Regeneration of Tooth Roots With Borrowed Stem Cells in Pigs


Because a recent post about tooth-making stem cells in alligators generated so much interest, I found another recent paper that reports the regeneration of the tooth root structure in pigs. This is a proof-of-concept paper that demonstrated the feasibility of such a procedure.

The journal is Stem Cells and Development and the research team is from the Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction in Beijing, China. The corresponding author is Songlin Wang from the Molecular Laboratory for Gene Therapy and Tooth Regeneration.

Tooth loss represents a growing problem in an aging population. Dental implants provide one solution, but without a good jaw bone into which these implants can be attached, implants have little chance of staying put. Regenerating a tooth root that can support a natural or artificial crown is the most important part of the tooth in maintaining tooth function.

In previous work, Wang and his collaborator Songtao Shi from UCLA have shown that stem cells from root apical papilla and periodontal ligament stem cells from exfoliated teeth can coat bioengineered surfaces and form tooth structures that can support artificial crowns in miniature pigs (see Sonoyama et al., PLoS One 1:e79-e92). However, aged patients sometimes have bone marrow stem cells that do not grow well in culture and respond poorly to bioengineering protocols. Therefore, Wang and his crew sought to demonstrate that mesenchymal stem cells from donor animals (allogeneic stem cells) could provide the same kind of benefit.

The two stem cell populations used in this paper was dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs). The DPSCs were cultured from exfoliated minipig teeth and grown in culture for two or three passages. The culture medium used, as far as I can tell, was the same one used the Gronthos in his PNAS paper that reported the isolation and characterization of DPSCs. That medium was a modified Eagle’s medium supplemented with 20% Fetal Calf Serum and 100 μM L-ascorbic acid 2-phosphate, 2 mM L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. Gronthos then grew his cells at 37°C in 5% CO2 (see S. Gronthos, et al PNAS 97(25): 13625–13630). After 2-3 passages, the DPSCs were seeded on a hydroxyapatite tricalcium phosphate scaffold and grown in a bioreactor for 5-7 days

PDLSCs were grown in culture with approximately the same cocktail as the DPSCs and then plated on 60 mm dishes with vinylene carbonate (Vc). Vc induces the PDLSCs to grow s sheets that could be used to wrap the hydroxyapatite tricalcium phosphate structures that had been seeded with DPSCs.

These wrapped structures were implanted into the gums of minipigs and then used to secure dental implants.

Tooth Root Regeneration

After 6 months, the implants were assessed as was the integrity and strength of the surrounding tissue.

Gross, radiographic, and histological analysis of the bio-root 6 months after transplantation. (A, C) Gross view of the general shape of HA/TCP and the bio-root 6 months after transplantation (ellipse). (B, D) X-rays revealed that HA/TCP formed tissues without an obvious dental structure (ellipses), but the HA/TCP/DPSC/PDLSC sheet implant formed a hard root structure (ellipses). (E, F) No obvious boundary was observed between newly regenerated tissue and bone in the microcomputed tomography scan image of the HA/TCP group. (G, H) A hard root structure (arrows) was present and a clear PDL space found between the implant and surrounding bony tissue (triangle arrows). (I–K) HE staining showed some bone formation and HA/TCP remaining in the HA/TCP group (I), and PDL-like tissues were generated parallel to the dentin-like matrix structure in the autologous group (J) and allogeneic group (K). (L) Semiquantitative analysis showed that mineralized tissue regeneration capacity of autologous or allogeneic groups was significantly higher compared with the HA/TCP group. Percentage of mineralized tissues at 6 months after crown restoration was significantly higher than that before crown restoration in both autologous and allogeneic groups. No significant difference of regenerated mineralized tissue percentages was found between autologous and allogeneic groups. Scale bar: (I–K) 200 μm. B, bone; HA/TCP, hydroxyapatite/tricalcium phosphate; PDL, periodontal ligament; MT, mineralized tissue. *P<0.01 compared with autologous or allogeneic groups; #P<0.01 compared with autologous or allogeneic groups after crown restoration.
Gross, radiographic, and histological analysis of the bio-root 6 months after transplantation. (A, C) Gross view of the general shape of HA/TCP and the bio-root 6 months after transplantation (ellipse). (B, D) X-rays revealed that HA/TCP formed tissues without an obvious dental structure (ellipses), but the HA/TCP/DPSC/PDLSC sheet implant formed a hard root structure (ellipses). (E, F) No obvious boundary was observed between newly regenerated tissue and bone in the microcomputed tomography scan image of the HA/TCP group. (G, H) A hard root structure (arrows) was present and a clear PDL space found between the implant and surrounding bony tissue (triangle arrows). (I–K) HE staining showed some bone formation and HA/TCP remaining in the HA/TCP group (I), and PDL-like tissues were generated parallel to the dentin-like matrix structure in the autologous group (J) and allogeneic group (K). (L) Semiquantitative analysis showed that mineralized tissue regeneration capacity of autologous or allogeneic groups was significantly higher compared with the HA/TCP group. Percentage of mineralized tissues at 6 months after crown restoration was significantly higher than that before crown restoration in both autologous and allogeneic groups. No significant difference of regenerated mineralized tissue percentages was found between autologous and allogeneic groups. Scale bar: (I–K) 200 μm. B, bone; HA/TCP, hydroxyapatite/tricalcium phosphate; PDL, periodontal ligament; MT, mineralized tissue. *P

As you can see in panel E and F, control implants that had no cells and only hydroxyapatite calcium triphosphate showed no tooth-like structures, but in G and F, the structures with cells showed a consistent tooth structure with a periodontal ligament (see broad arrow).  In panels J and K, there is obvious bone formation with periodontal ligament in the autologous and allogeneic stem cell transplants.

Cross sections of the implants also showed that not only did these structures look like real tooth root structures, but they contained structures proteins indicative of real tooth root structures.  Dentin sialophosphoprotein (mercifully abbreviated to DSPP) is present in the cell-seeded implants, but in on the hydroxyapatite calcium triphosphate-only implants.

Clinical assessment of implants failed to detect any gingivitis or periodontal disease associated with the implants.

This experiment shows that stem cell-seeded scaffolds can regenerate tooth root structures.  Since this worked in minipigs and not simply rodents, these results strongly suggest that such a strategy could work in humans.  Clinical trials anyone?

Stem Cells From Gum Tissue Help Replace Missing Teeth


Researchers from King’s College London, UK have developed a new method that replaced missing teeth with bioengineered material made from a patient’s own gum cells.

If a patient loses a tooth, the dentist or oral surgeon will typically replace it with an implant. The vast majority of dental implants used today are root-form endosseous implants. Such implants have a similar look to an actual tooth root and are placed within the bone of the jaw. The bone of the jaw fuses the surface of the implant with the surrounding bone (a process known as osseointegration). Because dental implants lack the periodontal ligament they will feel slightly different from natural teeth during chewing. Also, the friction from chewing and from other jaw movements can cause loss of bone around the implant.

??????????????

Research by members of Paul Sharpe‘s laboratory at King’s College London has brought us closer to the reality of bioengineered teeth to replace toss teeth. Bioengineered tooth research has focussed primarily on producing immature teeth that can grow into adult teeth. Typically, such tooth buds are grown in culture and then transplanted into the gums. The gum actually provides and adequate environment for embryonic tooth buds to develop and form adult teeth. Therefore, the prospect of forming bioteeth certainly seems viable. The only question is identifying the cells and materials that can combine to properly form a normal adult tooth.

TOOTH DEVELOPMENT

Sharpe noted, “What is required is the identification of adult sources of human epithelial and mesenchymal cells that can be obtained in sufficient numbers to make biotooth formation a viable alternative to dental implants.”

Sharpe and his colleagues surmised that gum tissue might provide the right cells for this project. Therefore, they isolated adult human gum tissue samples from patients at the Dental Institute at King’s College and grew it in culture in the laboratory. Next, Sharpe’s group combined this gum tissue with mouse embryonic tooth mesenchyme cells, which are stem cells that can induce tooth formation.  This gum-tooth combination created teeth with surrounding gum tissue that could be transplanted into the mouths of mice. The teeth had dentine, enamel and viable roots.

The epithelial cells from human gum were able to respond to tooth-inducing signals from the embryonic tooth mesenchymal cells in a manner that allowed them to contribute to the tooth crown and the roots, and formed all the available cell types necessary for normal tooth formation. Thus, it appears that gum biopsies can provide a realistic source for human biotooth production.

The next step in this research is the formidable challenge of finding a mesenchymal stem cell population that can induce tooth formation. Presently, only embryonic mesenchymal cells can do this, according to Sharpe, but it is possible that adult mesenchymal stem cells can be manipulated to become tooth-inducing cells.