Tissue-Nonspecific Alkaline Phosphatase-A Gatekeeper of Physiological Conditions in Health and a Modulator of Biological Environments in Disease.

Tissue-nonspecific alkaline phosphatase (TNAP) is a ubiquitously expressed enzyme that is best known for its role during mineralization processes in bones and skeleton. The enzyme metabolizes phosphate compounds like inorganic pyrophosphate and pyridoxal-5'-phosphate to provide, among others, inorganic phosphate for the mineralization and transportable vitamin B6 molecules. Patients with inherited loss of function mutations in the ALPL gene and consequently altered TNAP activity are suffering from the rare metabolic disease hypophosphatasia (HPP). This systemic disease is mainly characterized by impaired bone and dental mineralization but may also be accompanied by neurological symptoms, like anxiety disorders, seizures, and depression. HPP characteristically affects all ages and shows a wide range of clinical symptoms and disease severity, which results in the classification into different clinical subtypes. This review describes the molecular function of TNAP during the mineralization of bones and teeth, further discusses the current knowledge on the enzyme's role in the nervous system and in sensory perception. An additional focus is set on the molecular role of TNAP in health and on functional observations reported in common laboratory vertebrate disease models, like rodents and zebrafish.

Inhibition of Ape1 Redox Activity Promotes Odonto/osteogenic Differentiation of Dental Papilla Cells.

Dentinogenesis is the formation of dentin, a substance that forms the majority of teeth, and this process is performed by odontoblasts. Dental papilla cells (DPCs), as the progenitor cells of odontoblasts, undergo the odontogenic differentiation regulated by multiple cytokines and paracrine signal molecules. Ape1 is a perfect paradigm of the function complexity of a biological macromolecule with two major functional regions for DNA repair and redox regulation, respectively. To date, it remains unclear whether Ape1 can regulate the dentinogenesis in DPCs. In the present study, we firstly examed the spatio-temporal expression of Ape1 during tooth germ developmental process, and found the Ape1 expression was initially high and then gradually reduced along with the tooth development. Secondly, the osteo/odontogenic differentiation capacity of DPCs was up-regulated when treated with either Ape1-shRNA or E3330 (a specific inhibitor of the Ape1 redox function), respectively. Moreover, we found that the canonical Wnt signaling pathway was activated in this process, and E3330 reinforced-osteo/odontogenic differentiation capacity was suppressed by Dickkopf1 (DKK1), a potent antagonist of canonical Wnt signaling pathway. Taken together, we for the first time showed that inhibition of Ape1 redox regulation could promote the osteo/odontogenic differentiation capacity of DPCs via canonical Wnt signaling pathway.

Matrix metalloproteinases (mmp) in restorative dentistry and endodontics.

Matrix metalloproteinases (MMPs) seem to play a dual role in dentistry. While several MMPs have an important role to play in developmental defects of teeth and in caries, some MMPs also seem to have a defensive role. The main organic component of tooth structure is collagen and MMPs that degrade collagen and the extra cellular matrix have been implicated in progression of dental caries. MMPs have also been shown to be active in pulpitis and studies have shown that they can be used as diagnostic markers of pulpal inflammation. This paper reviews the role of MMPs in restorative dentistry and endodontics.

The influence of electrospun fibre scaffold orientation and nano-hydroxyapatite content on the development of tooth bud stem cells in vitro.

In stem cell-based dental tissue engineering, the goal is to create tooth-like structures using scaffold materials to guide the dental stem cells. In this study, the effect of fiber alignment and hydroxyapatite content in biodegradable electrospun PLGA scaffolds have been investigated. Fiber orientation of the scaffolds was random or aligned in bundles. For scaffolds with prefabricated orientation, scaffolds were fabricated from PLGA polymer solution containing 0, 10 or 20 % nano-hydroxyapatite. The scaffolds were seeded with porcine cells isolated from tooth buds (dental mesenchymal, dental epithelial, and mixed dental mesenchymal/epithelial cells). Samples were collected at 1, 3 and 6 weeks. Analyses were performed for cell proliferation, ALP activity, and cell morphology. Fiber alignment showed an effect on cell orientation in the first week after cell seeding, but had no long-term effect on cell alignment or organized calcified matrix deposition once the cells reach confluency. Scaffold porosity was sufficient to allow migration of mesenchymal cells. Hydroxyapatite incorporation did not have a positive effect on cell proliferation, especially of epithelial cells, but seemed to promote differentiation. Concluding, scaffold architecture is important to mesenchymal cell morphology, but has no long-term effect on cell alignment or organized ECM deposition. nHA incorporation does have an effect on cell proliferation, differentiation and ECM production, and should be regarded as a bioactive component of dental bioengineered scaffolds.

Peroxisomes in dental tissues of the mouse.

Patients with mild forms of peroxisomal biogenesis disorders show facial dysmorphism and exhibit dentition problems accompanied by enamel hypoplasia. However, no information is available on the role of peroxisomes in dental and paradontal tissues. Therefore, we studied the distribution of these organelles, their protein composition and the expression of corresponding genes during dental development and in mature decalcified teeth in mice. Perfusion-fixed heads of mice of different developmental stages (E13.5 to adult) were cut in sagittal direction into two halves and embedded in paraffin for serial sectioning and subsequent peroxidase-based immunohistochemistry or double-immunofluorescence preparations. Frozen, unfixed heads of newborn mice were used for cryosectioning and subsequent laser-assisted microdissection of ameloblasts and odontoblasts, RNA isolation and RT-PCR analysis. Our results revealed the presence of peroxisomes already in the bud stage of dental development. An increase in peroxisome abundance was noted during differentiation of ameloblasts and odontoblasts with the highest number of organelles in Tomes' processes of mature ameloblasts. A strong heterogeneity of peroxisomal enzyme content developed within differentiated dental cell types. A drastic down-regulation of catalase in maturing ameloblasts was noted in contrast to high levels of lipid metabolizing enzymes in peroxisomes of these cells. As known from the literature, differentiated ameloblasts are more prone to oxidative damage which could be explained by the low catalase levels inside of this cell type.

Boron enhances odontogenic and osteogenic differentiation of human tooth germ stem cells (hTGSCs) in vitro.

Stem cell technology has been a great hope for the treatment of many common problems such as Parkinson's disease, Alzheimer's disease, diabetes, cancer, and tissue regeneration. Therefore, the main challenge in hard tissue engineering is to make a successful combination of stem cells and efficient inductors in the concept of stem cell differentiation into odontogenic and osteogenic cell types. Although some boron derivatives have been reported to promote bone and teeth growth in vivo, the molecular mechanism of bone formation has not been elucidated yet. Different concentrations of sodium pentaborate pentahydrate (NaB) were prepared for the analysis of cell toxicity and differentiation evaluations. The odontogenic, osteogenic differentiation and biomineralization of human tooth germ stem cells (hTGSCs) were evaluated by analyzing the mRNA expression levels, odontogenic and osteogenic protein expressions, alkaline phosphatase (ALP) activity, mineralization, and calcium deposits. The NaB-treated group displayed the highest ALP activity and expression of osteo- and odontogenic-related genes and proteins compared to the other groups and baseline. In the current study, increased in vitro odontogenic and osteogenic differentiation capacity of hTGSCs by NaB application has been shown for the first time. The study offers considerable promise for the development of new scaffold systems combined with NaB in both functional bone and tooth tissue engineering.

Analysis of cathepsin-K activity at tooth and dental implant sites and the potential of this enzyme in reflecting alveolar bone loss.

BACKGROUND: Cathepsin-K is an enzyme involved in bone metabolism which may make this feature important for both natural teeth and dental implants. The aims of the present study are to comparatively analyze the gingival crevicular fluid (GCF)/peri-implant sulcus fluid (PISF) cathepsin-K levels of natural teeth and dental implants, and to assess the potential relationship between this biochemical parameter and alveolar bone loss around natural teeth and dental implants. METHODS: Probing depth, bleeding on probing, gingival index, and plaque index clinical parameters were assessed, and GCF/PISF samples were obtained from natural teeth/dental implants presenting with either clinical health, gingivitis/peri-implant mucositis, or chronic periodontitis/peri-implantitis. Cathepsin-K activity levels of 42 GCF samples and 54 PISF samples were determined, and marginal bone loss (MBL) measures were calculated from digitalized standardized intraoral periapical radiographs obtained from natural teeth and dental implants by using cemento-enamel junction and the actual distance between two consecutive threads of the dental implant as reference points for natural teeth and dental implants, respectively. RESULTS: Comparing the natural teeth group with dental implant group with regard to MBL measure, cathepsin-K activity, and GCF/PISF volume revealed no significant differences. In both natural teeth and dental implant groups, despite higher MBL measures, cathepsin-K activity, and GCF/PISF volumes with the presence of inflammation, it was the presence of alveolar bone loss that lead to significantly higher values for these parameters. CONCLUSION: We suggest cathepsin-K as a biochemical parameter for monitoring periodontal/peri-implant alveolar bone loss.

GTPases RhoA and Rac1 are important for amelogenin and DSPP expression during differentiation of ameloblasts and odontoblasts.

Morphogenesis and cytodifferentiation are distinct processes in tooth development. Cell proliferation predominates in morphogenesis; differentiation involves changes in form and gene expression. The cytoskeleton is essential for both processes, being regulated by Rho GTPases. The aim of this study was to verify the expression, distribution, and role of Rho GTPases in ameloblasts and odontoblasts during tooth development in correlation with actin and tubulin arrangements and amelogenin and dentin sialophosphoprotein (DSPP) expression. RhoA, Rac1, and Cdc42 were strongly expressed during morphogenesis; during cytodifferentiation, RhoA was present in ameloblasts and odontoblasts, Rac1 and its effector Pak3 were observed in ameloblasts; and Cdc42 was present in all cells of the tooth germ and mesenchyme. The expression of RhoA mRNA and its effectors RockI and RockII, Rac1 and Pak3, as analyzed by real-time polymerase chain reaction, increased after ameloblast and odontoblast differentiation, according to the mRNA expression of amelogenin and DSPP. The inhibition of all Rho GTPases by Clostridium difficile toxin A completely abolished amelogenin and DSPP expression in tooth germs cultured in anterior eye chamber, whereas the specific inhibition of the Rocks showed only a partial effect. Thus, both GTPases are important during tooth morphogenesis. During cytodifferentiation, Rho proteins are essential for the complete differentiation of ameloblasts and odontoblasts by regulating the expression of amelogenin and DSPP. RhoA and its effector RockI contribute to this role. A specific function for Rac1 in ameloblasts remains to be elucidated; its punctate distribution indicates its possible role in exocytosis/endocytosis.

Mechanism of cadmium induced crystal defects in developing rat tooth enamel.

It is well known that exposure to environmental cadmium causes itai-itai (ouch-ouch) disease. However, the exact mechanism underlying this bone disease remains unresolved. By focusing on the calcification mechanism, we examined developing tooth enamel in rats exposed to cadmium to test the hypothesis that cadmium exposure may cause defects in crystal formation. Electron microscopy revealed the presence of perforated crystals in developing tooth enamel, indicating that the process of crystal nucleation may have been interrupted by cadmium exposure. Furthermore, biochemical analyses revealed that the catalytic activity of carbonic anhydrase in the immature enamel matrix declined remarkably despite the fact that quantitative reduction of this enzyme was insignificant, suggesting that the decline of catalytic activity may have resulted from the replacement of zinc with cadmium ions. Therefore, we concluded that the poor catalytic activity of cadmium-binding carbonic anhydrase might hinder the nucleation process, leading to an impairment in mineralization that causes itai-itai disease.

Ectodermal Smad4 and p38 MAPK are functionally redundant in mediating TGF-beta/BMP signaling during tooth and palate development.

Smad4 is a central intracellular effector of TGF-beta signaling. Smad-independent TGF-beta pathways, such as those mediated by p38 MAPK, have been identified in cell culture systems, but their in vivo functional mechanisms remain unclear. In this study, we investigated the role of TGF-beta signaling in tooth and palate development and noted that conditional inactivation of Smad4 in oral epithelium results in much milder phenotypes than those seen with the corresponding receptor mutants, Bmpr1a and Tgfbr2, respectively. Perturbed p38 function in these tissues likewise has no effect by itself; however, when both Smad4 and p38 functions are compromised, dramatic recapitulation of the receptor mutant phenotypes results. Thus, our study demonstrates that p38 and Smad4 are functionally redundant in mediating TGF-beta signaling in diverse contexts during embryonic organogenesis. The ability of epithelium to utilize both pathways illustrates the complicated nature of TGF-beta signaling mechanisms in development and disease.

Functions of KLK4 and MMP-20 in dental enamel formation.

Two proteases are secreted into the enamel matrix of developing teeth. The early protease is enamelysin (MMP-20). The late protease is kallikrein 4 (KLK4). Mutations in MMP20 and KLK4 both cause autosomal recessive amelogenesis imperfecta, a condition featuring soft, porous enamel containing residual protein. MMP-20 is secreted along with enamel proteins by secretory-stage ameloblasts. Enamel protein-cleavage products accumulate in the space between the crystal ribbons, helping to support them. MMP-20 steadily cleaves accumulated enamel proteins, so their concentration decreases with depth. KLK4 is secreted by transition- and maturation-stage ameloblasts. KLK4 aggressively degrades the retained organic matrix following the termination of enamel protein secretion. The principle functions of MMP-20 and KLK4 in dental enamel formation are to facilitate the orderly replacement of organic matrix with mineral, generating an enamel layer that is harder, less porous, and unstained by retained enamel proteins.

Comparative properties of recombinant human and bovine matrix metalloproteinase-20.

INTRODUCTION: Matrix metalloproteinase-20 (MMP-20) is a predominant enzyme for the progressive processing of enamel extracellular matrix protein components (primarily amelogenin) during the early stages of enamel formation. So far, the recombinant porcine, mouse and bovine MMP-20 have been cloned and used extensively in the researches of tooth enamel development. The homology of these MMP-20s to human MMP-20 is approximately 80%. The effect of sequence differences on the properties of these enzymes is poorly understood even though they have been used to hydrolyse amelogenins from different species. OBJECTIVE: Our goal is to compare the characteristics between recombinant human MMP-20 (rhMMP-20) and bovine MMP-20 (rbMMP-20). DESIGN: rhMMP-20 and rbMMP-20 were parallelly expressed, purified and activated. The SDS-PAGE, zymography and quenched peptide assay were used for characterization and comparisons. RESULTS: Both proteases were activated by autocatalysis in a similar pattern of fragmentation. Dynamically, rbMMP-20 autoactivated faster and digested a fluorescence-quenched peptide Mca-PLGL-Dpa-AR, a non-amelogenin substrate, more efficiently than rhMMP-20. However, rhMMP-20 showed higher enzymatic activity for a human amelogenin substrate and in addition, it created an extra cleavage site at its C-terminus. CONCLUSIONS: The differences in their catalytic properties and substrate specificities may be attributed to the sequence divergence of MMP-20 between species, especially in the hinge region.

Expression of phosphorylated forms of ERK, MEK, PTEN and PI3K in mouse oral development.

Investigations into molecular mechanisms in vertebrates have examined which growth factors regulate many of the essential underlying cellular processes in development. Growth factors regulate cell proliferation and differentiation through diverse signaling pathways like the MEK (mitogen-activated protein kinase) and ERK (extracellular signal-regulated kinase) pathway. The MEK and ERK pathway can interact with the PI3K (phosphatidylinositol-3-kinase) and PTEN (phosphatase and tensin homologues deleted on chromosome 10) signaling pathway. Interactions between these pathways during development have been extensively studied in many organs; however, the importance of these pathways in oral development is not well known. In this study, we examined the expression of the phosphorylated forms of ERK (pERK), MEK (pMEK), PTEN (pPTEN) and PI3K during mouse development from E13.5 to E16.5. We found unique and overlapping expression of these factors in the craniofacial region, with pERK and pPTEN showing opposing activation patterns in both the tooth and the tongue.

Immunohistochemical detection of matrix metalloproteinase-1 in the periodontal ligament of equine cheek teeth.

The hypsodont equine cheek tooth erupts continuously throughout life. The collagen fibers of the periodontal ligament (PDL) have to remodel constantly to allow the tooth to move in an occlusal direction. Remodeling of the collagen fiber bundles needs to be well-coordinated in order to maintain functional tooth support. The aim of this study was to examine the role of matrix metalloproteinase-1 (MMP-1) in the collagen remodeling of the equine PDL under physiological conditions. Specimens containing the PDL interposed between the dental cementum and the alveolar bone were taken from nine Warmblood horses at three designated horizontal levels: subgingival, middle, and apical. The expression of MMP-1 was detected immunohistochemically. MMP-1 was found to be present in the specimens of all horses. Immunopositive fibroblasts/fibrocytes were accumulated within individual single collagen fascicles. Our results suggest that MMP-1 induced collagen degradation plays a central role in the physiological remodeling of the equine PDL. The distribution of MMP-1 positive fascicles indicates well-directed remodeling which occurs as an asynchronous process, so that only single collagen fascicles are remodeled at the same time. Due to this remodeling of one fascicle at a time, the overall anchorage of the tooth is preserved at all times.

Autosomal ichthyosis with hypotrichosis syndrome displays low matriptase proteolytic activity and is phenocopied in ST14 hypomorphic mice.

Human autosomal recessive ichthyosis with hypotrichosis (ARIH) is an inherited disorder recently linked to homozygosity for a point mutation in the ST14 gene that causes a G827R mutation in the matriptase serine protease domain (G216 in chymotrypsin numbering). Here we show that human G827R matriptase has strongly reduced proteolytic activity toward small molecule substrates, as well as toward its candidate epidermal target, prostasin. To further investigate the possible contribution of low matriptase activity to ARIH, we generated an ST14 hypomorphic mouse strain that displays a 100-fold reduction in epidermal matriptase mRNA levels. Interestingly, unlike ST14 null mice, ST14 hypomorphic mice were viable and fertile but displayed a spectrum of abnormalities that strikingly resembled ARIH. Thus, ST14 hypomorphic mice developed hyperproliferative and retention ichthyosis with impaired desquamation, hypotrichosis with brittle, thin, uneven, and sparse hair, and tooth defects. Biochemical analysis of ST14 hypomorphic epidermis revealed reduced prostasin proteolytic activation and profilaggrin proteolytic processing, compatible with a primary role of matriptase in this process. This work strongly indicates that reduced activity of a matriptase-prostasin proteolytic cascade is the etiological origin of human ARIH and provides an important mouse model for the exploration of matriptase function in ARIH, as well as multiple other physiological and pathological processes.

MMP-20 is predominately a tooth-specific enzyme with a deep catalytic pocket that hydrolyzes type V collagen.

Matrix metalloproteinase-20 (MMP-20, enamelysin) has a highly restricted pattern of expression. In healthy tissues, MMP-20 is observed in the enamel organ and pulp organ of developing teeth and is present only as an activated enzyme. To identify other tissues that may express MMP-20, we performed a systematic mouse tissue expression screen. Among the non-tooth tissues assayed, MMP-20 transcripts were identified only in minute quantities within the large intestine. The murine Mmp20 promoter was cloned, sequenced, and assessed for potential tooth-specific regulatory elements. In silico analysis identified four promoter modules that were common to Mmp20 and at least two of three coregulated predominantly tooth-specific genes that encode ameloblastin, amelogenin, and enamelin. We asked if the highly restricted MMP-20 expression pattern was associated with a broad substrate specificity that might preclude its expression in other tissues. An iterative mixture-based random doedecamer peptide library screen with Edman sequencing of MMP-20 cleavage products revealed that, among MMPs previously screened, MMP-20 had unique substrate preferences. These preferences indicate that MMP-20 has a deep and wide catalytic pocket that can accommodate substrates with large aromatic residues in the P1' position. On the basis of matrices derived from the peptide library data, we identified and then confirmed that type V collagen is an MMP-20 substrate. Since type V collagen is not present in dental enamel but is an otherwise widely distributed collagen, and since only active MMP-20 has been observed in teeth, our data suggest that control of MMP-20 activity is primarily regulated by transcriptional means.

[Primary culture of human periodontal ligament fibroblasts by explants with enzymatic digestion].

OBJECTIVE: To increase the success rate of primary culture of human periodontal ligament fibroblasts (HPLF), and to establish an experimental model for studying HPLF in vitro. METHODS: The primary cells were isolated from human periodontal ligament by explants with enzymatic digestion method. Morphological analysis and immunocytochemical staining were used to characterize the cell lineage, and growth curve assay to evaluate the biological features of HPLF. RESULTS: The success rate of primary culture of HPLF was 77.8%. Cultured cells were spindle-shaped, and had a positive reaction to antibodies against vimentin, and a negative reaction to antibodies against keratin. Their morphological and biological characteristics were similar to those of typical HPLF. Growth of HPLF obtained by this method was satisfactory. CONCLUSION: The success rate of primary culture of HPLF is significantly increased by explants combined with enzymatic digestion. The method is simple and feasible.

Temperospatial expression of matrix metalloproteinases 1, 2, 3, and 9 during early tooth development.

Odontogenesis involves a complex series of processes including epithelial-mesenchymal interactions, morphogenesis, differentiation, fibrillogenesis, and mineralization. Extracellular (ECM) remodeling plays a critical role in the rapid morphological changes that accompany these events. It is proposed that matrix metalloproteinases (MMPs) participate in the remodeling of tooth-specific matrices that accompanies the developmental events. MMPs are zinc-requiring endopeptidases that are centrally involved in the controlled turnover of ECM components and are key to a varied range of developmental processes. Thus, the aim of this study was to examine the expression of MMPs 1, 2, 3, and 9 within the developing tooth germ of Wistar rats, using immunohistochemical localisation. During the bud stage, MMPs 1, 2, 3, and 9 were expressed within both epithelial and mesenchymal cells. Later on, during the cap stage, differential expression was observed; of note was the expression of MMP 3 within the enamel knot. This study reports the temperospatial expression of MMPs 1, 2, 3, and 9 during early tooth development, and points to them having a key role during this important developmental period.

Conservation and variation in enamel protein distribution during vertebrate tooth development.

Vertebrate enamel formation is a unique synthesis of the function of highly specialized enamel proteins and their effect on the growth and organization of apatite crystals. Among tetrapods, the physical structure of enamel is highly conserved, while there is a greater variety of enameloid tooth coverings in fish. In the present study, we postulated that in enamel microstructures of similar organization, the principle components of the enamel protein matrix would have to be highly conserved. In order to identify the enamel proteins that might be most highly conserved and thus potentially most essential to the process of mammalian enamel formation, we used immunoscreening with enamel protein antibodies as a means to assay for degrees of homology to mammalian enamel proteins. Enamel preparations from mouse, gecko, frog, lungfish, and shark were screened with mammalian enamel protein antibodies, including amelogenin, enamelin, tuftelin, MMP20, and EMSP1. Our results demonstrated that amelogenin was the most highly conserved enamel protein associated with the enamel organ, enamelin featured a distinct presence in shark enameloid but was also present in the enamel organ of other species, while the other enamel proteins, tuftelin, MMP20, and EMSP1, were detected in both in the enamel organ and in other tissues of all species investigated. We thus conclude that the investigated enamel proteins, amelogenin, enamelin, tuftelin, MMP20, and EMSP1, were highly conserved in a variety of vertebrate species. We speculate that there might be a unique correlation between amelogenin-rich tetrapod and lungfish enamel with long and parallel crystals and enamelin-rich basal vertebrate enameloid with diverse patterns of crystal organization.

The localization of matrix metalloproteinase-20 (MMP-20, enamelysin) in mature human teeth.

MMP-20 (enamelysin), the matrix metalloproteinase family member discovered in the enamel organ, has also been detected in odontoblasts during dentin formation. We studied the presence and localization of MMP-20 in mature human teeth in health and disease. In immunohistochemistry, MMP-20-positive staining was observed most intensively in the radicular odontoblastic layer and also in dilated dentinal tubuli of caries lesions. By Western blotting, MMP-20 was detected in odontoblasts and pulp tissue of both sound and carious teeth, in dentinal fluid and dentin of sound teeth, but not in soft carious dentin. We conclude that MMP-20 produced during primary dentinogenesis is incorporated into dentin and may be released during caries progression. The main cellular source of MMP-20 in the dentin-pulp complex is the odontoblasts, which secrete MMP-20 into the dentinal fluid.