Phosphate: Known and potential roles during development and regeneration of teeth and supporting structures

Inorganic phosphate (P_i) is abundant in cells and tissues as an important component of nucleic acids and phospholipids, a source of high‐energy bonds in nucleoside triphosphates, a substrate for kinases and phosphatases, and a regulator of intracellular signaling. The majority of the body's P_i exists in the mineralized matrix of bones and teeth. Systemic P_i metabolism is regulated by a cast of hormones, phosphatonins, and other factors via the bone‐kidney‐intestine axis. Mineralization in bones and teeth is in turn affected by homeostasis of P_i and inorganic pyrophosphate (PPi), with further regulation of the P_i/PP_i ratio by cellular enzymes and transporters. Much has been learned by analyzing the molecular basis for changes in mineralized tissue development in mutant and knock‐out mice with altered P_i metabolism. This review focuses on factors regulating systemic and local P_i homeostasis and their known and putative effects on the hard tissues of the oral cavity. By understanding the role of P_i metabolism in the development and maintenance of the oral mineralized tissues, it will be possible to develop improved regenerative approaches. Birth Defects Research (Part C) 84:281–314, 2008. © 2008 Wiley‐Liss, Inc.     

Common mechanisms in development and disease: BMP signaling in craniofacial development

BMP signaling is one of the key pathways regulating craniofacial development. It is involved in the early patterning of the head, the development of cranial neural crest cells, and facial patterning. It regulates development of its mineralized structures, such as cranial bones, maxilla, mandible, palate, and teeth. Targeted mutations in the mouse have been instrumental to delineate the functional involvement of this signaling network in different aspects of craniofacial development. Gene polymorphisms and mutations in BMP pathway genes have been associated with various non-syndromic and syndromic human craniofacial malformations. The identification of intricate cellular interactions and underlying molecular pathways illustrate the importance of local fine-regulation of Bmp signaling to control proliferation, apoptosis, epithelial-mesenchymal interactions, and stem/progenitor differentiation during craniofacial development. Thus, BMP signaling contributes both to shape and functionality of our facial features. BMP signaling also regulates postnatal craniofacial growth and is associated with dental structures life-long. A more detailed understanding of BMP function in growth, homeostasis, and repair of postnatal craniofacial tissues will contribute to our ability to rationally manipulate this signaling network in the context of tissue engineering.     

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.     

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.     

Bone morphogenetic protein-1/Tolloid-like proteinases process dentin matrix protein-1

Bone morphogenetic protein-1 (BMP-1)/Tolloid-like metalloproteinases play key roles in formation of mammalian extracellular matrix (ECM), through the biosynthetic conversion of precursor proteins into their mature functional forms. These proteinases probably play a further role in formation of bone through activation of transforming growth factor beta-like BMPs. Dentin matrix protein-1 (DMP1), deposited into the ECM during assembly and involved in initiating mineralization of bones and teeth, is thought to undergo proteolysis in vivo to generate functional cleavage fragments found in extracts of mineralized tissues. Here, we have generated recombinant DMP1 and demonstrate that it is cleaved, to varying extents, by all four mammalian BMP-1/Tolloid-like proteinases, to generate fragments similar in size to those previously isolated from bone. Consistent with possible roles for the BMP-1/Tolloid-like proteinases in the physiological processing of DMP1, NH2-terminal sequences of products generated by BMP-1 cleavage of DMP1 match those predicted from processing at the predicted DMP1 site that shows greatest cross-species conservation of sequences. Moreover, fibroblasts derived from mouse embryos homozygous null for genes encoding three of the four mammalian BMP-1/Tolloid-like proteinases appear to be deficient in processing of DMP1. Thus, a further role for BMP-1-Tolloid-like proteinases in formation of mineralized tissues is indicated, via proteolytic processing of DMP1.     

Localization of transforming growth factor beta receptor II interacting protein-1 in bone and teeth: implications in matrix mineralization

Transforming growth factor beta receptor II (TGFβR-II) interacting protein 1 (TRIP-1) is a WD-40 protein that binds to the cytoplasmic domain of the TGF-β type II receptor in a kinase-dependent manner. To investigate the role of TRIP-1 in mineralized tissues, we examined its pattern of expression in cartilage, bone, and teeth and analyzed the relationship between TRIP-1 overexpression and mineralized matrix formation. Results demonstrate that TRIP-1 was predominantly expressed by osteoblasts, odontoblasts, and chondrocytes in these tissues. Interestingly, TRIP-1 was also localized in the extracellular matrix of bone and at the mineralization front in dentin, suggesting that TRIP-1 is secreted by nonclassical secretory mechanisms, as it is devoid of a signal peptide. In vitro nucleation studies demonstrate a role for TRIP-1 in nucleating calcium phosphate polymorphs. Overexpression of TRIP-1 favored osteoblast differentiation of undifferentiated mesenchymal cells with an increase in mineralized matrix formation. These data indicate an unexpected role for TRIP-1 during development of bone, teeth, and cartilage.     

Bone mineralization proceeds through intracellular calcium phosphate loaded vesicles: a cryo-electron microscopy study

Bone is the most widespread mineralized tissue in vertebrates and its formation is orchestrated by specialized cells - the osteoblasts. Crystalline carbonated hydroxyapatite, an inorganic calcium phosphate mineral, constitutes a substantial fraction of mature bone tissue. Yet key aspects of the mineral formation mechanism, transport pathways and deposition in the extracellular matrix remain unidentified. Using cryo-electron microscopy on native frozen-hydrated tissues we show that during mineralization of developing mouse calvaria and long bones, bone-lining cells concentrate membrane-bound mineral granules within intracellular vesicles. Elemental analysis and electron diffraction show that the intracellular mineral granules consist of disordered calcium phosphate, a highly metastable phase and a potential precursor of carbonated hydroxyapatite. The intracellular mineral contains considerably less calcium than expected for synthetic amorphous calcium phosphate, suggesting the presence of a cellular mechanism by which phosphate entities are first formed and thereafter gradually sequester calcium within the vesicles. We thus demonstrate that in vivo osteoblasts actively produce disordered mineral packets within intracellular vesicles for mineralization of the extracellular developing bone tissue. The use of a highly disordered precursor mineral phase that later crystallizes within an extracellular matrix is a strategy employed in the formation of fish fin bones and by various invertebrate phyla. This therefore appears to be a widespread strategy used by many animal phyla, including vertebrates.     

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.     

SPARC/osteonectin in mineralized tissue

Secreted protein acidic and rich in cysteine (SPARC/osteonectin/BM40) is one of the most abundant non-collagenous protein expressed in mineralized tissues. This review will focus on elucidating functional roles of SPARC in bone formation building upon results from non-mineralized cells and tissues, the phenotype of SPARC-null bones, and recent discoveries of human diseases with either dysregulated expression of SPARC or mutations in the gene encoding SPARC that give rise to bone pathologies. The capacity of SPARC to influence pathways involved in extracellular matrix assembly such as procollagen processing and collagen fibril formation as well as the capacity to influence osteoblast differentiation and osteoclast activity will be addressed. In addition, the potential for SPARC to regulate cross-linking of extracellular matrix proteins by members of the transglutaminase family of enzymes is explored. Elucidating defined biological functions of SPARC in terms of bone formation and turnover are critical. Further insight into specific cellular mechanisms involved in the formation and homeostasis of mineralized tissues will lead to a better understanding of disease progression.     

Tissue engineering: chondrocytes and cartilage

Tissue engineering offers new strategies for developing treatments for the repair and regeneration of damaged and diseased tissues. These treatments, using living cells, will exploit new developments in understanding the principles in cell biology that control and direct cell function. Arthritic diseases that affect so many people and have a major impact on the quality of life provide an important target for tissue engineering. Initial approaches are in cartilage repair; in our own programme we are elucidating the signals required by chondrocytes to promote new matrix assembly. These principles will extend to other tissues of the musculoskeletal system, including the repair of bone, ligament and tendon.     

THE EFFECTS OF FLUORIDE FEEDING ON THE ORGANIC MATRIX OF BONES AND TEETH OF PIGS AS OBSERVED BY AUTORADIOGRAPHY AFTER IN VITRO UPTAKE OF CA45 AND S35

Demineralized sections of fluorinated bones and teeth have been studied by autoradiography following in vitro uptake of Ca⁴⁵ or S³⁵O₄. The portions of tissue which do not become mineralized (cartilage, prebone, predentine, and precementum) show an increased Ca⁴⁵ uptake apparently related to an increase in chondroitin sulfate content in fluorosis. The tissues from the fluoride-fed animals show an increase of in vitro uptake of sulfur in the tissues which become mineralized (bone, dentine, cementum).     

Role of hyaluronan in angiogenesis and its utility to angiogenic tissue engineering

Angiogenesis represents the outgrowth of new blood vessels from existing ones, a physiologic process that is vital to supply nourishment to newly forming tissues during development and tissue remodeling and repair (wound healing). Regulation of angiogenesis in the healthy body occurs through a fine balance of angiogenesis-stimulating factors and angiogenesis inhibitors. When this balance is disturbed, excessive or deficient angiogenesis can result and contribute to development of a wide variety of pathological conditions. The therapeutic stimulation or suppression of angiogenesis could be the key to abrogating these diseases. In recent years, tissue engineering has emerged as a promising technology for regenerating tissues or organs that are diseased beyond repair. Among the critical challenges that deter the practical realization of the vision of regenerating functional tissues for clinical implantation, is how tissues of finite size can be regenerated and maintained viable in the long-term. Since the diffusion of nutrients and essential gases to cells, and removal of metabolic wastes is typically limited to a depth of 150-250 μ m from a capillary (3 - 10 cells thick), tissue constructs must mandatorily permit in-growth of a blood capillary network to nourish and sustain the viability of cells within. The purpose of this article is to provide an overview of the role and significance of hyaluronan (HA), a glycosaminoglycan (GAG) component of connective tissues, in physiologic and pathological angiogenesis, its applicability as a therapeutic to stimulate or suppress angiogenesis in situ within necrotic tissues in vivo, and the factors determining its potential utility as a pro-angiogenic stimulus that will enable tissue engineering of neo-vascularized and functional tissue constructs for clinical use.     

The Identification of Proteoglycans and Glycosaminoglycans in Archaeological Human Bones and Teeth

Bone tissue is mineralized dense connective tissue consisting mainly of a mineral component (hydroxyapatite) and an organic matrix comprised of collagens, non-collagenous proteins and proteoglycans (PGs). Extracellular matrix proteins and PGs bind tightly to hydroxyapatite which would protect these molecules from the destructive effects of temperature and chemical agents after death. DNA and proteins have been successfully extracted from archaeological skeletons from which valuable information has been obtained; however, to date neither PGs nor glycosaminoglycan (GAG) chains have been studied in archaeological skeletons. PGs and GAGs play a major role in bone morphogenesis, homeostasis and degenerative bone disease. The ability to isolate and characterize PG and GAG content from archaeological skeletons would unveil valuable paleontological information. We therefore optimized methods for the extraction of both PGs and GAGs from archaeological human skeletons. PGs and GAGs were successfully extracted from both archaeological human bones and teeth, and characterized by their electrophoretic mobility in agarose gel, degradation by specific enzymes and HPLC. The GAG populations isolated were chondroitin sulfate (CS) and hyaluronic acid (HA). In addition, a CSPG was detected. The localization of CS, HA, three small leucine rich PGs (biglycan, decorin and fibromodulin) and glypican was analyzed in archaeological human bone slices. Staining patterns were different for juvenile and adult bones, whilst adolescent bones had a similar staining pattern to adult bones. The finding that significant quantities of PGs and GAGs persist in archaeological bones and teeth opens novel venues for the field of Paleontology.     

Biomimetic self-assembly of apatite hybrid materials: From a single molecular template to bi-/multi-molecular templates

The self-assembly of apatite and proteins is a critical process to induce the formation of the bones and teeth in vertebrates. Although hierarchical structures and biomineralization mechanisms of the mineralized tissues have been intensively studied, most researches focus on the self-assembly biomimetic route using one single-molecular template, while the natural bone is an outcome of a multi-molecular template co-assembly process. Inspired by such a mechanism in nature, a novel strategy based on multi-molecular template co-assembly for fabricating bone-like hybrid materials was firstly proposed by the authors. In this review article we have summarized the new trends from single-molecular template to bi-/multi-molecular template systems in biomimetic fabrication of apatite hybrid materials. So far, many novel apatite hybrid materials with controlled morphologies and hierarchical structures have been successfully achieved using bi-/multi-molecular template strategy, and are found to have multiple common features in comparison with natural mineralized tissues. The carboxyl, carbonyl and amino groups of the template molecules are identified to initiate the nucleation of calcium phosphate during the assembling process. For bi-/multi-molecular templates, the incorporation of multiple promotion sites for calcium and phosphate ions precisely enables to regulate the apatite nucleation from the early stage. The roles of acidic molecules and the synergetic effects of protein templates have been significantly recognized in recent studies. In addition, a specific attention is paid to self-assembling of apatite nanoparticles into ordered structures on tissue regenerative scaffolds due to their promising clinical applications ranging from implant grafts, coatings to drug and gene delivery.     

Is there more to enamel matrix proteins than biomineralization?

Enamel proteins are proteins synthesized by ameloblast cells. These proteins are secreted into the enamel extracellular matrix where they nucleate and regulate the growth of hydroxyapatite crystals to form the mineralized enamel covering the crown of the teeth. Although the exact role of these proteins in enamel mineralization is just beginning to be elucidated, new studies suggest that these proteins might have functions outside enamel formation. Furthermore, extracts of enamel proteins are currently being used to regenerate periodontal tissues destroyed by periodontal disease and new studies suggest that they might have chondrogenic and osteogenic properties. These new functions of enamel proteins will be the focus of this review.     

Influence of Methionine and Vitamin E on Fluoride Concentration in Bones and Teeth of Rats Exposed to Sodium Fluoride in Drinking Water

Increased exposure to fluorine-containing compounds leads to accumulation of fluorides in hard tissues of bones and teeth, which may result in numerous skeletal and dental disorders. This study evaluates the influence of methionine and vitamin E on fluoride concentration in bones and teeth of rats subjected to long-term exposure to sodium fluoride in drinking water. The study was conducted in 30 3-month-old female Wistar FL rats. The animals were divided into five groups, six rats per group. The control group consisted of rats receiving only distilled water as drinking water. All other groups received NaF in the amount of 10 mg/kg of body mass/day in their drinking water. In addition, respective animal groups received: NaF + Met group—10 mg of methionine/kg of body mass/day, NaF + Met + E group—10 mg of methionine/kg of body mass/day and 3 mg of vitamin E (tocopheroli acetas)/rat/day and NaF + E group—3 mg of vitamin E/rat/day. Femoral bones and incisor teeth were collected for the study, and the fluoride concentration was determined using a fluoride ion-selective electrode. Fluoride concentration in both bones and teeth was found to be higher in the NaF and NaF + Met groups compared to the control group. In groups NaF + Met + E and NaF + E, the study material contained much lower fluoride concentration compared to the NaF group, while the effect was more prominent in the NaF + E group. The results of the studies indicate that methionine and vitamin E have opposite effects on accumulation of fluorides in hard tissue in rats. By stimulating fluoride accumulation, methionine reduces the adverse effect of fluorides on soft tissue, while vitamin E, which prevents excessive accumulation of fluorides in bones and teeth, protects these tissues from fluorosis. Therefore, it seems that combined application of both compounds would be optimal for the prevention of the adverse effects of chronic fluoride intoxication.     

Stained Ground Sections of Teeth and Bone

Formalin-fixed rat hemimandibles were ground to approximately 80μ with the aid of a diamond disc attached to a dental handpiece. A 1/10-hp motor attached to the handpiece by a pulley powered the disc to speeds up to 4000 rev/min. Contact between the hemimandible, which was placed on water-moistened cork, and the diamond disc was made possible through manipulation of the handpiece. The ground sections obtained by this procedure were stained with hematoxylin and eosin and periodic acid-Schiff for microscopic study. The fine structure of mineralized tissues (teeth and bones) as well as related soft structures, e.g., blood vessels, endosteum, pulp, etc., were demonstrated. The procedure did not require the embedding of the tissue nor were complicated grinding devices necessary.     

The Study of Barium Concentration in Deciduous Teeth,Impacted Teeth,and Facial Bones of Polish Residents

The study determines the concentration of Ba in mineralized tissues of deciduous teeth, permanent impacted teeth, and facial bones. The study covers the population of children and adults (aged 6–78) living in an industrial area of Poland. Teeth were analyzed in whole, with no division into dentine and enamel. Facial bones and teeth were subjected to the following preparation: washing, drying, grinding in a porcelain mortar, sample weighing (about 0.2 g), and microwave mineralization with spectrally pure nitric acid. The aim of the study was to determinate the concentration of Ba in deciduous teeth, impacted permanent teeth, and facial bones. The concentration of barium in samples was determined over the ICP OES method. The Ba concentration in the tested bone tissues amounted to 2.2-15.5 μg/g (6.6 μg/g?±?3.9). The highest concentration of Ba was present in deciduous teeth (10.5 μg/g), followed by facial bones (5.2 μg/g), and impacted teeth (4.3 μg/g) (ANOVA Kruskal-Wallis rank test, p?=?0.0002). In bone tissue and impacted teeth, Ba concentration increased with age. In deciduous teeth, the level of Ba decreased with children’s age.