Long noncoding RNA BDNF-AS protects local anesthetic induced neurotoxicity in dorsal root ganglion neurons

BackgroundLong noncoding RNA (lncRNA) BDNF-AS was recently identified to regulate neurotrophin signaling pathway. In this study, we examined the functional role of BDNF-AS in regulating local anesthetic-induced neurotoxicity in dorsal root ganglion (DRG) neurons.MethodsNeonatal mouse DRG neurons were cultured in vitro, and treated with local anesthetic, bupivacaine, to induce neurotoxicity. The corresponding change in BDNF-AS expression level in DRG was probed by qRT-PCR. BDNF-AS was knocked down by siRNA in DRG. The effects of BDNF-AS downregulation on neurite regrowth, neuronal apoptosis and activating TrkB signaling pathway in bupivacaine-injured DRG neurons were probed by neurite outgrowth assay, TUNEL assay and western blot assay, respectively.ResultsDuring the process of bupivacaine-induce neurotoxicity in DRG, BDNF-AS was significantly upregulated in both dosage- and time- dependent manners. In DRG neurons, siRNA-mediated BDNF-AS downregulation promoted neurite outgrowth, reduced neuronal apoptosis, and phosphorylated TrkB signaling pathway after bupivacaine-induce neurotoxicity.ConclusionsBDNF-AS downregulation rescued local anesthetic-induce neurotoxicity in DRG neurons, probably through the activation of neurotrophin TrkB signaling pathway.     

Survival,proliferation and differentiation enhancement of neural stem cells cultured in three‐dimensional polyethylene glycol–RGD hydrogel with tenascin

Polyethylene glycol hydrogel (PEG) conjugated with arginyl glycyl aspartic acid (RGD) (PEG–RGD) has been considered to be a scaffold in three‐dimensional (3D) culture that improves neurite outgrowth; on the other hand, tenascin C controls neural growth and differentiation. In this study, the effect of a combined RGD and tenascin C mixture in 3D culture (3D‐PEG–RGD–TnC) on the survival, growth and differentiation of neural stem cells. The viability of the culture has been evaluated by live/dead assay and the results show that the viability of NSCs in 3D‐PEG–RGD–TnC is significantly higher than its value in 3D‐PEG–RGD. The proliferation was evaluated by MTS test and was found to be slightly improved but statistically not significant. Accordingly, the differentiation was evaluated by immunoreactivity to nestin, neurofilament 68, neurofilament 160, neurofilament 200 and GFAP; and the expression of nestin, neuro D, musashi1, β‐tubulin III, GFAP, MBP and Oct4 was studied using RT–PCR. The results showed enhancement of the differentiation of NSCs into the neuronal phenotype in 3D‐PEG–RGD–TnC. The morphology of NSCs cultured in 3D‐PEG–RGD–TnC showed neurite outgrowths and increase in the contact between the differentiated cells' extensions. The conclusion of this study was that NSC survival, proliferation and differentiation are enhanced when the cells are cultured in 3D‐PEG–RGD–TnC. Copyright © 2014 John Wiley & Sons, Ltd.     

Laminin‐modified and aligned poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate)/polyethylene oxide nanofibrous nerve conduits promote peripheral nerve regeneration

Poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) (PHBV) has received much attention for its biodegradability and biocompatibility, characteristics that are required in tissue engineering. In this study, polyethylene oxide (PEO)‐incorporated PHBV nanofibres with random or aligned orientation were obtained by electrospinning. For further use in vivo, the nanofibre films were made into nerve conduits after treatment with NH₃ plasma, which could improve the hydrophilicity of inner surfaces of nerve conduits and then facilitate laminin adsorption via electrostatic interaction for promoting cell adhesion and proliferation. Morphology of the surfaces of modified PHBV/PEO nanofibrous scaffolds were examined by scanning electron microscopy. Schwann cell viability assay was conducted and the results confirmed that the functionalized nanofibres were favourable for cell growth. Morphology of Schwann cells cultured on scaffolds showed that aligned nanofibrous scaffolds provided topographical guidance for cell orientation and elongation. Furthermore, three‐dimensional PHBV/PEO nerve conduits made from aligned and random‐oriented nanofibres were implanted into 12‐mm transected sciatic nerve rat model and subsequent analysis were conducted at 1 and 2 months postsurgery. The above functionalized PHBV/PEO scaffolds provide a novel and promising platform for peripheral nerve regeneration. Copyright © 2016 John Wiley & Sons, Ltd.     

Nanofibrous nerve conduit‐enhanced peripheral nerve regeneration

Fibre structures represent a potential class of materials for the formation of synthetic nerve conduits due to their biomimicking architecture. Although the advantages of fibres in enhancing nerve regeneration have been demonstrated, in vivo evaluation of fibre size effect on nerve regeneration remains limited. In this study, we analyzed the effects of fibre diameter of electrospun conduits on peripheral nerve regeneration across a 15‐mm critical defect gap in a rat sciatic nerve injury model. By using an electrospinning technique, fibrous conduits comprised of aligned electrospun poly (ε‐caprolactone) (PCL) microfibers (981 ± 83 nm, Microfiber) or nanofibers (251 ± 32 nm, Nanofiber) were obtained. At three months post implantation, axons regenerated across the defect gap in all animals that received fibrous conduits. In contrast, complete nerve regeneration was not observed in the control group that received empty, non‐porous PCL film conduits (Film). Nanofiber conduits resulted in significantly higher total number of myelinated axons and thicker myelin sheaths compared to Microfiber and Film conduits. Retrograde labeling revealed a significant increase in number of regenerated dorsal root ganglion sensory neurons in the presence of Nanofiber conduits (1.93 ± 0.71 × 10³ vs. 0.98 ± 0.30 × 10³ in Microfiber, p < 0.01). In addition, the compound muscle action potential (CMAP) amplitudes were higher and distal motor latency values were lower in the Nanofiber conduit group compared to the Microfiber group. This study demonstrated the impact of fibre size on peripheral nerve regeneration. These results could provide useful insights for future nerve guide designs. Copyright © 2012 John Wiley & Sons, Ltd.     

Fast clearing RGD‐based near‐infrared fluorescent probes for in vivo tumor diagnosis

A fast clearing hydrophilic near‐infrared (NIR) dye ICG‐Der‐02 was used to constitute tumor targeting contrast agents. Cell adhesion molecule integrin α_vβ₃ served as the target receptor because of its unique expression on almost all sprouting tumor vasculatures. The purpose of this study was to synthesize and compare the properties of integrin α_vβ₃‐targeted, fast clearing NIR probes both in vitro and in vivo for tumor diagnosis. ICG‐Der‐02 was covalently conjugated to three kinds of RGD peptide including linear, monoeric cyclic and dimeric RGD to form three RGD‐based NIR probes. The integrin receptor specificities of these probes were evaluated in vitro by confocal microscopy. The dynamic bio‐distribution and elimination ratse were in vivo real‐time monitored by a near‐infrared imaging system in normal mice. Further, the in vivo tumor targeting abilities of the RGD‐based NIR probes were compared in α_vβ₃‐positive MDA‐MB‐231, U87MG and α_vβ₃‐negtive MCF‐7 xenograft mice models. Three RGD‐based NIR probes were successfully synthesized with good optical properties. In vitro cellular experiments indicated that the probes have a clear binding affinity to α_υβ₃‐positive tumor cells, with a cyclic dimeric RGD probe owing the highest integrin affinity. Dynamic bio‐distributions of these probes showed a rapid clearing rate through the renal pathway. In vivo tumor targeting ability of the RGD‐based porbes was demonstrated on MDA‐MB‐231 and U87MG tumor models. As expected, the c(RGDyK)₂‐ICG‐Der‐02 probe displayed the highest tumor‐to‐normal tissue contrast. The in vitro and in vivo block experiments confirmed the receptor binding specificity of the probes. The hydrophilic dye‐labeled NIR probes exhibited a fast clearing rate and deep tissue penetration capability. Further, the α_υβ₃ receptor affinity of the three RGD‐based NIR probes followed the order of dimer cyclic > monomer cyclic > linear. The results demonstrate potent fast clearing probes for in vivo early tumor diagnosis. Copyright © 2012 John Wiley & Sons, Ltd.     

Small-molecule GSK-3 inhibitor rescued apoptosis and neurodegeneration in anesthetics-injured dorsal root ganglion neurons

BackgroundApplication of general anesthetics may induce neurotoxicity in dorsal root ganglia (DRG) neurons. In this study, we examined the possible protective mechanism and associated signaling pathways of small-molecule glycogen synthase kinase-3 (GSK-3) inhibitor, SB216763, in bupivacaine-injured mouse DRG neurons in vitro.MethodsIn vitro DRG explant of 6-week old mice was treated with 5 mM bupivacaine to induce neurotoxicity. The explants were also pre-treated with SB216763 for 72 h. Neural protection of SB216763 on bupivacaine-injured DRG neurons was investigated by TUNEL assay, neurite outgrowth assay and western blot assay, respectively. Possible downstream gene of GSK-3 signaling pathway, protein kinase C (PKC) was knocked down by siRNA in DRG explant. Its function in regulating GSK-3 inhibition induced DRG neural protection was also examined by TUNEL, neurite outgrowth and western blot assays.ResultsPre-treatment of SB216763 significantly ameliorated bupivacaine induced apoptosis and neurite loss in DRG neurons. Western blot showed that, in addition to the decrease of phosphorylated-GSK-3 α/β protein, SB216763 increased PKC and decreased caspase-3 (Casp-3) in bupivacaine-injured DRG neurons. SiRNA-mediated PKC knockdown was able to reverse the neural protection of SB216763 in bupivacaine-injured DRG neurons. Western blot showed that PKC knockdown increased phosphorylated-GSK-3 α/β and Casp-3 protein in DRG neurons, confirming that PKC was directly involved in GSK-3-inhibition induced neural protection in DRG.ConclusionsGSK-3 inhibitor SB216763, through PKC, is effective in protecting anesthetics-induced neurotoxicity in DRG.     

Progenitor‐derived endothelial cell response,platelet reactivity and haemocompatibility parameters indicate the potential of NaOH‐treated polycaprolactone for vascular tissue engineering

The haemocompatibility of NaOH‐treated poly(ε‐caprolactone) (PCL) has been evaluated in vitro by analysing several parameters, including plasma recalcification time, whole blood clotting time and platelet adhesion/activation. NaOH‐treated PCL films showed a significant decrease in the clot formation speed and a reduced number of adhered platelets, which mainly exhibited non‐activated morphologies. Furthermore, mature endothelial cells derived from peripheral endothelial progenitor cells were cultured on the polymer to investigate the effects of the endothelial lining on polymer haemocompatibility. Interestingly, cells cultured on NaOH‐treated PCL films showed a significant stimulation of NO production. Although further research is required, NaOH treatment could be an interesting and simple strategy to modify PCL‐based materials in order to enhance endothelial NO production, where compromised, and provide a better interaction of the scaffold with the blood components. In conclusion, these results reinforce the use of NaOH‐treated PCL as a haemocompatible polymer for vascular tissue‐engineering applications. Copyright © 2010 John Wiley & Sons, Ltd.     

A new optical imaging probe targeting αVβ3 integrin in glioblastoma xenografts

α_Vβ₃ Integrins are a widely recognized target for in vivo molecular imaging of pathological conditions such as inflammation, cancer and rheumatoid arthritis. We have evaluated the sensitivity of a new, near‐infrared fluorescence (NIRF), RGD cyclic probe (DA364) in noninvasive detection of α_Vβ₃ integrin‐overexpressing tumors. DA364's binding affinity for α_Vβ₃ integrin was first evaluated in vitro. Human α_Vβ₃ integrin‐positive, U‐87 MG glioblastoma cells were then xenografted in nude mice, and DA364 was injected intravenously (i.v.) to evaluate its in vivo distribution, specificity and sensitivity in comparison with a commercially available probe. DA364 bound α_Vβ₃ integrin on U‐87 MG cells with high affinity and specificity, both in vitro and in vivo. This binding specificity was corroborated by the strong inhibition of its tumor uptake induced by nonfluorescent, cyclic‐RGD peptides. Ex vivo analysis showed that DA364 accumulated at the tumor site, whereas very low levels were detected in liver and spleen. In conclusion, DA364 allows sensitive and specific detection of transplantable glioblastoma by NIRF imaging, and is thus a promising candidate for the elaboration of imaging and therapeutic probes for α_Vβ₃ integrin‐overexpressing tumors. Copyright © 2011 John Wiley & Sons, Ltd.     

Phosphate glass fibres promote neurite outgrowth and early regeneration in a peripheral nerve injury model

Three‐dimensional (3D) scaffolds, which are bioactive and aid in neuronal guidance, are essential in the repair and regeneration of injured peripheral nerves. In this study, we used novel inorganic microfibres guided by phosphate glass (PG). PG fibres (PGfs) were aligned on compressed collagen that was rolled into a nerve conduit. In vitro tests confirmed that adult dorsal root ganglion (DRG) neurons showed active neurite outgrowth along the fibres, with a maximum number and length of neurites being significantly higher than those cultured on tissue culture plastic. In vivo experiments with nerve conduits that either contained PGfs (PGf/Col) or lacked them (Col) were conducted on transected sciatic nerves of rats for up to 12 weeks. One week after implantation, the PGf/Col group showed many axons extending along the scaffold, whereas the Col group showed none. Eight weeks after implantation, the PGf/Col group exhibited greater recovery of plantar muscle atrophy than the Col group. Electrophysiological studies revealed that some animals in the PGf/Col group at 6 and 7 weeks post‐implantation (5.3% and 15.8%, respectively) showed compound muscle action potential. The Col group over the same period showed no response. Motor function also showed faster recovery in the PGf/Col group compared to the Col group up to 7 weeks. However, there was no significant difference in the number of axons, muscle atrophy or motor and sensory functions between the two groups at 12 weeks post‐implantation. In summary, phosphate glass fibres can promote directional growth of axons in cases of peripheral nerve injury by acting as physical guides. Copyright © 2012 John Wiley & Sons, Ltd.     

Parathyroid hormone‐related peptide activates and modulates TRPV1 channel in human DRG neurons

Parathyroid hormone‐related peptide (PTHrP) is associated with advanced tumor growth and metastasis, especially in breast, prostate and myeloma cancers that metastasize to bones, resulting in debilitating chronic pain conditions. Our recent studies revealed that the receptor for PTHrP, PTH1R, is expressed in mouse DRG sensory neurons, and its activation leads to flow‐activation and modulation of TRPV1 channel function, resulting in peripheral heat and mechanical hypersensitivity. In order to verify the translatability of our findings in rodents to humans, we explored whether this signalling axis operates in primary human DRG sensory neurons. Analysis of gene expression data from recently reported RNA deep sequencing experiments performed on mouse and human DRGs reveals that PTH1R is expressed in DRG and tibial nerve. Furthermore, exposure of cultured human DRG neurons to PTHrP leads to slow‐sustained activation of TRPV1 and modulation of capsaicin‐induced channel activation. Both activation and modulation of TRPV1 by PTHrP were dependent on PKC activity. Our findings suggest that functional PTHrP/PTH1R‐TRPV1 signalling exists in human DRG neurons, which could contribute to local nociceptor excitation in the vicinity of metastatic bone tumor microenvironment.     

Osteogenesis of adipose‐derived stem cells on polycaprolactone–β‐tricalcium phosphate scaffold fabricated via selective laser sintering and surface coating with collagen type I

The current study aimed to fabricate three‐dimensional (3D) polycaprolactone (PCL), polycaprolactone and β‐tricalcium phosphate (PCL–TCP) scaffolds via a selective laser‐sintering technique (SLS). Collagen type I was further coated onto PCL–TCP scaffolds to form PCL–TCP–COL scaffolds. The physical characters of these three scaffolds were analysed. The osteogenic potential of porcine adipose‐derived stem cells (pASCs) was compared among these three scaffolds in order to find an optimal scaffold for bone tissue engineering. The experimental results showed no significant differences in pore size and porosity among the three scaffolds; the porosity was ca. 75–77% and the pore size was ca. 300–500 µm in all three. The compressive modulus was increased from 6.77 ± 0.19 to 13.66 ± 0.19 MPa by adding 30% β‐TCP into a 70% PCL scaffold. No significant increase of mechanical strength was found by surface‐coating with collagen type I. Hydrophilicity and swelling ratios showed statistical elevation (p < 0.05) after collagen type I was coated onto the PCL–TCP scaffolds. The in vitro study demonstrated that pASCs had the best osteogenic differentiation on PCL–TCP–COL group scaffolds, due to the highest ALP activity, osteocalcin mRNA expression and mineralization. A nude mice experiment showed better woven bone and vascular tissue formation in the PCL–TCP–COL group than in the PCL group. In conclusion, the study demonstrated the ability to fabricate 3D, porous PCL–TCP composite scaffolds (PCL:TCP = 70:30 by weight) via an in‐house‐built SLS technique. In addition, the osteogenic ability of pASCs was found to be enhanced by coating COL onto the PCL–TCP scaffolds, both in vitro and in vivo. Copyright © 2013 John Wiley & Sons, Ltd.     

An additive manufacturing‐based PCL–alginate–chondrocyte bioprinted scaffold for cartilage tissue engineering

Regenerative medicine is targeted to improve, restore or replace damaged tissues or organs using a combination of cells, materials and growth factors. Both tissue engineering and developmental biology currently deal with the process of tissue self‐assembly and extracellular matrix (ECM) deposition. In this investigation, additive manufacturing (AM) with a multihead deposition system (MHDS) was used to fabricate three‐dimensional (3D) cell‐printed scaffolds using layer‐by‐layer (LBL) deposition of polycaprolactone (PCL) and chondrocyte cell‐encapsulated alginate hydrogel. Appropriate cell dispensing conditions and optimum alginate concentrations for maintaining cell viability were determined. In vitro cell‐based biochemical assays were performed to determine glycosaminoglycans (GAGs), DNA and total collagen contents from different PCL–alginate gel constructs. PCL–alginate gels containing transforming growth factor‐β (TGFβ) showed higher ECM formation. The 3D cell‐printed scaffolds of PCL–alginate gel were implanted in the dorsal subcutaneous spaces of female nude mice. Histochemical [Alcian blue and haematoxylin and eosin (H&E) staining] and immunohistochemical (type II collagen) analyses of the retrieved implants after 4 weeks revealed enhanced cartilage tissue and type II collagen fibril formation in the PCL–alginate gel (+TGFβ) hybrid scaffold. In conclusion, we present an innovative cell‐printed scaffold for cartilage regeneration fabricated by an advanced bioprinting technology. Copyright © 2013 John Wiley & Sons, Ltd.     

Calcification of primary human osteoblast cultures under flow conditions using polycaprolactone scaffolds for intravascular applications

Total atherosclerotic occlusion is a leading cause of death. Recent animal models of this disease are devoid of cell‐mediated calcification and arteries are often not occluded gradually. This study is part of a project with the objective of developing a new model featuring the above two characteristics, using a tissue‐engineering scaffold. The amount and distribution of calcium deposits in primary human osteoblast (HOB) cultures on polycaprolactone (PCL) scaffolds under flow conditions were investigated. HOBs were cultured on PCL scaffolds with TGF‐β1 loadings of 0 (control), 5 and 50 ng. HOB–PCL constructs were cultured in spinner flasks. Under flow conditions, cell numbers present in HOB cultures on PCL scaffolds increased from day 7 to day 14, and most calcification was induced at day 21. TGF‐β1 loadings of 5 and 50 ng did not show a significant difference in ALP activity, cell numbers and amount of calcium deposited in HOB cultures, but calcium staining showed that 50 ng TGF‐β1 had higher calcium deposited on both days 21 and 28 under flow conditions compared with 5 ng of loading. Amount of calcium deposited by HOBs on day 28 showed a decrease from their levels on day 21. PCL degradation may be a factor contributing to this loss. The results indicate that cell‐induced calcification can be achieved on PCL scaffolds under flow conditions. In conclusion, TGFβ1–HOB loaded PCL can be applied to create a model for total atherosclerotic occlusion with cell‐deposited calcium in animal arteries. Copyright © 2011 John Wiley & Sons, Ltd.     

INS‐1 cell glucose‐stimulated insulin secretion is reduced by the downregulation of the 67 kDa laminin receptor

Understanding β cell–extracellular matrix (ECM) interactions can advance our knowledge of the mechanisms that control glucose homeostasis and improve culture methods used in islet transplantation for the treatment of diabetes. Laminin is the main constituent of the basement membrane and is involved in pancreatic β cell survival and function, even enhancing glucose‐stimulated insulin secretion. Most of the studies on cell responses towards laminin have focused on integrin‐mediated interactions, while much less attention has been paid on non‐integrin receptors, such as the 67 kDa laminin receptor (67LR). The specificity of the receptor‐ligand interaction through the adhesion of INS‐1 cells (a rat insulinoma cell line) to CDPGYIGSR‐, GRGDSPC‐ or CDPGYIGSR + GRGDSPC‐covered surfaces was evaluated. Also, the effects of the 67LR knocking down over glucose‐stimulated insulin secretion were investigated. Culture of the INS‐1 cells on the bioactive surfaces was improved compared to the low‐fouling carboxymethyl dextran (CMD) surfaces, while downregulation of the 67LR resulted in reduced cell adhesion to surfaces bearing the CDPGYIGSR peptide. Glucose‐stimulated insulin secretion was hindered by downregulation of the 67LR, regardless of the biological motif available on the biomimetic surfaces on which the cells were cultured. This finding illustrates the importance of the 67LR in glucose‐stimulated insulin secretion and points to a possible role of the 67LR in the mechanisms of insulin secretion. Copyright © 2013 John Wiley & Sons, Ltd.     

In vitro evaluation of gel‐encapsulated adipose derived stem cells: Biochemical cues for in vivo peripheral nerve repair

Adipose‐derived stem cells (ASC) are becoming one of the most exploited cells in peripheral nerve repair. They are fast‐growing and able to protect neurons from apoptosis; they can reduce post‐injury latency and the risk of muscle atrophy. This study evaluates laminin‐loaded fibrin gel as an ASC‐carrying scaffold for nerve repair. In vitro, ASC retained their proliferative activity but showed significant increase in proliferation rate when encapsulated in gels with low laminin concentrations (i.e., 1 μg/mL). We observed a linear decrease of ASC proliferation rate with increasing laminin concentration from 1 to 100 μg/mL. We next examined the effect of the ASC‐carrying fibrin gels on in vitro dorsal root ganglia (DRG) neurite extension, then in vivo sciatic nerve regeneration in adult rats. The ASC‐carrying gel was embedded in 15‐mm‐long, 1.5‐mm‐diameter polydimethylsiloxane regenerative conduits for in vivo evaluation. At 8‐week post implantation, robust regeneration was observed across the long gap. Taken together, these results suggest ASC‐carrying gels are a potential path to improve the efficacy of nerve regeneration through artificial guidance conduits and electrode nerve interfaces.     

Vector‐mediated release of GABA attenuates pain‐related behaviors and reduces NaV1.7 in DRG neurons

Pain is a common and debilitating accompaniment of neuropathy that occurs as a complication of diabetes. In the current study, we examined the effect of continuous release of gamma amino butyric acid (GABA), achieved by gene transfer of glutamic acid decarboxylase (GAD67) to dorsal root ganglia (DRG) in vivo using a non‐replicating herpes simplex virus (HSV)‐based vector (vG) in a rat model of painful diabetic neuropathy (PDN). Subcutaneous inoculation of vG reduced mechanical hyperalgesia, thermal hyperalgesia and cold allodynia in rats with PDN. Continuous release of GABA from vector transduced cells in vivo prevented the increase in the voltage‐gated sodium channel isoform 1.7 (Na_V1.7) protein that is characteristic of PDN. In vitro, infection of primary DRG neurons with vG prevented the increase in Na_V1.7 resulting from exposure to hyperglycemia. The effect of vector‐mediated GABA on Na_V1.7 levels in vitro was blocked by phaclofen but not by bicuculline, a GABA_B receptor effect that was blocked by pertussis toxin‐(PTX) interference with Gα(_i/o) function. Taken in conjunction with our previous observation that continuous activation of delta opioid receptors by vector‐mediated release of enkephalin also prevents the increase in Na_V1.7 in DRG exposed to hyperglycemia in vitro or in vivo, the observations in this report suggest a novel common mechanism through which activation of G protein coupled receptors (GPCR) in DRG neurons regulate the phenotype of the primary afferent.     

Chemical surface modification of poly‐ε‐caprolactone improves Schwann cell proliferation for peripheral nerve repair

Poly‐ε‐caprolactone (PCL) is a biodegradable and biocompatible polymer used in tissue engineering for various clinical applications. Schwann cells (SCs) play an important role in nerve regeneration and repair. SCs attach and proliferate on PCL films but cellular responses are weak due to the hydrophobicity and neutrality of PCL. In this study, PCL films were hydrolysed and aminolysed to modify the surface with different functional groups and improve hydrophilicity. Hydrolysed films showed a significant increase in hydrophilicity while maintaining surface topography. A significant decrease in mechanical properties was also observed in the case of aminolysis. In vitro tests with Schwann cells (SCs) were performed to assess film biocompatibility. A short‐time experiment showed improved cell attachment on modified films, in particular when amino groups were present on the material surface. Cell proliferation significantly increased when both treatments were performed, indicating that surface treatments are necessary for SC response. It was also demonstrated that cell morphology was influenced by physico‐chemical surface properties. PCL can be used to make artificial conduits and chemical modification of the inner lumen improves biocompatibility. Copyright © 2012 John Wiley & Sons, Ltd.     

Modification of MR molecular imaging probes with cysteine‐terminated peptides and their potential for in vivo tumour detection

One of the challenges facing superparamagnetic iron oxide (SPIO) nanoparticles is to improve their biological compatibility. While highly uniform SPIOs can be manufactured, the surfaces are hydrophobic as a result of the surfactants used in their fabrication. In this study, we developed a general strategy to fabricate an MR molecular imaging probe in one step by replacing hydrophobic surfactants with small peptides terminated with cysteine. The hydrophobic SPIO surface was transformed into a hydrophilic one by exchanging surface oleic acids with the peptides RGD–Cys or RGD–PEG–Cys. After the RGD–Cys and RGD–PEG–Cys peptide exchange, both RGD–Cys–SPIO and RGD–PEG–Cys–SPIO specifically targeted α_vβ₃‐expressing cells (A549) in vitro, with RGD–Cys–SPIO achieving this more efficiently. Furthermore, MR imaging of A549 tumors receiving RGD–Cys–SPIO or RGD–PEG–Cys–SPIO demonstrated that both the targeted particles could reach and label the α_vβ₃‐expressing tumor, much more efficiently than the non‐targeted particles (Cys–SPIO). Histology showed that the probes not only target the tumor neovasculature but also extravasate from vessels and address the tumor cells. Our study shows that directly replacing oleic acid with cysteine or cysteine‐terminated small peptides is a general strategy to transforming the hydrophobic surface of SPIO into a hydrophilic one, as well as providing targeting ligands. Such SPIOs are of interest as MR molecular imaging probes to detect for cancer in vivo. Copyright © 2010 John Wiley & Sons, Ltd.     

Cell‐free artificial implants of electrospun fibres in a three‐dimensional gelatin matrix support sciatic nerve regeneration in vivo

Surgical repair of larger peripheral nerve lesions requires the use of autologous nerve grafts. At present, clinical alternatives to avoid nerve transplantation consist of empty tubes, which are only suitable for the repair over short distances and have limited success. We developed a cell‐free, three‐dimensional scaffold for axonal guidance in long‐distance nerve repair. Sub‐micron scale fibres of biodegradable poly‐ε‐caprolactone (PCL) and collagen/PCL (c/PCL) blends were incorporated in a gelatin matrix and inserted in collagen tubes. The conduits were tested by replacing 15‐mm‐long segments of rat sciatic nerves in vivo. Biocompatibility of the implants and nerve regeneration were assessed histologically, with electromyography and with behavioural tests for motor functions. Functional repair was achieved in all animals with autologous transplants, in 12 of 13 rats that received artificial implants with an internal structure and in half of the animals with empty nerve conduits. In rats with implants containing c/PCL fibres, the extent of recovery (compound muscle action potentials, motor functions of the hind limbs) was superior to animals that had received empty implants, but not as good as with autologous nerve transplantation. Schwann cell migration and axonal regeneration were observed in all artificial implants, and muscular atrophy was reduced in comparison with animals that had received no implants. The present design represents a significant step towards cell‐free, artificial nerve bridges that can replace autologous nerve transplants in the clinic. Copyright © 2017 John Wiley & Sons, Ltd.     

RGD‐ligand mimetic antagonists of integrin αIIbβ3 paradoxically enhance GPVI‐induced human platelet activation

Summary. Background: The integrin α_IIbβ₃ is the major mediator of platelet aggregation and has, therefore, become an important target of antithrombotic therapy. Antagonists of α_IIbβ₃, for example abciximab, tirofiban and eptifibatide, are used in the treatment of acute coronary syndromes. However, in addition to effective blockade of the integrin, binding of can induce conformational changes in the integrin and can also induce integrin clustering. This class effect of RGD‐ligand mimetics might, therefore, underlie paradoxical platelet activation and thrombosis previously reported. Objectives: To examine the components of signaling pathways and functional responses in platelets that may underlie this phenomenon of paradoxical platelet activation. Methods: We assessed the effect of lotrafiban, and other α_IIbβ₃ antagonists including the clinically used drug tirofiban, on tyrosine phosphorylation of key signaling proteins in platelets by immunoblotting and also platelet functional outputs such as cytosolic calcium responses, phosphatidylserine exposure (pro‐coagulant activity) and dense granule release. Results: In all cases, no effect of α_IIbβ₃ antagonists were observed on their own, but these integrin antagonists did lead to a marked potentiation of glycoprotein VI (GPVI)‐associated FcR γ‐chain phosphorylation, activation of Src family kinases and Syk kinase. This correlated with increased dense granule secretion, cytosolic calcium response and exposure of phosphatidylserine on the platelet surface. P2Y₁₂ antagonism abolished the potentiated phosphatidylserine exposure and dense granule secretion but not the cytosolic calcium response. Conclusions: These data provide a mechanism for enhancement of platelet activity by α_IIbβ₃ inhibitors, but also reveal a potentially important signaling pathway operating from the integrin to GPVI signaling.