Regenerating Whole Teeth With A Tissue-Engineered Scaffold


As tissues go, teeth are relatively simple. They only consist of a few cell types, arranged in a rather straight-forward manner. Therefore, regenerating teeth, while more difficult than it seems, should represent a tractable problem for stem cell biologists and tissue engineers. While some progress has been made, tooth regeneration procedures will require more fine-tuning before they will be hailed as successful.

Tzong-Fu Kuo and others from the School of Veterinary Medicine, National Taiwan University, Taipei, Taiwan have examined the feasibility of whole-tooth regeneration in minipigs. Kuo and his group used a tissue-engineered tooth germ-like construct.

To construct their tooth germ constructs, Kuo and his colleagues extracted dental pulp from upper incisors, canines, premolars, and molars from mature miniature pigs. They grew the dental pulp tissue in culture in order to expand the faster-growing dental pulp stem cells (DPSCs) that can outgrowth everything else from the pulp in culture. They differentiated the DPSCs into odontoblasts, which make the dentine of the tooth, and osteoblasts, which make bone. Kuo’s team also acquired gingival epithelial cells from the gums of the minipigs.

Next the gum epithelial cells, odontoblasts, and osteoblasts were implanted onto the surface (upper, and lower layers, respectively), of a bioactive scaffold. This scaffold had the odontoblasts inside, the osteoblasts outside, and the gum epithelial cells outside the osteoblasts. Then Kuo and his coworkers transplanted these seeded bioactive scaffolds into the tooth sockets of the lower jaw of a minipig whose lower first and second molar tooth germs were removed.

13.5 months after the scaffolds were implanted, seven of eight pigs had formed two new teeth that had crowns, roots, and pulp. When the newly-formed teeth were extracted and sectioned, they had enamel-like tissues, dentin, cementum, odontoblasts, and periodontal tissues.

A fascinating finding in this study was that all the pigs, without exception, had regenerated molar teeth regardless of the original tooth from which the DPSCs were isolated. As an important control, minipigs that had their tooth germs removed or received empty scaffolds did not develop teeth.

This study from Kuo’s laboratory showed that implantation of a tooth germ-like structure can produce a complete tooth can do so successfully and efficiently. This study also established that the location of the implant seemed to deeply influence the morphology of the regenerated tooth.

The Isolation of Dental Stem Cell Lines and How They Repair Teeth


Research teams at INSERM and Paris Descartes University have isolated dental stem cell lines and detailed the natural mechanism by which such cells repair lesions in teeth. This discovery could provide the foundation for therapeutic strategies that mobilize resident dental stem cells and amplify their intrinsic capacity for repair.

Teeth are mineralized organs that are anchored within the gums by roots. The outermost layer of the tooth is the enamel, which is one of the hardest substances in the body. Beneath the enamel is the dentine (sometimes known as the ivory), and the dentin (dentine if you are British) is filled with microscopic tubes that radiate from the inner pulp to the surface. These dentinal tubules contain cells called odontoblasts, which are the cells that made the dentin in the first place, and dentinal fluid (contains a mixture of albumin, transferrin, tenascin and proteoglycans). The odontoblasts maintain the dentin, but as we age, the dentin tubules calcify. Dentin is a yellowish, bone-like matrix that is porous. It is softer than enamel and decays more rapidly and is subject to severe cavities if not properly treated. Beneath the dentin is the pulp, which contains blood vessels and nerves and also houses a resident stem cell population.

Tooth Anatomy

When a dental lesion appears, the dormant stem cells in the pulp awaken and try to repair the tooth, but the means by which these cells do this is unknown.

To address these gaps in our knowledge, researchers from INSERM (French Institute of Health and Medical Research or Institut national de la santé et de la recherche), and the Paris Descartes University have extracted and isolated dental stem cells from the pulp of mouse molars and analyzed them further.

In the midst of their characterization of these cells, the French teams discovered that these dental pulp stem cells possess five different cell surface receptors for the neurotransmitters dopamine and serotonin. The present of these receptors suggested that the response of dental pulp stem cells to tooth injury was mediated by these neurotransmitters. Blood platelets, for example, are activated by binding serotonin and dopamine. Could these dental be activated by similar means?

The first set of experiments examined tooth repair in mice that lacked platelets that produced serotonin or dopamine. Such mice failed to repair tooth lesions, suggesting that serotonin and dopamine are important to inducing stem cell-mediated tooth repair.

Next, these laboratories characterized these five receptors and found that four of them were intimately involved in tooth repair; knocking out any one of the would abrogate tooth repair responses.

“In stem cell research, it is unusual to be simultaneously able to isolate cell lines, identify the markers that allow them to be recognized (here the receptors), discover the signal that recruits them (serotonin and dopamine), and discover the source of that signal (blood platelets). In this work, we have been able, unexpectedly, to explore the entire mechanism,” said Odile Kellermann, the principal author of this work.

Dentists use pulp capping materials like calcium hydroxide and tricalcium phosphate-based biomaterials to repair the tooth and fill lesions. These new findings, however, could produce new therapeutic strategies aimed at mobilizing the resident dental pulp stem cells to magnify the natural reparative capacity of teeth without the use of replacement materials.

Healing Corneal Blindness with Stem Cells from Extracted Teeth


Scientists at the University of Pittsburgh have found a new way to treat corneal blindness, which affects millions of people around the world.

James Funderburgh and his colleagues at the University of Pittsburgh School of Medicine showed that stem cells isolated from the dental pulp of extracted wisdom teeth can be differentiated into specialized cells that can maintain the health and integrity of corneas and rid them of the scars caused by illness or injury that compromise the ability to see clearly.  These cells can be safely injected into the corneas of mice.

According to Funderburgh, who is a professor of ophthalmology, this new approach can replace damaged corneal eye tissue with tissue made from the patient’s own cells rather than cells from a donor. Such a procedure circumvents the problems of immunological rejection that dog the reconstruction of corneal tissue with cells from donors. Furthermore, donor corneas are in short supply in certain parts of the world (e.g., Africa and Asia).

“Our work is promising because using the patient’s own cells for treatment could help avoid these problems,” said Dr. Funderburgh, who is the senior author of a new paper describing the research, in a written statement.

A post-doctoral research fellow in Dr. Funderburgh’s laboratory, Dr. Fatima Syed-Picard, took cells from the pulp of extracted wisdom teeth and chemically processed them to differentiate them into specialized corneal cells. Then Syed-Picard and others injected these “keratocytes” into the corneas of healthy mice. Once in the eyes of laboratory mice, the tooth pulp-derived cells integrated with the existing tissue with no sign of rejection even after several weeks.

Could such a treatment work in human patients? “We are thinking that in the future people may ‘bank’ their extracted wisdom teeth or the cells from those teeth,” Funderburgh told The Huffington Post in an email. “For someone who did not do that it is possible to extract dental pulp with a root canal procedure, but this is still hypothetical. In the worst-case scenario, someone might consider having a tooth extracted to provide cells for this procedure.”

Last year more than 70,000 corneal transplants were performed in the U.S., According to Kevin Corcoran, president and CEO of the Washington, D.C.-based Eye Bank Association of America (EBAA), there were more than 70,000 corneal transplants performed in the US alone.

“There’s a lot of exciting research being done in the area of [corneal] transplant, and EBAA is interested in any outcome that can help restore sight to the blind or visually impaired,” said Corcoran, who was unfamiliar with the Pitt research.

Dr. Syed-Picard stressed that this research is still in the formative stages and the results are preliminary, and added that it would probably take a few years before human testing could begin. The next step, she said, would be to conduct a similar set of experiments in rabbits.

Gum Nerve Cells Become Tooth-Specific Mesenchymal Stem Cells


Stem cells self-renew and also produce progeny that differentiate into more mature cell types. The neurons and glia that compose nervous systems are examples of mature cells and these cells can be produced from embryonic stem cells, induced pluripotent stem cells, or neural stem cells. However, the reverse does not occur during development; more mature cells do not de-differentiate into less mature cells types. Development tends to be a one-direction event.

However, researchers have now discovered that inside teeth, nervous system cells can transform back into stem cells. This unexpected source of stem cells potentially offers stem cell scientists a new starting point from which to grow human tissues for therapeutic or research purposes without using embryos.

“More than just applications within dentistry, this finding can have very broad implications,” says developmental biologist Igor Adameyko of the Karolinska Institute in Stockholm, who led this new work. “These stem cells could be used for regenerating cartilage and bone as well.”

The soft “tooth pulp” in the center of teeth has been known to contain a small population of tooth-specific mesenchymal stem cells, which can typically differentiate into tooth-specific structures, bones, and cartilage. However, no one has conclusively determined where these stem cells came from. Adameyko hypothesized that if he could trace their developmental lineage, he should be able to recapitulate their development in the laboratory. This might offer new ways of growing stem cells for tissue regeneration.

Adameyko and his and colleagues had already studied glial cells, which are nervous system cells that surround neurons and support them. Several of the nerves that wind through the mouth and gums help transmit pain signals from the teeth to the brain are associated with glial cells.

Adameyko and others used fluorescent labels to mark the glial cells in the gum. When the gum-specific glial cells were observed over time, some of these cells migrated away from neurons in the gums into teeth, where they differentiated into mesenchymal stem cells. These same cells then matured into tooth cells. This work was reported in the journal Nature.

a–c, Incisor traced for 3 days from adult PLP-CreERT2/R26YFP mouse. Note protein gene product 9.5 (PGP9.5)+ nerve fibres (a). b, c, Magnified areas from a. d, e, Incisor traced for 30 days from adult PLP-CreERT2/R26YFP mouse. Note collagen IV+ blood vessels (d). e, YFP+ odontoblasts and adjacent pulp cells. f, Incisor traced for 30 days from Sox10-CreERT2/R26YFP mouse. g–k, Incisor traced for 40 days from PLP-CreERT2/R26Confetti incisor. h–j, Magnified areas from g. Arrow in h indicates a cluster of odontoblasts; arrow in j points at CFP+ and RFP+ cells in proximity to a cervical loop at the base of CFP+ and RFP+ streams shown in g and i. k, Streams of CFP+ and RFP+ pulp cells next to i and j. l, m, Incisor traced for 40 days from PLP-CreERT2/R26Confetti mouse with YFP+ and RFP+ pulp cells adjacent to clusters of odontoblasts with corresponding colours. m, Magnified region from l. n, Stream of pulp cells (arrows) in proximity to the cervical loop; yellow and red isosurfaces mark YFP+ and RFP+ cells. o, p, Progenies of individual MSCs intermingle with neighbouring clones in pulp (o) and odontoblast layer (p), projections of confocal stacks. q, r, Clonal organization of mesenchymal compartment in adult incisor. a–n, Dotted line, enamel organ and mineralized matrix. Scale bars, 100 µm (a, d, f, g, k, l); 50 µm (b, c, e, m–p). CL1 and CL2 indicate labial and lingual aspects of cervical loop. d.p.i., days post-injection. s, Incidence of mesenchymal clones depending on fraction of odontoblasts within the clone. t–v, Proximity of dental MSCs (dMSCs) to cervical loop (CL) correlates with clonal size and proportion of odontoblasts in clone.
a–c, Incisor traced for 3 days from adult PLP-CreERT2/R26YFP mouse. Note protein gene product 9.5 (PGP9.5)+ nerve fibres (a). b, c, Magnified areas from a. d, e, Incisor traced for 30 days from adult PLP-CreERT2/R26YFP mouse. Note collagen IV+ blood vessels (d). e, YFP+ odontoblasts and adjacent pulp cells. f, Incisor traced for 30 days from Sox10-CreERT2/R26YFP mouse. g–k, Incisor traced for 40 days from PLP-CreERT2/R26Confetti incisor. h–j, Magnified areas from g. Arrow in h indicates a cluster of odontoblasts; arrow in j points at CFP+ and RFP+ cells in proximity to a cervical loop at the base of CFP+ and RFP+ streams shown in g and i. k, Streams of CFP+ and RFP+ pulp cells next to i and j. l, m, Incisor traced for 40 days from PLP-CreERT2/R26Confetti mouse with YFP+ and RFP+ pulp cells adjacent to clusters of odontoblasts with corresponding colours. m, Magnified region from l. n, Stream of pulp cells (arrows) in proximity to the cervical loop; yellow and red isosurfaces mark YFP+ and RFP+ cells. o, p, Progenies of individual MSCs intermingle with neighbouring clones in pulp (o) and odontoblast layer (p), projections of confocal stacks. q, r, Clonal organization of mesenchymal compartment in adult incisor. a–n, Dotted line, enamel organ and mineralized matrix. Scale bars, 100 µm (a, d, f, g, k, l); 50 µm (b, c, e, m–p). CL1 and CL2 indicate labial and lingual aspects of cervical loop. d.p.i., days post-injection. s, Incidence of mesenchymal clones depending on fraction of odontoblasts within the clone. t–v, Proximity of dental MSCs (dMSCs) to cervical loop (CL) correlates with clonal size and proportion of odontoblasts in clone.

Before this experiment, it was generally believed that nervous system cells were unable to de-differentiate or revert back to a flexible stem cell state. Therefore, Adameyko said that it was very surprising to see such a process in action. He continued: “Many people in the community were convinced … that one cell type couldn’t switch to the other. But what we found is that the glial cells still very much maintain the capacity” to become stem cells. If stem cell researchers and physicians could master those chemical cues in the teeth pulp that signals glial cells to transform into mesenchymal stem cells, they could generate a new way to grow and make stem cells in the lab.

“This is really exciting because it contradicts what the field had thought in terms of the origin of mesenchymal stem cells,” says developmental biologist Ophir Klein of the University of California, San Francisco, who was not involved in the new work. But it’s also just the first step in understanding the interplay between the different cell populations in the body, he adds. “Before we really put the nail in the coffin in terms of where mesenchymal stem cells are from, it’s important to confirm these findings with other techniques.” If that confirmation comes, a new source of stem cells for researchers will be invaluable, he says.

Stem Cells From Teeth Make Brain-Like Cells


Researchers from the Centre for Stem Cell Research at the University of Adelaide have shown that stem cells taken from teeth can differentiate in culture into cells that resemble brain cells. This work suggests that stem cells from teeth might someday be sources of regenerative material to treat brain-specific maladies, such as stroke, for instance.

According to Dr. Kylie Ellis, Commercial Development Manager with the University’s commercial arm, Adelaide Research & Innovation (ARI), these stem cells do not form full-fledged neurons, but it is only a matter of time before this group figures out the right culture conditions that will make these cells form true neurons.

“Stem cells from teeth have great potential to grow into new brain or nerve cells, and this could potentially assist with treatments of brain disorders, such as stroke,” said Ellis. This work has been published in the journal Stem Cell Research & Therapy.

“The reality is, treatment options available to the thousands of stroke patients every year are limited,” Dr. Ellis says. “The primary drug treatment available must be administered within hours of a stroke and many people don’t have access within that timeframe, because they often can’t seek help for some time after the attack.”

“Ultimately, we want to be able to use a patient’s own stem cells for tailor-made brain therapy that doesn’t have the host rejection issues commonly associated with cell-based therapies. Another advantage is that dental pulp stem cell therapy may provide a treatment option available months or even years after the stroke has occurred,” she says.

Dr. Ellis and her colleagues, Professors Simon Koblar, David O’Carroll and Stan Gronthos, have been working on a laboratory-based model for to test potential treatments in humans. Ellis’ initial observations were part of this research venture, when she discovered that stem cells derived from teeth developed into cells that closely resembled neurons.

“We can do this by providing an environment for the cells that is as close to a normal brain environment as possible, so that instead of becoming cells for teeth they become brain cells,” Dr. Ellis says.

“What we developed wasn’t identical to normal neurons, but the new cells shared very similar properties to neurons. They also formed complex networks and communicated through simple electrical activity, like you might see between cells in the developing brain.”

This work with dental pulp stem cells opens up the potential for modelling many more common brain disorders in the laboratory. Such modeling systems could help in developing new treatments and diagnostic or therapeutic techniques for patients.

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?