Transplantation of bone-marrow-derived cells into a nerve guide resulted in transdifferentiation into Schwann cells and effective regeneration of transected mouse sciatic nerve

Peripheral nerves possess the capacity of self-regeneration after traumatic injury. Nevertheless, the functional outcome after peripheral-nerve regeneration is often poor, especially if the nerve injuries occur far from their targets. Aiming to optimize axon regeneration, we grafted bone-marrow-derived cells (BMDCs) into a collagen-tube nerve guide after transection of the mouse sciatic nerve. The control group received only the culture medium. Motor function was tested at 2, 4, and 6 weeks after surgery, using the sciatic functional index (SFI), and showed that functional recovery was significantly improved in animals that received the cell grafts. After 6 weeks, the mice were anesthetized, perfused transcardially, and the sciatic nerves were dissected and processed for transmission electron microscopy and light microscopy. The proximal and distal segments of the nerves were compared, to address the question of improvement in growth rate; the results revealed a maintenance and increase of nerve regeneration for both myelinated and non-myelinated fibers in distal segments of the experimental group. Also, quantitative analysis of the distal region of the regenerating nerves showed that the numbers of myelinated fibers, Schwann cells (SCs) and g-ratio were significantly increased in the experimental group compared to the control group. The transdifferentiation of BMDCs into Schwann cells was confirmed by double labeling with S100/and Hoechst staining. Our data suggest that BMDCs transplanted into a nerve guide can differentiate into SCs, and improve the growth rate of nerve fibers and motor function in a transected sciatic-nerve model.     

Two-dimensional differential adherence of neuroblasts in laser micromachined CAD/CAM agarose channels

Laser micromachining of hydrophobic gels into CAD/CAM patterns was used to develop differentially adherent surfaces and induce the attachment of B35 rat neuroblasts that would later form engineered nerve bundles. Narrow channels, 60-400 μm wide, were micromachined in a 2% agarose gel using an ArF laser, and subsequently filled with an extracellular matrix gel. Upon the addition of 1 ml of a 2 × 104 cells/ml neuroblast suspension, the cells selectively adhered to the ECM-lined channels in a non-confluent manner and we monitored their growth at various time points. The adherent neuroblasts were fluorescently imaged with a propidium iodide live/dead assay, which revealed that the cells were alive within the channels. After 72 h growth, the neuroblasts grew, proliferated, and differentiated into nerve bundles. The fully grown 1 cm long nerve bundle organoids maintained an aspect ratio on the order of 100. The results presented in this paper provide the foundation for laser micromachining technique to develop bioactive substrates for development of three-dimensional tissues. Laser micromachining offers rapid prototyping of substrates, excellent resolution, control of pattern depth and dimensions, and ease of fabrication.     

The effects of morphology and exchange on proton N.M.R. relaxation in agarose gels

The effects of morphology and exchange on N.M.R. relaxation times in agarose gels are interpreted within a unified theoretical framework based on the generalized Bloch equations. By acknowledging the spacial dependence of the N.M.R. parameters it is shown how the relaxation behaviour depends on the distance scale characterizing the heterogeneity of the gel. If this distance scale is sufficiently small to allow complete diffusive averaging we recover the traditional results based on the Bloch-McConnell equations describing relaxation in a homogeneous system. This is the case for fresh agarose gels which show monoexponential relaxation and has been widely interpreted in terms of the rapid exchange of protons between populations of ‘free’ and ‘bound’ water. Conversely, if the distance scale characterizing the heterogeneity is sufficiently large to prevent complete diffusive averaging our model predicts multiexponential relaxation. This is the case with the transverse magnetization in agarose gels that have been slowly frozen then thawed. These results show how it is possible to probe the degree of microheterogeneity in gel samples using N.M.R. For the purpose of deriving simple analytical expressions for the N.M.R. relaxation times we only consider one-dimensional solutions to our model. More realistic morphologies can be treated using numerical methods.     

Apparatus for rapid adjustment of the degree of alignment of NMR samples in aqueous media: verification with residual quadrupolar splittings in (23)Na and (133)Cs spectra

NMR spectra of (23)Na(+) and (133)Cs(+) in gelatine in a silicone rubber tube that was stretched to various extents showed remarkably reproducible resonance multiplicity. The relative intensities of the components of the split peaks had ratios, 3:4:3, and 7:12:15:16:15:12:7, respectively, that conformed with those predicted using a Mathematica program. The silicone-rubber tube was sealed at its lower end by a small rubber stopper and placed inside a thick-walled glass tube. Gelatine was injected in solution into the silicone tube and 'set' by cooling below 30 degrees C. A plastic thumb-screw held the silicone tube at various degrees of extension, up to approximately 2-fold. After constituting the gel in buffers containing NaCl and CsCl, both (23)Na and (133)Cs NMR spectroscopy revealed that after stretching the initial single Lorentzian line was split into a well-resolved triplet and a heptet, respectively. This was interpreted as being due to coupling between the electric quadrupoles of the nuclei and the average electric field gradient tensor of the collagen molecules of gelatine; these molecules became progressively more aligned in the direction of the main magnetic field, B(0), of the vertical bore magnet, as the gel was stretched. This apparatus provides a simple way of demonstrating fundamental physical characteristics of quadrupolar cations, some characteristics of gelatine under stretching, and a way to invoke static distortion of red blood cells. It should be useful with these and other cell types, for studies of metabolic and membrane transport characteristics that may change when the cells are distorted, and possibly for structural studies of macromolecules.     

Computational framework for the prediction of transcription factor binding sites by multiple data integration

Background

Activation of extracellular signal-regulated protein kinase (ERK), a member of mitogen-activated protein kinase (MAPK) family, has been proposed to mediate neurite outgrowth-promoting effects of several neurotrophic factors in vitro. However, the precise activity of ERK during axonal regeneration in vivo remains unclear. Peripheral axotomy has been shown to activate ERK in the cell bodies of primary afferent neurons and associated satellite cells. Nevertheless, whether ERK is also activated in the axons and surrounded Schwann cells which also play a key role in the regeneration process has not been clarified.

Results

Phosphorylation of ERK in the sciatic nerve in several time-points after crush injury has been examined. Higher phosphorylation of ERK was observed in the proximal and distal nerve stumps compared to the contralateral intact nerve from one day to one month after crush. The activation of ERK was mainly localized in the axons of the proximal segments. In the distal segments, however, active ERK was predominantly found in Schwann cells forming Bungner's bands.

Conclusion

The findings indicate that ERK is activated in both the proximal and distal nerve stumps following nerve injury. The role of activated ERK in Wallerian degeneration and subsequent regeneration in vivo remains to be elucidated.     

Agarose as a tissue equivalent phantom material for NMR imaging

Phantoms for evaluation of nuclear magnetic resonance (NMR) imaging systems were made from water-based agarose gels, according to a standard procedure herein described. Copper sulfate (CuSO4) was included in the gels to further affect their proton relaxation characteristics. The proton relaxation rates of each batch of gel are dependent on the concentrations of agarose and copper ions in it, with T1 depending more on copper than on agarose, and T2 depending more strongly on agarose than on copper. The wide range of T1 and T2 which can be covered, and the stability and physical characteristics of the agarose gel material make it well-suited for phantom use.     

碳纳米管与石墨烯协同提高导热硅脂热性能

以甲基硅油为基础油,碳纳米管、石墨烯或碳纳米管/石墨烯混杂物为导热填料,制备复合导热硅脂.研究结果表明:以碳纳米管为单一导热填料,碳纳米管管径越小,管长越长,越有利于导热硅脂的导热性能提升。当总填充量在6%、碳纳米管和石墨烯配比为2:1时,导热硅脂的热导率提高19%。碳纳米管对石墨烯纳米片起到了分隔和桥连的作用,提高了石墨烯纳米片的分散性,有利于三维热传导网络的形成,进而提高导热硅脂的热传导性能。     

Noninvasive diffusion MRI to determine the severity of peripheral nerve injury

ObjectivePrimary repair of peripheral nerves is recommended following transection; however, patient management following repair is challenged by a lack of biomarkers to nerve regeneration. Previous studies have demonstrated that diffusion magnetic resonance imaging (MRI) may provide viable biomarkers of nerve regeneration in injury models; though, these methods have not been systematically evaluated in graded partial transections and repairs.MethodsEx vivo diffusion MRI was performed in fixed rat sciatic nerve samples 4 or 12 weeks following partial nerve transection and repair (25% cut = 12, 50% cut = 12 and 75% cut = 11), crush injuries (n = 12), and sham surgeries (n = 9). Behavioral testing and histologic evaluation were performed in the same animals and nerve samples for comparison.ResultsDiffusion tractography provided visual characterizations of nerve damage and recovery consistent with the expected degree of injury within each cohort. In addition, quantitative indices from diffusion MRI correlated with both histological and behavioral evaluations, the latter of indicated full recovery for sham and crush nerves and limited recovery in all partially transected/repaired nerves. Nerve recovery between 4 and 12 weeks was statistically significant in partial transections 50% and 75% depth cuts (p = 0.043 and p = 0.022) but not for 25% transections.InterpretationOur findings suggest that DTI can i) distinguish different degrees of partial nerve transection following surgical repair and ii) map spatially heterogeneous nerve recovery (e.g., due to collateral sprouting) from 4 to 12 weeks in partially transected nerves.     

VI. Preparation of agarose gels as reference substances for NMR relaxation time measurement

A standard method has been developed for the production of reference materials for NMR relaxation time measurement. These are based on agarose gels doped with gadolinium. The reproducibility and temporal stability of the gels have been found to be excellent. Electron microscope studies have shown good homogeneity. The relaxation behavior of such gels has been mathematically modeled with good agreement with experiment.     

Peripheral nerve diffusion tensor imaging

We examined if MR diffusion tensor imaging with fiber tracking could be performed on the sciatic nerve. In 3 healthy volunteers the proximal thigh was examined on a 1.5-Tesla MR-scanner with a 2-channel phased-array coil. Though inherently susceptible to motion, field inhomogeneities, and fast T(2)-relaxation of the diffusion imaging technique, the sciatic nerve could be detected and correlated to T(2)-weighted images in all 3 subjects. Further investigations need to be done to improve the signal-to-noise ratio and to see if smaller peripheral nerves than the sciatic nerve can be detected.     

Relaxation times in paramagnetically doped agarose gels as a function of temperature and ion concentration

Tissue equivalent gels of NMR phantoms have been investigated at 3.4 MHz. The proton T1 and T2 relaxation times have been measured in Ni++ and Cu++ doped agarose gels as a function of temperature and ion concentration. Ni-agarose gels have the lower T1 temperature dependence, but gels containing both Cu++ and Ni++ can be produced for which T1 has virtually no temperature dependence.     

MR Imaging of total hip arthroplasty: comparison among sequences to study the sciatic nerve at 1.5 T

Purpose

This study was done to test a series of MR sequences for evaluating the sciatic nerve after total hip arthroplasty (THA).

Material and Methods

The study protocol was approved by the institutional review board. Informed consent was obtained from all patients. Twenty-five patients (11 men and 14 women mean age: 62.3±5.7 years) with THA were included in this prospective study. MRI protocol included sequences that were preliminarily tailored for nerve imaging in patients with THA: proton density (PD)-weighted turbo SE, T1-weighted turbo SE (TSE) 3 mm thickness, T1-weighted turbo SE (TSE) 6 mm thickness, T1-weighted turbo SE with high bandwidth (TSE hBW), T2- weighted TSE, T2-weighted with fat saturation and short-tau inversion recovery (STIR). For each sequence, we evaluated the visibility of the sciatic nerve using a semiquantitative score (0=total masking; 1=insufficient visibility; 2=sufficient visibility; 3=optimal visibility). The sum of the scores given to each sequence was divided by the maximal sum, obtaining a percentage visibility index. Friedman and sign tests were used for statistical analysis.

Results

MR examination time was approximately 40 min. No patients reported pain, heat or symptoms related to nerve stimulation. The visibility index ranged between 88% and 70% for the first four sequences. The T1-weighted TSE hBW sequence had the best visibility index (P<.05). The visibility indexes of the first four sequences were significantly higher (P<.004, sign test) than those of the remaining three sequences.

Conclusion

The sciatic nerve could be studied at 1.5 T in patients following THA. The nerve is better visualized with T1-weighted TSE hBW sequences. On T2-weighted sequences and STIR, the visibility of the nerve is low.     

Monodisperse Branched Molybdenum‐Based Bioactive Nanoparticles Significantly Promote Osteogenic Differentiation of Adipose‐Derived Stem Cells

Adipose‐derived stem cells (ADSCs) are considered to be ideal stem cell sources for bone‐tissue regeneration owing to their ease of collection and high activity. However, the regulation of osteogenic differentiation of ADSCs using biomaterials without adding growth factors is still not satisfactory. For the first time, molybdenum‐doped bioactive glass nanoparticles with a radial porous morphology (Mo‐rBGNs) are reported and their role in the osteogenic differentiation of ADSCs is investigated. The results show that Mo‐rBGNs exhibit radially porous and spherical morphology, relatively homogeneous particle size (200–400 nm), and excellent apatite‐forming bioactivity. They do not affect the proliferation of ADSCs, but significantly regulate their osteogenic differentiation and biomineralization. 5% Mo‐rBGNs significantly enhance the alkaline phosphatase activity and biomineralization ability and promote the osteogenic gene expressions of collagen I secretion and bone sialo protein in ADSCs. A reasonable and promising strategy for designing nanoscale bioactive materials with the excellent osteogenic ability for stem cell–based bone tissue regeneration is provided.     

Influence of Addition of Hyaluronic Acid (HA) on the Properties of Collagen/HA Composite Scaffolds

In order to develop a scaffolding material for tissue regeneration, porous matrices containing varying composites of collagen and hyaluronic acid (HA) (from 1:0 to 0:1) were fabricated using a freeze-drying method. The effect of the composition on the morphology, hydrophilicity, swelling behavior, mechanical properties, and in vitro cytotoxicity was investigated. The results showed that all the scaffolds had an interconnected pore structure with sufficient pore size for use as a support for the growth of fibroblasts. The addition of HA improved the swelling property, but reduced the compressive strength. The contact angle decreased with increasing HA content. In in vitro cytotoxicity tests using fibroblastic cells, the collagen/HA scaffolds showed no toxicity. All these results suggest that collagen/HA composite scaffolds are a potential candidate for tissue engineering scaffolds.     

Efficient delivery of 60 J pulse energy of long pulse Nd:YAG laser through 200 μm core diameter optical fibre

Most of today’s industrial Nd:YAG lasers use fibre-optic beam delivery. In such lasers, fibre core diameter is an important consideration in deploying a beam delivery system. Using a smaller core diameter fibre allows higher irradiances at focus position, less degradation of beam quality, and a larger stand-off distance. In this work, we have put efforts to efficiently deliver the laser output of ‘ceramic reflector’-based long pulse Nd:YAG laser through a 200 μm core diameter optical fibre and successfully delivered up to 60 J of pulse energy with 90% transmission efficiency, using a GRADIUM (axial gradient) plano-convex lens to sharply focus down the beam on the end face of the optical fibre and fibre end faces have been cleaved to achieve higher surface damage thresholds.     

LDA侧面泵浦固体激光器泵浦结构参数优化

为LDA侧面泵浦Nd∶YAG棒状固体激光器的优化设计、参数选取及后续试验提供了理论依据和参考,建立了环形侧面泵浦棒状介质泵浦光场分布数值计算模型,研究了LDA侧面泵浦固体Nd∶YAG激光器泵浦光场的分布特点。模拟分析了LDA的bar间距、玻璃管厚度、激光晶体半径等几个主要泵浦结构参数对泵浦光场分布的影响。通过对不同泵浦参数下泵浦光分布特性的数值计算,优化了LDA侧面泵浦固体激光器的泵浦结构参数,优化后的仿真结果表明:在泵浦距离为2 mm条件下,晶体直径为4 mm,玻璃管套筒厚度为1 mm,冷却水层厚度为1 mm时,泵浦光在晶体中心处强度相对值为40.8%,在晶体轴心附近分布比较均匀,且均匀分布区域相对较大。     

Sorption and Desorption of PVA-Pyrene Chains in and out of Agarose Gel

In situ steady-state fluorescence (SSF) measurement technique was applied to investigation of pyrene labeled Poly(vinyl alcohol) (PVA-Py) molecules diffusion in and out of agarose gels. Gel samples with four different concentration of agarose were prepared. PVA-Py was synthesized by "click" chemistry method and dissolved in water to use in diffusion experiments. The results were analyzed by using Fickian type diffusion model, and it was found that sorption and desorption processes of PVA-Py molecules in and out of agarose gel have two distinct regions for short and long diffusion times. Sorption and desorption coefficients were measured and it was seen that the diffusion rates were much larger at short times and at lower agarose concentrations.     

Cryopreservation of in vitro shoot tips of Dioscorea deltoidea Wall., an endangered medicinal plant: effect of cryogenic procedure and storage duration

In vitro shoot tips of Dioscorea deltoidea Wall., an endangered medicinal plant, were successfully cryopreserved using the vitrification and the encapsulation-dehydration techniques with subsequent high frequency plant regeneration. Using vitrification, post-liquid nitrogen (LN) shoot regeneration up to 83% was recorded when excised shoot tips were pretreated overnight on MS medium containing 0.3 M sucrose followed by loading with MS containing 2 M glycerol plus 0.4 M sucrose for 20 min at 25 degree C, dehydration with PVS2 for 90 min at 0 degree C and quenching in LN. After 1 h of storage in LN, the shoot tips were rewarmed in a water-bath at 40 degrees C, unloaded with 1.2 M sucrose solution for 20 min and cultured on recovery growth medium. While using encapsulation-dehydration, the highest regeneration frequency recorded was 76% when sucrose-pretreated shoot tips were encapsulated with 3% calcium alginate, precultured in 0.75 M sucrose for 3 days, dehydrated to 25% moisture content (FW basis) under the laminar air flow, stored in LN for 1h and rewarmed at 40 degree C. The cryopreserved shoot tips maintained their viability and an unaltered level of regeneration capability after up to one year of storage in LN.     

Ultrasonic wave properties in the particle compounded agarose gels

Ultrasonic wave properties (attenuation and velocity) in the particle compounded agarose gels have been experimentally studied in the range from 1 to 30 MHz. The particles used were talc, glass beads and graphite. The effects of size and volume concentration of particles were clearly observed as changes of ultrasonic wave properties. Applying the Urick's theory for viscous liquid suspensions, the specific curves of velocity in the gels were observed as a function of a beta, where a is the radius of the particles and beta is described by angular frequency omega, density rho and fluid viscosity eta. This indicates that the particle behavior in the gels seems to be similar with that in the viscous fluid. The estimated eta in the gels was higher than that of the free water, showing the high viscosity in the gels.