Future Perspectives of Biomimetics in Restorative Dentistry

The main goal of tooth restoration aims at achieving mineralization of initial enamel and dentinal lesions in native form. Most of the restorative materials and remineralization adjuvants for enamel and dentin mineralization are evidenced in the literature. Although commercially available restorative materials exhibit superior esthetics, mechanical properties and cost effectiveness, durability of the restoration threatened by the occurrence of inadequate strength, long-term solubility, and weaker adhesion to tooth and accelerated degradation after being bonded to tooth structure. Recently, the role of biomimetic science in restorative dentistry aims at creating a restoration that can be highly compatible with the structural, functional and biologic properties of dental tissues to reproduce and emulate the original performance of the intact tooth with high durability. In order to recover the prismatic structure in mineral-depleted enamel and to achieve interfibrillar mineralization in dentin, non-collagenous protein analogues have been proposed as templates for apatite deposition. Biomimetic analogues must be necessary to achieve functional mineralization and to recover the dynamic mechanical properties of teeth. The use of these analogues associated with ion-releasing materials seems to be a promising approach for both Review Article Basheer et al.; JPRI, 32(25): 19-28, 2020; Article no.JPRI.61573 20 enamel and dentin remineralization. This review enlightens the current and future perspectives of biomimetic analogues used for enamel and dentin remineralization as the clinical translation of this biomimetic research can be considered as the boon to restorative dentistry.


INTRODUCTION
From the beginning of mankind, humans have been inspired by principles of nature. The act of 'copying' the nature and giving 'life like', which imitates the nature is called as Biomimetics or biomimicry [1]. Biomimetics is the study of formation of structure or function of biologically produced substances and materials and its biological mechanisms for the purpose of synthesizing similar products by artificial mechanisms that mimic natural structures [2].
The role of biomimetic science in restorative dentistry aims at creating a restoration that is highly compatible with the structural, functional and biologic properties of dental tissues to reproduce and emulate the original performance of the intact tooth. The concept of just "drill and fill" is changed to the application of physical and mechanical tests combined with fundamentals of engineering science to design the restorative materials to simulate the native tissue. Thus the field of restorative dentistry has come a long way from just restoration of cavity to the concept of regenerating the enamel or dentin or the whole tooth itself [3].
Many restorative materials are commercially available with superior esthetics, mechanical properties and cost effectiveness. However durability of these restorations can be often compromised by occurrence of inadequate strength, long-term solubility, weaker adhesion and accelerated degradation after being bonded to tooth structure, fracture leading to secondary caries, and pulp necrosis [4]. While there are enormous development and improvement in the material aspect, no material has the ability to restore back the natural structural architecture of enamel and dentin which accounts for regaining the complete dynamic mechanical strength of the tooth [5]. The future of successful restorative dentistry lies in the emergence of material with remineralization or regeneration capacity of the lost tooth structure with resistance to further degradation. Current researches on enamel and dentin remineralization are focussed on the quality of apatite precipitation in the demineralized tissue. Biomimetic restorative materials can play a vital role in guiding the mineral deposition in order to restore the tissues to its native structure, organization and functionality. Thus, the ideal properties of biomimetic restorative material should be as follows:  Maintain as much natural tooth structure as possible.  Mimic or form natural enamel and dentin  Absorb and distribute stress like tooth structure and  Improve durable bond with natural tooth structure.
Biomimetic science is an emerging concept that helps to focus the dental research on simulating the nature and translate this scientific knowledge into future clinical treatments. This review enlightens the current and future perspectives of biomimetic science used for enamel and dentin remineralization.

BIOMIMETIC STRATEGY FOR ENAMEL RESTORATION
The acellular enamel does not regenerate itself unlike other bio-mineralized tissues in the body (dentin and bone) [6]. Traditional restorative materials such as amalgam, GIC or composite resin are used to substitute the lost enamel but none of these materials could achieve the exact physical, mechanical, and aesthetic properties of enamel [7]. The concept of incorporating remineralizing agents in restorative materials to reinforce the tooth has also been tried. However, the lack of ability to guide the formation of hydroxyapatite crystals to have ordered mineral architecture under physiological conditions is still questionable with conventional materials. Therefore, there is a need for an alternative approach that can safely and effectively promote organized mineral deposition onto the underlying tissues.
Dental enamel is a unique mineralized substance which mainly comprises of 96% hydroxyapatite and 4% breakdown products of organic component of non-collagenous proteins(NCPs). Approximately 90% of these NCPs are amelogenins [8]. These NCPs acts as a key factor in controlling the oriented elongation of enamel rod growth during the mineralization process and responsible for guiding the deposition of hydroxyapatite in its characteristic prismatic structure. These are negatively charged structure that interacts with calcium ions to control the orientation of the growing crystals [9,10]. Remineralization and regeneration of enamel can be possible to a certain extent, by the use of biomimetic template analogues of the natural proteins involved in bio-mineralization [11].
Although the calcium and phosphate ions in the saliva permit the recovery of mineral lost, amorphous calcium phosphate (ACP) has been proposed to be an essential precursor phase during the formation of mineralized tissue [12]. ACP is an unstable component than its crystalline polymorphs of calcium phosphate, which can readily dissolve and converts to HAP in aqueous solution. The role of NCPs which are secreted by ameloblast during the secretory stage of amelogenesis, particularly amelogenin, [13] has been believed to play an important role in the stabilization of ACPs and to control the crystal growth, including its size, shape, and organization during enamel biomineralization. In addition, the guidance for hierarchical structural assembly of HAP can be achieved with the help of protein peptide [14]. The peptide may act as Ca 2+ carrier as well as regulating factor. The stabilized calcium and phosphorus ions, binds with the peptide and become biologically available for the remineralization. It has been regulated by the NCPs to transform into ordered hydroxyapatite crystals.Thus the development of natural protein analogues which could substitute amelogenin and imitate the process of biomineralization would be an effective strategy to restore enamel in its original form. Thus the main requirements of functional remineralization of enamel are: 1. ACP precursor/ source 2. Amelogenin analogue 3. Protein peptide. The combination of non-classical crystallization mechanisms and development of synthetic amelogenin analogues, thereby emulating remaining biomineralization steps can serve for future trends to improve the repair of initial enamel defects. There are many biomimetic systems developed recently with various protein analogues which serve the function as follows:

Amelogenin [15]
It promotes the oriented bundle formation and also mediate the formation of orderly enamel-like structure. It has the control over the organized growth of apatite crystals and form transitional complexes with ACP. It promotes the hydrolysis of ACP to stable HAP by stabilizing the Ca-P clusters and guide their arrangement into linear chains.

Enamel Matrix Protein [16]
It guides the continuous growth of crystals and prevents the fusion of premature crystals thereby controlling nucleation, growth of the crystals, morphology and subsequent elongation.

Polyamidoamine Dendrimers (PAMAM) PAMAM-COOH, ALN-PAMAM-COOH, PAMAM-PO 3 H 2 [17,18]
It absorbs onto the etched enamel surface and provide nucleation sites and mineralization template for HAP. The phosphate-terminated dendrimer has been shown to be capable of forming HA layer of 11.23μm thickness on the acid-etched tooth enamel with crystals oriented along Z-axis, when treated with in artificial saliva for 3weeks. Alendronate-conjugated dendrimer regulates the growth of crystals and induces nanorod-like HAP with high uniformity. It also showed a recovery of 95.5% hardness of the enamel which makes a promising biomimetic material as well as potential remineralizing agent for etched enamel.

3DSS [19,20]
It forms 3D biomimetic scaffolds capable of nucleating HAP and has a high affinity to bind with the HAP surface. It promotes HAP with a small average crystalline size and induces formation of stable ACP nano-precursors preventing them from aggregation and precipitation.

Glutamic Acid [21]
It induces the nano apatite assembly on enamel and induces the oriented aggregation of nanoapatite and provides the active sites to control enamel formation. It has strong calcium complex forming potential in high concentration. The orientation of crystals mimics the natural enamel rod direction with the packs of crystals tightly arranged in a "fish-scale" manner proving its biomimetic ability to form enamel.

Polyacrylic Acid [22]
It sequester calcium and phosphate ions and forms amorphous calcium phosphate nanoprecursors. It was shown to improve the microhardness of enamel when treated onto artificial lesion,with enhanced mineralization, mechanical properties and higher phosphate content in combination with bio active glass.

Phosphorylated Chitosan (Pchi-ACP) [23]
It binds calcium ions, initiates nucleation and stabilizes ACP to form the nano-complexes of Pchi-ACP. It get adsorbs onto the surfaces of HAP crystals and shows similar remineralization of enamel lesions to that of fluoride, with higher remineralizing rate. It is a natural analogue which can be used as potential remineralizing agent for incipient lesion of enamel.

Casein Phosphopeptide [24]
It stabilizes calcium, phosphate and hydroxide ions and prevents spontaneous precipitation of calcium phosphate and it has proven to promote enamel subsurface lesion remineralization. It can also maintain the gradient of ions in high concentration on subsurface lesion along with high rate of remineralization.

RECENT STUDIES ON ENAMEL BIOMIMETIC REMINERALIZATION SYSTEMS
In recent years, treatment of caries lesions by the application of different combinations of biomimetic systems has been ACP based systems though repair and prevention of initial enamel lesions have not yet been confirmed in clinical trial. and phosphate-terminated dendrimer (PAMAM-PO 3 H 2 ) showed the ability to remineralize acid-etched human tooth enamel. It has strong tendency to selfassemble into hierarchical structures such as the preferential orientation of the c-axis of the HAP crystals along the amelogenin aggregates.
Alendronate-conjugated PAMAM dendrimer (ALN-PAMAM-COOH) also induces in situ remineralization of tooth enamel due to the combined effect of the HA-anchored property of the ALN moiety and the remineralization capability of the -COOH moiety [17,18].  Amelogenin-releasing agar hydrogel: Repetitive application of amelogeninreleasing agar hydrogel containing calcium, phosphate, and fluoride was evaluated to remineralize etched enamel in a cyclic treatment model and multispecies oral biofilm model. It significantly improved enamel hardness continuously over time and offered promising possibility of remodeling complex enamel minerals in an amelogenin-containing system [30].  CS-AMEL hydrogel: Development of a new amelogenin-containing chitosan (CS-AMEL) hydrogel significantly improved in situ regrowth of apatite crystals, generated a robust enamel-restoration interface, which is important for ensuring the efficacy and durability of restorations. Amelogenin assemblies carried in chitosan hydrogel could stabilize Ca-P clusters and arrange them into linear chains, which fuse with enamel crystals and then develop into enamel like co-aligned crystals with potential antimicrobial activity of chitosan [31].  Leucine-rich amelogenin peptide (LRAP): LRAP is a 59-residue splice variant of amelogenin and contains the N-and Cterminal charged regions of the full-length amelogenin. LRAP, like full-length amelogenin, also has a great potential for biomimetic regrowth of tooth enamel. LRAP selectively promotes linear growth along the c-axis of enamel crystals. It could stabilize ACP and guide ACP transformation into ordered bundles of apatite crystals [32].  Chitosan based systems : -Synthetic hybrid chitosan (CS)hydroxyapatite (HAP) -Chitosan causes major morphological modifications, subtle changes in HAP. CS macromolecules act as matrix for orderly embedding the structured amelogenin-HAP nanocrystals by selectively binding Ca 2+ from HAP faces via coordination interactions and amelogenin molecules adsorbed onto hydrophobic contacts [33].
-HAP@ACP Nanoparticles Guided by Glycine: CMC-ALN matrix stabilizes amorphous calcium phosphate (ACP) to form CMC/ACP nanoparticles. Sodium hypochlorite decompose amelogenin in vivo to degrade and generate HAP@ACP coreshell nanoparticles. HAP@ACP, which on the introduction of Gly lead to subsequent formation of oriented mineral crystal bundles orderly which transform from an amorphous phase to well-ordered rod-like apatite crystals [34]. Chimaeric peptide-mediated nanocomplexes of carboxymethyl chitosan/amorphous calcium phosphate (CMC/ACP) guided by amelogenin also can transform into enamel-like crystals which tightly bounds onto the demineralized surface. The newly formed enamel-like crystals were nearly well-organized with strong mechanical properties [35].  Other enamel biomimetic systems Other biomimetic systems such as glycerine-enriched gelatin system agarose hydrogel system (30), CS-EMD hydrogel [31] multifunctional biomimetic CS-HAP has been used to form layers containing ordered enamel-like structures of fluoride-substitute hydroxyapatite microcrystals on a human enamel surface using ethylene diamine tetra acetic acid disodium salt dehydrate (EDTA) as the mediating agent. Under near-physiological materials, it could provide highly organized enamel-like structure for teeth remineralization conditions [36].

BIOMIMETIC STRATEGY FOR DENTIN RESTORATION
Dentin is a more complex structure than enamel. The matrix composition consists of mineralized tissue with proteinaceous content by almost 50% in volume and comprised of collagen fibrils. The mineral phase in dentin can be described as intrafibrillar mineral which confines within or immediately adjacent to the gap zones of the collagen fibrils and interfibrillar mineral which has been located within the interstitial spaces separating the fibrils [37].
The mechanical properties of dentin are largely driven from its intrafibrillar mineralization. Apatite mineral in dentin needs to be bound and/or structurally incorporated in between the collagen fibrils to mechanically reinforce the dentin in terms of elastic modulus, hardness, and compressive strength [38]. Dentin remineralization is less effective than enamel remineralization on using conventional fluoride therapies and other enamel remineralizing agents [39]. Remineralization of partiallydemineralized dentin with conventional dentin remineralization strategy such as calcium hydroxide, Mineral Trioxide Aggregate and other calcium silicate based materials cannot provide complete remineralization within hybrid layers or the superficial part of a caries-affected dentin lesion due to the fewer amounts of residual mineral crystals. Also remineralization cannot occur in locations where seed crystallites are absent [40]. Thus, there can be no intrafibrillar apatite deposition expected from conventional top-down mineralization approach.

NON-CLASSICAL CRYSTALLIZATION PATHWAY
This is a multistage process (Bottom Up approach) [40]. Characterized by the selfassembly of amorphous nano-precursor particles and their subsequent mesoscopic transformation in biomineralization. Initially, the calcium and phosphate ions self-assemble into pre-nucleation clusters in the presence of calcium and phosphate source. Following this, these prenucleation clusters aggregate into amorphous ACP nano-precursors by the influence of NCP and its peptide component. These nanoprecursors have the ability to penetrate into the gap zones of collagen fibrils and further crystalize into apatite along the intrafibrillar space of collagen. The intrafibrillar remineralization of collagen fibrils results in interfibrillar remineralization between adjacent collagen fibrils. Apatite mineral in dentin needs to be bound to the collagenous matrix or ideally be incorporated into the collagen fibrils to mechanically reinforce the tissue. Thus, processdirecting agents along coupled with NCP analogue on remineralization agents can only induce intrafibrillar mineral deposition in collagen [10].

NCP Analogues
As collagen matrix serves as a scaffold for crystal deposition in dentin with lack of nucleation for hydroxyapatite deposition, the biomimetic remineralization process can be modulated by a series of NCPs. Thus, current research focuses on developing the NCP analogues which can play a vital role in biomimetic remineralization of dentin. The role of NCPs analogues are as follows:  They have high affinity for calcium ions and collagen fibrils.  They can act as modulator of elegant hierarchical structure of dentin.  They bind to collagen matrix, and induce ACP nano-precursors into collagen matrix.  They stabilize ACP precursors.
 They mainly regulates biomineralization of dentin by acting as a nucleator or inhibitor.
Some of the commonly tried NCP analogue systems are mentioned as follows:

Polyacrylic
Acid and Polyvinylphosphonic Acid (PVPA) [41,42] PAA simulates CaPO 4 binding sites of DMP1 and stabilizes ACP. It also helps in inhibiting nucleation for ACP stabilization and prolongs the lifetime of ACP. PVPA also can simulates the collagen-binding function of DMP1and inhibits the activity of MMPs. It also helps in recruiting ACP nano-precursors into collagen matrix. PAA and PVPA together, the nanoprecursors binds to the acid-demineralised collagen matrix and forms poly electrolyte-stabilised apatite nanocrystals initiating intrafibrillar and interfibrilllar remineralisation in collgen. The remineralization occurs through bottom up approach from mesocrytals to nanocrystal formation, thereby proving them to be a promising biomimetic component for guided remineralization in dentin.

Sodium Trimetaphosphate (STMP) [43]
STMP has phosphate groups which get adsorbed on demineralized collagen under an alkaline pH. It phosphorylates type I collagen and binds to demineralized collagen matrix forming covalent bonds. Studies have shown that STMP can remineralize resin-bonded dentin with intrafibrillar deposition of nanoapatite around phosphorylated collagen. It also attracts ACPnanoprecursors by electrostatic force to initiate nuclei of crystallization within the gap zones of collagen fibrils.

Phosphorylated Chitosan (P-Chi), Carboxymethyl Chitosan/Amorphous Calcium Phosphate (CMC/ACP) [44]
It binds to collagen by introducing functional groups onto the collagen using P-chi-collagen matrix complex and induces homogenous nucleation. CMC stabilizes dentine matrix protein 1 (DMP1) in ACP to form nanocomplexes of CMC/ACP, which can be made into scaffolds. The nanoclusters formed can infiltrate into gap zones of collagen leading to intrafibrillar mineralization. It has been proved to follow biomimetic strategy, thus aiding in biomineralization of dentin.

Polydopamine [45]
It is a synthetic mimic of marine adhesive protein consisting of 3, 4-dihydroxy-L-phenylalanine which helps in providing new nucleation site for HAp and binds to collagen fiber. This can adhere to almost all substrates and has good stability in many environments. It has surfaceanchored catecholamine moieties which binds to calcium ions, enabling the formation of HA crystals along c-axes. It also can cause tubular occlusion due to densely packed HA crystals. Hence, it can be used as dentin remineralizing agent as well as therapeutic agent for dentin hypersensitivity.

Polyamidoamine Dendrimer (PAMAM) [46,47]
It binds to collagen fibrils by recruiting ACP nanoprecursors into collagen matrix. It guides mesocrystals to assemble into large ones and also induces the periodicity of the mineralized fibrils. It promotes biomimetic intrafibrillar mineralization of dentin by following structural hierarchy. It can cause sequestration of the mineral ions and can act as template for nucleation, proving it to be a potent remineralizing material.

CONCLUSION
In future, restorative dentistry would no longer be using inert materials that simply fill the space left by cavity preparation, but instead, should stimulate dental tissues regeneration and/or by themselves self-mending. Evidence for the most biomimetic approaches for enamel and dentin remineralization is still in their laboratory stage. Clinical translation of this biomimetic research can be considered as the boon to restorative dentistry. New mechanisms for adhesion, integration, and sealing of dentin using biomimetic technologies can emerge as a major turnover in restorative field.

CONSENT
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ETHICAL APPROVAL
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