Effects of lignocellulosic biomass type on the economics of hydrothermal treatment of digested sludge for solid fuel and soil amendment applications.

Digested sludge is a waste stream from anaerobic digestion (AD) in wastewater treatment plants. Hydrothermal treatment (HTT) of sludge mixed with lignocellulosic biomass is an attractive approach to improve sludge dewaterability and generate value-added products. However, process economics has not been well understood. In this study, firstly, the effect of biomass type on the energy properties of hydrochars was studied. Secondly, two scenarios were simulated to evaluate the effects of biomass type on the economics (processing 50,000 tonnes of sludge per year) of HTT of digested sludge for solid fuel and soil amendment applications. The two HTT scenarios included sludge alone and sludge-biomass mixtures (four cases for four biomass feedstocks) at 180 °C for 60 min. In both scenarios, HTT liquids were returned to existing AD facilities for biomethane production to offset the energy cost of the HTT process. The results showed that the higher heating value significantly increased from 16.0-17.0 MJ kg-1 in the sludge alone case to 18.0-23.0 MJ kg-1 in sludge-biomass mixtures (except for rice husk). With the use of saved transport cost as a revenue source, HTT of sludge-biomass led to a net present value (NPV) range of AU$ 9.9-20.3 million (20 years) and an internal rate of return (IRR) range of 25.0 %-45.2 % for solid fuel application of resulting hydrochar compared to an NPV of AU$ 18.4 million and an IRR of 55.0 % from HTT of sludge alone scenario. HTT of sludge-biomass led to a NPV range of AU$ 4.5-14.5 million and an IRR range of 17.2 %-35.7 % for soil amendment application while the hydrochar from HTT of sludge alone was not recommended for soil application due to the high contents of heavy metals. This study provides useful and critical information for process scale-up and commercialization for integration into wastewater treatment plants.

Effects of hydrochar derived from hydrothermal treatment of sludge and lignocellulose mixtures on soil properties, nitrogen transformation, and greenhouse gases emissions.

In this study, hydrochar samples derived from hydrothermal treatment (HTT) of sludge and sludge-biomass mixtures were applied to a sandy soil and their effects on soil properties, soil nutrients, greenhouse gas (GHG) emissions, and soluble heavy metals were investigated. The application of untreated sludge and hydrochar derived from HTT of sludge at 180 °C led to the highest soluble nitrate, CO2 and N2O emissions, followed by the application of hydrochar samples derived from HTT of sludge-biomass mixtures at 180 °C. Although the application of hydrochar samples derived from HTT of sludge alone and sludge-biomass mixtures at 240 °C in sandy soil led to the lowest emissions of CO2 and N2O, it resulted in lower levels of soil electrical conductivity (EC), cation exchange capacity (CEC) and soluble phosphorus. The application of hydrochar samples derived from HTT at 240 °C led to the production of CH4 and lower nitrate-N contents than hydrochar samples derived from HTT at 180 °C. These results indicated that the soils containing hydrochar samples from HTT at 240 °C were anaerobic, which might inhibit the growth of plants. The application of hydrochar samples derived from HTT of sludge-biomass at 180 °C led to significantly improved contents of soil soluble phosphorus (2.56 and 2.84 g kg-1 soil) and soil nitrate-N (160.2 and 263.2 mg kg-1 soil) at the end of 60 days of incubation. However, these contents were lower than the contents of soluble phosphorus (3.71 and 4.45 g kg-1 soil) and nitrate-N (528.3 and 583.2 mg kg-1 soil) with the application of untreated sludge and sludge derived from HTT of sludge alone at 180 °C. Although more studies are needed to understand the mechanisms and effects on different soils, this study provides useful insights into the application of hydrochar derived from sludge-biomass mixture in soil.

Effects of lignocellulosic biomass type on nutrient recovery and heavy metal removal from digested sludge by hydrothermal treatment.

Sludge is a nutrient-rich organic waste generated from wastewater treatment plants. However, the application of sludge as a nutrient source is limited by its high contents of water and pollutants. In this study, the effects of biomass type on nutrient recovery and heavy metal removal from digested sludge by hydrothermal treatment (HTT) were investigated. Blending biomass with digested sludge for HTT at 180-240 °C increased the recovery of nitrogen in the treated solids. At the HTT temperature of 240 °C, HTT with hardwood sawdust led to the highest nitrogen recovery of 70.6%, compared to the lowest nitrogen recovery of 36.5% without biomass. Blending biomass slightly decreased the recovery of phosphorus compared to those without biomass. Nevertheless, the lowest phosphorus recovery of 91.3% with the use of hardwood sawdust at the HTT temperature of 240 °C was only ∼7.0% less than that without biomass. Blending biomass reduced the contents of macro-metals such as Ca, Fe, Mg and Al in treated solids but the metal contents varied with different biomasses. Regarding the heavy metals, the use of rice husk did not decrease the contents of Ni and Co while blending bagasse did not decrease the content of Cr at HTT temperatures of 210 °C and 240 °C compared to the use of other biomasses. The different effects of biomass type on nutrient recovery and heavy metals were likely related to the types and abundances of organic acids such as acetic acid, oxygen-containing functional groups such as C-OH and COOH, oxide minerals such as silica from biomasses and the overall effects of these factors. This study provides very useful information in selection of lignocellulosic biomass for HTT of sludge for nutrient recovery and heavy metal removal.

Effect of hydrothermal treatment on deep dewatering of digested sludge: Further understanding the role of lignocellulosic biomass.

In this study, lignocellulose-assisted hydrothermal treatment (HTT) of digestated sludge was studied to further understand the role of biomass in HTT and its effect on subsequent sludge dewatering. HTT of sludge-biomass mixtures at 180 °C for 60 min at a sludge/biomass total solids (TS) ratio of 1:1 led to solid residue moistures of 36%-40% after dewatering using a hydraulic press at 24 MPa, compared to 69.5% without biomass. Further investigation showed that organic acids, especially acetic acid generated from lignocellulosic biomass hydrolysed extracellular polymeric substances (EPS), especially EPS-protein, and improved sludge dewaterability. The role of organic acids was further verified with the addition of 10.0 g/L acetic acid for HTT of sludge at 180 °C in the absence of biomass. It was also observed that in HTT of sludge with 10.0 g/L acetic acid, protein nitrogen was converted to more stable forms of nitrogen such as pyrrole­nitrogen and quaternary­nitrogen. However, HTT with acetic acid alone resulted in dewatered solids with high ash contents, which may limit their applications as soil amendments. Combination of biomass and acetic acid with a sludge/biomass TS ratio of 3:1 and acetic acid loading of 10.0 g/L at a HTT temperature of 180 °C for 60 min led to solid moistures of 50.5% with hardwood sawdust and 57.7% with sugarcane bagasse after dewatering at 3 MPa, corresponding to total weight reductions of 66.3% and 55.7%, respectively. In contrast, HTT of sludge at 180 °C for 60 min without acetic acid and biomass resulted in a solid moisture of 76.6% after dewatering at 3 MPa and a corresponding weight reduction of 49.5%. With the use of biomass and acetic acid in HTT, the treated and dewatered solids also had increased carbon content and reduced ash content. These dewatered solids may be used as potential soil amendments though the properties related to soil applications need to be considered in future studies.

Electrophysiological analysis of healthy and dystrophic 3-D bioengineered skeletal muscle tissues.

Recently, methods for creating three-dimensional (3-D) human skeletal muscle tissues from myogenic cell lines have been reported. Bioengineered muscle tissues are contractile and respond to electrical and chemical stimulation. In this study, we provide an electrophysiological analysis of healthy and dystrophic 3-D bioengineered skeletal muscle tissues, focusing on Duchenne muscular dystrophy (DMD). We enlist the 3-D in vitro model of DMD muscle tissue to evaluate muscle cell electrical properties uncoupled from presynaptic neural inputs, an understudied aspect of DMD. Our data show that previously reported electrophysiological aspects of DMD, including effects on membrane potential and membrane resistance, are replicated in the 3-D muscle tissue model. Furthermore, we test a potential therapeutic compound, poloxamer 188, and demonstrate capacity for improving the membrane potential in DMD muscle. Therefore, this study serves as a baseline for a new in vitro method to examine potential therapies for muscular disorders.

Effect of ferrous iron loading on dewaterability, heavy metal removal and bacterial community of digested sludge by Acidithiobacillus ferrooxidans.

Acidithiobacillus ferrooxidans ILS-2 was adapted in digested sludge and used to treat sludge for dewaterability improvement. Results showed that increasing ferrous iron loading increased sludge dewaterability, but the inoculation of the bioleaching strain had little effect on sludge dewaterability compared to controls without the strain. The total extracellular polymeric substances (EPS) contents of sludges with and without bioleaching treatment were similar except for bioleaching treatment at 10% ferrous iron loading (on sludge total solids) where total EPS was higher with bioleaching treatment. However, bioleaching treatment for 48 h had a notable effect on removal of heavy metals, such as Mn, Ni and Zn, especially at the high loadings of ferrous iron. In the presence of A. ferrooxidans, the removal of Ni, Mn and Zn reached 93%, 88% and 80%, respectively, at a ferrous iron loading of 21%. The sequencing of 16S rRNA genes indicated that increasing ferrous iron loadings to 15% and 21% increased the relative abundance of Acidithiobacillus, Acidocella (with A. ferrooxidans) and Carboxylicivirga (without A. ferrooxidans) but decreased the abundance of Pseudomonas and Acinetobacter after 48 h treatment. This study enhanced the understanding of the correlations between bioleaching treatment of digested sludge, sludge dewaterability, heavy metal removal and bacterial communities.

De novo revertant fiber formation and therapy testing in a 3D culture model of Duchenne muscular dystrophy skeletal muscle.

The biological basis of Duchenne muscular dystrophy (DMD) pathology is only partially characterized and there are still few disease-modifying therapies available, therein underlying the value of strategies to model and study DMD. Dystrophin, the causative gene of DMD, is responsible for linking the cytoskeleton of muscle fibers to the extracellular matrix beyond the sarcolemma. We posited that disease-associated phenotypes not yet captured by two-dimensional culture methods would arise by generating multinucleated muscle cells within a three-dimensional (3D) extracellular matrix environment. Herein we report methods to produce 3D human skeletal muscle microtissues (hMMTs) using clonal, immortalized myoblast lines established from healthy and DMD donors. We also established protocols to evaluate immortalized hMMT self-organization and myotube maturation, as well as calcium handling, force generation, membrane stability (i.e., creatine kinase activity and Evans blue dye permeability) and contractile apparatus organization following electrical-stimulation. In examining hMMTs generated with a cell line wherein the dystrophin gene possessed a duplication of exon 2, we observed rare dystrophin-positive myotubes, which were not seen in 2D cultures. Further, we show that treating DMD hMMTs with a ß1-integrin activating antibody, improves contractile apparatus maturation and stability. Hence, immortalized myoblast-derived DMD hMMTs offer a pre-clinical system with which to investigate the potential of duplicated exon skipping strategies and those that protect muscle cells from contraction-induced injury. STATEMENT OF SIGNIFICANCE: Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disorder that is caused by mutation of the dystrophin gene. The biological basis of DMD pathology is only partially characterized and there is no cure for this fatal disease. Here we report a method to produce 3D human skeletal muscle microtissues (hMMTs) using immortalized human DMD and healthy myoblasts. Morphological and functional assessment revealed DMD-associated pathophysiology including impaired calcium handling and de novo formation of dystrophin-positive revertant muscle cells in immortalized DMD hMMTs harbouring an exon 2 duplication, a feature of many DMD patients that has not been recapitulated in culture prior to this report. We further demonstrate that this "DMD in a dish" system can be used as a pre-clinical assay to test a putative DMD therapeutic and study the mechanism of action.

Assessing Functional Metrics of Skeletal Muscle Health in Human Skeletal Muscle Microtissues.

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study skeletal muscle reformation and function in a scalable format that is amenable to experimental manipulations. 3D muscle culture systems are desirable as they enable scientists to study skeletal muscle ex vivo in the context of human cells. 3D in vitro models closely mimic aspects of the native tissue structure of adult skeletal muscle. However, their universal application is limited by the availability of platforms that are simple to fabricate, cost and user-friendly, and yield relatively high quantities of human skeletal muscle tissues. Additionally, since skeletal muscle plays an important functional role that is impaired over time in many disease states, an experimental platform for microtissue studies is most practical when minimally invasive calcium transient and contractile force measurements can be conducted directly within the platform itself. In this protocol, the fabrication of a 96-well platform known as 'MyoTACTIC', and en masse production of 3D human skeletal muscle microtissues (hMMTs) is described. In addition, the methods for a minimally invasive application of electrical stimulation that enables repeated measurements of skeletal muscle force and calcium handling of each microtissue over time are reported.

TGFß signaling curbs cell fusion and muscle regeneration.

Muscle cell fusion is a multistep process involving cell migration, adhesion, membrane remodeling and actin-nucleation pathways to generate multinucleated myotubes. However, molecular brakes restraining cell-cell fusion events have remained elusive. Here we show that transforming growth factor beta (TGFß) pathway is active in adult muscle cells throughout fusion. We find TGFß signaling reduces cell fusion, regardless of the cells' ability to move and establish cell-cell contacts. In contrast, inhibition of TGFß signaling enhances cell fusion and promotes branching between myotubes in mouse and human. Exogenous addition of TGFß protein in vivo during muscle regeneration results in a loss of muscle function while inhibition of TGFßR2 induces the formation of giant myofibers. Transcriptome analyses and functional assays reveal that TGFß controls the expression of actin-related genes to reduce cell spreading. TGFß signaling is therefore requisite to limit mammalian myoblast fusion, determining myonuclei numbers and myofiber size.

Global and local tension measurements in biomimetic skeletal muscle tissues reveals early mechanical homeostasis.

Tension and mechanical properties of muscle tissue are tightly related to proper skeletal muscle function, which makes experimental access to the biomechanics of muscle tissue formation a key requirement to advance our understanding of muscle function and development. Recently developed elastic in vitro culture chambers allow for raising 3D muscle tissue under controlled conditions and to measure global tissue force generation. However, these chambers are inherently incompatible with high-resolution microscopy limiting their usability to global force measurements, and preventing the exploitation of modern fluorescence based investigation methods for live and dynamic measurements. Here, we present a new chamber design pairing global force measurements, quantified from post-deflection, with local tension measurements obtained from elastic hydrogel beads embedded in muscle tissue. High-resolution 3D video microscopy of engineered muscle formation, enabled by the new chamber, shows an early mechanical tissue homeostasis that remains stable in spite of continued myotube maturation.

3D Bioprinting in Skeletal Muscle Tissue Engineering.

Skeletal muscle tissue engineering (SMTE) aims at repairing defective skeletal muscles. Until now, numerous developments are made in SMTE; however, it is still challenging to recapitulate the complexity of muscles with current methods of fabrication. Here, after a brief description of the anatomy of skeletal muscle and a short state-of-the-art on developments made in SMTE with "conventional methods," the use of 3D bioprinting as a new tool for SMTE is in focus. The current bioprinting methods are discussed, and an overview of the bioink formulations and properties used in 3D bioprinting is provided. Finally, different advances made in SMTE by 3D bioprinting are highlighted, and future needs and a short perspective are provided.

Epstein-Barr virus is not detected in mucosal lichen planus

BACKGROUND: Lichen planus (LP) is a chronic inflammatory, immunological, mucocutaneous disease can affect skin, genital and oral mucosa. Oral lichen planus (OLP) is the most common noninfectious, chronic inflammatory oral disease affecting 1-2% of the general adult population. World Health Organization (WHO) classifies OLP as a potentially malignant disorder. Epstein Barr virus or human herpesvirus-4, is a member of the herpes virus family and one of the most ubiquitous viruses known to human, infecting approximately 90% of the world's adult population. The virus often infects B lymphocytes resulting in a wide spectrum of mucocutaneous and systemic diseases, ranging from mild lesions to aggressive malignancies. The aim of this study was to investigate expression of the EBV encoded RNAs EBER1 and EBER2 in oral and genital lichen planus and compare results with normal tissues in situ hybridization which is considered the golden standard for detection of EBER. MATERIAL AND METHODS: A total of 68 biopsies, 25 oral LP, 26 genital LP, 10 oral controls and finally 7 genital controls were analysed using situ hybridization. RESULT: All samples had RNA as shown by the control slide, whereas no case contained neither EBER1 nor EBER2. CONCLUSIONS: Based on results from our study EBV is not involved in aetiology of lichen planus

Enhanced skeletal muscle formation on microfluidic spun gelatin methacryloyl (GelMA) fibres using surface patterning and agrin treatment.

Bioengineered functional muscle tissues are beneficial for regenerative medicine due to their treatment potential for various debilitating disorders, including myopathy and traumatic injuries. However, the contractile properties of engineered muscle constructs are lacking compared with their native counterparts. Here, we used microfluidic spinning to fabricate photocrosslinkable gelatin methacryloyl (GelMA) hydrogel fibres with well-defined surface morphologies for engineering muscle tissues. We examined whether the combination of topographical cues from surface micropatterning and biochemical stimulation with recombinant agrin can improve the generation of bioengineered muscle tissue. Topographical cues on micropatterned fibres promoted alignment of C2C12 myoblasts and augmented myotube formation during differentiation, as assessed by increased myotube length, aspect ratio, and the elevated mRNA expression of myogenic genes. Moreover, agrin treatment significantly increased acetylcholine receptor expression/clustering and myotube formation and upregulated dystrophin expression in differentiated C2C12 myotubes. Interestingly, the combination of topographical cues with agrin treatment further enhanced myotube maturation and functionality as shown by improved contractility under electrical stimulation. Thus, combining topographical cues and agrin treatment improved functions of engineered muscle tissue, which has potential in biorobotics, drug screening, tissue engineering, and regenerative medicine.

Increased expression of p16 in both oral and genital lichen planus

BACKGROUND: Lichen Planus, LP, is an inflammatory disease of possible autoimmune origin affecting mainly oral and genital mucosa and skin. According to the WHO oral LP is considered a potentially malignant disorders. The p16 tumour suppressor protein can act as an inhibitor of cyclin dependent kinases 4 and 6 and thus down regulate cell cycle progression. Since the discovery of p16 several studies have evaluated its expression in various forms of human cancers. The aim of this study was to evaluate and compare the expression of p16 in oral and genital LP and corresponding healthy mucosa. MATERIAL AND METHODS: A total of 76 cases of oral LP (OLP), 34 cases of genital LP (GLP), 12 cases of healthy oral and 9 cases of healthy genital mucosa were analysed by the use of immunohistochemistry. RESULTS: Data showed p16 to be highly expressed in both oral and genital LP, higher than in oral (p = 0.000), and genital controls (p = 0.002). CONCLUSIONS: Results suggest that the over-expression of p16 seen in LP play a part in the histopathology of the disease

Electrical stimulation of microengineered skeletal muscle tissue: Effect of stimulus parameters on myotube contractility and maturation.

Skeletal muscle tissues engineered in vitro are aneural, are short in the number of fibres required to function properly and degenerate rapidly. Electrical stimulation has been widely used to compensate for such a lack of neural activity, yet the relationship between the stimulation parameters and the tissue response is subject to debate. Here we studied the effect of overnight electrical stimulation (training) on the contractility and maturity of aligned C2C12 myotubes developed on micropatterned gelatin methacryloyl (GelMA) substrates. Bipolar rectangular pulse (BRP) trains with frequency, half-duration and applied pulse train amplitudes of f = 1 Hz, ton  = 0.5 ms and Vapp  = {3 V, 4 V, 4.5 V}, respectively, were applied for 12 h to the myotubes formed on the microgrooved substrates. Aligned myotubes were contracting throughout the training period for Vapp  ≥ 4 V. Immediately after training, the samples were subjected to series of BRPs with 2 ≤ Vapp  ≤ 5 V and 0.2 ≤ ton  ≤ 0.9 ms, during which myotube contraction dynamics were recorded. Analysis of post-training contraction revealed that only the myotubes trained at Vapp  = 4 V displayed consistent and repeatable contraction profiles, showing the dynamics of myotube contractility as a function of triggering pulse voltage and current amplitudes, duration and imposed electrical energy. In addition, myotubes trained at Vapp  = 4 V displayed amplified expression levels of genes pertinent to sarcomere development correlated with myotube maturation. Our findings are imperative for a better understanding of the influence of electrical pulses on the maturation of microengineered myotubes.

Gelatin-Polyaniline Composite Nanofibers Enhanced Excitation-Contraction Coupling System Maturation in Myotubes.

In this study, composite gelatin-polyaniline (PANI) nanofibers doped with camphorsulfonic acid (CSA) were fabricated by electrospinning and used as substrates to culture C2C12 myoblast cells. We observed enhanced myotube formation on composite gelatin-PANI nanofibers compared to gelatin nanofibers, concomitantly with enhanced myotube maturation. Thus, in myotubes, intracellular organization, colocalization of the dihydropyridine receptor (DHPR) and ryanodine receptor (RyR), expression of genes correlated to the excitation-contraction (E-C) coupling apparatus, calcium transients, and myotube contractibility were increased. Such composite material scaffolds combining topographical and electrically conductive cues may be useful to direct skeletal muscle cell organization and to improve cellular maturation, functionality, and tissue formation.

Effects of acidified aqueous glycerol and glycerol carbonate pretreatment of rice husk on the enzymatic digestibility, structural characteristics, and bioethanol production.

Rice husk as an abundant biomass was used in this study, and it contained 30.1% glucan and 13.5% xylan, 22.4% lignin. The pretreated rice husk with glycerol carbonate and acidified aqueous glycerol (10% water) at 90°C and 130°C for 60min had the maximum yield of glucan digestibility which was 78.2% and 69.7% respectively, using cellulase for 72h. The simultaneous saccharification and fermentation was conducted anaerobically at 37°C with Saccharomyces cerevisiae, 5% w/v glucan and 10FPU/g glucan of cellulase. 11.58 and 8.84g/L was the highest ethanol concentration after 3days of incubation form pretreated rice husk with glycerol carbonate and acidified aqueous glycerol respectively.

Development of Flexible Cell-Loaded Ultrathin Ribbons for Minimally Invasive Delivery of Skeletal Muscle Cells.

Cell transplantation therapy provides a potential solution for treating skeletal muscle disorders, but cell survival after transplantation is poor. This limitation could be addressed by grafting donor cells onto biomaterials to protect them against harsh environments and processing, consequently improving cell viability in situ. Thus, we present here the fabrication of poly(lactic-co-glycolic acid) (PLGA) ultrathin ribbons with "canal-like" structures using a microfabrication technique to generate ribbons of aligned murine skeletal myoblasts (C2C12). We found that the ribbons functionalized with a solution of 3,4-dihydroxy-l-phenylalanine (DOPA) and then coated with poly-l-lysine (PLL) and fibronectin (FN) improve cell attachment and support the growth of C2C12. The viability of cells on the ribbons is evaluated following the syringe-handling steps of injection with different needle sizes. C2C12 cells readily adhere to the ribbon surface, proliferate over time, align (over 74%), maintain high viability (over 80%), and differentiate to myotubes longer than 400 µm. DNA content quantification carried out before and after injection and myogenesis evaluation confirm that cell-loaded ribbons can safely retain cells with high functionality after injection and are suitable for minimally invasive cell transplantation.

Alterations in Factors Involved in Differentiation and Barrier Function in the Epithelium in Oral and Genital Lichen Planus.

Lichen planus is a chronic recurrent inflammatory disease affecting both skin and mucosa, mainly in oral and/or genital regions. Keratinocytes go through a well-regulated process of proliferation and differentiation, alterations in which may result in defects in the protective epithelial barrier. Long-term barrier impairment might lead to chronic inflammation. In order to broaden our understanding of the differentiation process in mucosal lichen planus, we mapped the expression of 4 factors known to be involved in differentiation. Biopsies were collected from oral and genital lichen planus lesions and normal controls. Altered expression of all 4 factors in epithelium from lichen planus lesions was found, clearly indicating disturbed epithelial differentiation in lichen planus lesions.

Expression of neutrophil gelatinase-associated lipocalin (NGAL) in peripheral nerve repair.

INTRODUCTION: Trauma is one of the causes of peripheral nerve injuries. Free radicals increase after tissue damage. Free radicals are usually scavenged and detoxified by antioxidants. In this study, we assessed the antioxidative role of the NGAL molecule in sciatic nerve repair in rats. MATERIALS AND METHODS: The sciatic nerves of 40 rats were crushed and the total mRNA of samples from day 1 and 3 and week 1, 3, 5 post injury was extracted. The expression of the NGAL gene was confirmed by RT-PCR. For immunohistochemistry analysis, the samples were fixed in paraformaldehyde and cut in 20 micrometer slices by cryostat. RESULTS: The expression of NGAL significantly upregulated in day 1, 3 and week 1 following the crushing of sciatic nerves in comparison with the intact nerves. Immunohistochemistry results also confirmed the protein expression of this gene. DISCUSSION: The NGAL molecule showed upregulation in the degeneration process after nerve injury, so it may play an important role in nerve repair.