Establishment and neural differentiation of neural crest-derived stem cells (NCSCs) from human dental pulp in serum-free conditions

The potential of obtaining cell cultures with neural crest resemblance (neural crest-derived stem cells; NCSCs) from dental-related tissues, including human dental pulp cells (hDPCs) has been discussed in the literature. However, most reports include the use of serum-rich conditions and do not describe the potential for neural differentiation, slowing translation to the clinic. Therefore, we aimed to culture and characterize NCSCs from the human dental pulp in vitro and evaluate their ability to differentiate into neurons; we also investigated the effectiveness of the addition of BMP4 to enhance this potential. Cultures were established from a varied cohort of patient samples and grown, as monolayers, in serum, serum-free and also under sphere-aggregation conditions to induce and identify a NCSC phenotype. hDPC cultures were characterized by immunocytochemistry and reverse transcription quantitative polymerase chain reaction. Monolayer cultures expressed stem cell, neural progenitor and neural crest-related markers. Culturing hDPCs as neurospheres (hDPCNCSCs) resulted in an increased expression of neural crest-related genes, while the addition of BMP4 appeared to produce better NCSC characteristics and neural differentiation. The neural-like phenotype was evidenced by the expression of TUJ1, peripherin, NFH, TAU, SYN1 and GAP43. Our results describe the establishment of hDPC cultures from a large variety of patients in serum-free medium, as NCSC that differentiate into neural-like cells, as well as an important effect of BMP4 in enhancing the neural crest phenotype and differentiation of hDPCs

Chemokines and pain in the trigeminal system

Chemotactic cytokines or chemokines are a large family of secreted proteins able to induce chemotaxis. Chemokines are categorized according to their primary amino acid sequence, and in particular their cysteine residues that form disulphide bonds to maintain the structure: CC, CXC, CX3C, and XC, in which X represents variable amino acids. Among their many roles, chemokines are known to be key players in pain modulation in the peripheral and central nervous systems. Thus, they are promising candidates for novel therapeutics that could replace current, often ineffective treatments. The spinal and trigeminal systems are intrinsically different beyond their anatomical location, and it has been suggested that there are also differences in their sensory mechanisms. Hence, understanding the different mechanisms involved in pain modulation for each system could aid in developing appropriate pharmacological alternatives. Here, we aim to describe the current landscape of chemokines that have been studied specifically with regard to trigeminal pain. Searching PubMed and Google Scholar, we identified 30 reports describing chemokines in animal models of trigeminal pain, and 15 reports describing chemokines involved in human pain associated with the trigeminal system. This review highlights the chemokines studied to date at different levels of the trigeminal system, their cellular localization and, where available, their role in a variety of animal pain models

A tuneable, photocurable, poly(caprolactone)-based resin for tissue engineering—synthesis, characterisation and use in stereolithography

Stereolithography is a useful additive manufacturing technique for the production of scaffolds for tissue engineering. Here we present a tuneable, easy-to-manufacture, photocurable resin for use in stereolithography, based on the widely used biomaterial, poly(caprolactone) (PCL). PCL triol was methacrylated to varying degrees and mixed with photoinitiator to produce a photocurable prepolymer resin, which cured under UV light to produce a cytocompatible material. This study demonstrates that poly(caprolactone) methacrylate (PCLMA) can be produced with a range of mechanical properties and degradation rates. By increasing the degree of methacrylation (DM) of the prepolymer, the Young’s modulus of the crosslinked PCLMA could be varied from 0.12–3.51 MPa. The accelerated degradation rate was also reduced from complete degradation in 17 days to non-significant degradation in 21 days. The additive manufacturing capabilities of the resin were demonstrated by the production of a variety of different 3D structures using micro-stereolithography. Here, β-carotene was used as a novel, cytocompatible photoabsorber and enabled the production of complex geometries by giving control over cure depth. The PCLMA presented here offers an attractive, tuneable biomaterial for the production of tissue engineering scaffolds for a wide range of applications

Porphyromonas gingivalis lipopolysaccharide rapidly activates trigeminal sensory neurons and may contribute to pulpal pain

Aim To determine whether Porphyromonas gingivalis lipopolysaccharide (LPS) can directly activate trigeminal neurons, to identify which receptors are involved, and to establish whether activation leads to secretion of the neuropeptide calcitonin‐gene related peptide (CGRP) and/or the translocation of NF‐κB. Methodology Mouse trigeminal ganglion (TG) cells were cultured in vitro for 2 days. The effect of P. gingivalis LPS (20 μg/mL) on calcium signalling was assessed (by calcium imaging using Cal‐520 AM) in comparison to the transient receptor potential channel A1 (TRPA1) agonist cinnamaldehyde (CA; 100 μM), the TRP channel V1 (TRPV1) agonist capsaicin (CAP; 1 μM), and high potassium (60 mM KCl). TG cultures were pre‐treated with either 1 μM CLI‐095 to block Toll‐like receptor 4 (TLR4) signalling or with 3 μM HC‐030031 to block TRPA1 signalling. CGRP release was determined using ELISA, and nuclear translocation of NF‐κB was investigated using immunocytochemistry. Data were analysed by one‐way analysis of variance, followed by Bonferroni’s post‐hoc test as appropriate. Results P. gingivalis LPS directly exerted a rapid excitatory response on sensory neurons and non‐neuronal cells (p<0.001 to p<0.05). The effects on neurons appear to be mediated via TLR4‐ and TRPA1‐dependent pathways. The responses were accompanied by an increased release of CGRP (p<0.001) and by NF‐κB nuclear translocation (p<0.01). Conclusions P. gingivalis LPS directly activates trigeminal sensory neurons (via TLR4 and TRPA1 receptors) and non‐neuronal cells, resulting in CGRP release and NF‐κB nuclear translocation. This indicates that P. gingivalis can directly influence activity in trigeminal sensory neurons and this may contribute to acute and chronic inflammatory pain

The role of miRNAs in neuropathic pain

Neuropathic pain is a debilitating condition affecting around 8% of the adult population in the UK. The pathophysiology is complex and involves a wide range of processes, including alteration of neuronal excitability and synaptic transmission, dysregulated intracellular signalling and activation of pro-inflammatory immune and glial cells. In the past 15 years, multiple miRNAs–small non-coding RNA–have emerged as regulators of neuropathic pain development. They act by binding to target mRNAs and preventing the translation into proteins. Due to their short sequence (around 22 nucleotides in length), they can have hundreds of targets and regulate several pathways. Several studies on animal models have highlighted numerous miRNAs that play a role in neuropathic pain development at various stages of the nociceptive pathways, including neuronal excitability, synaptic transmission, intracellular signalling and communication with non-neuronal cells. Studies on animal models do not always translate in the clinic; fewer studies on miRNAs have been performed involving human subjects with neuropathic pain, with differing results depending on the specific aetiology underlying neuropathic pain. Further studies using human tissue and liquid samples (serum, plasma, saliva) will help highlight miRNAs that are relevant to neuropathic pain diagnosis or treatment, as biomarkers or potential drug targets

Additive Manufactured Biodegradable Poly(glycerol sebacate methacrylate) Nerve Guidance Conduits

Entubulating devices to repair peripheral nerve injuries are limited in their effectiveness particularly for critical gap injuries. Current clinically used nerve guidance conduits are often simple tubes, far stiffer than that of the native tissue. This study assesses the use of poly(glycerol sebacate methacrylate) (PGSm), a photocurable formulation of the soft biodegradable material, PGS, for peripheral nerve repair. The material was synthesized, the degradation rate and mechanical properties of material were assessed and nerve guidance conduits were structured via stereolithography. In vitro cell studies confirmed PGSm as a supporting substrate for both neuronal and glial cell growth. Ex vivo studies highlight the ability of the cells from a dissociated dorsal root ganglion to grow out and align along the internal topographical grooves of printed nerve guide conduits. In vivo results in a mouse common fibular nerve injury model show regeneration of axons through the PGSm conduit into the distal stump after 21 days. After conduit repair levels of spinal cord glial activation (an indicator for neuropathic pain development) were equivalent to those seen following graft repair. In conclusion, results indicate that PGSm can be structured via additive manufacturing into functional NGCs. This study opens the route of personalized conduit manufacture for nerve injury repair. STATEMENT OF SIGNIFICANCE: This study describes the use of photocurable of Poly(Glycerol Sebacate) (PGS) for light-based additive manufacturing of Nerve Guidance Conduits (NGCs). PGS is a promising flexible biomaterial for soft tissue engineering, and in particular for nerve repair. Its mechanical properties and degradation rate are within the desirable range for use in neuronal applications. The nerve regeneration supported by the PGS NGCs is similar to an autologous nerve transplant, the current gold standard. A second assessment of regeneration is the activation of glial cells within the spinal cord of the tested animals which reveals no significant increase in neuropathic pain by using the NGCs. This study highlights the successful use of a biodegradable additive manufactured NGC for peripheral nerve repair

A novel role for lymphotactin (XCL1) signaling in the nervous system: XCL1 acts via its receptor XCR1 to increase trigeminal neuronal excitability

Chemokines are known to have a role in the nervous system, influencing a range of processes including the development of chronic pain. To date there are very few studies describing the functions of the chemokine lymphotactin (XCL1) or its receptor (XCR1) in the nervous system. We investigated the role of the XCL1-XCR1 axis in nociceptive processing, using a combination of immunohistochemical, pharmacological and electrophysiological techniques. Expression of XCR1 in the rat mental nerve was elevated 3 days following chronic constriction injury (CCI), compared with 11 days post-CCI and sham controls. XCR1 coexisted with neuronal marker PGP9.5, leukocyte common antigen CD45 and Schwann cell marker S-100. In the trigeminal root and white matter of the brainstem, XCR1-positive cells co-expressed the oligodendrocyte marker Olig2. In trigeminal subnucleus caudalis (Vc), XCR1 immunoreactivity was present in the outer laminae and was colocalized with vesicular glutamate transporter 2 (VGlut2), but not calcitonin gene-related peptide (CGRP) or isolectin B4 (IB4). Incubation of brainstem slices with XCL1 induced activation of c-Fos, ERK and p38 in the superficial layers of Vc, and enhanced levels of intrinsic excitability. These effects were blocked by the XCR1 antagonist viral CC chemokine macrophage inhibitory protein-II (vMIP-II). This study has identified for the first time a role for XCL1-XCR1 in nociceptive processing, demonstrating upregulation of XCR1 at nerve injury sites and identifying XCL1 as a modulator of central excitability and signalling via XCR1 in Vc, a key area for modulation of orofacial pain, thus indicating XCR1 as a potential target for novel analgesics

A novel role for lymphotactin (XCL1) signaling in the nervous system: XCL1 acts via its receptor XCR1 to increase trigeminal neuronal excitability

Chemokines are known to have a role in the nervous system, influencing a range of processes including the development of chronic pain. To date there are very few studies describing the functions of the chemokine lymphotactin (XCL1) or its receptor (XCR1) in the nervous system. We investigated the role of the XCL1-XCR1 axis in nociceptive processing, using a combination of immunohistochemical, pharmacological and electrophysiological techniques. Expression of XCR1 in the rat mental nerve was elevated 3 days following chronic constriction injury (CCI), compared with 11 days post-CCI and sham controls. XCR1 coexisted with neuronal marker PGP9.5, leukocyte common antigen CD45 and Schwann cell marker S-100. In the trigeminal root and white matter of the brainstem, XCR1-positive cells co-expressed the oligodendrocyte marker Olig2. In trigeminal subnucleus caudalis (Vc), XCR1 immunoreactivity was present in the outer laminae and was colocalized with vesicular glutamate transporter 2 (VGlut2), but not calcitonin gene-related peptide (CGRP) or isolectin B4 (IB4). Incubation of brainstem slices with XCL1 induced activation of c-Fos, ERK and p38 in the superficial layers of Vc, and enhanced levels of intrinsic excitability. These effects were blocked by the XCR1 antagonist viral CC chemokine macrophage inhibitory protein-II (vMIP-II). This study has identified for the first time a role for XCL1-XCR1 in nociceptive processing, demonstrating upregulation of XCR1 at nerve injury sites and identifying XCL1 as a modulator of central excitability and signalling via XCR1 in Vc, a key area for modulation of orofacial pain, thus indicating XCR1 as a potential target for novel analgesics

Chronic tooth pulp inflammation induces persistent expression of phosphorylated ERK (pERK) and phosphorylated p38 (pp38) in trigeminal subnucleus caudalis

Background: Extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase are transiently phosphorylated (activated) in the spinal cord and trigeminal nucleus by acute noxious stimuli. Acute stimulation of dental pulp induces short-lived ERK activation in trigeminal subnucleus caudalis (Vc), and p38 inhibition attenuates short-term sensitization in Vc induced by acute pulpal stimulation. We have developed a model to study central changes following chronic inflammation of dental pulp that induces long-term sensitization. Here, we examine the effects of chronic inflammation and acute stimulation on the expression of phosphorylated ERK (pERK), phosphorylated p38 (pp38) and Fos in Vc. Results: Chronic inflammation alone induced bilateral expression of pERK and pp38 in Vc, but did not induce Fos expression. Stimulation of both non-inflamed and inflamed pulps significantly increased pERK and pp38 bilaterally; expression was greatest in inflamed, stimulated animals, and was similar following 10-min and 60-min stimulation. Stimulation for 60 min, but not 10 min, induced Fos in ipsilateral Vc; Fos expression was significantly greater in inflamed, stimulated animals. pERK was present in both neurons and astrocytes; pp38 was present in neurons and other non-neuronal, non-astrocytic cell types. Conclusions: This study provides the first demonstration that chronic inflammation of tooth pulp induces persistent bilateral activation of ERK and p38 within Vc, and that this activation is further increased by acute stimulation. This altered activity in intracellular signaling is likely to be linked to the sensitization that is seen in our animal model and in patients with pulpitis. Our data indicate that pERK and pp38 are more accurate markers of central change than Fos expression. In our model, localization of pERK and pp38 within specific cell types differs from that seen following acute stimulation. This may indicate specific roles for different cell types in the induction and maintenance of pulpitic and other types of pain