Medium chain length polyhydroxyalkanoates as potential matrix materials for peripheral nerve regeneration.

Polyhydroxyalkanoates are natural, biodegradable, thermoplastic and sustainable polymers with a huge potential in fabrication of bioresorbable implantable devices for tissue engineering. We describe a comparative evaluation of three medium chain length polyhydroxyalkanoates (mcl-PHAs), namely poly(3-hydroxyoctanoate), poly(3-hydroxyoctanoate-co-3-hydoxydecanoate) and poly(3-hydroxyoctanoate-co-3-hydroxydecanoate-co-3-hydroxydodecanoate), one short chain length polyhydroxyalkanoate, poly(3-hydroxybutyrate), P(3HB) and synthetic aliphatic polyesters (polycaprolactone and polylactide) with a specific focus on nerve regeneration, due to mechanical properties of mcl-PHAs closely matching nerve tissues. In vitro biological studies with NG108-15 neuronal cell and primary Schwann cells did not show a cytotoxic effect of the materials on both cell types. All mcl-PHAs supported cell adhesion and viability. Among the three mcl-PHAs, P(3HO-co-3HD) exhibited superior properties with regards to numbers of cells adhered and viable cells for both cell types, number of neurite extensions from NG108-15 cells, average length of neurite extensions and Schwann cells. Although, similar characteristics were observed for flat P(3HB) surfaces, high rigidity of this biomaterial, and FDA-approved polymers such as PLLA, limits their applications in peripheral nerve regeneration. Therefore, we have designed, synthesized and evaluated these materials for nerve tissue engineering and regenerative medicine, the interaction of mcl-PHAs with neuronal and Schwann cells, identifying mcl-PHAs as excellent materials to enhance nerve regeneration and potentially their clinical application in peripheral nerve repair.

Aminosilane Functionalized Aligned Fiber PCL Scaffolds for Peripheral Nerve Repair.

Silane modification is a simple and cost-effective tool to modify existing biomaterials for tissue engineering applications. Aminosilane layer deposition has previously been shown to control NG108-15 neuronal cell and primary Schwann cell adhesion and differentiation by controlling deposition of ─NH2 groups at the submicron scale across the entirety of a surface by varying silane chain length. This is the first study toreport depositing 11-aminoundecyltriethoxysilane (CL11) onto aligned Polycaprolactone (PCL) scaffolds for peripheral nerve regeneration. Fibers are manufactured via electrospinning and characterized using water contact angle measurements, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). Confirmed modified fibers are investigated using in vitro cell culture of NG108-15 neuronal cells and primary Schwann cells to determine cell viability, cell differentiation, and phenotype. CL11-modified fibers significantly support NG108-15 neuronal cell and Schwann cell viability. NG108-15 neuronal cell differentiation maintains Schwann cell phenotype compared to unmodified PCL fiber scaffolds. 3D ex vivo culture of Dorsal root ganglion explants (DRGs) confirms further Schwann cell migration and longer neurite outgrowth from DRG explants cultured on CL11 fiber scaffolds compared to unmodified scaffolds. Thus, a reproducible and cost-effective tool is reported to modify biomaterials with functional amine groups that can significantly improve nerve guidance devices and enhance nerve regeneration.

Selectively inhibiting malignant melanoma migration and invasion in an engineered skin model using actin-targeting dinuclear RuII-complexes.

Due to the poor prognosis of metastatic cancers, there is a clinical need for agents with anti-metastatic activity. Here we report on the anti-metastatic effect of a previously reported Ru(ii) complex [{(phen)2Ru}2(tpphz)]4+, 14+, that has recently been shown to disrupt actin fiber assembly. In this study, we investigated the anti-migratory effect of +14+ and a close structural analogue+, 24+, on two highly invasive, metastatic human melanoma cell lines. Laser scanning confocal imaging was used to investigate the structure of actin filament and adhesion molecule vinculin and results show disassembly of central actin filaments and focal adhesions. The effect of both compounds on actin filaments was also found to be reversible. As these results revealed that the complexes were cytostatic and produced a significant inhibitory effect on the migration of both melanoma cell lines but not human dermal fibroblasts their effect on 3D-spheroids and a tissue-engineered living skin model were also investigated. These experiments demonstrated that the compounds inhibited the growth and invasiveness of the melanoma-based spheroidal tumor model and both complexes were found to penetrate the epidermis of the skin tissue model and inhibit the invasion of melanoma cells. Taken together, the cytostatic and antimigratory effects of the complexes results in an antimetastatic effect that totally prevent invasion of malignant melanoma into skin tissue.

Aligned Polyhydroxyalkanoate Blend Electrospun Fibers as Intraluminal Guidance Scaffolds for Peripheral Nerve Repair.

The use of nerve guidance conduits (NGCs) to treat peripheral nerve injuries is a favorable approach to the current "gold standard" of autografting. However, as simple hollow tubes, they lack specific topographical and mechanical guidance cues present in nerve grafts and therefore are not suitable for treating large gap injuries (30-50 mm). The incorporation of intraluminal guidance scaffolds, such as aligned fibers, has been shown to increase neuronal cell neurite outgrowth and Schwann cell migration distances. A novel blend of PHAs, P(3HO)/P(3HB) (50:50), was investigated for its potential as an intraluminal aligned fiber guidance scaffold. Aligned fibers of 5 and 8 µm diameter were manufactured by electrospinning and characterized using SEM. Fibers were investigated for their effect on neuronal cell differentiation, Schwann cell phenotype, and cell viability in vitro. Overall, P(3HO)/P(3HB) (50:50) fibers supported higher neuronal and Schwann cell adhesion compared to PCL fibers. The 5 µm PHA blend fibers also supported significantly higher DRG neurite outgrowth and Schwann cell migration distance using a 3D ex vivo nerve injury model.

Polyhydroxyalkanoates and their advances for biomedical applications.

Polyhydroxyalkanoates (PHAs) are sustainable, versatile, biocompatible, and bioresorbable polymers that are suitable for biomedical applications. Produced via bacterial fermentation under nutrient-limiting conditions, they are uncovering a new horizon for devices in biomedical applications. A wide range of cell types including bone, cartilage, nerve, cardiac, and pancreatic cells, readily attach grow and are functional on PHAs. The tuneable physical properties and resorption rates of PHAs provide a toolbox for biomedical engineers in developing devices for hard and soft tissue engineering applications and drug delivery. The versatility of PHAs and the vast range of different PHA-based prototypes are discussed. Current in vitro, ex vivo, and in vivo development work are described and their regulatory approvals are reviewed.

A Dinuclear Osmium(II) Complex Near-Infrared Nanoscopy Probe for Nuclear DNA.

With the aim of developing photostable near-infrared cell imaging probes, a convenient route to the synthesis of heteroleptic OsII complexes containing the Os(TAP)2 fragment is reported. This method was used to synthesize the dinuclear OsII complex, [{Os(TAP)2}2tpphz]4+ (where tpphz = tetrapyrido[3,2-a:2',3'-c:3″,2''-h:2‴,3'''-j]phenazine and TAP = 1,4,5,8- tetraazaphenanthrene). Using a combination of resonance Raman and time-resolved absorption spectroscopy, as well as computational studies, the excited state dynamics of the new complex were dissected. These studies revealed that, although the complex has several close lying excited states, its near-infrared, NIR, emission (λmax = 780 nm) is due to a low-lying Os → TAP based 3MCLT state. Cell-based studies revealed that unlike its RuII analogue, the new complex is neither cytotoxic nor photocytotoxic. However, as it is highly photostable as well as live-cell permeant and displays NIR luminescence within the biological optical window, its properties make it an ideal probe for optical microscopy, demonstrated by its use as a super-resolution NIR STED probe for nuclear DNA.

Harnessing Polyhydroxyalkanoates and Pressurized Gyration for Hard and Soft Tissue Engineering.

Organ dysfunction is a major cause of morbidity and mortality. Transplantation is typically the only definitive cure, challenged by the lack of sufficient donor organs. Tissue engineering encompasses the development of biomaterial scaffolds to support cell attachment, proliferation, and differentiation, leading to tissue regeneration. For efficient clinical translation, the forming technology utilized must be suitable for mass production. Herein, uniaxial polyhydroxyalkanoate scaffolds manufactured by pressurized gyration, a hybrid scalable spinning technique, are successfully used in bone, nerve, and cardiovascular applications. Chorioallantoic membrane and in vivo studies provided evidence of vascularization, collagen deposition, and cellular invasion for bone tissue engineering. Highly efficient axonal outgrowth was observed in dorsal root ganglion-based 3D ex vivo models. Human induced pluripotent stem cell derived cardiomyocytes exhibited a mature cardiomyocyte phenotype with optimal calcium handling. This study confirms that engineered polyhydroxyalkanoate-based gyrospun fibers provide an exciting and unique toolbox for the development of scalable scaffolds for both hard and soft tissue regeneration.

Cell guidance on peptide micropatterned silk fibroin scaffolds.

Guiding neuronal cell growth is desirable for neural tissue engineering but is very challenging. In this work, a self-assembling ultra-short surfactant-like peptide I3K which possesses positively charged lysine head groups, and hydrophobic isoleucine tails, was chosen to investigate its potential for guiding neuronal cell growth. The peptides were able to self-assemble into nanofibrous structures and interact strongly with silk fibroin (SF) scaffolds, providing a niche for neural cell attachment and proliferation. SF is an excellent biomaterial for tissue engineering. However neuronal cells, such as rat PC12 cells, showed poor attachment on pure regenerated SF (RSF) scaffold surfaces. Patterning of I3K peptide nanofibers on RSF surfaces significantly improved cellular attachment, cellular density, as well as morphology of PC12 cells. The live / dead assay confirmed that RSF and I3K have negligible cytotoxicity against PC12 cells. Atomic force microscopy (AFM) was used to image the topography and neurite formation of PC12 cells, where results revealed that self-assembled I3K nanofibers can support the formation of PC12 cell neurites. Immunolabelling also demonstrated that coating of I3K nanofibers onto the RSF surfaces not only increased the percentage of cells bearing neurites but also increased the average maximum neurite length. Therefore, the peptide I3K could be used as an alternative to poly-l-lysine for cell culture and tissue engineering applications. As micro-patterning of neural cells to guide neurite growth is important for developing nerve tissue engineering scaffolds, inkjet printing was used to pattern self-assembled I3K peptide nanofibers on RSF surfaces for directional control of PC12 cell growth. The results demonstrated that inkjet-printed peptide micro-patterns can effectively guide the cell alignment and organization on RSF scaffold surfaces, providing great potential for nerve regeneration applications.

Cost effective optimised synthetic surface modification strategies for enhanced control of neuronal cell differentiation and supporting neuronal and Schwann cell viability.

Enriching a biomaterial surface with specific chemical groups has previously been considered for producing surfaces that influence cell response. Silane layer deposition has previously been shown to control mesenchymal stem cell adhesion and differentiation. However, it has not been used to investigate neuronal or Schwann cell responses in vitro to date. We report on the deposition of aminosilane groups for peripheral neurons and Schwann cells studying two chain lengths: (a) 3-aminopropyl triethoxysilane (short chain-SC) and (b) 11-aminoundecyltriethoxysilane (long chain-LC) by coating glass substrates. Surfaces were characterised by water contact angle, AFM and XPS. LC-NH2 was produced reproducibly as a homogenous surface with controlled nanotopography. Primary neuron and NG108-15 neuronal cell differentiation and primary Schwann cell responses were investigated in vitro by S100ß, p75, and GFAP antigen expression. Both amine silane surface supported neuronal and Schwann cell growth; however, neuronal differentiation was greater on LC aminosilanes versus SC. Thus, we report that silane surfaces with an optimal chain length may have potential in peripheral nerve repair for the modification and improvement of nerve guidance devices.

A Tuneable, Photocurable, Poly(Caprolactone)-Based Resin for Tissue Engineering-Synthesis, Characterisation and Use in Stereolithography.

Stereolithography is a useful additive manufacturing technique for the production of scaffolds for tissue engineering. Here we present a tuneable, easy-to-manufacture, photocurable resin for use in stereolithography, based on the widely used biomaterial, poly(caprolactone) (PCL). PCL triol was methacrylated to varying degrees and mixed with photoinitiator to produce a photocurable prepolymer resin, which cured under UV light to produce a cytocompatible material. This study demonstrates that poly(caprolactone) methacrylate (PCLMA) can be produced with a range of mechanical properties and degradation rates. By increasing the degree of methacrylation (DM) of the prepolymer, the Young's modulus of the crosslinked PCLMA could be varied from 0.12-3.51 MPa. The accelerated degradation rate was also reduced from complete degradation in 17 days to non-significant degradation in 21 days. The additive manufacturing capabilities of the resin were demonstrated by the production of a variety of different 3D structures using micro-stereolithography. Here, ß-carotene was used as a novel, cytocompatible photoabsorber and enabled the production of complex geometries by giving control over cure depth. The PCLMA presented here offers an attractive, tuneable biomaterial for the production of tissue engineering scaffolds for a wide range of applications.

Bioresorbable and Mechanically Optimized Nerve Guidance Conduit Based on a Naturally Derived Medium Chain Length Polyhydroxyalkanoate and Poly(ε-Caprolactone) Blend.

Severe peripheral nerve injuries represent a large clinical problem with relevant challenges such as the development of successful synthetic scaffolds as substitutes to autologous nerve grafting. Numerous studies have reported the use of polyesters and type I collagen-based nerve guidance conduits (NGCs) to promote nerve regeneration through critical nerve defects while providing protection from external factors. However, none of the commercially available hollow bioresorbable NGCs have demonstrated superior clinical outcomes to an autologous nerve graft. Hence, new materials and NGC geometries have been explored in the literature to mimic the native nerve properties and architecture. Here, we report a novel blend of a natural medium chain length polyhydroxyalkanoate (MCL-PHA) with a synthetic aliphatic polyester, poly(ε-caprolactone) (PCL), suitable for extrusion-based high-throughput manufacturing. The blend was designed to combine the excellent ability of PHAs to support the growth and proliferation of mammalian cells with the good processability of PCL. The material exhibited excellent neuroregenerative properties and a good bioresorption rate, while the extruded porous tubes exhibited similar mechanical properties to the rat sciatic nerve. The NGCs were implanted to treat a 10 mm long sciatic nerve defect in rats, where significant differences were found between thin and thick wall thickness implants, and both electrophysiological and histological data, as well as the number of recovered animals, provided superior outcomes than the well-referenced synthetic Neurolac NGC.

Biomimetic surface delivery of NGF and BDNF to enhance neurite outgrowth.

Treatment for peripheral nerve injuries includes the use of autografts and nerve guide conduits (NGCs). However, outcomes are limited, and full recovery is rarely achieved. The use of nerve scaffolds as a platform to surface immobilize neurotrophic factors and deliver locally is a promising approach to support neurite and nerve outgrowth after injury. We report on a bioactive surface using functional amine groups, to which heparin binds electrostatically. X-ray photoelectron spectroscopy analysis was used to characterize the presence of nitrogen and sulfur. Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were bound by electrostatic interaction to heparin, and the release profile evaluated by enzyme-linked immunosorbent assay, which showed that ca. 1% of NGF was released from each of the bioactive surface within 7 days. Furthermore, each surface showed a maximum release of 97% of BDNF. Neurotrophin release on neurite outgrowth was evaluated by primary dorsal root ganglion with a maximum neurite growth response in vitro of 1,075 µm detected for surfaces immobilized with NGF at 1 ng/ml. In summary, the study reports on the design and construction of a biomimetic platform to deliver NGF and BDNF using physiologically low concentrations of neurotrophin. The platform is directly applicable and scalable for improving the regenerative ability of existing NGCs and scaffolds.

UV-Casting on Methacrylated PCL for the Production of a Peripheral Nerve Implant Containing an Array of Porous Aligned Microchannels.

Peripheral nerves are basic communication structures guiding motor and sensory information from the central nervous system to receptor units. Severed peripheral nerve injuries represent a large clinical problem with relevant challenges to successful synthetic nerve repair scaffolds as substitutes to autologous nerve grafting. Numerous studies reported the use of hollow tubes made of synthetic polymers sutured between severed nerve stumps to promote nerve regeneration while providing protection for external factors, such as scar tissue formation and inflammation. Few approaches have described the potential use of a lumen structure comprised of microchannels or microfibers to provide axon growth avoiding misdirection and fostering proper healing. Here, we report the use of a 3D porous microchannel-based structure made of a photocurable methacrylated polycaprolactone, whose mechanical properties are comparable to native nerves. The neuro-regenerative properties of the polymer were assessed in vitro, prior to the implantation of the 3D porous structure, in a 6-mm rat sciatic nerve gap injury. The manufactured implants were biocompatible and able to be resorbed by the host's body at a suitable rate, allowing the complete healing of the nerve. The innovative design of the highly porous structure with the axon guiding microchannels, along with the observation of myelinated axons and Schwann cells in the in vivo tests, led to a significant progress towards the standardized use of synthetic 3D multichannel-based structures in peripheral nerve surgery.

Modulation of neuronal cell affinity of composite scaffolds based on polyhydroxyalkanoates and bioactive glasses.

The biocompatibility and neuron regenerating properties of various bioactive glass (BG)/polyhydroxyalkanoate (PHA) blend composites were assessed in order to study their suitability for peripheral nerve tissue applications, specifically as lumen structures for nerve guidance conduits. BG/PHA blend composites were fabricated using Bioactive glass® 45 S5 (BG1) and BG 1393 (BG2) with the 25:75 poly(3-hydroxyoctanoate/poly3-hydroxybutyrate), 25:75 P(3HO)/P(3HB) blend (PHA blend). Various concentrations of each BG (0.5 wt%, 1.0 wt% and 2.5 wt%) were used to determine the effect of BG on neuronal growth and differentiation, in single culture using NG108-15 neuronal cells and in a co-culture along with RN22 Schwann cells. NG108-15 cells exhibited good growth and differentiation on all the PHA blend composites showing that both BGs have good biocompatibility at 0.5 wt%, 1.0 wt% and 2.5 wt% within the PHA blend. The Young's modulus values displayed by all the PHA blend/BG composites ranged from 385.6 MPa to 1792.6 MPa, which are able to provide the required support and protective effect for the regeneration of peripheral nerves. More specifically, the tensile strength obtained in the PHA blend/BG1 (1.0 wt%) (10.0 ± 0.6 MPa) was found to be similar to that of the rabbit peroneal nerve. This composite also exhibited the best biological performance in supporting growth and neuronal differentiation among all the substrates. The neurite extension on this composite was found to be remarkable with the neurites forming a complex connection network.

A Dinuclear Ruthenium(II) Complex Excited by Near-Infrared Light through Two-Photon Absorption Induces Phototoxicity Deep within Hypoxic Regions of Melanoma Cancer Spheroids.

The dinuclear photo-oxidizing RuII complex [{Ru(TAP2)}2(tpphz)]4+ (TAP = 1,4,5,8- tetraazaphenanthrene, tpphz = tetrapyrido[3,2-a:2',3'-c:3″,2''-h:2‴,3'''-j]phenazine), 14+, is readily taken up by live cells localizing in mitochondria and nuclei. In this study, the two-photon absorption cross section of 14+ is quantified and its use as a two-photon absorbing phototherapeutic is reported. It was confirmed that the complex is readily photoexcited using near-infrared, NIR, and light through two-photon absorption, TPA. In 2-D cell cultures, irradiation with NIR light at low power results in precisely focused phototoxicity effects in which human melanoma cells were killed after 5 min of light exposure. Similar experiments were then carried out in human cancer spheroids that provide a realistic tumor model for the development of therapeutics and phototherapeutics. Using the characteristic emission of the complex as a probe, its uptake into 280 µm spheroids was investigated and confirmed that the spheroid takes up the complex. Notably TPA excitation results in more intense luminescence being observed throughout the depth of the spheroids, although emission intensity still drops off toward the necrotic core. As 14+ can directly photo-oxidize DNA without the mediation of singlet oxygen or other reactive oxygen species, phototoxicity within the deeper, hypoxic layers of the spheroids was also investigated. To quantify the penetration of these phototoxic effects, 14+ was photoexcited through TPA at a power of 60 mW, which was progressively focused in 10 µm steps throughout the entire z-axis of individual spheroids. These experiments revealed that, in irradiated spheroids treated with 14+, acute and rapid photoinduced cell death was observed throughout their depth, including the hypoxic region.

Correction: A dinuclear ruthenium(ii) phototherapeutic that targets duplex and quadruplex DNA.

[This corrects the article DOI: 10.1039/C8SC05084H.].

Unidirectional neuronal cell growth and differentiation on aligned polyhydroxyalkanoate blend microfibres with varying diameters.

Polyhydroxyalkanoates (PHAs) are a family of prokaryotic-derived biodegradable and biocompatible natural polymers known to exhibit neuroregenerative properties. In this work, poly(3-hydroxybutyrate), P(3HB), and poly(3-hydroxyoctanoate), P(3HO), have been combined to form blend fibres for directional guidance of neuronal cell growth and differentiation. A 25:75 P(3HO)/P(3HB) blend (PHA blend) was used for the manufacturing of electrospun fibres as resorbable scaffolds to be used as internal guidance lumen structures in nerve conduits. The biocompatibility of these fibres was studied using neuronal and Schwann cells. Highly aligned and uniform fibres with varying diameters were fabricated by controlling electrospinning parameters. The resulting fibre diameters were 2.4 ± 0.3, 3.7 ± 0.3, and 13.5 ± 2.3 µm for small, medium, and large diameter fibres, respectively. The cell response to these electrospun fibres was investigated with respect to growth and differentiation. Cell migration observed on the electrospun fibres showed topographical guidance in accordance with the direction of the fibres. The correlation between fibre diameter and neuronal growth under two conditions, individually and in coculture with Schwann cells, was evaluated. Results obtained from both assays revealed that all PHA blend fibre groups were able to support growth and guide aligned distribution of neuronal cells, and there was a direct correlation between the fibre diameter and neuronal growth and differentiation. This work has led to the development of a family of unique biodegradable and highly biocompatible 3D substrates capable of guiding and facilitating the growth, proliferation, and differentiation of neuronal cells as internal structures within nerve conduits.

A dinuclear ruthenium(ii) phototherapeutic that targets duplex and quadruplex DNA.

With the aim of developing a sensitizer for photodynamic therapy, a previously reported luminescent dinuclear complex that functions as a DNA probe in live cells was modified to produce a new iso-structural derivative containing RuII(TAP)2 fragments (TAP = 1,4,5,8-tetraazaphenanthrene). The structure of the new complex has been confirmed by a variety of techniques including single crystal X-ray analysis. Unlike its parent, the new complex displays Ru → L-based 3MLCT emission in both MeCN and water. Results from electrochemical studies and emission quenching experiments involving guanosine monophosphate are consistent with an excited state located on a TAP moiety. This hypothesis is further supported by detailed DFT calculations, which take into account solvent effects on excited state dynamics. Cell-free steady-state and time-resolved optical studies on the interaction of the new complex with duplex and quadruplex DNA show that the complex binds with high affinity to both structures and indicate that its photoexcited state is also quenched by DNA, a process that is accompanied by the generation of the guanine radical cation sites as photo-oxidization products. Like the parent complex, this new compound is taken up by live cells where it primarily localizes within the nucleus and displays low cytotoxicity in the absence of light. However, in complete contrast to [{RuII(phen)2}2(tpphz)]4+, the new complex is therapeutically activated by light to become highly phototoxic toward malignant human melanoma cell lines showing that it is a promising lead for the treatment of this recalcitrant cancer.

Additive manufactured biodegradable poly(glycerol sebacate methacrylate) nerve guidance conduits.

Entubulating devices to repair peripheral nerve injuries are limited in their effectiveness particularly for critical gap injuries. Current clinically used nerve guidance conduits are often simple tubes, far stiffer than that of the native tissue. This study assesses the use of poly(glycerol sebacate methacrylate) (PGSm), a photocurable formulation of the soft biodegradable material, PGS, for peripheral nerve repair. The material was synthesized, the degradation rate and mechanical properties of material were assessed and nerve guidance conduits were structured via stereolithography. In vitro cell studies confirmed PGSm as a supporting substrate for both neuronal and glial cell growth. Ex vivo studies highlight the ability of the cells from a dissociated dorsal root ganglion to grow out and align along the internal topographical grooves of printed nerve guide conduits. In vivo results in a mouse common fibular nerve injury model show regeneration of axons through the PGSm conduit into the distal stump after 21 days. After conduit repair levels of spinal cord glial activation (an indicator for neuropathic pain development) were equivalent to those seen following graft repair. In conclusion, results indicate that PGSm can be structured via additive manufacturing into functional NGCs. This study opens the route of personalized conduit manufacture for nerve injury repair. STATEMENT OF SIGNIFICANCE: This study describes the use of photocurable of Poly(Glycerol Sebacate) (PGS) for light-based additive manufacturing of Nerve Guidance Conduits (NGCs). PGS is a promising flexible biomaterial for soft tissue engineering, and in particular for nerve repair. Its mechanical properties and degradation rate are within the desirable range for use in neuronal applications. The nerve regeneration supported by the PGS NGCs is similar to an autologous nerve transplant, the current gold standard. A second assessment of regeneration is the activation of glial cells within the spinal cord of the tested animals which reveals no significant increase in neuropathic pain by using the NGCs. This study highlights the successful use of a biodegradable additive manufactured NGC for peripheral nerve repair.

An Improved In Vivo Methodology to Visualise Tumour Induced Changes in Vasculature Using the Chick Chorionic Allantoic Membrane Assay.

BACKGROUND/AIM: Decreasing the vascularity of a tumour has proven to be an effective strategy to suppress tumour growth and metastasis. Anti-angiogenic therapies have revolutionized the treatment of advanced-stage cancers, however there is still demand for further improvement. This necessitates new experimental models that will allow researchers to reliably study aspects of angiogenesis. The aim of this study was to demonstrate an in vivo technique in which the highly vascular and accessible chorioallantoic membrane (CAM) of the chick embryo is used to study tumour-induced changes in the macro and microvessels. MATERIALS AND METHODS: Two cancer cell lines (human melanoma (C8161) and human prostate cancer (PC3)) were selected as model cells. Human dermal fibroblasts were used as a control. One million cells were labelled with green fluorescent protein and implanted on the CAM of the chick embryo at embryonic development day (EDD) 7 and angiogenesis was evaluated at EDDs 10, 12 and 14. A fluorescently-tagged lectin (lens culinaris agglutinin (LCA)) was injected intravenously into the chick embryo to label endothelial cells. The LCA is known to label the luminal surface of endothelial cells, or dextrans, in the CAM vasculature. Macrovessels were imaged by a hand-held digital microscope and images were processed for quantification. Microvessels were evaluated by confocal microscopy. Tumour invasion was assessed by histological and optical sectioning. RESULTS: Tumour cells (C8161 and PC3) produced quantifiable increases in the total area covered by blood vessels, compared to fibroblasts when assessed by digital microscopy. Tumour invasion could be demonstrated by both histological and optical sectioning. The most significant changes in tumour vasculature observed were in the microvascular structures adjacent to the tumour cells, which showed an increase in the endothelial cell coverage. Additionally, tumour intravasation and tumour thrombus formation could be detected in the areas adjacent to tumour cells. The fragility of tumour blood vessels could be demonstrated when tumour cells seeded on a synthetic scaffold were grown on CAM. CONCLUSION: We report on a modification to a well-studied CAM in vivo assay, which can be effectively used to study tumour induced changes in macro and microvasculature.