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 Male 26 (86.67%) 7 (70%) Female 4 (13.33%) 3 (30%) Age 7.13 ± 0.97 7.1 ± 0.74 Time between trauma and hDPSC implantation/apexification <2 months 3 (10%) 1 (10%) 2 to 7 months 19 (63.33%) 3 (30%) >7 months 8 (26.67%) 6 (60%) History of drug
allergy0 0 Laser Doppler
flowmetry (PU)2.81 ± 0.41 3.01 ± 0.44 Length of root (mm) 10.69 ± 1.32 9.99 ± 0.82 Width of apical
foramen (mm)3.17 ± 0.69 3.54 ± 0.44 - Table 2 Safety assessment of 20 patients 24 months after hDPSC implantation
ALT, alanine aminotransferase; AST, aspartate aminotransferase.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Normal Unit ALT 16 12 21 33 24 33 10 35 22 17 23 31 15 39 40 41 25 21 21 31 9–50 IU/liter AST 19 15 27 32 26 23 16 35 24 20 22 19 27 23 25 38 31 23 29 32 15–40 IU/liter Total protein 70.6 70.2 79.0 82.5 66.4 71.6 66.0 72.7 68.9 70.1 69.2 77.1 72.4 80.1 78.2 78.2 78.4 78.4 77.6 74.3 65.0–85.0 g/liter Globulin 24.5 28.2 30.5 30.7 21.8 23.0 22.7 25.7 23.5 31.2 32.4 29.4 31.8 28.2 31.5 30.4 28.1 27.3 30.4 36.5 20.0–40.0 g/liter Albumin 46.1 42.0 48.5 51.8 44.6 48.6 43.3 47.0 45.4 48.9 49.6 43.7 50.9 48.6 49.4 47.5 48.9 50.2 50.3 47.4 40.0–55.0 g/liter Total bilirubin 10.6 11.2 12.8 11.4 9.3 9.5 11.9 7.9 7.4 11.5 8.5 13.6 12.7 8.5 10.5 10.2 10.3 9.7 12.6 11.6 3.4–20.5 μM Direct bilirubin 4.4 5.2 6.7 5.1 4.9 5.1 5.9 3.4 2.9 5.2 4.3 2.4 5.5 4.9 3.8 5.6 5.6 5.8 5.6 5.0 0.0–6.8 μM Indirect bilirubin 6.2 6.0 6.1 6.3 4.4 4.4 6.0 4.5 4.5 6.3 4.7 4.9 5.4 5.2 5.3 5.3 4.9 4.9 5.2 5.6 4.3–6.4 μM Alkaline phosphatase 200 170 195 201 210 196 205 212 188 175 79 86 147 85 78 78 100 150 101 174 20–220 IU/liter γ-Glutamyltransferase 12 39 49 56 12 27 35 41 16 45 35 37 47 51 50 49 55 54 54 58 10–60 IU/liter Albumin/globulin 1.9 1.5 1.6 1.7 2.1 2.1 1.9 1.8 1.9 1.3 1.8 1.7 1.5 2.0 2.1 2.0 2.0 1.9 2.0 1.9 1.2–2.4 Urea 4.10 4.70 5.70 4.90 2.85 2.95 2.77 3.10 2.98 3.0 4.84 4.89 5.20 5.43 5.51 5.01 6.01 5.21 6.01 6.01 2.50–6.30 mM Creatinine 112 62 90 58 67 101 82 95 57 89 79 77 68 102 98 111 109 98 87 98 53–115 μM Uric acid 310 336 356 335 198 194 184 236 168 255 188 355 322 283 301 305 299 241 314 354 150–430 μM Lactate dehydrogenase 198 213 209 145 160 206 198 191 132 217 172 172 174 179 198 179 187 197 178 241 120–250 IU/liter Creatine kinase 124 105 230 207 76 92 226 61 67 223 154 211 198 233 211 204 254 241 213 274 50–310 IU/liter Creatine kinase
isoenzyme19 19 13 12 13 11 17 9 12 16 11 18 14 19 20 19 17 16 22 17 0–24 IU/liter Cystatin C 0.72 0.95 0.79 0.60 0.66 0.62 0.78 0.63 0.56 0.98 0.74 0.88 0.72 0.92 0.87 1.01 0.83 1.01 0.78 0.87 0.55–1.05 mg/liter T cell (%) 79.70 74.35 71.08 68.31 64.32 80.01 71.03 68.70 64.90 81.04 70.25 70.12 72.04 85.00 71.00 69.05 84.05 69.25 75.14 70.45 56–86 % CD4+ T (%) 51.27 48.28 49.32 48.29 51.27 54.32 53.45 55.71 56.11 56.02 55.03 53.10 49.56 48.24 54.00 49.56 49.32 55.25 54.23 48.72 33–58 % CD8+ T (%) 18.23 18.13 20.94 16.23 20.94 16.03 15.94 15.07 15.04 16.44 17.98 17.22 17.09 14.23 16.01 17.00 15.09 17.04 15.98 17.22 13–19 % B cell (%) 11.57 12.83 10.89 15.21 11.57 10.43 14.21 17.17 18.13 19.04 20.00 19.06 19.45 21.01 19.00 20.00 20.88 18.11 18.11 18.04 5–22 % NK cell (%) 5.43 5.21 5.67 7.63 5.43 6.09 7.43 7.43 7.01 10.23 19.05 14.55 15.01 20.12 15.23 21.36 16.21 20.01 14.10 18.36 5–26 % CD4+/CD8+ 2.45 2.16 2.24 1.97 2.45 2.06 2.01 2.08 1.98 2.07 2.27 2.09 2.02 2.41 2.00 1.99 1.86 1.45 2.00 1.99 0.71–2.78 Lymphocyte count 2143 2521 2019 2513 2143 2512 2201 2571 2963 2732 2905 2689 3012 2901 3014 3100 3215 2159 2401 3104 1530–3700 /μl Total T cell count 1705 1489 1397 1469 1705 1953 1802 1985 2471 2019 2601 2441 2401 2543 2601 2400 1987 2447 2221 2210 723–2737 /μl CD4+ T cell count 1320 1180 1338 1402 1318 1403 1340 1408 1065 1477 1258 1501 1278 1500 1300 1368 1379 1398 1365 1357 404–1612 /μl CD8+ T cell count 539 428 567 697 489 506 448 641 765 739 845 801 804 700 700 801 1001 987 871 803 220–1129 /μl B cell count 198 201 182 231 154 132 276 251 274 301 515 403 401 310 414 400 201 314 2581 358 80–616 /μl NK cell count 93 98 97 103 99 95 105 109 121 116 105 202 212 120 134 150 231 289 198 177 84–724 /μl Antistreptolysin O 69.7 54 32.7 44.2 55.8 50.7 19.90 69.70 20.00 56.30 47.7 24.4 77.4 38.9 52.9 76.3 47.6 32.7 66.3 51.5 <116 IU/ml C-reactive protein 0.25 0.30 0.35 0.17 0.29 0.15 0.18 0.25 0.70 0.37 0.51 0.19 0.45 0.62 0.66 0.61 0.44 0.22 0.34 0.43 <0.80 mg/dl Rheumatoid factor 4.92 4.50 4.02 3.64 5.90 8.92 2.55 4.92 10.90 10.05 8.12 15.7 13.7 8.9 12.40 13.40 8.40 8.65 16.5 7.78 <20 IU/ml IgA 110.4 132.4 99.8 89.4 110 125.5 100.9 110.4 335.2 129.4 325 78.4 223.8 307.4 112.7 228.6 90.6 98.4 240.7 164.7 82–453 mg/dl IgG 843.5 833.8 798.5 801.5 855.5 806.5 946.2 843.5 1110.5 891 1154 859.5 919.1 874.5 866.8 1239.5 874.3 944.2 1012.8 1321.2 751–1560 mg/dl IgM 62.4 52.7 55.1 78.6 74.6 68.2 78.5 62.4 190.3 162.6 176.9 96.7 64.3 196.8 186.4 174.3 154.2 78.5 62.6 123.6 40–274 mg/dl C3 83.9 92.9 79.5 81.9 107.1 86.1 120.1 83.9 88.7 113.1 123.7 102.4 107.6 100.7 92.1 97.8 87.3 100.4 90.7 88.4 79–152 mg/dl C4 16.9 23 18.6 18 19.9 19 22.7 16.9 27.6 18.5 21.6 20.3 28.3 27.6 25.8 30.2 22.9 19.7 29.1 28.4 16–38 mg/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)
Additional Files
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)
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.
