Research ArticleStem Cells

Deciduous autologous tooth stem cells regenerate dental pulp after implantation into injured teeth

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Science Translational Medicine  22 Aug 2018:
Vol. 10, Issue 455, eaaf3227
DOI: 10.1126/scitranslmed.aaf3227
  • Fig. 1 hDPSCs regenerate dental pulp in immunocompromised mice.

    (A) hDPSCs derived from deciduous canine tooth pulp from two patients (sample 1 and sample 2) formed single CFU clusters in culture. (B) hDPSCs derived from the teeth of two patients formed mineralized nodules when induced in osteogenic culture medium for 28 days. Uninduced hDPSCs failed to form mineralized nodules. Scale bar, 200 μm. (C) hDPSCs derived from the teeth of two patients were able to differentiate into Oil red O–positive adipocytes when cultured under adipogenic induction conditions for 21 days. Uninduced hDPSCs failed to form adipocytes. Scale bar, 50 μm. (D) Immunofluorescence staining showed that hDPSCs expressed CD146, CD105, NeuN, nestin, CGRP, TRPM8, and TRPV1, but not CD34. Scale bar, 20 μm. (E) hDPSC aggregates inserted into empty root canals of human teeth and implanted subcutaneously into immunocompromised mice (n = 12) for 8 weeks were stained with hematoxylin and eosin (H&E) and Masson stain. Implanted hDPSC aggregates regenerated pulp tissue. In the control group, calcium hydroxide was inserted into empty root canals of human teeth and implanted subcutaneously into immunocompromised mice for 8 weeks. After 8 weeks, no pulp tissue was regenerated and only calcified tissue was observed. Scale bar, 50 μm. Enlarged regions of the images show that odontoblasts (black arrows) were present at the margin of the regenerated pulp tissue. Blood vessels (open arrows) were also observed in the regenerated pulp tissue. Scale bar, 20 μm. (F) Left: Image shows dentin sialoprotein–positive odontoblasts (open black arrows) revealed by immunohistochemical staining. Scale bar, 20 μm. Right: Calcein staining showed newly formed dentin (white arrows) in the empty root canal of a human tooth. Scale bar, 0.1 mm.

  • Fig. 2 Histological analysis of pig DPSCs implanted into minipigs.

    (A) Pig DPSCs (pDPSCs) were implanted into permanent incisors of minipigs after pulpectomy (n = 3). H&E staining (left) and Masson staining (right) showed that pulp tissue was regenerated 3 months after pDPSC implantation. In the control group, calcium hydroxide instead of pDPSCs was inserted into young permanent incisors in minipigs (n = 3). After 3 months, no pulp tissue was regenerated and only calcium hydroxide was observed. Normal pulp tissue of minipigs was stained for comparison (top). Scale bar, 50 μm. Enlarged images show odontoblasts (black arrow) and blood vessels (open arrow) in select regions of regenerated pulp tissue. Scale bar, 20 μm. (B) Representative histological images showing that pig normal pulp tissue lacks NeuN-positive cells. Scale bar, 100 μm. (C) Pig DPSCs regenerated dental pulp that contained NeuN-positive cells (green); DAPI (blue) was used as a counterstain for nuclei. Scale bar, 200 μm. Two regions of regenerated pulp tissue were selected for higher magnification. Scale bar, 20 μm.

  • Fig. 3 Clinical trial profile and study timeline.

    (A) Design of clinical trial to examine dental pulp regeneration by autologous hDPSCs. (B) Study events and timeline of procedures and testing are shown for participants randomly allocated to the hDPSC implantation group or control group. At the first visit, dental pulp from deciduous teeth was removed and then cultured to obtain hDPSCs for implantation. All participants received radiovisiography (RVG), CBCT, continuous-wave Doppler, and root canal disinfection at the first visit. One month later, the hDPSC implantation group received hDPSCs and the control group received apexification treatment. All participants underwent RVG, CBCT, electric pulp vitality testing, and laser Doppler flowmetry at 6 and 12 months after treatment. Data from the electric pulp vitality test and the laser Doppler flowmetry test were compared between the hDPSC implantation and control groups. *Four patients in the hDPSC implantation group were excluded from analysis.

  • Fig. 4 Pulp regeneration in the incisor teeth of patients after hDPSC implantation.

    (A) Representative CBCT images of hDPSC-implanted incisor teeth at 6 and 12 months after treatment. The length of the root (red line) was increased at 6 and 12 months after hDPSC implantation. The apical foramen (blue line) was closed 12 months after hDPSC implantation. In the control group, the length of the root was not increased and the apical foramen was not closed at 12 months after apexification treatment. (B) Representative 3D images of a traumatized immature permanent human incisor tooth before and after implantation of hDPSCs. Frontal images (top) and lateral images (bottom) were constructed using Materialise’s interactive medical image control system (Mimics). Roots were elongated at 6 and 12 months after hDPSC implantation compared to before treatment (white stippled circles). In addition, the amount of dentin was increased at 6 and 12 months after treatment in the hDPSC implantation group (white arrows). (C to F) hDPSC implantation into patient incisor teeth improved vascular formation (C), sensation measured by the electric pulp test (D), root length (E), and width of the apical foramen (F) at 6 and 12 months after implantation. (G) Dentin thickness in implanted incisor teeth was increased at 6 and 12 months after hDPSC implantation compared to baseline. Error bars are means ± SD. Data were analyzed using Student’s t test.

  • Fig. 5 Histological analysis of hDPSC-regenerated dental pulp.

    (A) Representative histological images show that normal pulp tissue of human teeth lacks NeuN-positive cells. Scale bar, 200 μm. (B) Representative image of a human incisor 12 months after hDPSC implantation shows regenerated pulp tissue with a similar tissue structure to that of normal human pulp tissue. Odontoblasts (black arrows) were observed at the margin of the regenerated pulp tissue. Scale bar, 200 μm. (C) Pulp tissue regenerated after hDPSC implantation contained NeuN-positive cells (red); DAPI (blue) was used to stain nuclei. Odontoblasts were observed at the margin of the regenerated pulp tissue. Two regions of regenerated pulp tissue are shown at higher magnification. Scale bar, 20 μm.

  • Fig. 6 RVG images 12 and 24 months after hDPSC implantation.

    To further evaluate the safety of hDPSC implantation, we continued follow-up of 20 patients for 24 months after treatment. Digital RVG images of hDPSC-implanted incisors are shown for 10 patients at 12 and 24 months after hDPSC implantation (images for the remaining 10 patients are shown in fig. S5). The images show no inflammation at the periapical area in any of the incisor teeth after hDPSC implantation. Images show that root length was increased and the apical foramen was closed at 12 and 24 months after treatment. Red arrows indicate incisor teeth before and after hDPSC implantation.

  • Fig. 7 Dental pulp regeneration after hDPSC implantation into human incisors.

    To test the viability of hDPSC-implanted incisor teeth, we performed laser Doppler flowmetry and an electric pulp test for the implanted incisor teeth of 25 patients at 12 months and 20 patients at 24 months after treatment. hDPSC implantation into traumatized patient incisors improved vascular formation as shown by laser Doppler flowmetry (A) and sensation as shown by responses to the electric pulp test (B) at 24 months after treatment compared to 12 months after treatment. Error bars are means ± SD. Data were analyzed using Student’s t test.

  • Table 1 Baseline characteristics of patients.
    hDPSC group
    (n = 30)
    Control group
    (n = 10)
    Sex
      Male26 (86.67%)7 (70%)
      Female4 (13.33%)3 (30%)
    Age7.13 ± 0.977.1 ± 0.74
    Time between trauma and hDPSC implantation/apexification
      <2 months3 (10%)1 (10%)
      2 to 7 months19 (63.33%)3 (30%)
      >7 months8 (26.67%)6 (60%)
    History of drug
    allergy
    00
    Laser Doppler
    flowmetry (PU)
    2.81 ± 0.413.01 ± 0.44
    Length of root (mm)10.69 ± 1.329.99 ± 0.82
    Width of apical
    foramen (mm)
    3.17 ± 0.693.54 ± 0.44
  • Table 2 Safety assessment of 20 patients 24 months after hDPSC implantation

    ALT, alanine aminotransferase; AST, aspartate aminotransferase.

    1234567891011121314151617181920NormalUnit
    ALT16122133243310352217233115394041252121319–50IU/liter
    AST191527322623163524202219272325383123293215–40IU/liter
    Total protein70.670.279.082.566.471.666.072.768.970.169.277.172.480.178.278.278.478.477.674.365.0–85.0g/liter
    Globulin24.528.230.530.721.823.022.725.723.531.232.429.431.828.231.530.428.127.330.436.520.0–40.0g/liter
    Albumin46.142.048.551.844.648.643.347.045.448.949.643.750.948.649.447.548.950.250.347.440.0–55.0g/liter
    Total bilirubin10.611.212.811.49.39.511.97.97.411.58.513.612.78.510.510.210.39.712.611.63.4–20.5μM
    Direct bilirubin4.45.26.75.14.95.15.93.42.95.24.32.45.54.93.85.65.65.85.65.00.0–6.8μM
    Indirect bilirubin6.26.06.16.34.44.46.04.54.56.34.74.95.45.25.35.34.94.95.25.64.3–6.4μM
    Alkaline phosphatase200170195201210196205212188175798614785787810015010117420–220IU/liter
    γ-Glutamyltransferase123949561227354116453537475150495554545810–60IU/liter
    Albumin/globulin1.91.51.61.72.12.11.91.81.91.31.81.71.52.02.12.02.01.92.01.91.2–2.4
    Urea4.104.705.704.902.852.952.773.102.983.04.844.895.205.435.515.016.015.216.016.012.50–6.30mM
    Creatinine11262905867101829557897977681029811110998879853–115μM
    Uric acid310336356335198194184236168255188355322283301305299241314354150–430μM
    Lactate dehydrogenase198213209145160206198191132217172172174179198179187197178241120–250IU/liter
    Creatine kinase1241052302077692226616722315421119823321120425424121327450–310IU/liter
    Creatine kinase
    isoenzyme
    1919131213111791216111814192019171622170–24IU/liter
    Cystatin C0.720.950.790.600.660.620.780.630.560.980.740.880.720.920.871.010.831.010.780.870.55–1.05mg/liter
    T cell (%)79.7074.3571.0868.3164.3280.0171.0368.7064.9081.0470.2570.1272.0485.0071.0069.0584.0569.2575.1470.4556–86%
    CD4+ T (%)51.2748.2849.3248.2951.2754.3253.4555.7156.1156.0255.0353.1049.5648.2454.0049.5649.3255.2554.2348.7233–58%
    CD8+ T (%)18.2318.1320.9416.2320.9416.0315.9415.0715.0416.4417.9817.2217.0914.2316.0117.0015.0917.0415.9817.2213–19%
    B cell (%)11.5712.8310.8915.2111.5710.4314.2117.1718.1319.0420.0019.0619.4521.0119.0020.0020.8818.1118.1118.045–22%
    NK cell (%)5.435.215.677.635.436.097.437.437.0110.2319.0514.5515.0120.1215.2321.3616.2120.0114.1018.365–26%
    CD4+/CD8+2.452.162.241.972.452.062.012.081.982.072.272.092.022.412.001.991.861.452.001.990.71–2.78
    Lymphocyte count214325212019251321432512220125712963273229052689301229013014310032152159240131041530–3700/μl
    Total T cell count17051489139714691705195318021985247120192601244124012543260124001987244722212210723–2737/μl
    CD4+ T cell count13201180133814021318140313401408106514771258150112781500130013681379139813651357404–1612/μl
    CD8+ T cell count5394285676974895064486417657398458018047007008011001987871803220–1129/μl
    B cell count198201182231154132276251274301515403401310414400201314258135880–616/μl
    NK cell count939897103999510510912111610520221212013415023128919817784–724/μl
    Antistreptolysin O69.75432.744.255.850.719.9069.7020.0056.3047.724.477.438.952.976.347.632.766.351.5<116IU/ml
    C-reactive protein0.250.300.350.170.290.150.180.250.700.370.510.190.450.620.660.610.440.220.340.43<0.80mg/dl
    Rheumatoid factor4.924.504.023.645.908.922.554.9210.9010.058.1215.713.78.912.4013.408.408.6516.57.78<20IU/ml
    IgA110.4132.499.889.4110125.5100.9110.4335.2129.432578.4223.8307.4112.7228.690.698.4240.7164.782–453mg/dl
    IgG843.5833.8798.5801.5855.5806.5946.2843.51110.58911154859.5919.1874.5866.81239.5874.3944.21012.81321.2751–1560mg/dl
    IgM62.452.755.178.674.668.278.562.4190.3162.6176.996.764.3196.8186.4174.3154.278.562.6123.640–274mg/dl
    C383.992.979.581.9107.186.1120.183.988.7113.1123.7102.4107.6100.792.197.887.3100.490.788.479–152mg/dl
    C416.92318.61819.91922.716.927.618.521.620.328.327.625.830.222.919.729.128.416–38mg/dl

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/455/eaaf3227/DC1

    Fig. S1. hDPSCs differentiate into sensory neurons after intraganglion injection in rats.

    Fig. S2. Characteristics of minipig DPSCs.

    Fig. S3. Implantation of hDPSC aggregates in patients.

    Fig. S4. Number of CD3+, CD4+, and CD8+ T cells in minipigs after pDPSC implantation.

    Fig. S5. RVG images 12 and 24 months after hDPSC implantation.

    Table S1. Source data for Fig. 1.

    Table S2. Source data for Table 1.

    Table S3. Source data for Fig. 4.

    Table S4. Source data for Fig. 7.

    Table S5. Source data for figs. S2 and S4.

    Project protocol

    References (39, 40)

  • The PDF file includes:

    • Fig. S1. hDPSCs differentiate into sensory neurons after intraganglion injection in rats.
    • Fig. S2. Characteristics of minipig DPSCs.
    • Fig. S3. Implantation of hDPSC aggregates in patients.
    • Fig. S4. Number of CD3+, CD4+, and CD8+ T cells in minipigs after pDPSC implantation.
    • Fig. S5. RVG images 12 and 24 months after hDPSC implantation.
    • Legends for tables S1 to table S5
    • Project protocol
    • References (39, 40)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Source data for Fig. 1.
    • Table S2 (Microsoft Excel format). Source data for Table 1.
    • Table S3 (Microsoft Excel format). Source data for Fig. 4.
    • Table S4 (Microsoft Excel format). Source data for Fig. 7.
    • Table S5 (Microsoft Excel format). Source data for figs. S2 and S4.

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