration due to its well-defined recirculation region within the aneurysmal sac, allowing species concentration to vary slowly with time. The experimental results were obtained from fluid samples extracted from a glass aneurysm model, using the direct spectrophometric concentration measurement technique. The computational analysis was conducted using the four convection-diffusion discretisation schemes available to the Fluent user, including the First-Order Upwind, the Power Law, the Second-Order Upwind and the Quadratic Upstream Interpolation for Convective Kinetics (QUICK) schemes. The fluid has a diffusivity of 3.125 x 10-10 m2/s in water, resulting in a Peclet number of 2,560,000, indicating strongly convection-dominated flow. RESULTS: The discretisation scheme applied to the solution of the convection-diffusion equation, for blood-side mass-transport within the vasculature, has a significant influence on the resultant species concentration field. The First-Order Upwind and the Power Law schemes produce similar results. The Second-Order Upwind and QUICK schemes also correlate well but differ considerably from the concentration contour plots of the First-Order Upwind and Power Law schemes. The computational results were then compared to the experimental findings. An average error of 140% and 116% was demonstrated between the experimental results and those obtained from the First-Order Upwind and Power Law schemes, respectively. However, both the Second-Order upwind and QUICK schemes accurately predict species concentration under high Peclet number, convection-dominated flow conditions. CONCLUSION: Convection-diffusion discretisation scheme selection has a strong influence on resultant species concentration fields, as determined by CFD. Furthermore, either the Second-Order or QUICK discretisation schemes should be implemented when numerically modelling convection-dominated mass-transport conditions. Finally, care should be taken not to utilize computationally inexpensive discretisation schemes at the cost of accuracy in resultant species concentration.

New approaches to abdominal aortic aneurysm rupture risk assessment: engineering insights with clinical gain.

Abdominal aortic aneurysm (AAA) rupture remains a significant cause of death in the developed world. Current treatment approaches rely heavily on the size of the aneurysm to decide on the most appropriate time for clinical intervention and treatment. However, in recent years, several alternative rupture risk indicators have been proposed. This brief review examines some of these new approaches to AAA rupture risk assessment, from both numeric and experimental aspects and also what the future may hold for AAA rupture risk. Although numerically predicted wall stress, finite element analysis rupture index, rupture potential index, severity parameter, and geometric factors, such as asymmetry, have all been developed and show promise in possibly helping to predict AAA rupture risk, validation of these tools remains a significant challenge. Validation of biomechanics-based rupture indicators may be feasible by combining in vitro modeling of realistic AAA analogues with both retrospective and prospective monitoring and modeling of AAA cases. Peak wall stress is arguably the primary result obtained from numeric analyses; however, as the majority of ruptures occur in the posterior and posterior-lateral regions, the role of posterior wall stress has also recently been highlighted as potentially significant. It is also known that wall stress alone is not enough to cause rupture, as wall strength plays an equal role. Therefore, should a biomechanics-based rupture risk be implemented? There have been some significant steps, both numerically and experimentally, toward answering this and other questions relating to AAA rupture risk prediction, yet regardless of the efforts that are under way in several laboratories, the introduction of a numerically predicted rupture risk parameter into the clinicians' decision-making process may still be quite some time away.

Acoustic radiation force impulse imaging on ex vivo abdominal aortic aneurysm model.

A method for reliable, noninvasive estimation of abdominal aortic aneurysms (AAA) wall mechanics may be a useful clinical tool for rupture prediction. An in vitro AAA model was developed from an excised porcine aorta with elastase treatment. The AAA model behaviour was analysed using acoustic radiation force impulse (ARFI) imaging techniques to generate and measure wave propagation in both aneurysmal and normal aortic tissue. Opening angle measurement showed a fourfold decrease from healthy aorta to AAA model and pathologic analysis verified this elastin degradation. Maximum wave velocity at 180 mm Hg was 7 mm/ms for healthy tissue and 8.26 mm/ms for the aneurysmal tissue. The mechanical changes produced in the artificially induced aneurysm were found to be detectable using ARFI imaging.

Identification of rupture locations in patient-specific abdominal aortic aneurysms using experimental and computational techniques.

In the event of abdominal aortic aneurysm (AAA) rupture, the outcome is often death. This paper aims to experimentally identify the rupture locations of in vitro AAA models and validate these rupture sites using finite element analysis (FEA). Silicone rubber AAA models were manufactured using two different materials (Sylgard 160 and Sylgard 170, Dow Corning) and imaged using computed tomography (CT). Experimental models were inflated until rupture with high speed photography used to capture the site of rupture. 3D reconstructions from CT scans and subsequent FEA of these models enabled the wall stress and wall thickness to be determined for each of the geometries. Experimental models ruptured at regions of inflection, not at regions of maximum diameter. Rupture pressures (mean+/-SD) for the Sylgard 160 and Sylgard 170 models were 650.6+/-195.1mmHg and 410.7+/-159.9mmHg, respectively. Computational models accurately predicted the locations of rupture. Peak wall stress for the Sylgard 160 and Sylgard 170 models was 2.15+/-0.26MPa at an internal pressure of 650mmHg and 1.69+/-0.38MPa at an internal pressure of 410mmHg, respectively. Mean wall thickness of all models was 2.19+/-0.40mm, with a mean wall thickness at the location of rupture of 1.85+/-0.33 and 1.71+/-0.29mm for the Sylgard 160 and Sylgard 170 materials, respectively. Rupture occurred at the location of peak stress in 80% (16/20) of cases and at high stress regions but not peak stress in 10% (2/20) of cases. 10% (2/20) of models had defects in the AAA wall which moved the rupture location away from regions of elevated stress. The results presented may further contribute to the understanding of AAA biomechanics and ultimately AAA rupture prediction.

New pulsatile hydrostatic pressure bioreactor for vascular tissue-engineered constructs.

Mechanical conditioning represents a potential means to enhance the biochemical and biomechanical properties of tissue-engineered cell constructs. Bioreactors that can simulate physiologic conditions can play an important role in the preparation of tissue-engineered constructs. Although various forms of bioreactor systems are currently available, these have certain limitations, particularly when these are used for the creation of vascular constructs. The aim of the present report is to describe and validate a novel pressure bioreactor system for the creation of vascular tissue. Here, we present and discuss the design concepts, criteria, as well as the development of a novel pressure bioreactor. The system is compact and easily housed in an incubator to maintain sterility of the construct. Moreover, the proposed bioreactor, in addition to mimicking in vivo pressure conditions, is flexible, allowing different types of constructs to be exposed to various physiologic pressure conditions. The core bioreactor elements can be easily sterilized and have good ergonomic assembly characteristics. This system is a fundamental tool, which may enable us to make further advances in bioreactor technology and tissue engineering. The novel system allows for the application of pressure that may facilitate the growth and development of constructs needed to produce a tissue-engineered vascular graft.

An experimental and numerical comparison of the rupture locations of an abdominal aortic aneurysm.

PURPOSE: To identify the rupture locations of idealized physical models of abdominal aortic aneurysm (AAA) using an in-vitro setup and to compare the findings to those predicted numerically. METHODS: Five idealized AAAs were manufactured using Sylgard 184 silicone rubber, which had been mechanically characterized from tensile tests, tear tests, and finite element analysis. The models were then inflated to the point of rupture and recorded using a high-speed camera. Numerical modeling attempted to confirm these rupture locations. Regional variations in wall thickness of the silicone models was also quantified and applied to numerical models. RESULTS: Four of the 5 models tested ruptured at inflection points in the proximal and distal regions of the aneurysm sac and not at regions of maximum diameter. These findings agree with high stress regions computed numerically. Wall stress appears to be independent of wall thickness, with high stress occurring at regions of inflection regardless of wall thickness variations. CONCLUSION: According to these experimental and numerical findings, AAAs experience higher stresses at regions of inflection compared to regions of maximum diameter. Ruptures of the idealized silicone models occurred predominantly at the inflection points, as numerically predicted. Regions of inflection can be easily identified from basic 3-dimensional reconstruction; as ruptures appear to occur at inflection points, these findings may provide a useful insight into the clinical significance of inflection regions. This approach will be applied to patient-specific models in a future study.

Experimental modelling of aortic aneurysms: novel applications of silicone rubbers.

A range of silicone rubbers were created based on existing commercially available materials. These silicones were designed to be visually different from one another and have distinct material properties, in particular, ultimate tensile strengths and tear strengths. In total, eleven silicone rubbers were manufactured, with the materials designed to have a range of increasing tensile strengths from approximately 2 to 4 MPa, and increasing tear strengths from approximately 0.45 to 0.7 N/mm. The variations in silicones were detected using a standard colour analysis technique. Calibration curves were then created relating colour intensity to individual material properties. All eleven materials were characterised and a 1st order Ogden strain energy function applied. Material coefficients were determined and examined for effectiveness. Six idealised abdominal aortic aneurysm models were also created using the two base materials of the study, with a further model created using a new mixing technique to create a rubber model with randomly assigned material properties. These models were then examined using videoextensometry and compared to numerical results. Colour analysis revealed a statistically significant linear relationship (p<0.0009) with both tensile strength and tear strength, allowing material strength to be determined using a non-destructive experimental technique. The effectiveness of this technique was assessed by comparing predicted material properties to experimentally measured methods, with good agreement in the results. Videoextensometry and numerical modelling revealed minor percentage differences, with all results achieving significance (p<0.0009). This study has successfully designed and developed a range of silicone rubbers that have unique colour intensities and material strengths. Strengths can be readily determined using a non-destructive analysis technique with proven effectiveness. These silicones may further aid towards an improved understanding of the biomechanical behaviour of aneurysms using experimental techniques.

Vessel asymmetry as an additional diagnostic tool in the assessment of abdominal aortic aneurysms.

OBJECTIVE: Abdominal aortic aneurysm (AAA) rupture is believed to occur when the local mechanical stress exceeds the local mechanical strength of the wall tissue. On the basis of this hypothesis, the knowledge of the stress acting on the wall of an unruptured aneurysm could be useful in determining the risk of rupture. The role of asymmetry has previously been identified in idealized AAA models and is now studied using realistic AAAs in the current work. METHODS: Fifteen patient-specific AAAs were studied to estimate the relationship between wall stress and geometrical parameters. Three-dimensional AAA models were reconstructed from computed tomography scan data. The stress distribution on the AAA wall was evaluated by the finite element method, and peak wall stress was compared with both diameter and centerline asymmetry. A simple method of determining asymmetry was adapted and developed. Statistical analyses were performed to determine potential significance of results. RESULTS: Mean von Mises peak wall stress +/- standard deviation was 0.4505 +/- 0.14 MPa (range, 0.3157-0.9048 MPa). Posterior wall stress increases with anterior centerline asymmetry. Peak stress increased by 48% and posterior wall stress by 38% when asymmetry was introduced into a realistic AAA model. CONCLUSION: The relationship between posterior wall stress and AAA asymmetry showed that excessive bulging of one surface results in elevated wall stress on the opposite surface. Assessing the degree of bulging and asymmetry that is experienced in an individual AAA may be of benefit to surgeons in the decision-making process and may provide a useful adjunct to diameter as a surgical intervention guide.

A comparison of modelling techniques for computing wall stress in abdominal aortic aneurysms.

BACKGROUND: Aneurysms, in particular abdominal aortic aneurysms (AAA), form a significant portion of cardiovascular related deaths. There is much debate as to the most suitable tool for rupture prediction and interventional surgery of AAAs, and currently maximum diameter is used clinically as the determining factor for surgical intervention. Stress analysis techniques, such as finite element analysis (FEA) to compute the wall stress in patient-specific AAAs, have been regarded by some authors to be more clinically important than the use of a "one-size-fits-all" maximum diameter criterion, since some small AAAs have been shown to have higher wall stress than larger AAAs and have been known to rupture. METHODS: A patient-specific AAA was selected from our AAA database and 3D reconstruction was performed. The AAA was then modelled in this study using three different approaches, namely, AAA(SIMP), AAA(MOD) and AAA(COMP), with each model examined using linear and non-linear material properties. All models were analysed using the finite element method for wall stress distributions. RESULTS: Wall stress results show marked differences in peak wall stress results between the three methods. Peak wall stress was shown to reduce when more realistic parameters were utilised. It was also noted that wall stress was shown to reduce by 59% when modelled using the most accurate non-linear complex approach, compared to the same model without intraluminal thrombus. CONCLUSION: The results here show that using more realistic parameters affect resulting wall stress. The use of simplified computational modelling methods can lead to inaccurate stress distributions. Care should be taken when examining stress results found using simplified techniques, in particular, if the wall stress results are to have clinical importance.