|Year : 2011 | Volume
| Issue : 1 | Page : 122-131
|Regenerative endodontics: A state of the art
Rashmi Bansal1, Rajesh Bansal2
1 Department of Conservative Dentistry, Teerthankar Mahaveer Medical College, Moradabad, India
2 Department of Pediatrics, Teerthankar Mahaveer Medical College, Moradabad, India
Click here for correspondence address and email
|Date of Submission||19-Dec-2009|
|Date of Decision||29-Oct-2010|
|Date of Acceptance||21-Nov-2010|
|Date of Web Publication||25-Apr-2011|
| Abstract|| |
Scientific advances in the creation of restorative biomaterials, in vitro cell culture technology, tissue grafting, tissue engineering, molecular biology and the human genome project provide the basis for the introduction of new technologies into dentistry. Non-vital infected teeth have long been treated with root canal therapy (for mature root apex) and apexification (for immature root apex), or doomed to extraction. Although successful, current treatments fail to re-establish healthy pulp tissue in these teeth. But, what if the non-vital tooth could be made vital once again? That is the hope offered by regenerative endodontics, an emerging field focused on replacing traumatized and diseased pulp with functional pulp tissue. Restoration of vitality of non-vital tooth is based on tissue engineering and revascularization procedures. The purpose of this article is to review these biological procedures and the hurdles that must be overcome to develop regenerative endodontic procedures.
Keywords: Growth factors, regenerative endodontics, revascularization, scaffolds, stem cells
|How to cite this article:|
Bansal R, Bansal R. Regenerative endodontics: A state of the art. Indian J Dent Res 2011;22:122-31
Regenerative endodontic procedures can be defined as biologically based procedures designed to create and deliver tissues to replace diseased, missing and traumatized pulp-dentin complex. The science of regenerative endodontics has a long history dating back to 1952 when Dr. BW Hermann reported on the application of calcium hydroxide in a case report of vital pulp amputation.  Presently, two concepts exist in regenerative endodontics to treat non-vital infected teeth - one is the active pursuit of pulp-dentine regeneration to implant or regrow pulp (tissue engineering technology), and the other in which new living tissue is expected to form from the tissue present in the teeth itself, allowing continued root development (revascularization).
Tissue engineering can be defined as 'an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function.' 
The three key components for tissue engineering are:
- Stem cells - to respond to growth factors.
- Scaffold of extracellular matrix (ECM).
- Growth factors (signals for morphogenesis).
| Stem Cells|| |
They are defined as clonogenic cells capable of both self-renewal and multilineage differentiation since they are thought to be undifferentiated cells with varying degrees of potency and plasticity.  They differentiate into one daughter stem cell and one progenitor cell. There are basically two types of stem cells: 
- Embryonic stem cells - located within the inner cell mass of the blastocyst stage of development.
- Postnatal stem cells - that have been isolated from various tissues including bone marrow, neural tissue, dental pulp and periodontal ligament.
Since the sourcing of embryonic stem cells is controversial and is surrounded by ethical and legal issues, many researchers are now focussing attention on developing stem cell therapy using postnatal stem cells donated by the patients themselves or their close relatives. Stem cells are often categorized by their source:
- Autologous stem cells - are obtained from the same individual to whom they will be implanted.
- Allogenic stem cells - originate from a donor of the same species.
- Xenogenic cells - are those isolated from individuals of another species.
For endodontic regeneration, the most promising cells are autologous postnatal dental stem cells because there are less chances of immune rejection.  They show more striking odontogenic capability (typical tooth-shaped tissue with balanced amelogenesis and dentinogenesis) as compared to non-dental stem cell population like bone marrow stromal stem cell. , Various sources for postnatal dental stem cells have been successfully studied:
- Permanent teeth - Dental pulp stem cells (DPSC): derived from third molar. 
- Deciduous teeth - Stem cells from human-exfoliated deciduous teeth (SHED): stem cells are present within the pulp tissue of deciduous teeth. 
- Periodontal ligament - Periodontal ligament stem cells (PDLSC). 
- Stem Cells from apical papilla (SCAP). 
- Stem cells from supernumerary tooth - Mesiodens. 
- Stem cells from teeth extracted for orthodontic purposes. 
- Dental follicle progenitor cells. 
- Stem cells from human natal dental pulp- (hNDP). 
Stem cells from various sources and their features studied by various researchers ,,,,,,,,,,,,,,,,,,,,,,,,,,, are shown in [Table 1].
All types of postnatal dental stem cells studied have mesenchymal stem cell-like qualities, such as capacity for self-renewal and multilineage differentiation. Immunocytochemistry has proved the existence of stem cells in these cell populations using STRO-1 as a stem cell marker. These cells also express the mesenchymal stem-cell markers CD29 and CD44.  These stem cells are isolated from specialized tissue with potent capacities to differentiate into odontogenic cells; however, they also have the ability to give rise to other cell lineages similar to but different in potency from that of bone marrow stem cells.  From both DPSCs and SHED, tissue similar to normal dentin-pulp is reported to be regenerated which can be later on used for regenerative endodontics. But SHED are retrieved from a tissue that is 'disposable' and readily accessible. The best candidates for SHED are moderately resorbed canine and incisors with the presence of healthy pulp. In children, other sources of easily accessible stem cells are supernumerary teeth, mesiodens, over-retained deciduous teeth associated with congenitally missing permanent teeth and prophylactically removed deciduous molars for orthodontic indications. SHED also show higher proliferation capability, abundant cell supply and painless stem-cell collection with minimal invasion, so SHED could be a desirable option as a cell source for regenerative endodontics; , however, in comparision, DPSCs show higher inclination towards neuronal lineage.  Stem cells are isolated from aging teeth, also but it is observed that number of cells and their proliferation rate decreases with age and it is maximum when only crown is formed (germ stage). SCAP have higher proliferation rate as compared to DPSCs. They appear to be the source of primary odontoblasts that are responsible for root dentin formation whereas DPSCs are the likely source of replacement odontoblast. SCAP represent early progenitor cells,  so whether SCAP are a more suitable stem-cell source than DPSCs and SHED require further investigation. For regeneration of periodontium, PDLSCs are better source as stem cell as compared to cells isolated from pulp. Viable periodontal ligament is reported to be generated from PDLSCs.  Instead of forming entire tooth, even a bio-root with periodontal ligament tissues has been generated by utilizing SCAP along with the PDLSCs. This bio-root is encircled with periodontal ligament tissue and has natural relationship with the surrounding bone. 
Autologous stem cells are relatively easy to harvest and easy to inject by syringe (injectable postnatal stem-cell therapy). But in this technique, cells have low survival rate and they might migrate to different locations within the body possibly leading to aberrant patterns of mineralization.  A solution to the above problem is to apply the cells together with scaffold material- the second component of tissue engineering.
| Scaffold|| |
A scaffold used for regeneration should provide the framework for cell growth, differentiation and organization at a local site. A scaffold should be porous to allow for placement of cells and also be biocompatible with host tissue.  It should be biodegradable and should degrade gradually so that it is replaced by regenerative tissue.  It should be effective for transport of nutrients and waste.  Most tissue engineering efforts use biomaterials for scaffolds already approved by the FDA. They can be natural (collagen, dentin, fibrin, silk, alginate) or synthetic (various polymers like PLA, PGA, etc.). Synthetic polymers are generally degraded by simple hydrolysis while natural polymers are mainly degraded enzymatically. Various scaffolds studied by different researchers ,,,,,,,,,,,,,, are shown in [Table 2].
Collagen is the most widely studied natural scaffold. The most widely used synthetic scaffolds are polymers of lactide and glycolide. In regenerative endodontics, a tissue-engineered pulp is not required to provide structural support to the tooth. So, engineered pulp tissue can be administered in a soft three-dimensional scaffold matrix, such as polymer hydrogel,  which can be injected at the site (injectable scaffold delivery). Hydrogels have similar physical properties as that of living tissue, which is due to their high water content, soft and rubbery consistency and low interfacial tension with water or biological fluids. Research is focusing on making hydrogels photo-polymerizable  or self-hardening e.g., silanized hydroxyl-propyl-methyl cellulose,  so that they form rigid structures once they are implanted into the tissue sites. Another injectable scaffold studied is ?-tricalcium phosphate.β-tricalcium phosphate. It is alginate in gel phase and forms beads in solid phase. Treated dentin matrix also provides suitable environment for regeneration of dental tissue.  Silk scaffolds may be used for mineralized osteo-dentin formation. The size and shape of silk scaffold pores guide mineralized tissue.  Enamel matrix derivatives (Emdogain), whose major component is amelogenins, have also been used as potential scaffolds. 
The seeding of cells on tissue engineering scaffolds is known as 'creating a tissue construct'. To promote the formation of higher order tissue structures, tissue constructs are maintained in cell culture in the presence of bioactive molecules called growth factors-the third component of tissue engineering.
| Growth Factors|| |
Growth factors are proteins that bind to receptors on the cell and induce cellular proliferation and/or differentiation.  Many growth factors are quite versatile, stimulating cellular division in numerous cell types, while others are more cell specific.  Various growth factors studied ,,,,,,,,,,,,,,,,,,,,, in regeneration of pulp-dentin complex are depicted in [Table 3].
Growth factors play a role in signalling many events in pulp-dentine regeneration. Two important families of growth factor that play a vital role are transforming growth factor (TGF) and bone morphogenetic protein (BMP). TGF-β1 and β3 are important in cellular signalling 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 stimulates an increase in tertiary dentin matrix secretion suggesting that TGF-β1 is involved in injury signalling and tooth-healing reaction. BMPs induce higher quantity and more homogeneous reparatory dentin with the presence of many tubes with defined odontoblastic process as compared to that with calcium hydroxide. BMP-2, BMP-4 and BMP-7 have been shown to direct stem cell differentiation into odontoblasts and result in dentin formation making the BMP family the most likely candidate as growth factors. Some natural materials like dentin are also used because they release bio-active molecules. Enamel matrix derivative is also capable of inducing dentin formation when applied to dentin pulp complex.
Poor angiogenesis is a major roadblock for tissue regeneration. Following approaches are currently being studied for the development of vasculature to support the metabolic needs of engineered tissue:
- Transplanted endothelial cells can increase the vasculature in polymer scaffolds and integrate with growing host capillaries. 
- Localized delivery of inductive angiogenic factors (VEGF, PDGF, EGF) at the site of the engineered tissue. 
- Co-transplantation of hematopoietic and mesenchymal stem cells. 
Although we are aware of the role played by these growth factors, for tissue engineering to be successful it is critical to deliver appropriate growth factors to the desired site at the appropriate dose and for appropriate time for which further research is required. Many of these proteins have short half life in the body, yet they need to be present for an extended period to be effective. For this, an alternate approach is to deliver a gene that encodes for the growth instead of delivering factor itself, called gene therapy.
| Gene Therapy|| |
Genes can stimulate or induce a natural biological process by expressing a molecule involved in regenerative response for the tissue of interest.  Precise delivery and efficient transfer of genes into target tissue cells, prompt assessment of gene expression at required times and appropriate levels and/or minimization of undesirable systemic toxicity are essential prerequisites for successful gene therapy. Either viral or non-viral vectors are used to enable the cellular uptake and expression of genes. Viral vectors are genetically altered to eliminate their disease-causing ability. The viruses can replicate genes of interest together with their own genome, through the use of host cell genetic machinery. Various viral vectors studied ,,,,,,, are depicted in [Table 4].
Non-viral techniques involve either electroporation or ultrasound method for gene delivery. Ultrasound-mediated gene delivery is found to be successful both in vivo and in vitro but electroporation method is found successful only in vitro. This may be because of the lack of erythrocytes in the plasma clot due to thermal changes during electroporation in vivo. In the in vivo approach, the gene is delivered systematically into the bloodstream or locally to target tissues by injection or inhalation. In this approach, the healing potential of pulp tissue is enhanced by genes inducing dentin directly applied on the exposed amputated dental pulp. The ex vivo approach involves genetic manipulation of cells in vitro, which are subsequently transplanted to the regeneration site. The ex vivo gene therapy stimulates reparative dentin formation more optimally and rapidly in comparison to in vivo gene therapy.  From these very few available data there are certain challenges to the gene therapy:
- Need for establishment of isolation, identification and expansion protocol of pulp stem cells.
- Safe and efficient gene delivery system needs to be optimized.
- Potential serious health hazards exist with the use of gene therapy. These arise from the use of the vector (gene transfer) system, rather than the genes expressed.  The FDA did approve research into gene therapy involving terminally ill humans, but approval was withdrawn in 2003 after a 9-year old boy receiving gene therapy was found to have developed tumors in different parts of his body. 
- Researchers must learn how to accurately control gene therapy and make it very cell specific so that it is safe to use clinically.
- Requirements to demonstrate that gene therapy can provide cost-effective and safe long-term treatment for conditions that would otherwise lead to significant pulp necrosis.
Numerous regenerative studies have demonstrated that stem cells can attach to and grow on tissue-engineered scaffolds but there are few studies on potential of stem cells to create dental pulp constructs within human cleaned and shaped root canals.  Recent study has reported for the first time regeneration of dental pulp-like tissue in endodontically treated root canals of real-size, native human teeth. This newly formed tissue appeared dense with disconnected cells surrounded by extracellular matrix. Erythrocyte filled blood vessels were formed with endothelial-like cell lining. There was complete fill of dental pulp-like tissue in entire root canal from root apex to pulp chamber with tissue integration to dentinal walls.  Dental pulp construct growing in root canal without functional connection is meaningless; hence, further research is required to regenerate a replacement vital pulp attached to the circulatory system and the old dentin as well as produce new dentin matrix.
| Revascularization|| |
Regeneration of tissue from cells in teeth itself.
- Basically, body tissue is composed of two components: cells and the surrounding environment. The latter includes the ECM for cell proliferation and differentiation (natural scaffold). Revascularization approach in young permanent infected teeth with immature root apex and apical periodontitis was first attempted in 1971,  but it was not successful due to limitations in technologies, material and instruments available in those times. But with the currently available technologies, several case reports , have documented revascularization of necrotic root canal systems by disinfection followed by establishing bleeding into the canal system via over-instrumentation. The revascularization method assumes that the root canal space has been disinfected and that the formation of blood clot yields a matrix (e.g., fibrin) that traps cells capable of initiating new tissue formation. It is different from apexification because not only the apex is closed but the canal walls are thicker as well. It is also different from apexogenesis which also accomplishes a closed apex and thicker dentinal walls, but, by the use of remaining vital root pulp. The revascularization studies have established following prerequisites:
- Revascularization occurs most predictably in teeth with open apices and necrotic pulp secondary to trauma
- Apex open > 1.5 mm.
- Bacteria should be removed from canal by any of the following methods:
- '3 mix-MP' triple antibiotic paste consisting of ciprofloxacin, metronidazole and minocycline 
- Calcium hydroxide,  formocresol. 
- Effective coronal seal.
- Matrix into which new tissue can grow.
- Patients should be young.
- Use of anaesthetic without a vasoconstrictor when trying to induce bleeding. 
- No instrumentation of the canals.
- Sodium hypochlorite is used as an irrigant.
- Formation of a blood clot probably serves as a protein scaffold permitting 3-dimensional ingrowth of tissue.
All the studies report continued thickening of the dentinal walls and subsequent apical closure. The root length is increased by the growth of cementum. Connective tissue similar to periodontal ligament was also present in the canal space  .
The success of root canal revascularization is mainly due to the following facts: firstly, the immature avulsed tooth has an open apex, short root and intact but necrotic pulp tissue. Therefore, the new tissue has easy access to the root canal system and a relatively short distance for proliferation to reach the coronal pulp horn. The speed with which the tissue completely revascularizes the pulp space is important because bacteria from outside are continually attempting to enter the pulp space. The ischemically necrotic pulp acts as a scaffold into which the new tissue grows, and the fact that the crown is usually intact slows bacterial penetration because their only access to the pulp is through cracks or enamel defects. Thus, the race between proliferation of new tissue and infection of the pulp space favors the new tissue. Secondly, minimum instrumentation preserves viable pulp tissue which contributes to further development of open apex root. Thirdly, young patients have greater healing capacity and more stem cell regenerative potential.
Advantages of root canal revascularization:
- The greatest benefit of these biological approaches for dental tissue restoration over many conventional dental materials is that the reparative matrices become an integral part of the tooth, overcoming any of the problems of retention of a restoration and possible marginal bacterial microleakage.
- This treatment approach strengthens the root walls of immature teeth.
Further studies required in root canal revascularization:
- Radiographical findings of continued dentinal wall thickening do not address the cellular nature of this calcified material. In contrast source of cells regenerating the replacement pulp tissue in implanting dental pulp construct is endodontic in origin.
- Although these case reports primarily involve treating the immature permanent teeth, it is quite possible that knowledge gained from this clinical application will have value in developing regenerative endodontic procedures for the fully developed permanent teeth.
- It is more likely that the tissue in pulp space is more similar to periodontal ligament than to pulp tissue. 
| Conclusions|| |
Future regenerative endodontics may involve the cleaning and shaping of root canals followed by the implantation of vital dental pulp tissue constructs created in laboratory. The success of regenerative endodontic therapy is dependent on the ability of researchers to create a technique that will allow clinicians to create a functional pulp tissue within cleaned and shaped root canal systems. The source of pulp tissue may be from root canal revascularization, stem-cell therapy and pulp implantation.
Clinical success of regenerative endodontic therapy will depend on following clinical outcomes:
- Vascular blood flow
- Mineralizing odontoblastoid cells
- Intact afferent innervations
- Lack of signs or symptoms
Limitations (Concern for researchers)
- Although the replacement pulp has the potential to revitalize teeth, it may also become susceptible to further pulp disease and may require retreatment; the implantation of engineered tissue also requires enhanced microbiological control methods required for adequate tissue regeneration.
- The success of clinical applications of pulp stem cells is limited by the culture conditions and the nature of microenvironment in which the primitive multipotent pulp stem cells are maintained and expanded.
- To improve the ability of dental pulp constructs to adhere to root canal walls, it seems that the ideal scaffold design is in the same shape as gutta-percha cones. Researchers had used single-canal teeth and cylindrical scaffolds in an attempt to simplify the transplantation process. A more complex root canal anatomy will require more complex scaffolds or the use of more flexible scaffolds to perform regenerative endodontics.
- Dental pulp tissue constructs adhered more completely to the coronal aspects of the root canal and less completely to the middle and apical aspects. This likely was caused by the increasing complexity of root canal anatomy toward the apex and the physical constraints of the scaffold materials, as well as the placement method.
- Since most of the tissue-engineered parts have been developed using very potent signal molecules to induce the transformation the growth of the stem cells, a way has to be found to insure that these transformation and growth will not continue beyond control when implanted.
- Matching the aging of the implanted tissue-engineered parts with that of the surrounding tissues and organs is a great obstacle too.
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Department of Conservative Dentistry, Teerthankar Mahaveer Medical College, Moradabad
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3], [Table 4]
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