Tissue engineering can be defined as an interdisciplinary field that applies the principles of engineering and life sciences towards the development of biological substitutes that restore, maintain or improve tissue function (Langer and Vacanti 1993).
Endodontic therapy accounts for a high rate of success in retention of teeth. However, many teeth are not restorable because of apical resorption, fracture, incompletely formed roots or carious destruction of coronal structures. To tackle this issue, a novel approach to restore tooth structure based on biology, i.e., regenerative endodontic procedures by the application of tissue engineering can be carried out. Presently, two concepts exist in regeneration endodontics to treat non-vital infected teeth One is the active pursuit of pulp-dentin regeneration to implant or regrow pulp (tissue engineering technology) and the other, in which new living tissue is expected to form from the tissue present in the teeth itself, allowing continued root development (revascularization) (Banshal and Banshal 2011).
History of Tissue Engineering
The regenerative capacity of a real living creature was recorded as early as 330 BC when Aristotle observed that a lizard could grow back the lost tip of its tail. In the late 1700s, the scientist Spallanzani reported that a newt could regenerate an entire limb. Since then, the study of regeneration in lower life forms has laid the grounds for understanding the regenerative capabilities and potential of humans Bacteria and the single-celled protozoans regenerate complete organisms with each cell division. Many multicellular invertebrates also exhibit extensive regenerative abilities. When cut in half, the Atworm planaria can grow a new head from one piece and a new tail from the other. However, progression up the evolutionary ladder is generally accompanied by a reduction in regenerative capacity.
With the introduction of a new scientific method came new understanding of the natural world. The systematic unravelling of the secrets of biology was coupled with the scientific knowledge of disease and trauma. Artificial or prosthetic materials for replacing limbs, teeth and other tissues resulted in the partial restoration of lost function. Also, the concept of using one tissue as a replacement for another was developed. This concept was termed tissue engineering and was loosely applied to the use of prosthetic devices and the surgical manipulation of tissues. In essence, new and functional living tissue is fabricated using living cells, which are usually associated, in one way or another, with a matrix or scaffolding to guide tissue development (Vacanti 2006). New sources of cells, including many types of stem cells, have been identified in the past several years, igniting a new interest in the field. The emergence of stem cell biology has led to a new term, regenerative medicine and dentistry.
Components of Tissue Engineering
The three key components (Fig. 14.1) for tissue engineering are (Langer and Vacanti 1993).
I. Stem cells—to respond to growth factors
II. Scaffolds to represent extracellular matrix (ECM)
III. Growth factors—signals for morphogenesis
Stem cell biology has become an essential field for the understanding of tissue regeneration and implementation of regeneration. For many years the promise of stem cells has been talked about but, like the child who doesn’t get a penny, we have been left disappointed with heightened expectation and nothing to show. In today’s era, a fascinating area of medical science is stem cell research. The stem cell is the origin of life. As stated first by the great pathologist Rudolph Virchow, “All cells come from pre-existing cells.”
Stem cells can be defined as clonogenic cells capable of both self-renewal and multiple-lineage differentiation by Grothos et al. 2002.
The plasticity of the stem cell defines its ability to produce cells of different tissues (Rendon and Watt 2003). In short, stem cells are unprogrammed cells in the human body that can be described as ‘shape-shifters’. These cells can change into other types of cells.
Types of Stem Cells
• Embryonic stem cells—located within the inner cell mass of the blastocyte stage of development.
• Postnatal cells—that have been isolated from various tissues including bone marrow, neural tissues, dental pulp and periodontal ligament.
Embryonic/fetal Stem Cells
This type of cell is derived from embryos that are typically 4-5 days old called blastocytes. These cells are pluripotent, i.e., they have capacity to form all tissues (MacArthur and Oreffo 2005). Thriving culture of stem cells from human embryos was reported for the first time in 1988 (Thompson et al.
Pluripotent embryonic stem cells could be directly implanted into the patient’s tissues, where they would then differentiate into specific cell types after encountering the appropriate niche (MacArthur and Oreffo 2005).
The greater plasticity of embryonic stem cells makes these cells more valuable among researchers for developing new therapies (Gardner 2002).
The main problem with embryonic stem cells is ethical and legal issues, so now the hot topic among researchers/scientists is stem cell therapy using postnatal stem cells donated either by patients themselves or their close relatives.
Adult Stem Cells/Post-natal Stem Cells
• Autologous stem cells—are obtained from the same individual to whom they will be implanted.
The sources include bone marrow, peripheral blood, fat removed by liposuction, periodontal ligament, oral mucosa and dental pulp.
Various sources for postnatal dental stem cells (Simon et al. 2008):
• Permanent teeth-: Dental Pulp Stem Cells (DPSC): derived from the third molar.
• Deciduous teeth-: Stem cells from human-exfoliated deciduous teeth (SHED), i.e., stem cell present within the pulp tissue of deciduous teeth.
• Periodontal ligament-: periodontal ligament stem cells (PDLSCs).
• Stem Cells from Apical Papilla (SCAP).
• Stem cells from a supernumerary tooth—mesiodens or stem cells from teeth extracted for orthodontic purposes.
• Dental Follicle Progenitor Cells (DFPCs) (Vacanti 2006).
• Stem cells from human Natal Dental Pulp (hNDP).
Unique characteristics of adult stem cells (Langer and Vacanti 1993):
1) They exist as undifferentiated cells and maintain this phenotype by the environment and/or the adjacent cell populations until they are exposed to and respond to appropriate signals
2) They have an ability to self-replicate for prolonged periods.
3) They maintain their multiple differentiation potential throughout the life of the organism.
Newly discovered ‘chameleon’ cells are modified by the environment when the cells cross the border between two tissues (Grove 1921).
Dental Pulp Stem Cells (DPSCS)
The first type of dental pulp stem cell was isolated from the human pulp tissue and termed as ‘post-natal’ Dental Pulp Stem Cells (DPSCs) by Gronthos et al. 2000. The stem cell population in the pulp is tiny: approximately 1% of the total cells (Smith et al. 1995).
Isolation of heterogeneous populations of DPSCs
One crucial feature of pulp cells is their odontoblastic differentiation potential which is why they are called odontoblastoid cells, as these cells appear to synthesize and secrete dentin matrix like the odontoblasts cells they replace. Human pulp cells can be induced in vitro to differentiate into cells of odontoblastic phenotype, characterized by polarized cell bodies and accumulation of mineralized nodules (Couble et al. 2000). DPSCs isolated with enzyme treatment of pulp tissues form CFU Fs with various characteristics (Gronthos et al. 2000; Huang et al. 2006). If seeded onto dentin, some DPSCs are capable of generating new stem cells or multilineage differentiation into odontoblasts, adipocytes and neural-like cells. This stem cell behavior occurs following cryopreservation, signifying the potential use of frozen tissues for stem cell isolation (Zhang et al. 2006). Pulp cells can proliferate and differentiate into odontoblast-like cells processes, extending into dentinal tubules when in contact with chemo-mechanically treated dentine surface in an in vitro situation. This is a requirement for the secretion of new dentine (Huang et al. 2006).
Transplanted ex vivo expanded DPSCs mixed with hydroxyapatite/tricalcium phosphate (HA/ TCP) form ectopic pulp-dentin like tissue complexes in immunocompromised mice (Gronthos et al. 2000; Batouli et al. 2003).
These pools of heterogeneous DPSCs form vascularized pulp-like tissue and are surrounded by a layer of odontoblast-like cells expressing dentin sialophosphoprotein (DSPP), which produces dentin containing dentinal tubules similar to those in natural dentin. Over time, the amount of dentin gets thicker. When DPSCs are seeded onto human dentin surfaces and implanted into immunocompromised mice, a reparative dentin-like structure is deposited on the dentin surface (Batouli et al. 2003).
Carinci and his colleagues identified a subpopulation of stem cells from human dental pulp with osteogenic potential forming bonelike tissue in vivo. They termed these cells ‘Osteoblasts Derived from Human Pulpar Stem Cells’ (ODHPSCs) and used microarrays to compare the genetic profiles of these cells with those of normal osteoblasts (Carinci et al. 2008).
Human Dental Pulp Stem Cells (hDPSCs) have been isolated from adult tooth pulp of the third molars. Takeda et al. in 2008 successfully isolated hDPSCs from developing third molars, extracted before their eruption. They observed that the hDPSCs separated from the crown—completed stage showed higher proliferation potential compared with those from later stages. Combined with previous data (Batouli et al. 2003), these facts suggest that the dental pulp tissues contained stem cells capable of generating dentin-like structures, even when isolated at the early stage of tooth development.
Dental pulp cells can be reprogrammed into induced-pluripotent stem cells (iPS) at a higher rate compared with other cell types of human origin tried so far (Yan et al. 2010).
Stem Cells From Exfoliated Deciduous Teeth (SHED)
Types of stem cells from exfoliated deciduous teeth include:
• Adipocytes: they can be used to treat cardiovascular disease, spine and orthopedic conditions, congestive heart failure, Crohn’s disease, and to be used in plastic surgery (Perry et al. 2008).
• Chondrocytes and Osteoblasts: have successfully been used to grow bone and cartilage suitable for transplant (Miura et al. 2003).
• Mesenchymal: have the potential to treat degenerative neuronal disorders such as Alzheimer’s and Parkinson’s diseases, cerebral palsy (Perry et al. 2008).
Potential Clinical Applications of Stem Cell Therapy with SHED
This comprehensive list of diseases and conditions currently being treated using stem cells include Stem Cell Disorders, Acute and chronic Leukaemias, Myeloproliferative Disorders, Myelodysplastic Syndromes, Lymphoproliferative Disorders, Inherited Erythrocyte Abnormalities, Liposomal Storage Diseases, Histiocytic Disorders, Phagocyte Disorders, Congenital Immune System Disorders, Inherited Platelet Abnormalities, Plasma Cell Disorders and malignancies (Mao et al. 2006).
Advantages of banking SHED cells
• Provides a guaranteed matching donor (autologous transplant) for life. There are many advantages of autologous transplantation including no immune reaction and tissue rejection of the cells, no immunosuppressive therapy needed, and significantly reduced risk of infectious diseases.
• Saves cells before natural damage occurs.
• Painless and straightforward for both child and parent.
• Less than one-third of the cost of cord blood storage.
• SHED are adult stem cells and are not the subject of the same ethical concerns as embryonic stem cells (Jay 2008).
• SHED cells are complementary to stem cells from cord blood. While cord blood stem cells have proven valuable in the regeneration of blood cell types, SHED are able to regenerate solid tissue types that cord blood cannot—such as potentially repairing connective tissues, dental tissues, neuronal tissue and bone (Mao et al. 2006).
• SHED may also be useful for close relatives of the donor such as grandparents, parents, uncles and siblings (Jay 2008).
Collection, Isolation, and Preservation of SHED
The technique is non-invasive and straightforward involving collection, isolation and storage of SHED.
Step 1: Tooth Collection (Casagrande et al. 2010)
Since, SHED banking is a proactive decision made by the parents, the first step is to inform them to keep the tooth (fulfilling the above mentioned criteria) in sterile saline solution and inform the tooth bank or attending dentist of the bank The tooth exfoliated should have a pulp red in color, indicating that the pulp received blood flow up until the time of removal, which is indicative of cell viability. If the pulp is gray, the blood flow to the pulp has likely been compromised, and thus the stem cells are susceptibly necrotic and are no longer viable for recovery. Teeth that have become very mobile, either through trauma or disease (e.g., Classes III or IV mobility), often have a severed blood supply, and are not candidates for stem cell recovery. This is why recovery of stem cells from primary teeth is preferred after extraction than the tooth that is ‘hanging on by a thread’ with mobility. Pulpal stem cells should not be harvested from teeth with apical abscesses, tumors or cysts.
In the event of a scheduled procedure, the dentist visually inspects the freshly-extracted tooth to confirm the presence of a healthy pulpal tissue and the tooth or teeth is transferred into the vial containing a hypotonic phosphate-buffered saline solution, which provides nutrients and helps to prevent the tissue from drying out during transport (up to four teeth in the one vial). Placing a tooth into this vial at room temperature induces hypothermia.
The vial is then carefully sealed and placed into the thermite, a temperature phase change carrier, after which the carrier is then placed into an insulated metal transport vessel. The thermette, along with the insulated transport vessel maintains the sample in a hypothermic state during transportation. This procedure is described as sustentation
Store-A-Tooth, a company involved in tooth banking, uses the Save-A-Tooth device same as that used for transportation of avulsed teeth for transporting stem cells from the dental office to the laboratory.
The viability of the stem cells is both time and temperature-sensitive, and careful attention is required to ensure that the sample will remain viable. The time from harvesting to arrival at the processing storage facility should not exceed 40 hours.
The same steps are performed by the attending assistant of the tooth bank, if it is not a scheduled extraction for the collection of specimens.
Step 2: Stem Cell Isolation (Freshney et al. 2007):
When the tooth bank receives the vial, the following protocol is followed.
A) The tooth surface is cleaned by washing three times with Dulbecco’s Phosphate Buffered Saline without Ca++ and Mg++ (PBSA).
B) Disinfection is done with disinfection reagent such as povidone iodine and again washed with PB SA.
C) The pulp tissue is isolated from the pulp chamber with small sterile forceps or dental excavator. Stem cell-rich pulp can also be flushed out with salt-water from the center of the tooth.
D) Contaminated pulp tissue is placed in a sterile petri dish which is washed at least thrice with PB SA.
E) The tissue is then digested with collagenase Type I and Dispase for 1 hour at 37°C. Trypsin-EDTA can also be used.
F) Isolated cells are passed through a 70 µm filter to obtain single-cell suspensions.
G) Then the cells are cultured in a Mesenchymal Stem Cell Medium (MSC) medium which consists of alpha modified minimal essential medium with 2 mM glutamine and supplemented with 15% fetal bovine serum (FBS), 0.1 Mm L-ascorbic acid phosphate, 100 U/ml penicillin and 100 ug/ ml streptomycin at 37°C and 5% CO2 in air. Usually, isolated colonies are visible after 24 hours.
H) Different cell lines can be obtained such as odontogenic, adipogenic and neural by making changes in the MSC medium.
I) If cultures are obtained with unselected preparation, colonies of cells with a morphology resembling epithelial cells or endothelial cells can be established. Usually cells disappear during the course of successive cell passages. If contamination is extensive, three procedures can be performed:
1) Retfypsinizing culture for a short time so that only stromal cells are detached because epithelial or endothelial-like cells are more strongly attached to the culture flask or dish.
2) Changing the medium 4-6 hours after subculture because stromal cells attach to the culture surface earlier than contaminating cells.
3) Separate stem cells using Fluorescence Activated Cell Sorting (FACS), in which STRO-1 OR CD 146 can be used. This is considered the most reliable.
Confirmation of the current health and viability of these cells are given to the donor’s parents.
Step 3: Stem Cell Storage
In the light of present research, either of the following two approaches is used for stem cell storage.
b) Magnetic freezing
It is the process of preserving cells or whole tissues by cooling them to sub-zero temperatures. At these freezing temperatures, biological activity is stopped, as are any cellular processes that lead to cell death (Oh et al. 2005). SHED can be successfully stored long-term with cryopreservation and remain viable for use. These cells can be cryopreserved for an extended period of time, and when needed, carefully thawed to maintain their viability. Cells harvested near the end of log-phase growth (approximately 80-90% confluent) are best for cryopreservation. The sample is divided into four cryo-tubes, and each part is stored in a separate location in a cryo-genic system so that even in the unlikely event of a problem with one of storage units, there will be another sample available for use. The cells are preserved in liquid nitrogen vapor at a temperature of less than –150°C. This protects the cells and maintains their latency and potency. In a vial, 1-2 x 10 cells in 1.5 ml of freezing medium is optimum. Too low or high cell number may decrease recovery rate.
Suchânek et al. (2007) established a protocol of Dental Pulp Stem Cells (DPSCs) isolation and to cultivate DPSCs either from adult and exfoliated tooth and compared these cells with Mesenchymal Progenitor Cell (MPCs) cultures. The results proved that the DPSCs and MPCs were highly proliferative, clonogenic cells that can be expanded beyond Hayflick’s limit and remain cytogenetically stable.
Hiroshima University uses magnetic freezing rather than cryogenic freezing. This technology is called CAS and exploits the little-known phenomenon that applying even a weak magnetic field to water or cell tissue will lower the freezing point of that body by up to 6-7 degrees celsius. The idea of CAS is to completely chill an object below the freezing point without freezing occurring, thus ensuring, distributed low temperature without the cell wall damage caused by ice expansion and nutrient drainage due to capillary action, as generally caused by conventional freezing methods. Then, once the object is uniformly chilled, the magnetic field is turned off, and the object snap-freezes (TT-450—Stem Cells and Teeth Banks—www.japaninc.com/tt450).
The Hiroshima University company was the first to formulate this new technology. Using CAS, Hiroshima University claims that it can increase the cell survival rate in teeth to a high of 83%. This compares to 63% for liquid nitrogen (-196 degrees C), 45% for ultra-cold freezing (-80 degrees C), and just 21.5% for a household freezer (-20 degrees C) Maintaining a CAS system is a lot cheaper than cryogenics and more reliable as well.
The best candidates for SHED are moderately resorbed canine and incisors with the presence of healthy pulp. In children, other sources of easily accessible stem cells are supernumerary teeth, mesiodens, over-retained deciduous teeth associated with congenitally missing permanent teeth and prophylactically removed deciduous molars for orthodontic indications (Thomson et al. 1998).
According to Miura et al. 2003; Mao et al. 2006; Seo et al. 2008, deciduous tooth stem cells are an easily accessible stem cell source and capable of robust ex vivo expansion for several potential clinical applications.
In vivo characterization of SHED—Production of dentin pulp-like structures but without a complex formation. Ex vivo expanded SHED transplanted into immunocompromised mice yield human-specific odontoblast-like cells directly associated with a dentin-like structure. The regenerated dentin expresses dentin-specific DSPP. However, unlike DPSCs, SHED is unable to regenerate a complete dentin-pulp like complex in vivo (Miura et al. 2003).
Osteo-inductive capacity—One striking feature of SHED is that they are capable of inducing recipient murine cells to differentiate into bone-forming cells, which is not a property attributed to DPSCs following transplantation in vivo. When single-colony-derived SHED clones were transplanted into immunocompromised mice, only one-fourth of the clones had the potential to generate ectopic dentin-like tissue equivalent to that produced by multicolony-derived SHED (Miura et al. 2003).
However, all single-colony derived SHED clones tested are capable of inducing bone formation in immunocompromised mice. While SHED could not differentiate directly into osteoblasts, they appeared to induce new bone formation by forming an osteo-inductive template to recruit murine host osteogenic cells (Miura et al. 2003). With the osteo-inductive potential, SHED can repair critical-sized calvarial defects in mice with substantial bone formation (Seo et al. 2008). These findings imply that deciduous teeth may not only provide guidance for the eruption of permanent teeth, as generally assumed, but may also be involved in inducing bone formation during the eruption of permanent teeth.
Zheng et al. 2009 provided the first evidence that stem cells derived from miniature pig deciduous teeth (SPD) are capable of regenerative critical-size defects in the oro-facial bone in large animal models, specifically swine, and may potentially serve as an alternative stem-cell-based approach in the reconstruction of alveolar and orofacial bone defects.
SHED appear to represent a population of multipotent stem cells that are perhaps more immature than other post-natal stromal stem cell populations. SHED express neuronal and glial cell markers, which may be related to the neural-crest-cell origin of the dental pulp (Chai et al. 2000). Scientists believe that these stem cells behave differently than post-natal (adult) stem cells. SHED are capable of extensive proliferation and multipotent differentiation, which makes them an essential resource of stem cells for the regeneration and repair of craniofacial defects, tooth loss and bone regeneration (Chai et al. 2000).
Periodontal Ligament Stem Cells (PDLSCs)
This was first identified by Seo et al. 2004. Earlier evidence has shown that PDL contains cell populations that can differentiate into either cementum-forming cells (cementoblasts) or bone-forming cells (osteoblasts) (Isaka et al. 2001).
The presence of multiple cell types within PDL suggests that this tissue contains progenitor cells that maintain tissue homeostasis and regeneration of periodontal tissue. Enzyme digestion treatment of PDL releases a population of clonogenic cells with characteristics of postnatal stem cells (Seo et al. 2004).
The successful isolation and characterization of PDLSCs have led to the identification of tendon MSCs by the same approaches (Bi et al. 2007).
Instead of forming the entire tooth, even a bio-root with periodontal ligament tissues has been generated by utilizing SCAP along with the PDLSCs. This bio-root is encircled with periodontal ligament tissue and has a natural relationship with the surrounding bone.
Stem Cells from Apical Papilla (SCAP)
This was first identified by Sonoyama et al. 2008. Dental stem cells can also be extracted from the apical papilla of exfoliated primary teeth (SCAP) (Bluteau et al. 2008).
Apical papilla refers to the soft tissue at the apices of developing permanent teeth (Sonoyama et al. 2006). Apical papilla is more apical to the epithelial diaphragm, and there is an apical cell-rich zone lying between the apical papilla and the pulp (Rubio et al. 2005).
Similar to DPSCs and SHED, ex vivo expanded SCAP can undergo odontogenic differentiation in vitro. SCAP express lower levels of DSP, in comparison with DPSCs. SCAP represent early progenitor cells.
Conservation of these stem cells when treating immature teeth may allow the continuous formation of the root to its completion.
The tissue is loosely attached to the apex of the developing root and can be easily detached with a pair of tweezers. Importantly these are stem/progenitor cells located in both dental pulp and the apical papilla, but have somewhat different characteristics. Because of the apical location of the apical papilla, this tissue may be beneficial by its collateral circulation, which enables it to survive during the process of pulp necrosis.
The Potential Role of SCAP in Continued Root Formation: The role of the apical papilla in root formation may be observed in clinical cases. An immature human incisor was injured and the crown fractured with pulp exposure. During treatment, the apical papilla was retained while the pulp was extirpated. The continued root-tip formation was observed after root canal treatment. But still further investigation is needed to verify the radiographic evidence of continued apical papilla or if it is merely cementum formation. Although the finding suggests that apical root papilla is likely to play a pivotal role in root formation (Seo et al. 2007).
The Potential Role of SCAP in Pulp Healing and Regeneration: The open apex provided an excellent communication from the pulp space to the periapical tissues; therefore, it may be possible for periapical disease to occur while the pulp is only partially necrotic and infected. Along the same line of reasoning, stem cells in pulp tissue and in apical papilla may also have survived the infection and allowed regeneration of pulp and root maturation to occur. The infection could have spread through survived pulp tissue reaching the periapex. It should be noted that prolonged infection may eventually lead to total necrosis of the pulp and apical papilla; under these conditions, apexogenesis or maturogenesis, a term that encompasses not just the completion of root-tip formation but also the dentin of the root, would then be unlikely.
The Potential Role of SCAP in Replantation and Transplantation: The fate of human pulp space after dental trauma has been observed in clinical radiographs. Andreasen et al. 1995 and Kling et al. 1986 showed excellent radiographic images of the ingrowth of bone and periodontal ligament (PDL) (next to the inner dentinal wall) into the canal space with arrested root formation after the replantation of avulsed maxillary incisors, suggesting a complete loss of the viability of pulp, apical papilla and/or HERS. Some cases showed the partial formation of the root accompanied with ingrowth of bone and PDL into the canal space, and in some cases the teeth continued to develop roots to their completion, suggesting that there was partial or total pulp survival after the reimplantation (Seo et al. 2008).
SCAP can be used for bioroot engineering, in one of the animal studies, it showed negative results, whereas another animal study showed 33.3% success rate (Seo et al. 2008).