3.2. Disadvantages of the Revascularization Approach
-
The origin of where the tissue has been regenerated from is yet to be known.
-
According to researchers, effective composition and concentration of cells are mandatory for tissue engineering. However, these cells are entombed in fibrin clots; therefore, researchers do not rely on blood-clot formation for tissue engineering function.
-
Treatment outcomes will be variable by the variations in the composition and concentration of the cells
[36][37][38][39].
3.3. Prerequisites for Revascularization Approach (Figure 1)
Revascularization studies have established the following prerequisites:
-
There should be open apices and necrotic pulp secondary to trauma.
-
In addition, open apex should be less than 1.5 mm.
-
The following agents can be incorporated to remove microorganisms from the canal.
-
The coronal seal should be effective.
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There should be a matrix or the growth of new tissues.
-
When trying to induce bleeding, anaesthesia should be used without a vasoconstrictor
[42].
-
Canals should not be instrumented.
-
Sodium hypochlorite should be used as the irrigant.
-
There should be blood-clot formation.
Figure 1. Requisite preconditions for pulp regeneration (root canal disinfection and enlargement of the apical foramen)
[43].
4. Postnatal Stem Cell Therapy
Bone, buccal mucosa, fat, and skin are the common sources of postnatal-stem cells. After the apex is opened, the disinfected root-canal system is injected with postnatal-stem cells. This treatment is considered the simplest technique
[44]. There are numerous benefits of this type of tissue-engineering technique. Postnatal-stem cells are rationally easy to harvest, and these cells can persuade the regeneration of the pulp. Moreover, these cells are easy to deliver by syringe. In addition, application of these stem-cell therapy is used in regenerative medicine since past many years, for example, bone-marrow replacement and endodontic applications
[45]. However, low survival rates are one of the major disadvantages of this technique. Moreover, these cells can migrate into different locations of the body, which presents peculiar forms of mineralization
[46]. For the development of dental tissues by the differentiation of stem cells, bioactive-signalling molecules, growth factors, and scaffolds are required
[47]. Consequently, with only stem cells that exclude the growth factors or scaffolds, the chance of pulpal regeneration of new tissues is very low. In this approach, the chief identification of a postnatal-stem-cell source that must be able to differentiate into the diverse cell population can be obtained
[46]. However, this technique is not approved yet.
4.1. Pulp Implantation
In this procedure, after cleaning and shaping the root canal, the substituted pulp tissue is transplanted. Purified pulp-stem-cells line is among one the sources of the pulp tissue. This pulp tissue can also grow in the laboratory by cell biopsy. For this invitro technique, pulp tissues can be cultured by biodegradable-polymer nanofibers. Moreover, these tissues can be obtained from collagen I or fibronectin-extracellular-matrix proteins
[48]. It has been found that further investigations are required for the proteins, such as vitronectin and laminin. However, it has been proved that for growing pulpal cells, collagens I and III are not fruitful
[49]. In the root-canal system, the localization of postnatal-stem cells is a major advantage of pulp implantation. However, there are several disadvantages to this technique. It is a restriction of this technique that the apical portion of the root canal should be harvested with pulp cells. The reason behind this concept is that the sheets of the extracellular matrix are very thin, fragile, and they lack vascularity. Therefore, a scaffold that must have cellular proliferation is required for coronal delivery. If the cells are located 200 μm from a capillary- blood supply which is the maximum oxygen-diffusion distance, these cells are in danger of anoxia and necrosis.
4.2. Scaffold Implantation
For vascularization and cell organization, pulp-stem cells must be systematized into a three-dimensional assembly. This objective can be achieved by seeding pulp-stem cells with a porous-polymer scaffold
[50]. Distribution of therapeutic medicines to precise tissues can successfully be accomplished by these nano scaffolds
[51]. Moreover, the biological and mechanical properties needed for proper functioning are also provided by these scaffolds
[52]. In teeth that have pulp exposure, dentin chips have been introduced which accelerate dentin-bridge formation
[53]. These dentin chips aid in the reservoir of growth factors and they offer a matrix for the attachment of pulp-stem cells
[54][55]. In reaction to the dentine chip and the use of scaffolds, the regeneration of the pulp-dentin complex occurs. To provide structural support to the tooth it is not necessary to have a tissue-engineered pulp in the root-canal systems
[56]. Polymer hydrogel, a soft three-dimensional injectable scaffold matrix, will be administrated by syringe in tissue-engineered pulp tissues
[57]. They are easy to deliver into the root-canal systems and are non-invasive.
4.3. Three-Dimensional Cell Printing
The three-dimensional cell printing technique is considered the final approach for the replacement of pulp tissues
[58]. This approach can be used to position cells precisely
[59]. This technique mimics the natural pulp-tissue structure. In tissue-engineering technique, to maintain and repair dentine, odontoblastoid cells should be positioned around the periphery of the pulp.
4.4. Gene Therapy
To promote tissue mineralization, mineralizing genes would be delivered into the pulp tissues. However, Rutherford worked on this specific field of gene delivery into the pulp tissues, although there is a dearth of literature in this context
[60]. He suggested further research to improve the possible gene therapy inside the pulp after he failed in his work when he transduced pulps of ferret animals with cDNA-transfected mouse BMP-7. Researchers used the electroporation method to insert mineralizing genes into the pulp space by culturing of pulpal-stem cells. Initially, the FDA approved the gene therapy research on terminally ill humans; however, after the development of numerous tumours in a nine-year-old boy, the FDA withdrew this decision in 2003. Gene therapy arising from the use of vector systems is posing serious health hazards in contrast to gene expressions
[61][62].
4.5. Nitric Oxide
Among many wound healing and pathological processes, angiogenesis is considered an important process. The most potent and critical inducer of angiogenesis is the vascular endothelial growth factor (VEGF). A variety of stimuli take part in the regulation of gene expression of VEGF. The transcription factor is a key factor for hypoxia-mediated VEGF- gene upregulation, which is achieved by hypoxia-inducible factor 1 (HIF-1). Nitric oxide (NO) is a potent vasodilator. Nitric oxide (NO) can simply pervade natural membrane obstacles because it is lipophilic in nature. This VEGF regulates the amount of nitric oxide
[63]. Hypoxia as well as nitric oxide upregulate the VEGF genes by enhancing HIF-1 activity. Moreover, dendrimers are released by nitric oxide which acts as antibacterial agents
[64][65].
4.6. Platelet-Rich Plasma (PRP)
Special challenges are faced by clinicians for the treatment of an immature tooth with necrotic pulp and open apex. One of the strategies for its treatment is the traditional apexification procedure. This treatment process requires the formation of the apical barrier by multiple applications of calcium hydroxide. This apical barrier can also be formed by placing mineral trioxide aggregate (MTA) into the canal, which is followed by the conventional root-canal procedure
[20]. Due to incomplete root formation with these procedures, the chances of root fracture are very common
[10][20]. Platelet-rich plasma (PRP) has been suggested as probably the greatest platform for RET that will overcome all these problems
[20][66]. Platelet-derived growth factor, transforming growth factor b, and insulin-like growth factor form an integral part of the PRP
[5]. PRP can be utilized as a scaffold as it can form a three-dimensional fibrin matrix. It is easily prepared from the patient’s autologous whole blood
[20][67][68][69]. Growth factors and cytokines are 4-fold higher in platelets than found in whole blood
[70]. Mandibular-continuity defects, for the first time, were healed by the PRP and the placement of cancellous-bone grafts by the dental community
[71]. Human dental pulp stem cells (DPSCs), when treated with PRP, resulted in an increase in the differentiation and proliferation of these cells
[72].
There are numerous advantages of PRP treatment. During the preparation of the PRP, erythrocytes that would be responsible for necrosis after clot formation was removed
[20]. For cell migration, fibrin, fibronectin, and vitronectin are required which is obtained from the formation of PRP clots
[69]. Moreover, in regenerative-endodontic procedures, the optimal level of MTA placement is mandatory which can be done by the collagen matrix present in the PRP
[42][73]. Before clot formation, PRP does not release growth factor until it is activated. As soon as it is activated either endogenously or through the exogenous, such as by incorporation of calcium chloride or thrombin that acts as a clotting factor, PRP will start secreting growth factors that contribute to the repair and regeneration of the tissues
[69][74].
4.7. Cell Homing
In tissue regeneration, the first concept of cell homing was presented in Lancet in 2010. The concept was based on the delivery of transforming growth factor-b3 (TGFb3) without cell transplantation. This approach was first used for the regeneration of the articular cartilage [75]. However, for dental-tissue regeneration, the idea of cell homing was introduced in 2010 [76]. During cell homing, root canal of the extracted human teeth was shaped and cleaned followed by the delivery of the growth factors, scaffold, and stem cells. Residual proteins in the root canal or dentinal tubules were deactivated in the first phase. This can be done by sterilization of extracted teeth in an autoclave. This was followed by the infusion of collagen gel into a shaped and cleaned root canal that might be with or without basic fibroblast growth factors (bFGFs), vascular endothelial growth factors (VEGFs), platelet-derived growth factors (PDGFs), nerve growth factors (NGFs), or bone morphogenetic proteins (BMPs).
The prime difference between the cell homing and cell transplantation approaches is that, in the latter case, for dentine/pulp regeneration, the isolated cells (stem/progenitor) from the host are transplanted into the root canal of the host. Dental pulp-like cells have been differentiated in the cell-homing approach when growth factors are recruited into the root-canal system. Cell-homing-technique-dental-organ regeneration presents a harmonizing and/or balancing approach to cell-transplantation technique and, at the same time, this strategy has shown auspicious results in animal models [14][76][77]. Hematopoietic-stem cells were militarized and transferred to different tissues or organs using active navigation in the cell-homing approach. The ultimate outcome of this process is pulp-dentin re-cellularization and revascularization. Numerous growth factors along with cell homing will result in pulp-dentin regeneration. Tissue revascularization and regeneration-cell homing consist of two distinctive cellular processes. They are differentiation and recruitment [78][79].