Mesenchymal stem cell-mediated functional tooth regeneration in swine

Abstract

Mesenchymal stem cell-mediated tissue regeneration is a promising approach for regenerative medicine for a wide range of applications. Here we report a new population of stem cells isolated from the root apical papilla of human teeth (SCAP, stem cells from apical papilla). Using a minipig model, we transplanted both human SCAP and periodontal ligament stem cells (PDLSCs) to generate a root/periodontal complex capable of supporting a porcelain crown, resulting in normal tooth function. This work integrates a stem cell-mediated tissue regeneration strategy, engineered materials for structure, and current dental crown technologies. This hybridized tissue engineering approach led to recovery of tooth strength and appearance.

Figure 1. Isolation of Stem Cells from Root Apical Papilla (SCAP).

(A) Human apical papilla tissue was positive for STRO-1, an early mesenchymal progenitor marker, staining by immunofluorescence (arrows). (B) Single colonies were formed after human SCAP were plated at a low density (5×10³/T25 flask) and cultured for 10 days. (C) When human SCAP were cultured in odontogenic/osteogenic inductive conditions containing L-ascorbate-2-phosphate, dexamethasone, and inorganic phosphate for 4 weeks, mineralized nodules were found by Alizarin red S staining. (D) Cultured human SCAP formed Oil red O positive lipid clusters following 5 weeks of adipogenic induction in the presence of 0.5 mM isobutylmethylxanthine, 0.5 µM hydrocortisone, 60 µM indomethacin, and 10 µg/ml insulin. (E) Eight weeks after transplantation in immunocompromised mice, human SCAP differentiated into odontoblasts (arrows) that formed dentin (D) on the surfaces of a hydroxyapatite tricalcium (HA) carrier. (F) Immunohistochemical staining showed that human SCAP differentiated into odontoblasts (arrows) that were positive for anti-human specific mitochondria antibody staining. (G) Immunohistochemical staining showed that human SCAP-generated dentin (D) was positive for anti-DSP antibody staining (arrows). (H) Pre-immunoserum negative control of human SCAP transplant. (I) SCAP isolated from swine were capable of forming a single colony cluster when plated at a low cell density. (J) When transplanted into immunocompromised mice for 8 weeks, swine SCAP differentiate into odontoblasts (arrows) to regenerate dentin (D) on the surface of the hydroxyapatite carrier (HA). (K) Swine PDLSCs were capable of forming a single colony cluster. (L) After transplantation into immunocompromised mice, swine PDLSCs formed cementum (C) on the surface of hydroxyapatite carrier (HA). Collagen fibers were found to connect to newly formed cementum.

Figure 2. Characterization of human SCAP in comparison with DPSCs.

(A) Western blot analysis to confirm protein expression of genes identified in microarray studies showed greater abundance of survivin in SCAP than in DPSCs, with similar levels of DSP and Cbfa1/Runx2. (B) Flow cytometric analysis showed that ex vivo expanded SCAP contained approximately 7.5% CD24-positive cells, but DPSCs exhibited 0.5% positive staining for CD 24. (C) The proliferation rates of SCAP and DPSCs, derived from the same tooth, were assessed by co-culture with BrdU for 6 hours. The number of BrdU-positive cells was presented as a percentage of the total number of cells counted from five replicate cultures. SCAP showed a significantly higher proliferation rate in comparison with DPSCs (* P = 0.0042). (D) Single colony-derived SCAP were able to proliferate to over 70 population doublings, which was significantly higher than DPSCs (* P = 0.0192). (E) Dentin regeneration capacity of SCAP was significantly higher than that of DPSCs when transplanted into the same immunocompromised mice (* P = 0.0489) using Scion Image analysis system (Scion Image, Rockville, MD). (F) SCAP showed a significant higher telomerase activity than DPSCs at passage 1 (* P = 0.015). Cultured BMMSCs at passage 1 were used as a negative control to show an absence of telomerase activity. The telomerase activity was assessed by real time PCR based quantitative telomerase detection kit as described in Materials and Methods. (G) Cell motility assessed by a scratch assay. A representative area of SCAP and DPSCs at 72 hours after scratch was presented. Red arrows indicate the ranges of cell migration during 72 hours (* P = 0.0033).

Figure 3. Surface Molecule Characterization of human SCAP.

(A) Flow cytometric analysis of cultured SCAP at passage 1 revealed expression of STRO-1 (18.12%), CD146 (72.3%), CD24 (7.56%), CD166 (93.74%), CD73 (94.14%), CD90 (95.54%), CD105 (9.23%), CD106 (32.7%), CD29 (88.1%) and ALP (11.43%), but was negative for surface molecules CD18, CD14, CD34, CD45, and CD 150. (B) After 2 weeks osteo-induction in vitro with L-ascorbate-2-phosphate, dexamethasone, and inorganic phosphate, SCAP differentiated into odontoblasts with a decrease in CD24 expression from 7.56% to 0.22%. In contrast, ALP expression increased significantly from 11.43% to 86.59%.

Figure 4. Comined human SCAP/PDLSC-mediated tissue regeneration.

(A) On the outside of the HA/TCP carrier (HA), PDLSCs can form structures resembling Sharpey's fibers (arrows) connecting with newly formed cementum (C) on the surface of HA/TCP particles (HA). (B) Immunohistochemical staining showed that SCAP/PDLSC-generated tissues were positive for human specific mitochondria antibody staining (arrows).

Figure 5. Swine SCAP/PDLSC-mediated root/periodontal structure as an artificial crown support for the restoration of tooth function in swine.

(A) Extracted minipig lower incisor and root-shaped HA/TCP carrier loaded with SCAP. (B) Gelfoam containing 10×10⁶ PDLSCs (open arrow) was used to cover the HA/SCAP (black arrow) and implanted into the lower incisor socket (open triangle). (C) HA/SCAP-Gelfoam/PDLSCs were implanted into a newly extracted incisor socket. A post channel was pre-created inside the root shape HA carrier (arrow). (D) The post channel was sealed with a temporary filling for affixing a porcelain crown in the next step. (E) The HA/SCAP-Gelfoam/PDLSC implant was sutured for 3 months. (F) The HA/SCAP-Gelfoam/PDLSC implant (arrow) was re-exposed and the temporary filling was removed to expose the post channel. (G) A pre-made porcelain crown was cemented to the HA/SCAP-Gelfoam/PDLSC structure. (H) The exposed section was sutured. (I and J) Four weeks after fixation, the porcelain crown was retained in the swine after normal tooth use as shown by open arrows. (K) After 3 months implantation, the HA/SCAP-Gelfoam/PDLSC implant had formed a hard root structure (open arrows) in the mandibular incisor area as shown by CT scan image. A clear PDL space was found between the implant and surrounding bony tissue (triangle arrows). (L and M) H&E staining showed that implanted HA/SCAP-Gelfoam/PDLSC contains newly regenerated dentin (D) inside the implant (L) and PDL tissue (PDL) on the outside of the implant (M). (N) Compressive strength measurement showed that newly formed bio-roots have much higher compressive strength than original HA/TCP carrier (* P = 0.0002), but lower than that in natural swine root dentin (* P = 0.003) (NR: natural minipig root, BR: newly formed bio-root, HA: original HA carrier).