Stem cells….Rejuvenate, Resuscitate, Revitalise the dead tooth - a review

Introduction      

Stem cells (SC) are biological cells found in almost all multi-cellular organisms that can divide and differentiate into diverse specialized cell types and can self-renew to produce more stem cells. Stem cells have the remarkable potential to develop into many different cell types in the body during early life and growth.¹ 

Stem cells also known as “progenitor or precursor” cells are defined as clonogenic cells capable of both self-renewal and multi-lineage differentiation.² 

Characteristics of Stem cells

Stem cells are characterized by their three special properties.

1. Self renewal

   Stem cells are capable of dividing and renewing themselves for long periods. Stem cells may replicate many times, or proliferate.

2. Unspecialized nature

   Stem cells are unspecialized in that they do not have any tissue-specific structures that allow it to perform specialized functions. However, unspecialized stem cells can give rise to specialized cells, including heart muscle cells, blood cells, or nerve cells.

3. Differentiation

   When unspecialized stem cells give rise to specialized cells, the process is called differentiation. While differentiating, the cell usually goes through several stages, becoming more specialized at each step.

Stem cell therapies are playing a major role in cell transplantation biology. The potential use of embryonic and adult stem cells for basic and applied research, including testing the origin of human cancer, attempts have been made to characterize markers that would identify these stem cells. The promise of stem cell-based therapy for advancement of research in regenerative medicine has stimulated a great number of clinical trials, particularly for previously untreatable diseases.

Potency of stem cells

Potency of the stem cell specifies the differentiation potential i.e., the potential to differentiate into different cell types.

• Totipotent stem cells can differentiate into embryonic and extraembryonic cell types. Such cells can construct a complete, viable organism.
• Pluripotent stem cells are the descendants of totipotent cells and can differentiate into nearly all cells, i.e. cells derived from any of the three germ layers.
• Multipotent stem cells can differentiate into a number of cells, but only those of a closely related family of cells. These are true stem cells but can only differentiate into a limited number of types. For example, the bone marrow contains multi-potent stem cells that give rise to all the cells of the blood but not to other types of cells.
• Oligopotentstem cells can differentiate into only a few cells, such as lymphoid or myeloid stem cells.
• Unipotentcells can produce only one cell type, their own, but have the property of selfrenewal, which distinguishes them from nonstem cells.

Stem cells have different names depending on the tissue of origin; thus there are hematopoietic, mesenchymal, endothelial, mammary, intestinal, neural, skin, muscle, and hair follicle stem cells, among others. Among these stem cells, mesenchymal stem cells (MSCs) are noteworthy for their pluripotency, which means that they can differentiate into cells of any type, including those of the three embryonic germ layers. MSCs are very attractive tools for tissue repair.⁴  

Human dental stem cells that have been isolated and characterized are⁵:

1. DPSCs.

2. SHED.

3. Stem cells from apical papilla (SCAP).

4. Periodontal ligament stem cells (PDLSCs).

In particular, it has been shown that human dental pulp stem cells (DPSCs) can generate mineralized tissue, an extracellular matrix and structures type dentin, dental pulp, and periodontal ligament in xenograft models.⁴

Role of stem cells in Dentistry

Most research is directed toward regeneration of damaged dentin, pulp, resorbed root, periodontal regeneration and repair perforations. Whole tooth regeneration to replace the traditional dental implants is also in pipeline. Tissue engineering applications using dental stem cells that may promote more rapid healing of oral wounds and ulcers as well as the use of gene-transfer methods to manipulate salivary proteins and oral microbial colonization patterns are promising and possible.⁵

For osseous regeneration: Adult MSCs recently identified in the gingival connective tissues (gingival mesenchymal stem cells [GMSCs]) have osteogenic potential and are capable of bone regeneration in mandibular defects. GMSCs also suppress the inflammatory response by inhibiting lymphocyte proliferation and inflammatory cytokines and by promoting the recruitment of      regulatory T-cells and anti-inflammatory cytokines. Thus, GMSCs potentially promote the “right” environment for osseous regeneration and is currently being therapeutically explored.⁵   

Tooth stem cells Banking

Although tooth banking is currently not very popular the trend is gaining acceptance mainly in the developed countries. BioEden (Austin, Texas, USA), has international laboratories in UK (serving Europe) and Thailand (serving South East Asia) with global expansion plans.  Stemade biotech introduced the concept of dental stem cells banking in India recently by launching its operations in Mumbai and Delhi.⁵

Dental Pulp stem cells     

DPSCs, also known as postnatal dental pulp stem cells, were first isolated by Gronthos et al from third molars and were characterized as cells with a high level of clonogenicity and proliferation and the ability to generate densely calcified colonies and occasional nodules.⁶ These cells are located within the dental crown, in a “niche sealing” or “pulp chamber” that houses the connective tissue known as pulp.⁴

DPSCs are stem cells derived from human exfoliated deciduous teeth (SHED), from permanent secondary dentition systems (properly known as DPSCs), from teeth extracted by orthodontist due to impaction or irreversible periodontitis, or from inflamed pulp tissue. SHED cells were isolated by Miura et al from primary dentition systems and were characterized as cells with a high proliferation rate and the ability to differentiate into osteoblasts, neural cells, adipocytes, and odontoblasts.⁷ The DPSCs of permanent teeth, impacted third molars, and supernumerary teeth, which today are considered medical waste, are particularly interesting stem cells. Dental pulp is a potential source of stem cells for orthopedic, oral, and maxillofacial reconstruction. The general procedure for human DPSC administration is to implement them in scaffold or porous biomaterial to reinforce the graft site and induce tissue regeneration.⁴

Dental tissue engineering

The main goal of tissue engineering is to reconstruct natural tissues by combining progenitor or stem cells with growth factors and different biomaterials to serve as a scaffold for novel tissue growth. DSCs also produce a wide variety of neurotrophic factors and therefore they can be used in tissue engineering as a growth factor delivery system. These neurotrophic factors play a pivotal role in protecting neurons from apoptosis and inducing endogenous neural repair and neurite formation.⁸ 

Preconditioning of Dental Stem Cells to enhance their angiogenic and neurogenic Properties

One of the major hurdles in the field of tissue engineering is the survival of transplanted cells in vivo. To overcome this obstacle, several strategies have been developed to modulate the stem cells prior to transplantation in order to improve cellular survival and engraftment. Genetic modification offers a potential strategy to increase stem cell survival, for example, by over-expressing antiapoptotic genes such as Bcl- 2 or Akt.⁸

Preparing stem cells for transplantation by exposing them to a hypoxic environment may be a useful technique to improve the stem cell secretome, since hypoxia is a potent stimulus for the secretion of a variety of trophic factors.⁸

Dental Stem Cells and Pulp regeneration

Over the past decade, substantial advances have been made regarding the potential application of DSCs in the regeneration of viable dental tissues. Not only has successful dental pulp regeneration been reported for DPSCs, but also SCAPs and FSCs have proven to be effective in different in vivo models of pulp regeneration. Another important aspect which definitely needs to be taken into account during differentiation and tissue engineering is the specific microenvironment at the time of regeneration.⁸

As the dental follicle during tooth development gives rise to the periodontium, research has mainly focused on the ability of FSCs to regenerate cementum and periodontal ligament. In an attempt to engineer a complete tooth root, transplantation of similar constructs containing rat or human FSCs led to the regeneration of a dentin- pulp complex as well as cementum and periodontal ligament (PDL).

Despite the promising outcomes of DSC transplantation in a preclinical setting, the progression of DSCs from bench to bedside still holds some major challenges such as standardized cell isolation procedures and culture, moreover the intrinsic behaviour of the stem cells, as it can be influenced by a broad range of different donorrelated factors, such as (oral) health, age, and orthodontic tooth movement.⁸   

Although numerous studies have elaborately described the immunomodulatory effects of DSCs in vitro, little is known concerning the effects of allogeneic DSC transplantation in vivo. More research is thus required with respect to the immunomodulatory behaviour of allogeneic DSCs in vivo and potential graft-versus-host responses. DSCs are considered suitable candidates for cellbased treatment strategies and tissue engineering applications. There is abundant evidence supporting the angiogenic, neuroprotective, and neurotrophic actions of the DSC secretome.

Based on their origin, DSCs are expected to be ideal candidates for the regeneration of dental tissues such as the dental pulp and the periodontal ligament. Successful dental pulp regeneration has already been reported for DPSCs, SCAPs, and FSCs, whereas PDLSCs hold great potential for the regeneration of periodontal tissues. Furthermore, DPSCs, SHEDs, and PDLSCs have already been reported to improve regeneration after peripheral nerve injury by promoting remyelination, blood vessel formation, and nerve regeneration. These encouraging results contributed to approval of clinical studies to introduce DSC based therapies into the clinic.⁸

Induced pluripotent stem cells

Stem cells generated from adult somatic cells through a process of cellular reprogramming were termed induced pluripotentstem cells (iPSCs)⁹

Dental Applications of iPSCs

potential utility of iPSCs in dental applications are,

• Periodontal regeneration¹⁰

• Tooth development and regeneration^11-13

Stem cells in regenerative endodontic procedures [REP].

Endodontics uses cell therapy strategies to treat pulpal and periapical diseases. During these therapies, surgeons aim to reconstruct the natural microenvironments that regulate the activity of dental stem cells.¹¹

Dental stem cells may createa repair-conducive microenvironment, stimulating therecruitment of endogenous stem cells or progenitors atthe injury site. This insinuates that accurately designedbioactive scaffolds could generate effective dental tissuerepair responses through activation and mobilisation ofendogenous stem and progenitor cells, thus avoiding exogenous stem cell administration.¹⁴

Regenerative dentistry aims to restore tooth anatomy and function after dental damage. In this discipline, regenerative endodontics is a set of biology-based procedures designed to heal periapical lesions and replace cells and dentin of the pulp-dentin complex in a damaged tooth. Regenerative endodontic procedures (REPs) are bioengineering therapies that seek to restore the physiological functions of the dental pulp. These techniques involve a triad of elements: stem cells, growth factors, and biomaterials, also named scaffolds or template.¹⁵

Angiogenic and anti-angiogenic factors to dental pulp regeneration

Angiogenesis is defined as the formation of new blood vessels from preexisting capillaries, which has great importance in pulp regeneration and homeostasis. Dental pulp regeneration is a part of regenerative endodontics, which includes isolation, propagation, and re-transplantation of stem cells inside the prepared root canal space. The formation of new blood vessels through angiogenesis is mandatory to increase the survival rate of re-transplanted tissues. Here the contribution of human dental pulp stem cells and pro-angiogenic and anti-angiogenic factors to angiogenesis process and regeneration of dental pulp is under research (16).

Human dental pulp is a highly vascularized tissue, which because of its vascular network and progenitor or postnatal dental pulp stem cells (DPSCs) has an impressive naturally inherent regenerative capacity. Dental pulp regeneration is part of the regenerative endodontic concept, which provides replacements for damaged tooth structures including pulp-dentin complex. It is a field in regenerative medicine and a branch of tissue engineering, which uses stem cells, biochemical factors, and engineering materials to replace lost or impaired biological tissues.¹⁶

Gronthos et al have recombined human DPSCs with hydroxypatiteptricalcium phosphate (HATCP) ceramic powder, and transplanted them subcutaneously into immunocompromised mice. The recovered tissues contain the typical dentin structures surrounded by odontoblast-like cells with long cytoplasmic process deeply into the mineralized matrix.⁶

The biochemical stimulation of angiogenesis is related to the production of pro-angiogenic and anti-angiogenic factors including growth factors. The growth factors such as bone morphogenetic proteins have an impeccable role in tissue engineering.. Roberts-Clark and Smith acclaimed that dentin matrix can act as a reservoir for these angiogenic growth factors, and after any injury to pulp-dentin complex, these substances are released to promote angiogenesis in regenerating pulp tissue. However, the molecular and cellular mechanisms involved remain poorly understood.¹⁷

The DPSCs can secrete proangiogenic factors such as VEGF, FGF-2, PDGF, MMP-9, IGF-1, TGF-b, interleukin-8, and MCP-1¹⁶

DPSCs can also produce antiangiogenic factors including endostatin, IGFBP3, uPA, TIMP-1, and PAI-1¹⁶

Roberts-Clark and Smith indicated that the dentin matrix components in low concentrations have pro-angiogenic impact, whereas in high concentrations they have inhibitory effects on angiogenesis events of dental pulp.¹⁷ 

Conclusion

Dental stem cells are considered suitable candidates for cell-based treatment strategies and tissue engineering applications. Dental stem cells are expected to be ideal candidates for the regeneration of dental tissues such as the dental pulp and the periodontal ligament. Several stem cell lines with significant variability in potency have been isolated from human adult teeth. Identification and purification of stem cell subpopulations with improved potency is a necessary step before application of cell-based treatment in dental clinics.  Although applications using dental stem cells for pulp and periodontal regeneration have been reported in animal models, the number of clinical trials with long-term followup is very limited, if not inexistent. Several clinical trials using autologous stem cells for pulp and periodontal tissue regeneration have already been approved and are on course. Stem cell-based regenerative approaches in dentistry are just at the beginning and has the potential to benefit millions of patients.Stem cells therapy in general,is promising, bit is slightly far from being a mature clinical technology.