Bioactive Nerve Growth Factor Research Articles

Development and Biodistribution of a Nerve Growth Factor Radioactive Conjugate for PET Imaging.

The purpose of these studies was to develop a nerve growth factor (NGF) radiometal-chelator conjugate to determine the biodistribution and brain uptake of NGF by positron emission tomography/computerized tomography (PET-CT). Purified NGF from llama seminal plasma was conjugated with FITC, and the chelator NOTA or DFO. NGF conjugates were evaluated for bioactivity. NOTA- and DFO-conjugated NGF were radiolabeled with gallium-68 or zirconium-89 ([68Ga]GaCl3, half-life = 68min; [89Zr]Zr(oxalate)4, half-life = 3.3days). [89Zr]Zr-NGF was evaluated for biodistribution (0.5, 1, or 24h), PET imaging (60min), and brain autoradiography in mice. Cell-based in vitro assays confirmed that the NGF conjugates maintained NGF receptor-binding and biological activity. Zirconium-89 and gallium-68 radiolabeling showed a high efficiency; however, only[89Zr]Zr-NGF was stable in vitro. Biodistribution studies showed that, as with most small proteins < 70kDa, [89Zr]Zr-NGF uptake was predominantly in the kidney and was cleared rapidly with almost complete elimination of NGF at 24h. Dynamic PET imaging from 0-60min showed a similar pattern to ex vivo biodistribution with some transient liver uptake. Interestingly, although absolute brain uptake was very low, at 24h after treatment, cerebral cortex uptake was higher than any other brain area examined and blood. We conclude that conjugation of DFO to NGF through a thiourea linkage allows effective radiolabeling with zirconium-89 while maintaining NGF bioactivity. Following intravenous administration, the radiolabeled NGF targets non-neuronal tissues (e.g., kidney, liver), and although absolute brain uptake was very low, the brain uptake that was observed was restricted to the cortex.

Propionibacterium acnes contributes to low back pain via upregulation of NGF in TLR2-NF-κB/JNK or ROS pathway

Propionibacterium acnes infection in intervertebral discs (IVDs) is a newly identified cause of low back pain (LBP). In the present study, we aimed to determine whether the nerve growth factor (NGF), a critical pro-algesic factor, is involved in P. acnes-induced LBP. After co-culturing with P. acnes, nucleus pulposus cells (NPCs) produced NGF, which was upregulated after inoculation of P. acnes into IVDs of rats. In addition, administration of P. acnes into rat IVDs leads to significant mechanical allodynia and cold hyperreflexia, and significant upregulation of the pain-related factors, including substance P (SP), calcitonin gene-related peptide (CGRP), and Transient Receptor Potential Vanilloid 1 (TRPV1), in rat dorsal root ganglia (DRG), suggesting that P. acnes-inoculated rats had obvious discogenic LBP. However, inhibition of NGF bioactivity significantly ameliorated P. acnes-induced discogenic LBP, suggesting that P. acnes induced LBP via NGF. Finally, an in vitro mechanism study demonstrated that P. acnes stimulated NPCs to secrete NGF via TLR-2 receptor and NF-κB p65/JNK pathway, or ROS-related pathway. Therefore, P. acnes had a strong association with LBP by stimulating NPCs to secrete NGF via the TLR2-NF- κB/JNK or ROS-related pathway. These findings propose a novel potential therapeutic strategy for LBP.

Small Endogenous Ligands Modulation of Nerve Growth Factor Bioactivity: A Structural Biology Overview.

Experiments with cell cultures and animal models have provided solid support for the assumption that Nerve Growth Factor (NGF) plays a key role in the regulation of neuronal cell survival and death. Recently, endogenous ligands have been proposed as physiological modulators of NGF biological activity as part of this regulatory cascade. However, the structural and mechanistic determinants for NGF bioactivity remain to be elucidated. We recently unveiled, by an integrated structural biology approach, the ATP binding sites of NGF and investigated the effects on TrkA and p75NTR receptors binding. These results pinpoint ATP as a genuine endogenous modulator of NGF signaling, paving the way to the characterization of not-yet-identified chemical diverse endogenous biological active small molecules as novel modulators of NGF. The present review aims at providing an overview of the currently available 3D structures of NGF in complex with different small endogenous ligands, featuring the molecular footprints of the small molecules binding. This knowledge is essential for further understanding the functional role of small endogenous ligands in the modulation of neurotrophins signaling in physiological and pathological conditions and for better exploiting the therapeutic potentialities of NGF.

Endogenous modulators of neurotrophin signaling: Landscape of the transient ATP-NGF interactions

The Nerve Growth Factor (NGF) neurotrophin acts in the maintenance and growth of neuronal populations. Despite the detailed knowledge of NGF’s role in neuron physiology, the structural and mechanistic determinants of NGF bioactivity modulated by essential endogenous ligands are still lacking. We present the results of an integrated structural and advanced computational approach to characterize the extracellular ATP-NGF interaction. We mapped by NMR the interacting surface and ATP orientation on NGF and revealed the functional role of this interaction in the binding to TrkA and p75NTR receptors by SPR. The role of divalent ions was explored in conjunction with ATP. Our results pinpoint ATP as a likely transient molecular modulator of NGF signaling, in health and disease states.

Therapeutic Effects of Simultaneous Delivery of Nerve Growth Factor mRNA and Protein via Exosomes on Cerebral Ischemia

Stroke is the leading neurological cause of death and disability all over the world, with few effective drugs. Nerve growth factor (NGF) is well known for its multifaceted neuroprotective functions post-ischemia. However, the lack of an efficient approach to systemically deliver bioactive NGF into ischemic region hinders its clinical application. In this study, we engineered the exosomes with RVG peptide on the surface for neuron targeting and loaded NGF into exosomes simultaneously, with the resultant exosomes denoted as NGF@ExoRVG. By systemic administration of NGF@ExoRVG, NGF was efficiently delivered into ischemic cortex, with a burst release of encapsulated NGF protein and de novo NGF protein translated from the delivered mRNA. Moreover, NGF@ExoRVG was found to be highly stable for preservation and function efficiently for a long time in vivo. Functional study revealed that the delivered NGF reduced inflammation by reshaping microglia polarization, promoted cell survival, and increased the population of doublecortin-positive cells, a marker of neuroblast. The results of our study suggest the potential therapeutic effects of NGF@ExoRVG for stroke. Moreover, the strategy proposed in our study may shed light on the clinical application of other neurotrophic factors for central nervous system diseases.

The fabrication and related properties study of chitosan-poly (lactide-co-glycolide) double-walled microspheres loaded with nerve growth factor

To evaluate the feasibility of the chitosan-poly (lactide-co-glycolide) (PLGA) double-walled microspheres for sustained release of bioactive nerve growth factor (NGF) in vitro. NGF loaded chitosan-PLGA double-walled microspheres were prepared by emulsion-ionic method with sodium tripolyphosphate (TPP) as an ionic cross-linker. The double-walled microspheres were cross-linked by different concentrations of TPP [1%, 3%, 10% ( W/ V)]. NGF loaded PLGA microspheres were also prepared. The outer and inner structures of double-walled microspheres were observed by light microscopy, scanning electron microscopy, confocal laser scanning microscopy, respectively. The size and distribution of microspheres and fourier transform infra red spectroscopy (FT-IR) were analyzed. PLGA microspheres with NGF or chitosan-PLGA double-walled microspheres cross-linked by 1%, 3%, and 10%TPP concentration (set as groups A, B, C, and D respectively) were used to determine the degradation ratio of microspheres in vitro and the sustained release ratio of NGF in microspheres at different time points. The bioactivity of NGF (expressed as the percentage of PC12 cells with positive axonal elongation reaction) in the sustained release solution of chitosan-PLGA double-walled microspheres without NGF (set as group A1) was compared in groups B, C, and D. The chitosan-PLGA double-walled microspheres showed relative rough and spherical surfaces without aggregation. Confocal laser scanning microscopy showed PLGA microspheres were evenly uniformly distributed in the chitosan-PLGA double-walled microspheres. The particle size of microspheres ranged from 18.5 to 42.7 μm. The results of FT-IR analysis showed ionic interaction between amino groups and phosphoric groups of chitosan in double-walled microspheres and TPP. In vitro degradation ratio analysis showed that the degradation ratio of double-walled microspheres in groups B, C, and D appeared faster in contrast to that in group A. In addition, the degradation ratio of double-walled microsphere in groups B, C, and D decreased when the TPP concentration increased. There were significant differences in the degradation ratio of each group ( P<0.05). In vitro sustained release ratio of NGF showed that when compared with PLGA microspheres in group A, double-walled microspheres in groups B, C, and D released NGF at a relatively slow rate, and the sustained release ratio decreased with the increase of TPP concentration. Except for 84 days, there was significant difference in the sustained release ratio of NGF between groups B, C, and D ( P<0.05). The bioactivity of NGF results showed that the percentage of PC12 cells with positive axonal elongation reaction in groups B, C, and D was significantly higher than that in group A1 ( P<0.05). At 7 and 28 days of culture, there was no significant difference between groups B, C, and D ( P>0.05); at 56 and 84 days of culture, the percentage of PC12 cells with positive axonal elongation reaction in groups C and D was significantly higher than that in group B ( P<0.05), and there was no significant difference between groups C and D ( P>0.05). NGF loaded chitosan-PLGA double-walled microspheres have a potential clinical application in peripheral nerve regeneration after injury.

Tissue-engineered nerve grafts using a scaffold-independent and injectable drug delivery system: a novel design with translational advantages

Objective. Currently commercially available nerve conduits have demonstrated suboptimal clinical efficacy in repairing peripheral nerve defects. Although tissue-engineered nerve grafts (TENGs) with sustained release of neurotrophic factors (NTFs) are experimentally proved to be more effective than these blank conduits, there remains a lack of clinical translation. NTFs are typically immobilized onto scaffold materials of the conduit via adsorption, specific binding or other incorporation techniques. These scaffold-based delivery strategies increase complexity and cost of conduit fabrication and lack flexibility in choosing different drugs. Therefore, to facilitate clinical translation and commercialization, we construct a TENG using a scaffold-independent drug delivery system (DDS). Approach. This study adopted a scaffold-independent DDS based on methoxy-poly (ethylene glycol)-b-poly(γ-ethyl-L-glutamate) (mPEG-PELG) thermosensitive hydrogels that undergo sol-to-gel transition at body temperature. In addition, TENG, a chitosan scaffold filled with nerve growth factor (NGF)-loaded mPEG-PELG that gel in the lumen upon injection during surgery and function as a drug-releasing conduit-filler, was designed. Subsequently, the efficacy of DDS and therapeutic effects of TENG were assessed. Main results. The results demonstrated that NGF-loaded mPEG-PELG controllably and sustainably released bioactive NGF for 28 d. When bridging a 10 mm rat sciatic nerve gap, the morphological, electrophysiological, and functional analyses revealed that NGF-releasing TENG (Scaffold + NGF/mPEG-PELG) achieved superior regenerative outcomes compared to plain scaffolds and those combined with systemic delivery of NGF (daily intramuscular injection (IM)), and its effects were relatively similar to autografts. Significance. This study has proposed a TENG using thermosensitive hydrogels as an injectable implant to controllably release NGF, which has promising therapeutic potential and translatability. Such TENGs obviate the need for conduit modification, complex preloading or binding mediators, therefore they allow the ease of drug switching in clinical practice and greatly simplify the manufacturing process due to the independent preparation of drug delivery system.

Silk Fibroin Improves the Release of Nerve Growth Factor from Hydroxyapatite Particles Maintaining its Bioactivity.

Hydroxyapatite (HA) was emerging as the most promising biomaterial owing to its excellent bioactivity, biocompatibility, and high compressive strength for segmental bone defects. However, due to the lack of synergistic signals of osteogenic cells or growth factors, attempts concerning HA have not achieved an ideal therapeutical effect. This work was intended to combine HA particles with nerve growth factor (NGF) to coaccelerate the bone repair. To deal with the strong absorption on HA particles and short half-life of exogenous NGF, bovine serum albumin (BSA) and silk fibroin (SF) were introduced. Protected with SF, maximum release rate was 49.8% at 48 h for 2 mg/ml SF used group while the release rate was less than 5% without SF. PC-12 cells cultured in the NGF release fluid quit proliferation and differentiated to neural phenotype, and approximately 9.5% of NGF loading released from the particles within 48 h. Protected with BSA, in vitro release showed no obvious effect on strong absorption for the surface of HA particles, however, the drug loading of model protein decreased. Fortunately, SF demonstrated the capacity of adjusting the release profile of protein by varying the amount of SF embedded with no influence on drug loading and maintaining the bioactivity of NGF absorbed on HA particles.

Preparation of NGF encapsulated chitosan nanoparticles and its evaluation on neuronal differentiation potentiality of canine mesenchymal stem cells.

Sustained and controlled release of neurotrophic factors in target tissue through nanomaterial based delivery system could be a better strategy for nerve tissue regeneration. The present study aims to prepare the nerve growth factor (NGF) encapsulated chitosan nanoparticles (NGF-CNPs) and its evaluation on neuronal differentiation potentiality of canine bone marrow derived mesenchymal stem cells (cBM-MSCs). The NGF-CNPs were prepared by ionotropic gelation method with tripolyphosphate (TPP) as an ionic cross-linking agent. Observations on physiochemical properties displayed the size of nanoparticles as 80-90 nm with positive zeta potential as well as an ionic interaction between NGF and nanoparticle. NGF loading efficiency was found to be 61% while its sustained release was observed by an in vitro release kinetics study. These nanoparticles were found to be cytocompatible to cBM-MSCs when supplemented at a concentration upto 4 mg/ml in culture media. The NGF-CNP supplemented culture media was able to transdifferentiate the preinduced cBM-MSCs into neurons in a better way than unbound NGF supplementation. Further, it was also noticed that NGF-CNPs were able to transdifferentiate cBM-MSCs without any chemical based preinduction. In conclusion, our findings propose that NGF-CNPs are capable of releasing bioactive NGF with the ability to transdifferentiate mesenchymal stem cells into neurons, suggesting its potential future application in nerve tissue regeneration.

Establishment of nerve growth factor gradients on aligned chitosan-polylactide /alginate fibers for neural tissue engineering applications

Nerve conduits containing aligned fibrous fillers with gradiently distributed signal molecules are essential for long-gap nerve repair. This study was to develop an approach for establishing nerve growth factor (NGF) gradients onto the aligned chitosan-polylactide (CH-PLA) fibers. CH-PLA containing 37wt% of PLA was spun into fibers using a wet-spinning technique. CH-PLA fibers showed much higher wet-state tensile strength, enhanced degradation tolerance and significantly lower swelling degree in comparison to chitosan fibers. The CH-PLA fibers with diameters from 40 to 60μm were selected and segmentally coated in bundles using NGF-contained alginate solutions to establish NGF gradients lengthwise along fibers. The diameter of resulting NGF-loaded CH-PLA/alginate fibers was well controlled within a range between 60 and 120μm. Calcium ion crosslinked alginate coating layers on fibers showed abilities to administer the sustainable NGF release in a gradient distribution manner for at least 5 weeks. NGF-induced neurite outgrowth of PC12 cells confirmed that bioactivity of NGF released from fibers was well retained.

Heparin/collagen encapsulating nerve growth factor multilayers coated aligned PLLA nanofibrous scaffolds for nerve tissue engineering.

Biomimicing topological structure of natural nerve tissue to direct axon growth and controlling sustained release of moderate neurotrophic factors are extremely propitious to the functional recovery of damaged nervous systems. In this study, the heparin/collagen encapsulating nerve growth factor (NGF) multilayers were coated onto the aligned poly-L-lactide (PLLA) nanofibrous scaffolds via a layer-by-layer (LbL) self-assembly technique to combine biomolecular signals, and physical guidance cues for peripheral nerve regeneration. Scanning electronic microscopy (SEM) revealed that the surface of aligned PLLA nanofibrous scaffolds coated with heparin/collagen multilayers became rougher and appeared some net-like filaments and protuberances in comparison with PLLA nanofibrous scaffolds. The heparin/collagen multilayers did not destroy the alignment of nanofibers. X-ray photoelectron spectroscopy and water contact angles displayed that heparin and collagen were successfully coated onto the aligned PLLA nanofibrous scaffolds and improved its hydrophilicity. Three-dimensional (3 D) confocal microscopy images further demonstrated that collagen, heparin, and NGF were not only coated onto the surface of aligned PLLA nanofibrous scaffolds but also permeated into the inner of scaffolds. Moreover, NGF presented a sustained release for 2 weeks from aligned nanofibrous scaffolds coated with 5.5 bilayers or above and remained good bioactivity. The heparin/collagen encapsulating NGF multilayers coated aligned nanofibrous scaffolds, in particular 5.5 bilayers or above, was more beneficial to Schwann cells (SCs) proliferation and PC12 cells differentiation as well as the SC cytoskeleton and neurite growth along the direction of nanofibrous alignment compared to the aligned PLLA nanofibrous scaffolds. This novel scaffolds combining sustained release of bioactive NGF and aligned nanofibrous topography presented an excellent potential in peripheral nerve regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1900-1910, 2017.

Prolonged controlled delivery of nerve growth factor using porous silicon nanostructures

Although nerve growth factor (NGF) is beneficial for the treatment of numerous neurological and non-neurological diseases, its therapeutic administration represents a significant challenge, due to the difficulty to locally deliver relevant doses in a safe and non-invasive manner. In this work, we employ degradable nanostructured porous silicon (PSi) films as carriers for NGF, allowing its continuous and prolonged release, while retaining its bioactivity. The PSi carriers exhibit high loading efficacy (up to 90%) of NGF and a continuous release, with no burst, over a period of>26days. The released NGF bioactivity is compared to that of free NGF in both PC12 cells and dissociated dorsal root ganglion (DRG) neurons. We show that the NGF has retained its bioactivity and induces neurite outgrowth and profound differentiation (of >50% for PC12 cells) throughout the period of release within a single administration. Thus, this proof-of-concept study demonstrates the immense therapeutic potential of these tunable carriers as long-term implants of NGF reservoirs and paves the way for new localized treatment strategies of neurodegenerative diseases.

Development of multifunctional films for peripheral nerve regeneration.

This manuscript focuses on the development of multifunctional degradable polymer films that provide topographic cues for guided growth, surface gradients of growth factors as well as nanoparticles in the films for tunable release of growth factors to enable peripheral nerve regeneration. The combination of cues was designed to overcome limitations of current strategies to facilitate peripheral nerve regeneration. These multifunctional films successfully provided high protein loading capacities while persevering activity, protein gradients on the surface, and tunable release of bioactive nerve growth factor that promoted directional and guided neurite extension of PC12 cells of up to 10μm in 2days. These multifunctional films can be made into conduits for peripheral nerve regeneration.

신경세포에서 신경성장인자(nerve growth factor)의 조절에 미치는 천문동(Asparagus cochinchinensis) 열수추출물의 영향

Asparagus cochinchinensis is a medical plant that has long been used to treat fever, cough, kidney disease, breast cancer, inflammatory disease and brain disease in northeast Asian countries. Although several studies have been conducted on the anti-neuroinflammatory effects of A. cochinchinensis, the correlation between these effects and nerve growth factor (NGF) has not yet been examined. In this study, we investigated the effects of an aqueous extract of A. cochinchinensis (AEAC) on the secretion and action mechanism of NGF in neuronal cells. The concentration of the NGF protein in the supernatant collected from cultured cells increased significantly in B35 cells treated with AEAC in comparison with the vehicle-treated group without any specific cytotoxicity. Furthermore, the mRNA expression of NGF showed a very similar pattern to its protein concentration. To examine the bioactivity of NGF secreted from B35 cells, undifferentiated PC12 cells were cultured in an AEAC-conditioned medium and neuritic outgrowth was observed. The dendrite length of PC12 cells in the AEAC-treated group was significantly higher than that in the vehicle-treated group. Moreover, the level of the downstream effectors p-TrkA and p-ERK of the high-affinity NGF receptor was significantly higher in the AEAC-treated group, while the expression of the downstream effectors of the low-affinity NGF receptor was significantly lower in the same group. These results suggest that AEAC may contribute to the regulation of NGF expression and secretion in neuronal cells; it is therefore an excellent candidate for further investigation as a therapeutic drug for neurodegenerative diseases.

Enhanced bioavailability of nerve growth factor with phytantriol lipid-based crystalline nanoparticles in cochlea.

PurposeSupplementation of exogenous nerve growth factor (NGF) into the cochlea of deafened animals rescues spiral ganglion cells from degeneration. However, a safe and potent delivery of therapeutic proteins, such as NGF, to spiral ganglion cells remains one of the greatest challenges. This study presents the development of self-assembled cubic lipid-based crystalline nanoparticles to enhance inner ear bioavailability of bioactive NGF via a round window membrane route.MethodsA novel nanocarrier-entrapped NGF was developed based on phytantriol by a liquid precursor dilution, with Pluronic® F127 and propylene glycol as the surfactant and solubilizer, respectively. Upon dilution of the liquid lipid precursors, monodispersed submicron-sized particles with a slight negative charge formed spontaneously.ResultsBiological activity of entrapped NGF was assessed using pheochromocytoma cells with NGF-loaded reservoirs to induce significant neuronal outgrowth, similar to that seen in free NGF-treated controls. Finally, a 3.28-fold increase in inner ear bioavailability was observed after administration of phytantriol lipid-based crystalline nanoparticles as compared to free drug, contributing to an enhanced drug permeability of the round window membrane.ConclusionData presented here demonstrate the potential of lipid-based crystalline nanoparticles to improve the outcomes of patients bearing cochlear implants.

Using NGF heparin-poloxamer thermosensitive hydrogels to enhance the nerve regeneration for spinal cord injury

ObjectiveNerve growth factor (NGF) has potential in spinal cord injury (SCI) therapy, but limited by the poor physicochemical stability and low ability to cross the blood spinal cord barrier. Novel heparin-poloxamer (HP) thermo-sensitive hydrogel was constructed to enhance the NGF regeneration on SCI. MethodNGF–HP thermo-sensitive hydrogel was prepared and related characteristics including gelation temperature, rheological behavior and micromorphology were measured. Local NGF delivery to the injured spinal cord was achieved by in situ injection in the injured space. The cellular uptake of NGF–HP hydrogel was evaluated with PC12 cells in vitro. Pathologic characteristics and neuron regeneration effects on the SCI rats were studied to evaluate the enhanced therapy of NGF–HP hydrogel. Endoplasmic reticulum (ER) stress-induced apoptosis was analyzed to explore the related mechanism in SCI regeneration. ResultsNGF–HP hydrogel showed good morphology and stable bioactivity of NGF in vitro. NGF–HP hydrogel combined treatment significantly enhanced the efficiency of NGF cellular uptake (P<0.05) without obvious cytotoxicity. Significant improvements in both neuron functions and tissue morphology on the SCI rats were observed in NGF–HP hydrogel group. Compared with free HP hydrogel and NGF treatment groups, NGF–HP hydrogel group showed significant inhibition on the formation of glial scars in the extreme crushed rat SCI model. The neuroprotective effects of NGF–HP were related to the inhibition of chronic ER stress-induced apoptosis. ConclusionsHP hydrogel combined with orthotopic injection technique might be an effective method to deliver NGF into the injured site, which will provide an effective strategy for SCI regeneration. Statement of significanceSpinal cord injury (SCI) is a devastating condition that can lead to sudden loss of sensory and autonomic function. Current treatment includes decompression surgery, injury stabilization, secondary complications prevention and rehabilitation. However, neurological recovery is limited. Nerve growth factor (NGF) has potential in SCI therapy, but limited by the poor physicochemical stability and low ability to cross the blood spinal cord barrier. Hydrogels have good affinity and compatibility to biological tissue. In this study, we developed a novel heparin-poloxamer (HP) thermo-sensitive hydrogel to enhance the spinal cord regeneration of NGF. From SCI rat experiment, HP hydrogel combined with orthotopic injection technique showed best neuroprotective effects among experimental groups. This novel combined technique will provide an effective strategy for SCI regeneration.

The synergistic effect of nanotopography and sustained dual release of hydrophobic and hydrophilic neurotrophic factors on human mesenchymal stem cell neuronal lineage commitment.

A combination of nanotopography and controlled release is a potential platform for neuronal tissue engineering applications. Previous studies showed that combining both physical and chemical guidance was more effective than individual cues in the directional promotion of neurite outgrowth. Nanotopography can direct human mesenchymal stem cells (hMSCs) into neuronal lineage, while controlled release of neurotrophic factors can deliver temporally controlled biochemical signals. Hypothesizing that the synergistic effect will enhance neuronal lineage commitment of hMSCs, a fabrication method for multiple neurotrophic factors delivery from a single nanopatterned (350 nm gratings), poly-ɛ-caprolactone (PCL) film was developed and evaluated. Our results showed a synergistic effect on hMSC differentiation cultured on substrates with both nanotopographical and biochemical cues. The protein/drug encapsulation into PCL nanopatterned films was first optimized using a hydrophilic model protein, bovine serum albumin. The hydrophobic retinoic acid (RA) molecule was directly incorporated into PCL films. To achieve sustained release, hydrophilic nerve growth factor (NGF) was first encapsulated within polyelectrolyte complexation fibers before they were embedded within the nanopatterned PCL film. Our results showed that nanotopography on the fabricated polymer films remained intact, while release of bioactive RA and NGF was sustained over a period of 3 weeks. Under the combinatorial effect of physical and biochemical cues, we observed an enhanced upregulation of neuronal genes such as microtubule-associated protein 2 (MAP2) and neurofilament light (NFL) as compared with sustained delivery of individual cues and bolus delivery. Quantitative polymerase chain reaction analysis showed that MAP2 and NFL gene upregulation in hMSCs was most pronounced on the nanogratings with sustained release of both RA and NGF. The fabricated platforms supported the sustained delivery of multiple neurotrophins, including both hydrophobic and hydrophilic therapeutic agents, while providing surface patterning versatility for application in neural regeneration and tissue engineering.

Polyethlyene glycol microgels to deliver bioactive nerve growth factor.

Delivery of bioactive molecules is a critical step in fabricating materials for regenerative medicine, yet, this step is particularly challenging in hydrated scaffolds such as hydrogels. Although bulk photocrosslinked poly(ethylene glycol) (PEG) hydrogels have been used for a variety of tissue engineering applications, their capability as drug delivery scaffolds has been limited due to undesirable release profiles and reduction in bioactivity of molecules. To solve these problems, this article presents the fabrication of degradable PEG microgels, which are micron-sized spherical hydrogels, to deliver bioactive nerve growth factor (NGF). NGF release and activity was measured after encapsulation in microgels formed from either 3 kDa or 6 kDa PEG to determine the role of hydrogel mesh size on release. Microgels formed from 6 kDa PEG were statistically larger and had a higher swelling ratio than 3 kDa PEG. The 6 kDa PEG microgels provided a Fickian release with a reduced burst effect and 3 kDa microgels provided anomalous release over ≥20 days. Regardless of molecular weight of PEG, NGF bioactivity was not significantly reduced compared to unprocessed NGF. These results demonstrate that microgels provide easy mechanisms to control the release while retaining the activity of growth factors. As this microgel-based delivery system can be injected at the site of nerve injury to promote nerve repair, the potential to deliver active growth factors in a controlled manner may reduce healing time for neural tissue engineering applications.

Dienogest inhibits nerve growth factor expression induced by tumor necrosis factor-α or interleukin-1β

Dienogest (DNG), a selective P receptor (PR) agonist, is used to treat endometriosis. To investigate whether DNG affects nerve growth factor (NGF) expression, we stimulated human endometrial epithelial cells (hEECs) with inflammatory cytokines. Prospective basic research study using immortalized hEEC lines. Development Research, Mochida Pharmaceutical Co., Ltd., Japan. None. Not applicable. In immortalized hEECs, NGF production induced by tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β) was evaluated in the presence or absence of the synthetic progestin DNG or endogenous P. The NGF messenger RNA (mRNA) and protein were measured using real-time reverse transcriptase-polymerase chain reaction (PCR) and ELISA, respectively. The NGF bioactivity in the culture medium was measured by assaying neurite outgrowth of PC-12 cells. Tumor necrosis factor-α and IL-1β induced NGF mRNA and protein and increased NGF bioactivity in the culture medium. These activities were inhibited by DNG in a hEEC line that stably expresses PR. In contrast, in an hEEC line that constitutively expresses faint levels of PR, no inhibitory effect of DNG on NGF mRNA was detected. The NGF mRNA was also inhibited in hEEC lines that express only PR-A or only PR-B. Nerve growth factor is one of the key mediators that generates the pain associated with endometriosis. Dienogest inhibits NGF expression through PR-A and PR-B in hEEC, which may contribute to the pharmacological mechanisms of how DNG relieves pain in endometriosis.

Influence of biological matrix and artificial electrospun scaffolds on proliferation, differentiation and trophic factor synthesis of rat embryonic stem cells

Two-dimensional vs three-dimensional culture conditions, such as the presence of extracellular matrix components, could deeply influence the cell fate and properties. In this paper we investigated proliferation, differentiation, survival, apoptosis, growth and neurotrophic factor synthesis of rat embryonic stem cells (RESCs) cultured in 2D and 3D conditions generated using Cultrex® Basement Membrane Extract (BME) and in poly-(l-lactic acid) (PLLA) electrospun sub-micrometric fibres. It is demonstrated that, in the absence of other instructive stimuli, growth, differentiation and paracrine activity of RESCs are directly affected by the different microenvironment provided by the scaffold. In particular, RESCs grown on an electrospun PLLA scaffolds coated or not with BME have a higher proliferation rate, higher production of bioactive nerve growth factor (NGF) and vascular endothelial growth factor (VEGF) compared to standard 2D conditions, lasting for at least 2weeks. Due to the high mechanical flexibility of PLLA electrospun scaffolds, the PLLA/stem cell culture system offers an interesting potential for implantable neural repair devices.