Assessing student perception of the integration of portable wireless ultrasound imaging in undergraduate anatomy education.

Anatomy is the foundation of many physiology and healthcare-related degrees. With limited access to cadavers in many universities, it is essential to investigate techniques that could be utilized to support and enhance the teaching of anatomy. Ultrasound is used clinically to aid the diagnosis of a wide range of conditions by visualizing the anatomy of the patient. While research has investigated the advantages of ultrasound in medical education, the potential benefits of ultrasound in undergraduate bioscience degrees remain to be investigated. The aim of this study was to identify if a portable ultrasound probe that wirelessly attaches to a smartphone or tablet was perceived by students as beneficial for their understanding and learning of anatomy, and to identify if there were any barriers for students partaking in ultrasound sessions. Following five ultrasound-teaching sessions, 107 undergraduate students completed a 5-point likert questionnaire on their perception of the integration of portable ultrasound machines in anatomy education. The data indicated that 93% of students perceived that the ultrasound teaching sessions improved their anatomical understanding, 94% perceived that ultrasound increased their ability to understand the clinical relevance of learning anatomy, 97% enjoyed the sessions, and 95% of students believed that ultrasound should be integrated into anatomy teaching. In this study, we also found several barriers for students taking part in ultrasound sessions, including religious beliefs, and lacking adequate background knowledge. In conclusion, these findings demonstrate, for the first time, that students perceive portable ultrasound to enhance their anatomy studies, demonstrating the potential benefit the integration of ultrasound into the anatomy curriculum may serve within undergraduate bioscience courses.

Decellularisation and histological characterisation of porcine peripheral nerves.

Peripheral nerve injuries affect a large proportion of the global population, often causing significant morbidity and loss of function. Current treatment strategies include the use of implantable nerve guide conduits (NGC's) to direct regenerating axons between the proximal and distal ends of the nerve gap. However, NGC's are limited in their effectiveness at promoting regeneration Current NGCs are not suitable as substrates for supporting either neuronal or Schwann cell growth, as they lack an architecture similar to that of the native extracellular matrix (ECM) of the nerve. The aim of this study was to create an acellular porcine peripheral nerve using a novel decellularisation protocol, in order to eliminate the immunogenic cellular components of the tissue, while preserving the three-dimensional histoarchitecture and ECM components. Porcine peripheral nerve (sciatic branches were decellularised using a low concentration (0.1%; w/v) sodium dodecyl sulphate in conjunction with hypotonic buffers and protease inhibitors, and then sterilised using 0.1% (v/v) peracetic acid. Quantitative and qualitative analysis revealed a ≥95% (w/w) reduction in DNA content as well as preservation of the nerve fascicles and connective tissue. Acellular nerves were shown to have retained key ECM components such as collagen, laminin and fibronectin. Slow strain rate to failure testing demonstrated the biomechanical properties of acellular nerves to be comparable to fresh controls. In conclusion, we report the production of a biocompatible, biomechanically functional acellular scaffold, which may have use in peripheral nerve repair. Biotechnol. Bioeng. 2016;113: 2041-2053. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.

An anatomical study of porcine peripheral nerve and its potential use in nerve tissue engineering.

Current nerve tissue engineering applications are adopting xenogeneic nerve tissue as potential nerve grafts to help aid nerve regeneration. However, there is little literature that describes the exact location, anatomy and physiology of these nerves to highlight their potential as a donor graft. The aim of this study was to identify and characterise the structural and extracellular matrix (ECM) components of porcine peripheral nerves in the hind leg. Methods included the dissection of porcine nerves, localisation, characterisation and quantification of the ECM components and identification of nerve cells. Results showed a noticeable variance between porcine and rat nerve (a commonly studied species) in terms of fascicle number. The study also revealed that when porcine peripheral nerves branch, a decrease in fascicle number and size was evident. Porcine ECM and nerve fascicles were found to be predominately comprised of collagen together with glycosaminoglycans, laminin and fibronectin. Immunolabelling for nerve growth factor receptor p75 also revealed the localisation of Schwann cells around and inside the fascicles. In conclusion, it is shown that porcine peripheral nerves possess a microstructure similar to that found in rat, and is not dissimilar to human. This finding could extend to the suggestion that due to the similarities in anatomy to human nerve, porcine nerves may have utility as a nerve graft providing guidance and support to regenerating axons.

Effects of Chemical and Radiation Sterilisation on the Biological and Biomechanical Properties of Decellularised Porcine Peripheral Nerves.

There is a clinical need for novel graft materials for the repair of peripheral nerve defects. A decellularisation process has been developed for porcine peripheral nerves, yielding a material with potentially significant advantages over other devices currently being used clinically (such as autografts and nerve guidance conduits). Grafts derived from xenogeneic tissues should undergo sterilisation prior to clinical use. It has been reported that sterilisation methods may adversely affect the properties of decellularised tissues, and therefore potentially negatively impact on the ability to promote tissue regeneration. In this study, decellularised nerves were produced and sterilised by treatment with 0.1% (v/v) PAA, gamma radiation (25-28 kGy) or E Beam (33-37 kGy). The effect of sterilisation on the decellularised nerves was determined by cytotoxicity testing, histological staining, hydroxyproline assays, uniaxial tensile testing, antibody labelling for collagen type IV, laminin and fibronectin in the basal lamina, and differential scanning calorimetry. This study concluded that decellularised nerves retained biocompatibility following sterilisation. However, sterilisation affected the mechanical properties (PAA, gamma radiation), endoneurial structure and basement membrane composition (PAA) of decellularised nerves. No such alterations were observed following E Beam treatment, suggesting that this method may be preferable for the sterilisation of decellularised porcine peripheral nerves.

Ex-vivo recellularisation and stem cell differentiation of a decellularised rat dental pulp matrix.

Implementing the principles of tissue engineering within the clinical management of non-vital immature permanent teeth is of clinical interest. However, the ideal scaffold remains elusive. The aim of this work was to assess the feasibility of decellularising rat dental pulp tissue and evaluate the ability of such scaffold to support stem cell repopulation. Rat dental pulps were retrieved and divided into control and decellularised groups. The decellularisation protocol incorporated a low detergent concentration and hypotonic buffers. After decellularisation, the scaffolds were characterised histologically, immunohistochemistry and the residual DNA content quantified. Surface topography was also viewed under scanning electron microscopy. Biocompatibility was evaluated using cytotoxicity assays utilising L-929 cell line. Decellularised scaffolds were recellularised with human dental pulp stem cells up to 14 days in vitro. Cellular viability was assessed using LIVE/DEAD stain kit and the recellularised scaffolds were further assessed histologically and immunolabelled using makers for odontoblastic differentiation, cytoskeleton components and growth factors. Analysis of the decellularised scaffolds revealed an acellular matrix with histological preservation of structural components. Decellularised scaffolds were biocompatible and able to support stem cell survival following recellularisation. Immunolabelling of the recellularised scaffolds demonstrated positive cellular expression against the tested markers in culture. This study has demonstrated the feasibility of developing a biocompatible decellularised dental pulp scaffold, which is able to support dental pulp stem cell repopulation. Clinically, decellularised pulp tissue could possibly be a suitable scaffold for use within regenerative (reparative) endodontic techniques.

Development and characterisation of a full-thickness acellular porcine bladder matrix for tissue engineering.

The aim of this study was to produce a natural, acellular matrix from porcine bladder tissue for use as a scaffold in developing a tissue-engineered bladder replacement. Full-thickness, intact porcine bladders were decellularised by distention and immersion in hypotonic buffer containing 0.1% (w/v) SDS and nuclease enzymes. Histological analysis of the resultant matrices showed they were completely acellular; that the major structural proteins had been retained and that there were some residual poorly soluble intracellular proteins. The amount of DNA per mg dry weight of fresh porcine bladder was 2.8 (+/-0.1) microg/mg compared to 0.1 (+/-0.1) microg/mg in decellularised bladder and biochemical analysis showed proportional differences in the hydroxyproline and glycosaminoglycan content of the tissue before and after decellularisation. Uniaxial tensile testing indicated that decellularisation did not significantly compromise the ultimate tensile strength of the tissue. There was, however, an increase in the collagen and elastin phase slopes indicating decreased extensibility. Cytotoxicity assays using porcine smooth muscle cell cultures excluded the presence of soluble toxins in the biomaterial. In summary, a full-thickness natural acellular matrix retaining the major structural components and strength of the urinary bladder has been successfully developed. The matrix is biocompatible with bladder-derived cells and has potential for use in urological surgery and tissue-engineering applications.

Production of an acellular amniotic membrane matrix for use in tissue engineering.

A clinical need exists for an immunologically compatible surgical patch with a wide range of uses including soft tissue replacement, body wall repair, cardiovascular applications, and as a wound dressing. This study aimed to produce an acellular matrix from human amniotic membrane for future assessment as a surgical patch and a delivery system for epithelial cells. A novel detergent-based protocol was modified to remove all cellular components from amnion to render it non-immunogenic. Amnion was harvested within 24 h after elective caesarean section (n = 12). One sample group remained fresh, whereas the other was treated with 0.03% (w/v) sodium dodecyl sulphate, with hypotonic buffer and protease inhibitors, nuclease treatment, and terminal sterilization, using peracetic acid (0.1% v/v). Fresh and treated amnion was analyzed histologically for the presence of cells, deoxyribonucleic acid (DNA), collagen, glycosaminoglycans (GAGs), and elastin. Quantitative analysis was performed to determine levels of GAGs, elastin, hydroxyproline, denatured collagen, and DNA. The biomechanical properties of the membrane were determined using uniaxial tensile testing to failure. Histological analysis of treated human amnion showed complete removal of cellular components from the tissue; the histoarchitecture remained intact. All major structural components of the matrix were retained, including collagen type IV and I, laminin, and fibronectin. Differences were observed between fresh and decellularized amnion in matrix hydroxyproline (34.7 microg/mg vs 49.7 microg/mg), GAG (42.5 microg/mg vs 85.4 microg/mg), denatured collagen (2.2 microg/mg vs 1.7 microg/mg), and elastin (359.2 microg/mg vs 490.8 microg/mg) content. DNA content was diminished after treatment. Acellular matrices were biocompatible, cells grew in contact, and there was no decrease in cell viability after incubation with soluble tissue extracts. In addition, no significant reduction in ultimate tensile strength, extensibility, or elasticity was found after decellularization. Removal of the cellular components should eliminate immunological rejection. The resulting matrix was biocompatible in vitro and exhibited no adverse effects on cell morphology or viability.

Development and characterization of acellular porcine pulmonary valve scaffolds for tissue engineering.

Currently available replacement heart valves all have limitations. This study aimed to produce and characterize an acellular, biocompatible porcine pulmonary root conduit for reconstruction of the right ventricular outflow tract e.g., during Ross procedure. A process for the decellularization of porcine pulmonary roots was developed incorporating trypsin treatment of the adventitial surface of the scraped pulmonary artery and sequential treatment with hypotonic Tris buffer (HTB; 10 mM Tris pH 8.0, 0.1% (w/v) EDTA, and 10 KIU aprotinin), 0.1% (w/v) sodium dodecyl sulfate in HTB, two cycles of DNase and RNase, and sterilization with 0.1% (v/v) peracetic acid. Histology confirmed an absence of cells and retention of the gross histoarchitecture. Immunohistochemistry further confirmed cell removal and partial retention of the extracellular matrix, but a loss of collagen type IV. DNA levels were reduced by more than 96% throughout all regions of the acellular tissue and no functional genes were detected using polymerase chain reaction. Total collagen levels were retained but there was a significant loss of glycosaminoglycans following decellularization. The biomechanical, hydrodynamic, and leaflet kinematics properties were minimally affected by the process. Both immunohistochemical labeling and antibody absorption assay confirmed a lack of α-gal epitopes in the acellular porcine pulmonary roots and in vitro biocompatibility studies indicated that acellular leaflets and pulmonary arteries were not cytotoxic. Overall the acellular porcine pulmonary roots have excellent potential for development of a tissue substitute for right ventricular outflow tract reconstruction e.g., during the Ross procedure.

The use of antithrombotic therapies in reducing synthetic small-diameter vascular graft thrombosis.

BACKGROUND: Thrombosis of synthetic small-diameter bypass grafts remains a major problem. The aim of this article is to review the antithrombotic strategies that have been used in an attempt to reduce graft thrombogenicity. METHODS: A PubMed/MEDLINE search was performed using the search terms "vascular graft thrombosis," "small-diameter graft thrombosis," "synthetic graft thrombosis" combined with "antithrombotic," "antiplatelet," "anticoagulant," "Dacron," "PTFE," and "polyurethane." RESULTS: The majority of studies on antithrombotic therapies have used either in vitro models or in vivo animal experiments. Many of the therapies used in these settings do show antithrombotic efficacy against synthetic graft materials. There is however, a distinct lack of human in vivo studies to further delineate the performance and limitations of therapies displaying good antithrombotic characteristics. CONCLUSION: Very few antithrombotic therapies have translated into clinical use. More human in vivo studies are required to assess the efficacy and safety of such therapies.

Development and characterization of acellular allogeneic arterial matrices.

Surgeons have used cryopreserved vascular allografts successfully for many years to treat arterial occlusive disease and to repair arterial aneurysms. Vascular allografts demonstrate high patency rates but contain viable cells, which may evoke a rejection response following implantation. Removing the cells could prevent such a response and negate the need for cryopreservation and ultra-low temperature storage. The objectives of the study were to characterize human common femoral arteries and develop a decellularization protocol with a view to the generation of biocompatible and biomechanically functional vascular grafts for use in vascular bypass and arteriovenous access. The arteries were decellularized by subjecting the tissue to a single freeze-thaw cycle followed by sequential incubation in hypotonic tris buffer and low concentration sodium dodecyl sulphate. Each artery was disinfected using 0.1% (v/v) peracetic acid. Histological analysis demonstrated a lack of cells following decellularization and confirmed the integrity of the tissue histioarchitecture and retention of major structural proteins. There was a >95% reduction in DNA levels. The acellular tissues and extracts were not cytotoxic to either mouse 3T3 or baby hamster kidney cells. Biomechanical properties were determined by burst pressure, compliance, and tensile tests, which confirmed the retention of biomechanical properties following decellularization. In conclusion the study has developed a suitable protocol for the removal of cells from human common femoral arteries without adversely affecting the biochemical or biomechanical properties. These properties indicate the potential use for acellular human common femoral arteries for vascular bypass or arteriovenous access.

Investigation of the antiadhesive properties of human mesothelial cells cultured in vitro on implantable surgical materials.

The aim of the study was to evaluate the interactions of Permacol, Prolene mesh, Surgisis Gold, and Alloderm with human mesothelial cells in vitro. The capacity of primary human mesothelial cells to adhere to the surface of Alloderm, Surgisis Gold, Prolene mesh, and Permacol as well as support the proliferation and viability of the seeded cells was determined. Production of antifibrinolytic, fibrinolytic, and inflammatory mediators (IL-8, TPA, MMP-1, PAI-1, and TGF-beta) was assessed over an 8-day period. The adhesive nature of the implantable materials was determined by assessment of the strength of any fibrin clots formed between two surfaces of each implant material. Surgisis Gold and Permacol were capable of supporting the attachment and proliferation of primary human mesothelial cells and maintaining viability over an 8-day culture period. Mesothelial cells were shown to have covered the surface of Permacol and Surgisis Gold in a monolayer. The viability of cells cultured on Permacol was significantly greater than the other implant materials tested. Mesothelial cells cultured on Permacol or Surgisis were shown to be producing high levels of fibrinolytic compounds and low levels of antifibrinolytic and inflammatory mediators. Alloderm was shown to produce high levels of IL-8 and antifibrinolytic mediators when compared with the other implantable materials. Permacol was shown to be an unreliable surface for clot formation in vitro and any clots formed were shown to be significantly weaker than the clots produced between two surfaces of tissue culture plastic, Prolene mesh, Alloderm, and Surgisis Gold. This in vitro study indicated that Permacol and Surgisis Gold supported the growth and fibrinolytic activity of human mesothelial cells; however, Permacol was shown to be superior in this respect.

Cytocompatibility of poly(1,2 propandiol methacrylate) copolymer hydrogels and conetworks with or without alkyl amine functionality.

The cytocompatibility and adhesion of cells to biomaterials are key to their success in the clinic. Here we report a study of the toxicity, cell-adhesive properties and biocompatibility of a range of alkyl-aminated hydrogels and amphiphilic conetworks comprising 1,2-propandiol-3-methacrylate (GMA) as the hydrophilic component. Previously we had shown that addition of amines containing alkyl spacers of at 3-6 carbons or addition of oligo(butyl methacrylate) sequences to crosslinked polyGMA hydrogels could be used to produce a step change in cell adhesion. In this work we produced two series of polymer networks, based on polyGMA, which contained both of these structural features and we examined the effects that these materials had on A549 epithelial cells and human dermal fibroblasts. No toxicity was observed from either direct contact or from supernatants extracted over 48h. Each of the alkyl-aminated materials provided a good substrate for adhesion of both cell types whereas the non-alkyl-aminated materials were essentially non-cell-adhesive. Peritoneal murine macrophages were present on all of the materials and the activation of these adhered macrophages was investigated by determining the production of the pro-inflammatory cytokines, TNF-alpha, IL1beta and IL-6. All of the materials behaved similarly to a clinically acceptable control, Permacol (a decellularized collagen-based porcine derived material), and each material was much less activating than when the macrophages were in contact with lipopolysaccharide endotoxin. There were no differences in the capacity of the materials to activate TNF-alpha production by macrophages however, there was a trend towards stimulation of lower levels of IL-6 and IL-1beta by the alkyl-aminated materials.

Factors influencing the oxygen consumption rate of aortic valve interstitial cells: application to tissue engineering.

This study investigated the factors influencing the oxygen metabolism of aortic valve interstitial cells (VICs). Porcine VICs in cell suspension at different passages, and adhered to coverslips at different confluencies, as well as fresh porcine valve leaflets, were incubated in an oxygen respiration chamber at 37 degrees C in Dulbecco's modified Eagle's medium. The consumption rates at different oxygen concentrations were evaluated based on the Michaelis-Menten equation, and the corresponding maximum consumption rate (V(max)) and the Michaelis-Menten equation constant K(m) were determined. In all cases, the oxygen consumption rate was relatively constant until the concentration dropped to 5% (v/v). The metabolic activity of VICs in terms of oxygen consumption was dependent upon their in vitro passage number and proliferation status. These findings will provide valuable input to the selection of VICs with respect to their age and proliferation status for tissue engineering applications, as well as important input parameters for developing computational models of oxygen transport and optimization of the bioreactor conditions for heart valve tissue engineering.

Biocompatibility and potential of acellular human amniotic membrane to support the attachment and proliferation of allogeneic cells.

The aim of this study was to determine the biocompatibility of an acellular human amniotic membrane biomaterial, which may have clinical utility for cell delivery. Human amniotic membrane was decellularized using 0.03% (w/v) sodium dodecyl sulfate (SDS), with hypotonic tris buffer and protease inhibitors and nuclease treatment. The membrane was terminally sterilized using an optimal concentration of peracetic acid. Residual SDS present within the acellular membrane was quantified using radio-labeled C14 SDS. In vivo biocompatibility was assessed by implantation of acellular human amniotic membrane subcutaneously into mice for 3 months and comparison with fresh and glutaraldehyde-fixed tissue. Cellular infiltrate into the explanted tissues was characterized using monoclonal antibodies against the following cell surface markers: CD3, CD4, CD34, and F4/80. Calcification was determined using the Von Kossa's stain. The potential of acellular human amniotic membrane to support the attachment and proliferation, and maintain viability of primary human dermal fibroblasts and primary human dermal keratinocytes was assessed in vitro, using a static culture system. Peracetic acid at a concentration of 0.1% (v/v) was sufficient for the sterilization of acellular amniotic membrane. Levels of SDS present within the acellular tissue were 0.62 +/- 0.13 microg/mg. Analysis of explanted samples from the mice indicated that acellular amniotic membrane contained low numbers of T-cells and high numbers of fibroblastic cells, macrophages, and endothelial cells, indicative of a wound-healing response. There was no evidence of calcification present within explanted acellular amniotic membrane compared to explanted glutaraldehyde-fixed amniotic membrane. Acellular amniotic membrane was shown to be capable of supporting the attachment and proliferation of primary human fibroblasts and keratinocytes. The viability of the cells was maintained for up to 4 weeks. Cell-seeded acellular amniotic membrane has the potential for delivering autologous or allogeneic cells to treat a variety of conditions, including diabetic foot ulcers, corneal defects, and severe skin burns.