Local anesthetics induce autophagy in young permanent tooth pulp cells

Pulp cells are essential for tooth development, and dentin repair and regeneration. In addition these cells have been identified as an important stem cell source. Local anesthetics are widely used in dental clinics, as well as the other clinical disciplines and have been suggested to interfere with human permanent tooth development and induce tooth agenesis through unknown mechanisms. Using pig model and human young permanent tooth pulp cells, our research has identified that the local anesthetics commonly used in clinics can affect cell proliferation. Molecular pathway profiling suggested that LC3II is one of the earliest molecules induced by the agents and p62 is the only common downstream target identified for all the drugs tested. The effect of the drugs could be partially recovered by V-ATPase inhibitor only if early intervention is performed. Our results provide novel evidence that local anesthetics could affect tooth cell growth that potentially can have impacts on tooth development.

Ethics Committee of the Peking University School and Hospital of Stomatology, Beijing, China. Specifically, young permanent tooth pulp cells were obtained from two patients (12 and 16 years old) whose upper first premolars were extracted for orthodontic reasons. Following extraction, the tooth chamber was first opened using a dental drill. Pulp tissue was isolated using a 23-gauge fine needle and digested with 3% collagenase I (C0130-1G; Sigma) dissolved in Hank's balanced salt solution (Gibco, Cat. No.14175-053) supplemented with 1% penicillin-streptomycin (SV30079.01, Hyclone) using a final volume of 2ml enzyme solution per gram of tissue for 1 hour at 37°C with constant agitation. The enzyme reaction was then stopped by adding an equal volume of complete cell culture medium, comprising Dulbecco modified Eagle's medium/F12 (31331-028, Gibco) supplemented with 20% fetal bovine serum (F7524, Sigma) and 1% penicillin-streptomycin. The culture medium was changed every 2 days and cells were passaged when they reached 70-80% confluence by digestion with 0.05% Trypsin-EDTA (25300-054, Gibco).
Passage 7-9 cells at 70% confluence were used for all experiments.

Plasmid preparation and transfection
Plasmids were amplified and extracted using Plasmid Maxi Kit (12162, Qiagen), according to the manufacturer's recommendations. Cells were seeded at 2x10 5 cells/ml into 6-well plates and cultured for 24 hours until 60-80% confluence. For each transfection, 2µg of plasmid DNA was mixed with 200µL of jetPRIME buffer PolyPlus) and the mixture was added to an equal volume of jetPRIME solution and added to cells. Transfected cells were treated with anesthetic drug treatment after 24 hours.

Immunostaining
Cells cultured on glass coverslips were fixed in 4% ice--cold paraformaldehyde (diluted in 10mM PBS), incubated at room temperature for 20-30 minutes, and then rinsed three times for 5 min in PBST (10mM PBS containing 0.1% Triton--X100). Non--specific binding sites were first blocked by incubation in PBST containing 5% donkey serum, 0.25% cold water fish gelatin and 0.25% BSA for 60 minutes. Immunofluorescence images were obtained using either a Leica SP5

Mitochondrial energetic assay
The

Compounds used in XF Cell Mito Stress Test
Firstly, optimal concentrations of three compounds (final concentration 1uM) and cell seeding density were empirically determined prior to the assay. Cells were seeded at 10×10 3 cells/well in 100 µl of DMEM/F12 with 20%FBS in XF96 Cell Culture Microplates using 8 replicates and incubated for 24 hours at 37 °C in 5% CO 2 atmosphere. Five local anesthetics of 0.5mM and 2mM concentration were added with one control group. After 2/4/8/16 hours, the microplates were ready for check in the machine. The drug injections ports of the XF Assay Cartridge were loaded with the assay reagents in assay medium. 25 µl of the three compounds were added sequentially. Culture medium was exchanged with assay medium prior to measurements. Culture medium was aspirated and 80 µl pre-warmed assay medium added twice, aspirated and 175 µl pre-warmed assay medium added. The microplate was equilibrated in a CO 2 free incubator at 37ºC for 60 minutes. During this equilibration period, the XF96 Analyzer was calibrated with a calibration plate that had been hydrated at 37ºC overnight using the standard XF calibration protocol.
Following calibration, the calibration plate was replaced with the XF96 cell culture microplate containing pre-treated cells with local anesthetics and the experimental run started. Data were normalized by cell number and expressed as pmol of O 2 per minute per 10^ 4 cells.