International Journal of Contemporary Dental and Medical Reviews (2015), Article ID 060215, 7 Pages

REVIEW ARTICLE

Stem cell and the regenerative dentist K. Saraswathi Gopal, E. Madhubala Department of Oral Medicine and Radiology, Meenakshiammal Dental College and Hospital, Chennai, Tamil Nadu, India

Correspondence Dr. E. Madhubala, 15/16, “Rajkala,” Ganpathraj Nagar, Virugambakkam, Chennai - 600 092, Tamil Nadu, India. Phone: +91-9894147686, E-mail: [email protected] Received 18 February 2015; Accepted 30 March 2015 doi: 10.15713/ins.ijcdmr.60

Abstract Regenerative medicine and dentistry are making an impact with successful clinical trials using stem cells. The discovery that dental tissues are one of the storehouses for stem cells and the fact that many health issues can be sorted out by the therapeutic application of stem cells, the emphasis and significance of understanding this vital subject is gaining vast importance. At this juncture, where Regenerative medicine is progressing to become “The Future,” this article hopes to focus in simple terms on the basics that every individual dentist has to know to deliver the utmost benefit to the profession and the society. Keywords: Mesenchymal stem cells, plasticity, scaffold, tooth and tissue regeneration

How to cite the article: K. Saraswathi Gopal, E. Madhubala, “Stem cell and the regenerative dentist,” Int J Contemp Dent Med Rev, Vol. 2015. Article ID: 060215, 2015. doi: 10.15713/ins.ijcdmr.60

Introduction

cells with a specific function under certain physiological conditions.[4,5] Stem cell classification falls under three main categories: • Embryonic, germinal, and somatic[6,7]

Regenerative medicine has become the order of the day due to the promise it offers for new therapeutic modalities, which aim at repairing and replacing tissues and organs that have lost its potential, due to disease, damage, genetic defects and ageing. Dental regenerative therapy opens an arena to provide living, functional, and biocompatible tissues.[1] Tissues of the body (such as hematopoietic system, gastrointestinal system, dermal layers of skin) undergo rapid proliferation and were found to have the regenerative potential. This fact led to hypothesize that tissues, which regenerate may consist of cells that are responsible for the initialization of their replacement. These cells are the proposed “stem cells.”[2] Manipulation of these cells in vitro and using it for therapeutic purposes forms the basis for stem cell therapy.[3] Stem cells offer a great promise in treatment for diseases, in screening of new drugs in research, to develop a replica to study normal growth and investigate the unique regenerative abilities of stem cells offer a lot of optimistic hope amongst scientific researchers, therapeutics, and most importantly to the patients who will be the chief beneficiary of this boon to science. Two outstanding characteristics of stem cells make them unique [Figure 1]. 1. Stem cells are unspecialized cells, which have the capacity to renew themselves for prolonged periods by cell division. 2. And they can be induced to transform into specialized

Embryonic and Germinal Stem cells Embryonic stem cells (ESCs) are extracted from 2 to 11  days old embryos that are the blastocysts. ESCs are found to originate from the inner cell mass of the blastocyst [Figure 2].[8-11] ESCs are considered immortal. Their constant replicative life span can be attributed to their telomerase expression.[12] They can be propagated and maintained in an undifferentiated state indefinitely. Germinal stem cells are extracted from

Figure 1: Stem cells have the capacity to renew themselves and they can be induced to transform into specialized cells with specific function 1

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Figure 3: Somatic cells can be reprogrammed to adopt pluripotency by the enforced expression of a few embryonic transcription factors by retrovirus-mediated transduction

Figure 2: Embryonic stem cells are found to originate from the inner cell mass of the blastocyst

primary germinal layers of the embryo. They can differentiate into progenitor cells and have the potential to produce specific organ cells. They are best grown from supernumerary embryos obtained from in vitro fertilization centers. ESCs are totipotent, and they have the most desired potential of regeneration and repair of diseased tissue and organs in the body.[13,14] However, drawbacks like moral and ethical concerns, difficulty to control the growth and differentiation of the ESC which leads to a risk of tumorogenicity and teratoma formation have led to ESCs not so far being used therapeutically and have only remained an excellent platform for research.[13-16]

expressed, and expression of CD→ 45, 34,14, 11b, 79alpha or 19 and HLA-DR surface molecules should be lacking. • In vitro differentiation of MSC’s to osteoblasts, adipocytes and chondroblasts are considered necessary. Induced pluripotent stem cells (iPSCs)

The development of iPSCs from adult stem cells showed that these somatic cells can be reprogrammed to adopt pluripotency by the enforced expression of a few embryonic transcription factors by retrovirus-mediated transduction [Figure 3].[25,26] Takahashi and Yamanaka in 2006, practically derived iPSCs from embryonic and adult mouse fibroblasts by the ectopiccoexpression of genes: Oct4, Sox2, Klf4, and c-Myc, which transformed the somatic cells to an ESC-like pluripotent state.[26] In dental tissues, human DPSCs showed promise with higher reprogramming efficiency and generated iPSCs much more than the conventionally used dermal fibroblasts and this can be attributed to high expression of endogenous reprogramming factors such as c-Myc and KLF4and/or ESC marker genes.[27,28] Because the DPSCs are easily accessible by dentist, iPSCs generated from dental tissues are expected to be a promising cell source for tissue regeneration. Using iPSCs for regenerative medicine has several advantages, the prominent one being that their use can overcome the ethical issues associated with the use of ESCs. iPs can also be used as autologous patient-specific cells and this eliminates the threat of immune rejection of the graft.[28] The bottlenecks of iPS are inadequate cell number and tumor (teratoma) formation.[29]

Adult Stem Cells Adult stem cells are present in the postnatal population. Adult stem cells are multipotent – capable of differentiating into more than one cell type but not all cell types. Their life span is less replicative than that of ESCs. Mature tissues such as hematopoietic, gastrointestinal, neural, dental and other mesenchymal tissues demonstrate the presence of adult stem cells.[17-19] The plasticity of an adult stem cell is the ability of the stem cell to expand beyond its potential irrespective of the parent cell from which it is derived.[20] For example, dental pulp stem cells (DPSCs) not only develop into tooth tissue but also have the ability to differentiate into neuronal tissue.[21] Depending on where they originate, adult stem cells can be classified as hemopoietic stem cells, which are obtained from cord/peripheral blood and mesenchymal stem cells (MSCs).[22,23] which originate from the mesoderm layer and reside in a variety of adult tissues such as the bone marrow, limbal system, hepatic, dermal, dental tissues, etc. Criterias proposed by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy can be considered as guidelines, which define human MSC.[24] • MSC must continue to possess its property of plasticity when maintained in standard culture conditions. • Cell surface markers  -  CD105, 73 and 90, have to be

Stem Cell Markers Literatures prove it difficult to expand certain types of adult stem cells in culture, unlike ESCs. Adult stem cells reside in specific organs and tissues, but they are very few in number, and they may be buried deep in organs and tissues and diffusely spread out. Under the microscope, stem cells look like any other cell in the body,[30] and hence markers are proposed for MSCs and they fall 2

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et al. in 2008 attempted biological replacement of lost teeth in elderly patients found ADSCs to be a good alternative for tooth and related tissue regeneration[39] as the Odontogenic capacity of BMSCs significantly decreases with increasing age of the donors.[40]

into two categories: sole markers and stemness markers. A sole marker acts as a selection tool, which is sufficient to identify or purify MSC, which act by binding to unique receptors on the surface of the stem cell. Stro-1 is one of the most well-known markers for MSCs and is expressed in dental tissues,[30,31] synovial membrane, decidua parietalis-derived MSCs and multipotent dermal fibroblasts cell-surface glycoprotein on subsets Stro-1+ cells assists in isolating mesenchymal precursor cells (PCs).[30]

Dental stem cells

The most accessible stem cells are the dental stem cells. Stem cell-like qualities were found in certain cells derived from, dental pulp (DPSC), exfoliated deciduous teeth (stem cells from human exfoliated deciduous teeth [SHED]), PDL (PDL stem cell [PDLSC]), apical papilla (stem cells from apical papilla), and dental follicle (dental follicle progenitor cells)[41,42] [Figure 4]. Apart from regenerating dental tissues, biologically viable scaffolds can be used for the replacement of orofacial bone and cartilage, and defective salivary glands can be partially or completely regenerated. Their capacity for self-renewal and potential for multilineage differentiation, as in osteogenic, chondrogenic, and adipogenic differentiation as well as regeneration of tooth-specific structures such as cementum have made them the most sought after group of stem cell source.

Regeneration of tooth/tooth related tissues

For tooth-related tissue regeneration, the pre-requisites are: • Stem cells • Signaling molecules that can trigger the differentiation of unspecialized stem cells into specialized cell types, i.e. into osteoblasts, odontoblasts, cementoblasts, fibroblasts, etc. • Scaffold material which will act as a base to support and harvest cellular proliferation. Research has led to identification of MSC-like cells in adipose tissue,[32] placenta,[33] synovial membrane,[34] peripheral blood,[35] endometrial tissues, umbilical cord, umbilical cord blood,[35] dental tissues etc. The adult stem cell types that have been tried and explored in the arena of dentistry are: • Bone marrow stem cells (BMSCs), • Adipose tissue-derived stromal cells • Dental stem cells.

DPSCs

Gronthos et al. in 2000, were the first to successfully isolate stem cells from dental pulp (DPSCs), the experiment, which comprised of transplantation of these cells amalgamated with hydroxyapatite/tricalcium phosphate (HA/TCP) powder in an animal study (immune-compromised mice) showed formation of a structure mimicking dentin with odontblastlike cells that surrounded a pulp-like interstitial tissue(pulpdentin complex).[43] The markers expressed by DPSCs are similar to those of MSCs could differentiate in vitro into other mesenchymal cell derivatives the list of which is given in Table 1.

BMSCs

The bone marrow stem cells form colony-forming unit-fibroblasts in vitro and are capable of differentiating into many types of cells of mesenchymal lineages (e.g. osteoblasts, adipocytes, muscle PCs, tenocytes, chondrocytes or neurogenic cells). Ohazama et al. published a report in 2004, proved that the generation of tooth-like structures was possible by culturing bone marrowderived cells in combination with embryonic oral epithelium in the renal capsule.[36] Similar results were demonstrated in a study conducted by Li et al. that the amalgamation of oral epithelial cells from rat embryos with BMSSCs resulted in the expression of variety odontogenic genes and histologically produced toothlike structures.[37] Bone marrow stem cell implication in periodontal regeneration attracted a lot of attention when Kawaguch et al. in 2004 reported, after an animal study that auto-transplantation of these stem cells resulted in almost complete regeneration of periodontal defects in only four weeks and this was this was further confirmed histologically demonstrating cementum, periodontal ligament (PDL), and alveolar bone.[38] Adipose-derived stromal cells (ADSCs)

ADSCs have a clear advantage of accessibility with minimally invasive methods and the tissue abundance, e.g. liposuction procedure. They satisfy the criteria of defined by Dominici et al. and exhibit a multilineage differentiation potential in vitro. Jing

Figure 4: Anatomical position of dental stem cell sources 3

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Table 1: Multilineage differentiating potential of dental stem cells Stem cells from dental tissues DPSCs

Multilineage differentiating potential

SHEDs

Neuron‑like cells, odontoblasts, osteoblasts, and adipocytes

SCAPs

Osteoblasts, odontoblasts, and adipocytes

DFSCs

Osteoblasts, adipocytes , and nerve‑like cells

protocol. The extraction should be performed with strict control of the aseptic chain. Soft tissue remnants to be removed and the teeth to be immediately transferred to sterile chilled vials containing 20 mL of either of the three collection/ transport solutions: Hypothermosol, mesencult basal medium or phosphate-buffered saline. Transportation of the teeth on ice to the laboratory is preferred. In the laboratory, External sterilization of the tooth by several washes in sterile phosphatebuffered saline (PBS), followed by immersion in 1% povidoneiodine for 2 min, immersion in 0.1% sodium thiosulfate in PBS for 1 min and another wash in sterile PBS is mandatory. The roots of cleaned teeth is then separated from the crown to reveal the dental pulp, and the extirpated pulp is to be placed into an enzymatic bath consisting of type I and type II collagenase with thermolysin as the neutral protease. In order to digest the tissue and liberate the cells, pulps should then be allowed to incubate at 37°C for 40 min. Once the digestion is complete, to neutralize the digestion enzymes mesencult complete medium should be added to a final volume of 1.5 times the digestion volume. The mixture should be then centrifuged at 500 g for 5 min, and the supernatant aspirated. The cell pellet should then be re-suspended in fresh mesencult complete medium added with 0.25 μg/mL amphotericin B, 100  IU/mL penicillin-G, and 100 μg/mL streptomycin. Cells should be plated at an initial concentration of one tooth digest per 25 cm2 flask. Culture flasks should be monitored daily and any contaminated flasks removed immediately and recorded. Non-contaminated flasks should be monitored for cell growth, three times per week. After 14  days of growth, DPSC can be detached using 0.25% trypsin/1 mmole EDTA.[49] The extracted cells are then cryopreserved for future use. This process of cryopreservation involves equilibrating the cells with a solvent - the cryoprotectant solution that prevents cells forming ice crystals. At the same time, the integrity of cell membranes is preserved and reversed to normal on thawing. Programmable controlled-rate freezers are utilized to slowly bring down the temperature to freezing. The cells that are frozen by this method are then transferred for long-term storage to vapor-phase liquid nitrogen freezers to be stored at ultra-low temperatures (−150°C).[50] When the therapeutic need for these cells arises, reprogramming conditions/factors if required are introduced to induce self-renewal and pluripotency, with an aim of producing patient-specific iPSCs. Consecutive induction of these cells resultantly forms ectodermal epithelial cells and neural crestderived mesenchymal cells, which are further induced to form odontogenic cells in vitro. Combination by direct contact of both these cell populations, mimicking the in vivo arrangement provokes cellular interaction hence leading to the formation of an early-stage tooth germ. Once transplanted into the mouth, the recombinants develop and lead to functional recovery from tooth loss. Scaffold is an extracellular matrix and mold to culture the stem cell and induce the desired function. Provides physical support to the cells required for regeneration of any tissue. The

Odontoblasts, adipocytes, chondrocytes, and osteoblasts and functionally active Neurons

DPSCs: Dental pulp stem cells, SHEDs: Stem cells from human exfoliated deciduous teeth, SCAPs: Stem cells from the apical papilla, DFSCs: Dental follicle stem cells

SHED

Miura et al. in 2003 identified dental pulp from deciduous teeth (SHED) as a good source of high-quality human postnatal stem cells. The results of the experiment demonstrated that SHED’s yield dentin and bone on combination with HA/TCP powder; however they did not form a dentin-pulp complex. They have good plasticity for multilineage differentiation in vitro [Table 1], show higher rates of proliferation and population doublings in comparison with stem cells of the dental pulp.[44].Cordeiro et al. (2008) suggested that SHEDs could be the ideal source of stem cells for repairing damaged teeth or for induction of bone formation.[45] Stem cells from the apical papilla (SCAPs)

SCAPs are mostly found in the root apical papilla of third molars and teeth with open apices, i.e. developing teeth. The expression of surviving and telomerase, two important molecules in cell proliferation mediation, were found to be in high levels in these cells and this fact is attributed to the higher rates of proliferation in vitro compared with DPSCs.[46,47] SCAPs show a promising scope for cell-based therapy for root regeneration. Transplantation of these cells along with PDLSCs into tooth sockets of mini pigs demonstrated the formation of dentin and PDL.[47] Dental follicle stem cells (DFSCs)

PCs for cementoblasts, osteoblasts, and PDL cells, were demonstrated by Morsczeck et al. in 2005 from the ectomesenchymal covering of the tooth follicles of developing third molars (DFSCs). These cells also exhibit multilineage differentiation as depicted in Table 1. In vivo they form cementum and PDL.[48] Future treatment options for the restoration of damaged dentin, pulp, cementum and PDLs may make use of autologous stem cells such as DPSCs, SHEDs, SCAPs, and DFSCs that have been stored after removal from the patient. Isolation and culture of DPSCs

In a study conducted by Perry et al. in 2008, the methodology used by researchers for successful isolation of stem cells from the dental pulp can be used as a reference for the technique 4

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ideal requirement of scaffolds is that it should be biodegradable, and rate of degradation should coincide with the rate of tissue formation. They should be porous and allow appropriate differentiation of cells causing no damage to the characters of the progeny. In the root canal, where the pulp has blood supply only from apical end system it is preferable to have a scaffold that promotes vascularization of the implanted cells and to promote angiogenesis, growth factors that favor angiogenesis are impregnated. As a result of advanced technologies like robotics assisted tissue-engineering, constructs can be prepared that have a controlled spatial distribution of cells and growth factors, as well as engineered gradients of scaffold materials with a predicted microstructure.[51]

of damaged coronal dentine and pulp, regeneration of resorbed root, cervical or apical dentin and perforations, periodontal regeneration, craniofacial defects by osteogenesis,[53] whole tooth regeneration[5] and treatment of oral mucosal lesions (oral submucous fibrosis, oral lichen planus, dyskeratosis congenita, premalignant lesions such as leukoplakia, recurrent oral ulcers, graft versus host disease and oral cancers).[57,58] Conclusion The isolation and understanding of dental stem cells and their multilineage differentiating potential combined with their easy accessibility has brought a new light to the field of dentistry, paving a path for the progressive evolution to “regenerative dentistry.” This changing concept and definition of a “dental caregiver” to a “regenerative dentist” with scope to “regrow” lost dental tissues is a boon from science as a biological solution to a biological problem.

Potential Applications in Medicine Stem cells are being researched for a variety of chronic debilitating diseases that pose a therapeutic dilemma until date. Proposed cell therapy targets to repair, repopulate, and rewire tissues and organs with complete reversing of the pathology. In an experiment conducted by Seo et al. in 2009, SHEDs were successfully capable of repairing critical-size parietal defects in immunocompromised mice and they proposed a hypothesis that neural crest cell-derived SHED may offer optimal orofacial repairing with a matched neural crest origin.[52] Stem cells in cancer, according to a review by Sagar et al. in 2007, have expanded beyond its long-term usage for replenishment of blood and immune systems damaged by the cancer cells/chemotherapy/radiotherapy, but also to contribute in the tissue regeneration and as delivery vehicles in the cancer treatments.[53,54] DPSCs were proven to differentiate into functionally active neurons, and when implanted DPSCs induce endogenous axon guidance, suggesting their potential as cellular therapy for neuronal disorder. Thus, based on these findings, stem cell research has kindled hope in the field of medicine directed toward: brain damage, spinal cord injury, Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis, multiple sclerosis and other neurological and neurodegenerative disorders.[55] The Tissue regeneration concept hopes to repair heart damage, baldness, deafness, blindness, impaired vision, muscle damage, diabetes and related disorders, etc. Though not an established treatment yet, autologous hematopoietic stem cell therapy can induce remissions and even cure some selected patients with therapyrefractory autoimmune diseases.[56] Stem cell research is a boon to drug research as the application of drugs directly to human cells, and this will provide more relevant data than testing on animal models.

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