Enamel Regeneration - Current Progress and Challenges (2024)

Restoration: Synthetic Enamel Fabrication

Previous studies have proposed various methodologies for regenerating enamel like hydroxyapatite microstructures [9]. For example, a hydrothermal method using the controlled release of calcium from a Ca-EDTA, hydrothermal transformation of octacalcium phosphate rod to HA nanorods and using hydrogen peroxide containing pastes [10]. These approaches involve the methods that are performed under conditions of high temperature, high pressure, or extremely low acidity, which are not suitable for clinical application [9]. Recently research is being carried out under ambient conditions simulating oral cavity by using supersaturated solutions and the enamel derived protein amelogenin [2].

Based on the understanding of biological process involved in amelogenesis and advances in nanotechnology, Chen et al., fabricated fluoapatite nanorods, which resembles enamel prism like structures from a supersaturated chemical solution under physiological condition. These nanorods have similar characteristics to those of natural enamel crystals isolated from rat incisor enamel [11]. Yin et al., regenerated enamel like microstructures using a simple chemical approach, which may be a potential clinical application to repair enamel damage in dental clinics [12]. Zhang et al., have achieved an ordered dental enamel-like structure of hydroxyapatite (HAP) through a solution mediated solid-state conversion process with organic phosphate surfactant and gelatin as the mediating agent [13].

Few other studies have been done by immersion of scratched and demineralized tooth surface into solution. Chak Ryu et al., immersed an artificially scratched tooth in nanoscale HAP powder suspension distilled water for three months. SEM and AFM showed that the scratched surface was deposited with HAP crystals and the roughness increased which was similar to that of the innate layer [14]. Lianchen et al., using demineralized human enamel samples immersed in 10,000ppm PAMAM-COOH solution for 30 min and then in calcium phosphorous solution with or without fluoride for 20 h. SEM and FTIR showed that induced HAP crystal has similar structure and morphology resembles natural intact enamel. Hence, PAMAM-COOH may be used as an organic template on demineralized enamel to induce HAP crystals [15]. Here, the growth of the crystals occurred directly over the specimens but the duration is long which is unsuitable for development in an applied environment.

Stephen mann and colleagues prepared electrospun hydrogel mats of amoprphous calcium phosphate and polymer nano and micro fibres. Mats generated HAP crystals as an immediate layer, which covers the enamel surface. Hence, it could be used for regrowing enamel surfaces that have been lost due to erosion/or wear [10]. Ying et al., used an agarose hydrogel method, which mimics the natural enamel at secretory or matrix formation stage. This biomimetic mineralization model regenerates enamel like prismatic structure with hardness similar to natural enamel [9].

Hontsu et al., successfully fabricated a freestanding flexible HAP sheet, which was directly attached to enamel surface of extracted teeth using a calcium phosphate solution. The interface between sheet and surface was not completely adhered [16] later to improve the adhesiveness of the HAP sheet experimented with double layered HAP sheet coated with a tricalcium phosphate layer. The adhesive strength of the HAP/TCP sheet was markedly higher than that of the HAP sheet that indicates sheet may be used for restoration [17].

Initially Chen et al., showed that surfactants were used as reverse micelles or microemulsions to synthesize enamel [18]. Recently scientists from the university of Leeds found a way to mimic enamel matrix within enamel lesions and thus enabling a regeneration. They have developed a patented technology for the regeneration of enamel. The monomers of peptide p11-4 (curodont) forms a matrix enables denovo enamel crystal formation from saliva in constant equilibrium with demineralization [19]. Invivo studies revealed that the peptides were shown to decrease demineralization and show a strong trend toward increasing remineralization [20].

So far investigators demonstrated enamel like mineral structure using chemical solutions or by forming a HAP sheets. New strategies have been emerging based on the finding that amelogenin’s ability to function in critical phases of biomineralization [5]. Marinet proposed a cation selective membrane system to synthesize amelogenin based composite under biomimetic conditions [4]. In this method, the added effect of amelogenin protein on octacalcium crystals growth was tested. Elongated rod like crystals and protein adhering to side of crystals were found [5]. A new technique of electrolytic deposition method has been used to fabricate enamel mimicking composite coating from a solution containing calcium, phosphate ions, and soluble recombinant amelogenin proteins, at near physiological pH and ionic strength [4].

Regeneration: Cell based strategies

Presently investigators are interested in developing cell-based strategies to regenerate enamel. Regenerative treatment requires a stem cells, scaffold and growth factors.

Huang et al., studied the possibility of using synthetic and bioactive nanostructures that are known to self-assemble in physiologic environments into nanofibers network, in order to mimic the extracellular matrix that surrounds the ameloblasts [21]. Nanofibres with RGD epitope sequence as signaling function on their surfaces have been used to facilitate the attachment, proliferation, and differentiation of ameloblast-like cells [21]. Ameloblast-like cells (line LS8) and primary enamel organ epithelial (EOE) cells were cultured within PA hydrogels, and the PA was injected into the enamel organ epithelia of mouse embryonic incisors and transplanted under the kidney capsules in host mice for long-term culture [21]. Bioactive PA induced HAP structures similar to authentic enamel through its effect on cells by increasing their proliferation and their differentiation by delivering key integrin signals.

Further study was done to elucidate the coupling response of integrin receptors to the biomaterial and gene expression profiles. These cues provide an insight into molecular mechanisms involved in enamel formation, which helps in designing synthetic regenerative approaches and to manipulate pathways to control enamel regeneration [22].

Enamel tissue engineering

Developing a technique to manipulate EOE cells is a significant advance towards enamel replacement and therefore attempted to develop a strategy to generate enamel based on subcultured EOE cells using tissue-engineering technology [Table/Fig-2].

Honda et al., examined the enamel-forming capability of subcultured EOE cells, by transplanting cells onto a biodegradable scaffold in vivo [2]. Fresh dental pulp cells from the third molars of pigs during the early stage of crown formation were first plated on top of a scaffold and then subcultured EOE cells were seeded directly on top of the pulp cells. Four weeks after transplantation of EOE cells combined with dental pulp cells in scaffolds, several phenomena related to amelogenesis were distinguished in the implants [2]. In the most mature structures, enamel was readily found in the implants. Furthermore, amelogenin immunoreactivity was detected in tall columnar epithelial cells on the surface of the dentin or enamel, indicating that the tissue-engineered enamel contains well-developed ameloblasts. Together, these results indicate that the subcultured EOE cells have the potential to generate enamel.

Enamel production may have been facilitated in this culture model because EOE cells were maintained at an undifferentiated stage in the ameloblast-lineage cell phenotype by the 3T3 feeder layer. Interestingly, enamel formation was always observed after dentin was formed in the implants. On the other hand, when subcultured EOE cells were combined with subcultured dental pulp cells, enamel-dentin complexes were not observed in any of the implants [2]. This culture model provides a promising step towards a new therapy for reforming enamel.

As EOE cells disappear in adult teeth after tooth eruption. Alternative cell source for enamel forming cells are:

Epithelial cell rest of Malassez

Since ERM is a direct lineage from HERS, derived from the enamel organ through the cervical loop structures, recently reported that the epithelial cells from HERS can differentiate into ameloblasts and produce enamel-dentin complexes when combined with non-cultured dental pulp cells in the core of the dental pulp [2].

Bone marrow cells

In one experimental study bone marrow cells along with single cell suspension of dental epithelium were associated with dental mesenchyme obtained from tooth germ. After 20 days of culture, a tooth crown was generated from the constructs. The success of this study provides a new cell source for enamel tissue engineering [23].

Human embryonic stem cell derived epithelial cells

When human embryonic stem cells (hESCs) compared to human ameloblast-lineage cells (ALCs) found that hESCs as a potential alternative cell source for ameloblast regeneration [24].

Oral keratinocytes

It is possible that non-dental epithelial cells may be a new cell source for enamel tissue engineering technology. Of particular interest is whether post-natal non-dental oral epithelium can differentiate into ameloblasts to generate enamel [2].

Skin epithelial cells

Liu Y et al., studied the conversion potential of postnatal skin epithelial cells to ameloblasts. Study suggested that skin epithelial cells as the appropriate substitute for ameloblasts under effective induction. Advantages and limitations of the alternative cell sources is mentioned in the [Table/Fig-3] [25].

Summary – Future perspective

Some investigators fabricated HAP crystals by precipitation from supersaturated chemical solutions. Because of adhesive problem few others have demonstrated growth of HAP crystals directly over the etched or demineralized surface enamel surface by prolonged immersion into solutions. Further scientists developed hydrogel mats, which are able to generate crystals over tooth surfaces. Others have proved that adhering the flexible HAP sheet might be effective for restoration of eroded tooth surface. A very recent scientists from University of Leeds patented self-assembling peptide solution for initial carious lesion. Following recent advances in biomimetic synthesis of enamel a new strategy based on application of proteins that are known to control crystal initiation, crystal shape, and packing organization were experimented under biomimetic conditions.

So far studies have shown that enamel has been regenerated in the laboratory simulating physiological conditions. Advanced research is required to explore the use of stabilized supersaturated calcium phosphate solutions, where an enzyme can regulate mineralization. Based on our present knowledge of calcium phosphate chemistry and current understanding of how mineral deposition and organization are regulated in developing mineralized tissues, our overall working hypothesis is that the proper development of enamel like structure can be achieved through the regulation of mineral ion diffusion, crystal growth kinetics and crystal orientation. There is still a gap in understanding of detailed mechanism of ameloblast cell products assembling, nucleation and orientation of crystals.

In a cell based strategies enamel can be regenerated using a subculture enamel organ epithelial cells. However, further study is needed on how to combine the newly generated enamel with the original dental dentin or enamel in the tooth, and to control the shape and size of the tissue-engineered enamel. A further research is required to resolve the paucity of dental epithelial cells by the generation of induced pluripotent stem cells from somatic cells or by alternative source cells.

Another potential strategy to regenerate dental enamel involves the application of genes that are known to control the development of enamel-forming cells. Ameloblasts have an epithelial origin and their differentiation is tightly linked to odontoblast differentiation. Scientists have now understood the various signaling molecules controlling epithelial-mesenchymal interactions. Future trend may be the application of genes for enamel formation.

Financial or Other Competing Interests

None.

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