Human Teeth Is Useful Even after Its SHED! So, Why Discard It?

*Meghna Bhandary, Rachaita Chhabra, K. Ananya Rao and Mohammed Shahid*

#### **Abstract**

A few decades ago, if one underwent a knee injury that makes walking painful or had an atrophied kidney, then, he/she was condemned to a life hooked on to machines, or on constant medications. However, in today's era, teeth can be grown in a Petri dish; heart and liver replacements are possible with no risk of rejection because the organs are made of the patient's own cells. This is the promise of regenerative medicine and tissue engineering. The entire idea of regenerative medicine is based on the presence of stem cells in the body or the ability to introduce stem cells into the body without causing harm. These can be obtained from a variety of body and dental tissues. Deciduous teeth often discarded as biological waste is proven to possess Stem cells (SHED) that have promising applications in tissue engineering and regenerative medicine. Hence, their contribution toward the field of regenerative medicine and dentistry is immense. This chapter summarizes SHED's regenerative potentials and therapeutic applications; and also focuses on its potential future scope in regenerative dentistry. Furthermore, procedures involved in SHED-induced therapy, from SHED collection to SHED banking, have also been explained.

**Keywords:** stem cells, stem cell from exfoliated deciduous teeth (SHED), tooth, regeneration, repair, dentin pulp regeneration, therapeutic, biodentine

#### **1. Introduction**

*"The Regenerative Medicine revolution is upon us. Like iron and steel to the industrial revolution, like the microchip to the tech revolution, stem cells will be the driving force of this next revolution".*

*-Cade Hildreth.*

Staggering progress in the field of regenerative medicine has sowed the seeds of cell-based therapies for various diseases which cannot be cured by conventional methods. Stem cell therapy deals with the functional revival of specific tissue and/ or organ in patients who are suffering from severe injuries / chronic diseased conditions, in a state where the body's own regeneration feedback is not satisfactory.

The entire argument of regenerative medicine is built on the presence of stem cells in the body, or the ability to institute stem cells into the body without causing harm. Given that stem cells can be obtained from a variety of sources, the search for an ideal source that offers excellent therapeutic potential while requiring less invasiveness and immune rejection is unending. Even a tooth which is naturally discarded can be used as a great source for stem cells. Hence a better understanding of the nature and mechanism of stem cell is crucial for their application in cellbased therapy.

#### **1.1 What are stem cells and why is stem cell therapy so much of interest?**

Our bodies are the ultimate factory. Every cell has its own function to play, and the fate of each cell is determined at the embryo stage which then cannot be changed. However, the discovery of stem cells has paved the way for regenerative medicine. Stem cells are those immature cells which can differentiate into any type of cells as they are not specialized [1]. Therefore, they can be used in the repair and regeneration of dysfunctional tissues. For instance, they can help treat neurological diseases by making new brain cells to treat people with Parkinson's disease, or they could be used to repair the damaged immune system, or even reverse paralysis/ regrow lost limbs.

Therefore, stem cell research can help to:


## **2. Classification of stem cells**

Depending on the origin/source of the stem cells, stem cells are divided into various types;

#### **2.1 Embryonic stem cells (ESCs)**

They are pluripotent stem cells derived from the blastocyst's inner cell mass. The blastocyst stage, with 50–150 cells, occurs 4–5 days after conception. Embryonic stem cells are able to develop into any type of cell, except those of the placenta (**Figure 1**).

ESCs derived from mouse blastocysts have been studied for 2 decades and shown to differentiate into various cell types including fat cells, brain, nerve, insulin-producing cells of the pancreas, bone cells, endothelial cells, and heart muscle cells [3]. Human ESCs under appropriate culture conditions have demonstrated remarkable abilities to self-renew and produce multipotent cells. The cells are studied extensively in the treatment of diabetes, heart diseases, genetic disorders spinal cord injury, muscular dystrophy, heart illness, and vision/hearing loss. However, it possesses the risk of developing adverse effects such as tumors and unwanted immune responses. Hence the scope of ESCs is still under debate and research to understand how to prevent the rejection of transplanted cells is fundamental.

#### **2.2 Adult stem cells/ somatic or tissue-specific stem cells (ASCs)**

ASCs, also called somatic stem cells, are undifferentiated cells that can self-renew and generate all the cell types of the organ from which they originate. An adult stem cell is derived from adult tissue samples. Hence, their use in research and therapy is not considered to be controversial unlike embryonic stem cells derived from embryos.

ASCs are the gold standard for clinical applications and are being tested and accepted for a growing number of conditions. ASCs have been shown to have therapeutic benefits in clinical trials and progress towards fully tested and approved treatments. Phase I/II trials conducted suggest potential cardiovascular benefits from bone

**Figure 1.** *Human embryonic stem cells differentiation. Image source: Biorender.com.*

marrow–derived adult stem cells and umbilical cord blood–derived cells. Striking results have been reported using adult stem cells to treat neurological conditions, including chronic stroke. Positive long-term progression-free outcomes have been seen, including some remission for multiple sclerosis, as well as benefits in early trials for patients with type I diabetes mellitus and spinal cord injury. ASCs are also being used as vehicles for genetic therapies, such as for epidermolysis bullosa. One of the limitations of ASCs includes that they cannot be manipulated to produce all cell types, which limits their use in treating diseases (**Figure 2**).

#### **2.3 Induced pluripotent stem cells (iPSCs)**

The limitations in ASCs led to the creation of novel pluripotent cells termed induced pluripotent cells from the adult cells by the process of reprogramming the genes. ASCs can be fused with embryonic stem cells to generate induced pluripotent stem cells. Other somatic cells can also be altered to become pluripotent. iPSCs can differentiate from ESCs. Their gene expression and chromatin differ from ESCs. These cells are important because they may be utilized to create cells from almost all organs for each patient in therapeutic treatment. Besides, they also prevent the use of more ESCs which might cause ethical issues. It also helps to study new genetic diseases by generating iPSCs from their adult or somatic cells (**Figure 3**).

Hepatocyte–like cell derivatives, dendritic cells, macrophages, insulin–producing cell clusters similar to the duodenal islets of Langerhans, and hematopoietic and endothelial cells are currently produced from murine and human iPSCs, in addition to the already listed types of differentiated cells [4–7]. Reprogrammed iPSCs derived from peripheral blood cells could effectively develop into hematopoietic lineage cells [8]. Human β cell-derived iPSCs possess epigenetic memory and may develop into

**Figure 2.** *Sources of adult stem cells. Image source: Biorender.com.*

*Human Teeth Is Useful Even after Its SHED! So, Why Discard It? DOI: http://dx.doi.org/10.5772/intechopen.110769*

#### **Figure 3.**

*Evolution of induced pluripotent stem cells. Image source: Biorender.com.*

insulin-producing cells more readily [9]. Dopamine and motor neurons can also be produced from human iPSCs by directed differentiation *in vitro* [10, 11].

#### **2.4 Mesenchymal stem cells (MSCs)**

MSCs are a type of adult stem cell that can develop into mesodermal (osteocytes, adipocytes, and chondrocytes), ectodermal (neurocytes), and endodermal cell lines (hepatocytes). Some of the potent sources of MSCs include bone marrow, adipose tissue, synovial fluid, umbilical cord tissue, peripheral blood, placental tissue and dental pulp. MSCs can be extracted readily and yield more than other stem cells, making them beneficial for cell proliferation, differentiation, and tissue regeneration under severe immunological circumstances. These also have immunomodulatory features as they secrete cytokines and immune receptors, which regulate the microenvironment in the host tissue. MSCs can treat chronic diseases by producing cells of diverse cell lines, immunomodulating, and secreting anti-inflammatory chemicals. Thereby, showing promising results in preclinical studies for various medical conditions. Research continues to explore their potential in regenerative medicine (**Figure 4**).

MSCs have been studied for a wide range of therapeutic applications, including tissue repair, regenerative medicine, and cell-based therapies for various medical conditions. Some of the medical conditions that MSCs have been studied for include:


**Figure 4.** *Regenerating abilities of mesenchymal stem cells. Image source: Biorender.com.*


11.Osteoarthritis: Intra-articular injection of infrapatellar fat pad-derived mesenchymal stem cells is effective for reducing pain and improving knee function in patients being treated for knee osteoarthritis [16].

Various research on adult stem cells led to their discovery in different dental tissues. Stem cells extracted from dental tissue have been shown to possess similar properties to MSCs derived from other sources. Hence, considering dental stem cells are easily accessible. Currently, there is extensive research focusing on dental stem cells and their clinical applications.

#### **2.5 Dental stem cells**

Dental stem cells offer a very promising therapeutic approach to restoring structural defects. To date, eight unique populations of dental tissue-derived MSCs have been isolated and characterized. Postnatal dental pulp stem cells **(DPSCs)** were the first human dental MSCs to be identified from pulp tissue. Gradually, other dental MSC-like populations were also reported (**Figure 5**).


**Figure 5.**

*Sources of dental stem cells. Image source: Biorender.com.*

cementoblast, and osteoblast [20]. DFSCs are isolated from 3rd molars and expressed various biomarkers such as Notch 1, STRO-1 and Nestin. They demonstrated multilineage potential to undergo osteogenic, adipogenic, and neurogenic potential in vitro.


#### **3. Why there is such an interest in stem cells from exfoliated deciduous teeth?**

Pulp from naturally exfoliated deciduous teeth may be like an umbilical cord providing a rich and distinctive source of stem cells showing a multipotent nature. SHEDs exhibit a much higher proliferation rate, faster population doublings and greater osteoinductive capacity than DPSCs, adult MSCs and PDLSCs. They can differentiate into a variety of cell types including odontoblasts, osteoblasts, adipocytes, chondrocytes,

#### *Human Teeth Is Useful Even after Its SHED! So, Why Discard It? DOI: http://dx.doi.org/10.5772/intechopen.110769*

neural cells, hepatocytes, endothelial cells, β-cells, and dentin and pulp-like tissues. SHEDs express the same cell markers as ESCs such as OCT 4 and NONOG, which makes them have a significant impact on clinical applications. Evidence indicates that functional recovery and remodeling in lesions not only rely on their multipotency but also on their protective and anti-inflammatory action by the paracrine mechanism of grafted SHEDs. In this context, SHEDs have shown to function as an immunomodulator by suppressing T helper 17 cell functions. Transforming growth factors TGF-β1 and β2, fibroblast growth factor FGF-2, and Col I and III are highly expressed in SHED as compared to DPSCs.

The primary difference in the pulp of primary and permanent teeth is the occurrence of physiologic root resorption of the deciduous teeth. The transition from deciduous teeth to permanent teeth is a unique and dynamic process wherein the resorption of the deciduous teeth is coordinated with the development and eruption of permanent teeth. Deciduous teeth without any visible root resorption were unable to proliferate in vitro, whereas those in an advanced state of root resorption showed good proliferation and differentiation potential [31]. Due to its unique stemness of capability of multidifferentiation, self-renewal, developing into other cell lineages and easy accessibility, without major morbidity to host and minimal ethical concern, SHED has been widely investigated in the field of regenerative medicine and tissue engineering (**Figure 6**).

## **4. Applications of SHED in research**

Owing to its multipotent, no/reduced ethical conflicts and minimally invasive to obtain has opened a wide area for research. Research spans across categories—Dental materials, wound healing, dental tissue engineering, treatment of chronic diseases like diabetes, and Wilson's disease, treatment of autoimmune diseases like SLE, and encephalomyelitis, adjunct to surgical treatment e.g., cleft lip and palate, pediatric surgeries like biliary atresia.

#### **4.1 Cell culture studies**


potential (osteogenic/odontogenic) of various biomaterials on SHED and concluded that all the tested materials are bioinductive to SHED. Enamel Matrix Derivative (EMD) can be used in dentistry for various vital pulp therapies as that of Biodentine and Mineral Trioxide Aggregate (MTA) with predictable as well as enhanced success rates [33].


#### **4.2 Animal studies**


*Human Teeth Is Useful Even after Its SHED! So, Why Discard It? DOI: http://dx.doi.org/10.5772/intechopen.110769*


#### **5. Therapeutical applications of SHED**

It has been found that SHEDs showed alleviating effects on nervous system diseases, including Spinal cord injury, Parkinson's disease, Trigeminal neuralgia, Cerebral ischemia, Alzheimer's disease, and Encephalomyelitis. Owing to the capacity to interact with the local inflammatory microenvironment, SHEDs have also embraced remarkable modulatory effects in various autoimmune and inflammatory diseases such as rheumatoid arthritis, diabetes, acute kidney injury, liver fibrosis/ acute liver failure osteoarthritic, heatstroke, and acute respiratory distress syndrome (ARDS) could also benefit from SHEDs for the protective effects underlying immunomodulatory activities.

#### **5.1 Evidence supporting SHEDs potential in autoimmune and nervous diseases via paracrine and immunomodulatory**

Effects of SHED on Parkinson's disease: Transplantation of neural-like spheres derived from SHEDs into the striatum of parkinsonian rats significantly improved the behavioral disorders, the number of TH-positive (tyrosine hydroxylase) cells and the protective effect on endogenous dopaminergic neurons, indicating SHED spheres were of potential therapeutic value [45].

Effects of SHED on Acute liver failure: Intravenous administration of SHED-CM improved the condition of the injured liver and the animals' survival rate by induction of anti-inflammatory M2-like hepatic macrophages [46].

Effects of SHED on Heatstroke: Intravenous administration of SHED exhibited therapeutic benefits for heatstroke in mice, related to decreased inflammatory response, decreased oxidative stress, and increased hypothalamic pituitary adrenocortical axis activity.

Effects of SHEDs in the treatment of retinal degeneration: It has been confirmed through paracrine secreta that SHEDs exert neurotrophic, angiogenic, immunoregulatory, and antiapoptotic functions in injured tissues. SHEDs and SHED-CM showed therapeutic effects on Retinitis pigmentosa (RP) by improving retinal visual function and delaying the degeneration of photoreceptors by antiapoptotic activity. Therefore, SHEDs may be a promising stem cell source for treating retinal degeneration [47].

#### **5.2 Other therapeutic effects of SHED**


*Human Teeth Is Useful Even after Its SHED! So, Why Discard It? DOI: http://dx.doi.org/10.5772/intechopen.110769*


#### **6. Scope of tissue engineering in dentistry using SHED**

Recent advances in stem cell research especially in the field of dentistry have led to the onset of an entirely new era in which even an entire tooth can be regrown. This is just one of the several approaches that hold promise for tooth regeneration.

As far as pediatric dentistry is concerned, decay is one of the most common problems faced. Most often a pedodontist ends up performing a pulpectomy procedure

which involves the complete removal of the pulpal tissue and filling it with an ideal obturating material. Now, with technological evolution, researchers are using stem cell therapy for regenerative pulpotomies which can restore the vital pulp, which bypasses the need of going through painful invasive dental procedures. Following are some scope and potential applications of SHED in regenerative dentistry.

#### **6.1 Dentin Pulp complex regeneration (DPC)**

The DPC consists of the outer hard tissue layer, which is composed of orientated cells (odontoblasts) that secrete a specific matrix to form new dentin, and the inner soft tissue layer, which is composed of vital pulp tissue that comprises a network of microvasculature, nerves, and fibrous elements. Therefore, regeneration of the Dentin Pulp Complex entails a cascade of events involving odontogenesis and angiogenesis. SHEDs have a greater capacity for the formation of Dentin Pulp Complex cells, including osteoblasts, chondroblasts, adipocytes, endothelial cells, nerve cells, and odontoblasts [58–60]. SHEDs have demonstrated the ability to develop into functional odontoblasts and endothelial-like cells [61]. SHED's capacity to develop into odontoblasts is defined by the expression of dentin matrix protein-1 (DMP-1) and Dentin Sialophosphoprotein (DSPP) [60, 62]. The goal of DMP-1 is to maintain dentin mineralization, DSPP stimulates odontoblast development in stem cells by phosphorylation of SMAD 1/5/8 and nuclear translocation via the P38 and ERK1/2 pathways [63, 64]. Regenerating the missing interface between two distinct tissues (dentin and pulp), as seen in the Dentin Pulp Complex, is one of the main challenges of regenerative dentistry. It is crucial to provide a perfect environment that encourages the aggregation, proliferation, and differentiation of these disparate tissues. The ultimate regeneration of the dentin-pulp complex requires successful innervation and revascularization. In this context, various scaffolds have been employed to support cell growth and functionality in the transplanted area. Tissue engineering methods involving SHED, growth factors and scaffolds have been researched to regenerate DPC. SHED has shown the ability to regenerate pulp- and dentin-like tissues utilizing scaffolds and stem cells in animal models. SHEDs are also able to increase the angiogenesis process by forming vascular connective tissue structures and expressing and synthesizing VEGF. This ability is crucial in maintaining pulp viability as it can supply oxygen and nutrients needed for cell metabolism for tissue regeneration. Also, Exosomes extracted from SHED aggregates (SA-Exo) showed to significantly improve pulp tissue regeneration and angiogenesis in vivo, it also promoted endothelial differentiation and enhanced the angiogenic ability of HUVECs [65, 66].

#### **6.2 Dentin Pulp regeneration**

Dentin Pulp Regeneration aims to revitalize necrotic, infected, or lost pulp teeth by restoring the morphology and function of the pulp. Ideal pulp regeneration should possess natural structures such as nerve, fibers and blood supply, allowing nutritional, defense, sensation, and immunological functions to be restored. SHED have been utilized for pulp revascularization in regenerative endodontic procedures over the years. The conventional endodontic treatment in an immature tooth with pulpal necrosis is often challenging owing to its open root apex and thin root canal dentin. In addition, there is a risk of obturating material overflowing into the periapex. Regenerative endodontics (pulp revascularization) in an immature tooth aims to

*Human Teeth Is Useful Even after Its SHED! So, Why Discard It? DOI: http://dx.doi.org/10.5772/intechopen.110769*

promote continued root development by generating new tissues. SHED seeded onto the synthetic scaffolds formed well-vascularized pulp-like tissue in vivo on a tooth slice model [67].

To restore the vitality of a tooth, elements with regeneration properties in the pulp are required. SHED not only produced mesenchymal stem cell-specific markers, but it also caused odontoblastic differentiation and increased the formation of endothelium and fibroblasts. The regenerated pulp tissue built by SHED had similar cellularity and architecture of the physiological dental pulp [68]. The combination of SHED, Platelet Rich Fibrin, and Chitosan enhanced the migration, proliferation, and odontoblastic differentiation of dental pulp cells. Therefore, the combination of SHED, PRF, and Chitosan scaffold as a new method for pulp regeneration in a clinical environment appears promising as 3D tissue engineering. SHED were implanted in empty root canals of mini pigs to determine whether a full-length dental pulp is regenerated. After 3 months of implantation, the histological analysis showed that full-length dental pulp tissue was regenerated which contained the odontoblast layer and blood vessels. The regenerated pulp showed a similar tissue structure to the normal pulp. Furthermore, blood vessels and nerves were regenerated as confirmed by positive expressions of CD31 and neurofilament (NF) in regenerated pulp tissues. In addition, positive expressions of CGRP and TRPV1 cells indicated the regenerated pulp might have sensory nerves. These results indicated that implantation of SHED was capable of regenerating full-length dental pulp with blood vessels and nerves in a large animal model [69].

#### **6.3 Bio root regeneration**

Techniques based on cell-based tissue engineering have made significant advances in the field of tooth regeneration. However, regeneration of the complete tooth has proven to be laborious; consequently, tooth root regeneration is advocated as a more practical and promising alternative for tooth restoration than the regeneration of the entire tooth. The tooth root serves a vital role in chewing and maintaining the tooth's stability, which is the structural foundation of a functional tooth. Bio-engineered tooth root (bio root) mediated by stem cells has shown to be a promising treatment for tooth loss. Multiple studies have demonstrated that Dental Follicle cells are appropriate seeding cells for the development of bioroots. SHEDs can be regarded as a prospective seeding cell for use in bio root regeneration in the future [70]. The comparison of ultrastructure revealed that SHEDs participate in active cell metabolism and the autophagy process, which are essential for stem cell immunological defense, self-renewal, and apoptosis [71, 72]. In addition, they possessed protein synthesis and secretion capabilities that were superior to those of dental follicle cells, resulting in the establishment of a microenvironment that is favorable to tissue repair and regeneration, a breakthrough in the field of nerve regeneration [54, 73, 74].

#### **6.4 Periodontal regeneration**

SHEDs treated with dentin matrix can regenerate periodontal tissue composed of periodontal ligament fibers, blood vessels, and new alveolar bone [70]. Due to their high proliferative capacity, the strong immunosuppressive ability of multiple differentiation, and minimal carcinogenic potential, exfoliated human deciduous dental stem cells have been employed to restore periodontal tissue and repair alveolar bone abnormalities [75].

#### **6.5 Reconstruction of Cleft lip/palate (CL/P)**

Autogenous iliac bone grafting has been demonstrated to heal alveolar cleft defects; however, surgical intervention is required. Hence, the creation of a less invasive technique is anticipated. Consequently, Alveolar bone regeneration methods in patients with CL/P employing human bone marrow mesenchymal stem cells (hBMSCs) have been attempted, and the transplantation of hBMSC in a canine alveolar cleft model has demonstrated the ability to regenerate bone [76]. SHEDs have higher osteogenic potential compared to bone marrow stem cells [77]. SHEDs, human dental pulp stem cells (hDPSCs), and hBMSCs were utilized to induce bone regeneration in immunodeficient animals with calvarial bone abnormalities. However, animals treated with SHED scaffolds had the greatest amounts of osteoid and the widest distribution of collagen fibers. During cell culture, MSCs can secrete paracrine substances into a conditioned medium (CM). MSC-CM contain the growth factors insulin-like growth factor-1 (IGF-1), transforming growth factor 1 (TGF-1), and vascular endothelial growth factor (VEGF), which influence the features and behavior of regenerating bone cells [78–80]. It is possible that both transplanted MSCs and their paracrine actions contribute to tissue regeneration. SHED-CM demonstrated mature bone development and contained tissue-regenerating factors with functions in angiogenesis and osteogenesis. Deciduous dental pulp stem cells (DDPSC) associated with a hydroxyapatitecollagen sponge showed closure of alveolar defects during the secondary dental eruption in a clinical setting. Thus, SHED could be an ideal source of cells for alveolar cleft reconstruction due to its capacity to regenerate bone with minimal surgical invasion [81].

#### **6.6 Temporomandibular Joint Osteoarthritis (TMJOA)**

Exosomes secreted by SHEDs (SHED-Exos) demonstrated to suppress inflammation in TMJ chondrocytes. The anti-inflammatory effects of SHED-Exos were verified using western blotting and RT-qPCR. SHED-Exos down-regulated the expression of IL-6, IL-8, MMP1, MMP3, MMP9, MMP13, and dis-integrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5). Thus, they can be a novel therapeutic agent for TMJ inflammation [82].

#### **7. Limitations / challenges of stem cell research**

Even though stem cell offers a wide range of therapeutic potentials in regenerative medicine, one cannot deny the fact that it does possess limitations and challenges because of different ethical and other issues related to stem cell research. Some of them are listed below:


However, the advent of stem cells derived from less invasive tissues without immune rejection has widened the opportunities for cell-based therapies.

## **8. Then, why not discard primary teeth after they SHED?**

According to the data presented above, SHED have regenerative abilities comparable to umbilical cord stem cells, implying that they have a high potential for treating some life-threatening diseases. Exfoliated teeth are also an unexpectedly unique resource for stem-cell therapies such as autologous stem-cell transplantation and tissue engineering due to their ease of access and lack of ethical concerns. Banking SHED cells is thus extremely beneficial (**Figure 7**).

#### **9. SHED Banking and its advantages**

Storing your child's own teeth stem cells could give them access to a huge range of treatment opportunities throughout their lifetime

• It provides a guaranteed matching donor (autologous transplant) for life. There are many advantages of autologous transplant including no immune reaction and

**Figure 7.** *SHED and its importance.* tissue rejection of the cells, no immunosuppressive therapy needed, and significantly reduced risk of communicable diseases [83, 84].

	- Simple and painless 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 [83, 84].
	- SHED cells complement stem cells derived from cord blood. Even though cord blood stem cells have proven useful in the regeneration of blood cell types, SHED is capable of regenerating solid tissue types that cord blood cannot, such as potentially rebuilding connective tissues, dental tissue, neural tissue, and bone [85–88].

#### **Figure 8.**

*Isolation protocol of SHED. Image source: Biorender.com.*

○ SHED may also be beneficial for the donor's immediate relatives, such as grandparents, parents, uncles, and siblings [84].

Certain tooth selection criteria must be followed before SHED banking, which is crucial for the successful isolation and characterization of cell lines to maintain viability.


#### **10. Procedure**

We have outlined the detailed process involved in SHED banking for its application in stem cell therapy.

#### **10.1 Collection, isolation, and preservation of SHED**

1.Tooth collection


#### **Figure 9.** *Stem cell verification and validation.*

#### **Figure 10.**

*Typical step-by-step cryopreservation protocol. Image source: Cryopreservation Basics: Protocols and Best Practices for Freezing Cells. Stem cell technologies.*

*Human Teeth Is Useful Even after Its SHED! So, Why Discard It? DOI: http://dx.doi.org/10.5772/intechopen.110769*

The viability of the isolated cells is then tested (**Figure 9**).

3.Stem Cell Storage

Stem cell storage refers to the collection and cryopreservation of stem cells from source tissue for use in stem cell treatments or clinical trials in the future. Methods used for Stem cell storage are cryopreservation or magnetic freezing [89].

#### **10.2 Cryopreservation**

This routine procedure generally involves slow cooling in the presence of a cryoprotectant to avoid the damaging effects of intracellular ice formation (**Figure 10**).

**Figure 11.** *Magnetic freezing step by step protocol.*

#### **10.3 Magnetic freezing**

These above steps are followed by Stem cell differentiation, characterization, and validation of required cell types prior to their application in cell-based therapies (**Figure 11**).

#### **11. Conclusion**

Stem cell therapy has made significant advances in regenerative medicine and dentistry. It has simplified the treatment of many diseases that were previously difficult to treat. Cord blood stem cells are used to treat over 85 different blood and immune diseases, including Leukemia and Neuroblastoma. Considering the scope of stem cells in regenerative medicine, it is imperative to opt a source of stem cell which is less invasive and has promising future in regenerative therapies without immune rejection. SHED has been shown to be as distinct as cord blood stem cells. Multiple studies demonstrate that SHED can differentiate into odontoblasts, neurons, hepatocytes, endothelial cells, β-cells, and other cell types. This vast array of cell types generates an abundance of options for the application of SHED in tissue regeneration processes. SHED has demonstrated a wide range of therapeutic applications due to its ease of harvesting, lack of bioethical concerns, and excellent expansibility. Also, using one's own stem cells (SHED) reduces, if not eliminates, the risk of developing immune reactions or rejection after transplantation, as well as the risk of contracting disease from donor cells. However, Prior to SHED-based therapies becoming a clinical reality, it is necessary to have a deeper understanding of the mechanisms underpinning differentiation processes. Considering the tremendous use of SHED in stem cell therapy, will banking of SHED or the cost of stem cell banking justified and reasonable? The answer is—Why not, if money is not an issue? If a naturally discarded tooth can have such broad therapeutic applications in the future, why not save it? Regardless, the ultimate fate of SHED cell banking will be decided by the patient or parent.

#### **Acknowledgements**

We are extremely grateful for the generous contributions of our colleagues and friends to this work. Specially, Dr. B. Mohana Kumar, Associate Professor, Nitte University Centre for Stem Cell Research and Regenerative Medicine (NUCSReM), K. S. Hegde Medical Academy, Nitte (Deemed to be University), and his team for their expertise in stem cell isolation and characterization. Dr. Veena Shetty, PhD, Additional Professor, Department of Microbiology, K. S. Hegde Medical Academy, for providing emotional and scientific support for this work and also, Nitte (Deemed to be University) for providing access to all of the educational material required for this work.

*Human Teeth Is Useful Even after Its SHED! So, Why Discard It? DOI: http://dx.doi.org/10.5772/intechopen.110769*

#### **Author details**

Meghna Bhandary1 \*, Rachaita Chhabra2 , K. Ananya Rao1 and Mohammed Shahid3

1 Department of Pediatric and Preventive Dentistry, AB Shetty Memorial Institute of Dental Sciences, India

2 Pediatric Dental Practitioner, Mumbai, India

3 Department of Oral Pathology and Microbiology, A.J. Institute of Dental Sciences, India

\*Address all correspondence to: meghnabhandary92@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **References**

[1] Zakrzewski W, Dobrzyński M, Szymonowicz M, Rybak Z. Stem cells: Past, present, and future. Stem Cell Research & Therapy. 2019;**10**(1):68

[2] Ul Hassan A, Hassan G, Rasool Z. Role of stem cells in treatment of neurological disorder. International Journal of Health Science (Qassim). 2009;**3**(2):227-233

[3] Odorico JS, Kaufman DS, Thomson JA. Multilineage differentiation from human embryonic stem cell lines. Stem Cells. 2001;**19**(3):193-204

[4] Song Z, Cai J, Liu Y, Zhao D, Yong J, Duo S, et al. Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells. Cell Research. 2009;**19**(11):1233-1242

[5] Senju S, Haruta M, Matsunaga Y, Fukushima S, Ikeda T, Takahashi K, et al. Characterization of dendritic cells and macrophages generated by directed differentiation from mouse induced pluripotent stem cells. Stem Cells. 2009;**27**(5):1021-1031

[6] Tateishi K, He J, Taranova O, Liang G, D'Alessio AC, Zhang Y. Generation of insulin-secreting islet-like clusters from human skin fibroblasts. Journal of Biological Chemistry. 2008;**283**(46):31601-31607

[7] Choi KD, Yu J, Smuga-Otto K, Salvagiotto G, Rehrauer W, Vodyanik M, et al. Hematopoietic and endothelial differentiation of human induced pluripotent stem cells. Stem Cells. 2009;**27**(3):559-567

[8] Kim K, Doi A, Wen B, Ng K, Zhao R, Cahan P, et al. Epigenetic memory in induced pluripotent stem cells. Nature. 2010;**467**(7313):285-290

[9] Bar-Nur O, Russ HA, Efrat S, Benvenisty N. Epigenetic memory and preferential lineage-specific differentiation in induced pluripotent stem cells derived from human pancreatic islet Beta cells. Cell Stem Cell. 2011;**9**(1):17-23

[10] Karumbayaram S, Novitch BG, Patterson M, Umbach JA, Richter L, Lindgren A, et al. Directed differentiation of human-induced pluripotent stem cells generates active motor neurons. Stem Cells. 2009;**27**(4):806-811

[11] Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, Studer L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nature Biotechnology. 2009;**27**(3):275-280

[12] White SJ, Chong JJH. Mesenchymal stem cells in cardiac repair: Effects on myocytes, vasculature, and fibroblasts. Clinical Therapeutics. 2020;**42**(10):1880-1891

[13] Qu J, Zhang H. Roles of mesenchymal stem cells in spinal cord injury. Stem Cells International. 2017;**2017**:1-12

[14] Figueroa FE, Carrión F, Villanueva S, Khoury M. Mesenchymal stem cell treatment for autoimmune diseases: A critical review. Biological Research. 2012;**45**(3):269-277

[15] Sykova E, Cizkova D, Kubinova S. Mesenchymal stem cells in treatment of spinal cord injury and amyotrophic lateral sclerosis. Frontiers in Cell and Development Biology. 2021;**6**:9

[16] Koh YG, Choi YJ. Infrapatellar fat pad-derived mesenchymal stem cell

*Human Teeth Is Useful Even after Its SHED! So, Why Discard It? DOI: http://dx.doi.org/10.5772/intechopen.110769*

therapy for knee osteoarthritis. The Knee. 2012;**19**(6):902-907

[17] Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) *in vitro* and *in vivo*. Proceedings of the National Academy of Sciences. 2000;**97**(25):13625-13630

[18] Aydin S, Şahin F. Stem cells derived from dental tissues. 2019;123-132

[19] Takebe Y, Tatehara S, Fukushima T, Tokuyama-Toda R, Yasuhara R, Mishima K, et al. Cryopreservation method for the effective collection of dental pulp stem cells. Tissue Engineering. Part C, Methods. 2017;**23**(5):251-261

[20] Gan L, Liu Y, Cui D, Pan Y, Zheng L, Wan M. Dental tissue-derived human mesenchymal stem cells and their potential in therapeutic application. Stem Cells International. 2020;**2020**:1-17

[21] Liu J, Yu F, Sun Y, Jiang B, Zhang W, Yang J, et al. Concise reviews: Characteristics and potential applications of human dental tissue-derived mesenchymal stem cells. Stem Cells. 2015;**33**(3):627-638

[22] Matsubara T, Suardita K, Ishii M, Sugiyama M, Igarashi A, Oda R, et al. Alveolar bone marrow as a cell source for regenerative medicine: Differences between alveolar and iliac bone marrow stromal cells. Journal of Bone and Mineral Research. 2004;**20**(3):399-409

[23] Sonoyama W, Liu Y, Fang D, Yamaza T, Seo BM, Zhang C, et al. Mesenchymal stem cell-mediated functional tooth regeneration in swine. PLoS One. 2006;**1**(1):e79

[24] Sonoyama W, Liu Y, Yamaza T, Tuan RS, Wang S, Shi S, et al. Characterization of the apical papilla and its residing stem cells from human immature permanent teeth: A pilot study. Journal of Endodontia. 2008;**34**(2):166-171

[25] Patil R, Kumar BM, Lee WJ, Jeon RH, Jang SJ, Lee YM, et al. Multilineage potential and proteomic profiling of human dental stem cells derived from a single donor. Experimental Cell Research. 2014;**320**(1):92-107

[26] Kang J, Fan W, Deng Q, He H, Huang F. Stem cells from the apical papilla: A promising source for stem cell-based therapy. BioMed Research International. 2019;**2019**:1-8

[27] Krismariono A, Rubianto M, Maduratna E, Sari DR. A revolution of stem cell in periodontal regeneration. 2020;060013

[28] Ikeda E, Yagi K, Kojima M, Yagyuu T, Ohshima A, Sobajima S, et al. Multipotent cells from the human third molar: Feasibility of cell-based therapy for liver disease. Differentiation. 2008;**76**(5):495-505

[29] Zhang Q, Shi S, Liu Y, Uyanne J, Shi Y, Shi S, et al. Mesenchymal stem cells derived from human gingiva are capable of immunomodulatory functions and ameliorate inflammation-related tissue destruction in experimental colitis. The Journal of Immunology. 2009;**183**(12):7787-7798

[30] Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, et al. SHED: Stem cells from human exfoliated deciduous teeth. Proceedings of the National Academy of Sciences. 2003;**100**(10):5807-5812

[31] Bernardi L, Luisi SB, Fernandes R, Dalberto TP, Valentim L, Bogo Chies JA, et al. The isolation of stem cells from human deciduous teeth pulp is related

to the physiological process of resorption. Journal of Endodontia. 2011;**37**(7):973-979

[32] Junior AL, Pinheiro CCG, Tanikawa DYS, Ferreira JRM, Amano MT, Bueno DF. Mesenchymal stem cells from human exfoliated deciduous teeth and the orbicularis Oris muscle: How do they behave when exposed to a Proinflammatory stimulus? Stem Cells International. 2020;**2020**:1-15

[33] Dahake PT, Panpaliya NP, Kale YJ, Dadpe M, Kendre SB, Bogar C. Response of stem cells from human exfoliated deciduous teeth (SHED) to three bioinductive materials – An in vitro experimental study. Saudi. Dental Journal. 2020;**32**(1):43-51

[34] Athanasiadou E, Paschalidou M, Theocharidou A, Kontoudakis N, Arapostathis K, Bakopoulou A. Biological interactions of a calcium silicate based cement (biodentine™) with stem cells from human exfoliated deciduous teeth. Dental Materials. 2018;**34**(12):1797-1813

[35] Collado-González M,

García-Bernal D, Oñate-Sánchez RE, Ortolani-Seltenerich PS, Álvarez-Muro T, Lozano A, et al. Cytotoxicity and bioactivity of various pulpotomy materials on stem cells from human exfoliated primary teeth. International Endodontic Journal. 2017;**50**:e19-e30

[36] Araújo LB, Cosme-Silva L, Fernandes AP, de Oliveira TM, Cavalcanti B, Gomes Filho JE, et al. Effects of mineral trioxide aggregate, BiodentineTM and calcium hydroxide on viability, proliferation, migration and differentiation of stem cells from human exfoliated deciduous teeth. Journal of Applied Oral Science. 2018;**26**:6

[37] Nishino Y, Yamada Y, Ebisawa K, Nakamura S, Okabe K, Umemura E,

et al. Stem cells from human exfoliated deciduous teeth (SHED) enhance wound healing and the possibility of novel cell therapy. Cytotherapy. 2011;**13**(5):598-605

[38] Shimojima C, Takeuchi H, Jin S, Parajuli B, Hattori H, Suzumura A, et al. Conditioned medium from the stem cells of human exfoliated deciduous teeth ameliorates experimental autoimmune encephalomyelitis. The Journal of Immunology. 2016;**196**(10):4164-4171

[39] Rossato C, Brandão WN, Castro SBR, de Almeida DC, Maranduba CMC, Camara NOS, et al. Stem cells from human-exfoliated deciduous teeth reduce tissue-infiltrating inflammatory cells improving clinical signs in experimental autoimmune encephalomyelitis. Biologicals. 2017;**49**:62-68

[40] Xie J, Rao N, Zhai Y, Li J, Zhao Y, Ge L, et al. Therapeutic effects of stem cells from human exfoliated deciduous teeth on diabetic peripheral neuropathy. Diabetology and Metabolic Syndrome. 2019;**11**(1):38

[41] Fujiyoshi J, Yamaza H, Sonoda S, Yuniartha R, Ihara K, Nonaka K, et al. Therapeutic potential of hepatocytelike-cells converted from stem cells from human exfoliated deciduous teeth in fulminant Wilson's disease. Scientific Reports. 2019;**9**(1):1535

[42] Zhao L, Li Y, Kou X, Chen B, Cao J, Li J, et al. Stem cells from human exfoliated deciduous teeth ameliorate autistic-like Behaviors of *SHANK3* mutant beagle dogs. Stem Cells Translational Medicine. 2022;**11**(7):778-789

[43] Yamaza T, Alatas FS, Yuniartha R, Yamaza H, Fujiyoshi JK, Yanagi Y, et al. In vivo hepatogenic capacity and therapeutic potential of stem cells from human exfoliated deciduous teeth in liver *Human Teeth Is Useful Even after Its SHED! So, Why Discard It? DOI: http://dx.doi.org/10.5772/intechopen.110769*

fibrosis in mice. Stem Cell Research & Therapy. 2015;**6**(1):171

[44] Cao X, Wang C, Yuan D, Chen S, Wang X. The effect of implants loaded with stem cells from human exfoliated deciduous teeth on early osseointegration in a canine model. BMC Oral Health. 2022;**22**(1):238

[45] Wang J, Wang X, Sun Z, Wang X, Yang H, Shi S, et al. Stem cells from human-exfoliated deciduous teeth can differentiate into dopaminergic neuronlike cells. Stem Cells and Development. 2010;**19**(9):1375-1383

[46] Matsushita Y, Ishigami M, Matsubara K, Kondo M, Wakayama H, Goto H, et al. Multifaceted therapeutic benefits of factors derived from stem cells from human exfoliated deciduous teeth for acute liver failure in rats. Journal of Tissue Engineering and Regenerative Medicine. 2017;**11**(6):1888-1896

[47] Li XX, Yuan XJ, Zhai Y, Yu S, Jia RX, Yang LP, et al. Treatment with stem cells from human exfoliated deciduous teeth and their derived conditioned medium improves retinal visual function and delays the degeneration of photoreceptors. Stem Cells and Development. 2019;**28**(22):1514-1526

[48] Zhang X, Lei T, Chen P, Wang L, Wang J, Wang D, et al. Stem cells from human exfoliated deciduous teeth promote hair regeneration in mouse. Cell Transplantation. 2021;**30**:096368972110429

[49] Hattori Y, Kim H, Tsuboi N, Yamamoto A, Akiyama S, Shi Y, et al. Correction: Therapeutic potential of stem cells from human exfoliated deciduous teeth in models of acute kidney injury. PLoS One. 2015;**10**(11):e0143561

[50] Xie Y, Yu L, Cheng Z, Peng Y, Cao Z, Chen B, et al. SHED-derived exosomes promote LPS-induced wound healing with less itching by stimulating macrophage autophagy. Journal of Nanobiotechnology. 2022;**20**(1):239

[51] Li W, Jiao X, Song J, Sui B, Guo Z, Zhao Y, et al. Therapeutic potential of stem cells from human exfoliated deciduous teeth infusion into patients with type 2 diabetes depends on basal lipid levels and islet function. Stem Cells Translational Medicine. 2021;**10**(7):956-967

[52] Anoop M, Datta I. Stem cells derived from human exfoliated deciduous teeth (SHED) in neuronal disorders: A review. Current Stem Cell Research & Therapy. 2021;**16**(5):535-550

[53] Bai X, Xiao K, Yang Z, Zhang Z, Li J, Yan Z, et al. Stem cells from human exfoliated deciduous teeth relieve pain via downregulation of c-Jun in a rat model of trigeminal neuralgia. Journal of Oral Rehabilitation. 2022;**49**(2):219-227

[54] Sugimura-Wakayama Y, Katagiri W, Osugi M, Kawai T, Ogata K, Sakaguchi K, et al. Peripheral nerve regeneration by Secretomes of stem cells from human exfoliated deciduous teeth. Stem Cells and Development. 2015;**24**(22):2687-2699

[55] Li Y, Yang YY, Ren JL, Xu F, Chen FM, Li A. Exosomes secreted by stem cells from human exfoliated deciduous teeth contribute to functional recovery after traumatic brain injury by shifting microglia M1/M2 polarization in rats. Stem Cell Research & Therapy. 2017;**8**(1):198

[56] Seo B, Sonoyama W, Yamaza T, Coppe C, Kikuiri T, Akiyama K, et al. SHED repair critical-size calvarial defects in mice. Oral Diseases. 2008;**14**(5): 428-434

[57] Yamaza T, Kentaro A, Chen C, Liu Y, Shi Y, Gronthos S, et al. Immunomodulatory properties of stem cells from human exfoliated deciduous teeth. Stem Cell Research & Therapy. 2010;**1**(1):5

[58] Smojver I, Katalinić I, Bjelica R, Gabrić D, Matišić V, Molnar V, et al. Mesenchymal stem cells based treatment in dental medicine: A narrative review. International Journal of Molecular Sciences. 2022;**23**(3):1662

[59] Han Y, Zhang L, Zhang C, Dissanayaka WL. Guiding lineage specific differentiation of SHED for target tissue/organ regeneration. Current Stem Cell Research & Therapy. 2021;**16**(5):518-534

[60] Anggrarista KA, Cecilia P, Nagoro AA, Saskianti T, Surboyo MD. SHED, PRF, and chitosan as threedimensional of tissue engineering for dental pulp regeneration. Dental Hypotheses. 2021;**12**(1):43

[61] Kerkis I, Caplan AI. Stem cells in dental pulp of deciduous teeth. Tissue Engineering. Part B, Reviews. 2012;**18**(2):129-138

[62] Ganesh KishoreS O, Don KR, PriyaA J. Therapeutic potential of stem cells from human exfoliated deciduous teeth(Shed)-A review. Indian Journal of Forensic Medicine & Toxicology. **14**

[63] Khazaei S, Khademi A, Torabinejad M, Nasr Esfahani MH, Khazaei M, Razavi SM. Improving pulp revascularization outcomes with buccal fat autotransplantation. Journal of Tissue Engineering and Regenerative Medicine. 2020;**2020**:3094

[64] Vu HT, Han MR, Lee JH, Kim JS, Shin JS, Yoon JY, et al. Investigating the effects of conditioned media from stem cells of human exfoliated deciduous teeth on dental pulp stem cells. Biomedicine. 2022;**10**(4):906

[65] Wu M, Liu X, Li Z, Huang X, Guo H, Guo X, et al. SHED aggregate exosomes shuttled miR-26a promote angiogenesis in pulp regeneration via TGF-β/SMAD2/3 signalling. Cell Proliferation. 2021;**54**:7

[66] Rosa V, Zhang Z, Grande RHM, Nör JE. Dental pulp tissue engineering in full-length human root canals. Journal of Dental Research. 2013;**92**(11):970-975

[67] Demarco FF, Conde MCM, Cavalcanti BN, Casagrande L, Sakai VT, Nör JE. Dental pulp tissue engineering. Brazilian Dental Journal. 2011;**22**(1):3-13

[68] Sukarawan W, Osathanon T. Stem cells from human exfoliated deciduous teeth: Biology and therapeutic potential. In: Mesenchymal Stem Cells—Isolation, Characterization and Applications. London, UK: InTech; 2017

[69] Guo H, Zhao W, Liu A, Wu M, Shuai Y, Li B, et al. SHED promote angiogenesis in stem cell-mediated dental pulp regeneration. Biochemical and Biophysical Research Communications. 2020;**529**(4):1158-1164

[70] Yang X, Ma Y, Guo W, Yang B, Tian W. Stem cells from human exfoliated deciduous teeth as an alternative cell source in bioroot regeneration. Theranostics. 2019;**9**(9):2694-2711

[71] Guan JL, Simon AK, Prescott M, Menendez JA, Liu F, Wang F, et al. Autophagy in stem cells. Autophagy. 2013;**9**(6):830-849

[72] Boya P, Codogno P, Rodriguez-Muela N. Autophagy in stem cells: Repair, remodelling and metabolic *Human Teeth Is Useful Even after Its SHED! So, Why Discard It? DOI: http://dx.doi.org/10.5772/intechopen.110769*

reprogramming. Development. 2018;**145**(4)

[73] Taghipour Z, Karbalaie K, Kiani A, Niapour A, Bahramian H, Nasr-Esfahani MH, et al. Transplantation of undifferentiated and induced human exfoliated deciduous teeth-derived stem cells promote functional recovery of rat spinal cord contusion injury model. Stem Cells and Development. 2012;**21**(10):1794-1802

[74] Inoue T, Sugiyama M, Hattori H, Wakita H, Wakabayashi T, Ueda M. Stem cells from human exfoliated deciduous tooth-derived conditioned medium enhance recovery of focal cerebral ischemia in rats. Tissue Engineering. Part A. 2013;**19**(1-2):24-29

[75] Gao X, Shen Z, Guan M, Huang Q, Chen L, Qin W, et al. Immunomodulatory role of stem cells from human exfoliated deciduous teeth on periodontal regeneration. Tissue Engineering. Part A. 2018;**24**(17-18):1341-1353

[76] Abe T, Sumi K, Kunimatsu R, Oki N, Tsuka Y, Awada T, et al. Bone regeneration in a canine model of artificial jaw cleft using bone marrow– derived mesenchymal stem cells and carbonate hydroxyapatite carrier. The Cleft Palate-Craniofacial Journal. 2020;**57**(2):208-217

[77] Lee JM, Kim HY, Park JS, Lee DJ, Zhang S, Green DW, et al. Developing palatal bone using human mesenchymal stem cell and stem cells from exfoliated deciduous teeth cell sheets. Journal of Tissue Engineering and Regenerative Medicine. 2019;**13**(2):319-327

[78] Ando Y, Matsubara K, Ishikawa J, Fujio M, Shohara R, Hibi H, et al. Stem cell-conditioned medium accelerates distraction osteogenesis through

multiple regenerative mechanisms. Bone. 2014;**61**:82-90

[79] Inukai T, Katagiri W, Yoshimi R, Osugi M, Kawai T, Hibi H, et al. Novel application of stem cell-derived factors for periodontal regeneration. Biochemical and Biophysical Research Communications. 2013;**430**(2):763-768

[80] Ogata Y, Mabuchi Y, Yoshida M, Suto EG, Suzuki N, Muneta T, et al. Purified human synovium mesenchymal stem cells as a good resource for cartilage regeneration. PLoS One. 2015;**10**(6):e0129096

[81] Hiraki T, Kunimatsu R, Nakajima K, Abe T, Yamada S, Rikitake K, et al. Stem cell-derived conditioned media from human exfoliated deciduous teeth promote bone regeneration. Oral Diseases. 2020;**26**(2):381-390

[82] Luo P, Jiang C, Ji P, Wang M, Xu J. Exosomes of stem cells from human exfoliated deciduous teeth as an anti-inflammatory agent in temporomandibular joint chondrocytes via miR-100-5p/mTOR. Stem Cell Research & Therapy. 2019;**10**(1):216

[83] Mao JJ. Stem cells and the future of dental care. New York State Dental Journal. 2008;**74**(2):20

[84] Reznick JB. Continuing education: Stem cells: Emerging medical and dental therapies for the dental professional. Dentaltown Magazine. 2008;**2008**:42-53

[85] Arthur A, Rychkov G, Shi S, Koblar SA, Gronthos S. Adult human dental pulp stem cells differentiate toward functionally active neurons under appropriate environmental cues. Stem Cells. 2008;**26**(7):1787-1795

[86] Shi S, Bartold P, Miura M, Seo B, Robey P, Gronthos S. The efficacy of

mesenchymal stem cells to regenerate and repair dental structures. Orthodontics & Craniofacial Research. 2005;**8**(3):191-199

[87] Cordeiro MM, Dong Z, Kaneko T, Zhang Z, Miyazawa M, Shi S, et al. Dental pulp tissue engineering with stem cells from exfoliated deciduous teeth. Journal of Endodontia. 2008;**34**(8):962-969

[88] Mao JJ, Giannobile WV, Helms JA, Hollister SJ, Krebsbach PH, Longaker MT, et al. Craniofacial tissue engineering by stem cells. Journal of Dental Research. 2006;**85**(11):966-979

[89] Arora V, Arora P, Munshi A. Banking stem cells from human exfoliated deciduous teeth (SHED): Saving for the future. Journal of Clinical Pediatric Dentistry. 2009;**33**(4):289-294

#### **Chapter 21**
