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Gum Stem Cells
A Scientific Review
Introduction:
The periodontium is the area of specialized tissue surrounding the tooth and anchoring it to the underlying alveolar bone. It is composed of four regions; the cementum, periodontal ligament (PDL), gingival (gums), and the alveolar bone. The cementum is a layer of bone that surrounds the root of the tooth. Its main function is to serve as the site to which the PDL attaches. The other end of the PDL then attaches to the alveolar bone, therefore anchoring the tooth in place. The PDL is composed of cells that have either fibroblastic or osteoblastic properties (Ivanovski et al, 2006).
Figure 1: Cross Section of a Molar and the Surrounding Tissue.
Periodontal disease is a bacterial infection that leads to tissue damage of the periodontium and is the major cause of tooth loss. The PDL goes through limited regeneration and repair if there is no therapeutic involvement. The current therapeutic methods are also somewhat ineffective in that they have variable efficacy as well as safety issues among other things. There are novel approaches to PDL regeneration that involve tissue engineering. It is in these new methods that the potential for the use of stem cells can be applied.
Background:
Stem cell is a broad term that refers to any number of cells that are able to self renew and are not committed to being any one specific cell type. Adult stem cells are multipotent and can differentiate into any number of different cells, though this potential to differentiate is more limited than in embryonic stem cells. However, unlike embryonic stem cells there are fewer ethical and governmental restrictions when dealing with adult stem cells. Also, the use of adult stem cells can prove to be a more practical choice when dealing with tissue regeneration (Ivanovski et al, 2006). Embryonic stem cells also have a high potential for tumorigenesis making them less desirable to use than the adult stem cells. To date, no studies have been done to assess the tumorigenic potential of PDLSC’s.
Mesenchymal stem cells (MSC) differentiate into different types of connective tissue such as muscle, endothelial cells, fibroblasts, osteoblasts, adipocytes and chondroblasts Since, it is these types of cells that make up the tissue of the periodontium, it is determined that the PDL cells are derived from mesenchymal stem cells. The periodontal ligament stem cells (PDLSC) are indeed a specialized MSC’s.
Flow cytometry assays are often done in order to separate and identify certain cell populations. This is achieved by using a laser beam to separate cells based on the specific light-absorbing or fluorescing properties of these cells. The properties that are used to identify the specific populations of cells are cell surface markers that are unique to those populations. These markers are antigens that are expressed on certain cells and not others and can be identified using antibodies that are specific for those antigens. The flow cytometry method often used is Fluorescence-Activated Cell Sorting (FACS) (Nagatomo et al, 2006).
Generally when stem cells differentiate into committed cells, they go through different phases of development. Before they can become fully differentiated cells the stem cells go through an intermediate phase. At this point the cells are somewhat differentiated and are considered progenitor cells. These progenitor cells must undergo further replications and differentiation before they reach the stage at which they are considered fully differentiated and committed cells. The PDLSC differentiate into three different cells in order to make up the tissues of the cementum, PDL, gingival and alveolar bone. These include cementoblasts, fibroblasts and osteoblasts.
Fig 2: Differentiation of adult mesenchymal stem cells and progenitor cells into PDL cells.
Identification and Location of the PDLSC:
Scientists have believed for a couple of decades that there were progenitor cells located within the periodontal ligament that could differentiate into bone, cementum and extracellular matrix of the PDL. This is due to the observation of slow cycling time in populations of cells found in the PDL (McCullough et al, 1985). However, it was not until 2004 that a group of NIH scientists isolated these cells and were able to show that they were indeed stem cells. The PDLSC were isolated from the PDL of wisdom teeth and molars.
The stem cells required to form the types of tissues found in the periodontium, such as the cementum, ligament and bone are of mesenchymal origin. Also, the presence of all these varying cell types in the PDL led scientist to believe that they had a common origin which would be a progenitor stem cell. Therefore, markers for mesenchymal stem cells were used to isolate the stem cells found in the PDL. Progenitor cells were located in the paravascular regions of the PDL that originated from the spaces in the alveolar bone. These cells then differentiate and multiply and move to the different areas near the cementum and alveolar bone.
No specific cell markers are currently known in order to identify the exact location of these stem cells. However STRO-1 was used to identify a general group of mesenchymal stem cells in the paravascular regions of the PDL. STRO-1 is a cell surface marker for mesenchymal stem cells but is not found in hemopoietic stem cells. However, the use of only STRO-1 is not sufficient to identify and locate PDLSC’s. Therefore other MSC surface markers, such as CD146 and CD44 were also used. The cell staining for these various markers confirmed that the PDLSC were mainly located in the paravascular region of the PDL (Gronthos et al, 2006).
Two different cell morphologies were located in the paravascular region of the PDL. The first were cells that elongated nuclei and elongated cytoplasm and were typically found near the blood vessels of the PDL. These cells resemble endothelial cells and therefore could be the reason that studies vary in the number of PDL cells that are stem cells because they could in fact be endothelial cells. The second phenotype of cell seen in the paravascular region of the PDL that stained positive for MSC markers were round or oval shaped. They had prominent nuclei, very little cytoplasm and a great deal of hematoxylin staining. This second phenotype of cells are more likely the stem cells whereas the first type could be cells that have already gone through differentiation (Chen et al, 2006).
Properties of PDLSC:
Self-Renewal:
PDL fibroblast cell populations show a capacity for self-renewal. This is one of the criteria that mark them as stem cells. This self-renewal is seen in the ability for the PDL to rapidly turnover. Though there is little regeneration of the periodontium when there is mechanical damage due to periodontitis, therefore, the self-renewal could be responsible for maintaining the PDL. This ability to self-renew was studied by using colony forming assays. It was found that when the PDL cells were plated at low densities they were able to form colonies successfully (Nagatomo et al, 2006; Seo et al, 2004). Cells were also plated and used in transplantation into NOD/SCID mouse to show that they were able to regenerated issue that had been damaged (Seo et al, 2005).
Multipotency:
Another characteristic of PDLSC’s is that they are able to differentiate into a number of different cells. Their potential to differentiate was studied by running colony forming assays using different induction methods. PDLSC are able to form adipocytes, so adipogenesis was performed in a colony forming assay to determine the potential for the cells to differentiate into these types of cells. This was done by using a adipogenic medium and showed that there was indeed the formation of lipid rich vacuoles within the colonies. Similar colony forming assays were done to study calcification potential. These colony forming assays supported the evidence of stem cells in the PDL because of the cell’s ability to differentiate into different types of cells (Nagatomo et al, 2006).
Markers of PDLSC:
PDLSC are a specialized mesenchymal stem cell (MSC). Therefore, they express markers that are similar to those used to identify MSCs. A specific antigen marker does note exist for MSC’s or PDLSC’s. Therefore, a combination of markers that are found on MSC’s but not on hemopoietic stem cells, must be used in order to identify the cells.
Regulation of Differentiation:
During development, it is believed that the tissue of the periodontium originates from the dental follicle (Chen et al, 2006). Once the periodontium is formed, the exact factors that regulate growth and differentiation are not well understood, yet. There have been several protein factors that have been identified to play a role in regulating the type of tissue that PDL cells can differentiate to (Lallier et al, 2006). The growth factor FGF5, has been shown to induce the maintenance of the naïve state of the PDL cells and causes the cells to proliferate. The growth factor bone morphogenic protein-7 (BMP7) induces the PDL cells to mineralizes at the ends of the ligaments to connect the alveolar bone to the cementum. Growth differentiation factor-5 (GDF5), induces the PDL to differentiate into a non-mineralized ligament that is bundled together to form the body of the ligament. Interestingly, there is evidence that differentiation is regulated differently in progenitor cells of the PDL when compared to the mature cells within the PDL (Lallier et al, 2006). This knowledge is important if one attempts to induce a PDLSC to differentiate to a particular type of cell.
Transplantation:
A long term goal for dentist would be to use tissue engineering techniques and PDLSC to regenerate damaged areas within the oral cavity, in order to restore the original function and connection between the alveolar bone and the cementum in order to prevent tooth loss. PDLSC’s show potential in having an important role in clinical use because they are able to differentiate into the different forms of tissue found within the periodontium. On top of that researchers have been able to identify, isolate and expand ex vivo PDLSC cells in mouse models (Seo et al, 2004; Ivanovski et al, 2006). In these mouse models, PDLSC have been retrieved from PDL tissues that were isolated from an extracted wisdom tooth. The PDL tissue were then treated and expanded to favor the proliferation of PDLSC. Then using techniques that created a favorable microenvironment and with the combination of hydroxyapatite / tricalcium phosphate (HA/TCP) to induce differentiation, these stem cells were injected subcutaneously at the site of damage within the oral cavity to immune deficient mice. The results of these experiments saw evidence for regeneration of the tissue within the periodontium, including the PDL ligament (Seo et al, 2004; Shi et al, 2005) and gave great hope in having cell transplantation from donor to recipient one day. (Fig. 3) However, before such a treatment can be tested on humans, further research with higher animals has to be performed.
Storage of PDLSC:
It has recently been shown that PDLSC can be recovered from periodontal ligament tissues that have been cryopreserved for 3 or 6 months and still retain their stem cell characteristics and ability to regenerate PDL tissue when transplanted into immunocompromised mice. (Seo et al, 2005). The cryopreserved PDLSC also showed that they had maintained the ability to differentiate into its three lineages, showed no difference in histology when compared to the fresh PDL and also expressed positive markers associated with PDLSC. A notable difference was that cryopreserved PDLSC when compared to fresh cultured PDLSC formed less single colonies when cultured. Further research in determining whether it was the time of storage that caused this depletion in colonies and if there is a maximum time limit allowed to store these stem cells is still needed. This evidence of PDLSC being able to be cryopreserved and still maintain their characteristics opens up the possibility of storing these stem cells for an extended period of time in case they are needed in a future transplant to a patient. As an added bonus, the ease in which cryopreservation of the PDL tissue has been performed can give future insight into creating a protocol that can be used for storing other tissues that contain stem cells.
Future Studies:
There still remains a lot that has to be learned about PDLSC. An experiment on the exact number of PDLSC that exist and the exact location of these stem cells within PDL tissues is still needed. The experiments that have been performed so far, have taken PDL tissue from extracted teeth, cultured this tissue to favor the expansion of PDLSC and then identified markers of PDLSC within the culture to guarantee quality. However, a specific structure of the periodontal ligament at the cellular level has not been created. More information is still needed on what conditions and cytokines favor in vivo differentiation of PDLSC into the different types of tissues within the periodontium. Although there are some protein factors that are known to play a role in differentiation of PDLSC, the specific pathway is not understood yet.
Another study that still has to be performed is the discovery of specific markers for PDLSC. Currently, markers that are found within mesenchymal stem cells are used to distinguish PDLSC. These markers are also found at variable levels within the PDL tissue, further illustrating that the PDL tissue is heterogenic and composed of cells that are found at different points of the differentiation process. The heterogeneous nature of the PDL tissue also brings up another important topic that has to be looked into further, which is the difference in the properties of the stem cells in perspective donors and recipients. This difference in properties could be a possible source of an immune response in a case where the PDLSC is transplanted to two people who have a significant difference amongst the properties of the stem cell.
As mentioned earlier, experiments have been done to cryopreserve the PDL while still keeping the tissue viable for later use of the PDLSC’s.. Another piece of information that is still needed is the maximum length of cryopreservation storage that is possible for PDL tissue and still capable of supporting PDLSC that can differentiate into their lineages. Due to the fact that PDL tissue is so easy to obtain from a variety of donors, these cells can be used to optimize cryopreservation techniques and the techniques may later be applied to other types of stem cells. This makes the PDL very important not just for the study of PDLSC’s but also for other types of stem cells. PDLSC are mesenchymal stem cells so studies that are done with them may also apply to other mesenchymal stem cells which are more difficult to obtain due to more invasive procedures. People have their wisdom teeth removed everyday, which opens up an untapped source of stem cells from a variety of donors, without having to face the ethical issues involved with the procurement of many other types of stem cells.
Conclusion:
Periodontal disease is a worldwide problem that affects people of all classes. It is caused by a bacterial infection of the periodontium and results in a degeneration of tissue. Current methods used to repair any damage due to periodontal disease have poor efficacy, can be harmful, painful and expensive. The future therapeutic methods may involve tissue engineering using stem cells found in the Periodontal Ligament. The reason that the PDLSC’s hold such great importance is that most of the cells are able to differentiated into a mixture of periodontal ligament -including the specific fiber bundles that attach tooth to bone - and the mineralized tissue called cementum that covers the roots of our teeth. These cells are also beneficial in a clinical point of view because they are so easily accessible. In theory, people could one day preserve these stem cells and bank them from their own wisdom teeth that they have had extracted. These can then be used later in life to treat advanced periodontal disease. The idea of preserving one’s own cells for later use is the ideal for the future of tissue engineering via stem cells.
References:
- Chen SC, Marino V, Gronthos S, Bartold PM. “Location of putative stem cells in human periodontal ligament.” Journal of Periodontal Research. 2006: 547-553. 2006.
- Gronthos S, Mrozik K, Shi S, Bartold PM. “Ovine periodontal ligament stem cells: Isolation, characterization, and differentiation potential.” Calcif Tissue Int. 79(5): 3120-317, 2006
- Ivanovski S, Gronthos S, Shi S and Bartold PM. “Stem cells in the periodontal ligament.” Oral Disease. 12: 358-363, 2006.
- Lallier TE, Spencer A. “Use of microarrays to find novel regulators of periodontal ligament fibroblast differentiation.” Cell Tissue Res. 327(1) 93-109, 2006.
- McCulloch C.A.G. “Progenitor cell populations in the Periodontal Ligament of mice.” The Anatomical Record. 211: 258-262, 1985.
- Nagatomo K, Komaki M, Sekiya I, Sakaguchi Y, Noguchi K, Oda S, Muneta T, and Ishikawa I. “Stem cell properties of human periodontal ligament cells.” J Periodont Res. 41: 303-310, 2006.
- Seo BM, Miura M, Gronthos S, Bartold PM, Batouoli S, Brahim J, Young M, Robey PG, Wang CY and Shi S. “Investigation of multipotent postnatal stem cells from human periodontal ligament.” Lancet. 364: 149-155, 2004.
- Seo BM, Miura M, Sonoyama W, Coppe C, Stanyon R, Shi S. “Recovery of stem cells from cryopreserved periodontal ligament.” Dent Res. 84(10): 907-912, 2005.
- Shi S, Bartold PM, Miura M, Seo BM, Robey PG, Gronthos S. “The efficacy of mesenchymal stem cells to regenerate and repair dental structures.” Orthod Craniofacial Res. 8: 191-199, 2005.
Acknowledgements
This review was prepared by the following graduate students in the Stem Cell Biology Class, Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey: Mary Dovlatyan, Jorge Lau, Ruchika Mohla (in alphabetical order).
Teaching Assistant: Michael Riccardo
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