Dentin regeneration based on tooth tissue engineering: A review

Missing or damaged teeth due to caries, genetic disorders, oral cancer, or infection may contribute to physical and mental impairment that reduces the quality of life. Despite major progress in dental tissue repair and those replacing missing teeth with prostheses, clinical treatments are not yet entirely satisfactory, as they do not regenerate tissues with natural teeth features. Therefore, much of the focus has centered on tissue engineering (TE) based on dental stem/progenitor cells to create bioengineered dental tissues. Many in vitro and in vivo studies have shown the use of cells in regenerating sections of a tooth or a whole tooth. Tooth tissue engineering (TTE), as a promising method for dental tissue regeneration, can form durable biological substitutes for soft and mineralized dental tissues. The cell-based TE approach, which directly seeds cells and bioactive components onto the biodegradable scaffolds, is currently the most potential method. Three essential components of this strategy are cells, scaffolds, and growth factors (GFs). This study investigates dentin regeneration after an injury such as caries using TE and stem/progenitor cell-based strategies. We begin by discussing about the biological structure of a dentin and dentinogenesis. The engineering of teeth requires knowledge of the processes that underlie the growth of an organ or tissue. Then, the three fundamental requirements for dentin regeneration, namely cell sources, GFs, and scaffolds are covered in the current study, which may ultimately lead to new insights in this field.     

Mesenchymal progenitor cells in adult human dental pulp and their ability to form bone when transplanted into immunocompromised mice

The technique of tissue engineering is developing for the restoration of lost tissues. This new technique requires cells that fabricate tissue. Mesenchymal stem cells in bone marrow have been used as the cell source for this technique; however, dental pulp cells have recently been shown to possess stem-cell-like properties. We earlier demonstrated that dental pulp cells proliferate and produce an extracellular matrix that subsequently becomes mineralized in vitro. We now report that such dental pulp cells (first to eighth passage) produced bone instead of dentin when those cells were implanted into subcutaneous sites in immunocompromised mice with HA/TCP powder as their carrier. This evidence shows that dental pulp cells are the common progenitors of odontoblasts and osteoblasts, or dental pulp cells are mesenchymal stem cells themselves. It is expected that dental pulp cells can be a useful candidate cell source for tissue engineering, and contain the potential of new therapeutic approaches for the restoration of damaged or diseased tissue.     

Odontoblasts: the cells forming and maintaining dentine

Odontoblasts are tall columnar cells located at the periphery of the dental pulp. They derive from ectomesenchymal cells originated by migration of neural crest cells during the early craniofacial development. Odontoblasts form the dentine, a collagen-based mineralized tissue, through secretion of its collagenous and noncollagenous organic matrix components and by control the mineralization process. A conspicuous cell process arises from the cell body of odontoblasts and penetrates into the mineralized dentine. After dentinogenesis, odontoblasts deposit new layers of dentine throughout life and might also form a type of reactionary/reparative dentine in response to dental caries and other external factors may affect teeth.     

Dentin sialoprotein: biosynthesis and developmental appearance in rat tooth germs in comparison with amelogenins,osteocalcin and colagen type-I

A non-collagenous protein, extracted from rat incisor dentin, is a dentin sialoprotein (DSP). We examined immunohistochemically the developmental appearance and tissue distribution of DSP in 1 to 3-day-old rat molar and incisor tooth germs. The earliest staining for DSP was observed in newly differentiated odontoblasts. In more advanced stages, immunostaining for DSP gradually increased in pre-dentin, odontoblasts and dentin, and appeared in many cells of the dental papilla. In early stages of development before the breakdown of the dental basement membrane, pre-ameloblasts were also positive for DSP. This staining disappeared from the ameloblast cell body soon after deposition of the first layer of mineralized dentin. Radiolabelling of tooth matrix proteins with ¹⁴C-serine in vitro followed by immunoprecipitation and fluorography confirmed that DSP was synthesized by tooth-forming cells. The immunolocalization for DSP was different from that of either collagen type-I, osteocalcin or the amelogenins. Whereas collagen type-I and osteocalcin were restricted to the mesenchymal dental tissues, the amelogenins were detectable in both epithelial and mesenchymal dental cells and tissues at the epithelio-mesenchymal interface at early stages of development, prior to the onset of dentin mineralization. We conclude that DSP is expressed in and secreted by odontoblasts and some dental papilla cells from early stages of dentinogenesis onwards, i.e. later than type-I collagen, but before deposition of the first layer of mineralized dentin. In pre-mineralizing stages, some of the matrix proteins may be endocytosed from the pre-dentin by both cell types involved in the epithelio-mesenchymal interaction.     

Evolutionary genetics of vertebrate tissue mineralization: the origin and evolution of the secretory calcium-binding phosphoprotein family

Three principal mineralized tissues are present in teeth; a highly mineralized surface layer (enamel or enameloid), body dentin, and basal bone. Similar tissues have been identified in the dermal skeleton of Paleozoic jawless vertebrates, suggesting their ancient origin. These dental tissues form on protein matrix and their mineralization is controlled by distinctive proteins. We have shown that many secretory calcium-binding phosphoproteins (SCPPs) are involved in tetrapod tissue mineralization. These SCPPs all originated from the common ancestral gene SPARCL1 (secreted protein, acidic, cysteine-rich like 1) that initially arose from SPARC. The SCPP family also includes a bird eggshell matrix protein, mammalian milk casein, and salivary proteins. The eggshell SCPP plays crucial roles in rigid eggshell production, milk SCPPs in efficient lactation and in the evolution of complex dentition, and salivary SCPPs in maintaining tooth integrity. A comparative analysis of the mammalian, avian, and amphibian genomes revealed a tandem duplication history of the SCPP genes in tetrapods. Although these tetrapod SCPP genes are fewer in teleost genomes, independent parallel duplication has created distinct SCPP genes in this lineage. These teleost SCPPs are also used for enameloid and dentin mineralization, implying essential roles of SCPPs for dental tissue mineralization in osteichthyans. However, the SCPPs used for tetrapod enamel and teleost enameloid, as well as tetrapod dentin and teleost dentin, are all different. Thus, the evolution of vertebrate mineralized tissues seems to be explained by phenogenetic drift: while mineralized tissues are retained during vertebrate evolution, the underlying genetic basis has extensively drifted.     

Changes in the matrix proteins, fibronectin and collagen, during differentiation of mouse tooth germ.

The distribution of the matrix protein fibronectin was studied by indirect immunofluorescence in differentiating mouse molars from bud stage to the stage of dentin and enamel secretion, and compared to that of collagenous proteins procollagen type III and collagen type I. Fibronectin was seen in mesenchymal tissue, basement membranes, and predentin. The dental mesenchyme lost fibronectin staining when differentiating into odontoblasts. Fibronectin was not detected in mineralized dentin. Epithelial tissues were negative except for the stellate reticulum within the enamel organ. Particularly intense staining was seen at the epithelio-mesenchymal interface between the dental epithelium and mesenchyme. Fibronectin may here be involved in anchorage of the mesenchymal cells during their differentiation into odontoblasts. Procollagen type III was lost from the dental mesenchyme during odontoblast differentiation but reappeared with advancing vascularization of the dental papilla. Similarly, procollagen type III present in the dental basement membrane during the bud and cap stages disappeared from the cuspal area along with odontoblast differentiation. Weak staining was seen in predentin but not in mineralized dentin. The staining with anti-collagen type I antibodies was weak in dental mesenchyme but intense in predentin as well as in mineralized dentin.     

Identification of novel epithelial stem cell-like cells in human deciduous dental pulp

It is well known that interactions between epithelial components and mesenchymal components are essential for tooth development. Therefore, it has been postulated that both types of stem cells might be involved in the regeneration of dental hard tissues. Recently, mesenchymal dental pulp stem cells that have odontogenic potential were identified from human dental pulp. However, the existence of epithelial cells has never been reported in human dental pulp. In the present study, we isolated and characterized epithelial cell-like cells from human deciduous dental pulp. They had characteristic epithelial morphology and expressed epithelial markers. Moreover, they expressed epithelial stem cell-related genes such as ABCG2, Bmi-1, ΔNp63, and p75. Taken together, our findings suggest that epithelial stem cell-like cells might exist in human deciduous dental pulp and might play a role as an epithelial component for the repair or regeneration of teeth.     

Gingival-derived mesenchymal stem cells:An endless resource for regenerative dentistry

The gingiva, the masticatory portion of the oral mucosa, is excised and discarded frequently during routine dental treatments and following tooth extraction, dental crown lengthening, gingivectomy and periodontal surgeries. Subsequent to excision, healing eventually happens in a short time period after gingival surgery. Clinically, the gingival tissue can be collected very easily and, in the laboratory, it is also very easy to isolate gingival-derived mesenchymal stem cells (GMSCs) from this discarded gingival tissue. GMSCs, a stem cell population within the lamina propria of the gingival tissue, can be isolated from attached and free gingiva, inflamed gingival tissu-es, and from hyperplastic gingiva. Comparatively, they constitute more attractive alternatives to other dental-derived mesenchymal stem cells due to the availability and accessibility of gingival tissues. They have unique immunomodulatory functions and well-documented self-renewal and multipotent differentiation properties. They display positive signals for Stro-1, Oct-4 and SSEA-4 pluripotency-associated markers, with some co-expre-ssing Oct4/Stro-1 or Oct-4/SSEA-4. They should be considered as the best stem cell source for cell-based therapies and regenerative dentistry. The clinical use of GMSCs for regenerative dentistry represents an attrac-tive therapeutic modality. However, numerous biological and technical challenges need to be addressed prior to considering transplantation approaches of GMSCs as clinically realistic therapies for humans.     

Pro-inflammatory cytokines induce odontogenic differentiation of dental pulp-derived stem cells

Postnatal dental pulp stem cells (DPSCs) represent a unique precursor population in the dental pulp, which have multipotential and harbor great potential for tissue engineering purposes. However, for therapy applications, transplanted cells are often exposed to unfavorable conditions such as cytokines released from necrotic or inflammatory cells in injured tissues. It is not clear how stem cells exposed to these conditions changes in their characteristics. In this study, the effects of pro-inflammatory cytokines, such as IL-1 and TNF, on DPSCs were investigated. Cells were treated with IL-1, TNF, or both for 3, 7, and 12 days. The cultures were evaluated for cell proliferation, ALP activity, and real-time PCR. We found that a short treatment (3 days) of pro-inflammatory cytokines induced the odontogenic differentiation of DPSCs. Furthermore, post 3 days treatment with pro-inflammatory cytokines, the cell-scaffold complexes were implanted subcutaneously in mice for 8 weeks. Histological analysis demonstrated that the cultures gave obviously mineralized tissue formation, especially for both IL-1 and TNF applied. These data suggest that IL-1 and TNF produced in the early inflammatory reaction may induce the mineralization of DPSCs.     

Stem cells: their source,potency and use in regenerative therapies with focus on adipose-derived stem cells – a review

Stem cells can be defined as units of biological organization that are responsible for the development and the regeneration of organ and tissue systems. They are able to renew their populations and to differentiate into multiple cell lineages. Therefore, these cells have great potential in advanced tissue engineering and cell therapies. When seeded on synthetic or nature-derived scaffolds in vitro, stem cells can be differentiated towards the desired phenotype by an appropriate composition, by an appropriate architecture, and by appropriate physicochemical and mechanical properties of the scaffolds, particularly if the scaffold properties are combined with a suitable composition of cell culture media, and with suitable mechanical, electrical or magnetic stimulation. For cell therapy, stem cells can be injected directly into damaged tissues and organs in vivo. Since the regenerative effect of stem cells is based mainly on the autocrine production of growth factors, immunomodulators and other bioactive molecules stored in extracellular vesicles, these structures can be isolated and used instead of cells for a novel therapeutic approach called “stem cell-based cell-free therapy”. There are four main sources of stem cells, i.e. embryonic tissues, fetal tissues, adult tissues and differentiated somatic cells after they have been genetically reprogrammed, which are referred to as induced pluripotent stem cells (iPSCs). Although adult stem cells have lower potency than the other three stem cell types, i.e. they are capable of differentiating into only a limited quantity of specific cell types, these cells are able to overcome the ethical and legal issues accompanying the application of embryonic and fetal stem cells and the mutational effects associated with iPSCs. Moreover, adult stem cells can be used in autogenous form. These cells are present in practically all tissues in the organism. However, adipose tissue seems to be the most advantageous tissue from which to isolate them, because of its abundancy, its subcutaneous location, and the need for less invasive techniques. Adipose tissue-derived stem cells (ASCs) are therefore considered highly promising in present-day regenerative medicine.     

New insight into dental epithelial stem cells: Identification,regulation, and function in tooth homeostasis and repair

Tooth enamel, a highly mineralized tissue covering the outermost area of teeth, is always damaged by dental caries or trauma. Tooth enamel rarely repairs or renews itself, due to the loss of ameloblasts and dental epithelial stem cells (DESCs) once the tooth erupts. Unlike human teeth, mouse incisors grow continuously due to the presence of DESCs that generate enamel-producing ameloblasts and other supporting dental epithelial lineages. The ready accessibility of mouse DESCs and wide availability of related transgenic mouse lines make mouse incisors an excellent model to examine the identity and heterogeneity of dental epithelial stem/progenitor cells; explore the regulatory mechanisms underlying enamel formation; and help answer the open question regarding the therapeutic development of enamel engineering. In the present review, we update the current understanding about the identification of DESCs in mouse incisors and summarize the regulatory mechanisms of enamel formation driven by DESCs. The roles of DESCs during homeostasis and repair are also discussed, which should improve our knowledge regarding enamel tissue engineering.     

The tick tock of odontogenesis

Although a big deal of dental research is being focused to the understanding of early stages of tooth development, a huge gap exist on our knowledge on how the dental hard tissues are formed and how this process is controlled daily in order to produce very complex and diverse tooth shapes adapted for specific functions. Emerging evidence suggests that clock genes, a family of genes that controls circadian functions within our bodies, regulate also dental mineralized tissues formation. Enamel formation, for example, is subjected to rhythmical molecular signals that occur on short (24 h) periods and control the secretion and maturation of the enamel matrix. Accordingly, gene expression and ameloblast functions are also tightly modulated in regular daily intervals. This review summarizes the current knowledge on the circadian controls of dental mineralized tissues development with a special emphasis on amelogenesis.     

ALP,TRAcP and cathepsin K in elasmoid scales: a role in mineral metabolism?

Elasmoid scales from the common carp (and other teleostean fishes) appear to be an exciting new model in the research of mineralized tissues. The presence of alkaline phosphatase (ALP), a marker of mineralization, on both sides of the scale was demonstrated by means of enzyme histochemistry. Tartrate‐resistant acid phosphatase, a marker for mineral degradation and osteoclasts, was observed along the radii, at the same location as the ALP activity on the episquamal side. This points towards an active mineral metabolism, were scale cells are involved in both formation and degradation of the mineralized matrix. Cathepsin K staining revealed the presence of multinuclear osteoclasts along the grooves of the scale. Interestingly, the scales were taken from growing control fish; they were not induced to resorb their matrix. Presence of these enzymes in scale cells, together with the demineralized regions in the centre of the scale suggest a more dynamic mineral metabolism in cyprinid scales then previously observed in other species. Scales are derived from odontode tissues, their formation relies on many the same underlying mechanisms and genes as other mineralized tissues. Moreover, a single scale offers the possibility to culture scale‐forming and ‐degrading cells together on their original matrix. All of the these unique properties substantiate the potential of scales to yield new insights on osteoclasts and regulation of tissue mineralization.     

Human dental pulp cells exhibit bone cell-like responsiveness to fluid shear stress

Background aimsFor engineering bone tissue to restore, for example, maxillofacial defects, mechanosensitive cells are needed that are able to conduct bone cell-specific functions, such as bone remodelling. Mechanical loading affects local bone mass and architecture in vivo by initiating a cellular response via loading-induced flow of interstitial fluid. After surgical removal of ectopically impacted third molars, human dental pulp tissue is an easily accessible and interesting source of cells for mineralized tissue engineering. The aim of this study was to determine whether human dental pulp-derived cells (DPC) are responsive to mechanical loading by pulsating fluid flow (PFF) upon stimulation of mineralization in vitro.MethodsHuman DPC were incubated with or without mineralization medium containing differentiation factors for 3 weeks. Cells were subjected to 1-h PFF (0.7 ± 0.3Pa, 5Hz) and the response was quantified by measuring nitric oxide (NO) and prostaglandin E₂ (PGE₂) production, and gene expression of cyclooxygenase (COX)-1 and COX-2.ResultsWe found that DPC are intrinsically mechanosensitive and, like osteogenic cells, respond to PFF-induced fluid shear stress. PFF stimulated NO and PGE₂ production, and up-regulated COX-2 but not COX-1 gene expression. In DPC cultured under mineralizing conditions, the PFF-induced NO, but not PGE₂, production was significantly enhanced.ConclusionsThese data suggest that human DPC, like osteogenic cells, acquire responsiveness to pulsating fluid shear stress in mineralizing conditions. Thus DPC might be able to perform bone-like functions during mineralized tissue remodeling in vivo, and therefore provide a promising new tool for mineralized tissue engineering to restore, for example, maxillofacial defects.     

Human dental pulp stem cells: Applications in future regenerative medicine

Stem cells are pluripotent cells, having a property of differentiating into various types of cells of human body. Several studies have developed mesenchymal stem cells (MSCs) from various human tissues, peripheral blood and body fluids. These cells are then characterized by cellular and molecular markers to understand their specific phenotypes. Dental pulp stem cells (DPSCs) are having a MSCs phenotype and they are differentiated into neuron, cardiomyocytes, chondrocytes, osteoblasts, liver cells and β cells of islet of pancreas. Thus, DPSCs have shown great potentiality to use in regenerative medicine for treatment of various human diseases including dental related problems. These cells can also be developed into induced pluripotent stem cells by incorporation of pluripotency markers and use for regenerative therapies of various diseases. The DPSCs are derived from various dental tissues such as human exfoliated deciduous teeth, apical papilla, periodontal ligament and dental follicle tissue. This review will overview the information about isolation, cellular and molecular characterization and differentiation of DPSCs into various types of human cells and thus these cells have important applications in regenerative therapies for various diseases. This review will be most useful for postgraduate dental students as well as scientists working in the field of oral pathology and oral medicine.     

Hard tissue regeneration capacity of apical pulp derived cells (APDCs) from human tooth with immature apex

Recent studies indicate that dental pulp is a new source of adult stem cells. The human tooth with an immature apex is a developing organ, and the apical pulp of this tooth may contain a variety of progenitor/stem cells, which participate in root formation. We investigated the hard tissue regeneration potential of apical pulp derived cells (APDCs) from human tooth with an immature apex. APDCs cultured with a mineralization-promoting medium showed alkaline phosphatase activity in porous hydroxyapatite (HA) scaffolds. The composites of APDCs and HA were implanted subcutaneously in immunocompromised rats and harvested at 12 weeks after implantation. In histological analysis, the APDCs/HA composites exhibited bone- and dentine-like mineralized tissues in the pore areas of HA. This study suggests that the human tooth with an immature apex is an effective source of cells for hard tissue regeneration.     

Immunohistochemical analysis of two stem cell markers of α-smooth muscle actin and STRO-1 during wound healing of human dental pulp

Recent studies have employed two markers, alpha-smooth muscle actin (α-SMA) and STRO-1, to detect cells with mesenchymal stem cell properties in dental pulp. The present study aimed to explore the expression profile of α-SMA and STRO-1 in intact dental pulp as well as during wound healing in adult dental pulp tissue. Healthy pulps were mechanically exposed and capped with the clinically used materials MTA (ProRoot White MTA) or Ca(OH)(2) to induce a mineralized barrier at the exposed surface. After 7-42?days, the teeth were extracted and processed for immunohistochemical analysis using antibodies against α-SMA, STRO-1 and nestin (a neurogenic cytoskeletal protein expressed in odontoblasts). In normal pulp, α-SMA was detected in vascular smooth muscle cells and pericytes. Double immunofluorescent staining with STRO-1 and α-SMA showed that STRO-1 was localized in vascular smooth muscle cells, pericytes and endothelial cells, in addition to nerve fibers. During the process of dental pulp healing, numerous α-SMA-positive cells emerged at the wound margin at 14?days, and the initially formed mineralized barrier was lined with α-SMA-positive cells similar in appearance to reparative odontoblasts, some of which co-expressed nestin. STRO-1 was abundant in nerve fibers. In the advanced stage of mineralized barrier formation at 42?days, cells lining the barrier were stained with nestin, and no staining of α-SMA was detected in those cells. These observations indicate that α-SMA-positive cells temporarily appear along the wound margin during the earlier phase of mineralized barrier formation and STRO-1 is confined in vascular and neuronal elements.     

An immunohistochemical study on hard tissue formation in a subcutaneously transplanted rat molar

While dental pulp undergoes calcification following tooth replantation or transplantation, we actually know little about these mechanisms. We therefore conducted histological and immunohistochemical evaluations of mineralized tissue that formed in the pulp of rat maxillary molar transplanted into abdominal subcutaneous tissue. One, 2, 3, and 4 weeks post-transplantation, the teeth were investigated immunohistochemically using antibodies to osteocalcin (OCN), osteopontin (OPN), bone sialoprotein (BSP), dentin sialoprotein (DSP), and tissue non-specific alkaline phosphatase (TNAP). In the 1st week after transplantation, cell-rich hard tissue was formed at the root apex. At 2 weeks, formations of hard tissue, with few cells in the root canals and bone-like tissue in the coronal pulp chamber, were noted. After 3 and 4 weeks, the amounts of these hard tissues were increased. The immunolocalization of OCN, OPN, and BSP was seen strongly in coronal and apical hard tissues, but weakly in the root hard tissue. Conversely, DSP localized in the root hard tissue, but not in other newly formed hard tissues. At 1 week, TNAP localized along the periphery of the apical hard tissue and the lower surfaces of root predentin. These results demonstrate that the newly formed hard tissues in the pulp cavity of subcutaneously transplanted molars could be classified into three types, suggesting that these might be formed by type-specific cells.