Tooth Regeneration: Turning Hopes into Realities

Functional tooth regeneration has shown promising therapeutic strategy. Tooth regeneration is possible by combine use of adult stem cells, growth factors, and scaffold. In recent years, researchers have explored tooth regeneration. Significant effort has been made in recent decades to identify and characterize tooth stem cells and to unravel the developmental programs which these cells follow to generate a tooth.


Trilogy of Regeneration
The regeneration of orodental tissues is dependent on appropriate signals, cells, blood supply and scaffold that are needed to target the tissue at the site of defect. For reconstruction and healing of lost tissues, all the components are essential. [5]

Adult Stem Cells
The most valuable cells for regenerative medicine are stem cells. All tissues originate from stem cells. A stem cell is commonly defined as a cell that has the ability to continuously divide and produce progeny cells that differentiate (develop) into various other types of cells or tissues. Stem cells can be divided into two categoriesembryonic and adult. [6] Dental sources of adult stem cells are as follows: Periodontal ligament stem cells. [6] even caries lead to the release of growth factors resulting in demineralization of the dental tissues. Calcium hydroxide may result in extraction of growth factors from the dentin matrix. Once released, these growth factors may play key roles in signaling many of the events of tertiary dentinogenesis, a response of pulp-dentin repair. [11] Different growth factors with their sources and activity are described in Table 1. [12,13] Technologies for regenerative endodontics There are several major areas of research that might have application in the development of regenerative endodontic techniques. These techniques are as follows: Root canal revascularization through blood clotting, postnatal stem cell therapy, pulp implantation, scaffold implantation, injectable scaffold delivery, three-dimensional cell printing and gene delivery. [12]

Root Canal Revascularization through Blood Clotting
Several case reports have documented revascularization of necrotic canal by disinfection followed by establishing bleeding into the canal system via over-instrumentation. The use of intracanal irrigants (NaOCl and chlorhexidine) with placement of antibiotics (e.g., a mixture of ciprofloxacin, metronidazole and minocycline paste) for several weeks is important part of procedure.
Disinfection of root canal followed by the formation of blood clot matrix that trap cells capable of new tissue formation, although it is not clear the regenerated tissue's phenotype resembles dental pulp; however, case reports published to date do demonstrate continued root formation and the restoration of a positive response to thermal pulp testing. [2] Advantages • Simple, cost effective, completed with currently available instruments and medicaments. • Avoid immune rejection, pathogen transmission from replacing the pulp with a tissue engineered construct. [12] AAE Regenerative Endodontic Procedure [14] First appointment • Local anesthesia, dental dam isolation and access.

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Copious, gentle irrigation with 20 mL NaOCl with minimum extrusion of irrigants into the periapical space. About 1.5% NaOCl is recommended followed by irrigation with saline, with irrigation needle positioned about 1 mm from root end, to minimize cytotoxicity to stem cells in the apical tissues.  MTA alternate should also be used in teeth with esthetic concern as it results in discoloration.

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Anterior and premolar teeth -Use of CollaTape/CollaPlug and restoration with 3 mm of resin-modified glass ionomer (RMGI) followed by bonding a filled composite to the beveled enamel margin.

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Consider use of CollaTape/CollaPlug and restoration with 3 mm of MTA followed by RMGI or alloy in molar teeth or teeth with porcelain fused to metal crown.

Follow-up
Clinical and radiographic examination: • Absence of pain, soft-tissue swelling or sinus tract.

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Increased width of root walls 12-24 months after treatment.
Pulp vitality test.
A modification of the current clinical protocol was established to avoid crown discoloration. This novel approach seals the dentinal tubules of the chamber, thus avoiding any contact with triple antibiotic paste and the dentinal walls. [15]

Considerations for Clinical Regenerative Endodontic Procedures
At present, there is no single recommended protocol associated with successful clinical outcome till date. Common features of cases with successful clinical outcomes after REPs are: 1. Young patient (age and health status). 2. Apex size and immature apex. 3. Dentinal walls should be instrumented minimally. 4. Disinfection and placement of an intracanal medicament. 5. Creation of a blood clot or protein scaffold in canal. 6. Effective coronal seal. [16]

Age and Health Status of Patient
Revascularization procedure has been limited in patients with 8-16 years of age. The success depends on patient's ability to heal the dental pulp tissues. Not recommended in children younger than 8 years or older than 16 years, unless the tooth has an open apex with thin walls. [16]

Apex Size and Stage of Tooth Maturity
Traumatized immature permanent teeth with an apex that is open to a diameter of 1.1 mm or larger are the best candidates for regenerative endodontic procedures. Revascularization through an open apex allows the delivery of mesenchymal stem cells into the root canal space of necrotic immature teeth after a clinical regenerative endodontic procedure. This could allow host cell homing to form new tissues in the root canal space. [17]

Minimal or No Instrumentation of the Dentinal Walls
Root canal instrument not only increases fragility of dentin walls but also injures stem cells present in the apical area of these dentin walls and results in elimination of growth factors. [15]

Irrigants and chelating agents
If sodium hypochlorite is used in conjunction with regenerative endodontic treatments, it should be flushed from the root canals with saline in an attempt to reduce toxicity that reduces the regeneration responses. [18] Disinfection with chlorhexidine was used due to its antimicrobial properties and low cytotoxicity. If both of these irrigants are used in the canal, it is prudent to irrigate with sterile water or saline between applications. [19] Hydrogen Peroxide Hydrogen peroxide is antiseptic by release of oxygen radical. However, it requires a rinse to reduce pain and possible postoperative gaseous emphysema. [20] Chelating agents such as EDTA, citric acid and MTAD may also be used to remove the smear layer.

Intracanal Medications
Regeneration procedures use a triple antibiotic paste, sometimes called Hoshino's paste which contains 200 mg ciprofloxacin, 500 mg metronidazole and 100 mg minocycline, manipulated in propylene glycol vehicle. Paste is inserted through Lentulo spiral drill, syringe, or manual files.
The triple antibiotic paste is placed in contact with the necrotic pulp inside the root canal for up to 1 month before the revascularization procedure.
Recently, calcium hydroxide has been tested with pulp revascularization, showing clinical and radiographic success. It allows increase in thickness of dentinal walls. Ca(OH) 2 would increase the expression of some kind of kinases which indicates proliferation of stem cells from pulp and ligament. Therefore, it would not be cytotoxic for stem cells and would support their proliferation. [16]

Creation of a Blood Clot or Protein Scaffold in Canal
To create blood clot in the canal space, apical tissues should be lacerated just below the cement enamel junction. This can be performed with an endodontic explorer or file. If bleeding does not occur, explorer or file is dipped in 17% EDTA to prevent coagulation of the blood. If bleeding still does not occur, it may be necessary to place more antimicrobial in the canal to achieve additional healing and granulation tissue. Alternatives to a blood clot include (platelet-rich plasma [PRP]) and autologous fibrin matrix. [16]

Effective Coronal Seal
Regenerative endodontic procedures evoke bleeding into the root canal, which delivers undifferentiated mesenchymal stem cells in the root canal space. The survival of cells and regeneration of tissues is sensitive to the conditions within the intracanal environment, which must be biocompatible to allow new tissue formation. The placement of restorative materials in contact with the pulp tissue, root canal blood clot, or regenerating tissues is not recommended. These materials are not biocompatible to exposed pulp tissues because they can cause cell death and pulpitis and allow bacterial leakage. [16] Restorative materials should be used only after a thin protective liner of MTA or calcium hydroxide has been placed in contact with the coronal pulp tissue, root canal blood clot or regenerating tissues. [21]

Treatment of immature permanent teeth with necrotic pulps
Since the introduction of regenerative endodontic therapy for immature permanent teeth with apical periodontitis by Iwaya et al., regenerative endodontic therapy has become a treatment option for immature permanent teeth with necrotic pulps. Thickening of the canal walls and/or increased root length is considered to be the favorable outcomes of regenerative endodontic because they may strengthen the root and increase the root/crown ratio. Importantly, the vitality, immunity and sensibility of immature permanent teeth with necrotic pulps are restored after regenerative endodontic therapy. [22] Treatment of mature permanent teeth with necrotic pulps Thickening of the canal walls and/or continued root development does not occur in mature permanent teeth following regenerative endodontic therapy. However, apical closure can take place. Elimination of clinical symptoms and resolution of apical periodontitis is similar to traditional non-surgical root canal therapy. Therefore, regenerative endodontic therapy provides another treatment option for mature permanent teeth with necrotic pulp. [23] Treatment of teeth with persistent apical periodontitis after root canal therapy Endodontic surgery is usually indicated if non-surgical treatment is not feasible. The outcome of secondary root canal treatment is less favorable than that of primary root canal treatment because of several possibly complicated factors. Recently, regenerative endodontic therapy has also been used to manage teeth with persistent apical periodontitis after root canal therapy. [24] Clinical symptoms are eliminated and resolution of apical periodontitis occurs. Interestingly, thickening of the canal walls and apical closure was demonstrated after regenerative endodontic therapy of teeth with persistent apical periodontitis after root canal treatment. [25] Treatment of traumatized teeth with external inflammatory root resorption, horizontal root fracture, and avulsed teeth Traumatized teeth with external inflammatory root resorption, horizontal root fracture and complete avulsion are traditionally managed with control of root canal infection using chemomechanical debridement and root canal filling. [26] Recently, REPs are used to treat traumatized teeth with horizontal root fracture, external inflammatory root resorption, and complete avulsion.
For external inflammatory root resorption to take place, the protective layer of pre-cementum must be damaged, likely by trauma or inflammation, thus leading to exposure of the underlying dentin. [27] In addition, the canal space has to contain infected necrotic pulp tissue. The toxic products from bacteria and tissue breakdown in the canal space diffuse through the dentinal tubules, communicating with the root surface denuded of cementum, and initiate the inflammatory reaction. [28] Treatment of external inflammatory root resorption is usually carried out by complete chemomechanical debridement, long-term calcium hydroxide dressing and root canal filling. Calcium hydroxide would prevent osteoclast activity. Long-term calcium hydroxide dressing in the canal space of immature permanent teeth weakens the fragile thin root structure, thus increasing the likelihood of root fractures.
External inflammatory root resorption of immature permanent teeth and horizontal root fracture caused by trauma resulting in pulp necrosis get resolved by REPs achieving healing of the root fracture by hard tissue formation. [29] Avulsed teeth are treated with immediate replantation followed by chemomechanical debridement, calcium hydroxide dressing and root canal filling if the pulps become necrotic. Recently, an avulsed permanent mature incisor with more than 8 h extraoral dry time was replanted into the alveolar socket after complete chemomechanical debridement of the canal space and enlargement of the apical foramen to 1.5-2 mm. The tooth was then treated with regenerative endodontic therapy, using PRP instead of blood clot as a scaffold. At 12-month follow-up, the tooth showed resolution of apical periodontitis and arrest of internal and external inflammatory resorption. [30] Can Regenerative Endodontic Therapy be used for All Necrotic Teeth?
In some necrotic teeth, regenerative endodontics may not be suitable, for example, in teeth requiring a post for adequate techniques and it may be challenging in some others (e.g., tooth with pulp stones or pulp calcifications). [31] Retreatment in tooth with earlier failed treatment with regenerative endodontic procedures can be a challenge. The teeth treated with root canal filling may have a poorer survival rate than the teeth treated with regenerative procedures because of lack of defense mechanisms such as immunoinflammatory and sensory response.

chAllenges A n d lImItAtIons
Regenerative endodontics has encountered substantial challenges toward clinical translation. 1. Recent adoption by the American Dental Association of evoked pulp bleeding in immature permanent teeth is an important step for regenerative endodontics. However, majority of endodontic diseases cannot be treated by REPs. [32] 2. Simple recapitulation of cell therapy and tissue engineering has not led to clinical translation in regeneration endodontics. DPSCs may appear to be choice for dental pulp regeneration. However, DPSCs may not be available in a patient who is in need of pulp regeneration. 3. Transplantation of DPSCs will remain a scientific exercise, rather than a clinical reality, unless these issues (scientific, regulatory, and commercialization) are resolved. [33] conclusIon Regenerative endodontic has the potential for regenerating both pulp and dentin tissues and therefore may offer an alternative method to save teeth that may have compromised structural integrity. Several techniques have been described to accomplish endodontic regeneration. Endodontists will have to be dependent on tissue engineering therapies to regenerate pulp dentin tissue, to make REPs a success, the future development of regenerative endodontic procedures will require a comprehensive research program directed at each of these components and their application to the patients. It is believed that regenerative endodontics is an inevitable therapy, and they call for action from scientists, funding agencies, and the endodontic profession to pool resources to hasten its development. Millions of patients get benefited by REPs each year.