STEM CELLS IN BIOLOGICALLY- ENGINEERED TEETH BY ELIZABETH BLYTH. Grade awarded: Pass with Distinction

Transcription

1 STEM CELLS IN BIOLOGICALLY- ENGINEERED TEETH BY ELIZABETH BLYTH Grade awarded: Pass with Distinction RESEARCH PAPER BASED ON PATHOLOGY LECTURES AT MEDLINK and VET- MEDLINK 2014

2 Abstract This paper discusses the role of stem cells in biologically- engineered teeth ('bioteeth'). Bioteeth could become an attractive alternative to implants, dentures and bridges. Epithelial and mesenchymal cells can be combined to produce a tooth germ which can then be transplanted into the adult jaw to develop into a biotooth. One approach is to re- create the process of human odontogenesis. However a source of accessible epithelial and mesenchymal cells must be located. In 2013 a significant development was made in showing how human gingival epithelial cells could be combined to create a biotooth, and I propose that a population of dental stem cells would be the ideal source of mesenchyme. Another interesting approach is to research the mechanism by which teeth are seen to grow in the animal kingdom and stimulate this in humans; some species grow new teeth continuously throughout their lives. Finally the paper considers the challenges that still surround whole- tooth engineering. Introduction Stem cell research is an extremely exciting and topical field of scientific study. Stem cells are undifferentiated cells that are able to differentiate to form both specialised cells and more stem cells. They have the potential to aid treatments in many different areas of medicine because of two unique properties: they are unspecialised, which means they are able to differentiate into cells with specialised functions, and they are able to proliferate, meaning they can indefinitely divide to form more stem cells. There are two main types of stem cells: embryonic stem cells (from the embryo) and adult/somatic stem cells (from adult tissues). Stem cells can be categorised into groups depending on their ability to differentiate into other cell types, which range from unipotent stem cells (can divide to form only cells of their own type but can self- renew, e.g. skin cells), to multipotent stem cells (able to differentiate to form different cell types which are closely related, e.g. hematopoietic stem cells, which can only form blood cells), to totipotent stem cells (can give rise to all cell types, e.g. the zygote). Numerous populations of dental stem cells have been located which have multipotent abilities. This has posed the possibility of using stem cells to grow biologically- engineered teeth ('bioteeth'). With the correct growth factors, a combination of epithelial and mesenchymal cells has been shown to successfully develop into a tooth, but before whole- tooth engineering becomes a clinical reality we must locate an ethical and accessible source of epithelium and mesenchyme. Consequently populations in the oral cavity hold exciting prospects for use in bioteeth. If autologous gingival epithelial cells and dental stem cells could be combined to generate a biotooth by re- creating the natural process of human odontogenesis, bioteeth will turn from an unrealistic ideal into a feasible treatment option, and will hence transform modern dentistry. Teeth are often lost, commonly due to trauma, carious disease or periodontal disease. With the mean number of teeth amongst dentate adults in the United Kingdom being only 25.7 in 2009, clearly it is essential that we can replace lost teeth with functional, aesthetically- appropriate and durable new ones. A biologically- engineered tooth that is fully integrated in the jaw with a natural root structure and periodontal 2

3 ligament would undoubtedly be a better option than the prosthodontic devices currently available: dental implants, fixed partial dentures (bridges) or removable partial/complete dentures. Implants sometimes cause Peri- implantitis (an infection of the area which results in loss of surrounding bone). In an implant there is no natural connection between the jaw and the tooth with a periodontal ligament, meaning the implant is less stable and has a reduced life- span. The natural root structure of a canal with nerves and blood supply is not achieved with an implant because they are just screwed directly into the jaw. Fixed partial dentures can only be used in certain situations and the teeth either side of the pontic have to be crown- prepared, which may destroy healthy tooth structure. Dentures can make a cheaper solution when multiple teeth have been lost, but patients often find dentures hard to become accustomed to, as the foreign object in their mouths can cause discomfort. Dentures also lead to receding gums and bone loss. It is worth noting that these replacements for lost teeth all have a limited life- span, whereas a biotooth would be as durable as a natural tooth. Discussion There are several different approaches that are being adopted by scientists around the world to create a biotooth fit for the adult oral cavity. I am going to explore two of these approaches: A) re- create the natural process of human odontogenesis, or B) engineer the mechanisms behind the re- growth of teeth in some other animals who shed and re- grow teeth throughout their lives. A) Growing bioteeth by artificially inducing natural human odontogenesis. For odontogenesis, two types of cells are required: epithelial cells and mesenchymal cells. These cells interact using chemicals called growth factors to trigger the process of tooth development. When the foetus is six weeks old, the dental lamina forms. This is a C- shaped structure which arises from the base layer of the oral epithelium. During the bud stage, dental buds grow from the dental lamina. The deep surface of each bud turns in on itself, giving rise to a cap- like structure called the tooth germ (the cap stage). The tooth germ consists of an outer and inner dental epithelium and in between the stellate reticulum (a star- shaped layer of cells). Beneath the inner epithelium is the dental papilla (a small mass of mesenchymal tissue). Next the cap enlarges and elongates, causing the papilla to deepen, which forms a bell- shaped structure (the bell stage). The bell pinches off from the oral epithelium. Subsequently the unspecialised mesenchyme cells in the dental papilla differentiate to form odontoblasts, which give rise to the dentine. The remaining cells in the papilla form the dental pulp. The outer epithelial cells differentiate to form ameloblasts, which produce a layer of enamel on the outer surface of the developing tooth by depositing enamel prisms over the surface of the dentine. Root formation begins when the dental epithelial layers penetrate into the mesenchyme beneath, forming the epithelial root sheath. Odontoblasts lay down more and more dentine around the edges of the pulp chamber, causing it to narrow. Eventually the chamber forms a canal containing blood vessels and the nerves of the tooth. The mesenchymal cells differentiate to form cementoblasts (produce the cementum: a thin layer of bone on the outside of the root), 3

4 fibroblasts (give rise to the periodontal ligament) and osteoblasts (form alveolar bone around the tooth). Finally the tooth will erupt by pushing through the overlying gingival tissue into the oral cavity. In order to develop a biotooth, it would be necessary to assemble mesenchymal cells and epithelial cells in a laboratory to produce an engineered tooth germ, in such a way to mimic the cap stage of tooth development. The mesenchymal tissue would act as the dental papilla, which as described gives rise to odontoblasts, cementoblasts, fibroblasts, osteoblasts and the dental pulp, and the epithelial tissue would serve as a substitute for the dental outer and inner epithelium, which gives rise to ameloblasts. This tooth germ could then be transplanted back into the oral cavity and stimulated to develop into a fully living and natural tooth in the same way that permanent teeth develop in humans. We will now evaluate the potential sources of epithelial and mesenchymal cells that are essential to complete this. Obtaining a source of epithelial cells for whole- tooth engineering: Epithelial dental stem cells from humans have proved hard to source because the epithelial cells that differentiate to form ameloblasts in odontogenesis aren't retained after tooth eruption. There are several alternative populations of epithelial dental cells that could serve as a substitute: - Isolated precursor cells from the tooth germs of young children. In young children, the undifferentiated epithelial cells are still present because the teeth are still developing. Although it is hypothetically possible to create bioteeth using these cells, realistically it is far from practical in a clinical setting, because children would have to undergo surgery to provide cells for an adult patient's dental treatment. - Epithelial stem cells extracted from rodent incisors. Rodent incisors are an exciting prospect because they grow continuously throughout life. This source of stem cells is capable of creating ameloblasts and is highly proliferative. However cells from rodents could not be transplanted into a human mouth for ethical and immune- response rejection reasons. - Cells residing in embryonic oral epithelium. In 2004, these cells were shown to successfully create a tooth primordia which developed to form tooth structures in the adult jaw. Despite this achievement, the ethical controversy surrounding embryonic stem cells deems them inadequate to use in treatments. - Gingival epithelial cells from adult human oral mucosa. Cells from human gingival tissue can be combined with mouse embryonic tooth mesenchyme cells to form teeth. This development is significant because it shows that epithelial stem cells are not necessarily indispensable, and accessible autologous epithelial cells could be used. Obtaining a source of mesenchymal cells for whole- tooth engineering: Additionally a large enough source of mesenchymal cells must be found. There are a number of potential sources. Embryonic stem cells. Dental mesenchymal cells from mouse embryos have been used to create bioteeth, indicating that embryonic stem cells from humans could be used. However treatment would not be viable as embryos would have to be destroyed. 4

5 Stem cells from somatic bone marrow. A relatively large source of multipotent stem cells is found in the bone marrow. Yet the patient would need to have their bone marrow aspirated. No patient would consent to undertaking this complicated and painful to replace a missing tooth. Dental stem cells. Numerous populations of stem cells have been found in the oral cavity: - Adult dental pulp stem cells (DPSC): Stem cells are thought to exist in the dental pulp because the pulp can repair itself throughout its life. Precursors in the dental pulp have been found to form odontoblasts under the correct signals, although they have not yet been properly identified. - Stem cells from the dental follicle (DFSC): Stem cells have been extracted from the follicle of somatic third molars, which can differentiate to form cementoblasts (form cementum) and fibroblasts (form PDL). - Periodontal ligament stem cells (PDLSC): The periodontal ligament is the tissue that connects the cementum with the alveolar bone. It supports the tooth and acts as a shock absorber. Mesenchymal stem cells allow it to regenerate itself throughout life. PDLSC can be stimulated to create cementum and alveolar bone. PDLSC have the possibility to be used to treat currently- untreatable Periodontitis, where the periodontal ligament is destroyed, the alveolar bone recedes and the tooth is free to move in the socket. - Stem cells from the apical part of the papilla (SCAP): Stem cells have been found to exist in the pulp of the pointed terminal end of the tooth. They are easily accessible from adult third molars and have been found to exhibit more rapid growth and a greater capability for tooth formation than other dental stem populations, for example PDLSC. - Stem cells from human exfoliated teeth (SHED): SHED have been isolated from the pulp of human deciduous incisors, and they expressed a high capability to differentiate. SHED are one of the most exciting mesenchymal progenitors because they are so easily accessible. In fact several companies already offer services to harvest and store children's deciduous teeth so the stem cells within can be used in the patient and their family's later medical treatment. Such companies claim that these stem cells might later be able to cure conditions such as heart disease and diabetes, but it would be incredible if they could be used to create new biological teeth for the person they came from. B) Studying tooth regeneration in the animal kingdom. An alternative approach is to research the mechanism behind the re- growth of teeth in other species to see if these can be reproduced in the human oral cavity. Indeed, 5

6 in regenerative medicine it is often extremely valuable to learn about naturally occurring processes in other organisms which we want to stimulate artificially. Some species of reptile and fish grow new teeth throughout their lives. In fact most species show some mechanism of regeneration within the oral cavity. Humans possess several different types of dental stem cells which can differentiate to form types of tissue, but do not have the capacity to generate whole teeth by themselves. Many organisms possess teeth which grow continually throughout their lives, such as the incisors in mice. However again the stem cells present there are not able to undergo whole tooth generation. Whereas mammals can either be monophyodont (having only one permanent set of teeth with no deciduous precursor set) or diphyodont (having two dentitions: a deciduous one followed by a permanent one, as in humans), the dentitions of reptiles and fish can undergo episodic renewal. The American Alligator is one such example, and in the research paper titled 'Specialized stem cell niche enables repetitive renewal of alligator teeth', a team of scientists from America, China and Taiwan researched into how the model of tooth regeneration in the American Alligator could aid us in our quest to stimulate the growth of adult teeth in mammals. The American Alligator is a particularly interesting reptile to study because on top of undergoing life- long renewal, its teeth are in many respects similar to mammalian teeth, unlike many other reptiles, e.g. snakes. Alligators, like mammals, have thecodont teeth, meaning their teeth are situated in alveoli (cavities of the maxillary and mandibular arches that the roots of the teeth are embedded into). Embedded in the alligator's jaw is the current functional tooth, the developing tooth and the dental lamina. Alligators re- grow each tooth approximately 50 times in their lifetime. Using Micro- CT and X- ray scanning, the researchers observed the continuous development of the tooth family unit in the alligator, and structured it into 3 phases: pre- initiation (functional tooth, replacement tooth and undifferentiated dental lamina), initiation (next replacement tooth develops from the distal end of the dental lamina) and growth (loss of the functional tooth, replacement tooth becoming the new functional tooth and development of another replacement tooth from the dental lamina). The dental lamina contains odontogenic stem cells which can give rise to new teeth throughout life. When humans are born, we have a dental lamina of similar structure; however in mature adult dentitions, the few stem cells present are incapable of forming replacement teeth. Current developments To date, the concept of copying the mechanisms of tooth regeneration in animals like the American Alligator has not lead to any biological teeth being engineered. In reality, we are still far from this ultimate goal: not nearly enough is known about the details of these processes, and research in this field is still novel. However there are a handful of examples where biological teeth have been generated through following the mesenchymal and epithelial cell interactions displayed in human odontogenesis. In 2004 significant progress was made: for the first time, the source of mesenchymal cells was obtained from adult non- dental cells, instead of embryonic cells. Embryonic tooth epithelial cells were combined with stem cells from adult bone marrow and the resulting 6

7 primordia were then transplanted into adult renal capsules, which successfully produced developing teeth. In 2013 human gingival epithelial cells were combined with mouse embryonic stem cells to create teeth. Mesenchyme tissue was obtained from the tooth germ of the lower molars of mouse embryos. The human epithelial cells were injected into the top of the mesenchyme tissue, and the cell mass was cultured for a week, by which time tooth structures were clearly visible. The explants were transferred into the renal capsules (tissue around the kidneys) of SCID mice (mice who had no T or B lymphocytes, meaning no rejection could occur following the transplantation of the foreign material). After 6 weeks, following extraction of the kidneys, 20% of the tooth primordia had developed to have an outer enamel- like layer of high density mineralisation, an area of lower density mineralisation (similar to dentin) beneath, a dental pulp- like chamber and an root area. The future of whole- tooth engineering: where next? It is my view that not enough is yet known about tooth regeneration in the animal kingdom for us to be using this knowledge to grow teeth for adult humans. Nevertheless research in this field could still prove to be beneficial to the development of bioteeth because it allows us to see tooth regeneration from a new angle, and to broaden our knowledge about human odontogenesis. In other species, especially those from other classes, for example reptiles, often very different mechanisms for odontogenesis are used, and some aspects of these may be useful in devising an effective method for artificial tooth development. Overall I believe that the benefits of studying tooth regeneration in other species is limited to a certain degree because the tooth structure, arrangement and oral cavity in a species like the American Alligator is different to in humans. This means the environment in which the teeth develop is foreign to us, the mechanisms for tooth growth are unfamiliar and the cell and tissue types present are different to those in humans. Consequently it is clear that concentrating our efforts into stimulating human odontogenesis will be more effective in producing results. In the future, I think that human gingival cells will be the best source of epithelial cells for bioteeth. They could be obtained from the patient themselves rather than an outside source, such as a stem cell bank. This overcomes all problems surrounding rejection of the biotooth. A method of minor surgery would have to be devised to extract the gingival cells with minimal damage to the patient. However, I believe a better solution would be for few cells to be collected on a swab from the outer layer of gingival tissue. These could be sent to a laboratory, where they would divide in vitro by mitosis to form a large enough population of epithelial cells. This would eliminate the need for any oral surgery. It may be problematic to obtain sufficient numbers by this method, however. In my opinion, somatic stem cells from the oral cavity would be the most attractive source of mesenchymal cells for tooth engineering. Firstly they would be autologous (from the patient), meaning that the patient would have to give permission for the cells to be extracted, eliminating any ethical issues. Additionally, being autologous, the cells would have identical antigens to all of the other cells in the patient, so the generated tooth would be considered as 'self' and no rejection would occur. But above all, the advantage of using dental stem cells in generating bioteeth, or for any other medicinal/dental 7

8 treatment is that they are so easily accessible. This eliminates the extraction difficulties associated with other somatic stem cells. Invasive surgery would not be required to extract the mesenchyme, making the treatment a less disruptive experience for the patient. To date it has not been shown that somatic dental stem cells can be used as the mesenchyme for bioteeth, but I imagine if real progress is going to be made in ensuring that bioteeth become a reality, this source will be by far the best to use. SHED are for me a fascinating source because they express true odontogenic potential and absolutely no surgery would be required. I speculate that it would be incredible if our society could get to the stage where a certain number of exfoliated deciduous teeth were collected from each child and stored, frozen, in a central stem cell bank. Then later in life the stem cells could be re- cultured and used for the treatment of the child or a family member; this would be ground- breaking not just in dentistry, but also in various aspects of medicine. However the cost of such a wide- scale operation would be huge, and SHED must be proven to treat a very wide range of conditions before the investment is worthwhile. Conclusions Enormous developments have been made in the past few decades in the field of stem cell technology, and in dentistry such stem cell treatments hold exciting possibilities. If it was possible for both the mesenchymal and epithelial cells required to create the new tooth to be extracted from the patient's oral cavity, bioteeth could certainly become a feasible replacement treatment option for current prosthodontic devices. It would therefore be a much more attractive option to replace a lost tooth with a biologically- engineered tooth that forms a natural connection between the root and the bone with a natural periodontal ligament. A natural tooth would have a much longer life length than an implant, bridge or denture, and would be more durable. Despite these advancements, there are still numerous issues that need to be dealt with before bioteeth become a clinical reality. The main setbacks are listed below: 1. Obtaining a suitable source of epithelial cells and mesenchymal cells. They must be able to be extracted from the patient easily with minimal surgery and without destroying existing tooth structures. 2. Determining whether the generated tooth germs could develop in the adult jaw. Human gingival epithelial cells and mouse embryonic mesenchymal cells grew to form a tooth inside the renal capsules of a mouse, but this environment is very different from that of the human oral cavity. Somehow the same microenvironment that surrounds naturally developing teeth must be stimulated. Ozahama 2004 tranplanted embryonic tooth primordia into the adult jaw, which resulting in defined tooth structures developing, indicating that it is indeed possible for tooth germs to grow outside of their normal environment. 3. The cost factor. Although patients would probably pay slightly more for a biotooth than an implant, clearly no- one would pay in the tens of thousands, which is what it currently costs to grow a tooth in a laboratory. However as with all new 8

9 treatments, the cost of bioteeth will undoubtedly reduce significantly as further developments are made and the technology becomes more refined. 4. The success rate. Bioteeth will only be viable if teeth can be reliably grown every time. The success rate for Angelova Volponi, 2013 was only 20%. Due to these hurdles, whole- tooth engineering is not a treatment that will be making its way onto the market in the near future. Nonetheless, whole- tooth engineering is definitely an exciting ongoing area of research, and one that I would love to be involved with in the future. References 2009 Adult Dental Health Survey Stem cell basics, report by National Institutes of Health (Last updated 2014) Bluteau, G. (2008) Stem Cells for Tooth Engineering Regrowing teeth, report by Chu, J. (2007) teeth/page/2/ What are stem cells? report by Crosta, P. for Medical News Today (2008) Advantages and disadvantages of dentures, report by Duluth GA Dentures dentists.com/blog/dentures/advantages- and- disadvantages- of- dentures/ Langman, J. (1981) Medical embryology, Virginia, Williams and Wilkins Publishing Group, pg Mombelli, A. (1998) The diagnosis and treatment of Peri-implantitis Mrozik, K. (2010) A method to isolate, purify, and characterize human periodontal ligament stem cells Classifying stem cells, report by Murnaghan, I. (2014) Properties of a stem cell, report by Murnaghan, I. (2015) Ohazama, A. (2004) Stem- cell- based tissue engineering of murine teeth Syrbu, J. (2013) The Complete Pre-dental Guide to Modern Dentistry Volponi, A. (2013) Adult Human Gingival Epithelial Cells as a Source for Whole- tooth Bioengineering 9

10 Wu, P. (2013) Specialized stem cell niche enables repetitive renewal of alligator teeth 10