Mechanism Of Calcitonin Gene-related Peptide Positive Sensory Nerves In Regulating Reparative Dentin Formation

A crucial feature of dental pulpal defense is the formation of reparative dentin(RD)to create a mineralized barrier and prevent dental pulp from the site of bacterial invasion and injury.There are several biological events during the formation of RD.The distribution of dental nerves changes rapidly,which accompanies the increasing release of neurotransmitters and neuromodulators after irritation.Furthermore,the density and wall permeability of the vessels also change following pulpal injury.In the development and repair processes of different tissues,nerve fibers can affect these biological processes through modulating vessels.Dental nerves and vessels play an important role in RD formation.Whereas the specific regulatory mechanisms remain unclear,and the association between nerves and vessels during RD formation needs to be elucidated.In this study,we aimed to reveal the regulatory relation between dental nerves and vessels after injury,as well as the influence on RD formation.Chapter 1 The temporal sequence of neurovascular changes and reparative dentin formation during healing in dental pulpObjective:To detect the temporal sequence of innervation,vascularization,and RD formation after dental pulp damage.Methods:1.Animals and the construction of dentin injury model:The mice were deeply anesthetized with pentobarbital intraperitoneally.A cavity was prepared on the mesial surface of the mandibular first molars via a round bur,and the exposed dentin was etched with 37%phosphoric acid.2.Histological staining:The mice were transcardially perfused with paraformaldehyde at 1,3,7,14,and 28 days postoperatively.After fixation,the mandibles were isolated and decalcified.Then,the tissues were embedded in paraffin for sectioning,hematoxylin and eosin(H&E)staining,and Masson’s trichrome staining.3.Immunofluorescence staining:The paraffin sections were incubated with neurofilament(NF)antibody,CD31 antibody,and Endomucin(EMCN)antibody.Subsequently,the sections were incubated with the corresponding secondary antibodies.Results:1.Odontoblast layers under the injury site were disturbed one day after model establishment.On day 3,newly differentiated odontoblast-like cells were arranged along the pulp-dentin border.The staining also illustrated the normal timeline of the repair process over 28 days.The size of RD rose over time(P<0.01),and was first identified histologically on day 7 after dental pulp damage.2.At early time points preceding RD formation(days 1-3),NF+nerve density was increased obviously(P<0.01).At later time points following the beginning of nerve sprouting,CD31+ staining vascularity was increased,and vascularity had the highest quantitative value on day 3 after injury(P<0.05),a time point after nerve sprouting(days 1-3)and before the reparative dentinogenesis(days 7).3.Immunofluorescence staining of type H endothelium(CD31+EMCN+)indicated that the type H vessels in the injury sites were largely increased on day 7(P<0.01).Conclusion:Both nerve sprouting and vascularization patterns of dental pulp damage follow a temporospatial and predictable pattern of reaction to injury.And the increases in innervation precede angiogenesis and RD formation.Chapter 2 The influence of sensory or sympathetic denervation in the reparative dentin formationObjective:To explore the role of sensory and sympathetic innervation in RD formation.Methods:1.Inferior alveolar nerve axotomy(IANx):After removing the cortical bone of the mandibular ramus and exposing the IAN,the IAN was removed on the left side.To test whether the operation would be successful,measurements of the mechanical touch threshold were conducted via a set of Von Frey filaments.2.Superior cervical ganglionectomy(SCGx):The SCG was directly removed on the left side.Unilateral blepharoptosis after left SCGx was used as an indicator of the successful removal of the SCG.3.Construction of dentin injury model:Dental damage model was established on both sides 4 days after IANx and SCGx.4.Histological and immunofluorescence staining:The mice were transcardially perfused with paraformaldehyde after dental damage.After fixation,the mandibles were isolated and decalcified.Then,the tissues were embedded in paraffin for sectioning,histological staining,and the immunofluorescence of CD31 and EMCN.Results:1.The mechanical touch threshold rose significantly after IAN resection(P<0.001),and the blepharoptosis could be observed after SCGx.2.There was less RD beneath the affected dentin within IAN-resected molars(P<0.05).Notable,the densities of CD31+vessels and type H vessels were also significantly reduced in the IAN-resected molars(P<0.05).3.There was no statistical difference between sham-operated molars and SCG-resected molars.Conclusion:The sensory dental nerve fibers promote the formation of RD via enhancing local angiogenesis.In contrast,the sympathetic nerve fibers have no influence on RD formation.Chapter 3 Study on the regulatory mechanism of dental sensory nerves on the reparative dentin formationObjective:To explore the mechanism of sensory nerve fibers regulating the expression of cell junction proteins.Methods:1.Triple immunostaining staining:The paraffin sections were immunofluorescence stained with NF antibody,substance P(SP)antibody,and calcitonin gene-related peptide(CGRP)antibody.Subsequently,the sections were incubated with the corresponding secondary antibodies.2.Pharmacological treatments:Mice in the experimental group were treated with the CGRP receptor antagonist olcegepant(intravenous in acute and intraperitoneal in chronic treatment),with a single dose 10 minutes before cavity preparation and with once-daily doses over one week followed by a single dose before cavity preparation.Additionally,the application of vehicle was set as the vehicle-treated(VEH)group.3.Behavior test:The spontaneous pain-like behavior was monitored by a video camera one day before cavity preparation and on post-operative days 1 to 7.4.Histological and immunofluorescence staining:The mice were transcardially perfused with paraformaldehyde after dental damage.After fixation,the mandibles were isolated and decalcified.Then,the tissues were embedded in paraffin for sectioning,histological staining,and the immunofluorescence of CD31 and EMCN.Results:1.A marked increase in the density of CGRP+nerve fibers was seen on day 3(P<0.05),which was reduced but remained elevated over the baseline level on day 14.However,SP-positive nerve fibers did not change significantly.2.Spontaneous pain behavior was significantly decreased in the inhibitor-treated group as compared to the VEH group after cavity preparation.3.RD formation beneath the lesion was significantly decreased in the inhibitor-treated group compared with the VEH group(P<0.01).In the VEH group,CD31+vessels and type H vessels were again noted prominently within odontoblast and subodontoblastic layers on days 3 and 7 after injury respectively.In contrast,we detected a dramatic reduction in angiogenesis within injury sites in the oclegepant group compared to the VEH group(P<0.05).Conclusion:In the process of reparative dentinogenesis,the density of CGRP+nerve fibers dramatically increased at day 7 after dental damage,which corresponds to the timepoint of vascularization.Although the CGRP receptor antagonist can relieve the pain sensation of dentin injury,the healing process will be inhibited.