Demineralized Dentin Matrix for Dental and Alveolar Bone Tissues Regeneration: An Innovative Scope Review

Dentin is a permeable tubular composite and complex structure, and in weight, it is composed of 20% organic matrix, 10% water, and 70% hydroxyapatite crystalline matrix. Demineralization of dentin with gradient concentrations of ethylene diamine tetraacetic acid, 0.6 N hydrochloric acid, or 2% nitric acid removes a major part of the crystalline apatite and maintains a majority of collagen type I and non-collagenous proteins, which creates an osteoinductive scaffold containing numerous matrix elements and growth factors. Therefore, demineralized dentin should be considered as an excellent naturally-derived bioactive material to enhance dental and alveolar bone tissues regeneration. The PubMed and Midline databases were searched in October 2021 for the relevant articles on treated dentin matrix (TDM)/demineralized dentin matrix (DDM) and their potential roles in tissue regeneration. Several studies with different study designs evaluating the effect of TDM/DDM on dental and bone tissues regeneration were found. TDM/DDM was obtained from human or animal sources and processed in different forms (particles, liquid extract, hydrogel, and paste) and different shapes (sheets, slices, disc-shaped, root-shaped, and barrier membranes), with variable sizes measured in micrometers or millimeters, demineralized with different protocols regarding the concentration of demineralizing agents and exposure time, and then sterilized and preserved with different techniques. In the act of biomimetic acellular material, TDM/DDM was used for the regeneration of the dentin-pulp complex through direct pulp capping technique, and it was found to possess the ability to activate the odontogenic differentiation of stem cells resident in the pulp tissues and induce reparative dentin formation. TDM/DDM was also considered for alveolar ridge and maxillary sinus floor augmentations, socket preservation, furcation perforation repair, guided bone, and bioroot regenerations as well as bone and cartilage healing. To our knowledge, there are no standard procedures to adopt a specific form for a specific purpose; therefore, future studies are required to come up with a well-characterized TDM/DDM for each specific application. Likely as decellularized dermal matrix and prospectively, if the TDM/DDM is supplied in proper consistency, forms, and in different sizes with good biological properties, it can be used efficiently instead of some widely-used regenerative biomaterials.

as decellularized dermal matrix and prospectively, if the TDM/DDM is supplied in proper consistency, forms, and in different sizes with good biological properties, it can be used efficiently instead of some widely-used regenerative biomaterials.

Introduction
Dentin is chemically composed of approximately 70% mineral phase (40%-45% in vol), 20% organic matrix (30% in vol), and 10% water (20-25% in vol). Additionally, the organic component of dentin consists of 18% collagen and 2% noncollagenous proteins (NCP), proteoglycans, growth factors, phospholipids, and enzymes (Fig. 1A). The matrix is a repository for growth factors, such as basic fibroblast growth factor, insulin-like growth factor, transforming growth factor-b, and bone morphogenetic proteins (BMP). Several NCPs, such as osteopontin and osteocalcin, are common in dentin and bone; however, dentin phosphoprotein is an NCP found specifically in dentin [1]. The inorganic components of dentin are calcium and phosphate ions that form hydroxyapatite crystals that are larger compared with those found in bone and much smaller than those in enamel [2].
Regarding the histological structure of dentin, it is considered as a specialized mineralized avascular connective tissue that forms the main bulk of the tooth. It is covered by enamel on the crown and cementum on the root and surrounds the entire pulp tissue (Fig. 1B). Beneath the enamel, dentin has an outer mantle layer of 15-30 lm thickness whereas underneath the cementum, Tomes granular and/or the hyaline Hopewell-Smith layers are identified, and each of them represents approximately 15-30 lm thickness. The circumpulpal dentin forms the main bulk of the dentin, and its thickness continuously increases by about 4 lm/day at the expense of dental pulp space. Circumpulpal dentin includes the intertubular dentin and peritubular (intratubular) one. Compared with intertubular dentin, peritubular dentin has a relatively higher proportion of sulfated proteoglycans and minerals with lesser collagen fibrils; therefore, it is considered harder than intertubular dentin. Intertubular dentin results from the transformation of predentin into dentin and it is a composite consisting of collagen fibrils discontinuously reinforced with nanoplates of carbonated hydroxyapatite [3].
Dentin is highly permeable as it contains numerous dentinal tubules running from the pulp tissue to the dentino-enamel junction (DEJ) in the crown and till the dentino-cemental junction (DCJ) in the root. Dentin exhibits regional differences in tubule density and diameter wherein tubule diameter can vary from 0.9 lm peripherally to 2.5 lm at the pulp side. In the meantime, density is approximately 59,000-76,000 tubules/mm 2 at the pulp side whereas the number of these tubules decreases to half of such quantity at the area close to the DEJ. Dentinal tubules have collateral branches measuring 1 lm in diameter that form a three-dimensional network as they extended at specific angles crisscrossing intertubular dentin [4].
The dentinal tubule contains an odontoblast cell process, which is an extension of an odontoblast cell, and serumlike fluid which contains a mixture of proteoglycans, tenascin, transferrin, and albumin. Interestingly, odontoblast processes were seen only in the tubules near the pulp. Further, odontoblasts differentiate from ectomesenchymal cells of the dental papilla and are organized at the periphery of the pulp as a cellular palisade. They form the dentin matrix (predentin) by synthesizing collagen types I, III, and V; noncollagenous proteins as integrin-binding sialoprotein, matrix extracellular phosphoglycoprotein, Fig. 1 Representative diagrams created with BioRender.com for the chemical composition of A dentin, B component parts of the tooth and C histological structure of dentin osteopontin, dentin matrix protein 1, and sialophosphoprotein; glycoproteins as dermatan sulfate, keratan sulfate, heparan sulfate, and chondroitin sulfate [5]. They are also responsible for the deposition of minerals in the dentin matrix, which is not simply restricted in the mineralization front at the edge of predentin and dentin but occurs along the whole length of the odontoblast process [6].
During tooth development and organogenesis, odontoblasts form the primary dentin which comprises the main bulk of the circumpulpal dentin. After root completion and throughout the life of the tooth, odontoblasts form secondary dentin bordering the pulp at a slow rate (Fig. 1C). This type of dentin contains fewer dentinal tubules than primary dentin and there is usually a bend in the tubules at the interface between primary and secondary dentin. In response to environmental conditions and according to the severity of the stimuli, tertiary dentin (reactionary/reparative) is formed as odontoblasts forming the reactionary dentin while the dental pulp stem cells form the reparative one. Sometimes, the word synonyms of tertiary, reactionary, and reparative terms are used interchangeably [7].
At the micron length scale, crown dentin is similar to root dentin; however, unlike root dentin, the proportion of the tubular area is higher and tubules follow a gentle S-shaped curve in the crown part while they are straight in the root area. The dentinal tubules are surrounded by a 2-6 lm dense cuff of peritubular dentin, which suggests that the dentin mineral density is higher in the crown than in the root and the predentin is significantly wider in the crown than in the root [8].
Considering the physical and biological properties of dentin, the thickness is approximately 3-10 mm or even more, and this thickness differs in the regional parts of the same tooth, among different teeth and as a result of aging. The color of dentin greatly affects the color of the tooth due to the translucency of enamel as dentin is a yellowishhued material that becomes darker with age. In radiographic images, dentin is more radiolucent than enamel due to its lower mineral content whereas it is more radiopaque than cementum and bone. Dentin has no replacement mechanism for biologic turnover but it can be remodeled to a certain degree as odontoblasts and pulpal resident stem cells can produce secondary and tertiary dentin when it is damaged by excessive tooth wear, carious lesion, trauma, or through iatrogenic insult, such as accidental exposure. Dentin is a bone-like matrix that is a vital, sensitive and porous tissue, capable of responding to mechanical, thermal, chemical, evaporative, and osmotic environmental stimuli. Dentin can be regarded as both a barrier and permeable structure, depending upon its thickness, age, and other variables. The permeability to fluid flow through the dentinal tubules, as well as a directional design of these tubules, suggests that dentin has a sensory hydrodynamic function. Micropore-sized dentinal tubules that measure 0.9-2.5 lm diameter provide micropore spaces of 3.70%-5.88% porosity that increases the surface contact area of dentin [3].
Regarding the mechanical properties of dentin, the elastic modulus and hardness gradually increase from the pulp side toward DEJ and DCJ. The poorly mineralized intertubular dentin has a lower Young's modulus than the highly mineralized peritubular dentin. A hydrated environment affects the mechanical behavior of dentin, as the elastic modulus decreases by 35% and hardness decreases by 30% [9]. Young dentin has higher initial toughness and stable toughness values than aged dentin. The fatigue crack growth exponent is associated with the direction of the dentinal tubules [10]. Unlike enamel, dentin is less brittle and somewhat has viscoelastic properties. This elasticity is important to provide the flexibility that is required to support the overlying enamel and prevent its fracture. The tensile strength of dentin is attributed to the fibrous arrays of collagen type I, while high compressive strength and rigidity are provided by the crystals of the mineralized phase deposited within the collagen fibers [7].
To obtain tooth-derived substances, demineralization is required before clinical adaptation to open the dentinal tubules and release BMPs. Demineralization is the process of removing some of the highly crystalline inorganic substances from the dental hard tissues, which results in the loss of its structural integrity with the collapse and degradation of the supporting collagen matrix. Enamel and dentin contain calcium-deficient carbonate-rich hydroxyapatite crystallites with enamel having much larger crystals compared with dentin. The crystal sizes comprise 85 vol% of enamel structure compared to about 50 vol% in dentin. The larger surface area of the dentin apatite increases its dissolution susceptibility when exposed to acids and this susceptibility is further increased because of the higher carbonate content of the dentin mineral. Thus, the dentin minerals are dissolved more rapidly than the enamel ones, and because there is less total mineral in dentin than in enamel, the acid attack proceeds more quickly in dentin [11].
By preferentially removing peritubular dentin, acidetching agents used during dental restorative procedures and ethylenediamine tetraacetic acid (EDTA) used in endodontic treatments enlarge the openings of the dentinal tubules, making the dentin more permeable. Specific acids and chelating agents as 17% EDTA, sodium hypochlorite, and citric acid were used during root canal instrumentation to remove the smear layer and clean dentinal walls [11]. Moreover, bacterial acids (lactic acid), dietary acids (acetic acid, phosphoric acid, and citric acid), and gastric acid {hydrochloric acid (HCl)}, all demineralized the dentin through the processes of caries and acid-erosion. It has Tissue Eng Regen Med (2022) 19(4):687-701 689 become standard to use laboratory acids, such as formic acid [12, acetic and lactic acids [13], or chelating agents such as EDTA [14], to produce demineralized dentin models for use in in vitro remineralization studies. It is well known that each demineralizing agent has a unique effect as chelating agents, strong acids, and weak acids affecting both mineral and organic phases of dentin in significantly different ways. For example, the demineralizing agents caused some degree of collagen denaturation, citric acid caused the most damage and varying the concentrations of EDTA and citric acid affected collagen in a dissimilar manner [15].
In the past decade, teeth as graft material have been proposed with fascinating outcomes. Therefore, this study aimed to review evidence on treated dentin matrix (TDM)/ demineralized dentin matrix (DDM) for dental and bone tissues regeneration and summarize the in vitro and in vivo animal and human studies using TDM/DDM as an osteoinductive material for clinical applications.

Study design
We conducted a scoping review and searching for articles on TDM/DDM for tissue regeneration. For our scoping review, a five-stage framework was adopted following Arskey and O'Malley's design [16]. The five stages were: specifying research questions; identifying relevant studies; studies selection; extraction, mapping and charting the data; collating, summarizing, synthesizing and reporting results.

Stage I: identification of research questions
We aimed to answer the following questions; (1) is there is a difference between TDM and DDM, (2)   Abstract]) AND (regeneration [Title/Abstract])} and the following one for Medline (OVID) databases)treated dentin matrix and regeneration).af (all fields) and (demineralized dentin matrix and regeneration).af. Figure 2 demonstrates the studies' search procedure using the PRISMA flowchart.

Stage III: selection of studies
The relevant studies were selected according to the inclusion and exclusion criteria. The searches were not restricted by language type, however, were limited to original researches including in vitro and in vivo animal and human studies and excluding narrative reviews, systematic reviews, and meta-analyses.

Stage IV: extraction, mapping, and charting the data
A template was established and reviewed by each author. The authors were calibrated to extract the following data; authors, publication year, country of origin, study design, source of TDM/DDM (human or animal), tooth part (crown or root), matrix form, matrix size, demineralization protocol, ways of sterilization, and preservation and the outcome.

Stage V: collating, summarizing, synthesizing, and reporting results
Meta-synthesis and integration of findings from qualitative studies were performed to provide a new and more comprehensive interpretation of the findings.

Statistical analysis
Degree of chance-adjusted agreement (kappa coefficient value) was used to determine the inter-reviewer reliability.

Studies' selection and distribution of relevant articles according to date of publication, country origin and study designs
The initial search identified 113 unique references. No additional studies were recognized through hand searching. After filtering, 82 references were recorded and screened. After the eligibility criteria were applied and duplicates were removed, 68 in vitro, in vivo animal and human studies were obtained and were included in the present review (Fig. 2). The kappa value for inter-reviewer agreement was 0. 85 used interchangeably despite that the matrices were fabricated with the same protocols using gradient concentrations of EDTA, 0.6 N-HCl or 2% HNO 3 . In our opinion, it is devisable to use the term DDM for the dentin matrices prepared for regenerative purposes that obtained from different sources, fabricated in different forms and shapes, and demineralized with different protocols as the term TDD could mean physical treatment using laser therapy or mechanical one by means of abrasives.

Teeth and tooth parts involved in study designs
The majority of the published studies relied on harvesting vital and nonvital human permanent teeth. The human teeth were maxillary and mandibular premolars extracted for orthodontic reasons, nonfunctional third molars requiring removal for clinical reasons and exfoliated deciduous teeth. In addition, teeth obtained from other animal species were involved in a few study designs such as porcine deciduous incisors, rabbit permanent incisors, rat permanent molars, dog permanent premolars and rat permanent incisors, that were frequently selected whereas goat incisors, bovine posterior teeth, ovine lower anterior teeth and pig unerupted developing teeth were selected to less comparatively (Fig. 3).
Primarily and with the use of proper dental instruments, enamel, cementum, and periodontal tissues of the extracted teeth were completely removed from the outer tooth surface, and pulp tissue with predentin was completely removed from the internal one. Root dentin is the tooth part that is commonly used whereas crown dentin was used to a lesser degree (Fig. 4).

Methods of demineralization, sterilization and preservation
To our knowledge, no standard protocol for demineralization was established for the different forms and shapes of dentin matrices used for tissue regeneration. Therefore, the protocol requires optimization of the concentration and pH of the demineralizing agent, and the exposure time needs to be properly adjusted for each form and shape. Before demineralization, it is necessary to prepare dentin matrix to remove the debris resulting from mechanical instrumentation.
Regarding dentin-pulp complex regeneration, Holiel et al. [21] evaluated clinically the regenerative potential of DDM hydrogel as a direct pulp capping material in comparison with Biodentine and mineral trioxide aggregate (MTA). The study was performed on thirty intact fully erupted premolars scheduled to be extracted for orthodontic reasons and they found that hydrogel could achieve dentin regeneration and conserve pulp vitality and might serve as a reasonable natural substitute for Biodentine and MTA in restoring in vivo dentin defects. In addition, Mehrvarzfar et al. [75] compared in a clinical trial of thrity-three intact third molars of eleven healthy volunteers the pulpal responses to MTA and combination therapy of MTA and DDM as a pulp-dressing agent s for partial pulpotomy. They found that the dentin bridge was significantly thicker in MTA/DDM group than MTA group alone. Considering alveolar bone regeneration, Um et al. [52] evaluated in a case series study the efficacy of DDM loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) on ten experimental sites for socket preservation. They suggested that DDM may be a potential carrier for rhBMP-2 and it may be conceivable to reduce the concentration of rhBMP-2 to 0.2 mg/ml. In addition, Li et al. [53] conducted a clinical prospective study on forty patients and concluded that autogenous granules of DDM prepared at the chair side after extractions could act as an outstanding readily available alternative to bone graft material in guided bone regeneration, even for implantation of severe periodontitis cases. Moreover, Minamizato et al. [55] evaluated in a pilot study of sixteen patients underwent dental implant placement the clinical application of autogenous partially DDM prepared immediately after extraction for alveolar bone regeneration in implant dentistry and they considered partially DDM as an efficient, safe, and reasonable bone substitute. Furthermore, Pang et al. [57] conducted a prospective randomized clinical trial of a total 33thrity-three graft sites in twenty-four patients and they suggested that autogenous DDM is a viable option for alveolar bone augmentation following dental extraction, in comparison with anorganic bovine bone. Likewise, Elfana et al. [73] conducted a randomized controlled clinical trial to evaluate autogenous whole-tooth versus DDM grafts for alveolar ridge preservation and they concluded that the two grafts have similar clinical effects but histologically autogenous DDM grafts seems to demonstrate better graft remodeling, integration, and osteoinductive properties. Recently, Ouyyamwongs et al. [76] assessed clinically in a split-mouth randomized controlled clinical trial the potential of using autologous DDM in combination with platelet-rich fibrin (PRF) membrane or PRF membrane alone to preserve the ridge dimension. They concluded that the combination therapy reduced the horizontal ridge collapse, and promoted bone healing as shown clinically and radiographically.

Discussion
Regenerative dentistry has widely been recognized as a promising field in the provision of functional and biocompatible dental tissues as alternatives for conventional materials. Current progress in tissue engineering has offered new methods and technologies for dentin-pulp complex and bone regeneration. As there are several reasons for dental extraction, including caries, mobility, orthodontic tooth reasons, and trauma, DDM can be prepared with low risks of infection and rejection with noninvasive attainability; thus, it should be considered as a natural resource to be used to full advantage for other applications. DDM is autogenous tooth dentin that has osteoconductive and osteoinductive potential since dentin contains extracellular COL-1 and various growth factors. Based on the demineralization process, the factors remain available to the host environment; however, extracting proper concentrations of collagen and bioactive molecules from the extracted teeth is a challenging task and requires meticulous preparation of the tooth dentin. DDM is used in dental surgery in the treatment of extraction socket preservation and guided bone regenerations. It functions in a dual capacity: First as a scaffold to support bone regeneration and second as a carrier for bone morphogenic protein (BMP-2). When DDM serves as a carrier, it combines the properties of the grafting material with those of the delivered substances [38].
The type of demineralizing agents and time frames used to prepare DDM are dissimilar in different studies, and therefore they affect the amount of the mineral percentage remaining in the DDM. The DDM components have different inorganic/organic ratios compared to those originally found in the dentin matrix. Approximately, the powder form has mineral content of about 5%-10%, whereas block form has a mineral content of about 10%-30% [1]. ELISA performed on dentin particles showed a slightly higher amount of COL-1 in demineralized samples, as compared with untreated ones, although no significant difference was found and such a finding provides compelling evidence that the demineralization process didn't damage the extracellular matrix of dentin [85]. COL-1 was identified by electrophoresis approximately at 110-120 kDa [86]. Minor bands at 76-102 kDa were detected by electrophoresis, corresponding to dentin matrix protein-1, osteocalcin, osteopontin, proteoglycan, glycoprotein, sialoprotein, and phosphophoryn [87]. In vivo, osteonectin was found in the dentinal tubules of DDM [88]. Besides, the demineralization process is required for freeing the various growth factors and proteins which are essential for tissue regeneration and repair. Consequently, demineralization process is believed to induce release an abundant amount of transforming growth factor-b; an intermediate abundance of BMP-2, fibroblast growth factor-2, vascular endothelial growth factor, platelet-derived growth factor and insulinlike growth factor-1 with a lower abundance from BMP-4 and BMP-7 [89].
The degree of demineralization is critical for optimal dentin regeneration; the partially demineralized dentin matrix (PDDM) is thought to have optimal conditions for dentin regeneration. Partial demineralization results in the elimination of the major part of the mineral phase and immunogenic components while retaining a very low fraction of minerals (5-10 wt%), providing an osteoconductive and osteoinductive scaffold containing several growth factors [62]. On the other hand, the complete DDM (CDDM) showed bone resorption in the early stage of bone regeneration, probably because of the enzymatic digestion of exposed collagen [90]. PDDM probably promotes more osteogenic effects than CDDM does since several noncollagenous proteins were released from the dentin matrix during the process of demineralization. This may account for the more prominent bone formation in PDDM than CDDM in most previous studies.
In terms of particle size, the dentinogenic properties of DDM were greatly affected by the size and shape of dentin matrix particles. The larger-sized particles of the DDM, whose sizes ranged between 350 lm and 800 lm, were found to have better bone regeneration results than the smaller-sized particles that had more resorbability in the defect site before the initiation of new bone formation [89]. PDDM with larger particle sizes induced prominent bone regeneration, probably because PDDM possessed a suitable surface for cell attachment. There might be an exquisite balance between its resorption and bone formation on it. PDDM could be considered as a potential bone substitute.
Dentin and bone are mineralized tissues known to be an organic-inorganic hybrid. They are almost similar.
n their biochemical components but dentin is acellular matrix, while bone includes osteocytes. Autogenous bone is an ideal bone graft material as it has osteoconductive, osteoinductive and osteogenic capabilities but the major drawbacks of autogenous bone are that it requires a secondary donor site with an increase in the susceptibility risk of infection, therefore clinicians prefer commercially available non-autogenous graft materials. Allogenic and xenogeneic bone grafts have osteoinductive ability, but risk of viral infection still remains a considerable problem. Alloplastic bone grafts are clinically used, but they have disadvantages such as limited osteoinductivity and high cost [91]. Among these, demineralized freeze-dried bone allografts have been widely used for bone augmentation [92]. Demineralized bone matrix (DBM) likely as DDM is predominantly composed of COL-1 (95%) with the remainder comprising of NCPs with a small amount of growth factors. Consequently, DDM and DBM can be defined as acid-insoluble collagen bind with BMPs, which are members of the TGF-b super-family that enhances bone formation [93]. The quality and effectiveness of commercial DBM varies with processing techniques and several donor dependent factors likely as gender and age as they have an effect on osteoinductivity of DBM [94]. Therefore, differences in preparation and processing methods for bone can impact properties and clinical performance of DBM. In our opinion, the superiority of autogenous DDM over other graft materials should be confined to the dental applications facilitating the release of BMPs to induce the differentiation of undifferentiated mesenchymal cells into osteogenic and odontogenic cells, which have the potential to stimulate bone and dentin formation [95]. DDM is biocompatible, does not induce foreign body reactions and can be prepared by a standard treatment with very low cost.
It is beneficial to utilize extracted teeth as bone substitutes in implant dentistry, even though there are limited cases with available teeth, in addition to a limited volume. However, there is no risk of transmitting diseases as it is autogenous tissue and no additional surgery is needed to harvest tissues since unwanted teeth are utilized and this raises the need for tooth banking. The Korea Tooth Bank, which was established in Seoul in 2009, is one such toothbanking facility that can procure and store teeth, and then process them into bone graft substitutes. The Hospital Tooth Bank at Seoul National University Bundang Hospital, which was established in 2010 for performing storage and grafting of auto-tooth bone grafts based on experimental and clinical research.

Concluding remarks and future perspectives
Before clinical application and for each specific purpose, optimizing protocols of demineralization, characterization, developing a proper consistency, and applying a proper handling technique are necessary for standardization. Further studies are required to determine the most suitable conditions of demineralization and particle sizes for clinical application in implant dentistry.

Declarations
Conflict of interest The authors declare no conflict of interest.
Ethical statement There are no animal experiments carried out for this article.
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