Mohammad T. Abu-Rub

Regeneration and repair of tendon and ligament tissue using collagen fibre biomaterials.

Collagen fibres are ubiquitous macromolecular assemblies in nature, providing the structures that support tensile mechanical loads within the human body. Aligned type I collagen fibres are the primary structural motif for tendon and ligament, and therefore biomaterials based on these structures are considered promising candidates for mediating regeneration of these tissues. However, despite considerable investigation, there remains no collagen-fibre-based biomaterial that has undergone clinical evaluation for this application. Recent research in this area has significantly enhanced our understanding of these complex and challenging biomaterials, and is reinvigorating interest in the development of such structures to recapitulate mechanical function. In this review we describe the progress to date towards a ligament or tendon regeneration template based on collagen fibre scaffolds. We highlight reports of particular relevance to the development of the underlying biomaterials science in this area. In addition, the potential for tailoring and manipulating the interactions between collagen fibres and biological systems, as hybrid biomaterial-biological ensembles, is discussed in the context of developing novel tissue engineering strategies for tendon and ligament.

Nano-textured self-assembled aligned collagen hydrogels promote directional neurite guidance and overcome inhibition by myelin associated glycoprotein

The development of nerve guidance conduits is constantly evolving as the need arises for therapies for spinal cord injury. In addition to providing a path for regrowing axons to reconnect with their appropriate targets, the structural and biochemical cues provided by these conduits should be permissive for directional neurite outgrowth and be protective against inhibition in the vicinity of the injury site. Here, we adapted the use of iso-electric focusing to drive the alignment of supramolecular fibrils into self-assembled collagen hydrogels (∼300 µm diameter), and tested those hydrogels for the ability to direct and enhance the migration of neurites. Structural characterization revealed anisotropic alignment of nanofibrillar aggregates (∼20 nm diameter), arranged in micron-scale bundles (∼1 to 2 µm diameter) similar to the hierarchical size scales observed in native tissues. Neurite outgrowth extended bidirectionally along the axes of aligned hydrogels. Furthermore, it was shown that, as opposed to poly-D-lysine, neurite outgrowth on aligned hydrogels is not inhibited in the presence of myelin-associated glycoprotein (p > 0.05). These results highlight for the first time a structural and biochemical role for iso-electrically aligned collagen hydrogels in controlling neuronal growth, and indicate that the short-term signaling associated with these hydrogels can be used in adjunct therapy following injury to the spinal cord.

Essential modification of the Sircol Collagen Assay for the accurate quantification of collagen content in complex protein solutions

Collagen contains the unique imino acid hydroxyproline (HyPro), which is involved in the stabilization of this triple helical molecule. The concentration of HyPro is customarily used to calculate the total collagen content in a cell culture environment and in acid hydrolysates of normal and pathophysiological tissues. Radiolabelling, chromatographic and calorimetric assays have been developed over the years for the accurate determination of collagen content through HyPro estimation. Recently, the Sircol Collagen Assay (SCA) has been almost exclusively adopted as the fastest and simplest colorimetric method for the determination of collagen concentration in complex protein solutions. We show here that the colorimetric SCA, which is based on the binding of Sirius red (SR) to collagen, is flawed by interference of non-collagenous proteins (e.g. serum). In fact, we demonstrate that SCA in cell culture systems and tissue hydrolysates results in a dramatic overestimation of collagen content ranging from 3- to 24-fold. In order to rescue this otherwise very practical assay, we introduce a simple purification procedure that allows the removal of interfering non-collagenous proteins from culture media and tissue samples so that accurate measurements with SCA are now possible.

The neurotoxicity of gene vectors and its amelioration by packaging with collagen hollow spheres

Over the last twenty years there have been several reports on the use of nonviral vectors to facilitate gene transfer in the mammalian brain. Whilst a large emphasis has been placed on vector transfection efficiency, the study of the adverse effects upon the brain, caused by the vectors themselves, remains completely overshadowed. To this end, a study was undertaken to study the tissue response to three commercially available transfection agents in the brain of adult Sprague Dawley rats. The response to these transfection agents was compared to adeno-associated viral vector (AAV), PBS and naked DNA. Furthermore, the use of a collagen hollow sphere (CHS) sustained delivery system was analysed for its ability to reduce striatal toxicity of the most predominantly studied polymer vector, polyethyleneimine (PEI). The size of the gross tissue loss at the injection site was analysed after immunohistochemical staining and was used as an indication of acute toxicity. Polymeric vectors showed similar levels of acute brain toxicity as seen with AAV, and CHS were able to significantly reduce the toxicity of the PEI vector. In addition; the host response to the vectors was measured in terms of reactive astrocytes and microglial cell recruitment. To understand whether this gross tissue loss was caused by the direct toxicity of the vectors themselves an in vitro study on primary astrocytes was conducted. All vectors reduced the viability of the cells which is brought about by direct necrosis and apoptosis. The CHS delivery system reduced cell necrosis in the early stages of post administration. In conclusion, whilst polymeric gene vectors cause acute necrosis, administration in the brain causes adverse effects no worse than that of an AAV vector. Furthermore, packaging the PEI vector with CHS reduces surface charge and direct toxicity without elevating the host response.

GDNF Gene Delivery via a 2-(Dimethylamino)ethyl Methacrylate Based Cyclized Knot Polymer for Neuronal Cell Applications

Nonviral genetic therapeutic intervention strategies for neurological disorders hold great promise, but a lack of vector efficacy, coupled with vector toxicity, continue to hinder progress. Here we report the application of a newly developed class of polymer, distinctly different from conventional branched polymers, as a transfection agent for the delivery of glial cell line derived neurotrophic factor (GDNF) encoding gene. This new 2-(dimethylamino)ethyl methacrylate (DMAEMA) based cyclized knot polymer was studied for neuronal cell transfection applications, in comparison to branched polyethyleneimine (PEI). While showing a similar transfection profile over multiple cell types, the cyclized knot polymer showed far lower toxicity. In addition, transfection of Neu7 astrocytes with the GDNF encoding gene was able to cause neurite outgrowth when cocultured with dorsal root ganglia (DRGs). The cyclized knot polymer assessed here (PD-E 8%PEG), synthesized via a simple one-pot reaction, was shown to have great potential for neuronal gene therapy applications.

Spinal cord injury in vitro: modelling axon growth inhibition

Over the past three decades, tremendous progress has been made in elucidating mechanisms underlying regenerative failure after spinal cord injury and in devising therapeutic approaches to promote functional nerve regeneration. Various in vitro assays have been developed using brain and/or spinal cord neuronal cells to study axon growth in conditions that represent the post-injury environment. This review outlines the current models used to dissect, analyze and manipulate specific aspects of spinal cord injury leading to axon growth inhibition.

Electromechanical properties of dried tendon and isoelectrically focused collagen hydrogels

Assembling artificial collagenous tissues with structural, functional, and mechanical properties which mimic natural tissues is of vital importance for many tissue engineering applications. While the electro-mechanical properties of collagen are thought to play a role in, for example, bone formation and remodeling, this functional property has not been adequately addressed in engineered tissues. Here the electro-mechanical properties of rat tail tendon are compared with those of dried isoelectrically focused collagen hydrogels using piezoresponse force microscopy under ambient conditions. In both the natural tissue and the engineered hydrogel D-periodic type I collagen fibrils are observed, which exhibit shear piezoelectricity. While both tissues also exhibit fibrils with parallel orientations, Fourier transform analysis has revealed that the degree of parallel alignment of the fibrils in the tendon is three times that of the dried hydrogel. The results obtained demonstrate that isoelectrically focused collagen has similar structural and electro-mechanical properties to that of tendon, which is relevant for tissue engineering applications.

Non-viral xylosyltransferase-1 siRNA delivery as an effective alternative to chondroitinase in an in vitro model of reactive astrocytes

Reactive astrocytosis and the subsequent glial scar is ubiquitous to injuries of the central nervous system, especially spinal cord injury (SCI) and primarily serves to protect against further damage, but is also a prominent inhibitor of regeneration. Manipulating the glial scar by targeting chondroitin sulfate proteoglycans (CSPGs) has been the focus of much study as a means to improve axon regeneration and subsequently functional recovery. In this study we investigate the ability of small interfering RNA (siRNA) delivered by a non-viral polymer vector to silence the rate-limiting enzyme involved in CSPG synthesis. Gene expression of this enzyme, xylosyltransferase-1, was silenced by 65% in Neu7 astrocytes which conferred a reduced expression of CSPGs. Furthermore, conditioned medium taken from treated Neu7s, or co-culture experiments with dorsal root ganglia (DRG) showed that siRNA treatment resulted in a more permissive environment for DRG neurite outgrowth than treatment with chondroitinase ABC alone. These results indicate that there is a role for targeted siRNA therapy using polymeric vectors to facilitate regeneration of injured axons following central nervous system injury.