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Scaffolds are thought to be a key element needed for successful cartilage repair treatments, and this prospective extension study aimed to evaluate long-term structural and clinical outcomes following osteochondral defect treatment with a cell-free biphasic scaffold. Structural outcomes were assessed using quantitative 3-D magnetic resonance imaging (MRI) and morphological segmentation to determine the percentage of defect filling and repair cartilage T2 relaxation times, and clinical outcomes were determined with the modified Cincinnati Rating System, and the Knee Injury and Osteoarthritis Outcome Score (KOOS). Seventeen subjects with osteochondral defects in the knee were treated with ChondroMimetic scaffolds, from which 15 returned for long-term evaluation at a mean follow-up of 7.9 &plusmn, 0.3 years. The defects treated were trochlear donor sites for mosaicplasty in 13 subjects, and medial femoral condyle defects in 2 subjects. MRI analysis of scaffold-treated defects found a mean total defect filling of 95.2 &plusmn, 3.6%, and a tissue mean T2 relaxation time of 52.5 &plusmn, 4.8 ms, which was identical to the T2 of ipsilateral control cartilage (52.3 &plusmn, 9.2 ms). The overall modified Cincinnati Rating System score was statistically significant from baseline (p = 0.0065), and KOOS subscales were equivalent to other cartilage repair techniques. ChondroMimetic treatment resulted in a consistently high degree of osteochondral defect filling with durable, cartilage-like repair tissue at 7.9 years, potentially associated with clinical improvement.

Introduction In recent years, cell therapy has emerged as a promising therapeutic strategy for Parkinson’s disease. To increase cell viability, biomaterials are used to facilitate cell deposition, through injection, to the site of interest. However, the existing cell delivery approaches have shown limited success in clinical translation 1 .This study aims to develop a device for the delivery of a cell-embedded in situ forming hydrogel. Here, computational approaches on the delivery of collagen to the striatum are presented, to gain insight into different parameters affecting the delivery. Methods The delivery of collagen to the striatum was modelled computationally in the two-dimensional space. The striatum was modelled as a circular space, with an area of 3.98 cm2 corresponding to the mean volume of putamen in Parkinson’s disease patients2 . Within the finite volume method framework, the flow of collagen was considered incompressible, with non-Newtonian fluid behavior characterized experimentally, and constant inlet velocity corresponding to a maximum delivery volume. Results The effects of collagen injection on the velocity and pressure fields within the striatum were examined. Velocity streamlines and wall shear stress (WSS) values were also analysed near the edges of the needle, at the entrance of the collagen to the striatum. High WSS values may influence cell viability on the site of delivery. Conclusion Intrastriatal injection of a cell embedded hydrogel is a complex process which is not yet well characterized. Computational analysis of the delivery can assist to identify the obstacles facing clinical translation. Further analysis is required including 3D reconstruction from MRI images and computational modelling in the three-dimensional space.

Objectives:During recent years, microgels have emerged as an effective type of drug delivery system (DDS), due to their tuneable size, increased surface area and injectability. The current study focuses on the use of microfluidic techniques for the generation of monodisperse type-I collagen (col-I) microgels crosslinked with PEG-4S, encapsulating hollow collagen spheres with glial-derived neurotrophic factor (GDNF) and bone morphogenetic protein 2 (BMP-2) for regenerative therapies in Parkinson’s Disease and bone repair, respectively. Methods:Col-I (Collagen Solutions, UK) was crosslinked with 4S-Star-PEG succinimide glutarate (Jenkem, USA). Crosslinking occurred after merging col-I with PEG-4S crosslinker in the nozzle of a double-chamber capillary within an oil flow, gelification being completed in a coiled tube. Microgel size and mechanical properties were controlled with e.g. nozzle size and pH. Microgels were characterized with TNBS assay and their cytotoxicity with LIVE-DEAD® assay. Hollow spheres were prepared by covalently attaching collagen to silica templates which are later dissolved with hydrofluoric acid. Morphochemical characterization included: scanning electron microscopy, and Fourier-Transform Infrared. Hollow spheres were loaded with GDNF and BMP-2, seeded with different cell-types and encapsulated in the microgels within the microfluidic device. Results:Microgels with different size and stiffness were successfully synthetized in a glass microfluidic device. The microgels are non-cytotoxic to cells and foster cell growth at different crosslinker concentrations. In the synthesis of hollow spheres, no harmful by-products appeared, and their size ranged around 200 nm. Discussion:We demonstrate that microfluidics is an adequate technique for automatically generating monodisperse collagen microgels, and provides a simple tool for the posterior encapsulation of nanospheres and cells. The combination of microgels encapsulating hollow spheres is expected to provide a controlled and sustained delivery system of different therapeutic molecules.

Silica nanoparticles have been shown to have great potential as drug delivery systems (DDS), however, their fabrication often involves harsh chemicals and energy intensive laborious methods. This work details the employment of a bioinspired "green" method for the controlled synthesis of silica, use of the products to entrap and release drug molecules and their cytotoxicity in order to develop novel DDS. Bioinspired silica synthesis occurs at pH 7, room temperature and in less than 5 minutes, resulting in a rapid, cheaper and greener route. Drugs were loaded into silica during the silica formation, thus allowing a one step and one pot method for simultaneous silica synthesis and drug loading. We established that the drug release profile can be modulated by synthetic parameters, which can allow design of tailored DDS. A systematic investigation using a two level factorial design was adopted in order to identify the key synthetic parameters and quantify their effects on silica formation, drug loading and drug release. The observation that these new DDS are considerably less cytotoxic than their current counterparts, and exhibit additional benefits such as green synthesis and ease of functionalization, strengthens the argument for their future use in DDS and other biomedical applications. © 2014 the Partner Organisations.

Reconstituted collagen hydrogels are often used for in vitro studies of cell-matrix interaction and as scaffolds for tissue engineering. Understanding the mechanical and transport behaviours of collagen hydrogels is therefore extremely important, albeit difficult due to their very high water content (typically >99.5%). In the present study the mechanical behaviour of collagen hydrogels in confined compression was investigated using biphasic theory (J Biomechemical Engineering 102 (1980) 73), to ascertain whether the technique is sufficiently sensitive to determine differences in the characteristics of hydrogels of between 0.2% and 0.4% collagen. Peak stress, equilibrium stress, aggregate modulus and hydraulic permeability of the hydrogels exhibited sensitivity to collagen content, demonstrating that the technique is clearly able to discriminate between hydrogels with small differences in collagen content and may also be sensitive to factors that affect matrix remodelling. The results also offer additional insight into the deformation-dependent permeability of collagen hydrogels. This study suggests that confined compression, together with biphasic theory, is a suitable technique for assessing the mechanical properties of collagen hydrogels.

Conference submission on the development of a culture of 3T3 fibroblasts in collagen gels in the presence of ACE inhibitors presented at the annual congress of the british toxicology society

INTRODUCTION During recent years, microgels have emerged as an effective type of drug delivery system (DDS), showing advantages such as tuneable size, increased surface area and injectability1. Collagen has been extensively studied as a scaffold for regenerative medicine and drug delivery due to its biocompatibility, non-immunogenicity and degradability2,3. The current study focuses on the use of microfluidic techniques for the automated generation of monodisperse type-I collagen (col-I) microgels crosslinked with PEG-4S, encapsulating hollow collagen spheres with glial-derived neurotrophic factor (GDNF) and bone morphogenetic protein 2 (BMP-2) for regenerative therapies in Parkinson’s Disease and bone repair, respectively. MATERIALS AND METHODS Col-1 (Collagen Solutions, UK) stock was characterized in a rotational viscometer and UV-Vis spectrophotometer to determine the fibrillogenesis conditions. Collagen was crosslinked with 4S-Star-PEG succinimide glutarate (Jenkem, USA) to form hydrogels. Crosslinking occurred after merging col-I with PEG-4S crosslinker in the nozzle of a double-chamber capillary within an oil flow. A coiled tube was placed at the outlet for microgels to gelify inside the device. Microgel size was controlled varying the nozzle diameter and flow speeds, and the mechanical properties modulated through pH and concentration of collagen/crosslinker. Microgels were characterized with TNBS assay and their cytotoxicity with LIVE-DEAD® assay (Neu-7 astrocytes, n=3). Hollow spheres were prepared by covalently attaching collagen to silica templates that are later dissolved with hydrofluoric acid. They were characterized morphologically and chemically, namely: scanning electron microscopy, dynamic light scattering and Fourier-Transform Infrared (FTIR). Hollow spheres were loaded with GDNF and BMP-2 by diffusion, seeded with different cell-types and encapsulated in the microgels within the microfluidic device. RESULTS AND DISCUSSION Microgels with different size and stiffness were successfully synthetized in a glass microfluidic device. The microgels are non-cytotoxic to cells (fig.1C) and foster cell growth at different crosslinker concentrations. In the synthesis of hollow spheres, no harmful by-products appeared (fig.1D), and their size ranged around 200 nm (fig.1E). Microfluidic-generated hydrogels encapsulating hollow spheres can therefore be used for sustained delivery of GDNF and BMP-2. CONCLUSIONS We demonstrate that microfluidics is an adequate technique for automatically generating monodisperse collagen microgels and provides a simple and useful tool for the posterior encapsulation of nanospheres and cells. The combination of microgels encapsulating hollow spheres is expected to provide a controlled and sustained delivery system of different therapeutic factors. AKNOWLEDGEMENTS The authors would like to thank European Union H2020 Programme (H2020-MSCA-ITN-2015) and Grant No. 676408 for providing financial support to this project. &nbsp

Neurodegenerative diseases, such as Parkinson's disease, affect a large num- ber of the erderly population and still remain untreated. In recent years, cell therapy has emerged as a promising therapeutic strategy. To increase cell viability, biomaterials are of- ten used as scaffolds and facilitate cell deposition, through injection, to the site of interest. However, fluid forces acting on the cells during injection may lead to their disruption or death. This study aims to develop a novel device for the delivery of a cell-embedded, in situ forming, collagen hydrogel. A preliminary simulation study on constricted channels rep- resenting the syringe was performed to gain insight into the effect of needle diameter and syringe geometry. Straight needles emanating co-axially from syringes of various geome- tries were computationally modelled in the two-dimensional space, using OpenFOAMⓇ. The natural collagen solution was modelled as a continuum medium, without cells, and the flow was assumed incompressible, with non-Newtonian fluid constitutive behaviour. The effects of needle diameter and syringe geometry on velocity and shear stresses were examined. The results highlight the importance of geometric characteristics on the design of new cell delivery devices. If cells pass from the syringe barrel to the needle, the pressure drop and the increased velocity could damage them. This is more likely to occur using higher Gauge needles. Further analysis is required including simulations of cells during injection and analysis of their deformation.