|Year : 2015 | Volume
| Issue : 2 | Page : 82-85
Rajnish K Singhal1, Jyotsna Goyal2, Surinder Sachdeva3, Swantika Chaudhry3, Abha Sood3, Nishu Vakil3
1 Department of Conservative Dentistry and Endodontics, Eklavya Dental College and Hospital, Kotputli, Rajasthan, India
2 Department of Periodontics, Gian Sagar Dental College and Hospital, Banur, Patiala, Punjab, India
3 Department of Periodontics. MMCDSR, Mullana, Ambala, Haryana, India
|Date of Web Publication||2-Mar-2016|
MMCDSR, Mullana, Ambala, Haryana
Source of Support: None, Conflict of Interest: None
Tissue engineering is the science of design and manufacture of new tissues to replace impaired or damaged ones. The key ingredients for tissue engineering are stem cells, the morphogens, or growth factors that regulate their differentiation, and a scaffold of extracellular matrix that constitutes the microenvironment for their growth. Recently, there has been an increasing interest in applying the concept of tissue engineering to endodontics.
Keywords: Pulp regeneration, scaffolds, stem cell, tissue engineering
|How to cite this article:|
Singhal RK, Goyal J, Sachdeva S, Chaudhry S, Sood A, Vakil N. Regenerative endodontics. Saint Int Dent J 2015;1:82-5
Over the past century, endodontic therapy has shown a high rate of success in retention of teeth; however, this is not always possible. Many teeth remain unrestorable due to several reasons, and vital pulp therapy procedures are not always predictable, often resulting in the need for eventual endodontic therapy.
In view of the increasing demand for maintaining pulp vitality and the high cost of endodontic treatments being performed every day, there has been an increasing interest in investigating new methods for tissue replacement. With the expanding knowledge of developmental biological processes, and how they are mimicked during dental tissue repair, strategies to regenerate lost or diseased dental tissue will soon enter into clinical practice. Tissue engineering involves the development of functional tissue with the ability to replace missing or damaged tissue. This may be achieved either by transplanting cells seeded into a porous material or scaffold having open pores or by relying on ingrowth of cells into such a material, which in both cases develops into normal tissue. It has been consistently shown that the dental pulp contains cells that can differentiate into hard tissue forming cells following injury.
| The Principles of Tissue Engineering|| |
Tissue engineering approaches are conductive, inductive approaches, and cell transplantation. 
This approach makes use of a barrier membrane to exclude connective tissue cells that will interfere with the regenerative process, while enabling the desired host cells to populate the regeneration site. An example of this is the dental implants and guided tissue regeneration membranes.
This approach uses a biodegradable polymer scaffold as a vehicle to deliver growth factors and genes to the host site. The growth factors or genes can be released at a controlled rate, based on the breakdown of the polymer. The inductive approach uses a biodegradable scaffold to deliver growth factor/genes at a controlled rate, based on the breakdown of the polymer. One limitation of the inductive approach is that the inductive factors for a particular tissue may not be known.
This strategy uses a similar vehicle for delivery to transplant cells and partial tissues to the host site. The cell transplantation strategy truly reflects the multidisciplinary nature of tissue engineering that requires a clinician, a bioengineer, and a cell biologist. Tissue engineering is generally considered to consist of three key elements:
Stem cells are commonly defined as cells that have the ability to continuously divide and produce progeny cells that differentiate into various other types of cells that differentiate into various types of cells or tissues.
- Stem cells/progenitor cells
- Scaffolds or extracellular matrix
- Signaling molecules.
| Bioengineered Scaffolds|| |
The basic role of scaffolds in tissue engineering is to act as carriers for cells, to maintain the space, and to create an environment in which the cells can proliferate and produce the desired tissue matrix.
Types of scaffolds
- Natural scaffolds
- Mineral scaffolds
- Synthetic scaffolds.
The examples for natural scaffolds are collagen, hyaluronic acid, chitosan, and chitin. These natural scaffolds have been used in several craniofacial and dental applications. These lack the desired structural rigidity for use in the load bearing region.
These are composed of calcium phosphates in the form of hydroxyapatite or β tricalcium phosphate. These scaffolds are brittle and hence are prone to fracture.
The most widely used synthetic materials are polymers of polyglycolic acid, polylactic acid, and polydioxanone. These scaffolds lack critical cell signaling capabilities and can interfere with new tissue growth.
| Signaling Molecules|| |
These are the molecules that transmit signals between cells, functioning as stimulators/inhibitors of growth, as well as the modulators of differentiation. These consist of growth factors (Platelet-derived growth factor [PDGF] and transforming growth factor-β [TGF-β]), differentiation factors such as bone morphogenetic proteins (BMPs), and stimulating factors.
| Stem Cell|| |
The term stem cell was proposed for scientific use by Russian histologist Alexander Maksimov in 1908, while research on stem cells grew out of findings by Canadian scientists in the 1960s. In general, there are two broad types of stem cells which are embryonic stem cells and adult stem cells. In the year 2003, Dr. Songtao Shi who is a pediatric dentist discovered baby tooth stem cells by using the deciduous teeth of his 6-year-old daughter, he was luckily able to isolate, grow, and preserve these stem cells' regenerative ability, and he named them as stem cells from human exfoliated deciduous teeth (SHED). After the scientists studied the dental pulp looking for stem cells, they found that the dental pulp was rich in different stem cell types such as chondrocytes, osteoblasts, adipocytes, and mesenchymal stem cells.
| Regenerative Endodontic Techniques || |
Major areas of research that might have application in the development of regenerative endodontic techniques are: (a) Root canal revascularization via blood clotting, (b) postnatal stem cell therapy, (c) pulp implantation, (d) scaffold implantation, (e) injectable scaffold delivery, (f) three-dimensional cell printing, and (g) gene delivery. The revascularization method assumes that the root canal space has been disinfected effectively by the use of intracanal irrigants, with the placement of antibiotics for several weeks. Postnatal stem cells are injected into the disinfected root canal systems after the apex is opened. The postnatal stem cells can be derived from multiple tissues including skin, buccal mucosa, fat, and bone. In pulp implantation, the cultured pulp tissue is transplanted into cleaned and shaped root canal systems. The pulp tissue is grown in sheets in vitro on biodegradable polymer nanofibers or on the sheets of extracellular matrix proteins such as collagen I or fibronectin. Pulp stem cells must be organized into a three-dimensional structure that can support cell organization and vascularization. This can be accomplished by using a porous polymer scaffold, which is seeded with pulp stem cells. In pulp-exposed teeth, dentin chips have been found to stimulate reparative dentin bridge formation. Dentin chips may provide a matrix for pulp stem cell attachment and they may also be a reservoir of growth. Tissue engineered pulp tissue is seeded into the soft three-dimensional scaffold matrix, such as a polymer hydrogel, which may promote pulp regeneration by providing a substrate for cell proliferation and differentiation into an organized tissue structure The three-dimensional cell printing technique can be used to precisely position cells and this method has the potential to create tissue constructs that mimic the natural tooth pulp tissue structure. 
| Stem Cells from Human Exfoliated Deciduous Teeth|| |
The discovery of stem cell in deciduous teeth sheds a light on the intriguing possibility of using dental pulp stem cells (DPSCs) for tissue engineering . The obvious advantages of SHEDs are higher proliferation rate compared with stem cells from permanent teeth , easy to be expanded in vitro, high plasticity since they can differentiate into neurons, adipocytes, osteoblasts, and odontoblasts, and readily accessible in young patients. 
| Adult Dental Pulp Stem Cells|| |
The regenerative capacity of the human dentin/pulp complex enlightens scientists that dental pulp may contain the progenitors that are responsible for dentin repair. Gronthos first identified adult DPSCs in human dental pulp in 2000 and found DPSCs could regenerate a dentin-pulp-like complex,which is composed of mineralized matrix with tubules lined with odontoblasts and fibrous tissue containing blood vessels in an arrangement similar to the dentin-pulp complex found in normal human teeth. 
| Stem Cells from Dental Follicle|| |
The dental follicle is a mesenchymal tissue that surrounds the developing tooth germ. During tooth root formation, periodontal components, such as cementum, periodontal ligament, and alveolar bone, are created by dental follicle progenitors. Stem cells from dental follicle (DFSCs) have been isolated from the follicle of human third molars and express the stem cell markers: Notch1, STRO-1, and nestin. DFSCs werefound to be able to differentiate into osteoblasts/cementoblasts, adipocytes, and neurons. 
The seeding of cells on tissue engineering scaffolds is known as "creating a tissue construct." To promote the formation of higher-ordered tissue structures, tissue constructs are maintained in cell culture in the presence of growth factors or bioactive molecules. Growth factors, especially those of the TGF-β family, are important in cellular signaling for odontoblast differentiation and stimulation of dentin matrix secretion. These growth factors are secreted by odontoblasts and are deposited within the dentin matrix, where they remain protected in an active form through interaction with other components of the dentin matrix. The addition of purified dentin protein fractions has stimulated an increase in tertiary dentin matrix secretion, suggesting that TGF-β1 is involved in injury signaling and tooth-healing reactions.
| Discussion|| |
The production of dentin and dental pulp has also been achieved in animal and laboratory studies using tissue engineering strategies. The greatest potential for these engineered tissues is in the treatment of tooth decay. Dental caries remains one of the most prevalent young adult and childhood diseases. Tissue engineering of dental pulp itself may also be possible using cultured fibroblasts and synthetic polymer matrices. Further development and successful application of these strategies to regenerate dentin and dental pulp could one day revolutionize the treatment of our most common oral health problem, cavities.
The ability to regenerate new bone using stem cell therapy and biomimetic biomaterials is a major clinical need. Adult mesenchymal stem cells have been discovered in human dental pulp. These cells were found to be multipotent with a high osteogenic potential compared to human bone marrow mesenchymal stem cells. Human dental pulp stem/stromal cells have a high osteogenic capacity and the potential to be used in combination with 3D Bioglass ® scaffolds for bone tissue engineering in vitro. 
Viral vectors and nonviral techniques can be used for gene transfer in gene therapy. Although viral vectors provide gene transfer with high efficiency, attendant problems of cellular immunity due to adenoviruses or insertional mutagenesis due to retroviruses have been recognized. A potential method to overcome this dilemma is electroporation using pulsed electric fields to deliver DNA. Electroporation also has been used to deliver genes to living animals. However, electroporation yields only a transient gene expression and not as efficient as viral vectors. The in vivo electroporation of Gdf11 in the amputated pulp of canine teeth has shown that the pulp cells differentiated into osteodentinoblasts and secreted osteodentin matrix around them and formed osteodentin. ,
Pulp-like tissue can be regenerated de novo in emptied root canal space by stem cells from apical papilla and DPSCs that give rise to odontoblast-like cells producing dentin-like tissue on existing dentinal walls. 
Reconstructed, multiple microscopic images showed complete fill of dental pulp-like tissue in the entire root canal from root apex to pulp chamber with tissue integration to dentinal wall upon delivery of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), or PDGF with a basal set of NGF and BMP7. Quantitative ELISA showed that combinatory delivery of bFGF, VEGF, or PDGF with basal NGF and BMP7 elaborated von Willebrand factor, dentin sialoprotein, and NGF. The present chemotaxis-based approach has potent cell homing effects for recellularization and revascularization in endodontically treated root canals. 
Growing evidence demonstrates that stem cells are primarily found in niches and that certain tissues contain more stem cells than others. Among these tissues, the dental pulp is considered a rich source of mesenchymal stem cells that are suitable for tissue engineering applications. 
Dental pulp cells have shown promising potential in dental tissue repair and regeneration. However, during in vitro culture, these cells undergo replicative senescence and result in significant alteration in cell proliferation and differentiation. Recently, the transcription factors of Oct-4, Sox2, c-Myc, and Klf4 have been reported to play a regulatory role in the stem cell self-renewal process, namely cell reprogramming. 
Many of the invading bacteria are known to produce considerable amounts of hydrogen sulfide (H 2 S), which causes apoptosis in some tissues. H 2 S causes apoptosis in HPSCs by activating the mitochondrial pathway. It is suggested that H 2 S might be one of the factors modifying the pathogenesis of pulpitis by causing a loss of viability of HPSCs through apoptosis. 
A disintegrin and metalloproteinase 28 (ADAM28) could be correctly transcribed, translated, and expressed in human dental pulp-derived stem cells (HDPSCs). ADAM28 eukaryotic plasmid could significantly inhibit the HDPSC proliferation, promote specific differentiation of HDPSCs, induce apoptosis, and enhance the dentin sialophosphoprotein expression, whereas ADAM28 antisense oligodeoxynucleotides produce the opposite effects. 
Immature teeth with open apices treated with conventional nonsurgical root canal treatment often have a poor prognosis as a result of the increased risk of fracture and susceptibility to recontamination. Regenerative endodontics represents a new treatment modality that focuses on the reestablishment of pulp vitality and continued root development. This clinical procedure relies on the intracanal delivery of a blood clot (scaffold), growth factors (possibly from platelets and dentin), and stem cells. Evoked-bleeding step in regenerative procedures triggers the significant accumulation of undifferentiated stem cells into the canal space where these cells might contribute to the regeneration of pulpal tissues seen after antibiotic paste therapy of the immature tooth with pulpal necrosis. 
| Conclusion|| |
The science of tissue engineering exploits this valuable ability of stem cells/progenitor cells to regenerate or repair new tissues. In conservative dentistry, regenerative endodontic procedures aim to regenerate the pulp dentin complex utilizing dental stem cells, growth factors, and scaffold matrix. Ability to isolate and harvest stem cells, improvements in the design of scaffold materials, advances in cell culture technology, and commercial availability of growth factors have facilitated regeneration of pulp and dentin. Efforts have been continuously focused to overcome limitations associated with existing restorative materials and evolve a material that mimics natural tooth structure as closely as possible. Developing a treatment therapy that will eliminate the use of any artificial material and enable replacement of lost tissues with natural pulp and dentin is an attractive option. Applying tissue engineering concepts can help turn this dream into reality. A combination of stem cells, scaffold, and growth factors maintained in a controlled and regulated environment has shown immense potential to repair and generate tissues of the endodontium.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Kaigler D, Mooney D. Tissue engineering's impact on dentistry. J Dent Educ 2001;65:456-62.
Murray PE, Garcia-Godoy F, Hargreaves KM. Regenerative endodontics: A review of current status and a call for action. J Endod 2007;33:377-90.
Peng L, Ye L, Zhou XD. Mesenchymal stem cells and tooth engineering. Int J Oral Sci 2009;1:6-12.
El-Gendy R, Boccaccini AR, Bretcanu O, Kirkham J, Yang XB. Bone tissue engineering using human dental pulp stem cells and 3D Bioglass R scaffolds. Euro Cells Mater 2008;16:53.
Nakashima M, Mizunuma K, Murakami T, Akamine A. Induction of dental pulp stem cell differentiation into odontoblasts by electroporation-mediated gene delivery of growth/differentiation factor 11 (Gdf11). Gene Ther 2002;9:814-8.
Nakashima M. Tissue engineering in endodontics. Aust Endod J 2005;31:111-3.
Huang GT, Yamaza T, Shea LD, Djouad F, Kuhn NZ, Tuan RS, et al.
Stem/progenitor cell-mediated de novo regeneration of dental pulp with newly deposited continuous layer of dentin in an in vivo
model. Tissue Eng Part A 2010;16:605-15.
Kim JY, Xin X, Moioli EK, Chung J, Lee CH, Chen M, et al.
Regeneration of dental-pulp-like tissue by chemotaxis-induced cell homing. Tissue Eng Part A 2010;16:3023-31.
Casagrande L, Cordeiro MM, Nör SA, Nör JE. Dental pulp stem cells in regenerative dentistry. Odontology 2011;99:1-7.
Liu L, Wei X, Ling J, Wu L, Xiao Y. Expression pattern of Oct-4, Sox2, and c-Myc in the primary culture of human dental pulp derived cells. J Endod 2011;37:466-72.
Kobayashi C, Yaegaki K, Calenic B, Ishkitiev N, Imai T, Ii H, et al.
Hydrogen sulfide causes apoptosis in human pulp stem cells. J Endod 2011;37:479-84.
Zhao Z, Liu H, Wang D. ADAM28 manipulates proliferation, differentiation, and apoptosis of human dental pulp stem cells. J Endod 2011;37:332-9.
Lovelace TW, Henry MA, Hargreaves KM, Diogenes A. Evaluation of the delivery of mesenchymal stem cells into the root canal space of necrotic immature teeth after clinical regenerative endodontic procedure. J Endod 2011;37:133-8.