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?

Human Neural Stem Cell Line Heals Spinal Cord-Injured Rats


Spinal cord injuries represent one of the most intractable problems for regenerative medicine. When the spinal cord is injured, a tissue that is normally isolated from the bloodstream, now comes into contact with a variety of inflammatory factors and cells that increase the destruction of the original lesion. The spinal responds with a glial scar that plugs the lesion and prevents further exposure of the spinal cord to damaging inflammation, but the scar is also filled with molecules that repel neuronal axon growth cones. This spells curtains for neuronal regeneration, and finding a cell type that can negotiate around the glial scar and find the original muscle is a genuine tour de force.

Given this to be the case, there have been many experiments in rodents to examine the efficacy of various stem cell populations to as treatments for spinal cord injuries. A recent paper in Stem Cell Research and Therapy (van Gorp et al., 2013, 4:57) has examined human fetal spinal cord-derived neural stem cells (HSSCs) and their ability to restore motor function in rats with spinal cord injuries to the lower back. Because this group examined movement and spinal cord tissue samples, this paper contributes something significant to our knowledge of HSSC-mediate healing of spinal cord injuries.

The HSSC line used in this paper is neural stem cell line NSI566RSC, which was extracted from the spinal cord of an 8-week old “fetus.” I have placed fetus in quotes because at eight weeks, the fetus is actually a very old embryo, since the end of the eighth week is end of embryonic development. I realize that these types of age calculations have room for error, and therefore, the baby might very well have been at the early fetal stage. However, the baby’s mother terminated her pregnancy (yes it was an abortion and no I am not cool with that) and donated the dead baby’s tissue to UC San Diego for research purposes.

Sprague-Daley rats were subjected to spinal cord injuries at the level of the third lumbar vertebra. Three days later, half of the rats were given saline injections into their spinal cord and the other half were given HSSC injections into their spinal cords. The animals were evaluated for two months after the treatments on a daily basis. After two months, the rats were sacrificed (put down) and the spinal cord tissue was extensively analyzed.

Of the 35 animals employed in this study, 3 were excluded because of paw injuries or drug toxicity. Eight weeks after the cells were implanted, the rats were tested with a CatWalk apparatus to determine their gait. The rats injected with HSSCs showed a much more normal gait than those injected with saline. To give you some idea of the improvement, the rats that were not injured had a RCHPP or rostro-caudal hind paw positioning score of 0+/- 1.7mm, and the saline injected animals had an average RCHPP of -18 +/- 3.1 mm, and those injected with HSSCs had an RCHPP of -9.0 +/- 1.9 mm.

Despite these improvements, there were no significant differences in ladder climbing, stride length, overall coordination, or single-frame motion.

Next, Marsala and colleagues showed that the muscle spasms associated with spinal cord injury were slightly decreased by the implantation of HSSCs and not by injection of saline. To measure spasticity, the ankle or front paw is rotated and the electromyograph of the muscle is measured. The electromyograph or EMG measures the electrical activity of the muscle showed modest improvements in the HSSC-injected animals

Sensory sensitivity was improved in the HSSC-injected animals, and this improvement was progressive. When the rats were prodded below the level of the injury, where they should have no feeling, the HSSC-injected rats showed better response to the stimulation. This was the case with mechanical stimulation and thermal stimulation.

Post-mortem analysis also showed something interesting. When the fluid-filled cavity of the damaged spine was examined, the HSSC-injected animals had a significantly small cavity. Because the injected cells had been labeled with green fluorescent protein, they glowed under UV light and any neuronal cells derived from the injected HSSCs glowed green too. The lesioned areas in the HSSC-injected mice were repopulated with cells. Motorneurons, interneurons and glial cells were detected.

What to make of this study? The repopulation of the spinal cord and the growth of spinal nerve elements within the fluid-filled cavity is remarkable, but the lack of better motor function is disappointing. The recovery of sensory ability is significant, especially, since it is pretty clearly not due to spinal hypersensitivity.

There are two possibilities for the low motor recovery. First, there is a possibility that the these experiments were not conducted for as long a time period as they needed to be. Since the sensory ability improvement was progressive, maybe the motor recovery was too, perhaps? Secondly, maybe the grow and connection of motor neurons had trouble with the glial scar. Why the sensory nerves did not have such a problem and the motor neurons would is inexplicable at this time. However, another possibility is that the muscular targets of motor neurons are not as obvious in adult animals as they are in a developing animal. Finding ways to “paint” the muscles might be a way to increase motor neuron innervation in the future.

Thus, this cell line, NSI-566 RSC is certainly a potential treatment for spinal cord patients. A phase I trial is in the works.