Controlled Release of Bioactive Molecules from Collagen-Based Scaffolds for Bone Repair

Although bone has an intrinsic capacity for self repair, the healing of large bone defects that typically present in humans often involves complications which can result in failure to heal, leading to delayed union or non-union of the defect. Due to limitations of current therapeutic approaches of autografting and allografting, the use of tissue engineered scaffolds has emerged. Despite some success with this approach, a major limitation is creating functionally vascularised constructs. In addition, scaffolds often require an additional stimulus such as a growth factor (GF) to fully heal large fractures. However, current GF factor delivery approaches are often associated with limited success due to uncontrolled protein release. These issues arise due to the high concentrations of protein required to elicit healing which, in turn, is due to the result of the short half-life of proteins in vivo. These shortcomings have led to the emergence of scaffolds integrated with polymeric carriers which encapsulate, protect and control the release of GFs. In the context of producing superior bone graft substitutes, this thesis focused on fabricating a new generation of scaffolds by functionally enhancing collagen-hydroxyapatite (CHA) and collagen-glycosaminoglycan (CG) scaffolds, optimised specifically for bone repair in our laboratory, in order to make them capable of coupling both angio- and osteogenesis for promoting enhanced regeneration. Specifically, this research aimed to investigate the potential for controlled release of bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) encapsulated in alginate and poly (lactic-co-glycolic acid) (PLGA) microparticles (MPs) and subsequently, to investigate whether a series of functionalised scaffolds incorporating this system could promote enhanced bone repair in a critical size defect in vivo model. As a GF-free alternative approach, a final aim of this research was to develop and characterise a collagen-based scaffold incorporating pro-angiogenic and pro-osteogenic cobalt-bioactive glass (BG) and to assess the in vitro ability of the material to promote these processes. In the study shown in Chapter 2 of this thesis, feasible preparation methods for GF-eluting MPs were established. It was shown that PLGA MPs fabricated by double emulsion and spray dried alginate MPs were capable of not only controlling and prolonging the release of two of the main GFs pertinent to bone repair but also retaining their bioactivity. Additionally, it was demonstrated that the release kinetics of GFs can be tailored by using different polymers for encapsulation. In Chapter 3 it was shown that with an optimised fabrication process, it was possible to develop GF-releasing CHA scaffolds containing MPs without interfering with the structural properties of the scaffold previously optimised for bone repair. Additionally, sustained release of bioactive GFs from the optimised scaffolds was demonstrated with kinetics resembling the in vivo condition: the early expression of VEGF from alginate MPs and sustained release of BMP-2 from PLGA MPs. Hence, functionalised scaffolds were capable of eliciting a pro-angiogenic and pro-osteogenic response in vitro. Having demonstrated the functionality of these materials in vitro, VEGF and/or BMP-2-eluting scaffolds were implanted in a rat calvarial defect model where they enhanced healing compared to non-eluting scaffolds as well as non-treated animals. Ultimately, VEGF-releasing CHA scaffolds accelerated healing to the greatest extent offering an ideal platform to promote both vasculogenesis and bone repair. In Chapter 5 a GF-free biomaterial alternative, a novel scaffold was successfully fabricated by the incorporation of cobalt-eluting BG particles into a CG scaffold. This material was capable of stimulating angiogenesis in vitro via the release of cobalt, a known hypoxia mimic, as well as osteogenesis via the dissolution of osteoinductive BG particles. This study indicated that an angiogenic and osteogenic response may be achievable exclusively through a GF-free biomaterials based approach. Collectively, the research presented in this thesis has led to the development of a new generation of functionally enhanced collagen-based scaffolds. Specifically, polymeric MPs were developed that were capable of controlling the release of bioactive GFs pertinent to bone repair. Functionalised scaffolds containing MPs were capable of the sustained release of bioactive concentrations of VEGF and BMP-2 from MPs within the matrix. The alginate-VEGF scaffold emerged as the GF-eluting scaffold which promoted the most accelerated neovascularisation and bone repair in vivo. Furthermore this thesis has shown that novel cobalt-BG containing CG scaffolds enhanced cell-mediated osteogenesis and angiogenesis representing a desirable, economical, alternative to GF-based delivery for the regeneration of bone tissue. These novel functionalised scaffolds offer the advantage of off-the-shelf availability and lack the need to for in vitro cell culture. In addition, the delivery systems developed in this thesis have enormous potential in regenerative medicine, as they could be tuned in terms of the composition of the collagen-based scaffold and released therapeutic to promote healing of a diverse range of tissues and organs in addition to bone

Long-term controlled delivery of rhBMP-2 from collagen-hydroxyapatite scaffolds for superior bone tissue regeneration.

The clinical utilization of recombinant human bone morphogenetic protein 2 (rhBMP-2) delivery systems for bone regeneration has been associated with very severe side effects, which are due to the non-controlled and non-targeted delivery of the growth factor from its collagen sponge carrier post-implantation which necessitates supraphysiological doses. However, rhBMP-2 presents outstanding regenerative properties and thus there is an unmet need for a biocompatible, fully resorbable delivery system for the controlled, targeted release of this protein. With this in mind, the purpose of this work was to design and develop a delivery system to release low rhBMP-2 doses from a collagen-hydroxyapatite (CHA) scaffold which had previously been optimized for bone regeneration and recently demonstrated significant healing in vivo. In order to enhance the potential for clinical translation by minimizing the design complexity and thus upscaling and regulatory hurdles of the device, a microparticle and chemical functionalization-free approach was chosen to fulfill this aim. RhBMP-2 was combined with a CHA scaffold using a lyophilization fabrication process to produce a highly porous CHA scaffold supporting the controlled release of the protein over the course of 21days while maintaining in vitro bioactivity as demonstrated by enhanced alkaline phosphatase activity and calcium production by preosteoblasts cultured on the scaffold. When implanted in vivo, these materials demonstrated increased levels of healing of critical-sized rat calvarial defects 8weeks post-implantation compared to an empty defect and unloaded CHA scaffold, without eliciting bone anomalies or adjacent bone resorption. These results demonstrate that it is possible to achieve bone regeneration using 30 times less rhBMP-2 than INFUSE®, the current clinical gold standard; thus, this work represents the first step of the development of a rhBMP-2 eluting material with immense clinical potential

Hypoxia-mimicking bioactive glass/collagen glycosaminoglycan composite scaffolds to enhance angiogenesis and bone repair.

One of the biggest challenges in regenerative medicine is promoting sufficient vascularisation of tissue-engineered constructs. One approach to overcome this challenge is to target the cellular hypoxia inducible factor (HIF-1α) pathway, which responds to low oxygen concentration (hypoxia) and results in the activation of numerous pro-angiogenic genes including vascular endothelial growth factor (VEGF). Cobalt ions are known to mimic hypoxia by artificially stabilising the HIF-1α transcription factor. Here, resorbable bioactive glass particles (38 μm and 100 μm) with cobalt ions incorporated into the glass network were used to create bioactive glass/collagen-glycosaminoglycan scaffolds optimised for bone tissue engineering. Inclusion of the bioactive glass improved the compressive modulus of the resulting composite scaffolds while maintaining high degrees of porosity (\u3e97%). Moreover, in vitro analysis demonstrated that the incorporation of cobalt bioactive glass with a mean particle size of 100 μm significantly enhanced the production and expression of VEGF in endothelial cells, and cobalt bioactive glass/collagen-glycosaminoglycan scaffold conditioned media also promoted enhanced tubule formation. Furthermore, our results prove the ability of these scaffolds to support osteoblast cell proliferation and osteogenesis in all bioactive glass/collagen-glycosaminoglycan scaffolds irrespective of the particle size. In summary, we have developed a hypoxia-mimicking tissue-engineered scaffold with pro-angiogenic and pro-osteogenic capabilities that may encourage bone tissue regeneration and overcome the problem of inadequate vascularisation of grafts commonly seen in the field of tissue engineering

Controlled release of vascular endothelial growth factor from spray-dried alginate microparticles in collagen-hydroxyapatite scaffolds for promoting vascularization and bone repair.

A major limitation with current tissue-engineering approaches is creating functionally vascularized constructs that can successfully integrate with the host; this often leads to implant failure, due to avascular necrosis. In order to overcome this, the objective of the present work was to develop a method to incorporate growth factor-eluting alginate microparticles (MPs) into freeze-dried, collagen-based scaffolds. A collagen-hydroxyapatite (CHA) scaffold, previously optimized for bone regeneration, was functionalized for the sustained delivery of an angiogenic growth factor, vascular endothelial growth factor (VEGF), with the aim of facilitating angiogenesis and enhancing bone regeneration. VEGF was initially encapsulated in alginate MPs by spray-drying, producing particles of \u3c 10 µm in diameter. This process was found to effectively encapsulate and control VEGF release while maintaining its stability and bioactivity post-processing. These VEGF-MPs were then incorporated into CHA scaffolds, leading to homogeneous distribution throughout the interconnected scaffold pore structure. The scaffolds were capable of sustained release of bioactive VEGF for up to 35 days, which was proficient at increasing tubule formation by endothelial cells in vitro. When implanted in vivo in a rat calvarial defect model, this scaffold enhanced vessel formation, resulting in increased bone regeneration compared to empty-defect and VEGF-free scaffolds. This biologically functionalized scaffold, composed entirely of natural-based materials, may offer an ideal platform to promote angiogenesis and tissue regeneration. Copyright © 2015 John Wiley \u26 Sons, Ltd

Do TETRA (Airwave) base station signals have a short-term impact on health and well-being? A randomized double-blind provocation study

Background: "Airwave" is the new communication system currently being rolled out across the United Kingdom for the police and emergency services, based on the Terrestrial Trunked Radio Telecommunications System (TETRA). Some police officers have complained about skin rashes, nausea, headaches, and depression as a consequence of using their Airwave handsets. In addition, a small subgroup in the population self-report being sensitive to electromagnetic fields (EMFs) in general. Objectives: We conducted a randomized double-blind provocation study to establish whether short-term exposure to a TETRA base station signal has an impact on the health and well-being of individuals with self-reported "electrosensitivity" and of participants who served as controls.Methods: Fifty-one individuals with self-reported electrosensitivity and 132 age and sex-matched controls participated in an open provocation test; 48 sensitive and 132 control participants went on to complete double-blind tests in a fully screened semianechoic chamber. Heart rate, skin conductance, and blood pressure readings provided objective indices of short-term physiological response. Visual analog scales and symptom scales provided subjective indices of well-being.Results: We found no differences on any measure between TETRA and sham (no signal) under double-blind conditions for either controls or electrosensitive participants, and neither group could detect the presence of a TETRA signal at rates greater than chance (50%). When conditions were not double blind, however, the self-reported electrosensitive individuals did report feeling worse and experienced more severe symptoms during TETRA compared with sham.Conclusions: Our findings suggest that the adverse symptoms experienced by electrosensitive individuals are due to the belief of harm from TETRA base stations rather than to the low-level EMF exposure itself

Do threatening stimuli draw or hold visual attention in subclinical anxiety?

Biases in information processing undoubtedly play an important role in the maintenance of emotion and emotional disorders. In an attentional cueing paradigm, threat words and angry faces had no advantage over positive or neutral words (or faces) in attracting attention to their own location, even for people who were highly state-anxious. In contrast, the presence of threatening cues (words and faces) had a strong impact on the disengagement of attention. When a threat cue was presented and a target subsequently presented in another location, high state-anxious individuals took longer to detect the target relative to when either a positive or a neutral cue was presented. It is concluded that threat-related stimuli affect attentional dwell time and the disengage component of attention, leaving the question of whether threat stimuli affect the shift component of attention open to debate

Chaos and Noise in Galactic Potentials

ABBREVIATED ABSTRACT: This paper summarises an investigation of the effects of weak friction and noise in time-independent, nonintegrable potentials which admit both regular and stochastic orbits. The aim is to understand the qualitative effects of internal and external irregularities associated, e.g., with discreteness effects or couplings to an external environment, which stars in any real galaxy must experience. The two principal conclusions are: (1) These irregularities can be important on time scales much shorter than the natural relaxation time scale t_R associated with the friction and noise. For stochastic orbits friction and noise induce an average exponential divergence from the unperturbed Hamiltonian trajectory at a rate set by the value of the local Lyapunov exponent. Even weak noise can make a pointwise interpretation of orbits suspect already on time scales much shorter than t_R. (2) The friction and noise can also have significant effects on the statistical properties of ensembles of stochastic orbits, these also occurring on time scales much shorter than t_R. Potential implications for galactic dynamics are discussed, including the problem of shadowing.Comment: 45 pages, uuencoded PostScript (figures included), LA-UR-94-282

Tandem amplification of SCCmec can drive high level methicillin resistance in MRSA

Hospital-associated methicillin-resistant Staphylococcus aureus (MRSA) strains typically express high-level, homogeneous (HoR) beta-lactam resistance, whereas community-associated MRSA (CA-MRSA) more commonly express low-level heterogeneous (HeR) resistance. Expression of the HoR phenotype typically requires both increased expression of the mecA gene, carried on the staphylococcal cassette chromosome mec element (SCCmec), and additional mutational event(s) elsewhere on the chromosome. Here the oxacillin concentration in a chemostat culture of the CA-MRSA strain USA300 was increased from 8 mu g/ml to 130 mu g/ml over 13 days to isolate highly oxacillin-resistant derivatives. A stable, small-colony variant, designated HoR34, which had become established in the chemostat culture was found to have acquired mutations in gdpP, clpX, guaA, and camS. Closer inspection of the genome sequence data further revealed that reads covering SCCmec were similar to 10 times overrepresented compared to other parts of the chromosome. Quantitative PCR (qPCR) confirmed >10-fold-higher levels of mecA DNA on the HoR34 chromosome, and MinION genome sequencing verified the presence of 10 tandem repeats of the SCCmec element. qPCR further demonstrated that subculture of HoR34 in various concentrations of oxacillin (0 to 100 mu g/ml) was accompanied by accordion-like contraction and amplification of the SCCmec element. Although slower growing than strain USA300, HoR34 outcompeted the parent strain in the presence of subinhibitory oxacillin. These data identify tandem amplification of the SCCmec element as a new mechanism of high-level methicillin resistance in MRSA, which may provide a competitive advantage for MRSA under antibiotic selection