[Adenosine triphosphate induced odontoblastic differentiation of human dental pulp cells in vitro and in vivo].

Objective: To choose a suitable efficient concentration of adenosine triphosphate (ATP) which can induce human dental pulp cells (HDPC) differentiate into odontoblast successfully, and explore the role of this concentration of ATP in dentin regeneration in vivo. Methods: HDPC were treated with different concentrations (0, 10, 400, 600, 800 µmol/L) of ATP. Then cell counting kit-8 (CCK-8), quantitative real-time PCR, and Western blotting were used to detect the cell proliferation and the expressions of odontoblastic differentiation related markers, dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP). Alizarin red S staining experiment was used to analyze the effect of ATP on the mineralization ability of HDPC. By the above experiments, the suitable effective concentration of ATP was chosen to pretreat the HDPC for 7 days, then cells were seeded on gelfoam, inserted into the root canal fragment, and subsequently transplanted into the subcutaneous space on the back of immunodeficient mice, after three months, the samples were stained with HE for histological analysis. Results: The CCK-8 results in 5 d showed that 10 µmol/L ATP obviously promoted the proliferation of HDPC, while the 600 and 800 µmol/L ATP apparently inhibited the HDPC proliferation, however, the proliferation in 800 µmol/L ATP group was lower than that of 600 µmol/L ATP group (P<0.05). qPCR and Western blotting results showed that the 600 and 800 µmol/L ATP significantly up-regulated the DMP1 and DSPP expressions (P<0.05), furthermore, there was no significant difference in the two groups, but no changes were found in other groups (P>0.05). After 21 days of culturing, there were obvious mineralization nodules in 600 and 800 µmol/L ATP groups, but no mineralization nodules in other groups. Quantitative analysis of the staining results showed the A value in 0, 10, 400, 600, and 800 µmol/L ATP groups were respectively 1.05±0.15, 1.11±0.23, 1.15±0.17, 3.65±0.30, and 3.40±0.43, and the A value in 600 and 800 µmol/L ATP groups were higher than those of other groups; however, there was no difference in 600 and 800 µmol/L ATP groups. The histological analysis showed that 600 µmol/L ATP could induce the HDPC differentiate into dentin-like structure in the root canal fragment. Conclusions: Therefore, the suitable effective concentration of ATP is 600 µmol/L, which could induce HDPC differentiate into odontoblast-like cells, and form the dentin-like structure in vivo.

[Characteristics of orofacial operant test for orofacial pain sensitivity caused by occlusal interference in rats].

OBJECTIVE: To compare the orofacial pain sensitivity with operant test and mechanical hyperalgesia with von Frey filaments of two orofacial pain models (EOI: experimental occlusal interference; pIONX: partial infraorbital nerve transection). To investigate the operant and evoked characteristics of EOI-rats. METHODS: The orofacial operant behaviors were tested by Ugo Basile Orofacial Stimulation Test System. The mechanical thresholds of vibrissal pads were tested by von Frey filaments. Male Sprague-Dawley rats were randomly divided into eight groups: von Frey group: sham-EOI, EOI, sham-pIONX, pIONX (sham: sham-operated group); operant test group: sham-EOI, EOI, sham-pIONX, pIONX (sham: sham-operated group). The mechanical thresholds and orofacial operant behaviors were tested on pre-operation and post-operation days l, 3, 7, 10, 14 and 21. RESULTS: In pIONX of von Frey group, the mechanical withdrawal threshold decreased from days 1 to 21 (P<0.05), peaking from days 7 to 10, and lasted until the end of the experiment. There was no significant difference between the bilateral sides. In pIONX of operant test group, the total contact time decreased from days 10 to 21 (P<0.05), peaking from days 10 to 14, and lasted until the end of the experiment. In EOI of von Frey group, the mechanical withdrawal threshold decreased from days 3 to 21 (P<0.05), peaking on day 7, and lasted until the end of the experiment. There was no significant difference between the bilateral sides. In EOI of operant test group, the total contact time decreased from days 1 to 21 (P<0.05), peaking from days 7 to 10, and lasting until the end of experiment. CONCLUSION: Orofacial operant test is a stable method to evaluate orofacial pain behaviors, which could discriminate the feature of neuropathic and EOI orofacial pain. In these two animal models, both of the operant behaviors and the mechanical hyperalgesia exhibited different time courses. Orofacial operant test provides a novel method for evaluating the orofacial pain sensitivity and studying the orofacial pain mechanism thoroughly.

[Effect of long-term resistance exercise on masseter muscle mechanical hyperalgesia in rats].

OBJECTIVE: To investigate the effect of long-term resistance exercise of hindlimb on mechanical hyperalgesia of bilateral masseter muscle in rats with or without occlusal interference. METHODS: Six-teen male Sprague-Dawley rats (220-250 g) were randomly divided into four groups: the naive control group, naive exercise group, occlusal interference control group, and occlusal interference exercise group. The rats in occlusal interference groups (occlusal interference control group and occlusal interference exercise group) obtained occlusal interference with 0.4 mm-thick crowns bonded to the right maxillary first molars. The rats in exercise groups (naive exercise group and occlusal interference exercise group) performed squat-type resistance exercises for 30 minutes, once a day, 5 days/week, lasting for 14 weeks. Resistance exercise was recorded every day. Mechanical withdrawal thresholds of bilateral masseter muscle were tested per week by use of modified electronic von-frey anesthesiometer. The rats were weighed per week. After the 14-week exercise, the muscle strength of the hindlimb was tested with a grip strength meter. Muscle (gastrocnemius and soleus) weight of bilateral hindlimb and length of bilateral fibula of the rats were obtained. The muscle-mass/body-mass ratios and muscle-mass/fibula-length ratios were calculated. RESULTS: Between the naive control group and naive exercise group, there was no significant difference in the mechanical withdrawal thresholds of bilateral masseter muscle for the 0-4 weeks (P>0.05). During the 5-14 weeks, the mechanical withdrawal thresholds of the rats in the naive exercise group were higher than those in the naive control group (P<0.05). Between the occlusal interference control group and occlusal interference exercise group, there was no significant difference in the mechanical withdrawal thresholds of bilateral masseter muscle for the 0-6 weeks (P>0.05). During the 7-14 weeks, the mechanical withdrawal thresholds of rats in the naive exercise group were higher than those in the occlusal interference control group (P<0.05). After the 14week exercise, the body mass of the rats in nonexercise group (the naive control group and occlusal interference control group) were larger than those in exercise group [(462±6) g vs. (418±14) g, P<0.05]. And the muscle strength of hindlimb of the rats in exercise group were bigger than those in non-exercise group [(6.75±0.13) N vs. (5.41±0.15) N, P<0.01]. CONCLUSION: long-term resistance exercise can increase mechanical withdrawal thresholds of the bilateral masseter muscle in rats with or without masseter muscle mechanical hyperalgesia.

ACC Plasticity Maintains Masseter Hyperalgesia Caused by Occlusal Interference.

Acute occlusal interference following improper occlusal alteration in dental practice can induce chronic masticatory muscle pain. The underlying mechanism has not been clarified. Synaptic plasticity in the anterior cingulate cortex (ACC) plays a key role in the chronic pain state. This study investigated the role of synaptic plasticity in the ACC in acute occlusal interference-induced chronic masticatory muscle pain. A rat model of experimental occlusal interference (EOI) was established. In vivo local field potential (LFP) recording was conducted to evaluate the change of synaptic strength and plasticity from the medial thalamus (MT) to the ACC after EOI application. The effects of microdialysis of antagonists of glutamate receptors into the ACC on synaptic transmission from the MT to the ACC were examined. Furthermore, the influence of inhibiting glutamate receptors in the ACC on EOI-induced mechanical hyperalgesia in the masseter muscles of rats was investigated. The amplitude of LFP in the ACC evoked by MT stimulation was significantly potentiated since 14 d of EOI application. Long-term potentiation of LFP in the ACC was reliably induced by theta burst stimulation to the MT in control rats but was occluded in 14-d EOI rats. Microdialysis of AMPA/kainate receptor antagonist CNQX into the ACC attenuated LFP in the ACC evoked by stimulating the MT in control and EOI rats. Administration of NMDA receptor subunit NR2B antagonist Ro 25-6981 into the ACC significantly alleviated the potentiation of MT stimulation-evoked LFP in the ACC of EOI rats without affecting that in control rats. EOI-induced hyperalgesia in the bilateral masseter muscles of rats was dose-dependently relieved after microdialysis of Ro 25-6981 into ACC. These findings provide direct evidence that prolonged acute occlusal interference potentiates synaptic transmission in the ACC, which in turn mediates chronic masticatory muscle pain.