Dental pulp Stem Cells in Regenerative Medicine – A Literature Review
Chris Noel Timothy1, Samyuktha. P.S.1, Dr. M.P. Brundha2
1Undergraduate Student, Saveetha Dental College, Saveetha Institute of Medical and Technical Science, Chennai 600077 Tamil nadu, India
2Department of Pathology, Saveetha Institute of Medical and Technical Science, 162, Poonamallee High Road, Chennai 600077 Tamil nadu, India
Stem cells are unspecialized cells that have a property of differentiating into specific specialized cell types. They also have a self-renewal property. Stem cells are identified in various human adult tissues including adipose tissue, skin, blood, bone marrow, hair follicles and dental pulp. Stem cell research has undergone rapid development owing to its usefulness in regenerative therapies for various neurological and genetic disorders. Previous Studies have shown that the dental pulp tissue can also be used to derive Mesenchymal Stem Cells (MSCs) when tissue is grown in culture. These MSCs can differentiate into several cell types. It is proposed that dental pulp stem cells (DPSCs) can develop Induced Pluripotent Stem Cells (iPSCs) which can be used for therapies of various diseases .This study has a primary focus on Dental Pulp Stem Cells and its various uses and Advancements in regenerative medicine. Nevertheless, it is still a fairly recent discovery and therefore requires further research on its biological capability, regenerative property, etc. until it is introduced into everyday medical practices.
Stem cells are unspecialized cells that have a property of differentiating into specific specialised cell types. They also have a self-renewal property. Stem cells are identified in various human adult tissues including adipose tissue, skin, blood, bone marrow, hair follicles and dental pulp (1). Stem cell research has undergone rapid development owing to its usefulness in regenerative therapies for various neurological and genetic disorders. Previous Studies have shown that the dental pulp tissue can also be used to derive mesenchymal stem cells (MSCs) when tissue is grown in culture (2). These MSCs can differentiate into several cell types. It is proposed that dental pulp stem cells (DPSCs) can develop Induced Pluripotent Stem Cells (iPSCs) which can be used for therapies of various diseases (3).This review has a primary focus on Dental Pulp Stem Cells and its various uses and Advancements in regenerative medicine.
Odontogeny of dental pulp stem cells:
Development of teeth involves continuous interactions between the oral ectodermal epithelial cells that give rise to Enamel, Dental Papilla and Follicle and the mesenchymal cells that give rise to Dentin, Pulp, Cementum and Periodontal Ligament (4).
Five subtypes of mesenchymal stem cells have been described, these are: dental pulp stem cells (DPSC), periodontal ligament stem cells (PDLSC), stem cells from apical papilla (SCAP), dental follicle stem cells (DFSC) and gingival mesenchymal stem cells (GMSC) (5). Teeth are therefore an excellent source of stem cells for therapeutic procedures in the future and can be easily harvested following tooth extraction or natural shedding of deciduous teeth.
Dental Pulp Stem Cells (DPSC) were first isolated from third molars. These cells are found to have high clonogenicity and formed highly calcified colonies. DPSC have also been confirmed as mesenchymal stem cells by demonstrating their ability to form adipocytes, osteoblasts, odontoblasts, chondrocytes, neural ectodermal cells and myoblasts. From a developmental viewpoint the dental pulp is derived from ectomesenchyme arising in the periphery of the neural tube which migrates to the oral region where the cells differentiate into mesenchymal cells (6).
In normal physiology the dental pulp cells maintain and repair the periodontal tissue and respond to damage. Deep caries result in the dental pulp cells migrating to the damaged area and the creation of odontoblasts and dentine in an attempt to repair the damaged tooth. These observations led to the proposal that dental pulp stem cells could be active during reparative dentinogenesis.
Laboratory processing of dental pulp stem cells
There are many approaches to dental pulp stem cell collection and processing, the key is to ensure the quality and safety of the end product. The most commonly use done is described below: once exfoliated, or extracted, teeth are sent to the processing laboratory within 72 hours of exfoliation or extraction (7). The tooth is transported in sterile phosphate buffered saline with calcium and magnesium inside a validated and monitored collection kit which keeps the tooth between 4 and 26 °C. On arrival at the laboratory the tooth is opened in a Grade A clean room environment using a medical circular saw, the pulp exposed to 10% DMSO and the whole tooth is then frozen in a controlled rate freezer and stored in the vapor phase of liquid nitrogen.10 When the dental pulp stem cells are required then the tooth is thawed rapidly in a 37 °C water bath and then processed using either one of two standard techniques: the explant method and the enzymatic digestion method. In the explant method the dental pulp is dissected from the tooth in a Grade A clean room environment and the cells are then grown in vitro from these tissue fragments. In the enzymatic method the dental pulp tissue is digested in collagenase and dispase, in a Grade A clean room environment, and the resultant cells then grown in vitro (8). Both of these processing technologies yield good numbers of viable DPSC and future research will no doubt optimize these technologies to develop a gold standard.
Immunocytochemical identification of dental pulp mesenchymal stem cells
The International Society for Cellular Therapy (ISCT) state that mesenchymal stem cells express the following surface antigens: CD105 (endoglin: a putative novel endothelial cell specification gene), CD73 (5’ ectonucleotidase: an enzyme which metabolizes nucleotides to nucleosides) and CD90/Thy-1 (glycosylphosphatidylinositol-anchored glycoprotein) and a negative for CD11b, CD14, CD19, CD34, CD45, CD79a surface antigens and HLA-DR (9). These are assessed by the use of flow cytometry. Other workers propose that mesenchymal stem cells express STRO-1 (stromal precursor antigen 1), VCAM-1 (vascular cell adhesion molecule 1), SH2 (Src homology 2), SH3/SH4, CD271, GD2 (ganglioside 2), and SSEA-4 (stage-specific embryonic antigen-4) (10). Some workers even suggest that DPSC may have a different immunophenotype to those traditionally thought to be MSC. This variation in surface antigen expression may reflect the proliferative potential of DPSC. STRO-1 positive DPSC have been shown to have odontoosteogenic characteristics whereas CD34+, CD117+ and CD45- DPSC have a greater capacity for cell renewal and osteogenic differentiation. Other authors have referred to DPSC MSC expressing CD29+, CD44+ and CD73+.The expression of transcription factor genes Oct-4 and Nanog have also been used to identify DPSC MSC (11). The identification of DPSC mesenchymal stem cells is clearly a developing science which will no doubt be refined in the future to clearly describe each subpopulation of DPSC. The fact that DPSC have low expression of Class II HLA-DR (MHC) molecules means that they are immunologically privileged and it may be possible to transplant these cells from one person to another without the need for tissue matching. This raises the possibility of a public DPSC bank, perhaps in collaboration with key dental hospitals, to provide DPSC to anyone in need. Such donated DPSC could be extremely useful when using artificial bone to provide new bone for dental implants where the artificial bone could be used along with donated DPSC to enhance bone formation (12).
Sources and characteristics of dental pulp stem cells (DPSC) and stem cells from human exfoliated deciduous teeth (SHED)
DPSC have been isolated from exfoliated deciduous teeth (SHED: stem cells from human exfoliated deciduous teeth), from permanent secondary dentition, from teeth extracted due to impaction or periodontitis and from inflamed pulp tissue. SHED cells have been shown to have a high proliferative rate and are capable of producing osteoblasts, adipocytes, neuronal cells and odontoblasts. Some workers suggest that SHED cells have a greater proliferative capacity than DPSC obtained from adult third molars, incisors or supernumerary teeth on the basis that SHED cells represent a more immature type of stem cell. It has been shown that the properties of DPSC are directly related to the physical age of the tooth from which they are obtained (13). It is interesting to note that in terms of cell cycle 69.8% of SHED cells were found to be in the S and G2 stage, but only 56% of the DPSC were in those phases indicating increased proliferative capacity in SHED cells. The surface antigen expression of SHED cells also differs from that seen in DPSC. This is reflected in the fact that proliferation related and extracellular matrix (ECM) formation genes, for example genes encoding transforming growth factor (TGF) and fibroblast growth factor 2 (FGF), are expressed in SHED cells. Genes coding for collagen I and collagen III and pluripotency markers, such as Pou5f1, Oct3/Oct4, Sox2, and Nanog are also expressed higher in SHED cells. The expression of Nestin (a marker of neuroepithelial stem cells) is reduced in SHED resulting in their reduced ability to form neurospheres in comparison to DPSC (14). Permanent teeth, impacted third molars and supernumerary teeth are an excellent source of DPSC which have the following mesenchymal stem cell surface antigens: CD90+, CD146+, CD105+ and CD45−; and also express Oct4 and Nanog but lower expression than that seen in SHED cells. Both DPSC and SHED cells are an excellent source of mesenchymal stem cells for regenerative medicine procedures, in addition, SHED cells have recently been proposed as potential immuno-modulators in the treatment of autoimmune encephalomyelitis and other autoimmune pathologies of the central nervous system (15).
Clinical applications of DPSC in regenerative medicine
In a recent study, few researchers attempted cell-based therapy for bone regeneration using stem cells from deciduous teeth, dental pulp, and bone marrow. The Results obtained from their study demonstrated that stem cells from deciduous teeth, dental pulp, and bone marrow with platelet rich plasma have the ability to form bone. Bone formation with Deciduous and adult Tooth Stem Cells may possess the ability to generate a graft between a child and parent. This preclinical study could lay the path for stem cell therapy in orthopaedics and oral and maxillofacial reconstruction for clinical application (16). In studies involving human beings Dental Pulp Stem Cells have been transplanted along with some sort of framework or porous biomaterial to permit the cells to mature to enable osteogenesis (17, 18). The dental pulp stem cells have the capability of repairing skin lesions, ischemic tissue, bone damage, periodontal tissue, liver, skeletal muscle tissue, neuronal tissue and blood vessels (19-22). For these reasons dental pulp seems like a reliable and easy to obtain source of mesenchymal stem cells which can be used in regenerative medicine.
Rebirth of an entire new teeth from scratch for the substitution of missing or lost teeth is the most ambitious goal in dentistry and necessitates the use and recombination of dental pulp stem cells .Dental Mesenchymal Stem Cells can form all mesenchymal components of the tooth organ and the surrounding tissues such as dentin, cementum, and alveolar bone, while Dental enamel Stem Cells are crucial for the generation of enamel. Since most of the dental epithelial cell populations disappear shortly after tooth eruption and Dental Stem Cells are limited in human adult teeth, current knowledge on Dental Stem Cells have been obtained mainly from rodents, where they contribute to the renewal of the enamel and other tooth structures in the continuously growing incisors (23, 24). Although encouraging, this methodology also needs further research and investigation , as effective protocols for the differentiation of human Dental Stem Cells from inducible pluripotent stem cells are not available yet (25, 26).
Pulp and dentin regeneration
In order to produce functional pulp for clinical application, several concerns must be considered: first of all the regenerated pulp tissue must have sufficient vascularity, even though the blood supply occurs only from the apical foramen, second of all newly differentiated odontoblasts should form new dentin on the existing dentin wall of the root canal and finally new dentin must be produced by the differentiated odontoblasts from the stem cells on the existing dentin .Regeneration of dentin relies on having pulp vitality, however, regeneration of pulp tissue has been challenging as the tissue is covered by dentin without a guaranteed blood supply except from the apical foramen at the root end. With the dawn of modern tissue engineering concept and the unearthing of dental stem cells, regeneration of pulp and dentin is still under testing (27,28).
Stem Cell therapy as a treatment for liver disease requires operative stem cell derived hepatocyte cells. Dental Pulp Stem Cells have been differentiated to produce Hepatocyte-Like Cells (HLCs) with hepatocyte like functions that have been acquired, such as glycogen storage and urea production (29- 31).
In a recent study, Dental pulp cells were transplanted into collagen gels and infused within a silicon tube, which was positioned within a 7 mm gap in the buccal branch of rat facial nerve. The dental pulp cells formed blood vessels and myelinating tissue and contributed to the promotion of normal nerve regeneration (32). Considering the results, dental pulp stem cells could be used to treat nerve injury and neurological disorders. DPSC have been used as a graft into hemisected spinal cords in animal models resulting in an increased number of surviving motor neurons illustrating the promise of this technology in future clinical trials (33).
DPSC have also been shown to promote neurogenesis when co-cultured with rat retinal cells, this is thought to be related to the ability of DPSC to induce the expression of neurotrophins (neurotrophins are a family of proteins that induce the survival, development, and function of neurons) (34). The ultimate application in ophthalmology is to develop cell based regenerative technology which could repair or replace a damaged retina and therefore restore sight, This raises the possibility that DPSC could differentiate into functional photoreceptors which could in turn be used to restore sight in patients suffering from a damaged retina (35-37).
Stem cell hold great promise to solve a variety of health problems, diseases and disorders both inside the oral cavity and outside it. Although there are a variety of sources from which stem cells can be isolated, dental pulp is a very reliable and an easy to obtain source. Dental pulp stem cells can be collected, processed and cryogenically stored each time a deciduous tooth is exfoliated or a healthy adult tooth is extracted. In recent days the applications of the dental pulp stem cells have escalated, due to this the amount of study on their regenerative property has also increased. Nevertheless, it is still a fairly recent discovery and therefore requires further research on its biological capability, regenerative property, etc… until it is introduced into everyday medical practices.
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Accepted on 19.05.2019 © RJPT All right reserved
Research J. Pharm. and Tech 2019; 12(8):4052-4056.