Production and Preparation of Porcine Urinary Bladder Matrix (UBM) for Urinary Bladder Tissue-Engineering Purposes.

Surgical repair for the end stage bladder disease utilises vascularised, autogenous and mucus-secreting gastrointestinal tissue to replace the diseased organ or to augment inadequate bladder tissue. Post-operatively, the compliance of the bowel is often enough to restore the basic shape, structure and function of the urinary bladder; however, lifelong post-operative complications are common. Comorbidities that result from interposition of intestinal tissue are metabolic and/or neuromechanical, and their incidence approaches 100%. The debilitating comorbidities and complications associated with such urological procedures may be mitigated by the availability of alternative, tissue-engineered, animal-derived extracellular matrix (ECM) scaffolds such as porcine urinary bladder matrix (UBM). Porcine UBM is a decellularized biocompatible, biodegradable biomaterial derived from the porcine urinary bladder. This chapter aims to describe the production and preparation techniques for porcine UBM for urinary bladder regenerative purposes.

Xenogenic extracellular matrices as potential biomaterials for interposition grafting in urological surgery.

PURPOSE: The field of tissue engineering focuses on developing strategies for reconstructing injured, diseased, and congenitally absent tissues and organs. During the last decade urologists have benefited from remodeling and regenerative properties of bioscaffolds derived from xenogenic extracellular matrices. We comprehensively reviewed the current literature on structural and functional characteristics of xenogenic extracellular matrix grafting since it was first described in urological surgery. We also reviewed the clinical limitations, and assessed the potential for safe and effective urological application of extracellular matrix grafting in place of autogenous tissue. MATERIALS AND METHODS: We performed literature searches for English language publications using the PubMed® and MEDLINE® databases. Keywords included "xenogenic," "extracellular matrix" and "genitourinary tract applications." A total of 112 articles were scrutinized, of which 50 were suitable for review based on clinical relevance and importance of content. RESULTS: Since the mid 1990s xenogenic extracellular matrices have been used to successfully treat a number of pathological conditions that affect the upper and lower genitourinary tract. They are typically prepared from porcine organs such as small intestine and bladder. These organs are harvested and subjected to decellularization and sterilization techniques before surgical implantation. Bioinductive growth factors that are retained during the preparation process induce constructive tissue remodeling as the extracellular matrix is simultaneously degraded and excreted. However, recent documented concerns over durability, decreased mechanical strength and residual porcine DNA after preparation techniques have temporarily hampered the potential of extracellular matrices as a reliable replacement for genitourinary tract structures. CONCLUSIONS: Extracellular matrices are a useful alternative for successfully treating a number of urological conditions that affect the genitourinary tract. However, clinical concerns regarding mechanical limitations and biosafety need to be addressed before their long-term role in reconstructive urological surgery can be clearly established.

Engineering silicone rubbers for in vitro studies: creating AAA models and ILT analogues with physiological properties.

In vitro studies of abdominal aortic aneurysm (AAA) have been widely reported. Frequently mock artery models with intraluminal thrombus (ILT) analogs are used to mimic the in vivo AAA. While the models used may be physiological, their properties are frequently either not reported or investigated. This study is concerned with the testing and characterization of previously used vessel analog materials and the development of new materials for the manufacture of AAA models. These materials were used in conjunction with a previously validated injection molding technique to manufacture AAA models of ideal geometry. To determine the model properties (stiffness (beta) and compliance), the diameter change of each AAA model was investigated under incrementally increasing internal pressures and compared with published in vivo studies to determine if the models behaved physiologically. A FEA study was implemented to determine if the pressure-diameter change behavior of the models could be predicted numerically. ILT analogs were also manufactured and characterized. Ideal models were manufactured with ILT analog internal to the aneurysm region, and the effect of the ILT analog on the model compliance and stiffness was investigated. The wall materials had similar properties (E(init) 2.22 MPa and 1.57 MPa) to aortic tissue at physiological pressures (1.8 MPa (from literature)). ILT analogs had a similar Young's modulus (0.24 MPa and 0.33 MPa) to the medial layer of ILT (0.28 MPa (from literature)). All models had aneurysm sac compliance (2.62-8.01 x 10(-4)/mm Hg) in the physiological range (1.8-9.4 x 10(-4)/mm Hg (from literature)). The necks of the AAA models had similar stiffness (20.44-29.83) to healthy aortas (17.5+/-5.5 (from literature)). Good agreement was seen between the diameter changes due to pressurization in the experimental and FEA wall models with a maximum difference of 7.3% at 120 mm Hg. It was also determined that the inclusion of ILT analog in the sac of the models could have an effect on the compliance of the model neck. Ideal AAA models with physiological properties were manufactured. The behavior of these models due to pressurization was predicted using finite element analysis, validating this technique for the future design of realistic physiological AAA models. Addition of ILT analogs in the aneurysm sac was shown to affect neck behavior. This could have implications for endovascular AAA repair due to the importance of the neck for stent-graft fixation.

Using a National Register as a Sampling Frame from the Perspective of an Early Career Researcher.

BACKGROUND: Selection of a sampling frame is a key component of conducting survey-based research. This article discusses the use of a national register, the Dental Register, as a sampling frame from the perspective of an early career researcher. METHODS: While conducting a survey-based study of a nationally representative sample of general dentists in Ireland, I documented the difficulties I encountered while using a national register. As a research assistant and novice researcher, I recorded the advantages and disadvantages I discovered over the course of the project and its impact on the study. CONCLUSION: While using a national register has advantages such as a readily available sample of the target population, there are also inherent disadvantages depending on the manner in which records are recorded. KNOWLEDGE TRANSFER STATEMENT: This article can be used as an informative guide to researchers in selecting a sampling frame, with particular emphasis on the use of a national register in selecting a nationally representative sample of dentists.

Novel phase separated polycaprolactone/collagen scaffolds for cartilage tissue engineering.

Osteoarthritis is the leading cause of pain and disability worldwide. Despite treatment availability, fully functional and suitable long term treatments for large cartilage defects are yet to be sought. Cartilage tissue engineering provides an alternative treatment option with the potential of cartilage regeneration. Previously, scaffolds have been enhanced through coating with collagen type I, however, equal distribution of collagen and collagen penetration throughout the scaffold have been poor. Herein, this study aims to employ thermally induced phase separation to fabricate porous hybrid polycaprolactone (PCL)/collagen type I scaffolds, with equally distributed collagen type I throughout the scaffold. PCL/collagen scaffolds were produced using polycaprolactone, varying concentrations of collagen type I and acetic acid as the solvent. Scaffolds were seeded with chondrocytes for 14 days. Scaffolds possessed an interconnected and porous structure, which altered with an increase in collagen concentration. Collagen type I antibody staining revealed the presence of equally distributed collagen within the PCL fibres. A reduction in collagen type I concentration influenced the compressive properties of the PCL/collagen type 1 scaffolds. Specifically, 0.2% wt/vol concentration of collagen presented to have compressive properties similar to that of the native cartilage at 10% strain. All scaffolds allowed cell attachment and collagen scaffolds showed a greater number of viable chondrocytes after 14 days of culture, compared to PCL control. This study demonstrated the ability of fabricating PCL/collagen type I scaffolds which exhibit easily tuneable porosity and compressive properties. Further investigation on the long term feasibility of these scaffolds is warranted.

Optimization of SDS exposure on preservation of ECM characteristics in whole organ decellularization of rat kidneys.

Renal transplantation is well established as the optimal form of renal replacement therapy but is restricted by the limited pool of organs available for transplantation. The whole organ decellularisation approach is leading the way for a regenerative medicine solution towards bioengineered organ replacements. However, systematic preoptimization of both decellularization and recellularization parameters is essential prior to any potential clinical application and should be the next stage in the evolution of whole organ decellularization as a potential strategy for bioengineered organ replacements. Here we have systematically assessed two fundamental parameters (concentration and duration of perfusion) with regards to the effects of differing exposure to the most commonly used single decellularizing agent (sodium dodecyl sulphate/SDS) in the perfusion decellularization process for whole rat kidney ECM bioscaffolds, with findings showing improved preservation of both structural and functional components of the whole kidney ECM bioscaffold. Whole kidney bioscaffolds based on our enhanced protocol were successfully recellularized with rat primary renal cells and mesenchymal stromal cells. These findings should be widely applicable to decellularized whole organ bioscaffolds and their optimization in the development of regenerated organ replacements for transplantation. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1352-1360, 2017.

The effects of stent interaction on porcine urinary bladder matrix employed as stent-graft materials.

Deployment of stent-grafts, derived from synthetic biomaterials, is an established minimally invasive approach for effectively treating abdominal aortic aneurysms (AAAs). However, a notable disadvantage associated with this surgical technique is migration of the deployed stent-graft due to poor biocompatibility and inadequate integration in vivo. Recently, tissue-engineered extracellular matrices (ECMs) have shown early promise as integrating stabilisation collars in this setting due to their ability to induce a constructive tissue remodelling response after in vivo implantation. In the present study the effects of stent loading on an ECM׳s mechanical properties were investigated by characterising the compression and loading effects of endovascular stents on porcine urinary bladder matrix (UBM) scaffolds. Results demonstrated that the maximum stress was induced when the stent force was 8-times higher than a standard commercially available stent-graft and this represented about 20% of the failure strength of the UBM material. In addition, the influence of stent shape was also investigated. Findings demonstrated that the stress induced was higher for circular stents at low forces and a higher stress was induced on square stents when increased force was applied. Our findings demonstrate that porcine UBM possesses sufficient mechanical strength to withstand the compression and loading effects of commercially available stent-grafts in the setting of endovascular aneurysm repair.

Development of a rotational cell-seeding system for tubularized extracellular matrix (ECM) scaffolds in vascular surgery.

Tubularized porcine extracellular matrices (ECMs) are under investigation as adjuvant scaffolds for endovascular aneurismal repair (EVAR). Limitations with tubularized ECMs in this setting include difficulties in achieving a confluent endothelium on the scaffold's luminal surface prior to in vivo implantation. In this in vitro study a rotational "cell-seeding rig" (RCR) was constructed to assess the potential for endothelialization of tubular ECM constructs. Human aortic endothelial cells (HAECs) were cultured onto the luminal surfaces of tubular porcine urinary bladder matrix (UBM) scaffolds and rotated in the RCR at experimental rotational speeds. Results showed that endothelial attachment occurred at a rotation speed of six revolutions per hour. HAECs continued to proliferate after the initial attachment period of 24 h and formed a confluent endothelial monolayer after 14 days of growth. Our results demonstrate that RCRs facilitate attachment of HAECs in vitro at a speed of six revolutions per hour. The endothelialization technique presented in the current study may be important for advancing tissue-engineering approaches to address some of the current limitations in endovascular treatments of abdominal aortic aneurysms.

Combinatorial scaffold morphologies for zonal articular cartilage engineering.

Articular cartilage lesions are a particular challenge for regenerative medicine strategies as cartilage function stems from a complex depth-dependent organization. Tissue engineering scaffolds that vary in morphology and function offer a template for zone-specific cartilage extracellular matrix (ECM) production and mechanical properties. We fabricated multi-zone cartilage scaffolds by the electrostatic deposition of polymer microfibres onto particulate-templated scaffolds produced with 0.03 or 1.0mm(3) porogens. The scaffolds allowed ample space for chondrocyte ECM production within the bulk while also mimicking the structural organization and functional interface of cartilage's superficial zone. Addition of aligned fibre membranes enhanced the mechanical and surface properties of particulate-templated scaffolds. Zonal analysis of scaffolds demonstrated region-specific variations in chondrocyte number, sulfated GAG-rich ECM, and chondrocytic gene expression. Specifically, smaller porogens (0.03mm(3)) yielded significantly higher sGAG accumulation and aggrecan gene expression. Our results demonstrate that bilayered scaffolds mimic some key structural characteristics of native cartilage, support in vitro cartilage formation, and have superior features to homogeneous particulate-templated scaffolds. We propose that these scaffolds offer promise for regenerative medicine strategies to repair articular cartilage lesions.

On the potential of hydrated storage for naturally derived ECMs and associated effects on mechanical and cellular performance.

Tissue engineered acellular vascular grafts are an emerging concept in the development of vascular prostheses for the minimally invasive treatment of cardiovascular diseases. Extracellular matrix (ECM) scaffolds, such as small intestinal submucosa (SIS) and urinary bladder matrix (UBM), offer many advantages over currently available synthetic devices. However, storage of such biomaterials can unduly influence the scaffold properties. This study evaluated the effects of up to 16 weeks hydrated storage on the mechanical and cellular performance of stented and unstented tubular scaffolds. This study aimed to demonstrate the viability, mechanical integrity, and bioactive potential of xenogeneic ECMs as potential off-the-shelf vascular prosthetic devices. Rehydrated ECM samples versus the lyophilized controls showed an increase in UTS and stiffness. The mechanical strength of all samples evaluated was above the average reported aortic tissue failure strength and more compliant than current synthetic materials employed. Post-storage cellular bioactivity investigations indicated that both ECM scaffolds tested were unaffected by increased hydrated storage duration when compared with the controls. Overall, the results indicate that the biomechanical and biologic properties of ECMs are not negatively affected by long-term hydrated storage. Therefore, with further investigations, naturally derived ECM materials may offer potential as an off-the-shelf therapeutic treatment of cardiovascular diseases.

The effect of vessel material properties and pulsatile wall motion on the fixation of a proximal stent of an endovascular graft.

Migration is a serious failure mechanism associated with endovascular abdominal aortic aneurysm (AAA) repair (EVAR). The effect of vessel material properties and pulsatile wall motion on stent fixation has not been previously investigated. A proximal stent from a commercially available stent graft was implanted into the proximal neck of silicone rubber abdominal aortic aneurysm models of varying proximal neck stiffness (ß=25.39 and 20.44). The stent was then dislodged by placing distal force on the stent struts. The peak force to completely dislodge the stent was measured using a loadcell. Dislodgment was performed at ambient pressure with no flow (NF) and during pulsatile flow (PF) at pressures of 120/80 mmHg and 140/100 mmHg to determine if pulsatile wall motions affected the dislodgement force. An imaging analysis was performed at ambient pressure and at pressures of 120 mmHg and 140 mmHg to investigate diameter changes on the model due to the radial force of the stent and internal pressurisation. Stent displacement forces were ~50% higher in the stiffer model (7.16-8.4 N) than in the more compliant model (3.67-4.21 N). The mean displacement force was significantly reduced by 10.95-12.83% from the case of NF to the case of PF at 120/80 mmHg. A further increase in pressure to 140/120 mmHg had no significant effect on the displacement force. The imaging analysis showed that the diameter in the region of the stent was 0.37 mm greater in the less stiff model at all the pressures which could reduce the fixation of the stent. The results suggest that the fixation of passively fixated aortic stents could be comprised in more compliant walls and that pulsatile motions of the wall can reduce the maximum stent fixation.

Porcine extracellular matrix scaffolds in reconstructive urology: An ex vivo comparative study of their biomechanical properties.

Functional reconstruction of the human urinary bladder has been attempted by replacing defective bladder tissue with tissue-engineered xenogenic extracellular matrix (ECM) scaffolds. However, experimental studies that demonstrate the effects of implanted ECMs on important biomechanical properties such as total bladder capacity (TBC) and compliance (C) are lacking. In the current study, the effects of ECM scaffold surface area (SA) on TBC and C was assessed, ex vivo, in an ovine model (n=5). TBC and C were measured at pressures (P) of 5, 10, 15 and 20 mm Hg prior to performing a 3×3 cm (9 cm(2)) partial cystectomy defect. Equal-sized 3×3 cm (9 cm(2)) and larger 6×6 cm (36 cm(2)) urinary bladder matrix (UBM) scaffolds of porcine origin replaced the 3×3 cm cystectomy defect, and TBC and C were re-recorded for comparative analysis. The results showed that TBC decreased by 39.6%±0.005% (122.9 ml±15 ml, p<0.05) and C by 38.9%±0.51%, (ΔP=0-5mmHg, p<0.05) in ovine bladders reconstructed with 3×3 cm UBM scaffolds compared to their native values. It was also found that TBC increased by 25.6±0.64% (64.2 ml ± 8.8 ml, p>0.05) and C by 24.5±0.43% (ΔP=0-5mmHg, p>0.05) in the 6×6 cm UBM scaffold group compared to the 3×3 cm UBM scaffold group; however, these values were not statistically significant. The present work demonstrates that a fourfold increase in ECM scaffold SA relative to its intended defect does not lead to a significant improvement in TBC and C values.

Extracellular matrices as advanced scaffolds for vascular tissue engineering.

An alternative non-vascular extracellular material was obtained by decellularisation of porcine urinary bladder and examined for its potential as scaffold for vascular tissue engineering. Analysis using Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM) and Laser Scanning Microscopy (LSCM) showed a porous interconnective microarchitecture, an abundance of well preserved fibers on the abluminal side and a micropatterned flat luminal surface. Uniaxial tensile testing revealed a strength of 1.9+/-0.3 MPa for the rehydrated material in a phosphate buffered saline medium for the ECM-UBM sheet and these results comparable to those of native artery of a middle aged human. Multilamination of the UBM increases the tensile properties in general (9+/-0.45 MPa for 2 layer, 30+/-0.6 MPa for 4 layers construct), with no effect on elongation capacities (38-40%) of the material. Contact-angle measurements indicated that the ECM-UBM surface exhibited a hydrophylic characteristic and better wettability than any vascular synthetic materials. Comparison of the initial adhesion in the multiplayer ECM constructs was evaluated and was found not to be altered by the preparation. The HAECs and HSMC shown an excellent adherence, spread and proliferation on the ECM-UBM material with the preservation of the cell phenotype. The level of MMP-1 and MMP-9 produced by endothelial cells was evaluated in this study and provides some data on the remodelling capacity of the scaffold. Vascular cell seeded-UBM constructs may also provide a suitable and affordable in vitro model for cell-physiology and drug development studies, which may elucidate to the mechanisms of vascular disease formation, and its prevention and treatment.

3D reconstruction and manufacture of real abdominal aortic aneurysms: from CT scan to silicone model.

Abdominal aortic aneurysm (AAA) can be defined as a permanent and irreversible dilation of the infrarenal aorta. AAAs are often considered to be an aorta with a diameter 1.5 times the normal infrarenal aorta diameter. This paper describes a technique to manufacture realistic silicone AAA models for use with experimental studies. This paper is concerned with the reconstruction and manufacturing process of patient-specific AAAs. 3D reconstruction from computed tomography scan data allows the AAA to be created. Mould sets are then designed for these AAA models utilizing computer aided designcomputer aided manufacture techniques and combined with the injection-moulding method. Silicone rubber forms the basis of the resulting AAA model. Assessment of wall thickness and overall percentage difference from the final silicone model to that of the computer-generated model was performed. In these realistic AAA models, wall thickness was found to vary by an average of 9.21%. The percentage difference in wall thickness recorded can be attributed to the contraction of the casting wax and the expansion of the silicone during model manufacture. This method may be used in conjunction with wall stress studies using the photoelastic method or in fluid dynamic studies using a laser-Doppler anemometry. In conclusion, these patient-specific rubber AAA models can be used in experimental investigations, but should be assessed for wall thickness variability once manufactured.

Sotos syndrome with hypodontia.

Sotos syndrome, or cerebral gigantism, is a rare genetic condition characterized by tall stature, gigantism, dolichocephaly, advanced bone age and learning disability. The purpose of this case report is to highlight the dental management of a 10-year-old boy with Sotos syndrome who presented with hypodontia and dental caries.

3-D numerical simulation of blood flow through models of the human aorta.

A Spiral Computerized Tomography (CT) scan of the aorta were obtained from a single subject and three model variations were examined. Computational fluid dynamics modeling of all three models showed variations in the velocity contours along the aortic arch with differences in the boundary layer growth and recirculation regions. Further down-stream, all three models showed very similar velocity profiles during maximum velocity with differences occurring in the decelerating part of the pulse. Flow patterns obtained from transient 3-D computational fluid dynamics are influenced by different reconstruction methods and the pulsatility of the flow. Caution is required when analyzing models based on CT scans.