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Phosphatases are involved in bone and tooth mineralization, but their mechanisms of action are not completely understood. Tissue-nonspecific alkaline phosphatase (TNAP, ALPL) regulates inhibitory extracellular pyrophosphate through its pyrophosphatase activity to control mineral propagation in the matrix; mice without TNAP lack acellular cementum, and have mineralization defects in dentin, enamel, and bone. PHOSPHO1 is a phosphatase found within membrane-bounded matrix vesicles in mineralized tissues, and double ablation of Alpl and Phospho1 in mice leads to a complete absence of skeletal mineralization. Here, we describe mineralization abnormalities in the teeth of Phospho1-/- mice, and in compound knockout mice lacking Phospho1 and one allele of Alpl (Phospho1-/-;Alpl+/-). In wild-type mice, PHOSPHO1 and TNAP co-localized to odontoblasts at early stages of dentinogenesis, coincident with the early mineralization of mantle dentin. In Phospho1 knockout mice, radiography, micro-computed tomography, histology, and transmission electron microscopy all demonstrated mineralization abnormalities of incisor dentin, with the most remarkable findings being reduced overall mineralization coincident with decreased matrix vesicle mineralization in the Phospho1-/- mice, and the almost complete absence of matrix vesicles in the Phospho1-/-;Alpl+/- mice, whose incisors showed a further reduction in mineralization. Results from this study support prominent non-redundant roles for both PHOSPHO1 and TNAP in dentin mineralization. [ABSTRACT FROM PUBLISHER]

Sphingomyelin phosphodiesterase 3 (Smpd3) encodes a membrane-bound enzyme that cleaves sphingomyelin to generate several bioactive metabolites. A recessive mutation called fragilitas ossium (fro) in the Smpd3 gene leads to impaired mineralization of bone and tooth extracellular matrix (ECM) in fro/fro mice. In teeth from fro/fro mice at various neonatal ages, radiography and light and electron microscopy showed delayed mantle dentin mineralization and a consequent delay in enamel formation as compared with that in control +/fro mice. These tooth abnormalities progressively improved with time. Immunohistochemistry showed expression of SMPD3 by dentin-forming odontoblasts. SMPD3 deficiency, however, did not affect the differentiation of these cells, as shown by osterix and dentin sialophosphoprotein expression. Using a transgenic mouse rescue model (fro/fro; Col1a1-Smpd3) in which Smpd3 expression is driven by a murine Col1a1 promoter fragment active in osteoblasts and odontoblasts, we demonstrate a complete correction of the tooth mineralization delays. In conclusion, analysis of these data demonstrates that Smpd3 expression in odontoblasts is required for tooth mineralization. [ABSTRACT FROM PUBLISHER]

Abstract: Extracellular proteins play a key role in generating and maintaining cohesion and adhesion in biological tissues. These “natural glues” are involved in vital biological processes such as blood clotting, wound healing and maintaining the structural integrity of tissues. Macromolecular assemblies of proteins can be functionally stabilized in a variety of ways in situ that include ionic interactions as well as covalent crosslinking to form protein networks that can extend both within and between tissues. Within tissues, myriad cohesive forces are required to preserve tissue integrity and function, as are additional appropriate adhesive forces at interfaces both within and between tissues of differing composition. While the mechanics of some key structural adhesive proteins have been characterized in tensile experiments at both the macroscopic and single protein levels, the fracture toughness of thin proteinaceous interfaces has never been directly measured. Here, we describe a novel and simple approach to measure the cohesive behavior and toughness of thin layers of proteinaceous adhesives. The test is based on the standard double-cantilever beam test used for engineering adhesives, which was adapted to take into account the high compliance of the interface compared with the beams. This new “rigid double-cantilever beam” method enables stable crack propagation through an interfacial protein layer, and provides a direct way to measure its full traction–separation curve. The method does not require any assumption of the shape of the cohesive law, and the results provide abundant information contributing to understanding the structural, chemical and molecular mechanisms acting in biological adhesion. As an example, results are presented using this method for thin films of fibrin—a protein involved in blood clotting and used clinically as a tissue bio-adhesive after surgery—with the effects of calcium and crosslinking by Factor XIII being examined. Finally, a simple model is proposed, demonstrating how a bell-shaped cohesive law forms during the failure of the fibrin interface based on an eight-chain model whose structure degrades and changes configuration with stress. [Copyright &y& Elsevier]

Bone cell culture systems are essential tools for the study of the molecular mechanisms regulating extracellular matrix mineralization. MC3T3-E1 osteoblast cell cultures are the most commonly used in vitro model of bone matrix mineralization. Despite the widespread use of this cell line to study biomineralization, there is as yet no systematic characterization of the mineral phase produced in these cultures. Here we provide a comprehensive, multi-technique biophysical characterization of this cell culture mineral and extracellular matrix, and compare it to mouse bone and synthetic apatite mineral standards, to determine the suitability of MC3T3-E1 cultures for biomineralization studies. Elemental compositional analysis by energy-dispersive X-ray spectroscopy (EDS) showed calcium and phosphorus, and trace amounts of sodium and magnesium, in both biological samples. X-ray diffraction (XRD) on resin-embedded intact cultures demonstrated that similar to 1-month-old mouse bone, apatite crystals grew with preferential orientations along the (100), (101) and (111) mineral planes indicative of guided biogenic growth as opposed to dystrophic calcification. XRD of crystals isolated from the cultures revealed that the mineral phase was poorly crystalline hydroxyapatite with 10 to 20 nm-sized nanocrystallites. Consistent with the XRD observations, electron diffraction patterns indicated that culture mineral had low crystallinity typical of biological apatites. Fourier-transform infrared spectroscopy (FTIR) confirmed apatitic carbonate and phosphate within the biological samples. With all techniques utilized, cell culture mineral and mouse bone mineral were remarkably similar. Scanning (SEM) and transmission (TEM) electron microscopy showed that the cultures had a dense fibrillar collagen matrix with small, 100 nm-sized, collagen fibril-associated mineralization foci which coalesced to form larger mineral aggregates, and where mineralized sites showed the accumulation of the mineral-binding protein osteopontin. Light microscopy, confocal microscopy and three-dimensional reconstructions showed that some cells had dendritic processes and became embedded within the mineral in an osteocyte-like manner. In conclusion, we have documented characteristics of the mineral and matrix phases of MC3T3-E1 osteoblast cultures, and have determined that the structural and compositional properties of the mineral are highly similar to that of mouse bone. [ABSTRACT FROM AUTHOR]

Abstract: We investigated matrix–mineral relationships in the avian eggshell at the ultrastructural level using scanning and transmission electron microscopy combined with surface-etching techniques to selectively increase topography at the matrix–mineral interface. Moreover, we investigated the distribution of osteopontin (OPN) in the eggshell by colloidal-gold immunolabeling for OPN, and assessed the effects of this protein on calcite crystal growth in vitro. An extensive organic matrix network was observed within the calcitic structure of the eggshell that showed variable, region-specific organization including lamellar sheets of matrix, interconnected fine filamentous threads, thin film-like surface coatings of proteins, granules, vesicles, and isolated proteins residing preferentially on internal {104} crystallographic faces of fractured eggshell calcite. With the exception of the vesicles and granules, these matrix structures all were immunolabeled for OPN, as were occluded proteins on the {104} calcite faces. OPN inhibited calcite growth in vitro at the {104} crystallographic faces producing altered crystal morphology and circular growth step topography at the crystal surface resembling spherical voids in mineral continuity prominent in the palisades region of the eggshell. In conclusion, calcite-occluded and interfacial proteins such as OPN likely regulate eggshell growth by inhibiting calcite growth at specific crystallographic faces and compartmental boundaries to create a biomineralized architecture whose structure provides for the properties and functions of the eggshell. [Copyright &y& Elsevier]

Biomineralization is remarkably diverse and provides myriad functions across many organismal systems. Biomineralization processes typically produce hardened, hierarchically organized structures usually having nanostructured mineral assemblies that are formed through inorganic-organic (usually protein) interactions. Calcium‑carbonate biomineral predominates in structures of small invertebrate organisms abundant in marine environments, particularly in shells (remarkably it is also found in the inner ear otoconia of vertebrates), whereas calcium-phosphate biomineral predominates in the skeletons and dentitions of both marine and terrestrial vertebrates, including humans. Reconciliation of the interplay between organic moieties and inorganic crystals in bones and teeth is a cornerstone of biomineralization research. Key molecular determinants of skeletal and dental mineralization have been identified in health and disease, and in pathologic ectopic calcification, ranging from small molecules such as pyrophosphate, to small membrane-bounded matrix vesicles shed from cells, and to noncollagenous extracellular matrix proteins such as osteopontin and their derived bioactive peptides. Beyond partly knowing the regulatory role of the direct actions of inhibitors on vertebrate mineralization, more recently the importance of their enzymatic removal from the extracellular matrix has become increasingly understood. Great progress has been made in deciphering the relationship between mineralization inhibitors and the enzymes that degrade them, and how adverse changes in this physiologic pathway (such as gene mutations causing disease) result in mineralization defects. Two examples of this are rare skeletal diseases having osteomalacia/odontomalacia (soft bones and teeth) – namely hypophosphatasia (HPP) and X-linked hypophosphatemia (XLH) – where inactivating mutations occur in the gene for the enzymes tissue-nonspecific alkaline phosphatase (TNAP, TNSALP, ALPL) and phosphate-regulating endopeptidase homolog X-linked (PHEX), respectively. Here, we review and provide a concept for how existing and new information now comes together to describe the dual nature of regulation of mineralization – through systemic mineral ion homeostasis involving circulating factors, coupled with molecular determinants operating at the local level in the extracellular matrix. For the local mineralization events in the extracellular matrix, we present a focused concept in skeletal mineralization biology called the Stenciling Principle – a principle (building upon seminal work by Neuman and Fleisch) describing how the action of enzymes to remove tissue-resident inhibitors defines with precision the location and progression of mineralization. Unlabelled Image • A review on extracellular matrix mineralization in the skeleton. • Extracellular matrix composition and structure of bone is described. • Mechanisms of mineralization in bone are described. • TNAP enzyme degrades mineralization inhibitor PP i (reference to hypophosphatasia). • PHEX enzyme degrades mineralization inhibitor OPN (reference to X-linked hypophosphatemia). [ABSTRACT FROM AUTHOR]