Using Nanotechnology to Improve Soft Tissue Adhesion to Intraosseous Transcutaneous Amputation Prostheses (ITAP)

Intraosseous Transcutaneous Amputation Prosthesis (ITAP) is a new generation device in limb replacement that may solve the issues of the externally fixed stump socket prosthesis, helping to restore the original limb function. ITAP is implanted into the medullary cavity of bone with an abutment that protrudes through the skin for limb attachment. The skin-implant interface is maintained with a flange, which resides below the epithelium, and is designed with pores to enhance soft tissue sub-epithelial ingrowth. This goals of this design are to stabilises the soft tissue, reducing the relative movements and seals the skin ITAP interface preventing bacterial infection. The current ITAP design includes a bi-dimensional flange that results in poor soft tissue attachment and, consequently, in the failure of the implant. My thesis aims to investigate firstly the effect of a new three dimensional porous flange on soft tissue attachment and ingrowth both in-vivo and in a clinical study with animal patients. Secondly, to improve epithelial and sub-epithelial attachment to the flange in-vitro and ex-vivo by modifying the surface of the ITAP with TiO2 nanotubes which have been shown to enhance cell attachment and have the potential to prevent downgrowth and infection around ITAP. The key original contributions to knowledge from my thesis are that firstly porous flanges increase the soft tissue attachment and ingrowth, contributing to stabilize the implant (p-value = 0.01 comparing 1000µm and 1250µm porosity with smooth titanium).. Moreover the size of nanotubes around 110nm, significantly increase the epithelial and sub-epithelial tissue attachment compared to the currently used smooth titanium, in- vivo, in vitro and ex-vivo, in order to create a stronger bound on the interface with the ITAP. In conclusion the combination of the porosity of the flange and the TiO2 nanotubes can significantly increase the soft tissue attachment and ingrowth to the flange in comparison with the commercially used bi-dimensional and smooth ITAPs

Sequence learning induces selectivity to multiple task parameters in mouse somatosensory cortex

Sequential temporal ordering and patterning are key features of natural signals, used by the brain to decode stimuli and perceive them as sensory objects. To explore how cortical neuronal activity underpins sequence discrimination, we developed a task in which mice distinguished between tactile “word” sequences constructed from distinct vibrations delivered to the whiskers, assembled in different orders. Animals licked to report the presence of the target sequence. Mice could respond to the earliest possible cues allowing discrimination, effectively solving the task as a “detection of change” problem, but enhanced their performance when responding later. Optogenetic inactivation showed that the somatosensory cortex was necessary for sequence discrimination. Two-photon imaging in layer 2/3 of the primary somatosensory “barrel” cortex (S1bf) revealed that, in well-trained animals, neurons had heterogeneous selectivity to multiple task variables including not just sensory input but also the animal’s action decision and the trial outcome (presence or absence of the predicted reward). Many neurons were activated preceding goal-directed licking, thus reflecting the animal’s learned action in response to the target sequence; these neurons were found as soon as mice learned to associate the rewarded sequence with licking. In contrast, learning evoked smaller changes in sensory response tuning: neurons responding to stimulus features were found in naive mice, and training did not generate neurons with enhanced temporal integration or categorical responses. Therefore, in S1bf, sequence learning results in neurons whose activity reflects the learned association between target sequence and licking rather than a refined representation of sensory features

Optimising soft tissue in-growth in vivo in additive layer manufactured osseointegrated transcutaneous implants

Osseointegrated transcutaneous implants could provide an alternative and improved means of attaching artificial limbs for amputees, however epithelial down growth, inflammation, and infections are common failure modalities associated with their use. To overcome these problems, a tight seal associated with the epidermal and dermal adhesion to the implant is crucial. This could be achieved with specific biomaterials (that mimic the surrounding tissue), or a tissue–specific design to enhance the proliferation and attachment of dermal fibroblasts and keratinocytes. The intraosseous transcutaneous amputation prosthesis is a new device with a pylon and a flange, which is specifically designed for optimising soft tissue attachment. Previously the flange has been fabricated using traditional machining techniques, however, the advent of additive layer manufacturing (ALM) has enabled 3–dimensional porous flanges with specific pore sizes to be used to optimise soft tissue integration and reduce failure of osseointegrated transcutaneous implants. The study aimed to investigate the effect of ALM–manufactured porous flanges on soft tissue ingrowth and attachment in an in vivo ovine model that replicates an osseointegrated percutaneous implant. At 12 and 24 weeks, epithelial downgrowth, dermal attachment and revascularisation into ALM–manufactured flanges with three different pore sizes were compared with machined controls where the pores were made using conventional drilling. The pore sizes of the ALM flanges were 700, 1000 and 1250 μm. We hypothesised that ALM porous flanges would reduce downgrowth, improve soft tissue integration and revascularisation compared with machined controls. The results supported our hypothesis with significantly greater soft tissue integration and revascularisation in ALM porous flanges compared with machined controls

Customized biofilm device for antibiofilm and antibacterial screening of newly developed nanostructured silver and zinc coatings

Background Bacterial colonisation on implantable device surfaces is estimated to cause more than half of healthcare-associated infections. The application of inorganic coatings onto implantable devices limits/prevents microbial contaminations. However, reliable and high-throughput deposition technologies and experimental trials of metal coatings for biomedical applications are missing. Here, we propose the combination of the Ionized Jet Deposition (IJD) technology for metal-coating application, with the Calgary Biofilm Device (CBD) for high-throughput antibacterial and antibiofilm screening, to develop and screen novel metal-based coatings. Results The films are composed of nanosized spherical aggregates of metallic silver or zinc oxide with a homogeneous and highly rough surface topography. The antibacterial and antibiofilm activity of the coatings is related with the Gram staining, being Ag and Zn coatings more effective against gram-negative and gram-positive bacteria, respectively. The antibacterial/antibiofilm effect is proportional to the amount of metal deposited that influences the amount of metal ions released. The roughness also impacts the activity, mostly for Zn coatings. Antibiofilm properties are stronger on biofilms developing on the coating than on biofilms formed on uncoated substrates. This suggests a higher antibiofilm effect arising from the direct contact bacteria-coating than that associated with the metal ions release. Proof-of-concept of application to titanium alloys, representative of orthopaedic prostheses, confirmed the antibiofilm results, validating the approach. In addition, MTT tests show that the coatings are non-cytotoxic and ICP demonstrates that they have suitable release duration (> 7 days), suggesting the applicability of these new generation metal-based coatings for the functionalization of biomedical devices.Conclusions The combination of the Calgary Biofilm Device with the Ionized Jet Deposition technology proved to be an innovative and powerful tool that allows to monitor both the metal ions release and the surface topography of the films, which makes it suitable for the study of the antibacterial and antibiofilm activity of nanostructured materials. The results obtained with the CBD were validated with coatings on titanium alloys and extended by also considering the anti-adhesion properties and biocompatibility. In view of upcoming application in orthopaedics, these evaluations would be useful for the development of materials with pleiotropic antimicrobial mechanisms

Expression and characterization of two new alkane-inducible cytochrome P450s from Trichoderma harzianum

Abstract The inducibility CYPs by various carbon sources, including some n-alkanes and fatty acids, has been studied in Trichoderma harzianum. It was observed that n-dodecane and a mixture of fatty acids were good inducers of total CYP content and ω-hydroxylase of lauric acid, a marker for ω-hydroxylation of n-alkanes. By RACE it was isolated a cDNA containing an open reading frame of 1520 bp which encoded a CYP52 protein of 520 amino acids. Further, another n-alkane inducible CYP was identified in a library of T. harzianum by LC-MS/Ms analysis of a microsomal protein band induced by n-dodecane exposure. Thus, the filamentous fungus T. harzianum is expected to have a CYP dependent conversion of alkanes to fatty acids and their incorporation into cellular lipids