Traditionally immature necrosed teeth were being treated by apexification using calcium hydroxide and recently by using MTA. Drawbacks of this traditional approach were no continued root development and thin roots prone to fracture in future. Regenerative endodontics uses concept of tissue engineering to restore the root canals to a healthy state, allowing for continued root development and surrounding tissue. With the advances in tissue engineering and molecular sciences, the higher success rates can be achieved.
Tissue engineering is an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitute that restores, maintain or improve the tissue function or whole organ.1 (Langar and Vacanti)
Tissue engineering is ‘understanding the principles of tissue growth and applying this to produce functional replacement tissue for clinical use.2 (McArthur and Oreffo)
Regenerative endodontic procedures (REP) defined as biologically based procedures designed to replace damaged structures as dentin, root structure and cells of pulp dentin complex. 3 (Murray)
Dr B. W Hermann (early 1960): Application of Calcium Hydroxide [Ca(OH)2] for vital pulp therapy
Nygaard-Ostby, 1961: Established a blood clot to use as a scaffold to re-vascularize tissue within the root canals of teeth
Rule DC, 1966: Use of double antibiotic paste
Hoshino, 1993: Use of triple antibiotic paste
Iwaya, 2001: Evoked intracanal bleeding step
Triad of tissue engineering5
Characteristics of stem cells
Exist as undifferentiated cells and maintain this phenotype by the environment and/or the adjacent cell populations and they are exposed to and respond to appropriate signals.
Ability to self-replicate for prolonged period.
Maintain their multiple differentiation potential throughout the life of the organism.
Categories of stem cells
According to their source
Autologous cells: Obtained from same individual to whom it will be replanted.
Allogeneic cells: Obtained from body of the donor of same species.
Xenogeneic cells: Isolated from individuals of another species. Eg: Animal cells in construction of cardiovascular implants.
Syngeneic/ Isogenic cells: Isolated from genetically identical organisms. Eg. Twins, Clones.
According to their potency
Totipotent: Cells that can differentiate into new organisms. Eg. cells from early embryos.
Pluripotent: Cells that can differentiate into nearly all cells but not entire organism. Eg. Blastocyst
Multipotent: Cells that can differentiate into limited range of cells. Eg. Fetal tissues, Cord blood, Dental pulp stem cells (DPSCs)
Different populations of adult stem cells in the oral region
Stem cells markers
Cells coated with surface receptors such as proteins have capability of selectively binding to other signalling molecules. Stem cells markers have ability to fluoresce or emit light energy when activated by UV light or Laser beam. They help in identification and isolation of stem cells.
Isolation of stem cells
By staining the cells with specific antibody markers and using flow cytometer. This process is called as “Fluorescent antibody cell sorting (FACS)”.
Physiological/ Histological criteria of isolation of stem cells are by phenotype, chemotaxis, proliferation, differentiation and mineralization.
Immunomagnetic bead selection.
Growth factors / Morphogens / Signaling Molecules
These factors trigger the differentiation of selected mesenchymal stem cells into odontoblast like cells. Their functions are:
To stimulate division of neighbouring cells.
To stimulate differentiation of certain cells along a specified pathway.
To stimulate angiogenesis.
Dentin is considered as a reservoir of growth factor and cytokines. These growth factors /cytokines secreted by odontoblast during primary dentinogenesis become sequestrated and fossilized into the dentin after biomineralization. 6
Different growth factors
Growth hormone (Paracrine/ Autocrine role)
Insulin like growth factor (IGF-1, IGF-2)
Transforming growth factor β (TGF β-1, TGF β-2, TGF β-3)
Bone morphogenic proteins (BMP-2, BMP-4, BMP-6)
Fibroblast growth factors (FGFs)
Tumour necrotic factors (TNFs)
Colony stimulating factors
Platelet derived growth factors (PDGF)
Nerve growth factors (NGF)
Second level of regulation by transcription factor MSX-1 and MSX-2.
Toll like receptors (TLR-4 activated by lipopolysaccharides).
Patients taking long term corticosteroids present with dramatic reduction in radiographic size of pulp chamber and upto fivefold increase in thickness of predentin layer. 7
Scaffold / Matrix
Scaffold provides a physiochemical and a biological three dimensional micro-environment for cell growth and differentiation, promoting cell adhesion and migration. Scaffold is used to guide, organize and provide physical or chemical signals and help in growth and differentiation of cells.
Scaffold is necessary to,
Properties of ideal scaffold 8
Should be porous to allow placement of cells and growth factors.
Should allow effective transport of nutrients, oxygen and waste.
Should be biodegradable, leaving no toxic by-products.
Should be replaced by regenerative tissue while retaining the shape and form of the final tissue structure.
Should be biocompatible.
Should have adequate physical and mechanical strength.
Classification of scaffold
Overview of regenerative endodontics
Traditionally immature roots were treated by apexification using calcium hydroxide or apical plug of MTA. This procedure resolved the signs and symptoms but no continued root development or pulpal nociception was seen.
Regenerative endodontic procedures (REPs) are biologically based procedures designed to replace damaged structures as dentin, root structures and cells of pulp dentin complex. It aims to,
Heal apical periodontitis.
Promotes normal pulpal physiological functions.
Continued root development.
Blood clot could be the first step in healing of damaged dental pulp, similar to role of blood clot in healing of other areas. (Eg. Alveolar socket after extraction) 9
According to clinical studies on regenerative endodontics mentioned in Cohen’s pathways of pulp 12th edition, 5
Initially, there was strong focus on disinfection and intentional bleeding into the root canal. But there is paradigm shift from “disinfect canals at all cost” to “disinfect while creating a micro-environment conducive for tissue engineering.” Other terms for regenerative endodontics are: Revascularization, Revitalization, Maturogenesis.
According to the American association of Endodontists (AAE), clinical consideration for a regenerative procedure, primary goal of regenerative procedure is elimination of clinical symptoms and resolution of apical periodontitis. Secondary goal is thickening of canal walls and continued root maturation. Tertiary goal is to have a positive response from pulp vitality. 10
Different techniques of tissue engineering 3
Root canal vascularization via blood clotting
Incompletely formed roots (often blunderbuss) present difficulty in cleaning and shaping of the apical portion of the root canal. Presence of thin fragile dental walls may prone to fracture during instrumentation or obturation of root canal which possess risk of extruding materials into periradicular tissues. To treat this, traditionally apexification was choice of treatment which means creating apical barrier to prevent extrusion. But major disadvantages of apexification procedure are:
So, American Association of Endodontists recommends clinical considerations for Regenerative Endodontic procedures as follows: 10
Ideal case selection would be young patients with immature infected teeth where cessation of root maturation has occurred. Three measures advocated by American Association of Endodontists clinical considerations for Regenrative Endodontic procedures are.
Minimal or no instrumentation of dentinal walls
Regenerative endodontic procedures advocate minimal or no filing of the canal. Although some studies suggest formation of biofilms and penetration into dentinal tubules in histologic studies. 5 Some degree of mechanical debridement may also be required to disrupt biofilms on canal walls for continued root maturation. 11
Disinfection of the root canal system
Copious irrigation with 20ml sodium hypochlorite with close ended needle / side vented needle / Endovac / Max I probe is recommended. Lower concentration of sodium hypochlorite (1.5%) (20ml / canal for 5 mins) is advised followed by irrigation with saline / EDTA (20ml / canal for 5 mins) with irrigating needle positioned about 1 mm from root end to minimize cytotoxicity to stem cells in apical tissues. Higher concentrations of NaOCl decreases survival of SCAP.
Use of 1.5% NaOCl has minimal destructive effect on SCAP whereas use of 17% EDTA increases SCAP survival. 12 According to Galler 2015, EDTA demineralizes dentin which exposes dentin matrix which in turn releases growth factors. 13 Irrigation with Chlorhexidine should be avoided due to its cytotoxicity to stem cells. 14
Placement of an intracanal medicament
AAE Protocol is to use Triple Antibiotic Paste (TAP) at concentration no greater than 0.5mg/ml (Conducive to stem cell survival). TAP contains Metronidazole, Ciprofloxacine and Minocycline in the ratio 1:1:1.
Creation of blood clot / protein scaffold in canal after resolution of symptoms. REP involves lacerating periapical tissues to initiate bleeding or use of PRF / PRP.
Effective coronal seal is of importance after formation of blood clot. Premeasured piece of Collaplug on the top of blood clot to serve as internal matrix for placement of approximately 3 mm of MTA followed by 3-4 mm layer of GIC bonded by Reinforced composite over it.
The goal of REP is to have continued root development and reestablishment of pulp vitality. REP resulted in 31.6% increase in radiographic root area. (RRA) 16
According to Jeeruphan 2012, Survival rate of revascularization is 100% versus 95% for MTA apexification and 71.2%for calcium hydroxide apexification. 17
Chen et al. 2012 described 5 types of responses for teeth treated with REPs
Increased thickness of root canal walls and continued root maturation.
No significant continuation of root development with root apex becoming blunt and close.
Continued root development with apical foramen remaining open.
Severe calcification of canal space.
Hard tissue barrier formed in the canal between coronal MTA plus and root apex.
Possible mechanisms for continued root development followed by REPs 18
Few vital pulp cells remain at the apical end: They have ability to proliferate and differentiate into odontoblasts guided by the intact HERs.
Periodontal ligament stem cells: This possibility is valid in case of destruction of HERs and apical papillary tissues.
SCAP: instrumentation beyond apical limit of canal leads to transplantation of SCAP into tissue lumen
Blood Clot itself: Blood clot is reservoir of growth factors such as PDGF, TGF etc. It stimulates differentiation, growth, maturation of fibroblast, odontoblast and cementoblast from their undifferentiated precursors.
Complications associated with REPs
Discolouration is more associated with TAP due to minocycline. It is also associated with calcium hydroxide / MTA. To treat discolouration, walking bleach using sodium perborate can be performed.
Post natal stem cell therapy
Stem cell categories are
Embryonic (Pluripotent): Isolated from blastocyst. They can give rise to all derivatives of 3 germ layers.
Post natal cells: Collected from bone marrow / umbilical cord blood. They are less plastic, have limited potential of differentiation.
Stem cells of dental origin are:
DPSCs: Responsible for dental repair.
Can regenerate pulp-dentin like complex.
SHED: Can differentiate into odontonblast-like cells that form dentin like structures.
PDLSCs: Present in enzymatically digested PDL.
Can form cementum / PDL like structures.
SCAP: Found at apices of developing teeth at junction of apical papilla and dental pulp. They can undergo odontoblastic, osteogenic, neurogenic differentiation.
In post natal stem cell therapy, cells are injected in infected root canal.
During dental pulp regeneration, an ideal scaffold should also ensure good neurovascular supply to new pulp tissue. Eg. DPSCs are seeded on 3-D polyglycolic acid matrix, grown in vitro and surgically implanted.
Injectable scaffold delivery
3-D cell printing
It is used to precisely position the cells and thus constructed tissue mimics the natural dental pulp tissue structure.
Inkjet device is used to disperse the layers of cells suspended in hydrogel to recreate dental pulp tissue.
Precise 3-D models for individual pulp cavity and effective delivery system are required.
It is the means of delivering genes for growth factors, morphogens and ECM molecules to somatic cells of individual resulting in therapeutic effect.
Recent studies done in the field of Regenerative endodontics
Shah N. et al. 2012 19 introduced a recent novel technique named as “sealbio” which is regeneration based non obturation technique for management of pulp and periapical tissues involved matured permanent teeth. This technique incorporates the apical clearing meaning preparation larger than 3 sizes of master apical file. (MAF) also known as apical foramen widening followed by intentional over-instrumentation. (Scaffold of blood clot) It is hypothesized that endogeneous, locally residing stem cells will populate the scaffold, differentiate into forming cells and lay down fibrous / cemental tissue to achieve a biologic seal over the apical foramen, hence the term sealbio.
Roots et al. 2009 suggested endodontic grafting. It involves filling of root canal with ceramic sealer which acts as osteoconductive. It promotes the physiological closure of the root canal by cementoid hard tissue. (Eg. Bioaggregate and iRoot SP)
Nakahara et al. 2007 20 suggested whole tooth generation by tooth/ periodontal organ engineering.
Regenerative endodontics present a new era in biological and clinical endodontics. Further research in the area of stem cell based pulp engineering will allow for true regeneration.
Conflict of Interest
The author declares no potential conflicts of interest with respect to research, authorship, and/or publication of this article.