STEM CELL REGENERATIVE THERAPY IN ORAL AND MAXILLOFACIAL REGION: A SYSTEMATIC REVIEW

Dr. Saraswathi Gopal. K and Dr. Padma. M. 1. MDS Professor and Head, Dept of Oral Medicine and Radiology, Meenakshi Ammal Dental College and Hospital, Chennai, India. 2. Post Graduate student, Dept of Oral Medicine and Radiology, Meenakshi Ammal Dental College and Hospital, Chennai, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

In the modern era, where biology and biotechnology have replaced the chemistry, we are exploring "biological solutions to biological problems." The breakthrough of stem cells has produced new possibilities in the regeneration of different organs and tissues. The scope of application of stem cells in dentistry is vast and includes continued root formation, in pulp/dentin tissue regeneration, in replantation and transplantation, and reconstruction of the periodontium, in cancer management and research. In few years plenty of studies have been conceded which showed that stem cells and tissue engineering are giving rise to a separate branch termed "Regenerative Dentistry" that will have its own position in future dental practice. This article provides an insight into the various aspects of stem cells therapies in oral mucosal lesions and its future perspectives.

Introduction:-
Stem cells are the precursors of the body tissue which are undifferentiated biological cells that can differentiate into specialized cells and can divide (through mitosis) to produce extra stem cells. (1,2) They are also defined as immature or undifferentiated cells that are capable of generating daughter cells identical to themselves or of differentiating into diverse cellular phenotypes. These cells are originated in multicellular organisms (3,4) . Human dental tissues have a limited potential to regenerate but the discovery of dental stem cells have developed new and surprising scenario in regenerative dentistry.

(3), 1631-1643
1632 History Of Stem Cells:-Stem cells also known as "progenitor or precursor" cells and are clonogenic cells capable of both self-renewal and multi-lineage differentiation. In 1868 the term "stem cell" for the first time appeared in the works of German biologist Haeckel (4,5) . Wilson coined the term stem cell (5) . In 1908 Russian Histologist Alexander Maksimov postulated existence of hematopoietic stem cells at congress of hematologic society in Berlin (6) . Then the term "stem cell" was proposed for scientific use.

Stem Cell Properties:-
A classic stem cell should possess two properties namely self renewal and potency 7, 8, 9 . • Self-renewal is the capacity of the cell to undergo numerous cycles of cell division maintaining the undifferentiated state. An ideal stem cell should have the capacity of self renewal (the ability of the cell to proliferate to about 40-60 population doublings).
• Potency means the differentiation capacity of the stem cells.

Classification Of Stem Cells (10,11):-
There are different types of stem cells that are usually considered for their potential use in research. They can be classified on the basis of the following: 1. The extent to which they can differentiate into different cell types.

Source of stem cells.
Extent to which they can Differentiate:a. Totipotent cells: These stem cells have the potential to differentiate into any kind of cell in the body. b. Pluripotent cells: These are true stem cells, with the potential to differentiate into any differentiated cells in the body, except the cells of placenta or other supporting tissues of the uterus. c. Multipotent cells: These are true stem cells, but only differentiate into a limited number of cell types, e.g. the bone marrow contains multipotent stem cells that give rise to all the cells of the blood but not to other types of cells. Multipotent stem cells are of three major types -ectodermal (skin and nerves), mesodermal or mesenchymal (bone, cartilage, muscle and adipose tissue) and endodermal (intestines and other organs).
On the Basis of Source (  1633 b. Embryonic germ cells (or foetal germ cells):-Embryonic germ cells are pluripotent stem cells derived from the so-called primordial germ cells, which are the cells that give rise to the gametes (sperm and eggs) in adults. These can be isolated from the precursor to the gonads in aborted tissues.
c. Adult Stem Cells:-Adult stem cells are characterised as haematopoietic or mesenchymal . Hematopoietic stem cells are capable of forming all types of white blood cells and red cells. Sources of Haematopoeitic stem cells are Umbilical cord blood, Umbilical cord, bone marrow and peripheral blood. Haematopoietic stem cells have been used to treat blood cancers such as leukaemia and other blood disorders. Mesenchymal are capable of forming a wide variety of connective tissues such as bone, muscle, cartilage, fat, tendons-but not internal organs or skin , Sources of Non-haematopoietic Stem Cells or Mesenchymal Stem Cells are adipose tissue, bone marrow and almost all body tissues including the pulp tissue of the teeth (13) .
Bone marrow (14,15) :-Bone marrow stem cells (BMSCs) can be harvested from sternum or iliac crest. It is composed of both hematopoietic stem cells and mesenchymal stem cells (MSCs). The advantage of bone marrow is that it has a larger volume of stem cells and can be differentiated in to wide variety of cells. Isolation of BMSCs can be carried out only under general anesthesia. Bone marrow contains both types of stem cells and has been most extensively researched.
Adipose tissue (8,16) :-They can be harvested from the lipectomy or liposuction aspirate. Adipose derived stem cells (ADSCs) contain a group of pluripotent mesenchymal stem cells. Advantage of adipose tissue is that it is easily accessible and abundant in many individuals. (17) Induced pluripotent stem cells (iPSCs) are adult cells that have been genetically reprogrammed to an embryonic stem cell-like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells. They express stem cell markers also and are capable of generating cells characteristic of all three germ layers. iPSCs may prove to be useful tools for drug development and modeling of diseases and in transplantation medicine . In this evolving concept 3-4 genes found in the stem cells are transferred into the donor cells using appropriate vectors. The stem cells thus derived by culturing will have properties almost like embryonic stem cells. This path breaking discovery may have a major role in future stem cell therapy.

Source Of Stem Cells From Dental Tissues (18,19):-
Stem cells from oral and maxillofacial region predominantly contain mesenchymal stem cells. Deciduous teeth, wisdom teeth and tooth extracted for orthodontic treatment are the major sources of dental tissue. In oral and maxillofacial area different types of dental stem cells were isolated and characterized ( Figure : 2

). They include • Dental pulp stem cells (DPSCs) • Stem cells from exfoliated deciduous teeth (SHED) • Periodontal ligament stem cells (PDLSCs) • Stem cells from apical papilla (SCAP) • Dental follicle progenitor cells (DFPCs)
Dental pulp stem cells (DPSCs) were successfully isolated by Gronthos et al (18) ., in 2000. They were able to demonstrate odontoblast like cells from DPSCs producing ectopic dentin in the immunocompromized mice. (19) were identified to be cells of higher proliferation rate, with increased population doublings, immature multipotent clonogenic cells isolated from deciduous teeth that can differentiate into several cell types.

Periodontal ligament stem cells (PDLSCs)
were isolated from periodontal ligament of 25 human third molars by Seo BM et al in 2004 (20) . They demonstrated cementoid cells, adipocytes when transplanted into immunocompromized rodents. (21) isolated mesenchymal stem cells from apical papilla which are capable of forming odontoblast like cells in vivo.

Stem cells from apical papilla (SCAP) Sonoyama et al in 2008
1634 (22) obtained stem cells from dental follicle which can form cementoblasts, periodontal ligament cells and osteoblasts. The advantages of stem cells from oral and maxillofacial region is that 1.

Dental follicle progenitor cells (DFPCs) Morsczeck C et al in 2005
Have high plasticity.

2.
It can be cryopreserved for longer period.

3.
It showed good interaction with scaffold and growth factors.

4.
The dental pulp stem cells will be better fitting tool due to easy surgical access, the very low morbidity of the anatomical site.

Stem Cells Storage And Transport (24,25) :-
Tissue samples containing stem cells are placed in a screw top vial containing Hypothermosol, mesencult basal medium or phosphate-buffered saline media, which nourishes it during transport. The sample should reach the processing storage facility before 40 hours. In the laboratory the samples were trypsinized and cryopreserved at −150°C to yield colonies of stem cells. The required cell type can be manipulated by utilizing right inductive signals and appropriate growth factors to the stem cells.

Stem Cell Application In Oral Diseases:-
Clinical applications of dental stem cells will continue to emerge in the near term and longer term. Currently dental stem cell research focuses on regeneration of dentine, pulp and teeth, alveolar bone, regeneration of periodontal ligament, and various other applications of mesenchymal and hematopoietic stem cells in repair of craniofacial defects, oral mucosal lesions and salivary gland regeneration after radiation therapy.
Regeneration of teeth (25,26) :-Tooth regeneration represents a new era in dentistry as the concepts of repair is being shifted to regeneration. Regenerative dentistry restores a tissue defect to its original form and function by biological substitutes. Dental tissue stem cells/ progenitor cells can differentiate into dental cell lineages, and are used to regenerate some dental tissues in the treatment of tooth defects and tooth loss. Research focuses on whole tooth regeneration using a strategy of transplanting artificial tooth germ and allowing it to develop in the adult oral environment. Two means of regenerating teeth include conventional tissue engineering, in which the application of cells in a carrier material in vitro under the influence of a stimulus leads to tissue regeneration. The second process of tooth regeneration is using dental epithelium and mesenchymal cells (27,28) in vivo after direct implantation, which is based on knowledge of embryogenesis and physiological tooth development during childhood.
Handa K (2002) (29) isolated progenitor cells from bovine dental follicles. In invitro conditions these cells formed clusters of spheroid like cells and in invivo conditions, cementum matrix formation took place by these cultured dental follicle cells. Gronthoset al 2002 (25) demonstrated both in vivo and in vitro in animals that dental pulp stem cells (DPSCs) were able of forming ectopic dentin and related pulp tissue (25). Batouli et al. 2003 (30) demonstrated the capacity of dental pulp stem cells to form a dentin pulp-like complex by transplanting them in immunocompromised mice. After 16 weeks, the pulp-like tissue contained a fibrous connective tissue, blood vessels, and odontoblasts associated with newly formed dentin indicating the possibility of using those stem cells for the repair of injured dental structures. Duailibi et al. 2004 (31) , in their studies were able to form tooth from single cell suspensions of cultured rat tooth bud cells. They confirmed bioengineered rat teeth grown in 12 weeks with Poly Lactic co glycolic 1635 acid (PLGA) and Poly glycolic acid (PGA) scaffold. Honda et al 2005 (32) developed tissue engineered teeth, when implanted into omentum of rat utilizing porcine tooth bud cells and PGA fiber engage scaffold that reminds of the model of odontogenesis. Young et al 2005 (33) using porcine tooth bud cells, PLGA and PGA scaffolds produced a crossbreed tooth bone for the cure of tooth loss beside with alveolar bone resorption. Huang et al 2008 (34) in his review article summarized new protocol for endodontically involved immature permanent teeth in which minimal instrumentation was done in it followed by disinfection with triple antibiotic paste. Treated tooth is coated with mineral trioxide aggregate (MTA) and filled with glass ionomer cement. Periodical observation was done to ascertain root maturation.

Periodontal regeneration:-
Recently new techniques of cell transplantation have been developed to regenerate periodontium using Periodontal ligament cells (PDLCs) isolated from extracted human teeth and Dental follicle stem cells (DFSCs) which could become an alternative cell source for periodontal regeneration therapy. When transplanted into immunocompromised mice, PDLCs have shown to regenerate collagen fibers and cementum/periodontal ligamentlike structures. Periodontium can also be regenerated by culturing of periosteum, non-dental stem cells such as bone marrow mesenchymal stem cells, and adipose-derived stem cells. Studies have shown that grafted autologous cultured cell membrane derived from periosteum into a surgically created class III furcation defect in dogs, lead to the formation of cementum, periodontal ligament-like tissues, and alveolar bone in 3 months after the grafting procedure. Another research reported a treatment using cells derived from periosteum in human periodontal disease by Kawaguchi et al2004 (35) . It is also confirmed by Hasegawa et al 2005, that periodontal defects can be managed by reimplantation of these cells (36). Autologous mesenchymal stem cells from iliac crest in combination with platelet rich plasma from peripheral blood was used for periodontal regeneration. Significant closure of bone defect and improvement of attachment level was observed after one year follow up. It also showed good healing and regeneration of interdental papilla (37). Nagatomo et al2006 (38) in their experimental studies found that PDL cells having stem cell properties can regenerate periodontium. Transplantation of PDL derived cells into animal models were shown to regenerate periodontal tissue. Iwata et al2009 (39) harvested and expanded primary canine PDL cells in vitro and also made into transplantable constructs containing Poly glycolic acid (PGA) Scaffold and PDL cell sheets. The transplantable constructs in combination with porous tricalcium phosphate (b-TCP) induced regeneration of periodontal structures, including alveolar bone, cementum, and periodontal fibers. Liu et al2008 regenerated periodontal tissue in miniature swine using scaffolds seeded with periodontal ligament derived stem cells (40) . PDLSCs can differentiate into cells that can colonize on biocompatible scaffold, suggesting an easy and efficient autologous source of stem cells for regeneration of dental tissues (41) . Marie MK et al 2009in their experimental on goat was able to regenerate periodontal tissues around titanium implant using autologous bone marrow stem cells with scaffold . (42) Repair of craniofacial defects:-Craniofacial defects results from post-cancer ablative surgery, craniofacial osseous deficiencies can also arise from infection, trauma, congenital malformations and progressively deforming skeletal diseases. Although autologous bone graft is considered the best option, it has the limitation of donor sites. Use of skeletal or dental stem cells can be used in future to repair craniofacial region and may provide a promising alternative approach for reconstruction of craniofacial defects. Lagenbach et al2010 (43) in their in vitro studies used microspheres (scaffold free tissue construct) to close the bone defects. They found osteogenically differentiated microspheres with outgrowing cells can be used to fill up bone defects. This new procedure has added advantage of permitting the transplantation of more cells and better integrity compared with cell suspensions or gels (44) . Degenerative bone diseases including TMJ defects can be bioengineered with stem cells. Cells from various sources like articular cartilage cells, fibroblasts, mesenchymal stem cells have been used to reconstruct TMJ (45). Bone tissue engineering endeavours to repair large bone losses using three dimensional scaffolds to deliver vital cells to the defective site.

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The main mechanism behind the use of stem cells in these lesions is discussed in detail. Oral ulcers and wound healing (46) :-Optimum healing of a cutaneous wound requires a good integration of the complex biological and molecular events of cell migration and proliferation and extracellular matrix deposition, angiogenesis and remodeling. Large wounds take a long time to heal, and defective healing leaves behind unacceptable scars and strictures.
Bone marrow-derived mesenchymal stem cells (BM-MSCs) are self-renewing, expandable stem cells and are able to differentiate into adipocytes, osteoblasts, and chondrocytes. These are used possess the ability to engraft at the site of injury and promote tissue regeneration and wound healing through synergistic downregulation of proinflammatory cytokines and increased production of soluble factors with antioxidant, antiapoptotic, and proangiogenic properties.
In oral wounds, they exhibit increased re-epithelialization, cellularity, intracellular matrix formation and neoangiogenesis, thereby accelerate wound healing. Hence, MSC therapy can be a promising therapeutic modality for oral ulcers and wounds. El-Menoufy et al 2010 (47 ) . Submucosally injected autologous Bone Marrow-Mesenchymal Stem Cells suspended in phosphate buffered saline around formocresol induced oral ulcers in dogs. There was increased expression of both collagen and vascular endothelial growth factor (VEGF) genes in Mesenchymal Stem Cells-treated ulcers compared with controls. Similar results were seen by Aziz Aly et al 2012. (48) in a study on formocresol induced oral ulcers in dogs using Bone Marrow and adipose-derived stem cells. Hence He concluded Mesenchymal Stem Cells transplantation may help to accelerate the healing of oral ulcers [ Figure 3]. Oral mucositis:-Oral mucositis is one of the most debilitating side effects which occur with chemo or radiotherapy. Management of mucositis is fairly symptomatic. Presently mesenchymal stem cells have been explored in its management by virtue of their immunomodulatory, anti inflammatory functions as well as regenerative properties. Their therapeutic efficacy can further be increased by transgenic approach or preconditioning them with certain factors like proinflammatory cytokines.
Zhang et al 2012. (49) injected spheroid Gingiva-Derived Mesenchymal Stem Cells (GMSCs) in mice with chemotherapy-induced oral mucositis and found that treatment with GMSCs decreased the severity and incidence of ulceration and restored the papillae structure, the lining, and thickness of the epithelial layer as compared with those of untreated disease group. The improved therapeutic benefits of spheroid-derived GMSCs may be attributed to their increased capabilities for engraftment and survival at the injury sites, trans-differentiation into epithelial cells, and preconditioning to hypoxic and oxidative challenges. Hence GMSCs could prove a sure shot therapy in oral mucositis following the cancer therapies.  (51) . Stem cell therapy in pemphigus not only has shown promises in treatment but also brings about a shift towards non steroidal approach in autoimmune diseases. However the use of stem cell therapy for oral pemphigus is yet to be ascertained and needs new clinical trials.

Stem Cell Therapy In Potentially Malignant Disorders:-Oral submucous fibrosis:-
Oral submucous fibrosis is a chronic, insidious disease associated with both significant morbidity (including pain, reduced mouth opening) and an increased risk of malignancy. Various agents like Areca nut, gutkha, spices etc., are known to cause insult to oral mucosa by increasing cytokine production and release of reactive oxygen species; which in turn results in increased synthesis of collagen, decreased collagen breakdown, compromised vascularity and increased tissue oxidative stress, ultimately resulting in clinical OSMF (52) .
Stem cell therapy is primarily aimed at neoangiogenesis by releasing cytokines and growth factors (paracrine effect) (53) .This may result in increased free radical scavenging by antioxidants (either naturally occurring or extrageous). Neoangiogenesis may also facilitate the removal of senescent cells from the lesions by supplying more number of scavenging defense cells and reversal of hypoxia in the diseased tissue. Stem cell therapy may help to stimulate resident tissue stem cells to transform into new fibroblasts, which may help in the removal of disintegrated biochemically and morphologically altered collagen fibers( Figure 4) Sankaranarayanan et al 2013. (54) have demonstrated the effectiveness of stem cell treatment in OSMF patients by injecting 0.5-1 ml of marrow-derived stem cell concentrate into labial and buccal mucosa and tongue under local anesthesia. They found reduction in blanching, decrease in the burning sensation while consuming spicy food, significant increase in the mouth opening and the results were found to be sustained in the follow-up period from 6 months to 5 years Various factors like stress, genetics, systemic diseases, drugs, dental restorative materials and viruses are known to cause the disease either by an antigen-specific mechanisms like activating cytotoxic T-cells and nonspecific mechanisms like mast cell degranulation and matrix metalloproteinase activation. Both of these cause the disruption of the basement membrane, which in turn triggers apoptosis of basal epithelial cells (55) . Recent advance in the treatment of lichen planus employ the use of immunomodulating agents such as tacrolimus which may treat the disease. In the past several years, studies have focussed on immunosuppressive properties of mesenchymal stem cells on various immune cell type as shown in figure 5. Based on these studies it is proposed that mesenchymal stem cells can be utilized to treat oral lichen planus via systemic infusion or local application (56,57) 1639  (58) , contain the ability to undergo self-renewal and differentiation and hence have the ability to initiate tumorigenesis and support ongoing tumor growth. Stem cells here play a dual role-in carcinogenesis and in the development of possible new cancer treatment options in future. For past so many years stem cells have been used in the replenishment of blood and immune system damage during treatment of cancer by chemotherapy or radiotherapy.
Apart from their use in the immuno-reconstitution, the stem cells have been reported to contribute in the tissue regeneration as they have extraordinary capacity to regenerate and differentiate. Recently, a new model of carcinogenesis was suggested, which lead to the cancer stem cell hypothesis. According to this hypothesis, not all epithelial cells are capable of generating a tumour, and only long-surviving cells such as stem cells, can undergo the summative oncogenic alterations required for carcinogenesis (59.60) .

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The Mesenchymal stem cells (MSCs) have been used in the cell-based bone reconstruction following chemotherapy and surgery in malignancies like osteosarcoma and Ewing sarcoma (61) Another important aspect of their use in cancer therapy is the use as delivery vehicles. Systematic delivery of drug or gene therapy has promising future but is currently limited by various factors such as immune detection, nonspecific accumulation in normal tissues and poor permeation. Stem cells can act as cell-based carriers that may target the desired site (62) . The recent concept of use of stem cells as delivery vehicles came from the fact that the tumors send out chemo-attractants such as Vascular endothelial growth factor (VEGF) to recruit mesenchymal stem cells (MSC) to form the supporting stroma of the tumor (63,64) . However, further work is required to understand the role of stem cells in cancer therapies, with the eventual goal of eliminating the residual disease and recurrence.

Salivary gland regeneration after radiation therapy:-
Radiation induced hyposalivation is a major problem in the treatment of head and neck cancer. Studies have shown that stem cell therapy may provide a means to reduce radiation-induced hyposalivation and improve the quality of life of patients. The ability of salivary gland tissue to regenerate after atrophy has provided knowledge about location and isolation of cell populations that contain salivary gland stem cells. It has been determined that within the ducts of salivary glands reside cells capable of proliferation and differentiation known as stem/ progenitor cell population. Application of specific growth factors to these cells induces differentiation into functional units (61) . Stem cells isolated from mouse salivary glands have shown to rescue saliva production in irradiated salivary glands.
Pringle et al in 2016 (65) , showed engrafment and functional capabilities of human adult epithelial stem cells in salivary gland demonstatrd in vitro self renewal and differentiation into multilineage organoids. He also showed in vivo functionality, long term engrafment and functional restoration in Xenotransplantation model. (66) Dental pulp stem cells were proven to differentiate into functionally active neurons, and when implanted dental pulp stem cells induce endogenous axon guidance, suggesting their potential as cellular therapy for neuronal disorder. Thus based on these findings, stem cell research has hope in the field of medicine directed toward: brain damage, spinal cord injury, Parkinson's disease, Alzheimer's disease, multiple sclerosis and other neurological and neurodegenerative disorders. The Tissue regeneration concept hopes to repair heart damage, baldness, deafness, blindness, impaired vision, muscle damage, diabetes mellitus, etc. Stem cell research is a boon to drug research as the drug application can be directly given on to human cells, and this will provide more relevant data than testing on animal models.

Conclusion:-
Stem cell research in dentistry has given opportunity for dentists to assume a leading role not only in the treatment of dental pathosis and also in medical diseases. With the advances in the stem cell biology, dental stem cells will hopefully be able to correct cleft palate, periodontal defects, save injured teeth, jaw bones and most strikingly regenerate the entire teeth structures. Though certain studies have confirmed the effectiveness of stem cell therapy treatment in pemphigus vulgaris, potentially malignant disorder like Oral submucous fibrosis, the research is mainly confined to animal models in oral mucositis, cancer management and Lichenplanus, thus more human research trials are needed to ascertain the role of stem cells in their management. The evolution of more methods of stem cell therapy in the future will give simple, definitive and effective treatment of most of the oral mucosal diseases.