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.
Dr. Jeremy Mao is the Edward V. Zegarelli Professor of Dental Medicine at Columbia University Medical Center. Mao and his colleagues have published a novel technology that includes a growth factor-infused, three-dimensional scaffold that has the potential to regenerate an anatomically correct tooth in the mouth just nine weeks after implantation. By this procedure, which was developed in the university’s Tissue Engineering and Regenerative Medicine Laboratory, Mao can direct the body’s own stem cells to migrate to the scaffold and infiltrate it. Once these stem cells have colonized the scaffold, they will produce a tooth that can grow in the socket and merge with the surrounding tissue and integrate into it.
Mao’s technique not only eliminates the need to grow teeth in a culture, but it can regenerate anatomically correct teeth by using the body’s own resources. If you factor in the faster recovery time and the comparatively natural process of regrowth (as opposed to implantation), you have a massively appealing dental treatment.
Columbia University has already filed patent applications in regard to this technology. They are also seeking associates to aid in its commercialization. Mao is also considering the best approach for applying his technique to cost-effective clinical therapies.
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.
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.
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.
Massive injuries to the face can cause bone loss and “tooth avulsion.” Medically speaking, avulsion simply refers to the detachment of a body structure from its normal location by means of surgery or trauma. Dental implants and help with lost teeth, but if the facial bone has suffered so much loss that you cannot place implants in them, then you are out of luck. Dental prostheses can help, but these do not always fit very well.
Darnell Kaigler and his group at the University of Michigan Center for Oral Health wanted to help a 45-year-old woman who had lost seven teeth and a good portion of her upper jaw bone (maxilla) as a result of massive trauma to the face. This poor lady had some dentures that did not fit well and a mouth that did not work well.
Bone can be grown from stem cells, but getting those stem cells to survive and do what you want them to do is the challenge of regenerative medicine. Therefore, Dr. Kraigler and his group used a new technique to help this young lady, and their results are reported in the December 2014 issue of the journal Stem Cells Translational Medicine.
First, Kraigler and his co-workers extracted bone marrow stem cells from a bone marrow aspiration that was taken from the upper part of the hip bone (the posterior crest of the ilium for those who are interested). They used a product called ixmyelocel-T from Aastrom Biosciences in Ann Arbor , MI. This product is a patient-specific, expanded multicellular therapy, cell-processing system that selectively expands mesenchymal cells, monocytes and alternatively activated macrophages, up to several hundred times more than the number found in the patient’s bone marrow, while retaining many of the hematopoietic cells collected from only a small sample (50ml) of the patient’s bone marrow. Thus, the healing cells from the bone marrow are grown and made healthy, after which the cells were bagged and frozen for later use.
Then the patient was readied for the procedure by having the gum tissue cut and lifted as a flap of tissue (under anesthesia, or course). Then four holes were drilled into the bone and setting screws were inserted. This is an important procedure, because implanted stem cells will not survive unless they have blood vessels that can bring them oxygen and nutrients. By drilling these holes, the tissue responds by making new blood vessels. To this exposed surface, the bone marrow-derived stem cells were applied with a tricalcium phosphate (TCP). TCP is a salt that will induce mesenchymal stem cells to form bone. Once the TCP + stem cell mixture was applied to the gum, a collagen membrane was placed over it, and the gum was then sewn shut with sutures.
Four months later, the patient underwent a cone-beam computed tomography (CBCT) scan. The bone regrowth can be seen in the figure below.
According to the paper, there was an “80% regeneration of the original jawbone.”
Into this newly regenerated bone, permanent dental implants were placed. The results are shown below.
Pardon me, but permit me an unprofessional moment when I say that this is really cool. Of course, this patient will need to be observed over the next several years to determine the longevity of her bone regeneration, but the initial result is certainly something to be excited about.
Tricalcium phosphate or TCP has been used to induce the bone-making activities of mesenchymal stem cells. It has also been used in several animal studies as a delivery vehicle for mesenchymal stem cells (for example, see Rai B, et al., Biomaterials 2010, 31:7960–7970; KrebsbachPH, et al., Transplantation 1997, 63:1059–1069; ZhouJ, et al., Biomaterials 2010, 31:1171–1179). TCP also seems to support stem cell proliferation, survival, and differentiation into bone. Kresbach and others showed that TCP most consistently yielded bone formation when used as a delivery vehicle for mesenchymal stem cells compared to other biomaterials commonly used, such as gelatin sponges and demineralized bone matrix. However, there are no studies that have ascertained how well stem cells attach to TCP, and this attachment is an important factor in determining how many stem cells reach the site of injury. This study by Kaigler and his group (A. Rajan and others) showed that a 30-minute incubation of the cells with TCP gave sufficient attachment of the cells to the TCP for clinical use. The efficiency of this incubation period was also not affected by the temperature.
The other exciting features of this paper, is that most of the materials used in this study were commercially available. The bone marrow stem cell isolation technique was pioneered by DennisJE, and others in their 2007 article in the journalStem Cells (25:2575–2582). Effective commercialization of this technique has shown the efficacy of this procedure for clinical use. This paper also shows the clinical feasibility of using TCP as a delivery vehicle for mesenchymal stem cell-based bone treatments.
In conclusion, I will quote the authors: “Cell survival and seeding efficiency in the context of tissue engineering and cell-therapy strategies are critical parameters for success that have not been rigorously examined in a clinical context. This study defined optimized conditions for these parameters using an autologous stem cell therapy to successfully treat a patient who had a debilitating craniofacial traumatic deficiency. To our knowledge, there have been no other clinical reports of cell therapy for the treatment of craniofacial trauma defects. This clinical report serves as solid foundation on which to develop more expanded studies using this approach for the treatment of larger numbers of patients with other debilitating conditions (e.g., congenital disorders) to further evaluate efficacy and feasibility.”
Several different types of stem cells can regenerate tooth enamel, but regenerating the tooth root has proven quite difficult.
As you can see from the image above, the tooth root is covered with a tough, fibrous covering called the cementum. The cementum connects the tooth root to the alveolar bone of the upper and low jaw by means of the periodontal membrane. the cementum is a thin layer of bone-like material that covers the roots. It is yellowish and softer than either dentine or enamel. It is made by a layer of cementum-producing cells called cementoblasts that are adjacent to the dentine. The periodontal ligament is cellular and its fibers hold the tooth in its socket, which are embedded in the cementum, as shown in the micrograph below. The complexity of this structure shows you why regenerating this structure is so difficult.
Howwever, a new study from the laboratory of Weihua Guo at Sichuan University, China has shown that platelet-rich fibrin (PRF) and treated dentin matrix (TDM) can concentrate a variety of various growth factors that summon native stem cells to them, and induce them to regenerate the tooth root.
Guo’s laboratory examined the ability of PRF and TDM to summon endogenous stem cells to the site of an extracted tooth in order to initiate regeneration of the tooth root. Tooth roots contain soft and hard periodontal tissues, and if periodontal ligament stem cells (PDLSCs) and bone marrow mesenchymal stem cells (BMSCs) could be recruited to the site of tooth extraction by PRF and TDM, then maybe they could initiate tooth root regeneration.
Beagles were used as a transplantation model for this experiment. After tooth extraction PRF and TDM implants were embedded in the tooth socket. Also, these matrices were examined in cell culture with PDLSCs and BMSCs.
PRF significantly recruited and stimulated the growth of both PDLSCs and BMSCs in culture. In combination, PRF and TDM induced cell differentiation of these implanted stem cell populations. PRF and TDM induced the expression of mineralization-related genes, such as bone sialoprotein (BSP) and osteopotin (OPN) after only one week in culture.
When implanted into the tooth sockets of beagles that had teeth extracted, transplantation platelet-rich fibrin made from the dog’s own blood products, and TDM made from other animals into fresh tooth extraction socket successfully regenerated the tooth root 3 months after the surgery. The cementum and periodontal ligament (PDL)-like tissues with properly orientated fibers were clearly present, and the presence of these structures is indicative of functional restoration.
These results suggest that tooth root and the connection of the tooth root to the alveolar bone by cementum and peridontal ligaments can be effectively regenerated through the implantation of PRF and TDM in a tooth socket. It seems to achieve this regeneration by summoning BMSCs and PDLSCs. These cues provided by these matrices and the microenvironment provided by the tooth socket are key factors for this regeneration. This strategy provides a genuine clinical pathway toward tooth root regeneration in human patients with destroying human embryos.
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.
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.