Release Of Nerve Growth Factor Research Articles

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.

Analysis of leukocyte populations and nerves in developing murine corneas

Abstract The avascular cornea is the most densely innervated tissue in the body and is endowed with resident bone marrow-derived cells. We have identified a novel subset of resident corneal plasmacytoid dendritic cells (pDCs). We have shown pDCs support corneal nerves through release of nerve growth factor (NGF). This study was to examine the presence of pDCs in the developing cornea. We examined corneas from E17, P2, and 8 week adult C57BL/6 mice. Corneas were stained for the pan-leukocyte marker CD45, the pDC marker plasmacytoid dendritic cell antigen-1 (PDCA-1), the pan-neuronal marker bIII-tubulin. Corneal CD45+ and PDCA-1+ cells and nerves were quantified. Significance was assigned by ANOVA and post-hoc Tukey test. All data is reported as mean ± standard deviation. Corneal CD45+ leukocyte density increased from E17 (65.4±14 cells/mm2) to P2 pups (301.9±19 cells/mm2, p<0.05). Both E17 and P2 corneas had lower CD45+ density when compared to adults (345±34 cells/mm2, p<0.05). We observed pDCs in the anterior stroma proximal to corneal nerves in E17, P2, and adult corneas. In E17, few pDCs were present in the peripheral cornea (1.5±2.6 cells/mm2), with none in the central cornea. In P2, pDCs were found in the periphery (68.19±8.99 cells/mm2), but not central corneas. The density of pDCs in adult peripheral (82.2±5.1 cells/mm2) and central (51.6±4.9 cells/mm2) corneas was greater than E17 (p<0.001) and P2 (p<0.05) corneas. Nerve density increased from E17 (45.43±6.75 mm/mm2) when compared to P2 (83.15±.21.6 mm/mm2 p<0.05) and to adult (151.6±3.25 mm/mm2 p<0.05) corneas. Our data show pDC density increased from embryonic, to neonatal, and adult corneas. Concurrently corneal nerve density increased during development from embryonic to adult mice.

Pharmacological Manipulation of Trk, p75NTR, and NGF Balance Restores Memory Deficit in Global Ischemia/Reperfusion Model in Rats.

Long-term memory impairment is reported in more than 50% of cardiac arrest survivors. Monosialoganglioside (GM1) provided neuroprotection in experimental models of stroke but failed to replicate its promise clinically for unknown reasons. GM1 stimulates the release of nerve growth factor (NGF), which is synthesized as a precursor protein (pro-NGF) that either mediates apoptosis through the p75 neurotrophin receptor (p75NTR) or is cleaved by the protease furin (FUR) to yield mature NGF, the latter supporting survival through tropomyosin kinase receptor (Trk). The flavanol epicatechin (EPI) inhibits p75NTR-mediated signaling and apoptosis by pro-NGF. The aim of the current work is to test whether these two drugs affect, or communicate with, each other in the setting of CNS injuries. Using the two-vessel occlusion model of global ischemia/reperfusion (I/R), we tested if pharmacological modulation of Trk, p75NTR, and NGF balance with GM1, EPI, and their combination, can correct the memory deficit that follows this insult. Finally, we tested if FUR insufficiency and/or p75NTR-mediated apoptosis negatively affect the neurotherapeutic effect of GM1. Key proteins for Trk and p75NTR, FUR, and both forms of NGF were assessed. All treatment regiments successfully improved spatial memory retention and acquisition. A week after the insult, most Trk and p75NTR proteins were normal, but pro/mature NGF ratio remained sharply elevated and was associated with the poorest memory performance. Pharmacological correction of this balance was achieved by reinforcing Trk and p75NTR signaling. GM1 increased FUR levels, while concomitant administration of EPI weakened GM1 effect on pro-survival Trk and p75NTR mediators. GM1 neuroprotection is therefore not limited by FUR but could be dependent on p75NTR. Graphical Abstract "."

Changes in vitreal protein profile and retina mRNAs in Reeler mice: NGF, IL33 and Müller cell activation.

A possible link between Nerve Growth Factor (NGF) and Reelin might take place during impaired retinal development occurring in the Reelin deficient mouse model (Reeler). To better characterize NGF and retina impairments at the Reeler retina, vitreous and retina were investigated by means of protein expression and glial cell activation. Reeler (n = 9; RELN-/-) and WT (n = 9; wild-type RELN+/+, B6C3Fe) mice were analyzed at 14, 21 and 28 postnatal days (p). Retinas and vitreous were subjected to confocal analysis and protein array, followed by conventional analysis. A significant increase of NGF, IL33 and TIMP1, a trend to a decrease of IL12 and IL6, as well as a significant decrease of NT3 were detected in Reeler vitreous, particularly at p28 (p<0.05). MIP3β mRNA was decreased while IL33mRNA was significantly upregulated in Reeler retina. Increased number of GFAP+ and Nestin+ cells as well as upregulation of Glutamine Synthetase and Nestin mRNAs were observed in Reeler retinas (p<0.05). These findings extend our previous studies on Reeler retina showing a selective Müller cell activation. NGF and IL33 release into vitreous would suggest a local activation of Müller cells, in addition to retinal ganglion and accessory cells. Overall, the data from this experimental study would strength the potential neuroprotective role played by activated Muller cells through NGF release.

PLGA porous scaffolds by polydopamine-assisted immobilization of NGF for spinal cord injury repair

The degradable polymer biomaterials have great potential in the field of nerve regeneration, which have gradually been applied to spinal cord injury repair. In this study, a polylactic acid-glycolic acid copolymer (PLGA) porous scaffold was prepared by the process of phase inversion, then the surface of the scaffold was modified by polydopamine (PDA) as a substrate, followed by adsorption of nerve growth factor (NGF) to obtain a PDA-PLGA/NGF scaffold. The characterization and hydrophilicity of the scaffolds were evaluated by electron microscopy, EDX and contact angle measurement. The effect of PDA modification on the binding efficiency and release profile of NGF was observed by ELISA. The proliferation and differentiation of NSCs on the surface of biomaterials was evaluated by MTT, immunofluorescence staining and PCR. Subsequently, the rat T9 spinal cord transection model was established and the nerve scaffolds were implanted to injured site. Our results showed PDA modification could significantly improve the NGF adsorption capacity and provide sustained release of NGF. PDA-PLGA/NGF scaffolds could not only enhance NSCs proliferation and neuronal differentiation in vitro, but also promote recovery of spinal cord injury in vivo. Therefore, we believed that PDA-PLGA/NGF scaffolds could be a promising method to facilitate neurogenesis and repair spinal cord injury.

Bile acids induce visceral hypersensitivity via mucosal mast cell–to–nociceptor signaling that involves the farnesoid X receptor/nerve growth factor/transient receptor potential vanilloid 1 axis

Increased colonic bile acid (BA) exposure, frequent in diarrhea-predominant irritable bowel syndrome (IBS-D), can affect gut function. Nerve growth factor (NGF) is implicated in the development of visceral hypersensitivity (VH). In this study, we tested the hypothesis that BAs cause VH via mucosal mast cell (MMC)-to-nociceptor signaling, which involves the farnesoid X receptor (FXR)/NGF/transient receptor potential vanilloid (TRPV)1 axis. BAs were intracolonically administered to rats for 15 d. Visceral sensitivity to colorectal distention and colonic NGF expression were examined. BAs caused VH, an effect that involved MMC-derived NGF and was accompanied by enhanced TRPV1 expression in the dorsal root ganglia. Anti-NGF treatment and TRPV1 antagonism inhibited BA-induced VH. BAs induced NGF mRNA and protein expression and release in cultured mast cells. Colonic supernatants from patients with IBS-D with elevated colonic BA content transcriptionally induced NGF expression. In FXR-/- mice, visceral sensitivity and colonic NGF expression were unaltered after BA treatment. Pharmacological antagonism and FXR silencing suppressed BA-induced NGF expression and release in mast cells. Mitogen-activated protein kinase kinase (MKK) 3/6/p38 MAPK/NF-κB signaling was mechanistically responsible for FXR-mediated NGF expression and secretion. The findings show an MMC-dependent and FXR-mediated pronociceptive effect of BAs and identify the BA/FXR/NGF/TRPV1 axis as a key player in MMC-to-neuron communication during pain processing in IBS.-Li, W.-T., Luo, Q.-Q., Wang, B., Chen, X., Yan, X.-J., Qiu, H.-Y., Chen, S.-L. Bile acids induce visceral hypersensitivity via mucosal mast cell-to-nociceptor signaling that involves the farnesoid X receptor/nerve growth factor/transient receptor potential vanilloid 1 axis.

Long term effects of cigarette smoke extract or nicotine on nerve growth factor and its receptors in a bronchial epithelial cell line

Long-term exposure to cigarette smoke induces severe injuries to respiratory system through several mechanisms, some of them are well defined, but many others are not yet elucidated. Beside its classical role in nervous system, we have previously shown that Nerve Growth Factor (NGF) and its receptors have a crucial role in airway inflammatory diseases, such as Chronic Obstructive Pulmonary Disease. To expand our knowledge about the relevance of NGF and its receptors in airway diseases induced by cigarette smoking, we exposed for 16 weeks the bronchial epithelial cell line BEAS-2B to sub-toxic concentrations of whole cigarette smoke extract or pure nicotine. Viability, cell cycle gene expression, cell morphology and migration ability were tested and compared to NGF release and gene expression. Modulation of its receptors TrKA and p75NTR was also analyzed.The present study shows that long term exposure of BEAS-2B cells to cigarette smoke extract or nicotine induces: (A) differences: in cell viability, in the expression of cell cycle-related genes, in NGF release and in gene expression of NGF and its receptors; (B) similarities: in morphology and migration ability.Taken together, our data provide new insights about the biological role of NGF and its receptors in airway diseases induced by long-term cigarette smoking and, finally, our data evidence the opportunity not to use nicotine lozenges or e-cigarettes as anti smoking replacement therapy in patients with a previous airway disease according to the ability of nicotine to increase the amount of the pro-inflammatory cytokine NGF into the bronchial environment.

3D bioprinted rat Schwann cell-laden structures with shape flexibility and enhanced nerve growth factor expression.

Three-dimensional (3D) bioprinting composite alginate-gelatin hydrogel has encouraged the fabrication of cell-laden functional structures with cells from various tissues. However, reports focusing on printing this hydrogel for nerve tissue research are limited. This study aims at building in vitro Schwann cell 3D microenvironment with customized shapes through 3D bioprinting technology. Rat Schwann cell RSC96s encapsulated in composite alginate-gelatin hydrogel were printed with an extrusion-based bioprinter. Cells maintained high viability of 85.35 ± 6.19% immediately after printing and the printed hydrogel supported long-term Schwann cell proliferation for 2weeks. Furthermore, after 14days of culturing, Schwann cells cultured in printed structures maintained viability of 92.34 ± 2.19% and showed enhanced capability of nerve growth factor (NGF) release (142.41 ± 8.99pg/ml) compared with cells from two-dimensional culture (92.27 ± 9.30pg/ml). Specific Schwann cell marker S100β was also expressed by cells in printed structures. These printed structures may have the potential to be used as in vitro neurotrophic factor carriers and could be integrated into complex biomimetic artificial structures with the assistance of 3D bioprinting technology.

Spatio-temporal release of NGF and GDNF from multi-layered nanofibrous bicomponent electrospun scaffolds

Scaffolds capable of providing dual neurotrophic factor (NTF) delivery with different release kinetics, spatial delivery of NTFs at different loci and topographical guidance are promising for enhanced peripheral nerve regeneration. In this study, we have designed and fabricated multi-layered aligned-fiber scaffolds through combining emulsion electrospinning, sequential electrospinning and high-speed electrospinning (HS-ES) to modulate the release behavior of glial cell line-derived growth factor(GDNF) and nerve growth factor (NGF). GDNF and NGF were incorporated into poly(lactic-co-glycolic acid) (PLGA) fibers and poly(D,L-lactic acid) (PDLLA) fibers, respectively. Aligned fibers were obtained in each layer of multi-layered scaffolds and relatively thick tri-layered and tetra-layered scaffolds with controlled layer thickness were obtained. Their morphology, structure, properties, and the in vitro release of growth factors were examined. Dual and spatio-temporal release of GDNF and NGF with different release kinetics from multi-layered scaffolds was successfully demonstrated. High separation efficiency by PDLLA fibrous barrier layer for spatial neurotrophic factor delivery from both tri-layered scaffolds and tetra-layered scaffolds was achieved.

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.

NGF and PI3K/Akt signaling participate in the ventral motor neuronal protection of curcumin in sciatic nerve injury rat models

ObjectiveIt has been reported that sciatic nerve injury (SNI) leads to degeneration, damage, and apoptosis of motor neurons. Nerve growth factor (NGF) plays a pivotal role in regeneration and reestablishment of neuronal function via activating PI3K/Akt survival signaling pathways. Curcumin owns neuroprotective effect following brain injury. In the present study, we attempt to investigate underlying mechanism of neuroprotective effect of curcumin through elucidating its correlation with NGF and PI3K/Akt signaling pathways in vitro and in vivo. MethodsPC-12 cells were exposed H2O2 in order to induce neuron cell injury and cells were then treated with curcumin. Caspase-3, NGF level and Akt phosphorylation were determined using flow cytometry and western blotting. Then, cells were treated with NGF specific siRNA followed by measurement of apoptosis, NGF and Akt phosphorylation levels. In animal model, rats were subjected to SNI and then randomly designated into four different groups: curcumin, curcumin + LY294002, curcumin + NGF shRNA, and negative controls and 12 rats in each group (n = 12). After four weeks of continuous treatment, tissue samples were obtained and subjected to TUNEL, NeuN double staining and western blotting. ResultsCurcumin significantly reduced the number of apoptotic cells induced by H2O2 and this effect was associated with upregulation of TrkA, Akt and downregulation of p17. ProNGF level was significantly decreased while mature NGF level was increased with curcumin treatment. When NGF was suppressed, anti-apoptotic effect of curcumin was attenuated. In addition, inhibition of PI3K/Akt results in increased apoptotic rate compared to vehicles following curcumin treatment which was reflected by decreased p17, Ki67, and cyclin D1. Suppression of NGF and inhibition of PI3K led to increased neuron cell death through increasing proNGF and decreasing mNGF, Akt, TrkA, p75NTR, and p17. ConclusionOur findings revealed that curcumin exerts its protective effect against injured neurons through stimulating NGF release which further activates TrkA and PI3K/Akt cell survival signaling.

Asparagus cochinchinensis stimulates release of nerve growth factor and abrogates oxidative stress in the Tg2576 model for Alzheimer\u2019s disease

BackgroudUse of multifunctional drugs with neurotrophic supporting and oxidative stress suppressing activity may be considered a therapeutic strategy to protect or repair cellular damage caused during the progression of Alzheimer’s disease (AD). In this study, we investigated the therapeutic effects of aqueous extract of A. cochinchinesis root (AEAC), particularly its role as a nerve growth factor (NGF) stimulator and anti-oxidant in Tg2576 mice showing AD phenotypes of human.MethodsTg2576 mice were received 100 mg/kg/day AEAC via oral administration, while mice in the Vehicle treated group received dH2O for 4 weeks. Non-Tg littermates were used as a control group. Following AEAC treatment for 4 weeks, NGF function, anti-oxidantive status, Aβ-42 peptide level, γ-secretase expression and neuronal cell functions were analyzed in the brain of Tg2576 mice.ResultsAEAC containing flavonoids, phenols, saponins and protodioscin induced enhancement of NGF secretion and decreased intracellular ROS in the neuronal and microglial cell line. These effects as well as enhanced SOD levels were also detected in AEAC treated Tg2576 mice. The expression of p-Akt among downstream effectors of the high affinity NGF receptor was dramatically recovered in AEAC treated Tg2576 mice, while the expression of p75NTR was slightly recovered in the same group. Significant recovery on the level of Aβ-42 peptides and the expression of γ-secretase members including PS-2, APH-1 and NCT were detected in AEAC treated Tg2576 mice. Furthermore, AEAC treated Tg2576 mice showed decreased numbers of dead cells and suppressed acetyl choline esterase (AChE) activity.ConclusionsThese results suggest that AEAC contribute to improving the deposition of Aβ-42 peptides and neuronal cell injuries during the pathological progression stage of AD in the brain of Tg2576 mice through increased NGF secretion and suppressed oxidative stress.

Quantification of the Release of NGF from Osteoblasts and Osteocytes in Response to Mechanical Load

Prostate cancer (PCa) is the second most common type of cancer in men with an estimated 161,360 new cases to be diagnosed in 2017 according to the American Cancer Society. Approximately 80% of prostate cancer patients experience metastases to bone. Within the bone environment, prostate cancer metastases form osteosclerotic lesions that cause a reduction in bone strength and an increase in mechanical strains in the bone. This new environment leads to the release of signaling molecules and growth factors from bone cells that heighten cancer invasion and proliferation. Nerve Growth Factor (NGF) is a signaling molecule released from osteoblasts that we have previously shown can increase proliferation in bone‐derived PCa cells. From these previous studies, I hypothesized that mechanical load increases the amount of NGF released from osteoblasts and osteocytes. To determine the effects of mechanical load on bone cells, osteoblasts and osteocytes were subjected to different magnitudes (5, 12, and 20 dynes/cm2) of oscillatory fluid shear. At each magnitude, cells in culture were sheared for 2 hours. Media was collected and cells were lysed. NGF concentrations were quantified from the conditioned media using an ELISA assay. Protein concentrations were determined from the cell lysate using a BCA assay. Results were measured in NGF concentration per protein concentration to account for differences in cell density. Osteocytes released significantly more NGF during fluid shear than static cells, although NGF release was variable. The static osteocytes released approximately 0.022 pg/mg NGF while the maximum concentration of NGF released in sheared cells was 0.054 pg/mg at 5 dynes/cm2. However, osteoblastic MC3T3 cells had no significant response to shear in terms of NGF release. This indicates that while both types of cells exhibited a basal release of NGF, only the osteocytes increased this release in response to fluid shear. Additionally, these findings support the concept that the osteocytes are more sensitive than osteoblasts to mechanical load. Osteocytes are located in lacuna‐canalicular system and are exposed to comparable forces of fluids flowing through these small channels. Since osteocytes make up the majority of bone cells, their NGF release could have a significant impact on the bone microenvironment. We can then predict that the changes in load caused by ostesclerotic lesions could increase prostate cancer cell invasion and proliferation through upregulation of NGF release. Future studies include the treatment of prostate cancer cells with conditioned media from sheared osteocytes and observing any changes in proliferation. These data will further our understanding of the cellular mechanisms involved in prostate cancer proliferation and help to identify potential therapeutic targets for the treatment of metastatic prostate cancer in bone.Support or Funding InformationThis study was supported by the Milton H. Stetson Undergraduate Research Fellowship, a grant from the National Institutes for Health (DC014461) and a private donation from Dr. Jeremie Axe at First State Orthopedics. We also thank Nick Trompeter and Lindsey Stevens for their help and advice.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Link N as a therapeutic agent for discogenic pain.

Neurotrophins (NTs) are the major contributors of sensory axonal sprouting, neural survival, regulation of nociceptive sensory neurons, inflammatory hyperalgesia, and neuropathic pain. Intervertebral disc (IVD) cells constitutively express NTs. Their expression is upregulated by proinflammatory cytokines present in the IVD during degeneration, which can promote peripheral nerve ingrowth and hyperinnervation, leading to discogenic pain. Currently, there are no targeted therapies that decrease hyperinnervation in degenerative disc disease. Link N is a naturally occurring peptide with a high regenerative potential in the IVD. Therefore, the suitability of Link N as a therapeutic peptide for suppressing NTs, which are known modulators and mediators of pain, was investigated. The aim of the present study is to determine the effect of Link N on NTs expression, nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), and their cognate receptors TrkA and TrkB as they are directly correlated with symptomatic back pain. Furthermore, the neurotransmitter (substance P) was also evaluated in human annulus fibrosus (AF) cells stimulated with cytokines. Human AF cells isolated from normal IVDs were stimulated with interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) in the presence or absence of Link N. NGF release in the media was evaluated by Western blotting. Total RNA was isolated and gene expression was measured using real‐time PCR. Gene expression of NGF, BDNF, TrkA, and TrkB significantly decreased in human disc cells stimulated with either IL‐1β or TNF‐α supplemented with Link N when compared to the cells stimulated only with IL‐1β or TNF‐α. NGF protein expression was also suppressed in AF cells coincubated with Link N and IL‐1β when compared to the cells stimulated only with IL‐1β. Link N can suppress the stimulation of NGF, BDNF, and their receptors TrkA and TrkB in AF cells in an inflammatory milieu. Thus, coupled with previous observations, this suggests that administration of Link N has the potential to not only repair the discs in early stages of the disease but also suppress pain.

Integration of Phase-Change Materials with Electrospun Fibers for Promoting Neurite Outgrowth under Controlled Release.

We report a temperature-regulated system for the controlled release of nerve growth factor (NGF) to promote neurite outgrowth. The system is based upon microparticles fabricated using coaxial electrospray, with the outer solution containing a phase-change material (PCM) and the inner solution encompassing payload(s). When the temperature is kept below the melting point of the PCM, there is no release due to the extremely slow diffusion through a solid matrix. Upon increasing the temperature to slightly pass the melting point, the encapsulated payload(s) can be readily released from the melted PCM. By leveraging the reversibility of the phase transition, the payload(s) can be released in a pulsatile mode through on/off heating cycles. The controlled release system is evaluated for potential use in neural tissue engineering by sandwiching the microparticles, co-loaded with NGF and a near-infrared dye, between two layers of electrospun fibers to form a tri-layer construct. Upon photothermal heating with a near-infrared laser, the NGF is released with well-preserved bioactivity to promote neurite outgrowth. By choosing different combinations of PCM, biological effector, and scaffolding material, this controlled release system can be applied to a wide variety of biomedical applications.

Thermoresponsive- co-Biodegradable Linear-Dendritic Nanoparticles for Sustained Release of Nerve Growth Factor To Promote Neurite Outgrowth.

Thermoresponsive and biodegradable linear-dendritic nanoparticles containing poly( N-isopropylacrylamide), poly(l-lactic acid), and poly(l-lysine) dendrons were investigated for sustained release of nerve growth factor (NGF) in response to temperature change. The nanoparticles and their degradants were not cytotoxic to neuron-like PC12 cells for at least one month. The nanoparticles were preferentially taken up by PC12 cells 6-13-times more at temperatures above (37 °C) than below (25 °C) the lower critical solution temperature of the nanoparticles. NGF could be loaded into the nanoparticles in aqueous solution and slowly released from the nanoparticles for 12 and 33 days at 25 and 37 °C, respectively. The released NGF was biologically active by promoting neurite outgrowth of PC12 cells. This work demonstrates a new concept of using thermoresponsive and biodegradable linear-dendritic nanoparticles for thermally targeted and sustained release of NGF and other protein drugs for the treatment of Alzheimer's disease and other neurological disorders.

Nerve growth factor plays a role in the neurotherapeutic effect of a CD45+ pan-hematopoietic subpopulation derived from human umbilical cord blood in a traumatic brain injury model

Background aimsHuman umbilical cord blood (HUCB) is an important source of stem cells for therapy of hematopoietic disorders and is a potential therapy for various neurological disorders, including traumatic brain injury (TBI). The expression of nerve growth factor (NGF) and its receptors TrkA, p75NTR and α9β1 integrin on an HUCB CD45+ pan-hematopoietic subpopulation was investigated in the context of its neurotherapeutic potential after TBI. MethodsNGF and its receptors were detected on CD45+ cells by reverse transcriptase polymerase chain reaction, flow cytometry analysis and confocal microscopy. CD45+ cells were stimulated by TBI brain extracts, and NGF levels were measured by enzyme-linked immunosorbent assay. TBI mice were divided into six groups for xenogeneic intravenous transplantation, 1 day post-trauma, with 1 × 106 CD45+ cells untreated or treated with the anti-NGF neutralizing antibody K252a, a TrkA antagonist; VLO5, an α9β1 disintegrin; or negative (vehicle) and positive (NGF) controls. ResultsThe HUCB CD45+ subpopulation constitutively expresses NGF and its receptors, mainly TrkA and p75NTR and minor levels of α9β1. In vitro experiments provided evidence that trauma-related mediators from brain extracts of TBI mice induced release of NGF from HUCB CD45+ cell cultures. HUCB CD45+ cells induced a neurotherapeutic effect in TBI mice, abrogated by cell treatment with either anti-NGF antibody or K252a, but not VLO5. ConclusionsThese findings strengthen the role of NGF and its TrkA receptor in the HUCB CD45+ subpopulation's neurotherapeutic effect. The presence of neurotrophin receptors in the HUCB CD45+ pan-hematopoietic subpopulation may explain the neuroprotective effect of cord blood in therapy of a variety of neurological disorders.

Asymmetric Sensory-Motor Regeneration of Transected Peripheral Nerves Using Molecular Guidance Cues

Neural interfaces are designed to decode motor intent and evoke sensory precepts in amputees. In peripheral nerves, recording movement intent is challenging because motor axons are only a small fraction compared to sensory fibers and are heterogeneously mixed particularly at proximal levels. We previously reported that pain and myelinated axons regenerating through a Y-shaped nerve guide with sealed ends, can be modulated by luminar release of nerve growth factor (NGF) and neurotrophin-3 (NT-3), respectively. Here, we evaluate the differential potency of NGF, glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), pleiotrophin (PTN), and NT-3 in asymmetrically guiding the regeneration of sensory and motor neurons. We report that, in the absence of distal target organs, molecular guidance cues can mediate the growth of electrically conductive fascicles with normal microanatomy. Compared to Y-tube compartments with bovine serum albumin (BSA), GDNF and NGF increased the motor and sensory axon content, respectively. In addition, the sensory to motor ratio was significantly increased by PTN (12.7:1) when compared to a BDNF + GDNF choice. The differential content of motor and sensory axons modulated by selective guidance cues may provide a strategy to better define axon types in peripheral nerve interfaces.

Human Periodontal Ligament- and Gingiva-derived Mesenchymal Stem Cells Promote Nerve Regeneration When Encapsulated in Alginate/Hyaluronic Acid 3D Scaffold.

Repair or regeneration of damaged nerves is still a challenging clinical task in reconstructive surgeries and regenerative medicine. Here, it is demonstrated that periodontal ligament stem cells (PDLSCs) and gingival mesenchymal stem cells (GMSCs) isolated from adult human periodontal and gingival tissues assume neuronal phenotype in vitro and in vivo via a subcutaneous transplantation model in nude mice. PDLSCs and GMSCs are encapsulated in a 3D scaffold based on alginate and hyaluronic acid hydrogels capable of sustained release of human nerve growth factor (NGF). The elasticity of the hydrogels affects the proliferation and differentiation of encapsulated MSCs within scaffolds. Moreover, it is observed that PDLSCs and GMSCs are stained positive for βIII-tubulin, while exhibiting high levels of gene expression related to neurogenic differentiation (βIII-tubulin and glial fibrillary acidic protein) via quantitative polymerase chain reaction (qPCR). Western blot analysis shows the importance of elasticity of the matrix and the presence of NGF in the neurogenic differentiation of encapsulated MSCs. In vivo, immunofluorescence staining for neurogenic specific protein markers confirms islands of dense positively stained structures inside transplanted hydrogels. As far as it is known, this study is the first demonstration of the application of PDLSCs and GMSCs as promising cell therapy candidates for nerve regeneration.

Multi-tubule conduit-filler constructs loaded with gradient-distributed growth factors for neural tissue engineering applications.

Chitosan/silk fibroin/glycerophosphate gels were loaded with nerve growth factor (NGF) and further processed into multi-tubule fillers. NGF was loaded into the fillers in such a way so that a NGF gradient was established longitudinally along the filler length. A type of poly(lactide-co-glycolide)(PLGA)/chitosan(CH) porous conduit was fabricated using a pre-crosslinking method. The filler was fully filled into the lumen of conduits to build multi-tubule conduit-filler constructs that are intended for long-gap peripheral nerve repair. In vitro degradation in a lysozyme-contained medium revealed that constructs had degradation-tolerant features and the optimized multi-tubule filler was capable of maintaining its multi-tubules unblocked for around 10-week. After being degraded for various periods up to 8 weeks, the optimal conduit-filler constructs showed confirmative ability to retain their compressive load, deformation recovery and tensile strength at about 80N/m, 75% and 15N/cm2 in wet state, respectively. The constructs were able to administer NGF release and to maintain persistent NGF gradients longitudinally distributed inside the PLGA/CH conduit for about 6 weeks or even longer. The PC12 cell neurite extension assay confirmed that the presently developed multi-tubule conduit-filler constructs were reliable for effectively preserving the bioactivity of released NGF.