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Expression of Neurotrophic Factors in Human Dentin and Their Regulation of Trigeminal Neurite Outgrowth
Regenerative Endodontics
Expression of Neurotrophic Factors in Human Dentin and Their Regulation of Trigeminal Neurite Outgrowth Obadah Austah, BDS, MS,*† Matthias Widbiller, DDS,*‡ Phillip L. Tomson, BDS, PhD,§ and Anibal Diogenes, DDS, PhD* Abstract Introduction: Neurotrophic factors play a significant role in the innervation of the pulp-dentin complex during and after organogenesis. There have been numerous bioactive molecules identified in the dentin extracellular matrix; however, the expression of neurotrophic factors in the dentin matrix and their biological activity are largely unknown. The purpose of this study was to characterize the relative expression of neurotrophic factors in human dentin matrix proteins (DMPs) and their effect on neurite outgrowth of trigeminal (TG) neurons. Methods: Dentin was powdered in liquid nitrogen from noncarious human third molar teeth. DMPs were solubilized through an EDTA extraction method, dialyzed, and lyophilized until use. The relative expression of nerve growth factor, brain-derived neurotrophic factor, glial cell line derived neurotrophic factor, neurotrophin 3, and neurotrophin 4/5 was determined by the enzyme-linked immunosorbent assay. Rat TG neurons were cultured and exposed to different concentrations of DMPs (1–105 ng/mL) or vehicle, and a quantitative neurite outgrowth assay was performed. Results: Human DMPs contained all of the tested neurotrophic factors, with glial cell line derived neurotrophic factor and neurotrophin 4/5 found at the highest levels. DMPs were able to promote the neurite outgrowth of rat TG neurons at an optimum concentration of 10–102 ng/mL, whereas the effect was partially inhibited at higher concentrations (>103 ng/mL). Conclusions: The human dentin extracellular matrix is a rich reservoir for neurotrophic factors that are key components for neuronal homeostasis, differentiation, and regeneration. These data suggest that neurotrophins in DMPs could play an important role as signaling molecules for the innervation of the pulp-dentin complex during the processes of tooth formation, repair, and regeneration. (J Endod 2019;-:1–6)
Key Words Dentin matrix protein, dentinogenesis, neurite outgrowth, neurotrophic factors, neurotrophins, regeneration
D
entin has been tradiSignificance tionally thought to be Key neurotrophic factors (NGF, BDNF, GDNF, an inert mineralized tissue NT3, and NT4/5) found fossilized in the dentin that provides mechanical matrix are released with EDTA and promote support and protection. neurogenesis. Thus, they can be exploited in pulpal Recent advances in the repair and regenerative therapy. basic biological understanding of the pulpdentin complex unraveled a substantial array of bioactive molecules embedded in the mineralized matrix during dentinogenesis that can be released upon various stimuli (1–3). The presence of these molecules highlights their important roles in the repair and regeneration of the pulp-dentin complex throughout the life span of teeth. Dentin bioactivity was shown a few decades ago by the application of autologous demineralized dentin chips as a pulp cap (4) or apical plug (5) to promote pulpal repair and apical closure and healing in primate animals. Later, the promineralization and reparative dentinogenesis potentials were shown to be caused by the rich pool of noncollagenous proteins that were released upon demineralization of the tissue (6). Demineralization of dentin can occur by bacterial acids during caries attack activities or by using various dentin conditioning agents such as EDTA (7, 8) and materials used to promote pulp healing such as calcium hydroxide (9) and mineral trioxide aggregate (10). However, the understanding of their relative solubility and the control of their release in vivo is still limited. Dental pulp is 1 of the most densely innervated tissues in the body, and the innervating fibers have the capability to regenerate and restore tissue hemostasis after various injuries (11). Neurotrophic factors are a broad family of growth factors that are important in regulating development, differentiation, function, and survival of neuronal cells. Previous studies showed that certain neurotrophic factors including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), neurotrophin 4 (NT4), and glial cell–line derived neurotrophic factor (GDNF) are present in the dental structures (12, 13). These growth factors act in an orchestrated fashion to promote the complex innervation of the pulp-dentin complex.
From the *Department of Endodontics, University of Texas Health Science Center at San Antonio, San Antonio, Texas; †Department of Endodontics, Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia; ‡Department of Conservative Dentistry and Periodontology, University Hospital, Regensburg, Germany; and §Department of Oral Biology, Institute of Clinical Sciences, The University of Birmingham School of Dentistry, Birmingham, UK. Address requests for reprints to Dr Anibal Diogenes, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2018 American Association of Endodontists. https://doi.org/10.1016/j.joen.2018.12.011
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Neurotrophic Factors in Human Dentin
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Regenerative Endodontics It is well established that key growth factors are present and released from dentin and mediate important biological activities such as repair and regeneration. The transforming growth factor beta superfamily is 1 of the first identified factors and is known to stimulate mineralization and regenerative activity in the pulp-dentin complex (14). For any repair or regenerative process, angiogenesis and neurogenesis are integral parts of ideal outcomes to maintain key cellular and nutritional supply and provide neuroprotection. In fact, proangiogenic factors such as platelet-derived growth factor and vascular endothelial growth factor have been already identified in dentin (14, 15). However, there is limited knowledge regarding the presence and biological function of proneurogenic or neurotrophic factors in human dentin. Therefore, the purpose of this study was to characterize the relative expression and concentration of neurotrophic factors in human powdered dentin matrix proteins (DMPs) and evaluate their bioactivity on neurite outgrowth of trigeminal (TG) neurons.
enamel portion of the teeth were carefully removed. The remaining dentin structures were frozen in liquid nitrogen and ground using a freezer/mill (Spex 6700 Freezer/Mill; Glen Creston Ltd, London, UK) into a fine powder and passed through a 60-mm mesh sieve. Next, DMPs were solubilized using 10% EDTA in the presence of protease inhibitors, 10 mmol/L n-ethylmaleimide (Sigma-Aldrich, St Louis, MO), and 5 mmol/L phenylmethylsulfonyl fluoride (Sigma-Aldrich) with continuous agitation at 4 C for 14 days. Extracted supernatants were dialyzed for 10 days and lyophilized using a freezer dryer (Modulyo; Edwards, Crawley, UK) as previously described (10).
Materials and Methods
Quantification of Neurotrophic Factors In brief, the lyophilized DMPs were dissolved in phosphatebuffered saline at a concentration of 10 mg/mL. To optimize the detection range of the enzyme-linked immunosorbent assay (ELISA), undiluted and serial dilutions of the samples were used to evaluate the concentrations of NGF, BDNF, GDNF, NT3, and NT4/5 using a multineurotrophic rapid screening ELISA kit (Biosensis, South Australia, Australia) following the manufacturer’s instructions.
Isolation of Human Dentin Extracellular Matrix Proteins Extracted noncarious third molars were obtained from the Oral Surgery Department at Birmingham Dental Hospital, Birmingham, UK, after informed patient consent and ethical approval (14/EM/1128). The cementum and attached soft tissues were removed from the root surface of the teeth and sectioned longitudinally. Pulp tissue and the
Rat TG Ganglia Neuronal Primary Culture Adult male Sprague-Dawley rats (weight, 200–250 g each [Charles River, Wilmington, MA]) were used in this study. All animal study protocols were approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at
Figure 1. Mean concentrations (pg/mg) of neurotrophic factors in human dentin powdered extracellular matrix proteins (DMPs) as determined by ELISA. *P < .05, **P < .01; 1-way ANOVA with the Bonferroni post hoc test, P < .05. Data are presented as the mean standard error of the mean.
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Regenerative Endodontics
Figure 2. The mean percentage of neurite outgrowth when exposed to different concentrations of DMPs (1–105 ng/mL) for 72 hours. *P < .05, **P < .01; 1-way ANOVA with the Dunnett post hoc test, P < .05. Data are presented as the mean standard error of the mean.
San Antonio, San Antonio, TX, and conformed to the International Association for the Study of Pain and federal guidelines. Animals were housed for 1 week before the experiments with food and water available ad lib. Rats were euthanized by decapitation, and the TG ganglia were dissected and isolated through an enzymatic process as previously described (16). Single-cell neuronal suspensions at the concentrations of 5 105 neurons/mL were cultured in 12-well poly-D-lysine–coated culture plates (Corning Life Sciences, Corning, NY) in growth medium containing Dulbecco modified Eagle medium (Life Technologies, Carlsbad, CA), 10% heat-inactivated fetal bovine serum (Life Technologies), 3 mg/mL 5-fluoro-2-deoxyuridine, 7 mg/mL uridine (Sigma-Aldrich), and 1 glutamine-penicillinstreptomycin (Invitrogen, Carlsbad, CA). Neurons were allowed to attach for 24 hours in 5% CO2 at 37 C before neurite outgrowth experiments.
Neurite Outgrowth Experiments To assess the bioactivity of the sequestered neurotrophic factors in dentin, 2 experiments were performed to evaluate the effect of DMPs on the outgrowth of TG neurons. All experiments were performed in triplicate, and for each experiment, 3 rats (6 TG ganglia) were used. The lyophilized DMPs were resuspended in Dulbecco modified Eagle medium and added to the culture media in serial dilution of concentrations ranging from 1 ng/mL–105 ng/mL. For the first method, the neurite outgrowth assay kit (NS220; Millipore, Burlington, MA) was used. In brief, TG neurons were seeded onto culture inserts with 3-mm pores at the base in a 12-well plate at a density of 3 104 cells/well. TG neurons were cultured in either basal growth media alone (the control group) or in growth media in the presence of different concentrations of DMPs (1, 10, 102, 103, 104, or 105 ng/mL) (the experimental groups). Neurons were cultured JOE — Volume -, Number -, - 2019
with fresh media replaced daily for 3 days. Absorbance was measured at 490 nm using a FlexStation 3 Benchtop Multimode Microplate Reader (Molecular Devices, Sunnyvale, CA) following the manufacturer’s instructions. In the second method, we evaluated the effect of the optimum concentration of DMPs determined in the concentration response experiment on neurite outgrowth using immunocytochemistry and confocal microscopy. In brief, TG neurons were seeded onto poly-D-lysine–coated coverslips at a density of 3 104 cells/well in basal growth medium containing either the vehicle (the control group) or 102 ng/mL DMPs (the experimental group). Neurons were cultured for 3 days followed by fixation in 4% paraformaldehyde and immunostaining as described previously (17). Immunostaining was performed with primary antibody against beta III tubulin (cat# MAB 1195 at a 1:200 dilution in blocking solution; R&D Systems, Minneapolis, MN) followed by species-appropriate Alexa Fluor 488 secondary antibody at 1:200 dilution in blocking solution (Molecular Probes, Eugene, OR). Immunoreactivity was visualized with a Nikon Eclipse 90i microscope with a Nikon C1si confocal laser scan head (Nikon Instruments, Melville, NY), and images were collected using standardized settings at 10 and 20 magnifications. Four standardized selected images of each slide were acquired at 20 magnification and visualized in ImageJ software (Version 2.0.0-rc-67/1.52c; National Institutes of Health, Bethesda, MD) to determine the percentage of area occupied by neurites. Data were normalized to the percentage of the control group.
Statistical Analysis For the ELISA and the neurite outgrowth experiments, results were analyzed using 1-way analysis of variance (ANOVA) with the Bonferroni post hoc test and the Dunnett post hoc test, respectively. For the Neurotrophic Factors in Human Dentin
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Regenerative Endodontics
Figure 3. (A) Neurite outgrowth of TG neurons cultured in either the control (basal medium) or 102 ng/mL DMPs for 72 hours. Representative confocal microphotographs of beta III tubulin immunostaining (green) of primary TG neuronal incubated in (B) the control (basal medium) and (C) 102 ng/mL DMPs. *P < .05; unpaired 2-tailed Student t test, P < .05. Images were acquired by Nikon C1si laser scanning confocal microscopy with 20 magnification.
immunocytochemistry experiment, the difference in the percentage of area occupied by neurites between the control and DMP group was analyzed with the unpaired Student t test. All statistical analyses were performed using Prism Version 7.0a software (GraphPad, La Jolla, CA) with statistical significance set at P < .05. All data were expressed as mean standard error of the mean.
Results Relative Expression of Neurotrophic Factors in Human Dentin ELISA-based measurements of neurotrophic factors in dentin revealed variable detectable levels (pg/mg) of all screened neurotrophic factors. Interestingly, GDNF (10.71 3.91 pg/mg) and NT4/5 (11.43 0.52 pg/mg) were expressed in significantly higher levels than NT3 (8.12 0.54 pg/mg), BDNF (5.033 0.19 pg/mg), and NGF (4.84 0.064 pg/mg) (P < .05) (Fig. 1). In addition, NGF and BDNF were the least detected amounts found in dentin (Fig. 1). DMPs Mediate Neurite Outgrowth of TG Neurons Neurite outgrowth assay with different concentrations of DMPs resulted in a bell-shaped dose-response curve with optimum concentration at 10–102 ng/mL (Fig. 2). Lower concentrations of DMPs (1, 101, and 102 ng/mL) resulted in significant promotion of neurite extension (1.5-fold increase) compared with the control group (P < .01) (Fig. 2). Interestingly, this increase was reversed when higher concentrations were used (103, 104, and 105 ng/mL). Immunocytochemical analysis of cultured TG neurons in basal medium (control) or supplemented with 102 ng/mL DMPs for 3 days showed a significantly larger area occupied by neurites in the DMP 4
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group than the control (P < .05) (Fig. 3A). In addition, the substantial greater number of branching and length of neurites can be appreciated (Fig. 3B and C).
Discussion Dentin is well-known to be a rich resource of bioactive molecules such as growth factors, cytokines, and chemokines that are embedded in the mineralized ECM during tooth development and released upon demineralizing events (7). Our results showed that dentin represents a rich source of several neurotrophic factors (NGF, BDNF, GDNF, NT3, and NT4/5) with known profound effects in neuronal chemotaxis and function. Indeed, these neurotrophic factors promoted the robust increase in neurite outgrowth in vitro. Neurotrophic growth factors play key roles in innervation development by mediating organization, plasticity, axonal targeting, repair, survival, and regeneration of the free nerve terminals (12). Previous studies of odontogenesis examined the temporal and spatial expression of the neurotrophic factors and showed the participation of NGF, BDNF, and GDNF during teeth innervation and GDNF and NT3/4 in the epithelial-mesenchymal cross talk during early morphogenesis (13, 18, 19). Interestingly, our results showed a higher relative expression of GDNF, NT3, and NT4/5 than NGF and BDNF in dentin. This variation can be conceivably explained by the early expression of GDNF and neurotrophins during tooth formation and their sequestration in the dentin matrix before its mineralization. On the contrary, lower concentrations of NGF and BDNF can be explained by their late involvement during tooth innervation, which begins when root formation commences (20). Nevertheless, they were still present beforehand but in much lower levels and that reflects JOE — Volume -, Number -, - 2019
Regenerative Endodontics their minimal rate of sequestration. Thus, neurotrophins are sequestered in dentin during dental organogenesis and may play a role in the defensive and reparative capabilities of the pulp-dentin complex during life. The innervation of the pulp dentin complex is dynamic, and the arborization of the free nerve endings in response to insults such as caries has been elegantly shown (21). The overall function of this increased innervation in the repair and regeneration of the pulp-dentin complex is not fully understood. However, this increase of innervation in areas of injury suggest an active role of these nerve fibers that exceed their most accepted putative function as nociceptors. It has been hypothesized that this increase of innervation is mainly caused by the effect of NGF. It is well established that NGF is very critical for the differentiation of neural crest cells to dental organ and for tooth innervation (22). Tooth innervation was mostly absent in tyrosine kinase receptor (trkA) knockout and anti–NGF-treated animals (23, 24). Moreover, different models of tooth injury led to up-regulation of NGF adjacent to the injury site (25, 26). The source of neurotrophins such as NGF has been attributed solely to cells of the pulp-dentin complex such as odontoblasts and pulpal fibroblasts, emphasizing the importance of neurotrophic factors in regulating their functions (25). However, our study suggests that the release of both bioactive molecules could also be from the demineralized dentin. Similarly, BDNF, NT3, and NT4/5 promote neurite outgrowth, development, and survival of sensory neurons, and their effect is mediated by receptor binding followed by internalization and retrograde transportation to the primary neurons in TG (18). GDNF is another important factor, and it is mainly expressed postnatally in the subodontoblastic layer (27). It has also been implicated in the regulation of postnatal tooth innervation and promoting survival and proliferation of dental pulp cells (28). In this study, the release of neurotrophins from dentin can affect all major subtypes of nociceptors in the dental pulp (18, 29, 30), suggesting a strong role of the neuronal response to injury in the dental pulp. One of the limitations of our study is the variabilities of the collected samples in regard to donor age, sex, and stage of root development. This might affect the presented levels of neurotrophic factors and their effect on TG neurons. Further research is warranted to determine whether there are differences in these factors’ relative abundance and bioactivity in teeth from patients of different ages and sex. This is particularly important in the context of regenerative endodontic procedures (REPs). It has been shown that approximately 60% of all the reported cases of REPs report regaining tooth sensibility to vitality testing (31). Previous studies have histologically confirmed the presence of regenerated nerve fibers in teeth treated with REPs, thus providing the biological basis for such a clinical finding (31–33). The mechanism of axonal growth and targeting into the newly formed tissues is not fully understood. A study showed that stem cells of the apical papilla, when in the presence of TG neurons, release NGF, GDNF, and BDNF, with the latter being responsible for mediating axonal growth and targeting (34). Importantly, the beneficial effect of EDTA conditioning in regenerative endodontics has been well-documented (8, 9, 35). In this study, we showed that EDTA promoted the release of neurotrophic factors from dentin. Thus, DMP-derived neurotrophic factors are likely adjuvants, in addition to stem cells of apical papilla, in mediating axonal growth and targeting into the newly formed tissues after REPs. An in vitro neurite outgrowth in cultured neurons is considered as a study model for neuronal regeneration potential (36). In order to evaluate whether the neurotrophic factors released from dentin are bioactive, we used 2 well-characterized assays to quantify the neurite outgrowth of primary TG neurons. Our results showed a concentration-dependent response with higher stimulations of neurite outgrowth in lower concentrations (10–102 ng/mL). On the contrary, JOE — Volume -, Number -, - 2019
103 ng/mL DMPs or higher led to an inhibitory effect on TG neurons. There are few possibilities to explain the observed response. One possible reason is that a very high concentration of neurotrophic factors will activate low-affinity receptors with known divergent functions. For instance, the high-dose inhibitory effect of NGF is shown to be mediated by the low-affinity P75 receptor (37). Lower concentrations of NGF result in signaling through its high-affinity receptor (trkA). However, greater concentrations will lead to the occupancy of its low-affinity receptor (P75). Interestingly, the activation of the low-affinity receptor has an opposite effect to that seen with trkA regarding the promotion of neuronal survival (38). Another possible reason is the presence of inhibitory molecules not identified in this study at higher concentrations could inhibit neurite outgrowth. For instance, a higher dose of DMPs might contain higher levels of semaphoring 3a (a neuronal inhibitory factor that participates in dental innervation), which in turn led to the observed inhibitory effects (39). In conclusion, our study showed for the first time the expression of all dental tissue-associated neurotrophic factors in DMPs. Also, these factors retain their bioavailability, and the application of optimum concentration promoted the neurite outgrowth of primary cultured TG neurons. Our data highlight the potential role of these DMP-derived factors in neuronal responses to injury and their exploitation to enhance the current therapeutic approaches in both the repair and regeneration of the pulp-dentin complex. Future experiments evaluating the effect of DMPs on axonal targeting of TG neurons and elucidating the soluble factors regulating those activities are warranted. Additionally, experiments assessing the effect of known dentin solubilizers on the release of the neurotrophic factors are also needed.
Acknowledgments This research was supported in part by a research grant from the Foundation for Endodontics. The authors deny any conflicts of interest related to this study.
References 1. Smith AJ, Duncan HF, Diogenes A, Simon S, Cooper PR. Exploiting the bioactive properties of the dentin-pulp complex in regenerative endodontics. J Endod 2016;42:47–56. 2. Tomson PL, Lumley PJ, Smith AJ, Cooper PR. Growth factor release from dentine matrix by pulp-capping agents promotes pulp tissue repair-associated events. Int Endod J 2016;50:281–92. 3. Tomson PL, Lumley PJ, Alexander MY, Smith AJ, Cooper PR. Hepatocyte growth factor is sequestered in dentine matrix and promotes regeneration-associated events in dental pulp cells. Cytokine 2013;61:622–9. 4. Anneroth G, Bang G. The effect of allogeneic demineralized dentin as a pulp capping agent in Java monkeys. Odontol Revy 1972;23:315–28. 5. Tronstad L. Tissue reactions following apical plugging of the root canal with dentin chips in monkey teeth subjected to pulpectomy. Oral Surg Oral Med Oral Pathol 1978;45:297–304. 6. Begue-Kirn C, Smith AJ, Ruch JV, et al. Effects of dentin proteins, transforming growth factor beta 1 (TGF beta 1) and bone morphogenetic protein 2 (BMP2) on the differentiation of odontoblast in vitro. Int J Dev Biol 1992;36:491–503. 7. Smith AJ, Scheven BA, Ferracane J, Cooper PR. Dentine as a bioactive extracellular matrix. Arch Oral Biol 2012;57:109–21. 8. Galler KM, Buchalla W, Hiller KA, et al. Influence of root canal disinfectants on growth factor release from dentin. J Endod 2015;41:363–8. 9. Graham L, Cooper PR, Cassidy N, Nor JE, Sloan AJ, Smith AJ. The effect of calcium hydroxide on solubilisation of bio-active dentine matrix components. Biomaterials 2006;27:2865–73. 10. Tomson PL, Grover LM, Lumley PJ, Sloan AJ, Smith AJ, Cooper PR. Dissolution of bio-active dentine matrix components by mineral trioxide aggregate. J Dent 2007; 35:636–42. 11. Olgart L, Edwall L, Fried K. Cat dental pulp after denervation and subsequent re-innervation: changes in blood-flow regulation and distribution of neuropeptide-, GAP-43- and low-affinity neurotrophin receptor-like immunoreactivity. Brain Res 1993;625:109–19.
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Regenerative Endodontics 12. Lewin GR, Barde YA. Physiology of the neurotrophins. Annu Rev Neurosci 2003;19: 289–317. 13. Nosrat CA, Fried K, Ebendal T, Olson L. NGF, BDNF, NT3, NT4 and GDNF in tooth development. Eur J Oral Sci 2014;106:94–9. 14. Cassidy N, Fahey M, Prime S, Smith AJ. Comparative analysis of transforming growth factor-b isoforms 1–3 in human and rabbit dentine matrices. Arch Oral Biol 1997; 42:219–23. 15. Roberts-Clark DJ, Smith AJ. Angiogenic growth factors in human dentine matrix. Arch Oral Biol 2000;45:1013–6. 16. Patwardhan AM, Berg KA, Akopain AN, et al. Bradykinin-induced functional competence and trafficking of the d-opioid receptor in trigeminal nociceptors. J Neurosci 2005;25:8825–32. 17. Fehrenbacher JC, Sun XX, Locke EE, Henry MA, Hargreaves KM. Capsaicin-evoked iCGRP release from human dental pulp: a model system for the study of peripheral neuropeptide secretion in normal healthy tissue. Pain 2009;144:253–61. 18. Mitsiadis TA, Luukko K. Neurotrophins in odontogenesis. Int J Dev Biol 1995;39: 195–202. 19. Nosrat I, Seiger A, Olson L, Nosrat CA. Expression patterns of neurotrophic factor mRNAs in developing human teeth. Cell Tissue Res 2002;310:177–87. 20. Fristad I, Heyeraas KJ, Kvinnsland I. Nerve fibres and cells immunoreactive to neurochemical markers in developing rat molars and supporting tissues. Arch Oral Biol 1994;39:633–46. 21. Fried K, Gibbs JL. Dental pulp innervation. In: Goldberg M, ed. The Dental Pulp. Berlin, Germany: Springer-Verlag; 2014:75–95. 22. Amano O, Bringas P, Takahashi I, et al. Nerve growth factor (NGF) supports tooth morphogenesis in mouse first branchial arch explants. Dev Dyn 1999;216: 299–310. 23. Xiao-Bing Q, Naftel JP. Effects of neonatal exposure to anti-nerve growth factor on the number and size distribution of trigeminal neurones projecting to the molar dental pulp in rats. Arch Oral Biol 1996;41:359–67. 24. 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. 25. Woodnutt DA, Wager-Miller J, O’Neill PC, Bothwell M, Byers MR. Neurotrophin receptors and nerve growth factor are differentially expressed in adjacent nonneuronal cells of normal and injured tooth pulp. Cell Tissue Res 2000;299:225–36. 26. Byers MR, Wheeler EF, Bothwell M. Altered expression of NGF and P75 NGF-receptor by fibroblasts of injured teeth precedes sensory nerve sprouting. Growth Factors 1992;6:41–52.
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27. Kvinnsland IH, Luukko K, Fristad I, et al. Glial cell line-derived neurotrophic factor (GDNF) from adult rat tooth serves a distinct population of large-sized trigeminal neurons. Eur J Neurosci 2004;19:2089–98. 28. Gale Z, Cooper PR, Scheven BA. Effects of glial cell line-derived neurotrophic factor on dental pulp cells. J Dent Res 2011;90:1240–5. 29. Wheeler EF, Naftel JP, Pan M, von Bartheld CS, Byers MR. Neurotrophin receptor expression is induced in a subpopulation of trigeminal neurons that label by retrograde transport of NGF or fluoro-gold following tooth injury. Brain Res Mol Brain Res 1998;61:23–38. 30. Luukko K, Moshnyakov M, Sainio K, Saarma M, Sariola H, Thesleff I. Expression of neurotrophin receptors during rat tooth development is developmentally regulated, independent of innervation, and suggests functions in the regulation of morphogenesis and innervation. Dev Dyn 1996;206:87–99. 31. Diogenes A, Henry MA, Teixeira FB, Hargreaves KM. An update on clinical regenerative endodontics. Endod Topics 2013;28:2–23. 32. Lei L, Chen Y, Zhou R, Huang X, Cai Z. Histologic and immunohistochemical findings of a human immature permanent tooth with apical periodontitis after regenerative endodontic treatment. J Endod 2015;41:1172–9. 33. Meschi N, Hilkens P, Lambrichts I, et al. Regenerative endodontic procedure of an infected immature permanent human tooth: an immunohistological study. Clin Oral Investig 2016;20:807–14. 34. 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. 35. Martin David E, de Almeida Jose Flavio A, Henry Michael A, et al. Concentrationdependent effect of sodium hypochlorite on stem cells of apical papilla survival and differentiation. J Endod 2014;40:51–5. 36. Mitchel PJ, Smith RC. A quantitative method for analysis of in vitro neurite outgrowth. J Neurosci Methods 2007;164:350–62. 37. Kemp SW, Webb AA, Dhaliwal S, Syed S, Walsh SK, Midha R. Dose and duration of nerve growth factor (NGF) administration determine the extent of behavioral recovery following peripheral nerve injury in the rat. Exp Neurol 2011;229: 460–70. 38. Kohn J, Aloyz RS, Toma JG, Haak-Frendscho M, Miller FD. Functionally antagonistic interactions between the TrkA and p75 neurotrophin receptors regulate sympathetic neuron growth and target innervation. J Neurosci 1999; 19:5393–408. 39. Luukko K, Kvinnsland IH, Kettunen P. Tissue interactions in the regulation of axon pathfinding during tooth morphogenesis. Dev Dyn 2005;234:482–8.
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Expression of Neurotrophic Factors in Human Dentin and Their Regulation of Trigeminal Neurite Outgrowth Obadah Austah, BDS, MS,*† Matthias Widbiller, DDS,*‡ Phillip L. Tomson, BDS, PhD,§ and Anibal Diogenes, DDS, PhD* Abstract Introduction: Neurotrophic factors play a significant role in the innervation of the pulp-dentin complex during and after organogenesis. There have been numerous bioactive molecules identified in the dentin extracellular matrix; however, the expression of neurotrophic factors in the dentin matrix and their biological activity are largely unknown. The purpose of this study was to characterize the relative expression of neurotrophic factors in human dentin matrix proteins (DMPs) and their effect on neurite outgrowth of trigeminal (TG) neurons. Methods: Dentin was powdered in liquid nitrogen from noncarious human third molar teeth. DMPs were solubilized through an EDTA extraction method, dialyzed, and lyophilized until use. The relative expression of nerve growth factor, brain-derived neurotrophic factor, glial cell line derived neurotrophic factor, neurotrophin 3, and neurotrophin 4/5 was determined by the enzyme-linked immunosorbent assay. Rat TG neurons were cultured and exposed to different concentrations of DMPs (1–105 ng/mL) or vehicle, and a quantitative neurite outgrowth assay was performed. Results: Human DMPs contained all of the tested neurotrophic factors, with glial cell line derived neurotrophic factor and neurotrophin 4/5 found at the highest levels. DMPs were able to promote the neurite outgrowth of rat TG neurons at an optimum concentration of 10–102 ng/mL, whereas the effect was partially inhibited at higher concentrations (>103 ng/mL). Conclusions: The human dentin extracellular matrix is a rich reservoir for neurotrophic factors that are key components for neuronal homeostasis, differentiation, and regeneration. These data suggest that neurotrophins in DMPs could play an important role as signaling molecules for the innervation of the pulp-dentin complex during the processes of tooth formation, repair, and regeneration. (J Endod 2019;-:1–6)
Key Words Dentin matrix protein, dentinogenesis, neurite outgrowth, neurotrophic factors, neurotrophins, regeneration
D
entin has been tradiSignificance tionally thought to be Key neurotrophic factors (NGF, BDNF, GDNF, an inert mineralized tissue NT3, and NT4/5) found fossilized in the dentin that provides mechanical matrix are released with EDTA and promote support and protection. neurogenesis. Thus, they can be exploited in pulpal Recent advances in the repair and regenerative therapy. basic biological understanding of the pulpdentin complex unraveled a substantial array of bioactive molecules embedded in the mineralized matrix during dentinogenesis that can be released upon various stimuli (1–3). The presence of these molecules highlights their important roles in the repair and regeneration of the pulp-dentin complex throughout the life span of teeth. Dentin bioactivity was shown a few decades ago by the application of autologous demineralized dentin chips as a pulp cap (4) or apical plug (5) to promote pulpal repair and apical closure and healing in primate animals. Later, the promineralization and reparative dentinogenesis potentials were shown to be caused by the rich pool of noncollagenous proteins that were released upon demineralization of the tissue (6). Demineralization of dentin can occur by bacterial acids during caries attack activities or by using various dentin conditioning agents such as EDTA (7, 8) and materials used to promote pulp healing such as calcium hydroxide (9) and mineral trioxide aggregate (10). However, the understanding of their relative solubility and the control of their release in vivo is still limited. Dental pulp is 1 of the most densely innervated tissues in the body, and the innervating fibers have the capability to regenerate and restore tissue hemostasis after various injuries (11). Neurotrophic factors are a broad family of growth factors that are important in regulating development, differentiation, function, and survival of neuronal cells. Previous studies showed that certain neurotrophic factors including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), neurotrophin 4 (NT4), and glial cell–line derived neurotrophic factor (GDNF) are present in the dental structures (12, 13). These growth factors act in an orchestrated fashion to promote the complex innervation of the pulp-dentin complex.
From the *Department of Endodontics, University of Texas Health Science Center at San Antonio, San Antonio, Texas; †Department of Endodontics, Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia; ‡Department of Conservative Dentistry and Periodontology, University Hospital, Regensburg, Germany; and §Department of Oral Biology, Institute of Clinical Sciences, The University of Birmingham School of Dentistry, Birmingham, UK. Address requests for reprints to Dr Anibal Diogenes, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2018 American Association of Endodontists. https://doi.org/10.1016/j.joen.2018.12.011
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Regenerative Endodontics It is well established that key growth factors are present and released from dentin and mediate important biological activities such as repair and regeneration. The transforming growth factor beta superfamily is 1 of the first identified factors and is known to stimulate mineralization and regenerative activity in the pulp-dentin complex (14). For any repair or regenerative process, angiogenesis and neurogenesis are integral parts of ideal outcomes to maintain key cellular and nutritional supply and provide neuroprotection. In fact, proangiogenic factors such as platelet-derived growth factor and vascular endothelial growth factor have been already identified in dentin (14, 15). However, there is limited knowledge regarding the presence and biological function of proneurogenic or neurotrophic factors in human dentin. Therefore, the purpose of this study was to characterize the relative expression and concentration of neurotrophic factors in human powdered dentin matrix proteins (DMPs) and evaluate their bioactivity on neurite outgrowth of trigeminal (TG) neurons.
enamel portion of the teeth were carefully removed. The remaining dentin structures were frozen in liquid nitrogen and ground using a freezer/mill (Spex 6700 Freezer/Mill; Glen Creston Ltd, London, UK) into a fine powder and passed through a 60-mm mesh sieve. Next, DMPs were solubilized using 10% EDTA in the presence of protease inhibitors, 10 mmol/L n-ethylmaleimide (Sigma-Aldrich, St Louis, MO), and 5 mmol/L phenylmethylsulfonyl fluoride (Sigma-Aldrich) with continuous agitation at 4 C for 14 days. Extracted supernatants were dialyzed for 10 days and lyophilized using a freezer dryer (Modulyo; Edwards, Crawley, UK) as previously described (10).
Materials and Methods
Quantification of Neurotrophic Factors In brief, the lyophilized DMPs were dissolved in phosphatebuffered saline at a concentration of 10 mg/mL. To optimize the detection range of the enzyme-linked immunosorbent assay (ELISA), undiluted and serial dilutions of the samples were used to evaluate the concentrations of NGF, BDNF, GDNF, NT3, and NT4/5 using a multineurotrophic rapid screening ELISA kit (Biosensis, South Australia, Australia) following the manufacturer’s instructions.
Isolation of Human Dentin Extracellular Matrix Proteins Extracted noncarious third molars were obtained from the Oral Surgery Department at Birmingham Dental Hospital, Birmingham, UK, after informed patient consent and ethical approval (14/EM/1128). The cementum and attached soft tissues were removed from the root surface of the teeth and sectioned longitudinally. Pulp tissue and the
Rat TG Ganglia Neuronal Primary Culture Adult male Sprague-Dawley rats (weight, 200–250 g each [Charles River, Wilmington, MA]) were used in this study. All animal study protocols were approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at
Figure 1. Mean concentrations (pg/mg) of neurotrophic factors in human dentin powdered extracellular matrix proteins (DMPs) as determined by ELISA. *P < .05, **P < .01; 1-way ANOVA with the Bonferroni post hoc test, P < .05. Data are presented as the mean standard error of the mean.
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Figure 2. The mean percentage of neurite outgrowth when exposed to different concentrations of DMPs (1–105 ng/mL) for 72 hours. *P < .05, **P < .01; 1-way ANOVA with the Dunnett post hoc test, P < .05. Data are presented as the mean standard error of the mean.
San Antonio, San Antonio, TX, and conformed to the International Association for the Study of Pain and federal guidelines. Animals were housed for 1 week before the experiments with food and water available ad lib. Rats were euthanized by decapitation, and the TG ganglia were dissected and isolated through an enzymatic process as previously described (16). Single-cell neuronal suspensions at the concentrations of 5 105 neurons/mL were cultured in 12-well poly-D-lysine–coated culture plates (Corning Life Sciences, Corning, NY) in growth medium containing Dulbecco modified Eagle medium (Life Technologies, Carlsbad, CA), 10% heat-inactivated fetal bovine serum (Life Technologies), 3 mg/mL 5-fluoro-2-deoxyuridine, 7 mg/mL uridine (Sigma-Aldrich), and 1 glutamine-penicillinstreptomycin (Invitrogen, Carlsbad, CA). Neurons were allowed to attach for 24 hours in 5% CO2 at 37 C before neurite outgrowth experiments.
Neurite Outgrowth Experiments To assess the bioactivity of the sequestered neurotrophic factors in dentin, 2 experiments were performed to evaluate the effect of DMPs on the outgrowth of TG neurons. All experiments were performed in triplicate, and for each experiment, 3 rats (6 TG ganglia) were used. The lyophilized DMPs were resuspended in Dulbecco modified Eagle medium and added to the culture media in serial dilution of concentrations ranging from 1 ng/mL–105 ng/mL. For the first method, the neurite outgrowth assay kit (NS220; Millipore, Burlington, MA) was used. In brief, TG neurons were seeded onto culture inserts with 3-mm pores at the base in a 12-well plate at a density of 3 104 cells/well. TG neurons were cultured in either basal growth media alone (the control group) or in growth media in the presence of different concentrations of DMPs (1, 10, 102, 103, 104, or 105 ng/mL) (the experimental groups). Neurons were cultured JOE — Volume -, Number -, - 2019
with fresh media replaced daily for 3 days. Absorbance was measured at 490 nm using a FlexStation 3 Benchtop Multimode Microplate Reader (Molecular Devices, Sunnyvale, CA) following the manufacturer’s instructions. In the second method, we evaluated the effect of the optimum concentration of DMPs determined in the concentration response experiment on neurite outgrowth using immunocytochemistry and confocal microscopy. In brief, TG neurons were seeded onto poly-D-lysine–coated coverslips at a density of 3 104 cells/well in basal growth medium containing either the vehicle (the control group) or 102 ng/mL DMPs (the experimental group). Neurons were cultured for 3 days followed by fixation in 4% paraformaldehyde and immunostaining as described previously (17). Immunostaining was performed with primary antibody against beta III tubulin (cat# MAB 1195 at a 1:200 dilution in blocking solution; R&D Systems, Minneapolis, MN) followed by species-appropriate Alexa Fluor 488 secondary antibody at 1:200 dilution in blocking solution (Molecular Probes, Eugene, OR). Immunoreactivity was visualized with a Nikon Eclipse 90i microscope with a Nikon C1si confocal laser scan head (Nikon Instruments, Melville, NY), and images were collected using standardized settings at 10 and 20 magnifications. Four standardized selected images of each slide were acquired at 20 magnification and visualized in ImageJ software (Version 2.0.0-rc-67/1.52c; National Institutes of Health, Bethesda, MD) to determine the percentage of area occupied by neurites. Data were normalized to the percentage of the control group.
Statistical Analysis For the ELISA and the neurite outgrowth experiments, results were analyzed using 1-way analysis of variance (ANOVA) with the Bonferroni post hoc test and the Dunnett post hoc test, respectively. For the Neurotrophic Factors in Human Dentin
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Figure 3. (A) Neurite outgrowth of TG neurons cultured in either the control (basal medium) or 102 ng/mL DMPs for 72 hours. Representative confocal microphotographs of beta III tubulin immunostaining (green) of primary TG neuronal incubated in (B) the control (basal medium) and (C) 102 ng/mL DMPs. *P < .05; unpaired 2-tailed Student t test, P < .05. Images were acquired by Nikon C1si laser scanning confocal microscopy with 20 magnification.
immunocytochemistry experiment, the difference in the percentage of area occupied by neurites between the control and DMP group was analyzed with the unpaired Student t test. All statistical analyses were performed using Prism Version 7.0a software (GraphPad, La Jolla, CA) with statistical significance set at P < .05. All data were expressed as mean standard error of the mean.
Results Relative Expression of Neurotrophic Factors in Human Dentin ELISA-based measurements of neurotrophic factors in dentin revealed variable detectable levels (pg/mg) of all screened neurotrophic factors. Interestingly, GDNF (10.71 3.91 pg/mg) and NT4/5 (11.43 0.52 pg/mg) were expressed in significantly higher levels than NT3 (8.12 0.54 pg/mg), BDNF (5.033 0.19 pg/mg), and NGF (4.84 0.064 pg/mg) (P < .05) (Fig. 1). In addition, NGF and BDNF were the least detected amounts found in dentin (Fig. 1). DMPs Mediate Neurite Outgrowth of TG Neurons Neurite outgrowth assay with different concentrations of DMPs resulted in a bell-shaped dose-response curve with optimum concentration at 10–102 ng/mL (Fig. 2). Lower concentrations of DMPs (1, 101, and 102 ng/mL) resulted in significant promotion of neurite extension (1.5-fold increase) compared with the control group (P < .01) (Fig. 2). Interestingly, this increase was reversed when higher concentrations were used (103, 104, and 105 ng/mL). Immunocytochemical analysis of cultured TG neurons in basal medium (control) or supplemented with 102 ng/mL DMPs for 3 days showed a significantly larger area occupied by neurites in the DMP 4
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group than the control (P < .05) (Fig. 3A). In addition, the substantial greater number of branching and length of neurites can be appreciated (Fig. 3B and C).
Discussion Dentin is well-known to be a rich resource of bioactive molecules such as growth factors, cytokines, and chemokines that are embedded in the mineralized ECM during tooth development and released upon demineralizing events (7). Our results showed that dentin represents a rich source of several neurotrophic factors (NGF, BDNF, GDNF, NT3, and NT4/5) with known profound effects in neuronal chemotaxis and function. Indeed, these neurotrophic factors promoted the robust increase in neurite outgrowth in vitro. Neurotrophic growth factors play key roles in innervation development by mediating organization, plasticity, axonal targeting, repair, survival, and regeneration of the free nerve terminals (12). Previous studies of odontogenesis examined the temporal and spatial expression of the neurotrophic factors and showed the participation of NGF, BDNF, and GDNF during teeth innervation and GDNF and NT3/4 in the epithelial-mesenchymal cross talk during early morphogenesis (13, 18, 19). Interestingly, our results showed a higher relative expression of GDNF, NT3, and NT4/5 than NGF and BDNF in dentin. This variation can be conceivably explained by the early expression of GDNF and neurotrophins during tooth formation and their sequestration in the dentin matrix before its mineralization. On the contrary, lower concentrations of NGF and BDNF can be explained by their late involvement during tooth innervation, which begins when root formation commences (20). Nevertheless, they were still present beforehand but in much lower levels and that reflects JOE — Volume -, Number -, - 2019
Regenerative Endodontics their minimal rate of sequestration. Thus, neurotrophins are sequestered in dentin during dental organogenesis and may play a role in the defensive and reparative capabilities of the pulp-dentin complex during life. The innervation of the pulp dentin complex is dynamic, and the arborization of the free nerve endings in response to insults such as caries has been elegantly shown (21). The overall function of this increased innervation in the repair and regeneration of the pulp-dentin complex is not fully understood. However, this increase of innervation in areas of injury suggest an active role of these nerve fibers that exceed their most accepted putative function as nociceptors. It has been hypothesized that this increase of innervation is mainly caused by the effect of NGF. It is well established that NGF is very critical for the differentiation of neural crest cells to dental organ and for tooth innervation (22). Tooth innervation was mostly absent in tyrosine kinase receptor (trkA) knockout and anti–NGF-treated animals (23, 24). Moreover, different models of tooth injury led to up-regulation of NGF adjacent to the injury site (25, 26). The source of neurotrophins such as NGF has been attributed solely to cells of the pulp-dentin complex such as odontoblasts and pulpal fibroblasts, emphasizing the importance of neurotrophic factors in regulating their functions (25). However, our study suggests that the release of both bioactive molecules could also be from the demineralized dentin. Similarly, BDNF, NT3, and NT4/5 promote neurite outgrowth, development, and survival of sensory neurons, and their effect is mediated by receptor binding followed by internalization and retrograde transportation to the primary neurons in TG (18). GDNF is another important factor, and it is mainly expressed postnatally in the subodontoblastic layer (27). It has also been implicated in the regulation of postnatal tooth innervation and promoting survival and proliferation of dental pulp cells (28). In this study, the release of neurotrophins from dentin can affect all major subtypes of nociceptors in the dental pulp (18, 29, 30), suggesting a strong role of the neuronal response to injury in the dental pulp. One of the limitations of our study is the variabilities of the collected samples in regard to donor age, sex, and stage of root development. This might affect the presented levels of neurotrophic factors and their effect on TG neurons. Further research is warranted to determine whether there are differences in these factors’ relative abundance and bioactivity in teeth from patients of different ages and sex. This is particularly important in the context of regenerative endodontic procedures (REPs). It has been shown that approximately 60% of all the reported cases of REPs report regaining tooth sensibility to vitality testing (31). Previous studies have histologically confirmed the presence of regenerated nerve fibers in teeth treated with REPs, thus providing the biological basis for such a clinical finding (31–33). The mechanism of axonal growth and targeting into the newly formed tissues is not fully understood. A study showed that stem cells of the apical papilla, when in the presence of TG neurons, release NGF, GDNF, and BDNF, with the latter being responsible for mediating axonal growth and targeting (34). Importantly, the beneficial effect of EDTA conditioning in regenerative endodontics has been well-documented (8, 9, 35). In this study, we showed that EDTA promoted the release of neurotrophic factors from dentin. Thus, DMP-derived neurotrophic factors are likely adjuvants, in addition to stem cells of apical papilla, in mediating axonal growth and targeting into the newly formed tissues after REPs. An in vitro neurite outgrowth in cultured neurons is considered as a study model for neuronal regeneration potential (36). In order to evaluate whether the neurotrophic factors released from dentin are bioactive, we used 2 well-characterized assays to quantify the neurite outgrowth of primary TG neurons. Our results showed a concentration-dependent response with higher stimulations of neurite outgrowth in lower concentrations (10–102 ng/mL). On the contrary, JOE — Volume -, Number -, - 2019
103 ng/mL DMPs or higher led to an inhibitory effect on TG neurons. There are few possibilities to explain the observed response. One possible reason is that a very high concentration of neurotrophic factors will activate low-affinity receptors with known divergent functions. For instance, the high-dose inhibitory effect of NGF is shown to be mediated by the low-affinity P75 receptor (37). Lower concentrations of NGF result in signaling through its high-affinity receptor (trkA). However, greater concentrations will lead to the occupancy of its low-affinity receptor (P75). Interestingly, the activation of the low-affinity receptor has an opposite effect to that seen with trkA regarding the promotion of neuronal survival (38). Another possible reason is the presence of inhibitory molecules not identified in this study at higher concentrations could inhibit neurite outgrowth. For instance, a higher dose of DMPs might contain higher levels of semaphoring 3a (a neuronal inhibitory factor that participates in dental innervation), which in turn led to the observed inhibitory effects (39). In conclusion, our study showed for the first time the expression of all dental tissue-associated neurotrophic factors in DMPs. Also, these factors retain their bioavailability, and the application of optimum concentration promoted the neurite outgrowth of primary cultured TG neurons. Our data highlight the potential role of these DMP-derived factors in neuronal responses to injury and their exploitation to enhance the current therapeutic approaches in both the repair and regeneration of the pulp-dentin complex. Future experiments evaluating the effect of DMPs on axonal targeting of TG neurons and elucidating the soluble factors regulating those activities are warranted. Additionally, experiments assessing the effect of known dentin solubilizers on the release of the neurotrophic factors are also needed.
Acknowledgments This research was supported in part by a research grant from the Foundation for Endodontics. The authors deny any conflicts of interest related to this study.
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