Neural tissue engineering options for peripheral nerve regeneration

Tissue engineered nerve grafts (TENGs) have emerged as a potential alternative to autologous nerve grafts, the gold standard for peripheral nerve repair. Typically, TENGs are composed of a biomaterial-based template that incorporates biochemical cues. A number of TENGs have been used experimentally to bridge long peripheral nerve gaps in various animal models, where the desired outcome is nerve tissue regeneration and functional recovery. So far, the translation of TENGs to the clinic for use in humans has met with a certain degree of success. In order to optimize the TENG design and further approach the matching of TENGs with autologous nerve grafts, many new cues, beyond the traditional ones, will have to be integrated into TENGs. Furthermore, there is a strong requirement for monitoring the real-time dynamic information related to the construction of TENGs. The aim of this opinion paper is to specifically and critically describe the latest advances in the field of neural tissue engineering for peripheral nerve regeneration. Here we delineate new attempts in the design of template (or scaffold) materials, especially in the context of biocompatibility, the choice and handling of support cells, and growth factor release systems. We further discuss the significance of RNAi for peripheral nerve regeneration, anticipate the potential application of RNAi reagents for TENGs, and speculate on the possible contributions of additional elements, including angiogenesis, electrical stimulation, molecular inflammatory mediators, bioactive peptides, antioxidant reagents, and cultured biological constructs, to TENGs. Finally, we consider that a diverse array of physicochemical and biological cues must be orchestrated within a TENG to create a self-consistent coordinated system with a close proximity to the regenerative microenvironment of the peripheral nervous system.     

Characterization of heptapeptide toxins extracted from Microcystis aeruginosa (Egyptian isolate)

Four toxic peptides from local fresh water cyanobacterium Microcystis aeruginosa were purified and identified by high performance liquid chromatography (HPLC) and ion spray mass spectroscopic studies as: RR; YR; LR and LA with molecular weights of 1006.8, 1073, 984.8 and 910.6 respectively. Amino acid analysis indicated the presence of equimolar amounts of aspartic acid, glutamic acid, arginine, leucine and tyrosine, in addition to both alanine and dehydroalanine. Mouse assay toxicity indicated that the first two peptides, at the peak area of RR, YR. were highly toxic with LD₅₀ 20, 18.2 μg/kg body weight; however, the latter two, at the peak areas LR and I.A. have a lesser toxicity with LD₅₀ 36 and 40 μg/kg body weight respectively. Three linear peptide analogs to those naturally found devoid of Adda were synthesized using the continuous flow technique. HPLC pure synthesized analog products were tested for toxicity using male mice (i.p. injection). None of them induced toxic activity.     

骨组织工程支架材料充填下颌阻生智牙拔牙窝的临床效果

目的:探讨下颌阻生智牙拔除后,用骨组织工程支架材料充填拔牙窝,修复第二磨牙远中牙槽骨高度的临床效果。方法:13例患者在阻生智牙拔除后,即刻植入组织工程骨微粒(小牛无机松质骨Bio-oss、倍骼生PerioGlasfi)于牙槽窝内,术后定期随访,并从临床和X线影像检查比较术后1、12周时,第二磨牙远中牙龈附着水平和牙槽嵴高度的变化。采用SPSS10.0软件包对数据进行配对样本t检验。结果:所有患者术后未出现并发症,术后12周时,第二磨牙远中牙槽嵴高度显著降低(P<0.05)。结论:下颌阻生智牙拔除术后即刻植入组织工程骨支架材料,有利于保持第二磨牙远中牙槽嵴的高度。     

Gal/non-Gal antigens in pig tissues and human non-Gal antibodies in the GalT-KO era

Our knowledge regarding Gal and non-Gal antigens in GalT-KO pig tissues can be summarized as α3Galactosyl-tranferase gene knock out eliminates the Galα3Galβ4GlcNAc-R antigen expression in pig tissues as well as anti-Gal antibody binding. Other Galα-terminating saccharides (e.g. iGb3 glycolipids and Galα2 determinants) may be present but have not been documented. α3Galactosyl-tranferase gene knock out slightly changes the carbohydrate antigen expression but no "new" antigens recognized by the human immune system have been found. Non-Gal antigens are both of protein and carbohydrate nature but their exact chemical structures are poorly defined. Regarding human non-Gal antibodies our knowledge is as Non-Gal antibodies exist naturally and increase in humans/non-human primate (NHP) receiving WT or GalT-KO pig grafts. Non-Gal antibodies with new antigen epitope recognition can be induced in humans/NHP after challenge by WT or GalT-KO pig grafts. Non-Gal antibodies react with both carbohydrates and proteins. Part of the protein reactivity is directed to glycoprotein carbohydrates chains. Non-Gal antibodies reacting with neuraminic acid terminated saccharides (both N-Acetyl and N-Glycoloyl variants) are present in humans/NHP. Anti-neuraminic acid antibodies are increased, as well as induced, after grafting pig organs into humans/NHP. Non-Gal antibodies does not cause hyperacute xenorejection but can be cytotoxic and cause xenoorgan damage. If humans sensitized to HLA antigens are at a higher risk of rejecting pig xenograft compared with non-sensitized individuals is not fully clarified. Clinical trials are needed to evaluate the relevance of non-Gal antigens/antibodies and for the xenofield to advance.     

Dicer is essential for neuronal polarity

Dicer, an RNase III endonuclease, is the enzyme which cleaves microRNA (miRNA) and small interfering RNA (siRNA) precursors into 21–25 nucleotide species. This cleavage is an essential step in the biogenesis of these small noncoding RNA molecules. In their mature forms, siRNA and miRNAs function to regulate gene expression through different mechanisms (Bartel, 2004). To investigate the role of Dicer and microRNAs in neuronal polarity development, we used mice in which the RNase III domain of Dicer was conditionally floxed. To knockout Dicer gene, hippocampal neurons were electroporated with Cre together with pmaxGFP^® plasmid by Amaxa^® Mouse Neuron Nucleofector^® Kit. Neuronal polarity was analyzed at 3 days in vitro (DIV). Neurons expressing pmaxGFP^® showed normal polarity. In contrast, the majority of neurons transfected with Cre developed multiple axons. We found multiple axons were significantly increasing. Here we explore Dicer function in neuronal polarity by inactivating it in the hippocampal neuron using the Cre/loxP approach. Neurons which lack Dicer have multiple axons, demonstrating that Dicer is essential for neuron polarity, providing evidence that Dicer function is required to neuronal development.     

High-Temperature Oxidation of Fe3Al Intermetallic Alloy Prepared by Additive Manufacturing LENS

The isothermal oxidation of Fe-28Al-5Cr (at%) intermetallic alloy microalloyed with Zr and B (<0.08 at%) in air atmosphere, in the temperature range of 1000 to 1200 °C, was studied. The investigation was carried out on the thin-walled (<1 mm) elements prepared by Laser Engineered Net Shaping (LENS) from alloy powder of a given composition. Characterization of the specimens, after the oxidation, was conducted using X-ray diffraction (XRD) and scanning electron microscopy (SEM, with back-scatter detector (BSE) and energy-dispersive X-ray spectroscopy (EDS) attachments). The investigation has shown, that the oxidized samples were covered with a thin, homogeneous α-Al₂O₃ oxide layers. The intensity of their growth indicates that the material lost its resistance to oxidation at 1200 °C. Structural analysis of the thin-walled components’ has not shown intensification of the oxidation process at the joints of additive layers.     

肠炎沙门菌ΔspiCΔcrp基因工程疫苗候选株的构建

目的 构建肠炎沙门菌ΔspiCΔcrp双基因缺失株,以探索其作为新型基因工程疫苗的可能性。方法 以等位基因同源重组方法,在构建单基因缺失肠炎沙门菌ΔspiC基础上,运用λRed重组酶系统构建双基因缺失株肠炎沙门菌ΔspiCΔcrp。结果 PCR和抗生素抗性结果表明肠炎沙门菌ΔspiCΔcrp成功构建;生物学鉴定显示,与野生菌相比,其生长速度与生化特性发生了变化,LD₅₀提高约1 000倍,毒力显著降低。结论 双基因缺失株肠炎沙门菌ΔspiCΔcrp被成功构建,为其作为疫苗的免疫学评价奠定基础。     

In vitro engineering of cartilage: effects of serum substitutes, TGF-beta, and IL-1alpha

OBJECTIVES: Cartilage is avascular and relatively homogeneous, making it an attractive tissue for in vitro histogenesis and surgical use in patients. We developed novel platform technologies in order to define the requirements for optimal in vitro chondrogenesis by isolated cells. In this series of studies, we tested alternatives to fetal bovine serum (FBS) and the effects of growth factors on formation of cartilage in 3D porous collagen sponges. DESIGN: We used porous collagen sponges to assess the effects of serum substitutes and exogenous TGF-beta1 and IL-1alpha on chondrocytes (bovine articular chondrocytes, bACs) and on chondroinduced human dermal fibroblasts (hDFs). We determined the effects of low concentrations of FBS and two serum substitutes, Nutridoma and ITS(+3), on cellularity and matrix production. After culture for intervals, sponges were harvested for histological and biochemical measurement of cartilage-specific chondroitin 4-sulfate proteoglycan (C 4-S PG). RESULTS: Cultured bACs showed equivalent growth in Nutridoma (1%) and 10% FBS. Both TGF-beta1 and IL-1alpha significantly stimulated accumulation of C 4-S PG by bACs in 3D porous collagen sponges. Many endogenous growth factors were upregulated in hDFs cultured with chondroinductive DBP. Addition of TGF-beta1 and IL-1alpha for 11 days significantly stimulated accumulation of C 4-S PG by hDFs cultured in DMEM with 1% Nutridoma. CONCLUSION: Porous collagen sponges are supportive of chondrogenesis and of chondroinduction by DBP. Optimization of serum-free culture conditions, including growth factors, matrix components, and mechanical stimuli will expedite translation to wider clinical applications. Use of autogenous dermal fibroblasts pre-cultured with DBP and induced to chondrocytes offers an alternative to autogenous chondrocytes.     

A tan in a test tube - in vitro models for investigating ultraviolet radiation-induced damage in skin

Presently, global rates of skin cancers induced by ultraviolet radiation (UVR) exposure are on the rise. In view of this, current knowledge gaps in the biology of photocarcinogenesis and skin cancer progression urgently need to be addressed. One factor that has limited skin cancer research has been the need for a reproducible and physiologically-relevant model able to represent the complexity of human skin. This review outlines the main currently-used in vitro models of UVR-induced skin damage. This includes the use of conventional two-dimensional cell culture techniques and the major animal models that have been employed in photobiology and photocarcinogenesis research. Additionally, the progression towards the use of cultured skin explants and tissue-engineered skin constructs, and their utility as models of native skin's responses to UVR are described. The inherent advantages and disadvantages of these in vitro systems are also discussed.