- Email: [email protected]
Neurotrophic Proteins in Dentin and Their Effect on Trigeminal Sensory Neurons
Basic Research—Biology
Neurotrophic Proteins in Dentin and Their Effect on Trigeminal Sensory Neurons Matthias Widbiller, DDS,*† Obadah Austah, BDS, MS,*‡ Sophia R. Lindner, DDS,*§ Jenny Sun, DDS,* and Anibal Diogenes, DDS, MS, PhD* Abstract Introduction: A plethora of bioactive molecules present during tooth formation become sequestered in the mineralized dentin matrix and can be released into the pulp tissue after demineralization from carious lesions. However, neurotrophic factors are differentially expressed and secreted during various stages of odontogenesis. Thus, the aims of this study were (1) to investigate their presence and relative abundance in crown and root dentin and (2) to evaluate the bioactivity of dentin-derived proteins on neuronal cells. Methods: Dentin matrix proteins (DMPs) were isolated from matched roots and crowns of extracted healthy human third molars. The total protein amount as well as the concentration of growth factors and neurotrophic proteins were quantified. The impact on neuritogenesis was determined with mouse trigeminal neurons in vitro and by a hydrogel implant model in vivo. Transient receptor potential cation channel subfamily V member 1 (TRPV1) sensitization of DMP-conditioned neurons was evaluated by single-cell calcium imaging. Results: The relative concentration of neurotrophic molecules revealed that nerve growth factor is the most abundant neurotrophin with 3-fold increased expression in radicular dentin. Similarly, brain-derived neurotrophic factor and neurotrophin 3 are more abundant in radicular than coronal dentin. Conversely, glial cell line– derived neurotrophic factor is more abundant in coronal dentin, whereas neurotrophin 4 is equally distributed. Dentin matrix proteins promoted neurite outgrowth in vitro and axonal targeting in vivo, with a greater effect observed by radicular dentin extracts. Furthermore, DMPs sensitized TRPV1 responses in mouse trigeminal neurons with greater activity seen with extracts from root dentin. Conclusions: Neurotrophic factors are differentially distributed between coronal and radicular dentin with different effects of dentinderived proteins on axonal growth and targeting as well as the sensitization of TRPV1. Thus, extracellular
proteins from the dentin matrix are likely involved in neurogenic responses to caries and could be exploited in clinical regenerative endodontics to promote reinnervation and enhance tissue regeneration. (J Endod 2019;45:729–735)
Key Words Dentin, neuronal outgrowth, neurotrophins, regeneration, sensitization, trigeminal nerve
R
egenerative endodonSignificance tic procedures (REPs) Because of differential expression during odontoare accepted as a biologgenesis, neurotrophic proteins are present in ical treatment option for different amounts in the crown and root dentin. immature teeth with pulp Coronal and radicular dentin matrix proteins reveal necrosis (1, 2), with the considerable effects on neuritogenesis and TRPV1 primary goals of prosensitization; however, the impact of proteins from moting continued root radicular dentin prevails. Thus, bioactive proteins development and decreasfrom the dentin matrix might not only play a role ing the risk of tooth during carious decay but also could serve as a fracture associated with reservoir for signaling molecules to promote reinconventional apexification nervation in regenerative endodontics. procedures (3). Although studies, including prospective studies (4), have shown similar success rates for healing of apical periodontitis to apexification and greater continued root development for REPs, there is increasing evidence that these procedures fall short on restoring all structures and functions of the native dental pulp (5). Instead, true pulp regeneration should comprise full restoration of the pulp-dentin complex in its form and function (restitutio ad integrum). The possibility of neuronal regeneration was shown for in vivo models (6, 7), and the presence of neuronal fibers has been histologically demonstrated in cases treated with REPs (8, 9). Interestingly, only a fraction of the cases treated with these procedures regain responses to sensibility testing. The exact mechanism of axonal targeting into the newly formed tissue after these procedures is not fully understood but could include the release of brainderived neurotrophic factor (BDNF) from recruited stem cells of the apical papilla (10). To overcome the low predictability of reinnervation, supplementation of bioactive molecules to scaffold materials may support the ingrowth of neuronal structures (11, 12). Interestingly, the initial innervation during odontogenesis takes place at a relatively late developmental stage. Until the late bell stage, axons reside in a waiting position surrounding the dental follicle in a basketlike manner and only innervate the neighboring
From the *Department of Endodontics, University of Texas Health Science Center at San Antonio, San Antonio, Texas; Departments of †Conservative Dentistry and Periodontology and §Oral and Maxillofacial Surgery, University Hospital Regensburg, Regensburg, Germany; and ‡Department of Endodontics, Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia. Address requests for reprints to Dr Anibal Diogenes, Department of Endodontics, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2019 American Association of Endodontists. https://doi.org/10.1016/j.joen.2019.02.021
JOE — Volume 45, Number 6, June 2019
Neurotrophic Proteins in Dentin
729
Basic Research—Biology structures. Then, these axons rapidly start entering the dental papilla at the late bell stage through root formation and tooth eruption (13). This process occurs in a highly organized temporal and spatial fashion through the production of signaling molecules by odontoblasts and subodontoblast cells (14). In particular, nerve growth factor (NGF) and glial cell line–derived neurotrophic factor (GDNF) seem to play an important role in this process (15, 16). Although the presence of neurotrophins has been shown in tooth development, it is unclear if they are sequestered and “fossilized” in the extracellular matrix of human dentin as described for various other molecules (17). These molecules participate in the neuronal sprouting observed with demineralizing carious lesions and chemical agents, and are likely involved in the development of hyperalgesia after injury (18, 19). Importantly, their neurogenic potential could be exploited in pulp tissue engineering. Because these neurotrophins are differentially present during various stages of odontogenesis, in this study we tested the hypothesis that concentrations of neurotrophins in coronal and radicular dentin are different with different functional effects of dentin matrix proteins (DMPs) on trigeminal ganglia (TG) neurons.
Materials and Methods Extraction of DMPs Extracted healthy third molars from 9 patients (17–24 years old) were collected from the oral surgery clinic at the University of Texas Health Science Center at San Antonio, San Antonio, TX, after obtaining informed patient consent. After the removal of enamel and cementum, all crowns were separated from the roots at the cementoenamel junction, and pulp was removed. For each patient, DMPs were extracted from crowns and roots according to an established protocol (20). All subsequent experiments were performed in a paired design comparing the results of crown versus root for each donor sample. Animals Eighteen male 6- to 8-week-old C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) were used for the isolation of primary TG neurons, and 6 severe combined immunodeficient (SCID) mice (6–8 weeks old, NOD.CB17-Prkdcscid/J, Jackson Laboratories) were used for the in vivo innervation assay. All animals were housed with food and water offered ad libitum in a 12-hour light/dark cycle at the University of Texas Health Science Center at San Antonio according to the guidelines established by our Institutional Animal Care and Use Committee, the National Institutes of Health Guide and Public Health Service policy on humane care and the use of laboratory animals, and the ARRIVE guidelines of the National Center for the Replacement and Reduction of Animals in Research. Quantification of Neurotrophic Factors The total protein concentrations in the crown and root extracts were determined by a bicinchoninic acid assay (Pierce BCA Protein Assay Kit; Thermo Fisher Scientific, Waltham, MA). Neurotrophic factors and neurotrophins were quantified by enzyme-linked immunosorbent assays (ELISAs) as follows: GDNF (Human GDNF Rapid ELISA Kit; biosensis, Thebarton, Australia), NGF (Human beta-NGF ELISA Kit; Thermo Fisher Scientific), BDNF (Quantikine ELISA Total BDNF Immunoassay; R&D Systems, Minneapolis, MN), and neurotrophin 3 (NT3) (Human NT3 Rapid ELISA Kit; biosensis), neurotrophin 4 (NT4) (Human NT4/5 Rapid ELISA Kit; biosensis). In addition, 2 growth factors reported to be abundantly expressed in the dentin matrix were quantified as the positive control: transforming growth factor beta 1 (TGF-b1) (Quantikine ELISA Human TGF-b1; R&D Systems) and vascular endothelial growth factor (VEGF) (Quantikine ELISA Human VEGF; R&D Sys730
Widbiller et al.
tems). The concentration of detected molecules was normalized to the total protein amount of the respective crown or root dentin sample.
Primary Culture of Mouse TG Neurons TG neurons were isolated from C57BL/6 mice as described previously (21); single neurons were cultured on poly-D-lysine–coated culture dishes or onto coverslips (Corning BioCoat; Thermo Fisher Scientific) in Dulbecco modified Eagle medium containing 10% heatinactivated fetal bovine serum (Thermo Fisher Scientific), 3 mg/mL 5-fluoro-2-deoxyuridine and 7 mg/mL uridine (Sigma-Aldrich, St Louis, MO), 1 glutamine, and 1 penicillin-streptomycin (Thermo Fisher Scientific). Neurons were seeded in a density of 13,000 cells/cm2 and allowed to attach overnight at 5% CO2 and 37 C before further experimentation. Immunocytochemistry and Determination of Neurite Area The effects of coronal and radicular DMPs on neurite outgrowth of mouse TG neurons were evaluated by immunocytochemical staining and quantification of the neurite surface area. Neurons were cultured on coverslips in 24-well plates exposed to the respective crown or root dentin extracts at a final protein concentration of 75 mg/mL in medium for 3 days, washed with 0.1 mol/L phosphate-buffered saline, fixed with 4% formaldehyde for 10 minutes, and processed for immunocytochemistry with a primary antibody against beta III tubulin (1:1000 dilution, R&D Systems) and an Alexa Fluor 488 secondary antibody (1:200 dilution, Thermo Fisher Scientific). Controls included sections without primary or secondary antibody. In addition, F-actin was stained by phalloidin (Texas Red-X phalloidin; Thermo Fisher Scientific). Coverslips were mounted on slides (VECTASHIELD with DAPI; Vector Laboratories, Burlingame, CA). Z-stack images were taken using a Nikon C1si laser scanning confocal microscope (Nikon, Minato, Japan) using standardized settings at 20 magnification and processed in Fiji (National Institutes of Health, Bethesda, MD) (22). For quantification, Tubb3-positive fibers were thresholded and quantified as described previously (10). The surface area of nerve fibers was determined in 6 randomly selected regions per coverslip. Neurite Outgrowth Quantification A colorimetric cell-based ELISA kit was used for quantification of total neurite formation (Human TUBB3 Cell-Based ELISA Kit; Abnova, Taipei City, Taiwan). Briefly, TG neurons cultured in poly-D-lysine– coated 96-well plates were exposed to DMPs (75 mg/mL) for 5 days. Next, cells were fixed, and Tubb3 was quantified by ELISA according to the manufacturer’s instructions. Neurite outgrowth was also quantified using a transwell assay according to the manufacturer’s instructions (Neurite Outgrowth Assay Plus Kit; Merck Millipore, Billerica, MA). Photometric quantification of the labeled neurites was performed by measuring absorbance at 490 nm using a FlexStation 3 Benchtop Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA) after 48 hours of DMP exposure.
In Vivo Innervation Assay A Matrigel implant model (Corning Matrigel GFR Membrane Matrix; Thermo Fisher Scientific) was used to evaluate the effect of matrix proteins extracted from crown and root dentin on innervation and axonal targeting in vivo (10, 23). SCID mice under isoflurane inhalation anesthesia (3 %) received bilateral subcutaneous injections to the dorsum of 450 mL growth factor reduced Matrigel JOE — Volume 45, Number 6, June 2019
Basic Research—Biology (Corning Matrigel GFR Membrane Matrix; Thermo Fisher Scientific) containing paired crown or root DMPs (75 mg/mL). The implants were harvested 7 days postinjection and processed for immunohistochemical staining with a primary antibody against neurofilament heavy polypeptide (1:1000 dilution; Biolegend, San Diego, CA). Immunoreactivity was detected by an Alexa Fluor 488 secondary antibody (1:200 dilution, Thermo Fisher Scientific). After mounting, fluorescence staining was visualized by confocal microscopy as described earlier.
Single-cell Calcium Imaging To determine the effect of DMPs from crown and root dentin on neuronal function, changes in the activity of the transient receptor potential cation channel subfamily V member 1 (TRPV1) were evaluated. TG neurons were cultured on poly-D-lysine–coated coverslips and exposed to medium (control) or medium supplemented with coronal and radicular DMPs for 24 hours. Next, neurons were loaded with the calcium indicator fura-2 acetoxymethyl ester (2 mmol/L, Thermo Fisher Scientific) and exposed to either 100 nmol/L capsaicin (CAP) or vehicle for 60 seconds. Changes in fluorescence as a measure of intracellular calcium concentration were detected by a Nikon TE2000U microscope fitted with a 40/1.35 NA Fluor objective (Nikon). Data were collected and analyzed with MetaFluor software (MetaMorph; Universal Imaging Corporation, Downingtown, PA). At the end of the experiment, cells were exposed to a 50 mmol/L KCl pulse as a positive control to detect all excitable cells. The percentage of CAPresponsive neurons of all excitable cells and the mean response amplitude (D F340/F360) were calculated from 5 coverslips per group with at least 20 cells each. Data Treatment and Statistical Analysis All experiments were conducted with associated crown and root DMPs from 9 donors (n = 9). For ELISAs and neurite outgrowth experiments, data were not normally distributed and, therefore, analyzed nonparametrically by pair-wise Mann-Whitney U tests. For calcium imaging experiments, data were analyzed by 1-way analysis of variance followed by the Tukey multiple comparison test. All statistical analyses were computed with GraphPad Prism 7 (GraphPad Software, La Jolla, CA), and statistical significance (P # .05) was indicated by asterisks in the respective figures.
Results Quantification of Neurotrophic Factors The total protein mass in crown and root dentin extracts was equivalent (Fig. 1A). Furthermore, no significant differences of the abundant growth factors TGF-b1 and VEGF were detected between coronal and radicular dentin (Fig. 1A). Moreover, NGF, BDNF, and NT3 prevailed in root dentin, with NGF comprising the greatest proportion. On the contrary, NT4 was found to be equally distributed, whereas GDNF predominated in crown dentin (Fig. 1A). Interestingly, quantification of neurotrophic proteins revealed a 3-fold higher amount in radicular dentin compared with coronal parts (Fig. 1B). Immunocytochemistry and Determination of Neurite Area Immunostaining of mouse trigeminal neurons for beta III tubulin after 3 days revealed enhanced neurite formation when cultured with DMPs. Protein extracts from radicular dentin led not only to an increase in number but also in the maturity of neurites as evidenced by the thickness of axons (Fig. 2A). This observation was confirmed by quantificaJOE — Volume 45, Number 6, June 2019
tion of the neurite area, which was significantly increased in neuronal cultures exposed to DMPs (Fig. 2B).
Neurite Outgrowth and Targeting Neurite formation was additionally measured by a cell-based beta III tubulin immunoassay. Cultivation of mouse trigeminal neurons with dentin extracts resulted in a significantly increased Tubb3 expression per cell (Fig. 2C) with more Tubb3-positive neurites in cultures supplemented by radicular DMPs (165% 21% for radicular compared with 144% 18% coronal samples). The specific axonal targeting of trigeminal neurons was quantified in a transwell assay in which proteins from root dentin promoted outgrowth with significant differences compared with crown dentin and control (Fig. 2D).
In Vivo Innervation Supplementation of DMPs to hydrogels implanted subcutaneously in SCID mice increased the number of infiltrating axons, which were distributed equally throughout the implant after 7 days. Although a distinct difference between crown and root dentin was hard to detect, innervation density was considerably lower in control implants as shown by neurofilament heavy polypeptide immunostaining (Fig. 2E).
Single-cell Calcium Imaging Quantitative single-cell Ca2+ imaging was used to determine the impact of proteins extracted from coronal or radicular dentin on the TRPV1 activity of trigeminal neurons. Treatment with capsaicin resulted in Ca2+ influx in treated as well as untreated cells. Although only a small fraction of neurons in the control group was sensitive to capsaicin, significantly more cells responded after treatment with DMPs (Fig. 3A). Pretreatment with radicular dentin extracts led to the highest proportion of TG neurons responsive to capsaicin (84% 3%), which significantly exceeded the coronal dentin-treated group (68% 4%). However, the mean peak of calcium influx was similar in the control group and the DMP-treated groups (Fig. 3B).
Discussion Current regenerative endodontic procedures rely on the release of dentin matrix growth factors to promote the formation of a reparative intracanal tissue. The dentin matrix harbors a multitude of bioactive molecules that become sequestered and “fossilized” during tooth formation (17). Although their effects on angiogenesis (24), odontoblast differentiation (20), and dentin formation (25) have been described, little is known about the presence of neurotrophic factors and their bioactivity. In this study, we showed that the root dentin matrix has a higher concentration of known neurotrophic factors and that they retain their bioactivity, promoting greater neurogenesis than coronal dentin. In this study, all investigated neurotrophic proteins (NGF, BDNF, NT3, NT4, and GDNF) were detected in the human dentin matrix with their concentration approximately 3 times higher in root dentin compared with crown dentin. The predominating factors in radicular dentin were NGF followed by NT3 and BDNF, whereas GDNF predominated in crowns. Interestingly, innervation during tooth development has a strict neurotrophic factor–directed spatiotemporal control (13, 26). Thus, the differential entrapment of neurotrophic factors in the crown and root dentin matrix provides further evidence for the intense neurogenic activity around the time of root formation and that GDNF may have predominated during crown formation. The abundance of NGF, particularly in the root, agrees with its wellknown role during tooth innervation (14, 15, 27). Similarly, BDNF is a target-invading factor for trigeminal axons (26) that prevailed in
Neurotrophic Proteins in Dentin
731
Basic Research—Biology
Figure 1. Quantification of growth factors and neurotrophic proteins. (A) The total protein amount as well as the concentration of TGF-b1 and VEGF were equal in crown and root dentin. However, the neurotrophins NGF, BDNF and NT3 prevailed in radicular dentin. GDNF was increased in coronal dentin, whereas the neurotrophin NT4 was evenly distributed. Data are presented as the median with the interquartile range, and asterisks indicate significant differences (P # .05). (B) The total amount of neurotrophic proteins in the root dentin was 3 times higher compared with crown dentin, most of which is formed by NGF.
root dentin extracts. However, GDNF was reported to be expressed primarily in cuspal areas by odontoblasts or subodontoblast cells when trigeminal axons approach (16, 26), which is in line with our observations. It is thought to play a morphogenetic role and might have neuroprotective functions (26). Instead, NT3 and NT4 have been observed in early developmental stages throughout the whole mandibular mesenchyme and thus more likely play a role in odontogenesis than in pulpal innervation (26). Although this study did not identify and test all factors with possible neurotrophic activities within dentin, it supports their role of these well-known factors in organogenesis. DMPs are solubilized by carious lesions and may reach the dental pulp, participating in neuronal responses to injury. These responses include extensive neuronal arborization and axonal growth toward the site of injury and nociceptor sensitization (18, 19). The ligandgated cation channel TRPV1 is expressed exclusively in a subset of 732
Widbiller et al.
capsaicin-sensitive nociceptors and is essential for the development of inflammatory hyperalgesia (28, 29). It has been shown to be sensitized by bacterial lipopolysaccharides and be significantly up-regulated in injury and pulpitis (30–32). The effect of DMPs on TRPV1 responses was evaluated in this study for its translational significance to pulpitis- and injury-dependent increased expression in the dental pulp. The treatment of primary trigeminal cultures with DMP extracts for 24 hours resulted in an increase in the number of cells responding to CAP stimulation after exposure to DMPs, which was significantly more pronounced in neuronal cultures treated with radicular DMPs. It is important to emphasize that the individual neurotrophic factors within the dentin matrix mediating this increase in TRPV1 responsiveness were not identified in this study. Nonetheless, NGF, being the most abundant neurotrophic factor in radicular dentin with wellknown effects on TRPV1 expression (33–35) and sensitization (36), is JOE — Volume 45, Number 6, June 2019
Basic Research—Biology
Figure 2. DMPs promote neurite outgrowth in vitro and in vivo. (A) Neurite formation was enhanced by extracellular matrix proteins from crown and especially root dentin. Cells are visualized by fluorescence staining for Tubb3 (green), actin filaments (red), and nuclei (blue). (B) Quantification of the neurite surface area, which is positively stained for Tubb3 (green in A), confirmed the observations made. (C) An increase in neuritogenesis of sensory neurons cultured with DMPs was also detected in a cell-based immunoassay for Tubb3. (D) Axonal growth was similarly enhanced by extracts from root dentin with a significant difference compared with crown dentin. All data are depicted as the median with the interquartile range, and significant differences are labeled by asterisks (P # .05). (E) Innervation was also evaluated in vivo by hydrogel implants that were optionally supplemented with DMPs. Immunostaining for neurofilament heavy polypeptide (yellow) showed more nerve fibers sprouting into implants with crown and root DMPs compared with the control.
likely involved in the increased TRPV1 responsiveness observed. It is important to note that treatment with DMPs did not result in an increase in the response magnitude, suggesting that TRPV1 responses to capsaicin were not enhanced. Instead, the increase in the percentage of capsaicin-responsive neurons is likely caused by an overall increase in TRPV1 expression (37, 38), most likely through posttranslational modification (34, 39) that was not directly tested in this study. Thus, JOE — Volume 45, Number 6, June 2019
neurotrophic factors within DMPs may be involved in arborization and increased TRPV1 activities seen in carious injuries. Interestingly, there are many reports of the reestablishment of nociception in cases treated with REPs. In addition, histologic examination of teeth treated with these procedures have shown that the newly formed tissues are richly innervated with nociceptors (8, 9). Although the cold stimulus typically used in pulp sensibility tests is likely to activate the
Neurotrophic Proteins in Dentin
733
Basic Research—Biology
Figure 3. Sensitization of the TRPV1 response of trigeminal sensory neurons by DMPs. (A) The percentage of capsaicin-responsive neurons increased significantly after treatment with dentin extracts from coronal and, particularly, radicular dentin. (B) Capsaicin-evoked accumulation of intracellular calcium (D F340/F360) was similar after pretreatment of the neurons with matrix proteins. Data are presented as mean standard deviation, and asterisks mark significant differences (P # .05).
transient receptor ankyrin type 1 and not TRPV1, there is a robust coexpression of these 2 channels that is increased by NGF (33). The use of EDTA in REPs has the intent to expose and release DMPs, which according to our findings include all major neurotrophic proteins with a predominance of NGF from the treated radicular dentin. Also, stem cells from the apical papilla, an important kind of mesenchymal stem cells involved in REPs, have been shown to promote neuronal fiber recruitment through the secretion of BDNF (10). Thus, the reinnervation of a pulplike tissue after regenerative endodontics is likely mediated through the following 2 mechanisms: a noncellular-mediated mechanism by released DMPs and a cellular-mediated mechanism by stem cells of the apical papilla. Collectively, our data show that dentin contains all the major neurotrophic factors with differential distribution between radicular and coronal dentin. DMPs can be released with EDTA treatment, promote neurite outgrowth and increase TRPV1 responses. These effects were more pronounced in samples treated with root DMPs that were shown to have a predominance of NGF, a potent neurotrophin. The results of this study corroborate elegant studies showing the differential expression of these factors during various stages of tooth development. Also, their activity upon solubilization could explain neuronal reactions such as arborization and nociception to demineralizing events such as caries. Lastly, the robust effect on neurite outgrowth and axonal targeting could, at least in part, be one of the mechanisms by which the pulplike tissue becomes reinnervated in patients after REPs. The cocktail of DMPs may be exploited to optimally coordinate axonal ingrowth into the root canal space and facilitate pulpal reinnervation during REPs.
Acknowledgments Supported by a research grant from the American Association of Endodontists Foundation. The authors deny any conflicts of interest related to this study.
References 1. Diogenes A, Henry MA, Teixeira FB, Hargreaves KM. An update on clinical regenerative endodontics. Endod Topics 2013;28:2–23. 2. Galler KM. Clinical procedures for revitalization: current knowledge and considerations. Int Endod J 2016;49:926–36. 3. Cvek M. Prognosis of luxated non-vital maxillary incisors treated with calcium hydroxide and filled with gutta-percha. A retrospective clinical study. Dent Traumatol 1992;8:45–55.
734
Widbiller et al.
4. Lin J, Zeng Q, Wei X, et al. Regenerative endodontics versus apexification in immature permanent teeth with apical periodontitis: a prospective randomized controlled study. J Endod 2017;43:1821–7. 5. Diogenes A, Ruparel NB, Shiloah Y, Hargreaves KM. Regenerative endodontics: a way forward. J Am Dent Assoc 2016;147:372–80. 6. Kim JY, Xin X, Moioli EK, et al. Regeneration of dental-pulp-like tissue by chemotaxis-induced cell homing. Tissue Eng Part A 2010;16:3023–31. 7. Huang GT, Yamaza T, Shea LD, et al. Stem/progenitor cell-mediated de novo regeneration of dental pulp with newly deposited continuous layer of dentin in an in vivo model. Tissue Eng Part A 2010;16:605–15. 8. Lei L, Chen Y, Zhou R, et al. Histologic and immunohistochemical findings of a human immature permanent tooth with apical periodontitis after regenerative endodontic treatment. J Endod 2015;41:1172–9. 9. Austah O, Joon R, Fath WM, et al. Comprehensive characterization of 2 immature teeth treated with regenerative endodontic procedures. J Endod 2018;44: 1802–11. 10. De Almeida JF, Chen P, Henry MA, Diogenes A. Stem cells of the apical papilla regulate trigeminal neurite outgrowth and targeting through a BDNF-dependent mechanism. Tissue Eng Part A 2014;20:3089–100. 11. Widbiller M, Driesen RB, Eidt A, et al. Cell homing for pulp tissue engineering with endogenous dentin matrix proteins. J Endod 2018;44:956–62.e2. 12. Schmalz G, Widbiller M, Galler KM. Signaling molecules and pulp regeneration. J Endod 2017;43:S7–11. 13. Hildebrand C, Fried K, Tuisku F, Johansson CS. Teeth and tooth nerves. Prog Neurobiol 1995;45:165–222. 14. Fried K, Lillesaar C, Sime W, et al. Target finding of pain nerve fibers: neural growth mechanisms in the tooth pulp. Physiol Behav 2007;92:40–5. 15. Matsuo S, Ichikawa H, Henderson TA, et al. trkA modulation of developing somatosensory neurons in oro-facial tissues: tooth pulp fibers are absent in trkA knockout mice. Neuroscience 2001;105:747–60. 16. Nosrat CA, Fried K, Ebendal T, Olson L. NGF, BDNF, NT3, NT4 and GDNF in tooth development. Eur J Oral Sci 1998;106(Suppl 1):94–9. 17. Smith AJ, Scheven BA, Takahashi Y, et al. Dentine as a bioactive extracellular matrix. Arch Oral Biol 2012;57:109–21. 18. Kimberly CL, Byers MR. Inflammation of rat molar pulp and periodontium causes increased calcitonin gene-related peptide and axonal sprouting. Anat Rec 1988; 222:289–300. 19. Taylor PE, Byers MR, Redd PE. Sprouting of CGRP nerve fibers in response to dentin injury in rat molars. Brain Res 1988;461:371–6. 20. Widbiller M, Eidt A, Lindner SR, et al. Dentine matrix proteins: isolation and effects on human pulp cells. Int Endod J 2018;51(Suppl 4):e278–90. 21. Patwardhan AM, Berg KA, Akopain AN, et al. Bradykinin-induced functional competence and trafficking of the delta-opioid receptor in trigeminal nociceptors. J Neurosci 2005;25:8825–32. 22. Schindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 2012;9:676–82. 23. Lopatina T, Kalinina N, Karagyaur M, et al. Adipose-derived stem cells stimulate regeneration of peripheral nerves: BDNF secreted by these cells promotes nerve healing and axon growth de novo. PLoS One 2011;6:e17899. 24. Zhang R, Smith AJ, Cooper PR, et al. Angiogenic activity of dentin matrix components. J Endod 2011;37:26–30.
JOE — Volume 45, Number 6, June 2019
Basic Research—Biology 25. Tziafas D, Alvanou A, Panagiotakopoulos N, et al. Induction of odontoblast-like cell differentiation in dog dental pulps after in vivo implantation of dentine matrix components. Arch Oral Biol 1995;40:883–93. 26. Fried K, Nosrat C, Lillesaar C, Hildebrand C. Molecular signaling and pulpal nerve development. Crit Rev Oral Biol Med 2000;11:318–32. 27. Mitsiadis TA, Magloire H, Pagella P. Nerve growth factor signalling in pathology and regeneration of human teeth. Sci Rep 2017;7:1327. 28. Caterina MJ, Rosen TA, Tominaga M, et al. A capsaicin-receptor homologue with a high threshold for noxious heat. Nature 1999;398:436–41. 29. Caterina MJ, Leffler A, Malmberg AB, et al. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 2000;288:306–13. 30. Morgan CR, Rodd HD, Clayton N, et al. Vanilloid receptor 1 expression in human tooth pulp in relation to caries and pain. J Orofac Pain 2005;19:248–60. 31. Ruparel S, Hargreaves KM, Eskander M, et al. Oxidized linoleic acid metabolitecytochrome P450 system (OLAM-CYP) is active in biopsy samples from patients with inflammatory dental pain. Pain 2013;154:2363–71. 32. Watase T, Shimizu K, Komiya H, et al. Involvement of transient receptor potential vanilloid 1 channel expression in orofacial cutaneous hypersensitivity following tooth pulp inflammation. J Oral Sci 2018;60:8–13.
JOE — Volume 45, Number 6, June 2019
33. Diogenes A, Akopian AN, Hargreaves KM. NGF up-regulates TRPA1: implications for orofacial pain. J Dent Res 2007;86:550–5. 34. Price TJ, Louria MD, Candelario-Soto D, et al. Treatment of trigeminal ganglion neurons in vitro with NGF, GDNF or BDNF: effects on neuronal survival, neurochemical properties and TRPV1-mediated neuropeptide secretion. BMC Neurosci 2005;6:4. 35. Zhang X, Huang J, McNaughton PA. NGF rapidly increases membrane expression of TRPV1 heat-gated ion channels. EMBO J 2005;24:4211–23. 36. Zhuang Z-Y, Xu H, Clapham DE, Ji R-R. Phosphatidylinositol 3-kinase activates ERK in primary sensory neurons and mediates inflammatory heat hyperalgesia through TRPV1 sensitization. J Neurosci 2004;24:8300–9. 37. Shinoda M, Asano M, Omagari D, et al. Nerve growth factor contribution via transient receptor potential vanilloid 1 to ectopic orofacial pain. J Neurosci 2011;31: 7145–55. 38. Simonetti M, Fabbro A, D’Arco M, et al. Comparison of P2X and TRPV1 receptors in ganglia or primary culture of trigeminal neurons and their modulation by NGF or serotonin. Mol Pain 2006;2:11. 39. Ji R-R, Samad TA, Jin S-X, et al. p38 MAPK activation by NGF in primary sensory neurons after inflammation increases TRPV1 levels and maintains heat hyperalgesia. Neuron 2002;36:57–68.
Neurotrophic Proteins in Dentin
735
Neurotrophic Proteins in Dentin and Their Effect on Trigeminal Sensory Neurons Matthias Widbiller, DDS,*† Obadah Austah, BDS, MS,*‡ Sophia R. Lindner, DDS,*§ Jenny Sun, DDS,* and Anibal Diogenes, DDS, MS, PhD* Abstract Introduction: A plethora of bioactive molecules present during tooth formation become sequestered in the mineralized dentin matrix and can be released into the pulp tissue after demineralization from carious lesions. However, neurotrophic factors are differentially expressed and secreted during various stages of odontogenesis. Thus, the aims of this study were (1) to investigate their presence and relative abundance in crown and root dentin and (2) to evaluate the bioactivity of dentin-derived proteins on neuronal cells. Methods: Dentin matrix proteins (DMPs) were isolated from matched roots and crowns of extracted healthy human third molars. The total protein amount as well as the concentration of growth factors and neurotrophic proteins were quantified. The impact on neuritogenesis was determined with mouse trigeminal neurons in vitro and by a hydrogel implant model in vivo. Transient receptor potential cation channel subfamily V member 1 (TRPV1) sensitization of DMP-conditioned neurons was evaluated by single-cell calcium imaging. Results: The relative concentration of neurotrophic molecules revealed that nerve growth factor is the most abundant neurotrophin with 3-fold increased expression in radicular dentin. Similarly, brain-derived neurotrophic factor and neurotrophin 3 are more abundant in radicular than coronal dentin. Conversely, glial cell line– derived neurotrophic factor is more abundant in coronal dentin, whereas neurotrophin 4 is equally distributed. Dentin matrix proteins promoted neurite outgrowth in vitro and axonal targeting in vivo, with a greater effect observed by radicular dentin extracts. Furthermore, DMPs sensitized TRPV1 responses in mouse trigeminal neurons with greater activity seen with extracts from root dentin. Conclusions: Neurotrophic factors are differentially distributed between coronal and radicular dentin with different effects of dentinderived proteins on axonal growth and targeting as well as the sensitization of TRPV1. Thus, extracellular
proteins from the dentin matrix are likely involved in neurogenic responses to caries and could be exploited in clinical regenerative endodontics to promote reinnervation and enhance tissue regeneration. (J Endod 2019;45:729–735)
Key Words Dentin, neuronal outgrowth, neurotrophins, regeneration, sensitization, trigeminal nerve
R
egenerative endodonSignificance tic procedures (REPs) Because of differential expression during odontoare accepted as a biologgenesis, neurotrophic proteins are present in ical treatment option for different amounts in the crown and root dentin. immature teeth with pulp Coronal and radicular dentin matrix proteins reveal necrosis (1, 2), with the considerable effects on neuritogenesis and TRPV1 primary goals of prosensitization; however, the impact of proteins from moting continued root radicular dentin prevails. Thus, bioactive proteins development and decreasfrom the dentin matrix might not only play a role ing the risk of tooth during carious decay but also could serve as a fracture associated with reservoir for signaling molecules to promote reinconventional apexification nervation in regenerative endodontics. procedures (3). Although studies, including prospective studies (4), have shown similar success rates for healing of apical periodontitis to apexification and greater continued root development for REPs, there is increasing evidence that these procedures fall short on restoring all structures and functions of the native dental pulp (5). Instead, true pulp regeneration should comprise full restoration of the pulp-dentin complex in its form and function (restitutio ad integrum). The possibility of neuronal regeneration was shown for in vivo models (6, 7), and the presence of neuronal fibers has been histologically demonstrated in cases treated with REPs (8, 9). Interestingly, only a fraction of the cases treated with these procedures regain responses to sensibility testing. The exact mechanism of axonal targeting into the newly formed tissue after these procedures is not fully understood but could include the release of brainderived neurotrophic factor (BDNF) from recruited stem cells of the apical papilla (10). To overcome the low predictability of reinnervation, supplementation of bioactive molecules to scaffold materials may support the ingrowth of neuronal structures (11, 12). Interestingly, the initial innervation during odontogenesis takes place at a relatively late developmental stage. Until the late bell stage, axons reside in a waiting position surrounding the dental follicle in a basketlike manner and only innervate the neighboring
From the *Department of Endodontics, University of Texas Health Science Center at San Antonio, San Antonio, Texas; Departments of †Conservative Dentistry and Periodontology and §Oral and Maxillofacial Surgery, University Hospital Regensburg, Regensburg, Germany; and ‡Department of Endodontics, Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia. Address requests for reprints to Dr Anibal Diogenes, Department of Endodontics, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2019 American Association of Endodontists. https://doi.org/10.1016/j.joen.2019.02.021
JOE — Volume 45, Number 6, June 2019
Neurotrophic Proteins in Dentin
729
Basic Research—Biology structures. Then, these axons rapidly start entering the dental papilla at the late bell stage through root formation and tooth eruption (13). This process occurs in a highly organized temporal and spatial fashion through the production of signaling molecules by odontoblasts and subodontoblast cells (14). In particular, nerve growth factor (NGF) and glial cell line–derived neurotrophic factor (GDNF) seem to play an important role in this process (15, 16). Although the presence of neurotrophins has been shown in tooth development, it is unclear if they are sequestered and “fossilized” in the extracellular matrix of human dentin as described for various other molecules (17). These molecules participate in the neuronal sprouting observed with demineralizing carious lesions and chemical agents, and are likely involved in the development of hyperalgesia after injury (18, 19). Importantly, their neurogenic potential could be exploited in pulp tissue engineering. Because these neurotrophins are differentially present during various stages of odontogenesis, in this study we tested the hypothesis that concentrations of neurotrophins in coronal and radicular dentin are different with different functional effects of dentin matrix proteins (DMPs) on trigeminal ganglia (TG) neurons.
Materials and Methods Extraction of DMPs Extracted healthy third molars from 9 patients (17–24 years old) were collected from the oral surgery clinic at the University of Texas Health Science Center at San Antonio, San Antonio, TX, after obtaining informed patient consent. After the removal of enamel and cementum, all crowns were separated from the roots at the cementoenamel junction, and pulp was removed. For each patient, DMPs were extracted from crowns and roots according to an established protocol (20). All subsequent experiments were performed in a paired design comparing the results of crown versus root for each donor sample. Animals Eighteen male 6- to 8-week-old C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) were used for the isolation of primary TG neurons, and 6 severe combined immunodeficient (SCID) mice (6–8 weeks old, NOD.CB17-Prkdcscid/J, Jackson Laboratories) were used for the in vivo innervation assay. All animals were housed with food and water offered ad libitum in a 12-hour light/dark cycle at the University of Texas Health Science Center at San Antonio according to the guidelines established by our Institutional Animal Care and Use Committee, the National Institutes of Health Guide and Public Health Service policy on humane care and the use of laboratory animals, and the ARRIVE guidelines of the National Center for the Replacement and Reduction of Animals in Research. Quantification of Neurotrophic Factors The total protein concentrations in the crown and root extracts were determined by a bicinchoninic acid assay (Pierce BCA Protein Assay Kit; Thermo Fisher Scientific, Waltham, MA). Neurotrophic factors and neurotrophins were quantified by enzyme-linked immunosorbent assays (ELISAs) as follows: GDNF (Human GDNF Rapid ELISA Kit; biosensis, Thebarton, Australia), NGF (Human beta-NGF ELISA Kit; Thermo Fisher Scientific), BDNF (Quantikine ELISA Total BDNF Immunoassay; R&D Systems, Minneapolis, MN), and neurotrophin 3 (NT3) (Human NT3 Rapid ELISA Kit; biosensis), neurotrophin 4 (NT4) (Human NT4/5 Rapid ELISA Kit; biosensis). In addition, 2 growth factors reported to be abundantly expressed in the dentin matrix were quantified as the positive control: transforming growth factor beta 1 (TGF-b1) (Quantikine ELISA Human TGF-b1; R&D Systems) and vascular endothelial growth factor (VEGF) (Quantikine ELISA Human VEGF; R&D Sys730
Widbiller et al.
tems). The concentration of detected molecules was normalized to the total protein amount of the respective crown or root dentin sample.
Primary Culture of Mouse TG Neurons TG neurons were isolated from C57BL/6 mice as described previously (21); single neurons were cultured on poly-D-lysine–coated culture dishes or onto coverslips (Corning BioCoat; Thermo Fisher Scientific) in Dulbecco modified Eagle medium containing 10% heatinactivated fetal bovine serum (Thermo Fisher Scientific), 3 mg/mL 5-fluoro-2-deoxyuridine and 7 mg/mL uridine (Sigma-Aldrich, St Louis, MO), 1 glutamine, and 1 penicillin-streptomycin (Thermo Fisher Scientific). Neurons were seeded in a density of 13,000 cells/cm2 and allowed to attach overnight at 5% CO2 and 37 C before further experimentation. Immunocytochemistry and Determination of Neurite Area The effects of coronal and radicular DMPs on neurite outgrowth of mouse TG neurons were evaluated by immunocytochemical staining and quantification of the neurite surface area. Neurons were cultured on coverslips in 24-well plates exposed to the respective crown or root dentin extracts at a final protein concentration of 75 mg/mL in medium for 3 days, washed with 0.1 mol/L phosphate-buffered saline, fixed with 4% formaldehyde for 10 minutes, and processed for immunocytochemistry with a primary antibody against beta III tubulin (1:1000 dilution, R&D Systems) and an Alexa Fluor 488 secondary antibody (1:200 dilution, Thermo Fisher Scientific). Controls included sections without primary or secondary antibody. In addition, F-actin was stained by phalloidin (Texas Red-X phalloidin; Thermo Fisher Scientific). Coverslips were mounted on slides (VECTASHIELD with DAPI; Vector Laboratories, Burlingame, CA). Z-stack images were taken using a Nikon C1si laser scanning confocal microscope (Nikon, Minato, Japan) using standardized settings at 20 magnification and processed in Fiji (National Institutes of Health, Bethesda, MD) (22). For quantification, Tubb3-positive fibers were thresholded and quantified as described previously (10). The surface area of nerve fibers was determined in 6 randomly selected regions per coverslip. Neurite Outgrowth Quantification A colorimetric cell-based ELISA kit was used for quantification of total neurite formation (Human TUBB3 Cell-Based ELISA Kit; Abnova, Taipei City, Taiwan). Briefly, TG neurons cultured in poly-D-lysine– coated 96-well plates were exposed to DMPs (75 mg/mL) for 5 days. Next, cells were fixed, and Tubb3 was quantified by ELISA according to the manufacturer’s instructions. Neurite outgrowth was also quantified using a transwell assay according to the manufacturer’s instructions (Neurite Outgrowth Assay Plus Kit; Merck Millipore, Billerica, MA). Photometric quantification of the labeled neurites was performed by measuring absorbance at 490 nm using a FlexStation 3 Benchtop Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA) after 48 hours of DMP exposure.
In Vivo Innervation Assay A Matrigel implant model (Corning Matrigel GFR Membrane Matrix; Thermo Fisher Scientific) was used to evaluate the effect of matrix proteins extracted from crown and root dentin on innervation and axonal targeting in vivo (10, 23). SCID mice under isoflurane inhalation anesthesia (3 %) received bilateral subcutaneous injections to the dorsum of 450 mL growth factor reduced Matrigel JOE — Volume 45, Number 6, June 2019
Basic Research—Biology (Corning Matrigel GFR Membrane Matrix; Thermo Fisher Scientific) containing paired crown or root DMPs (75 mg/mL). The implants were harvested 7 days postinjection and processed for immunohistochemical staining with a primary antibody against neurofilament heavy polypeptide (1:1000 dilution; Biolegend, San Diego, CA). Immunoreactivity was detected by an Alexa Fluor 488 secondary antibody (1:200 dilution, Thermo Fisher Scientific). After mounting, fluorescence staining was visualized by confocal microscopy as described earlier.
Single-cell Calcium Imaging To determine the effect of DMPs from crown and root dentin on neuronal function, changes in the activity of the transient receptor potential cation channel subfamily V member 1 (TRPV1) were evaluated. TG neurons were cultured on poly-D-lysine–coated coverslips and exposed to medium (control) or medium supplemented with coronal and radicular DMPs for 24 hours. Next, neurons were loaded with the calcium indicator fura-2 acetoxymethyl ester (2 mmol/L, Thermo Fisher Scientific) and exposed to either 100 nmol/L capsaicin (CAP) or vehicle for 60 seconds. Changes in fluorescence as a measure of intracellular calcium concentration were detected by a Nikon TE2000U microscope fitted with a 40/1.35 NA Fluor objective (Nikon). Data were collected and analyzed with MetaFluor software (MetaMorph; Universal Imaging Corporation, Downingtown, PA). At the end of the experiment, cells were exposed to a 50 mmol/L KCl pulse as a positive control to detect all excitable cells. The percentage of CAPresponsive neurons of all excitable cells and the mean response amplitude (D F340/F360) were calculated from 5 coverslips per group with at least 20 cells each. Data Treatment and Statistical Analysis All experiments were conducted with associated crown and root DMPs from 9 donors (n = 9). For ELISAs and neurite outgrowth experiments, data were not normally distributed and, therefore, analyzed nonparametrically by pair-wise Mann-Whitney U tests. For calcium imaging experiments, data were analyzed by 1-way analysis of variance followed by the Tukey multiple comparison test. All statistical analyses were computed with GraphPad Prism 7 (GraphPad Software, La Jolla, CA), and statistical significance (P # .05) was indicated by asterisks in the respective figures.
Results Quantification of Neurotrophic Factors The total protein mass in crown and root dentin extracts was equivalent (Fig. 1A). Furthermore, no significant differences of the abundant growth factors TGF-b1 and VEGF were detected between coronal and radicular dentin (Fig. 1A). Moreover, NGF, BDNF, and NT3 prevailed in root dentin, with NGF comprising the greatest proportion. On the contrary, NT4 was found to be equally distributed, whereas GDNF predominated in crown dentin (Fig. 1A). Interestingly, quantification of neurotrophic proteins revealed a 3-fold higher amount in radicular dentin compared with coronal parts (Fig. 1B). Immunocytochemistry and Determination of Neurite Area Immunostaining of mouse trigeminal neurons for beta III tubulin after 3 days revealed enhanced neurite formation when cultured with DMPs. Protein extracts from radicular dentin led not only to an increase in number but also in the maturity of neurites as evidenced by the thickness of axons (Fig. 2A). This observation was confirmed by quantificaJOE — Volume 45, Number 6, June 2019
tion of the neurite area, which was significantly increased in neuronal cultures exposed to DMPs (Fig. 2B).
Neurite Outgrowth and Targeting Neurite formation was additionally measured by a cell-based beta III tubulin immunoassay. Cultivation of mouse trigeminal neurons with dentin extracts resulted in a significantly increased Tubb3 expression per cell (Fig. 2C) with more Tubb3-positive neurites in cultures supplemented by radicular DMPs (165% 21% for radicular compared with 144% 18% coronal samples). The specific axonal targeting of trigeminal neurons was quantified in a transwell assay in which proteins from root dentin promoted outgrowth with significant differences compared with crown dentin and control (Fig. 2D).
In Vivo Innervation Supplementation of DMPs to hydrogels implanted subcutaneously in SCID mice increased the number of infiltrating axons, which were distributed equally throughout the implant after 7 days. Although a distinct difference between crown and root dentin was hard to detect, innervation density was considerably lower in control implants as shown by neurofilament heavy polypeptide immunostaining (Fig. 2E).
Single-cell Calcium Imaging Quantitative single-cell Ca2+ imaging was used to determine the impact of proteins extracted from coronal or radicular dentin on the TRPV1 activity of trigeminal neurons. Treatment with capsaicin resulted in Ca2+ influx in treated as well as untreated cells. Although only a small fraction of neurons in the control group was sensitive to capsaicin, significantly more cells responded after treatment with DMPs (Fig. 3A). Pretreatment with radicular dentin extracts led to the highest proportion of TG neurons responsive to capsaicin (84% 3%), which significantly exceeded the coronal dentin-treated group (68% 4%). However, the mean peak of calcium influx was similar in the control group and the DMP-treated groups (Fig. 3B).
Discussion Current regenerative endodontic procedures rely on the release of dentin matrix growth factors to promote the formation of a reparative intracanal tissue. The dentin matrix harbors a multitude of bioactive molecules that become sequestered and “fossilized” during tooth formation (17). Although their effects on angiogenesis (24), odontoblast differentiation (20), and dentin formation (25) have been described, little is known about the presence of neurotrophic factors and their bioactivity. In this study, we showed that the root dentin matrix has a higher concentration of known neurotrophic factors and that they retain their bioactivity, promoting greater neurogenesis than coronal dentin. In this study, all investigated neurotrophic proteins (NGF, BDNF, NT3, NT4, and GDNF) were detected in the human dentin matrix with their concentration approximately 3 times higher in root dentin compared with crown dentin. The predominating factors in radicular dentin were NGF followed by NT3 and BDNF, whereas GDNF predominated in crowns. Interestingly, innervation during tooth development has a strict neurotrophic factor–directed spatiotemporal control (13, 26). Thus, the differential entrapment of neurotrophic factors in the crown and root dentin matrix provides further evidence for the intense neurogenic activity around the time of root formation and that GDNF may have predominated during crown formation. The abundance of NGF, particularly in the root, agrees with its wellknown role during tooth innervation (14, 15, 27). Similarly, BDNF is a target-invading factor for trigeminal axons (26) that prevailed in
Neurotrophic Proteins in Dentin
731
Basic Research—Biology
Figure 1. Quantification of growth factors and neurotrophic proteins. (A) The total protein amount as well as the concentration of TGF-b1 and VEGF were equal in crown and root dentin. However, the neurotrophins NGF, BDNF and NT3 prevailed in radicular dentin. GDNF was increased in coronal dentin, whereas the neurotrophin NT4 was evenly distributed. Data are presented as the median with the interquartile range, and asterisks indicate significant differences (P # .05). (B) The total amount of neurotrophic proteins in the root dentin was 3 times higher compared with crown dentin, most of which is formed by NGF.
root dentin extracts. However, GDNF was reported to be expressed primarily in cuspal areas by odontoblasts or subodontoblast cells when trigeminal axons approach (16, 26), which is in line with our observations. It is thought to play a morphogenetic role and might have neuroprotective functions (26). Instead, NT3 and NT4 have been observed in early developmental stages throughout the whole mandibular mesenchyme and thus more likely play a role in odontogenesis than in pulpal innervation (26). Although this study did not identify and test all factors with possible neurotrophic activities within dentin, it supports their role of these well-known factors in organogenesis. DMPs are solubilized by carious lesions and may reach the dental pulp, participating in neuronal responses to injury. These responses include extensive neuronal arborization and axonal growth toward the site of injury and nociceptor sensitization (18, 19). The ligandgated cation channel TRPV1 is expressed exclusively in a subset of 732
Widbiller et al.
capsaicin-sensitive nociceptors and is essential for the development of inflammatory hyperalgesia (28, 29). It has been shown to be sensitized by bacterial lipopolysaccharides and be significantly up-regulated in injury and pulpitis (30–32). The effect of DMPs on TRPV1 responses was evaluated in this study for its translational significance to pulpitis- and injury-dependent increased expression in the dental pulp. The treatment of primary trigeminal cultures with DMP extracts for 24 hours resulted in an increase in the number of cells responding to CAP stimulation after exposure to DMPs, which was significantly more pronounced in neuronal cultures treated with radicular DMPs. It is important to emphasize that the individual neurotrophic factors within the dentin matrix mediating this increase in TRPV1 responsiveness were not identified in this study. Nonetheless, NGF, being the most abundant neurotrophic factor in radicular dentin with wellknown effects on TRPV1 expression (33–35) and sensitization (36), is JOE — Volume 45, Number 6, June 2019
Basic Research—Biology
Figure 2. DMPs promote neurite outgrowth in vitro and in vivo. (A) Neurite formation was enhanced by extracellular matrix proteins from crown and especially root dentin. Cells are visualized by fluorescence staining for Tubb3 (green), actin filaments (red), and nuclei (blue). (B) Quantification of the neurite surface area, which is positively stained for Tubb3 (green in A), confirmed the observations made. (C) An increase in neuritogenesis of sensory neurons cultured with DMPs was also detected in a cell-based immunoassay for Tubb3. (D) Axonal growth was similarly enhanced by extracts from root dentin with a significant difference compared with crown dentin. All data are depicted as the median with the interquartile range, and significant differences are labeled by asterisks (P # .05). (E) Innervation was also evaluated in vivo by hydrogel implants that were optionally supplemented with DMPs. Immunostaining for neurofilament heavy polypeptide (yellow) showed more nerve fibers sprouting into implants with crown and root DMPs compared with the control.
likely involved in the increased TRPV1 responsiveness observed. It is important to note that treatment with DMPs did not result in an increase in the response magnitude, suggesting that TRPV1 responses to capsaicin were not enhanced. Instead, the increase in the percentage of capsaicin-responsive neurons is likely caused by an overall increase in TRPV1 expression (37, 38), most likely through posttranslational modification (34, 39) that was not directly tested in this study. Thus, JOE — Volume 45, Number 6, June 2019
neurotrophic factors within DMPs may be involved in arborization and increased TRPV1 activities seen in carious injuries. Interestingly, there are many reports of the reestablishment of nociception in cases treated with REPs. In addition, histologic examination of teeth treated with these procedures have shown that the newly formed tissues are richly innervated with nociceptors (8, 9). Although the cold stimulus typically used in pulp sensibility tests is likely to activate the
Neurotrophic Proteins in Dentin
733
Basic Research—Biology
Figure 3. Sensitization of the TRPV1 response of trigeminal sensory neurons by DMPs. (A) The percentage of capsaicin-responsive neurons increased significantly after treatment with dentin extracts from coronal and, particularly, radicular dentin. (B) Capsaicin-evoked accumulation of intracellular calcium (D F340/F360) was similar after pretreatment of the neurons with matrix proteins. Data are presented as mean standard deviation, and asterisks mark significant differences (P # .05).
transient receptor ankyrin type 1 and not TRPV1, there is a robust coexpression of these 2 channels that is increased by NGF (33). The use of EDTA in REPs has the intent to expose and release DMPs, which according to our findings include all major neurotrophic proteins with a predominance of NGF from the treated radicular dentin. Also, stem cells from the apical papilla, an important kind of mesenchymal stem cells involved in REPs, have been shown to promote neuronal fiber recruitment through the secretion of BDNF (10). Thus, the reinnervation of a pulplike tissue after regenerative endodontics is likely mediated through the following 2 mechanisms: a noncellular-mediated mechanism by released DMPs and a cellular-mediated mechanism by stem cells of the apical papilla. Collectively, our data show that dentin contains all the major neurotrophic factors with differential distribution between radicular and coronal dentin. DMPs can be released with EDTA treatment, promote neurite outgrowth and increase TRPV1 responses. These effects were more pronounced in samples treated with root DMPs that were shown to have a predominance of NGF, a potent neurotrophin. The results of this study corroborate elegant studies showing the differential expression of these factors during various stages of tooth development. Also, their activity upon solubilization could explain neuronal reactions such as arborization and nociception to demineralizing events such as caries. Lastly, the robust effect on neurite outgrowth and axonal targeting could, at least in part, be one of the mechanisms by which the pulplike tissue becomes reinnervated in patients after REPs. The cocktail of DMPs may be exploited to optimally coordinate axonal ingrowth into the root canal space and facilitate pulpal reinnervation during REPs.
Acknowledgments Supported by a research grant from the American Association of Endodontists Foundation. The authors deny any conflicts of interest related to this study.
References 1. Diogenes A, Henry MA, Teixeira FB, Hargreaves KM. An update on clinical regenerative endodontics. Endod Topics 2013;28:2–23. 2. Galler KM. Clinical procedures for revitalization: current knowledge and considerations. Int Endod J 2016;49:926–36. 3. Cvek M. Prognosis of luxated non-vital maxillary incisors treated with calcium hydroxide and filled with gutta-percha. A retrospective clinical study. Dent Traumatol 1992;8:45–55.
734
Widbiller et al.
4. Lin J, Zeng Q, Wei X, et al. Regenerative endodontics versus apexification in immature permanent teeth with apical periodontitis: a prospective randomized controlled study. J Endod 2017;43:1821–7. 5. Diogenes A, Ruparel NB, Shiloah Y, Hargreaves KM. Regenerative endodontics: a way forward. J Am Dent Assoc 2016;147:372–80. 6. Kim JY, Xin X, Moioli EK, et al. Regeneration of dental-pulp-like tissue by chemotaxis-induced cell homing. Tissue Eng Part A 2010;16:3023–31. 7. Huang GT, Yamaza T, Shea LD, et al. Stem/progenitor cell-mediated de novo regeneration of dental pulp with newly deposited continuous layer of dentin in an in vivo model. Tissue Eng Part A 2010;16:605–15. 8. Lei L, Chen Y, Zhou R, et al. Histologic and immunohistochemical findings of a human immature permanent tooth with apical periodontitis after regenerative endodontic treatment. J Endod 2015;41:1172–9. 9. Austah O, Joon R, Fath WM, et al. Comprehensive characterization of 2 immature teeth treated with regenerative endodontic procedures. J Endod 2018;44: 1802–11. 10. De Almeida JF, Chen P, Henry MA, Diogenes A. Stem cells of the apical papilla regulate trigeminal neurite outgrowth and targeting through a BDNF-dependent mechanism. Tissue Eng Part A 2014;20:3089–100. 11. Widbiller M, Driesen RB, Eidt A, et al. Cell homing for pulp tissue engineering with endogenous dentin matrix proteins. J Endod 2018;44:956–62.e2. 12. Schmalz G, Widbiller M, Galler KM. Signaling molecules and pulp regeneration. J Endod 2017;43:S7–11. 13. Hildebrand C, Fried K, Tuisku F, Johansson CS. Teeth and tooth nerves. Prog Neurobiol 1995;45:165–222. 14. Fried K, Lillesaar C, Sime W, et al. Target finding of pain nerve fibers: neural growth mechanisms in the tooth pulp. Physiol Behav 2007;92:40–5. 15. Matsuo S, Ichikawa H, Henderson TA, et al. trkA modulation of developing somatosensory neurons in oro-facial tissues: tooth pulp fibers are absent in trkA knockout mice. Neuroscience 2001;105:747–60. 16. Nosrat CA, Fried K, Ebendal T, Olson L. NGF, BDNF, NT3, NT4 and GDNF in tooth development. Eur J Oral Sci 1998;106(Suppl 1):94–9. 17. Smith AJ, Scheven BA, Takahashi Y, et al. Dentine as a bioactive extracellular matrix. Arch Oral Biol 2012;57:109–21. 18. Kimberly CL, Byers MR. Inflammation of rat molar pulp and periodontium causes increased calcitonin gene-related peptide and axonal sprouting. Anat Rec 1988; 222:289–300. 19. Taylor PE, Byers MR, Redd PE. Sprouting of CGRP nerve fibers in response to dentin injury in rat molars. Brain Res 1988;461:371–6. 20. Widbiller M, Eidt A, Lindner SR, et al. Dentine matrix proteins: isolation and effects on human pulp cells. Int Endod J 2018;51(Suppl 4):e278–90. 21. Patwardhan AM, Berg KA, Akopain AN, et al. Bradykinin-induced functional competence and trafficking of the delta-opioid receptor in trigeminal nociceptors. J Neurosci 2005;25:8825–32. 22. Schindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 2012;9:676–82. 23. Lopatina T, Kalinina N, Karagyaur M, et al. Adipose-derived stem cells stimulate regeneration of peripheral nerves: BDNF secreted by these cells promotes nerve healing and axon growth de novo. PLoS One 2011;6:e17899. 24. Zhang R, Smith AJ, Cooper PR, et al. Angiogenic activity of dentin matrix components. J Endod 2011;37:26–30.
JOE — Volume 45, Number 6, June 2019
Basic Research—Biology 25. Tziafas D, Alvanou A, Panagiotakopoulos N, et al. Induction of odontoblast-like cell differentiation in dog dental pulps after in vivo implantation of dentine matrix components. Arch Oral Biol 1995;40:883–93. 26. Fried K, Nosrat C, Lillesaar C, Hildebrand C. Molecular signaling and pulpal nerve development. Crit Rev Oral Biol Med 2000;11:318–32. 27. Mitsiadis TA, Magloire H, Pagella P. Nerve growth factor signalling in pathology and regeneration of human teeth. Sci Rep 2017;7:1327. 28. Caterina MJ, Rosen TA, Tominaga M, et al. A capsaicin-receptor homologue with a high threshold for noxious heat. Nature 1999;398:436–41. 29. Caterina MJ, Leffler A, Malmberg AB, et al. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 2000;288:306–13. 30. Morgan CR, Rodd HD, Clayton N, et al. Vanilloid receptor 1 expression in human tooth pulp in relation to caries and pain. J Orofac Pain 2005;19:248–60. 31. Ruparel S, Hargreaves KM, Eskander M, et al. Oxidized linoleic acid metabolitecytochrome P450 system (OLAM-CYP) is active in biopsy samples from patients with inflammatory dental pain. Pain 2013;154:2363–71. 32. Watase T, Shimizu K, Komiya H, et al. Involvement of transient receptor potential vanilloid 1 channel expression in orofacial cutaneous hypersensitivity following tooth pulp inflammation. J Oral Sci 2018;60:8–13.
JOE — Volume 45, Number 6, June 2019
33. Diogenes A, Akopian AN, Hargreaves KM. NGF up-regulates TRPA1: implications for orofacial pain. J Dent Res 2007;86:550–5. 34. Price TJ, Louria MD, Candelario-Soto D, et al. Treatment of trigeminal ganglion neurons in vitro with NGF, GDNF or BDNF: effects on neuronal survival, neurochemical properties and TRPV1-mediated neuropeptide secretion. BMC Neurosci 2005;6:4. 35. Zhang X, Huang J, McNaughton PA. NGF rapidly increases membrane expression of TRPV1 heat-gated ion channels. EMBO J 2005;24:4211–23. 36. Zhuang Z-Y, Xu H, Clapham DE, Ji R-R. Phosphatidylinositol 3-kinase activates ERK in primary sensory neurons and mediates inflammatory heat hyperalgesia through TRPV1 sensitization. J Neurosci 2004;24:8300–9. 37. Shinoda M, Asano M, Omagari D, et al. Nerve growth factor contribution via transient receptor potential vanilloid 1 to ectopic orofacial pain. J Neurosci 2011;31: 7145–55. 38. Simonetti M, Fabbro A, D’Arco M, et al. Comparison of P2X and TRPV1 receptors in ganglia or primary culture of trigeminal neurons and their modulation by NGF or serotonin. Mol Pain 2006;2:11. 39. Ji R-R, Samad TA, Jin S-X, et al. p38 MAPK activation by NGF in primary sensory neurons after inflammation increases TRPV1 levels and maintains heat hyperalgesia. Neuron 2002;36:57–68.
Neurotrophic Proteins in Dentin
735