Inge Fristad

Nerve fibres and cells immunoreactive to neurochemical markers in developing rat molars and supporting tissues

The distribution of nerve fibres immunoreactive to calcitonin gene-related peptide (CGRP), substance P (SP) and neuropeptide Y (NPY) was compared to the general neurochemical markers for nerves and neuroendocrine cells protein gene product 9.5 (PGP 9.5) and neurone-specific enolase (NSE), by use of the avidin-biotin peroxidase complex method in developing dental structures in rats aged 13 to 27 days. A substantially greater part of the nerve fibres was immunoreactive to CGRP and SP than to NPY. In the bell stage, nerve fibres immunoreactive to PGP 9.5, CGRP and SP were found in the dental follicle but not in the dental papilla and stellate reticulum. In the advanced bell stage, after initiation of dentine and enamel formation, PGP 9.5, CGRP- and SP-immunoreactive fibres were found in the dental papilla, while the first NPY-immunoreactive fibres were observed in the papilla when root formation started. Concomitant with the beginning of root development, a subodontoblastic nerve plexus was gradually formed and PGP 9.5-, CGRP- and SP-immunoreactive fibres were found within the dentinal tubules. From the start of root formation, CGRP-, SP- and NPY-immunoreactive nerves were shown in the developing periodontal ligament, although a mature distribution pattern was not observed until root formation was nearly completed. Ameloblasts, odontoblasts and cell-like structures in the outer enamel epithelium and within the dental lamina were PGP 9.5-immunoreactive at the bell stage. As the tooth matured, the immunolabelling gradually decreased, but was still present in some odontoblasts after tooth eruption. NSE-immunoreactive, cell-like structures were found in the periphery of the dental follicle, and persisted close to alveolar bone in the periodontal ligament when the tooth reached occlusion. Hence, it may be concluded that sensory nerves containing SP and CGRP are present in the pulp in advance of sympathetic nerves immunoreactive to NPY.

Cytokine signalling in rat pulp interstitial fluid and transcapillary fluid exchange during lipopolysaccharide-induced acute inflammation

The dental pulp consists of loose connective tissue encased in rigid dentinal walls. Because of its topography the tissue has low interstitial compliance and limited capacity to expand during fluid volume changes. Due to limitations regarding access to interstitial fluid, basic knowledge on transcapillary fluid transport parameters is lacking for this organ. The scope of this project was dual: first we aimed at establishing a method for isolation of pulp interstitial fluid (IF), and second we applied the method in rats subjected to lipopolysaccharide (LPS)‐induced endotoxaemia. The aim was to measure colloid osmotic pressure (COP) and pro‐inflammatory cytokines in the pulp IF during acute inflammation. Fluid volumes and pulpal blood flow (PBF) were measured to obtain more information about microcirculatory changes that take place in this pulpitis model. By centrifugation of incisor pulp at 239 g we were able to extract fluid representative for IF. Pulp IF had a relative high control COP (∼83% of plasma COP) and was similar to plasma COP 3 h after LPS challenge. The pulp exhibited a high content of IF (0.60 ± 0.03 ml (g wet weight)−1) and a vascular volume of 0.03 ± 0.01 ml (g w.w.)−1 No differences were observed in the distribution of fluid volumes after 1.5 and 3 h LPS exposure. PBF and systemic blood pressure dropped significantly after LPS administration. PBF remained low whereas systemic blood pressure was re‐established during the 3‐h period, implying organ dysfunction. There was a differential pattern of cytokine expression in pulp IF and serum with cytokines such as IL‐1α, IL‐1β and TNF‐α locally produced, whereas others such as IFN‐γ and IL‐6 were produced systemically and probably spilled over to the pulp IF after LPS exposure. Our findings show that pulp IF can be isolated by centrifugation and that this method is useful when studying fluid balance and extracellular signalling mechanisms in the dental pulp in normal and pathological conditions.

Glial cell line‐derived neurotrophic factor (GDNF) from adult rat tooth serves a distinct population of large‐sized trigeminal neurons

Glial cell line‐derived neurotrophic factor (GDNF) mediates trophic effects for specific classes of sensory neurons. The adult tooth pulp is a well‐defined target of sensory trigeminal innervation. Here we investigated potential roles of GDNF in the regulation of adult trigeminal neurons and the dental pulp nerve supply of the rat maxillary first molar. Western blot analysis and radioactive 35S‐UTP in situ hybridization revealed that GDNF in the dental pulp and its mRNAs were localized with Ngf in the coronal pulp periphery, in particular in the highly innervated subodontoblast layer. Retrograde neuronal transport of iodinated GDNF and Fluorogold (FG) from the dental pulp indicated that GDNF was transported in about one third of all the trigeminal dental neurons. Of the GDNF‐labelled neurons, nearly all (97%) were large‐sized (≥35 µm in diameter). Analysis of FG‐labelled neurons revealed that, of the trigeminal neurons supporting the adult dental pulp, ≈ 20% were small‐sized, lacked isolectin B4 binding and did not transport GDNF. Of the large‐sized dental trigeminal neurons ≈ 40% transported GDNF. About 90% of the GDNF‐accumulating neurons were negative for the high‐temperature nociceptive marker VRL‐1. Our results show that a subclass of large adult trigeminal neurons are potentially dependent on dental pulp‐derived GDNF while small dental trigeminal neurons seems not to require GDNF. This suggests that GDNF may function as a neurotrophic factor for subsets of nerves in the tooth, which apparently mediate mechanosensitive stimuli. As in dorsal root ganglia both small‐ and large‐sized neurons are known to be GDNF‐dependent; these data provide molecular evidence that the sensory supply in the adult tooth differs, in some aspects, from the cutaneous sensory system.

Effect of Intermittent Long-Lasting Electrical Tooth Stimulation on Pulpal Blood Flow and Immunocompetent Cells: A Hemodynamic and Immunohistochemical Study in Young Rat Molars

Release of sensory neuropeptides after stimulation of afferent nerve fibers has previously been shown to induce vasodilation and increased vascular permeability in the dental pulp, a condition recognized as neurogenic inflammation. In the present study a possible role for the sensory neuropeptides in transendothelial migration of immunocompetent cells was investigated. The dental pulp is an isolated tissue densely innervated with sensory fibers containing neuropeptides, and following electrical stimulation of the crown, the effect on pulpal blood flow and immunocompetent cells can be studied in a noninvasive model. A laser Doppler flowmeter was used to measure relative changes in pulpal blood flow during long-lasting intermittent stimulation of innervated and denervated rat first molars. In the innervated teeth, stimulation promptly increased pulpal blood flow by on average 45% at the start of the experiment, whereas almost no blood flow increase was recorded after 4 to 5 h stimulation. Surgical sectioning of the inferior alveolar nerve abolished blood flow increase upon stimulation. After stimulation, a quantitative analysis of CD43+, CD4+, CD11+, and I-A antigen-expressing cells was performed, and the effect of stimulation on calcitonin gene-related peptide (CGRP)-immunoreactive and substance P (SP)-immunoreactive (IR) nerve fibers was studied. Immunohistochemistry was performed by the avidin-biotin peroxidase method. Stimulation resulted in an almost complete depletion of CGRP- and SP-IR nerve fibers in the first molar pulp, whereas nerve fibers in the gingiva and neighboring teeth were unaffected. A significant increase in the number of CD43+ cells was found in the innervated tooth after stimulation compared to the stimulated denervated (P < 0.01) and unstimulated control (P < 0.05) first molars. For I-A antigen-expressing cells a significant increase (P < 0.05) was found between the innervated stimulated and unstimulated control, but not between the innervated and denervated stimulated first molars. Hence, from the present experiment it is concluded that the pulpal nerves participate in and facilitate transendothelial migration of CD43+ cells during acute neurogenic inflammation.

Endothelial microvascular networks affect gene-expression profiles and osteogenic potential of tissue-engineered constructs.

IntroductionA major determinant of the potential size of cell/scaffold constructs in tissue engineering is vascularization. The aims of this study were twofold: first to determine the in vitro angiogenic and osteogenic gene-expression profiles of endothelial cells (ECs) and mesenchymal stem cells (MSCs) cocultured in a dynamic 3D environment; and second, to assess differentiation and the potential for osteogenesis after in vivo implantation.MethodsMSCs and ECs were grown in dynamic culture in poly(L-lactide-co-1,5-dioxepan-2-one) (poly(LLA-co-DXO)) copolymer scaffolds for 1 week, to generate three-dimensional endothelial microvascular networks. The constructs were then implanted in vivo, in a murine model for ectopic bone formation. Expression of selected genes for angiogenesis and osteogenesis was studied after a 1-week culture in vitro. Human cell proliferation was assessed as expression of ki67, whereas α-smooth muscle actin was used to determine the perivascular differentiation of MSCs. Osteogenesis was evaluated in vivo through detection of selected markers, by using real-time RT-PCR, alkaline phosphatase (ALP), Alizarin Red, hematoxylin/eosin (HE), and Masson trichrome staining.ResultsThe results show that endothelial microvascular networks could be generated in a poly(LLA-co-DXO) scaffold in vitro and sustained after in vivo implantation. The addition of ECs to MSCs influenced both angiogenic and osteogenic gene-expression profiles. Furthermore, human ki67 was upregulated before and after implantation. MSCs could support functional blood vessels as perivascular cells independent of implanted ECs. In addition, the expression of ALP was upregulated in the presence of endothelial microvascular networks.ConclusionsThis study demonstrates that copolymer poly(LLA-co-DXO) scaffolds can be prevascularized with ECs and MSCs. Although a local osteoinductive environment is required to achieve ectopic bone formation, seeding of MSCs with or without ECs increases the osteogenic potential of tissue-engineered constructs.

NK1, NK2, NK3 and CGRP1 receptors identified in rat oral soft tissues, and in bone and dental hard tissue cells.

The distribution of the tachykinin receptors neurokinin-1 (NK1), neurokinin-2 (NK2) and neurokinin-3 (NK3), and the calcitonin gene-related peptide-1 (CGRP1) receptor were examined in rat teeth and tooth-supporting tissues by immunohistochemical methods and light and confocal microscopy. Western blot analysis was performed to identify the NK1- and the CGRP1-receptor proteins in the dental pulp. The results showed that odontoblasts and ameloblasts, cementoblasts and cementocytes, osteoblasts and osteocytes are all supported with the tachykinin receptors NK1 and NK2, but a distinct, graded cellular labeling pattern was demonstrated. The ameloblasts were also positive for CGRP1 receptor. Blood vessels in oral tissues expressed the tachykinin receptors NK1, NK2 and NK3, and the CGRP1 receptor. Both gingival and Malassez epithelium were abundantly supplied by NK2 receptor. Pulpal and periodontal fibroblasts demonstrated NK1 and NK2 receptors. Western blot analysis identified both the NK1- and the CGRP1-receptor proteins in the dental pulp. These results clearly indicate that the neuropeptides substance P, neurokinin A, neurokinin B and CGRP, released from sensory axons upon stimulation, directly modulate the function of the different types of bone and dental hard tissue cells, and regulate functions of blood vessels, fibroblasts and epithelial cells in oral tissues.

Neuropeptide Y expression in the trigeminal ganglion and mandibular division of the trigeminal nerve after inferior alveolar nerve axotomy in young rats

Neuropeptide Y (NPY) is a 36-amino-acid peptide residing in sympathetic nerve terminals, originating from the superior cervical ganglion in oral tissues. NPY exerts vasoconstrictor action together with noradrenalin and has been found to inhibit the release of neurotransmitters from primary afferent fibers. During regeneration of the axotomized inferior alveolar nerve (IAN), NPY-immunoreactive (IR) nerve fibers have been shown in the odontoblast layer and dentin, an area normally innervated by afferent nerve fibers. The dynamic shift in neuropeptide expression in the trigeminal ganglion and in the dental pulp was studied by immunohistochemistry 1, 2, 3, and 8 weeks after IAN axotomy. In the ipsilateral first mandibular molar a temporal loss of pulpal sensory nerves lasting for approximately 1 week was found after axotomy. An upregulation of NPY was shown in neurons located in the mandibular area of the trigeminal ganglion, concomitant to a reduction in number of neurons expressing substance P (SP). To study an alternate and possible trigeminal origin of some of the peripheral nerve fibers IR to NPY in the dental pulp, double immunofluorescence labeling was performed for NPY and calcitonin gene-related peptide (CGRP). Coexistence of NPY and CGRP was shown in neurons located in the trigeminal ganglion and in nerve fibers in the tooth pulp during IAN regeneration. Furthermore, retrograde tracing with Fluorogold revealed NPY-IR neurons projecting to the first molar pulp 3 weeks after axotomy. Hence, we conclude that after IAN axotomy NPY is produced in trigeminal ganglion neurons and transported in afferent regenerating fibers to the dental pulp. These results add further evidence for a plasticity in peptide transcription in sensory neurons after nerve injury and indicate a trigeminal origin of at least some of the pulpal NPY-IR fibers during nerve regeneration.

Dental Innervation: Functions and Plasticity After Peripheral Injury

Oral tissues including the periodontal ligament, gingiva, and tooth pulp have a relatively dense sensory innervation and a rich vascular supply. Teeth and supporting tissues are susceptible to tissue injury and inflammation, partly due to lack of collateral blood and nerve supply and to their low compliance. This review focuses on dental nerve functions and adaptive changes in the trigeminal ganglion and tooth pulp after peripheral injuries. An overview of the peptidergic innervation of oral tissues is presented, followed by a discussion of plasticity in neuropeptide expression in trigeminal peripheral neurons after local insults to teeth and peripheral nerve injuries. The functional implications of these adaptive changes are considered, with special reference to nerve regeneration, inflammation, and hemodynamic regulation.

Effect of inferior alveolar nerve axotomy on immune cells and nerve fibres in young rat molars

Denervation has been a useful approach to the investigation of interactions between nerve fibres and the pulp-dentine complex. Information on the immunological implications of axotomy is still lacking. The effect of axotomy on CD43+, CD4+, CD11b+ and I-A antigen-expressing cells in both the distal segment of the cut inferior alveolar nerve and in the first molar pulp of young rats was evaluated. Nerve fibres immunoreactive to protein gene product (PGP) 9.5, the neuropeptides substance P and calcitonin gene-related peptide (CGRP), and neuropeptide Y were visualized also by use of the avidin-biotin peroxidase complex method. Recruitment of macrophages was found in the distal segment of the sectioned inferior alveolar nerve 2 days after axotomy, with a further increase in number during the 6-day observation period. However, in the dental pulp, the number of CD43+, CD4+, CD11b+ and I-A antigen-expressing cells was almost unaffected. An almost complete sensory denervation of the first mandibular molar pulp was obtained 2 days after axotomy. After 6 days, the mesial part of the coronal pulp still remained denervated, while regenerated nerve fibres had reached both the root pulp and the distal part of the coronal pulp. Nerve fibres immunoreactive to neuropeptide Y were slightly reduced in density 2 days after axotomy, and after 6 days the localization of neuropeptide Y-immunoreactive fibres was changed compared to the control, with fibres also distributed in the odontoblast layer close to dentine. Hence, following axotomy in young rats, an almost complete sensory denervation is achieved in the first molar, whereas nerve fibres immunoreactive to neuropeptide Y change their distribution pattern, with fibres located close to the dentine after 6 days. Due to the almost unchanged number and distribution of immunocompetent cells in the pulp after axotomy, the young rat molar pulp may represent a suitable and useful experimental model to study neuro-immune interactions.

Experimental orthodontic tooth movement and extensive root resorption: periodontal and pulpal changes.

Previous studies have reported changes both in dental pulp and in periodontal ligament (PDL) following orthodontic tooth movement. However, pulpal changes following extensive root resorption after orthodontic tooth movement have not been studied in detail. The aim of this study was therefore to evaluate inflammatory changes, both in the dental pulp and in the compressed PDL, after experimentally induced extensive root resorption. Extensive root resorption was induced in rats by the activation and re-activation of orthodontic force, with a short intervening period of no force application. The distribution of immune cells, nerve fibres and blood vessels was studied immunohistochemically using antibodies against CD68-immunoreactive (IR) cells, major histocompatibility complex (MHC) class II Ia-expressing cells, CD43-IR cells, protein gene product 9.5 (PGP 9.5), and laminin. In the compressed PDL of experimental first molars, significantly increased density of CD68-IR cells and MHC class II Ia-expressing cells were found, whereas the density of CD43-IR cells were unchanged when compared with control second molars. In the compressed PDL, there was an increased density of blood vessels, but no sprouting of nerve fibres. In the dental pulp, however, no increased density of immune cells or sprouting of nerve fibres was recorded. In conclusion, inflammation after extensive root resorption was confined to the compressed PDL, whereas the dental pulp was unaffected.